Escherichia coli

Arch Oral Biol, 1997 Jan, 42(1), 19 - 24
Cloning and expression of FomA, the major outer-membrane protein gene from Fusobacterium nucleatum T18; Haake SK et al.; The major outer-membrane protein . FomA, of Fusobacterium nucleatum has been associated with porin activity, interbacterial adherence and stimulation of host immune cells . Until now, molecular analysis of FomA has not been possible because previous attempts to clone the fomA gene were not successful . The inability to clone F . nucleatum genes led to speculation that Escherichia coli may not be a suitable host . This report concerns the amplification of the fomA gene of F . nucleatum T18 using oligonucleotide primers containing restriction endonuclease sites that allow cloning of fomA into the E . coli expression vector pMMB67 . The resultant plasmid, pXWI, was transformed into E . coli DH5 alpha, providing high-level expression of recombinant FomA (rFomA) . Amino acid sequencing of rFomA demonstrated that the FomA signal peptide was correctly processed by E . coli signal peptidase I . rFomA was correctly localized to the outer membrane by the E . coli export pathway . The rFomA protein also displayed the heat-modifiable oligomeric and conformational properties of native FomA (nFomA) . This demonstration of rFomA expression, processing, export, and secondary and tertiary structure in E . coli provides support for the feasibility of molecular analysis of the structure and function of FomA and other F . nucleatum proteins using recombinant techniques.

Mikrobiologiia, 1997 Jan-Feb, 66(1), 38 - 41
{Participation of exometabolites in adaptation of Escherichia coli to stressors}; Nikolaev IuA; Escherichia coli cells exposed to heat (48 degrees C), cold (5-15 degrees C), oxidative (N-ethylmaleimide), and tetracycline-induced stresses secreted yet unidentified compounds (exometabolites) into the medium . These compounds protected E . coli cells against the same or other stresses . The activity spectra, i.e., the potency of protection against different stresses, varied depending on the origination conditions of the exometabolites.

Mikrobiologiia, 1997 Jan-Feb, 66(1), 101 - 6
{Phenotypic variability of a recombinant luminescent strain of Escherichia coli in water microcosms}; Kargatova TV et al.; The behavior of Escherichia coli Z905, carrying a recombinant plasmid pPHL7 with genes determining ampicillin resistance and bacterial luminescence, and the efficiency of expression of cloned genes were studied after introduction of the strain into model aqueous ecosystems with different trophic chain lengths . The E . coli Z905 variants isolated from ecosystems after different periods of time were found to vary in their resistance to ampicillin (from 50 to 0.05 micrograms/ml) and in the intensity of bioluminescence . An increase in the concentration of the selective factor (ampicillin) or in the extent of the aqueous microcosm blooming restored the expression of the recombinant plasmid genes in some clones.

Chirurg, 1997 Jan, 68(1), 84 - 6
{Retroperitoneal phlegmon after transanal endoscopic microsurgical excision of rectal adenoma}; Klaue HJ et al.; We report the case of a 55-year old male patient who underwent transanal endoscopic microsurgery for recurrent benign rectal adenoma . He developed severe postoperative retroperitoneal phlegmon and sepsis and died 28 days after the operation due to untreatable diffuse intraabdominal bleeding caused by persistent thrombocytopenia.

Mol Med, 1997 Jan, 3(1), 49 - 59
mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility; Zhang M et al.; BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice . In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin . The recombinant protein was produced and its activity in cell culture was assessed . MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe . Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas . Recombinant mMaspin protein was produced in E . coli . Invasion and motility assays were used to assess the biological function of mMaspin . RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level . Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines . The expression is altered at different developmental stages in mammary gland . Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner . As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility . Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions . CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene . The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland . mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.

Arch Insect Biochem Physiol, 1997, 35(1-2), 59 - 69
Expression of EcR and USP in Escherichia coli: purification and functional studies; Elke C et al.; The functional ecdysteroid receptor complex consists of a nuclear receptor heterodimer of ecdysteroid receptor (EcR) and ultraspiracle (USP) . EcR and USP of both Chironomus tentans and Drosophila melanogaster were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST) . Cell lysis and protein solubilization with the anionic detergent sarkosyl yielded preparations of EcR and USP with properties similar to those of the endogenous receptors in various respects . The heterodimer of the expressed proteins specifically bound the labeled ecdysteroid (Ec) {3H}ponasterone A . Furthermore, it preferentially recognized the palindromic ecdysone response element (EcRE) PALI . Interestingly, binding to the PAL1 element was also observed for EcR homodimers . USP homodimers, in turn, preferentially bound to the direct repeat element DR1 . When incubated with native polytene chromosomes of Chironomus, EcR/USP specifically accumulated at the early Ec-inducible puff site IV-2B.

Eur Urol, 1997, 31(3), 347 - 9
Neonatal adrenal abscesses; Steffens J et al.; Unilateral or bilateral suprarenal abscesses are rare in the neonate . We describe 2 cases, an 8-week-old Syrian female with a right, and a 6-week-old white female infant with a left adrenal abscess, both following adrenal hemorrhage . The etiology and treatment are discussed.

Life Sci, 1997, 60(19), 1669 - 77
Effects of low power laser-irradiation on differential blood count and body temperature in endotoxin-preimmunized rabbits; Schindl L et al.; Low power laser irradiation has been shown to have various immune-modulatory effects under in vitro conditions but little is known about such effects in animal models . Escherichia coli endotoxin-preimmunized rabbits were used to determine the influence of transcutaneously applied low power laser light on differential blood count and rectal temperature . After three initial immunizations animals were either boostered with 5 ng/kg of endotoxin or injected with pyrogen-free saline and subsequently underwent irradiation using two different wavelengths of red laser light and sham irradiation, respectively . Differential blood count of laser-treated animals was characterized by significantly higher lymphocyte values and lower neutrophil values at twenty hours (boostered rabbits) and twenty-three hours (non-boostered rabbits) after irradiation . Differential blood cell counts returned to baseline values within 23 hours in the boostered animals, whereas in the non-boostered rabbits lymphocytes showed a trend to further increase . Recording of rectal temperature revealed a further rise after laser application, changes being of greater magnitude and longer duration in the non-boostered animals . These results seem to indicate that a single low power laser irradiation can modulate immune-responses depending on the immunological status of the organism.

Life Sci, 1997, 60(18), 1535 - 44
Inhibition of human glutathione S-transferases by basic triphenylmethane dyes; Glanville SD et al.; Human glutathione S-transferases (GSTs) of the Alpha-, Mu- and Pi- classes were expressed in E . coli and isolated by affinity chromatography . They were tested for their susceptibility to inhibition by basic triphenylmethane dyes . hGSTA 1-1 was inhibited by Malachite Green with a Ki value of the order of 10 microM . The inhibitory species appeared to be the dye-GSH adduct . This isoenzyme was not inhibited by either Crystal Violet or Ethyl Violet at concentrations up to 50 microM . hGSTM 2-2 was weakly inhibited by all three dyes tested with Ki values being in the range 40-80 microM . For all dyes the inhibition was best characterised as non-competitive . hGSTP 1-1 was not inhibited by Crystal Violet or by Ethyl Violet but was strongly inhibited by Malachite Green (Ki = 0.3 microM) . The mode of inhibition appeared to be non-competitive but it seems probable that the mechanism is complex . There is at present no evidence to show clearly whether the dominant inhibitory species is the free dye or the adduct.

Parasite Immunol, 1997 Jan, 19(1), 41 - 6
Serological reactivity to heat shock protein 70 in patients with hydatid disease; Colebrook AL et al.; Heat shock protein 70 (hsp70) was chosen as a model antigen with which to investigate autoantibody production in humans with cystic hydatid disease . Levels of specific serum antibody were assessed in the sera of patients with surgically confirmed infection with E . granulosus and sera from non-infected controls . Antigens used were human hsp70, obtained from K562 cells grown in culture, and E . granulosus hsp70 obtained by expression of the full length protein in Escherichia coli following cloning of the associated mRNA . Antibody reactivity to human hsp70 was detected in the sera of only a small proportion of hydatid patients (10%) as well as a similar proportion of sera from age matched controls . Specific antibodies reactive with E . granulosus hsp70 were detected in 60% of hydatid patients, although some samples (21%) from healthy controls also reacted with E . granulosus hsp70, the level of reactivity was significantly higher in hydatid patients . This report identifies E . granulosus hsp70 as an immunogen during human hydatid infection but, despite its having a predicted 81% protein sequence homology with human hsp70, it does not appear to induce autoimmune reactivity against the homologous human protein.

J Dairy Sci, 1997 Jan, 80(1), 67 - 74
Preinfection in vitro chemotaxis, phagocytosis, oxidative burst, and expression of CD11/CD18 receptors and their predictive capacity on the outcome of mastitis induced in dairy cows with Escherichia coli; Van Werven T et al.; Four to 6 wk after parturition, 12 cows in second, fourth, or fifth lactation were experimentally infected in one gland with Escherichia coli . The capacity of chemotaxis, phagocytosis, oxidative burst, and expression of CD11/CD18 receptors to predict the severity of IMI was measured . Bacterial counts in the infected quarter, expressed as area under the curve, and residual milk production in the uninfected quarters were compared to determine severity of the infection . Although these two outcome parameters were highly negatively correlated, regression models with preinfection tests for leukocyte function fitted best with bacterial counts as an outcome parameter . Of the preinfection tests for leukocyte function, chemotaxis best predicted the outcome of the IMI that had been experimentally induced by E . coli . The number of circulating peripheral leukocytes just prior to inoculation was used to predict 52 and 45% of the severity of IMI for bacterial counts and residual milk production, respectively . As a categorical variable, parity predicted 75 and 56% of the severity of IMI expressed as bacterial counts and residual milk production, respectively . Because of the strong effect of parity on the outcome of the experimentally induced mastitis, analysis was performed to discriminate between second parity cows and older cows . Significant differences were found for the number of circulating peripheral leukocytes and for the expression of CD11b/CD18 and CD11c/CD18 receptors between younger and older cows.

Environ Mol Mutagen, 1997, 29(2), 180 - 8
The influence of DNA repair by Ogt alkyltransferase on the distribution of alkylnitrosourea-induced mutations in Escherichia coli; Vidal A et al.; To determine the influence of DNA repair by Ogt alkyltransferase on the distribution of alkylnitrosourea-induced mutations, we have analysed in Ogt-proficient and Ogt-deficient bacterial strains the DNA sequence changes of a total of 357 independent mutations occurring within the initial part of the lacl gene of Escherichia coli . The majority (>80%) of mutations induced by either N-ethyl-N nitrosourea (ENU) or N-methyl-N-nitrosourea (MNU) in the two genetic backgrounds were G:C --> A:T transitions, consistent with the predominant role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents . The analysis of the distribution of G:C --> A:T transitions induced by ENU in Ogt+ and Ogt bacteria reveals an influence of the 5'-flanking base at the level of repair by Ogt alkyltransferase . The Ogt protein appears more efficient at repairing O6-ethylguanine lesions, which are flanked 5' by a G or C, in agreement with previously reported data from our group for ethylmethane sulfonate . In contrast, no preference could be inferred for the repair of O6-methylguanine lesions by Ogt protein . These results seem to indicate that the preference of the Ogt alkyltransferase to repair certain DNA sequences might be a function of the size of the alkyl group . The importance of the alkyl group length has been described also at the level of the (A)BC excinuclease machinery that seems to have a DNA sequence specificity opposite to that of Ogt alkyltransferose.

Clin Exp Allergy, 1997 Jan, 27(1), 96 - 103
Pentoxifylline attenuates LPS-induced bronchial hyperresponsiveness but not the increase in exhaled nitric oxide; Rolla G et al.; BACKGROUND: Inhaled endotoxin (LPS) may cause a transient increase in airway responsiveness, possibly through a cytokine-mediated airway inflammation, which is associated with an increase in nitric oxide synthesis and release . OBJECTIVE: We wondered whether pentoxifylline (PTX), which may attenuate cytokine release induced by LPS, could inhibit LPS-induced increase in airway responsiveness . METHODS: Methacholine (Mch) bronchial responsiveness was assessed 2 and 24 h after saline or LPS inhalation in eight subjects with bronchial hyperresponsiveness (PD20FEV1 610 +/- 53 micrograms), treated with iv saline or PTX, in a double-blind crossover design . Nitric oxide (NO) in the exhaled air, which was expected to increase after LPS inhalation, and PEFR values were also measured at baseline, hourly for 6 h and 24 h later . RESULTS: After LPS inhalation PEFR decreased significantly compared with placebo inhalation, reaching a maximum decrease of 11.25 +/- 1.05 and 4.5 +/- 0.84% of baseline, at 2 h, respectively during saline and PTX infusion, P < 0.001 . Exhaled NO were elevated after LPS compared with placebo inhalation at 1 h (35.6 +/- 4.8 vs 18 +/- 2.8 ppb, P < 0.001), with no difference during saline or PTX infusion . Exhaled NO remained elevated until the 6th hour . PD20FEV1 2h after LPS inhalation was significantly lower than after placebo inhalation both during saline infusion (234 +/- 29 vs 625 +/- 62 micrograms, P < 0.001) and during PTX infusion (441 +/- 47 vs 616 +/- 48 micrograms, P < 0.001), the difference between saline and PTX being significant (P < 0.01) . At 24 h no difference in PEFR, PD20FEV1 and exhaled NO was observed in comparison with pre-study values . CONCLUSION: PTX attenuates both the decrease in airway patency and the increase in bronchial responsiveness induced by LPS inhalation, without any significant change in exhaled NO, which is increased by LPS inhalation.

Biochemistry (Mosc), 1997 Jan, 62(1), 95 - 103
Denaturation of uridine phosphorylase from Escherichia coli K-12 with guanidine hydrochloride: kinetics of inactivation, dissociation, and reactivation of the enzyme; Burlakova AA et al.; Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride is accompanied by the displacement of the maximum in the protein fluorescence spectrum (lambda max) from 331 to 348 nm . The half-maximal change in the lambda max position is observed at 1.18 M guanidine hydrochloride . For this concentration of denaturant, the sedimentation pattern consists of two boundaries, one of which corresponds to the motion of the hexameric enzyme form (s20,w = 8.2 S) and other represents a monomer (s20,w = 2.6 S) . In the presence of 2 M guanidine hydrochloride the enzyme moves as a monomer . The kinetics of inactivation of uridine phosphorylase by guanidine hydrochloride are complex (minima and maxima are observed on the kinetic curves) . The initial rate of the enzyme reactivation after dilution of the enzyme preincubated with guanidine hydrochloride is second order with respect to protein . It is assumed that the rate of the reactivation process is limited by the reassociation of low-activity monomers into dimers followed by a rapid hexamer formation . The second-order rate constant for the reassociation of the enzyme is 3.0.10(4) M-1.sec-1 (50 mM borate buffer, pH 7.7, containing 100 mM inorganic phosphate; 20 degrees C) . Thiol groups become accessible to titration by 5,5'-dithiobis-(2-nitrobenzoic acid) after treatment of uridine phosphorylase with guanidine hydrochloride . Uridine and uracil inhibit the unfolding of the protein globule by guanidine hydrochloride.

Reprod Fertil Dev, 1997, 9(1), 77 - 83
Progress towards using recombinant myxoma virus as a vector for fertility control in rabbits; Robinson AJ et al.; The history of myxoma virus, its use in Australia as a mortality agent and the development of the virus as a vector for controlling fertility in wild rabbit populations in Australia is reviewed . Myxoma virus recombinants have been constructed to express model antigens . Four potential insertion sites in the genome have been identified and two have been used to construct single and double recombinant viruses expressing Escherichia coli enzymes beta-galactosidase and beta-glucuronidase . Another recombinant expressing an influenza virus haemagglutinin gene (A/PR8/34) induced high and sustained antibody responses following intradermal inoculation in rabbits . To demonstrate the potential of introducing a recombinant virus into wild rabbit populations, a virus containing a natural deletion was released at four field locations . Preliminary analysis of the data has shown that the introduced virus spread well on 3 of the 4 locations . The steps being taken to address the ethical and safety implications of the introduction of a recombinant virus into the field are discussed.

Appl Biochem Biotechnol, 1997 Jan, 62(1), 15 - 27
Cloning and expression of the gene for xylose isomerase from Thermus flavus AT62 in Escherichia coli; Park BC et al.; The gene encoding xylose isomerase (xylA) was cloned from Thermus flavus AT62 and the DNA sequence was determined . The xylA gene encodes the enzyme xylose isomerase (XI or xylA) consisting of 387 amino acids (calculated Mr of 44,941) . Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene . The XI gene was stably expressed in E . coli under the control of tac promoter . XI produced in E . coli was simply purified by heat treatment at 90 degrees C for 10 min and column chromatography of DEAE-Sephacel . The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis . However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer . The enzyme has an optimum temperature at 90 degrees C . Thermostability tests revealed that half life at 85 degrees C was 2 mo and 2 h at 95 degrees C . The optimum pH is around 7.0, close to where by-product formation is minimal . The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes . The K(m) values for various sugar substrates were calculated as 106 mM for glucose . Divalent cations such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.

Radiats Biol Radioecol, 1997 Jan-Feb, 37(1), 30 - 4
{Changes in the activity level of tumor necrosis factor in the blood serum after irradiation and a combined radiation-thermal lesion}; Petrov VN et al.; To elucidate the role of the tumor-necrosis factor (TNF) in the pathogenesis of an additional aggravating effect of a thermal burn on the development and outcome of a radiation disease, the changes in the activity of cytokine in the blood serum of irradiated mice were studied . The mice were exposed to radiation at a dose of 7 Gy and subjected to a combined radiation-thermal injury (CRTI) . The level of endogenous and LPS-induced TNF was determined in a cytotoxic test on a mice fibroblast L-929 culture . It was found that the activity of TNF in the blood circulation of mice subjected to irradiation and CRTI did not increase much . As a result of an intravenous injection of a provoking stimulus (LPS from E . coli), an increase in the TNF activity in the early period after irradiation was higher than with CRTI . There is no correlation between the increase in the death rate for mice with a combined injury and the changes in the LPS-induced TNF activity.

Dig Dis, 1997 Jan-Apr, 15(1-2), 67 - 91
Enterohemorrhagic Escherichia coli: a family of emerging pathogens; Noel JM et al.; Enterohemorrhagic Escherichia coli (EHEC) have emerged over the last decade as important enteric pathogens because of their potential to induce both hemorrhagic colitis and fatal hemolytic uremic syndrome (HUS) . HUS following EHEC colitis has become the leading cause of pediatric renal failure requiring kidney transplant in North America . The ability for EHEC to induce disease is dependent upon their ability to adhere to the intestinal mucosa in an intimate fashion, and to produce potent cytotoxins . These virulence factors (toxin production and enteroadherence) have been implicated in the pathogenesis of EHEC-induced disease . In this review we will discuss the symptomatology, epidemiology, laboratory diagnosis, pathogenesis, complications, treatment, and prevention of EHEC disease . We will review HUS with emphasis on treatment and prevention . Finally we will review animal models for EHEC infection in order to discuss their role in developing new strategies for the treatment and prevention of EHEC-associated diseases.

Pathol Biol (Paris), 1997 Jan, 45(1), 34 - 40
{Routine detection of beta-lactamases TEM resistant to inhibitors (IRT) and oxacillinases (OXA) in Escherichia coli.}; Libert JM et al.; Comparative study of twenty two strains of Escherichia coli producing a well-characterized penicillinase of the OXA type (oxacillinase) or of the IRT type (TEM resistant to inhibitors) evidenced different criteria leading to the distinction of two groups of strains, according to the type of the harboured enzyme . Applied to eleven clinical strains, these criteria were always concordant with the determination of the isoelectric point, of hybridization and of oligotyping using probes for the classification into OXA or IRT type . We therefore propose the measurement of three inhibition diameters on antibiogram (cefepime, mecillinam and ceftazidime) for the routine distinction of the two enzymes . The interest of the easy characterization of these enzymes is reinforced by our findings that, as for all OXA strains, the inoculum effect which is displayed by some third-generation cephalosporins is very important and should be taken into account in the treatment of severe infections.

Haemostasis, 1997 Jan-Feb, 27(1), 16 - 24
Effects of prostacyclin substitution on systemic procoagulant turnover and cardiorespiratory variables in experimental hypercoagulability; Scherer R et al.; Endotoxin infusion (lipopolysaccharide from Escherichia coli 120 micrograms kg-1 i.v.) was titrated to produce hypercoagulability in rabbits and the effects of prostacyclin (PGI2) treatment (continuous infusion of 6 ng kg-1 min-1 i.v.) on coagulation variables, cardiorespiratory variables, and fibrin deposition in the microcirculation of vital organs were studied . PGI2 infusion did not influence the concentration of soluble fibrin, thrombelastographic variables, or systemic platelet aggregability . Fibrin deposition in the microcirculation of the liver and the lungs was reduced to 50% of that observed in untreated animals (p < 0.01) . The antiplatelet properties of PGI2 were unable to reduce experimental endotoxin-induced systemic procoagulant turnover but improved organ perfusion during the initial phase of disseminated intravascular coagulation.

Dev Neurosci, 1997, 19(2), 210 - 8
Astrocytic lineage analysis by detection of GFAP promoter activity in vitro; Morita N et al.; To survey the emergence and onset of differentiation of the astrocytic lineage in the developing mouse cerebral wall, the promoter activity of a 2.5 kb 5'-flanking region of glial fibrillary acidic protein (GFAP) was measured in individual developing brain cells using a retrovirus-mediated gene transfer system . We identified precursors for astrocytes in primary culture of embryonic mouse cerebral wall cells by detection of GFAP promoter activity, which was detected approximately 3 days prior to the appearance of GFAP immunoreactivity . Since retroviruses only integrate into the chromosomes of actively proliferating cells, cells detected by this method should have been mitotically active at the time of retroviral infection on day 15 postfertilization (E15) . Furthermore, we observed that cells activating GFAP promoter were located near the ventricular surface of cultured cerebral wall slices as a cluster of spherical cells . These results demonstrate that precursor cells for astrocytes exist within the germinative zone of developing cerebral wall, and that these cells are mitotically active on day E15, which is a late stage of neuronal production period in the mouse cerebral wall . The morphology, location and mitotic activity of these cells suggest that they are unlikely to be cells that have been transformed from radial glial cells.

Life Sci, 1997, 60(15), 1223 - 30
Role of nitric oxide in lipopolysaccharide-induced mortality from spontaneously hypertensive rats; Yen MH et al.; To investigate whether nitric oxide (NO) contributed to a higher mortality induced by lipopolysaccharide (LPS) in spontaneously hypertensive rats (SHR), NO synthase inhibitors were used to examine the mortality from LPS in SHR and normotensive Wistar-Kyoto (WKY) rats . We evaluated the mortality from LPS in a series of doses (5, 10, or 20 mg/kg, i.v.) in the anesthetized rat . Plasma nitrite was measured before and at 1, 2, and 3 h after treated rats with LPS (5 mg/kg, i.v.) . Pressure responses to N omega-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG) were performed in rats treated with or without LPS for 3 h . Thoracic aortic cyclic guanosine 3',5'-monophosphate (cGMP) levels were also assessed . Our results demonstrated that injection of LPS caused a dose-dependent mortality in both strains, having a more marked effect in SHR . The survival time of rats after injection of LPS (5 mg/kg, i.v.) was much shorter in SHR . A higher basal level of plasma nitrite was observed in SHR and this difference was further augmented by LPS . The administration of L-NAME (3 mg/kg, i.v.) and AG (15 mg/kg, i.v.) 3 h after LPS had no significant effects on the survival time of WKY rats, but significantly prolonged that of SHR to a similar time of WKY rats . The injecton of L-NAME prior to LPS increased blood pressure of WKY rats by 28+/-5 mmHg and increased that of SHR by 38+4 mmHg . At 3 h after LPS, L-NAME had a greater pressor effect in SHR than in WKY rats . By contrast, before rats injected with LPS, AG slightly increased blood pressure of SHR by 7+/-3 mmHg but not of WKY rats (3+/-2 mmHg), whereas it also had a greater pressor effect in SHR than in WKY rats after treated rats with LPS for 3 h . In addition, LPS induced a higher level of cGMP in SHR than in WKY rats, which was attenuated by in vitro treatment of aortic rings from LPS-rats with L-NAME or AG to a similar level in SHR and WKY rats . These results suggest that a higher level of NO evoked by LPS is associated with a higher mortality in SHR and we propose that the elevated NO synthesis in SHR may play an important role in the compensatory mechanisms activated to combat the hypertensive state.

Dtsch Tierarztl Wochenschr, 1997 Jan, 104(1), 29 - 33
{Measurement of skin temperature as a method of detecting febrile diseases in swine}; Wendt M et al.; The objective of the study was to determine whether precise estimation of rectal temperature of pigs is possible by taking their skin temperature in consideration of different factors as body weight, ambient temperature and relative air humidity . Therefore skin temperature of 272 pigs (7-222 kg BW) was measured by direct (thermo couple element) and indirect (infrared thermometer) methods at distinct localisations . In order to investigate different stages of pyretogenesis Escherichia coli endotoxin was administered to 30 of the pigs intravenously . A significant influence on skin temperature could be ascertained for the ambient temperature and the body weight, but not for the relative air humidity . From these data equations were established to estimate rectal temperature (RT) by measurement the skin temperature (ST) at the base of the ear: 1 . Weaners and fattening pigs: RT = ST + 21.867 - 0.089x1 - 0.432x2 + 0.009x3, (r = 0.703) 2 . Sows: RT = ST + 31.511 - 0.074x1 - 0.657x2 - 0.011x3, (r = 0.641) (x1 = ambient temperature, x2 = skin temperature, x3 = body weight) In spite of consideration of ambient temperature and body weight estimation of the rectal temperature was not suitable for clear detection of febrile pigs . Only in 35.45% of the younger pigs and in 29.30% of the sows a definite diagnose (fever yes/no) could be made . This method can only be used as a screening in the herd, if estimation of extreme values allowed the recognition of febrile illness . A follow-up control of the rectal temperature is always necessary in groups of animals, where estimations give no clear results.

J Basic Microbiol, 1997, 37(1), 53 - 69
Discoordinate gene expression of gyrA and gyrB in response to DNA gyrase inhibition in Escherichia coli; Neumann S et al.; The intracellular level of DNA supercoiling is regulated in Escherichia coli by a homeostatic control mechanism that includes DNA gyrase and topoisomerase I gene expression . Despite several biochemical and genetical evidence that supports the existence of a homeostatic regulation mechanism, there are only few studies focusing gyrA and gyrB gene expression in connection to the mechanism involved in the regulation of DNA supercoiling in vivo . To study DNA gyrase gene expression and to be able to isolate mutants with altered expression of DNA gyrase, we constructed a new chromosomal reporter system based on two translational fusions of gyrA and gyrB to lacZ Using this stable monitor system in a robust wild type, we simultaneously studied the influence of several inhibitors of DNA gyrase (quinolones and coumarins) on gyrA and gyrB gene expression as well as on the intracellular level of DNA supercoiling . Surprisingly, we found a delayed and differential response of gyrA and gyrB gene expression following inhibition of DNA gyrase by quinolones or coumarins . Whereas both groups of drugs were able to increase the expression of gyrA, the gyrB gene expression was only induced by the coumarins . Although the action of the quinolones was able to alter DNA supercoiling, we never observed any induction of gyrB from the chromosome . These results revealed that the gene expressio of gyrA appears to be more sensitive to alterations in DNA supercoiling than the gyrB gene expression and suggest that probably additional regulatory mechanisms on the post-translational level might be involved in the regulation of DNA supercoiling and DNA gyrase gene expression.

Immunogenetics, 1997, 45(6), 413 - 21
The gene for the ligand binding chain of the human interferon gamma receptor; Merlin G et al.; In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed . The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons . The extracellular domain is encoded by exons 1 to 5 and by part of exon 6 . The transmembrane region is also encoded by exon 6 . Exon 7 encodes the intracellular domain and the 3' untranslated portion . The gene was located on chromosome 6q23.1, as determined by in situ hybridization . The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity . No consensus-matching TATA or CAAT boxes in the 5' region were found . Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified . Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites . Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone . The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length . In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

Microbiol Immunol, 1997, 41(1), 43 - 50
Characterization of glycoprotein H and L of human herpesvirus 7; Mukai T et al.; The genes encoding the glycoproteins H (gH) and L (gL) of human herpesvirus 7 (HHV-7) have been identified . The gH open reading frame (ORF) was 2,070 base pairs in length and encoded a predicted 690 amino-acid protein . The gH contained characteristics of a transmembrane glycoprotein including 10 consensus N-linked glycosylation sites, 12 cysteine residues, a potential amino-terminal signal sequence and a predicted transmembrane segment located near the carboxyl terminus . The gL ORF was 738 base pairs in length and encoded a predicted 246 amino-acid protein . Four possible N-glycosylation sites and 6 cysteine residues existed within gL . The predicted amino-acid sequences of the HHV-7 gH and human herpesvirus 6 variant A (HHV-6A) gH gene products exhibited 23.6% identity to each other; and those of the gL gene products had 26.0% identity . Upon in vitro translation of the gL gene, the addition of microsomal membranes resulted in two modified products with molecular weights of 32 kDa and 35 kDa from the unmodified initial translation product of 26 kDa . An amino-terminal portion of gH and the full length of gL were expressed as glutathione S-transferase fusion proteins, and these proteins were used to raise immune sera in mice . Lysates of cells infected with HHV-7 were subjected to immunoprecipitation analysis . Approximate molecular weights of 33, 37, 80 and 90 kDa polypeptides were immunoprecipitated with antibodies against the gH protein . Antibodies against the gL protein polypeptides with the same molecular weights were also precipitated, and were observed with the antibodies against the gH protein . These results suggest that HHV-7 gH and gL may form a heterodimeric complex with each other in HHV-7 infected cells, as has been reported for other herpesviruses.

Microbiol Immunol, 1997, 41(1), 33 - 42
Monoclonal antibody #5-2-26 recognizes the phosphatase-sensitive epitope of rabies virus nucleoprotein; Kawai A et al.; We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein . The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein . The MAb did not recognize the N-protein analogues produced in Escherichia coli (E . coli), indicating that the N-gene products were not normally processed in E . coli after translation . On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum) . The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all . These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.

Microbiol Immunol, 1997, 41(2), 131 - 8
Father-to-mother-to-infant transmission of HIV-1: clonally transmitted isolate of infant mutates more rapidly than that of the mother and rapidly loses reactivity with neutralizing antibody; Okamoto Y et al.; The sequences of the V3 loop and surrounding regions of human immunodeficiency virus type-1 from a father-to-mother-to-infant trimmer were studied and the horizontal and vertical transmissions compared . The father's virus was variable for reactivity with neutralizing antibody and sequences of the V3 loop central core sequence . In contrast, the mother's viral sequences were much less diverse and reacted with a virus neutralizing antibody . The infant's viral sequences were also less diverse than those of the father, and N-glycosylation sites were conserved . By phylogenetic analysis, the major clone, of which V3-peptide reacted with the neutralizing antibody, was found to be transmitted from the mother to her infant; however, the mutated minor clones did not bind to the antibody . These findings suggest that both horizontal and vertical virus transmission were selective, and that the clonally transmitted virus in infants mutates more rapidly than viruses in the mother, to whom the virus was horizontally transmitted.

Microbiol Immunol, 1997, 41(2), 83 - 91
Consensus sequence on the genes encoding the major outer surface proteins (OspA and OspB) of Borrelia garinii isolate; Wang J et al.; Japanese Lyme borrelias classified as ribotype IV is predominant among isolates derived from clinical specimens, reservoir rodents and Ixodes persulcatus ticks, and has been characterized as Borrelia garinii . These B . garinii isolates have antigenic and genetic features apparently different from North American, European and other Asian isolates, especially in major outer surface proteins A (OspA) and B (OspB) . In this study, we cloned and sequenced the genes encoding OspA and OspB from B . garinii strain FujiP2 (ribotype IV strain) isolated from I . persulcatus in Shizuoka, Japan . A sequence analysis revealed significant differences to the previously published sequences of ospA and ospB of B . burgdorferi sensu lato . The open reading frames of ospA and ospB consist of 822 and 888 nucleotides corresponding to the proteins of 273 and 295 amino acids, with molecular weights of 29,643 and 31,786 daltons, respectively . The most interesting finding is that the two osp genes share a consensus 282 bp sequence in their carboxy-terminal portions and that the ospB gene is flanked by a 282 bp-long direct repeat sequence . The deduced amino-acid (aa) sequences of OspA and OspB of strain FujiP2 showed 60.1% homology, and have overall similarities of 70.5%, 70.3% and 75.6% to OspAB proteins of B . burgdorferi sensu stricto strain B31, Borrelia afzelii strain ACA1 and Borrelia garinii strain Ip90, respectively.

Microbiol Immunol, 1997, 41(2), 77 - 82
Prevalence and characteristics of enteropathogenic Escherichia coli with the eae gene in diarrhoeic rabbits; Blanco JE et al.; A field study was carried out with the objective of investigating the prevalence of enteropathogenic Escherichia coli (EPEC) with the eae gene in diarrhoeic rabbits . EPEC eae+ were isolated from 60 (74%) of 81 diarrhoeic rabbits sampled in 30 industrial fattening farms localized in the four provinces of Galicia (northwestern Spain) . Attaching and effacing lesions were found in 44 of 50 animals processed for histology . The 111 E . coli strains identified belonged to 19 different O serogroups and 13 biotypes . However, 53 (48%) of the strains belonged to serogroup O103 and 36 (32%) showed the serobiotype O103:B14 . The eae gene was significantly more frequent (100%; 47 of 47) among the highly pathogenic rhamnose-negative strains of serobiotypes O103:B6 and O103:B14 than among the E . coli strains belonging to other serobiotypes (36%; 23 of 64) (P < 0.001) . In this first report about the prevalence of EPEC with the eae gene in rabbits, we conclude that the class of E . coli strains observed is a common cause of diarrhoea in Galician rabbit farms, and that highly pathogenic rhamnose-negative strains of serotype O103:K-:H2 and biotype B14 are specially predominant.

Avian Dis, 1997 Jan-Mar, 41(1), 257 - 60
Isolation and identification of Ornithobacterium rhinotracheale from commercial broiler flocks on the Delmarva peninsula; Odor EM et al.; The growth and biological characteristics of isolates of Ornithobacterium rhinotracheale (ORT) from commercial broiler chickens in the mid-Atlantic region of the U.S.A . appear to be identical to those previously reported in the literature . The clinical disease and lesions are also similar to those reported from other poultry growing regions including South Africa and Europe . The diagnostic cases included in this report were often associated with known respiratory pathogens, namely, lentogenic Newcastle disease virus, and infectious bronchitis virus, and Escherichia coli bacteria . The role of ORT in the disease cases presented in this report is unclear.

Avian Dis, 1997 Jan-Mar, 41(1), 221 - 33
Dynamics of Escherichia coil infection in experimentally inoculated chickens; Pourbakhsh SA et al.; In order to study the dynamics of avian colibacillosis, commercial broiler chickens were inoculated with a pathogenic Escherichia coli strain (01:K1:H7) into the left caudal thoracic air sac . Chickens were euthanatized at different times from 3 to 48 hr postinoculation and examined for bacterial counts and macroscopic and microscopic lesions . The E . coli strain colonized the air sacs, lungs, and trachea and was recovered from blood and all tested extrarespiratory organs of inoculated birds . A gradual increase in bacterial counts in the trachea, lungs, air sacs, and liver was observed from 3 to 12 hr . Clinical signs and macroscopic lesions of colibacillosis were observed in all inoculated birds . Moderate to severe lesions of airsacculitis, pericarditis, perihepatitis, and splenic hypertrophy were observed . Microscopically, inflammatory cell infiltration, serious to fibrinous exudate, and cellular debris on serosal surfaces were present in the liver, spleen, and air sacs . In air sacs, heterophils were present in low numbers perivascularly 3 hr after inoculation and became more numerous by 24 hr postinoculation . Ultrastructurally, epithelial cells in the air sacs and in air capillary regions of the lung were swollen and vacuolated beginning at 3 hr postinoculation . Bacteria were adherent to and present within the epithelial cells at 3 hr postinoculation and were also seen in phagocytic cells and, rarely, in the connective tissue of these organs at 24 hr postinoculation . These results indicate that both air sacs and lungs can be the portal of entry for E . coli into the systemic circulation, probably via damaged epithelium.

Avian Dis, 1997 Jan-Mar, 41(1), 214 - 20
Experimental production of ascites in broiler chickens using infectious bronchitis virus and Escherichia coli; Tottori J et al.; Common commercial strain male broilers were intratracheally inoculated with 0.3 ml of fluid containing 10(3.7) embryo infective doses of infectious bronchitis virus (IBV) at 14 days of age and 7.5 x 10(6) colony-forming units of Escherichia coli at 18 days of age . Ascites was detected in 15 out of 100 infected birds, which was significantly higher than in a control group of 100 mock-infected birds (P < 0.01) . Some parabronchi were blocked by copious exudate containing heterophils and fibrin in the infected birds at 22 days of age although these findings were not seen in the infected birds at 35 days of age or in birds with ascites . The erythrocyte packed cell volume and right ventricle/total ventricle (RV/TV) ratio of birds with ascites were higher than in birds without ascites . The RV/TV weight ratio for the infected group at the age of 35 days was higher than that of the control group . No IBV or E . coli were recovered from the ascitic birds . These findings suggest that these infectious agents induce ascites in the broilers, and then disappear until the birds suffer from ascites.

Growth Factors, 1997, 14(1), 67 - 79
Specificity and functional effects of antibodies to human stem cell factor; Zannettino AC et al.; Three monoclonal antibodies (Mabs), 7H6, 4B10 and Genzyme Mab, and a commercially-available polyclonal antiserum (Genzyme) to human Stem Cell Factor (SCF) were compared for their ability to detect native and recombinant SCF in a variety of assays, and for blocking of SCF function . All antibodies were found to bind to the membrane bound isoform as well as soluble SCF and to bind to both glycosylated (yeast MGF) and unglycosylated (E . coli SCF) recombinant factor . Mabs 7H6 and 4B10, as well as the polyclonal antiserum could immunoprecipitate membrane-associated SCF and all the antibodies could detect recombinant soluble SCF on western blots, although the binding of all except 7H6 was partially sensitive to reduction . Titration of the antibodies on CHO cells expressing membrane-associated human SCF showed similar dose-dependence for all Mabs with 70% of maximum binding seen at 3, 5 and 8 micrograms/ml for 7H6, 4B10 and Genzyme Mab respectively, however the maximum binding seen with 7H6 was approximately 2-fold greater than with 4B10 and 7-fold greater than Genzyme Mab . Competitive binding experiments of the Mabs on cells expressing membrane SCF gave non-reciprocal blocking in all cases with 7H6 completely blocking 4B10 and Genzyme Mab binding . All antibodies except the Genzyme Mab effectively blocked SCF binding to c-Kit-expressing cells, and were strongly inhibitory in an assay of in vitro haemopoiesis which is believed to depend on adhesive interactions, as well as the "classical' cytokine-receptor interaction, mediated by SCF binding to c-Kit.

Biomed Pharmacother, 1997, 51(1), 5 - 12
Consequences of attachment of Helicobacter pylori to gastric cells; Segal ED; Helicobacter pylori, a human pathogen and type 1 carcinogen, causes gastritis, gastric ulcers and gastric cancer . In vivo, H pylori colonizes only gastric surface cells from the antral and fundal regions of the stomach, and heterotopic or metaplastic gastric epithelium present within the esophagus and duodenum . This review summarizes what is known about the association and consequences of attachment between H pylori and gastric cells in vitro, and compares this to the findings demonstrated in vivo . It has been shown that attachment of H pylori to gastric cells results in cup and pedestal formation and cytoskeleton rearrangement similar to that described for enteropathogenic Escherichia coli . Attachment of H pylori induces additional cellular changes in the host cell, including cytokine responses and induction of signal transduction pathways.

Mol Gen Mikrobiol Virusol, 1997, (1), 3 - 7
{Relationship between the UV-induction of exact exclusion of transposons and the function of umuDC, lexA, recA genes and plasmid pkM101}; Rusina OIu et al.; A pair of isogenic strains-E . coli K-12 and E . coli B/r differing by the status of umuDC genes and presence of pKM101 plasmid-were constructed and the relationship between UV induction of transposons Tn5 and Tn 10 and the gene umuDC function shown . This relationship is not absolute, in contrast to that of point mutations . Induction of precise excision of these transposons can be inhibited by pKM101 plasmid . Induction of precise excision of Tn5 and Tn 10 from the sites under study is absolutely lexA- and recA- dependent.

J Biomol NMR, 1997 Jan, 9(1), 79 - 93
Assessment of protein solution versus crystal structure determination using spin-diffusion-suppressed NOE and heteronuclear relaxation data; LeMaster DM; A spin-diffusion-suppressed NOE buildup series has been measured for E . coli thioredoxin . The extensive 13C and 15N relaxation data previously reported for this protein allow for direct interpretation of dynamical contributions to the 1H-1H cross-relaxation rates for a large proportion of the NOE cross peaks . Estimates of the average accuracy for these derived NOE distances are bounded by 4% and 10%, based on a comparison to the corresponding X-ray distances . An independent fluctuation model is proposed for prediction of the dynamical corrections to 1H-1H cross-relaxation rates, based solely on experimental structural and heteronuclear relaxation data . This analysis is aided by the demonstration that heteronuclear order parameters greater than 0.6 depend only on the variance of the H-X bond orientation, independent of the motional model in either one- or two-dimensional diffusion (i.e., 1-S2 = 3/4 sin2 2 theta sigma) . The combination of spin-diffusion-suppressed NOE data and analysis of dynamical corrections to 1H-1H cross-relaxation rates based on heteronuclear relaxation data has allowed for a detailed interpretation of various discrepancies between the reported solution and crystal structures.

Fold Des, 1997, 2(1), 23 - 33
Favourable native-like helical local interactions can accelerate protein folding; Viguera AR et al.; BACKGROUND: Extensive studies of peptide conformation have provided reasonable knowledge of the rules determining helix stability . This knowledge can be used to stabilize proteins against chemical and thermal denaturation . This has been done in two proteins: the chemotactic protein from Escherichia coli, Che Y (a 129 aa alpha/beta parallel protein with five alpha-helices, which shows an accumulating intermediate during refolding) and the activation domain of human procarboxypeptidase A2, ADA2h (a 81 aa alpha + beta protein domain, with two alpha-helices, which follows a two-state mechanism) . As the introduced stabilizing interactions are local in nature, the energy balance between the contribution of local and nonlocal interactions changes considerably . Recent theoretical analyses of protein folding using simplified models have indicated that optimization of folding speed requires this balance to be biased towards nonlocal interactions . To determine whether this is the case, we study here the folding kinetics of two ADA2h mutants in which alpha-helix 1 (mutant M1) or 2 (mutant M2) has been stabilized through local interactions, as well as the equilibrium and kinetic behaviour of a double mutant (DM) in which both helices have been stabilized . RESULTS: The stability of DM is considerably enhanced with respect to wild type (WI) and this mutant can be considered as a thermoresistant protein (Tm > 363 K) . The thermodynamic parameters obtained by chemical denaturation (urea and GdnHCl) show that DM is approximately 2.6 kcal mol-1 more stable than WT . The effects on folding kinetics are different in each of the single mutants . M1 shows very little effect in refolding, while its unfolding is greatly decelerated with respect to WT . M2 shows, together with a deceleration in unfolding, a significant acceleration in refolding . As with equilibrium parameters, the kinetics of the double mutant can be explained by the simple addition of the effects found in each single mutant . Interestingly enough, the refolding slope mkf in mutants M2 and DM is smaller than in the wild-type and M1 mutant . CONCLUSIONS: Thermoresistance can be achieved, in some cases, by increasing favourable native local interactions . The balance between local and nonlocal interactions can be significantly changed in some proteins and still keep a cooperative unfolding transition similar to that of the wild type . The introduction of favourable local interactions by mutational redesign can also be used to increase the folding speed of certain proteins, showing that not all proteins in nature have been optimized for rapid folding, contrary to what has been theoretically indicated . This behaviour is probably also shared by other polypeptides with highly unstructured denatured states . All these phenomena have been shown experimentally in ADA2h by mutations that increase helix stability . However, the effects promoted for such an approach in proteins with residual structure and/or intermediates in the denatured ensemble could be different . This has been shown by experiments performed on CheY in which the cooperativity of the folding process was greatly affected.

J Ind Microbiol Biotechnol, 1997 Jan, 18(1), 49 - 55
Nucleotide sequence of a gene encoding an organophosphorus nerve agent degrading enzyme from Alteromonas haloplanktis; Cheng T et al.; Organophosphorus acid anhydrolases (OPAA) catalyzing the hydrolysis of a variety of toxic organophosphorus cholinesterase inhibitors offer potential for decontamination of G-type nerve agents and pesticides . The gene (opa) encoding an OPAA was cloned from the chromosomal DNA of Alteromonas haloplanktis ATCC 23821 . The nucleotide sequence of the 1.7 -kb DNA fragment contained the opa gene (1.3 kb) and its flanking region . We report structural and functional similarity of OPAAs from A . haloplanktis and Alteromonas sp JD6.5 with the enzyme prolidase that hydrolyzes dipeptides with a prolyl residue in the carboxyl-terminal position . These results corroborate the earlier conclusion that the OPAA is a type of X-Pro dipeptidase, and that X-Pro could be the native substrate for such an enzyme in Alteromonas cells.

Acta Chir Belg, 1997 Jan-Feb, 97(1), 39 - 43
Infected abdominal aortic aneurysm associated with a psoas abscess, aorto-duodenal and sigmoid fistulas . Case report and review of the literature; Louagie YA et al.; A case of atherosclerotic abdominal aortic aneurysm, complicated by aortoenteric fistulizations and infected by Escherichia coli, is presented . Chronic contained rupture resulted in the formation of a huge left psoas abscess which was responsible for the symptoms . No similar case has been reported in the literature . Resection and extra-anatomic vascular reconstruction were curative.

Mech Dev, 1997 Jan, 61(1-2), 165 - 73
Molecular characterization and embryonic expression of the even-skipped ortholog of Tribolium castaneum; Brown SJ et al.; In short germ insects, the procephalon and presumptive anterior segments comprise most of the embryonic rudiment which lengthens as posterior segments are added during development (Sander, K . (1976) Adv . Insect Physiol . 12, 125-238) . The expression pattern of a grasshopper ortholog of the primary pair-rule gene even-skipped (eve) suggests that it is not relevant to segmentation in this short germ insect (Patel, N.H., Ball, E.E . and Goodman, C.S . (1992) Nature 357, 339-342) . However in Drosophila, a long germ insect that forms all segments simultaneously, eve plays a vital role in segment formation (Nusslein-Volhard, C., Wieschaus, E . and Kluding, H . (1984) Roux's Arch . Dev . Biol . 193, 267-282) . We have characterized the eve ortholog of the beetle Tribolium castaneum . The homeodomain sequence is highly conserved between beetle, fly, and grasshopper eve orthologs . Tc eve is expressed in stripes during segmentation, but in a pattern differing in some details from that of the fly gene . This pattern is coincident with that detected with a cross-reacting antibody (Patel, N.H., Condron, B.G . and Zinn, K . (1994) Nature 367, 429-434) . Thus, an ancestral even-skipped gene appears to have evolved a role in segmentation in a common ancestor of flies and beetles . Unlike vertebrate orthologs but similar to eve, Tc eve is not linked to the homeotic complex.

J Surg Res, 1997 Jan, 67(1), 54 - 7
The effect of endotoxin on canine jejunal motility and transit; Cullen JJ et al.; Intestinal transit is rapid during endotoxemia; however, little is known regarding the small intestinal motility changes which produce this rapid intestinal transit . The aim of our study was to determine the degree and duration of disrupted jejunal transit, and changes in jejunal motility following a sublethal dose of endotoxin . Eight dogs underwent construction of jejunal Thiry-Vella fistulas (TVF) with manometry catheters to record motility along the TVF . Following recovery, a 240-kcal liquid meal was given and the TVF was perfused with an isotonic solution . Liquid transit was assessed by bolus of a nonabsorbable marker instilled into the proximal end of the TVF . Recordings of gastrointestinal contractile activity were made digitally to determine postpandial motility . Following completion of the baseline studies, each dog was given a single dose of Escherichia coli lipopolysaccharide (200 micrograms/kg, iv) and the postprandial studies were repeated for the next 3 days . Endotoxin decreased the frequency of jejunal contractions for 2 days while the strength of jejunal contractions was diminished for 1 day . Jejunal transit of liquids was rapid on Postendotoxin Day 1 . The rapid transit was associated with a greater percentage of single pressure waves propagating aborally on Postendotoxin Day 1 than the baseline percentages established prior to endotoxin . We conclude that endotoxemia temporarily disrupts postprandial jejunal motility and transit . The rapid liquid intestinal transit seen with endotoxemia may be due to changes in contractile propagation.

Rapid Commun Mass Spectrom, 1997, 11(4), 405 - 9
Characterization of a mutant recombinant S100 protein using electrospray ionization mass spectrometry; Raftery MJ et al.; Two recombinant proteins derived by thrombin cleavage of a fusion protein between glutathione-S-transferase and CP10 (Chemotactic protein 10 kDa) were separated by C4 reversed-phase high-performance liquid chromatography (RP-HPLC) . Both proteins were recognised by a polyclonal antibody to native CP10 following sodium dodecyl sulphate/polyacryamide gel electrophoresis (SDS/PAGE) and Western blotting . The major form (approximately 90%) had a mass of 10308 Da, by electrospray mass spectrometry (ESI-MS), which compared well with the theoretical mass of rCP10 (10307.6 Da) whereas the minor component (approximately 10%) had a mass of 11333 Da, 1025 mass units greater than expected . One sequence was obtained by N-terminal sequencing, suggesting that the N-terminus was not modified . The mass of peptides isolated after Asp-N digestion and C18 RP-HPLC were determined by ESI-MS and each assigned a probable sequence based on the expected peptide man of rCP10 . The mutant protein produced one additional peak at 10.0 min with mass 1639 Da and the sequence DSHKEQQRGIPGNSS by Edman degradation . The first 5 amino acids corresponded to the last 5 C-terminal amino acids of rCP10 . Analysis of the cDNA sequence of the expression vector used to produce rCP10 indicated that the 10 additional C-terminal amino acids were translated after the insertion of glutamine at the normal TAG stop codon . Another stop codon (TGA) located 27 base pairs downstream halts translation . The calculated mass of the mutant protein is 11332.7 Da, in good agreement with the experimental mass . Readthrough occurs in strains of E . coli (eg JPA101) with the amber mutation supE, and this allowed substitution of glutamine at TAG codons in approximately 5-10% of transcripts.

Gene Ther, 1997 Jan, 4(1), 55 - 62
Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver; de Roos WK et al.; Targeted gene delivery is essential for gene therapy involving in vivo gene transfer . In the present study we analyzed the efficiency and tissue-specificity of gene transfer into the liver with recombinant adenoviruses . Adenovirus vectors carrying the E . coli lacZ gene (Ad.RSV.beta-gal) and the firefly luciferase gene (AdCMV-luc) as reporters were administered to the liver of adult Wistar rats, either via infusion into the portal vein (intraportal infusion; IPI) or via perfusion of the vascularity isolated liver (isolated liver perfusion; ILP) . Ex vivo liver perfusion experiments with Ad.RSV . beta-gal were used to optimize the conditions for hepatic gene transfer . Ex vivo perfusion of rat livers with 2 x 10(9) plaque forming units (p.f.u) Ad RSV.beta-gal was sufficient to infect about 20% of the liver parenchymal cells . Perfusion with chelating agents (1 mM EGTA, or 2 mM EDTA) prior to the administration of the vector increased the efficiency to at least 40% . Similar efficiencies were obtained in experiments with liver lobes of Rhesus monkeys . In vivo administration of AdCMV-luc via ILP resulted in a significantly more efficient (P = 0.028) and also more reproducible gene transfer when compared to IPI . Although detectable in both groups, extrahepatic luciferase expression was considerably reduced in the ILP group . Our data demonstrate that IPL can be used for efficient and reproducible liver-specific gene delivery . Therefore, we think that the perfusion of vascularly isolated organs is useful as a modality for the tissue-specific administration of recombinant adenovirus vectors.

Gene Ther, 1997 Jan, 4(1), 32 - 8
Polycations increase the efficiency of adenovirus-mediated gene transfer to epithelial and endothelial cells in vitro; Arcasoy SM et al.; Recombinant adenoviruses are being developed for gene therapy for cystic fibrosis and other lung diseases, and for prevention and treatment of vascular thrombosis . A major limitation to the clinical utility of adenoviruses is the low efficiency of gene transfer achieved in vivo . In addition, little is known about the initial interactions between adenoviruses and the target cell . To address the hypothesis that the negative charge presented by membrane glycoproteins reduces the efficiency of adenovirus-mediated gene transfer, primary cultures of human airway, Madin-Darby canine kidney cells, an immortalized cystic fibrosis airway epithelial cell line, and primary cultures of sheep pulmonary artery endothelium were infected with recombinant adenovirus containing the E . coli lacZ reporter gene (Ad2 beta gal2) in the presence of various polyions . For each cell type, adsorption of Ad2 beta gal2 in the presence of the polycations polybrene, protamine, DEAE-dextran, and poly-L-lysine significantly increased the percentage of cells that express lacZ . The polyanion heparin did not significantly alter gene transfer efficiency, but completely abrogated the effects of polycations . These data provide evidence that negatively charged moieties on the cell surface reduce the efficiency of adenovirus-mediated gene transfer, and that alteration of the charge interaction between adenoviruses and the cell surface may improve the potential clinical application of these vectors.

Cell Motil Cytoskeleton, 1997, 36(3), 246 - 52
Physical properties of dystrophin rod domain; Kahana E et al.; We have prepared two fragments of the human dystrophin rod domain, each containing eight spectrin-like repeating units, by expression in Escherichia coli . The first corresponds to the central portion of the rod, the other to three repeats from the N-terminal end, fused to five repeats from the C-terminal end . The latter makes up the entire mutant rod, found in a patient with mild (Becker-type) muscular dystrophy . Both fragments were found to possess an ordered, stable structure, and had the form of short rod-like particles in the electron microscope . Molecular weight determinations by sedimentation equilibrium revealed that both polypeptides were monomeric in solution, suggesting that the dystrophin rod domain is incapable of forming an antiparallel homodimer . This supports the inference from sequence analyses {Winder et al., 1995: FEBS Lett . 369:27-33, 1996: Biochem . Soc . Trans . 24:2805} that the dystrophin rod domain lacks the arrangement of sites required for lateral self-association, and that dystrophin, unlike the other known proteins of the spectrin superfamily, may thus exist as a monomer.

Electrophoresis, 1997 Jan, 18(1), 156 - 62
Mapping and identification of Brucella melitensis proteins by two-dimensional electrophoresis and microsequencing; Teixeira-Gomes AP et al.; Two-dimensional (2-D) gel electrophoresis was used to map Brucella melitensis proteins . The 2-D proteins map of B . melitensis B115 revealed 595 silver-stained protein spots separated by both isoelectric point and molecular mass . Twenty-five proteins were identified either by immunoblotting using monoclonal antibodies (MAbs) or by N-terminal microsequencing . The protein spots identified by MAbs were the 89 kDa outer membrane protein, DnaK, bacterioferritin, CP24, and BP26 . Some spots were identified by N-terminal microsequencing as proteins whose sequences had been reported previously from Brucella, such as three heat-shock proteins, namely DnaK, GroEL and GroES; bacterioferritin; Cu-Zn superoxide dismutase; and the 50S ribosomal protein L7/L12 . Other proteins had amino acid sequences homologous with those of various proteins from other bacteria found in protein databases: ClpP; the 10K-S protein; the ORFU phosphoprotein; succinyl-CoA synthetase alpha sub-unit; an inorganic pyrophosphatase; the Fe and/or Mn superoxide dismutase; the nucleoside diphosphate kinase, an amino acid ABC type transporter, and an electron transfer flavoprotein small subunit . Seven proteins were identified with N-terminal sequences not yet reported in databases . The 2-D map established in this study will be the basis for comparative studies of protein expression in Brucella.

Electrophoresis, 1997 Jan, 18(1), 6 - 11
Factors that affect the stability of protein-DNA complexes during gel electrophoresis; Fried MG et al.; The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids . Often it is found that complexes persist within electrophoresis gels for much longer than expected on the basis of their free-solution lifetimes . Volume exclusion, direct interaction with gel matrices and the reduction of water activity by the gel have been proposed as mechanisms enhancing the stability of complexes during electrophoresis . We have used the well-characterized interaction of the E . coli cyclic AMP receptor protein (CAP) with lactose promoter DNA to test these proposals . We found that the activity of water within polyacrylamide gels differs little from that of the buffer in which they were cast and that the dependence of the dissociation rate constant on water activity is too small for osmotic stabilization to contribute significantly to the lifetimes of CAP-DNA complexes . In addition, we found that a cross-linked gel matrix is not required for the stabilization of CAP-DNA complexes, that comparable stabilization is produced by three dissimilar polymers (linear polyacrylamide, dextran and polyethylene glycol), and that these polymers stabilize complexes more effectively than equivalent weight concentrations of their cognate monomers . While these results challenge the notion that direct interaction with the gel matrix contributes to the stability of protein-DNA complexes, they are all features expected of excluded volume mechanisms.

J Biochem (Tokyo), 1997 Jan, 121(1), 145 - 9
Identification of the Ca(2+)-binding domains in reticulocalbin, an endoplasmic reticulum resident Ca(2+)-binding protein with multiple EF-hand motifs; Tachikui H et al.; Reticulocalbin (RCN) is a member of the EF-hand Ca(2+)-binding protein family and is a luminal protein of the endoplasmic reticulum (ER) with a molecular weight of 44,000 {Ozawa, M . and Muramatsu, T . (1993) J . Biol . Chem . 268, 699-705} . Although RCN has six repeats of a domain containing an EF-hand motif, the varying degrees of divergence of the amino acid sequences of these domains from the EF-hand consensus sequences suggested that some domains might have lost their Ca(2+)-binding capability and adopted new functions . To identify the domains involved in Ca(2+)-binding, discrete domains of RCN were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system . 45Ca2+ blot analysis of the resultant fusion proteins revealed that the first, fourth, fifth, and sixth domains bind Ca2+, however, the second and third ones do not . The fusion proteins containing all six domains, and the first and second domains, respectively, showed Ca(2+)-dependent increases in their electrophoretic mobilities, suggesting that Ca2+ induces a conformational change in reticulocalbin.

J Biochem (Tokyo), 1997 Jan, 121(1), 138 - 44
A mutation study of the DNA binding domain of human papillomavirus type11 E2 protein; Matsumoto T et al.; A site-specific mutation study was performed on the C-terminal domain, containing a cloned DNA binding region, of the human papillomavirus type11 (HPV11) E2 protein to determine the specific properties of residues directly involved in the DNA binding . The effect of a point mutations on the DNA binding was assessed by means of a gel mobility shift assay . The mutagenesis was concentrated on the residues in the third helix from the N-terminal, that is known as the "recognition helix," in the crystal structure of the bovine papillomavirus (BPV) E2 protein . Most point mutations caused a great decrease in the DNA binding activity . The leucine repeat in the DNA binding region was proved not to be a leucine prerequisite, as the leucines could be substituted by valine without significant loss of the DNA binding ability . Substitution of Leu for Glu caused a significant decrease in the DNA binding, indicating that the hydrophobicity of the residue at this position is important . The results suggest that the individual contribution of each amino acid residue in the DNA binding region is essential for the DNA binding.

Biol Neonate, 1997, 71(2), 111 - 8
Reticuloendothelial system uptake of infused 125I-trypsin in newborn and adult rats; Levine JJ et al.; Previous studies have demonstrated enhanced intestinal trypsin uptake and decreased liver clearance of trypsin in newborn rats compared to adults . In order to examine the effectiveness of the reticuloendothelial system (RES) in clearing trypsin, bovine trypsin (1.25 mg/100 g body weight) plus trace 125I-trypsin were injected into the portal vein of 2-week-old (n = 57) and adult (n = 44) control rats or following RES stimulation using intraperitoneally injected lipopolysaccharide or RES suppression with intraperitoneally injected oleic acid emulsion . Plasma, liver and spleen 125I activities were assessed at 1, 5 or 15 min following infusion in control, stimulated and suppressed animals . Newborn control rats had significantly increased 125I plasma levels with decreased liver and spleen 125I activity compared to control adults . RES stimulation in the newborns did not lead to any change in liver or plasma levels although splenic values increased while adults had a decrease in liver 125I activity . RES suppression in the newborns led to increased plasma and decreased spleen 125I-trypsin values while adult rat levels were unchanged . The immature reticuloendothelial system in newborns is poorly responsive to RES stimulation although it can be made even further inefficient by RES suppression . The combination of RES immaturity and lack of response to stimulation may make newborns susceptible to proteolytic damage, especially during times of increased systemic levels of proteolytic enzymes.

Can J Microbiol, 1997 Jan, 43(1), 92 - 6
A rapid and efficient system for screening HIV-1 Pol mRNA-specific ribozymes; Ramezani A et al.; Hammerhead ribozymes are potentially important tools for suppressing intracellular expression of unwanted RNAs . However, the reports that exist on their activity against different targets have described mixed success . As an initial step towards developing a rapid and effective system for in vivo testing of ribozymes, two human immunodeficiency virus type-1 (HIV-1) polymerase (Pol) mRNA-specific ribozymes, RzPro directed against the protease (Pro) coding region and RzRT directed against the reverse transcriptase (RT) coding region, were designed and tested in Escherichia coli . Both ribozymes displayed similar efficiencies in cleaving their target RNAs in vitro . RNA polymerase chain reaction was adapted to demonstrate the in vivo cleavage of RzPro and RzRT target sites . The resultant drop in HIV-1 RT activity was measured as well . The degree of suppression of RT activity was more apparent in vivo in cells expressing RzRT . The RT activity in cells expressing RzRT was shown to decrease by up to 96% . This system will be useful for rapid screening of (i) other ribozyme target sites within the Pol mRNA so that multitargeted ribozymes could be designed for use in anti-HIV-1 gene therapy, (ii) ribozymes with improved stability and catalytic activity, and (iii) cofactors, if any that could enhance ribozyme activity in vivo.

J Protein Chem, 1997 Jan, 16(1), 37 - 49
Structure and bioactivity of recombinant human CTAP-III and NAP-2; Proudfoot AE et al.; Connective tissue-activating peptide III (CTAP-III) and neutrophil-activating peptide-2 (NAP-2) are both derived from a common precursor, platelet basic protein (PBP), which is stored in the alpha-granules of platelets and released upon their activation . CTAP-III is an 85-residue peptide which is converted to NAP-2 by enzymic removal of the 15 amino-terminal residues . Both peptides play a role in the early stages of wound healing and inflammation through different activities . We have cloned the cDNA for PBP and expressed constructs coding for the CTAP-III and NAP-2 polypeptides in Escherichia coli . We have purified and renatured these recombinant proteins . The integrity of the recombinant proteins has been ascertained by in vitro bioassays . CTAP-III causes 51% histamine release from the basophilic cell lin KU812 at 10(-7) M, whereas NAP-2 only causes 28% release at the same concentration . In assays on human neutrophils, NAP-2 had an EC50 of 2 x 10(-8) M in chemotaxis, an EC50 of 3 x 10(-8) M for shape change, and could displace IL-8 from neutrophils with a Kd of 7.5 x 10(-9) M . CTAP-III had no activity in these assays . The disulfide bonds have been identified by peptide mapping and sequence analysis, and are in the positions predicted by homology to interleukin-8 and platelet factor 4 . Measurement of the molecular mass at physiologic concentrations by gel permeation chromatography has shown that CTAP-III forms predominantly tetramers and dimers, whereas NAP-2 is only dimetric . SDS/PAGE analysis of samples cross-linked with disuccinimidyl suberate support these topologies . We postulate a mechanism for tetramer formation based on the interaction of the amino-terminal extension in CTAP-III involving a helix-helix interaction that could stabilize the association of two CTAP-III dimers.

Protein Eng, 1997 Jan, 10(1), 63 - 8
Combinatorial manipulation of three key active site residues in glycinamide ribonucleotide transformylase; Warren MS et al.; The enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2) has previously been shown to have three key polar active site residues important for catalysis: N106, H108 and D144 . Mutations of any of these three residues lead to substantially decreased catalytic activity, although none of them are completely irreplaceable . In order to determine whether any alternative arrangement of amino acids at these three positions could lead to an active protein, all three of these residues were simultaneously subjected to saturation site-directed mutagenesis . The resulting combinatorial library of mutant genes was screened for those encoding active proteins using functional complementation . Glycinamide ribonucleotide transformylase was found to be capable of tolerating no more than one mutation amongst these key residues, since the only proteins found to be sufficiently active to allow growth of auxotrophic cells on selective media were the wild-type and enzymes containing a single mutation to one of these residues . It seems likely that no enzymes containing two or more mutations of these three residues possess significant catalytic activity . The combinatorial approach used could prove to be quite useful in protein engineering and protein evolution experiments.

J Exp Biol, 1997 Jan, 200 ( Pt 2), 353 - 60
Regulation of Na+/H+ antiporter in trout red blood cells; Malapert M et al.; The trout red blood cell Na+/H+ antiporter (beta NHE) plays two interesting properties: it is the only NHE own to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself . Cloning and expression of beta NHE have provided inificant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter . Expression of beta NHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge a common kinase . Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses . NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a "NHE1 transmembrane domain/beta NHE cytoplasmic domain' chimera is fully activated by cyclic AMP . In red cells, activation of beta NHE is the result of phosphorylation by PKA of at least two independent sites . Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites . Furthermore, Calyculin A (CIA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter . It is suggested that CIA may be able to revive "sequestered' antiporters . We propose that the molecular events underlying beta NHE desensitisation could be similar to those involved in rhodopsin desensitisation . Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger . Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.

J Exp Biol, 1997 Jan, 200 ( Pt 2), 335 - 41
EmrE, the smallest ion-coupled transporter, provides a unique paradigm for structure-function studies; Schuldiner S et al.; EmrE is an Escherichia coli multidrug transporter which confers resistance to a wide variety of toxicants by actively removing them in exchange for hydrogen ions . EmrE is a highly hydrophobic 12 kDa protein which has been purified by taking advantage of its unique solubility in organic solvents . After solubilization and purification, the protein retains its ability to transport as judged from the fact that it can be reconstituted in a functional form . Hydrophobicity analysis of the sequence yielded four putative transmembrane domains of similar sizes . Results from transmission Fourier transform infrared measurements agree remarkably well with this hypothesis and yielded alpha-helical estimates of 78% and 80% for EmrE in CHCl3:MeOH and 1,2-dimyristoyl phosphocholine, respectively . Furthermore, the fact that most of the amide groups in the protein do not undergo amide-proton H/D exchange implies that most (approximately 80%) of the residues are embedded in the bilayer . These observations are only consistent with four transmembrane helices . A domain lined by Cys41 and Cys95 accessible only to substrates such as the organic mercurial 4-(chloromercuri)benzoic acid has been identified . Both residues are asymmetric in their location with respect to the plane of the membrane, Cys95 being closer than Cys41 to the outside face of the membrane . In co-reconstitution experiments of wild-type protein with three different inactive mutants, negative dominance has been observed . This phenomenon suggests that EmrE is functional as a homo-oligomer.

J Exp Biol, 1997 Jan, 200 ( Pt 2), 217 - 24
Coupling H+ transport and ATP synthesis in F1F0-ATP synthases: glimpses of interacting parts in a dynamic molecular machine; Fillingame RH; Reversible, F1F0-type ATPases (also termed F-ATP synthases) catalyze the synthesis of ATP during oxidative phosphorylation . In animal cells, the enzyme traverses the inner mitochondrial membrane and uses the energy of an H+ electrochemical gradient, generated by electron transport, in coupling H+ translocation to ATP formation . Closely related enzymes are found in the plasma membrane of bacteria such as Escherichia coli, where the enzymes function reversibly depending upon nutritional circumstance . The F1F0-type enzymes are more distantly related to a second family of H(+)-translocating ATPases, the vacuolar-type or V-ATPases . Recent structural information has provided important hints as to how these enzymes couple H+ transport to the chemical work of ATP synthesis . The simplest F1F0-type enzymes, e.g . as in E . coli, are composed of eight types of subunits in an unusual stoichiometry of alpha 3 beta 3 gamma delta epsilon (F1) and a1b2c12 (F0) . F1 extends from the membrane, with the alpha and beta subunits alternating around a central subunit gamma . ATP synthesis occurs alternately in different beta subunits, the cooperative tight binding of ADP + Pi at one catalytic site being coupled to ATP release at a second . The differences in binding affinities appear to be caused by rotation of the gamma subunit in the center of the alpha 3 beta 3 hexamer . The gamma subunit traverses a 4.5 nm stalk connecting the catalytic subunits to the membrane-traversing F0 sector . Subunit c is the H(+)-translocating subunit of F0 . Protonation/deprotonation of Asp61 in the center of the membrane is coupled to structural changes in an extramembranous loop of subunit c which interacts with both the gamma and epsilon subunits . Subunits gamma and epsilon appear to move from one subunit c to another as ATP is synthesized . The torque of such movement is proposed to cause the rotation of gamma within the alpha 3 beta 3 complex . Four protons are translocated for each ATP synthesized . The movement of gamma and epsilon therefore probably involves a unit of four c subunits . The organization of subunits in F0 remains a mystery; it will have to be understood if we are to understand the mechanism of torque generation.

Biol Chem, 1997 Jan, 378(1), 39 - 46
RNA packaging of yeast retrotransposon Ty1 in the heterologous host, Escherichia coli; Luschnig C et al.; Expression of components of the yeast retrotransposon Ty1 in E . coli was used to study early steps of retrotransposition . We find that polypeptides encompassing the capsid-forming component of Ty1 can assemble into particles in the heterologous host . Ty RNA can be detected in particle fractions . RNA packaging depends on features in the 5' part of Ty RNA, because deletion of 5' proximal sequences leads to decreased packaging efficiency . Protein domains required for the RNA packaging process reside between amino acids 146 and 394 of the capsid protein . The data presented also indicate that several early steps in the Ty1 life cycle can occur in a cellular environment which differs from yeast cytoplasm, supporting the notion that these steps are independent of host factors.

Mol Microbiol, 1997 Jan, 23(2), 387 - 98
DNA sequence of recombinase-binding sites can determine Xer site-specific recombination outcome; Blake JA et al.; Xer site-specific recombination functions in the stable inheritance of circular plasmids and bacterial chromosomes . Two related recombinases, XerC and XerD, mediate this recombination, which 'undoes' the potential damage of homologous recombination . Xer recombination on natural plasmid sites is preferentially intramolecular, converting plasmid multimers to monomers . In contrast, recombination at the Escherichia coli recombination site, dif, occurs both intermolecularly and intramolecularly, at least when dif is inserted into a multicopy plasmid . Here the DNA sequence features of a family of core recombination sites in which the XerC- and XerD-binding sites, which are separated by 6 bp, were analysed in order to ascertain what determines whether recombination will be preferentially intramolecular, or will occur both within and between molecules . Sequence changes in either the XerC- or XerD-binding site can alter the recombination outcome . Preferential intramolecular recombination between a pair of recombination sites requires additional accessory DNA sequences and accessory recombination proteins and is correlated with reduced affinities of recombinase binding to recombination core sites, reduced XerC-mediated cleavage in vitro, and an apparent increased overall bending in recombinase-core-site complexes.

Mol Microbiol, 1997 Jan, 23(2), 381 - 6
Increase in negative supercoiling of plasmid DNA in Escherichia coli exposed to cold shock; Mizushima T et al.; Negative supercoiling of plasmid DNA in Escherichia coli cells can decrease transiently when exposed to heat shock . The effect of cold shock on DNA supercoiling was examined, and analysis by agarose gel electrophoresis in the presence of chloroquine revealed that negative supercoiling of plasmid DNA in cells increased when cells were exposed to cold shock . This increase was transient and was nil when the cells were pretreated with nalidixic acid, an inhibitor of DNA gyrase . In a mutant deficient in expression of HU protein, the increase in negative supercoiling of DNA by cold shock is less apparent than in wild-type cells . It is proposed that DNA gyrase and HU protein have a role in the DNA supercoiling reaction seen with cold shock.

Mol Microbiol, 1997 Jan, 23(2), 365 - 79
Mutational analysis of receptor binding mediated by the Dr family of Escherichia coli adhesins; Carnoy C et al.; The fimbrial and afimbrial adhesins of the Dr family mediate the adherence of uropathogenic and diarrhoea-associated Escherichia coli to decay-accelerating factor (DAF) present on erythrocytes and other cell types . The Dr haemagglutinin binds type IV collagen and, unlike other members of the Dr family, mediates an adherence inhibited in the presence of chloramphenicol . We examined the ability of other members of the Dr family-AFAI, AFAIII, and F1845-to bind to type IV collagen, and demonstrated that the collagen-binding phenotype was unique to the Dr haemagglutinin . We employed site-directed mutagenesis to demonstrate the requirement of a negatively charged amino-acid at position 54 of the Dr haemagglutinin subunit for chloramphenicol sensitivity of binding . Mutations at position 32, 40, 54, 90, and 113 differently affected type IV collagen binding and chloramphenicol sensitivity of binding, while retaining DAF-binding capability . These results suggest the existence of a conformational receptor-binding domain in the major structural subunit of Dr family adhesins and demonstrate that chloramphenicol sensitivity of binding and adherence to type IV collagen were independent and separable phenotypes . Finally, we showed that the two conserved cysteine residues of Dr family structural subunits form a disulphide bond and that mutations of these residues abolish haemagglutination and binding to type IV collagen.

Mol Microbiol, 1997 Jan, 23(2), 355 - 64
Promoter-independent cold-shock induction of cspA and its derepression at 37 degrees C by mRNA stabilization; Fang L et al.; The gene for CspA, the major cold-shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift . Here, we report that three-base substitutions around the Shine-Dalgarno sequence in the 159-base 5'-untranslated region of the cspA mRNA stabilizes the mRNA 150-fold, resulting in constitutive expression of cspA at 37 degrees C . This stabilization was found to be at least partially due to resistance against RNase E degradation . The cold-shock induction of cspA was also achieved by exchanging its promoter with the non-cold-shock Ipp promoter . The results presented indicate that the cspA gene is efficiently transcribed even at 37 degrees C . However, the translation of the cspA mRNA is blocked because of its extreme instability at 37 degrees C . The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37 degrees C is prevented by destabilizing its mRNA.

Mol Microbiol, 1997 Jan, 23(2), 333 - 44
Transcription induction of the ferric citrate transport genes via the N-terminus of the FecA outer membrane protein, the Ton system and the electrochemical potential of the cytoplasmic membrane; Kim I et al.; Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE without entering the cells of Escherichia coli K-12 . Point mutants of the outer membrane-receptor protein FecA are affected in induction independent of the FecA transport activity, suggesting that FecA is directly involved in induction . Alignment of FecA with the other ferric siderophore receptors of E . coli reveals an N-terminal extension in FecA that is not found in the receptors whose synthesis is not induced by their cognate ferric siderophores . In this study, we show that excision of the N-terminal region abolished the inducing activity of FecA, but retained its transport activity . Overproduction of the N-terminal FecA fragment inhibited FecA-dependent induction, but not transport . Constitutive expression caused by C-terminally truncated FecR derivatives was not inhibited by the N-terminal FecA fragment . The N-terminal region of FecA was localized in the periplasm, which indicates that FecA probably interacts with FecR, which is involved in signal transduction across the cytoplasmic membrane . Transcription initiation of the fec transport genes required the Ton system, consisting of TonB, ExbB, and ExbD, and was inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), which dissipate the electrochemical potential of the cytoplasmic membrane . fec transcription of mutant fecA4, which displays constitutive fec transcription in the absence of TonB, was not affected by CCCP . The data support a model that proposes initiation of fec transport gene transcription by binding of ferric citrate to FecA . The transcription initiation signal is transferred across the outer membrane through the activity of the Ton system at the expense of the electrochemical potential of the cytoplasmic membrane . The N-terminus of FecA interacts in the periplasm with the C-terminus of FecR, through which the signal is transferred across the cytoplasmic membrane into the cytoplasm, where it increases the activity of the sigma factor Fecl, which then directs the RNA polymerase to the fec promoter upstream of fecA.

Mol Microbiol, 1997 Jan, 23(2), 255 - 65
A recombinational defect in the C-terminal domain of Escherichia coli RecA2278-5 protein is compensated by protein binding to ATP; Alexseyev AA et al.; RecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly-278 to Thr and Val-275 to Phe, in the alpha-helix H of the C-terminal subdomain of the protein . RecA2278-5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response . Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand . In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42 degrees C) in the absence of ATP resulted in inhibition at 42 degrees C of many activities related to homologous recombination including ss- and dsDNA binding, high-affinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA-RecA interaction, and strand transfer capability . The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e . dsDNA binding and high-affinity binding for ATP . Besides ATP, sodium acetate (1.5 M) was shown to be another factor that can stabilize the RecAmut protein at 42 degrees C, judging by restoration of its DNA-free ATPase activity . The similarity of influence of high salt (with its non-specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation . The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecA-mut protein . Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activity of the protein.

Mol Microbiol, 1997 Jan, 23(2), 247 - 53
Plasmid stability: comments on the dimer catastrophe hypothesis; Boe L et al.; Using a derivative of the plasmid pBR322 we have tested the dimer catastrophe hypothesis of plasmid instability . Most of the theory was confirmed by our observations, but our data suggest that some of the quantitative aspects need modification . In a recF strain of Escherichia coli we estimated the difference in loss rate between the plasmid in the monomeric and the dimeric state to be a factor of 13-14 and the difference in the loss rate between the plasmid in the monomeric and the trimeric state to be a factor of 14-50 . We were able to confirm that plasmid oligomers were heterogeneously distributed within a rec+ population, but we were unable to detect any pronounced difference in the level of growth inhibition exerted by the plasmid when in the monomeric, dimeric, or trimeric state . This leaves open the question as to whether runaway plasmid multimerization was prevented (i) by a small correlation between the inhibition of growth and the 'multimeric status' of the plasmid, (ii) by intramolecular homologous recombination, or (iii) whether the process of runaway multimerization is too slow to be recognized within the duration of the experiments, i.e . 200 generations of growth.

Mol Microbiol, 1997 Jan, 23(2), 237 - 45
The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo; Maguire BA et al.; Strain BM108 of Escherichia coli has a chromosomal mutation in the rpmB,G operon that prevents synthesis of ribosomal proteins L28 and L33 . The mutation was lethal unless synthesis of protein L28 was induced from a plasmid . Without protein L28, RNA and protein synthesis were linear rather than exponential . No 70S ribosomes were made . Instead, RNA accumulated in '30S material' and '47S particles'; the latter were distinct from 50S ribosomal subunits, lacked proteins L28 and L33 and had substoicheometric amounts of three other proteins . When L28 synthesis was induced (but protein L33 was still absent), the strain grew as well as, and assembled 70S ribosomes with similar kinetics to, a wild-type control . Thus, protein L28 is required for ribosome assembly in strain BM108 while protein L33 has no significant effect on ribosome synthesis or function.

Mol Microbiol, 1997 Jan, 23(2), 211 - 22
An RNA polymerase alpha subunit mutant impairs N-dependent transcriptional antitermination in Escherichia coli; Obuchowski M et al.; We show that the rpoA341 mutation in the gene encoding the alpha subunit of Escherichia coli RNA polymerase results in a decreased level of transcripts originating from the lytic promoters PL and PR of infecting lambda phage . However, using lacZ fusions we demonstrate that initiation of transcription from both PL and PR is not impaired in the rpoA341 host . Rather, it is the level of the longer, antiterminated PL- and PR-derived transcripts which is altered: the activity of beta-galactosidase in bacteria harbouring a source of N and a PL-nutL-tL1-tI-lacZ or PR-nutR-tR1-lacZ fusion is considerably lower in the rpoA341 mutant relative to the rpoA+ strain . In the absence of the antiterminator protein N no difference is observed in the level of longer PR-derived transcripts between wild-type (rpoA+) and mutant (rpoA341) hosts . Although synthesis of N appears to be similar in both phage-infected rpoA+ and rpoA341 cells, overexpression of the N gene leads to restoration of wild-type levels of the longer PL- and PR-derived transcripts in the mutant host . While this mutation does not appear to affect vegetative phage growth in nus+ backgrounds, in combination with certain nus mutations it retards lytic development . Therefore, we conclude that the rpoA341 mutation specifically interferes with the function of the N-antitermination complex, suggesting that the C-terminal domain of the RNA polymerase alpha subunit may play an important role in N-dependent transcriptional antitermination.

Mol Microbiol, 1997 Jan, 23(2), 191 - 202
Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4-dimethylamino-L-phenylalanine precursor of pristinamycin I; Blanc V et al.; Four pap genes (papA, papB, papC, papM) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases . Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4-dimethylamino-L-phenylalanine (DMPAPA) . This was first verified when disruption of papA in S . pristinaespiralis led to a PI- phenotype, which was reversed by the addition of DMPAPA into the culture medium . Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity . It catalysed the two successive N-methylation steps of 4-amino-L-phenylalanine leading to DMPAPA via 4-methylamino-L-phenylalanine . These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.

J Cell Sci, 1997 Jan, 110 ( Pt 2), 157 - 68
gamma-tubulin in trypanosomes: molecular characterisation and localisation to multiple and diverse microtubule organising centres; Scott V et al.; A genomic clone from Trypanosoma brucei, which contains a full length gamma-tubulin gene, was isolated using degenerate oligonucleotide primers . The sequence of this clone predicts a protein of 447 amino acids having a high degree of homology with gamma-tubulins from human and Xenopus laevis (67.2% amino acid identity) and only 57.7% identity with the Plasmodium falciparum gamma-tubulin . Northern blot analysis of poly(A)+ selected RNA from a procyclic culture detects a major transcript of approximately 2.2 kb plus a minor transcript of approximately 3.6 kb . A fusion protein comprising almost the full length gamma-tubulin gene product (amino acids 8-447) plus an amino-terminal histidine tag has been expressed and purified from Escherichia coli and used to raise a polyclonal antibody . Immunofluorescence, using this antibody, shows classical centrosomal localisation in mammalian cells . In T . brucei gamma-tubulin is present in the basal bodies which subtend the flagellum and also at the anterior tip of the cell body where many minus ends of microtubules are located . Furthermore the antibody reveals a small subset of the sub-pellicular microtubules and a discrete dot within the nucleus which alters form with progression through the mitotic cycle . Evidence is also presented for discrete punctate staining within the microtubules of the cell body which may represent the presence of gamma-tubulin on the ends of individual microtubules . Our results indicate that gamma-tubulin is associated with diverse microtubule organising centres and structures in trypanosomes.

J Antimicrob Chemother, 1997 Jan, 39(1), 99 - 101
The effect of artemisinin on granulocyte function assessed by flow cytometry; Wenisch C et al.; The effect of dihydroartemisinin, artemisinin and artesunate (0.1, 0.5, 5 and 50 mg/L) on phagocytic function and release of reactive oxygen products by neutrophils was studied by flow cytometry . Incubation with dihydroartemisinin, artemisinin and artemether resulted in a decreased capacity to phagocytose Escherichia coli (0.1-50 mg/L: 62-40%, 66-32% and 59-47% of the control values, respectively; P < 0.001 for all) . Conversely, the derivatives enhanced the intracellular generation of reactive oxygen intermediates (0.1-50 mg/L: 146-140%, 174-197% and 188-136% of the control values, respectively; P < 0.001 for all) . Artemisia derivatives enhance the reactive oxygen response of neutrophils but depress their phagocytic ability at therapeutic blood levels.

J Antimicrob Chemother, 1997 Jan, 39(1), 89 - 93
pH effects on the inhibition of extended-spectrum and classical TEM beta-lactamases by penicillanic acid sulphones; Fornara AM et al.; MICs of piperacillin with tazobactam or sulbactam (4 mg/L) were higher at pH 6.5 than at pH 8.0 for Escherichia coli transconjugants with TEM-1 or TEM-2 enzymes, but not for those with TEM-3 or TEM-10 enzymes . Investigation showed: (i) all four enzymes were less sensitive to sulphones at pH 6.5 than at pH 8.0; (ii) pH effects on MICs arose also for the TEM-3 and TEM-10 producers at lower sulphone concentrations; (iii) the TEM-3 and TEM-10 producers formed less enzyme than those with TEM-1 and TEM-2 and (iv) pH effects on the MICs for TEM-1 producers depended on enzyme quantity . We conclude that all four TEM enzymes have pH-dependent susceptibility to sulphones, but whether this affects MICs depends on the inhibitor:enzyme ratio achieved in the bacteria.

Biochem Mol Biol Int, 1997 Jan, 41(1), 169 - 77
Expression and reconstitution of calcineurin A and B subunits; Wei Q et al.; Calcineurin consists of two subunits, a catalytic A subunit of 60 kDa and a regulatory B subunit of 19 kDa . Both the A and B subunits of rat brain calcineurin were expressed in E . coli . The B-subunit was readily overexpressed in the pET-21a vector with yields of > 70 mg of purified B subunit per 1 culture, representing > 17% of the soluble E . coli protein . About 8 mg of purified A subunit was obtained . The enzyme activities of the A-subunit and the reconstituted AB complex were found to be comparable to that of the bovine brain enzyme . The reconstitution of the AB complex was studied and shown to be rapid.

Biochem Mol Biol Int, 1997 Jan, 41(1), 49 - 56
A mutant metallothionein which has inverse fragment composition exhibits high cadmium-binding ability; Yamaguchi R et al.; In order to investigate the role of the alpha-fragment of metallothionein (MT) in metal-binding, two mutant MTs, beta Ala alpha Cys- and alpha Cys beta Cys-mutant MTs, expressed in Escherichia coli were analyzed for their metal-binding ability . A mutant MT where all of the cysteine residues in the beta-fragment of MT were substituted by alanine residues and another mutant MT that had the inverse fragment composition (alpha-beta, i.e., beta-alpha in wild-type MT) were designated as the beta Ala alpha Cys and the alpha Cys beta Cys-mutant MT's, respectively . Both expressed Cd-binding mutant MTs were identified by amino acid analyses . From their metal-binding capacities, the two mutant MTs exhibited higher Cd-binding abilities than wild-type MT . The results suggested that the alpha-fragment plays a key role in the Cd-binding of MT, and that the metal-binding tightness of MT is dependent on the metal-binding ability of a prior fragment in MT.

J Photochem Photobiol B, 1997 Jan, 37(1-2), 26 - 30
Transformation of Escherichia coli cells by pH 2.3 plasmid DNA treated with psoralens plus near-UV light; Repanovici R et al.; The effect of two psoralens, 8-methoxypsoralen (8MOP) and angelicin (ANG), plus near-UV (PUVA) on the transformation capacity of pH 2.3 plasmid DNA on Escherichia coli was studied . Under identical experimental conditions the 8MOP linking to plasmid DNA drastically decreased its transformation capacity compared with the ANG linking . In the case of 8MOP, the decrease depends on the UV dose, as well as on the molar ratios of psoralen and DNA nucleotides . When the effect of short-wavelength UV (UVB) was tested, the higher the molar ratios, the more the combined effects of PUVA and UVB were negative.

Vaccine, 1997 Jan, 15(1), 79 - 84
The B cell epitope of paramyosin recognized by a protective monoclonal IgE antibody to Schistosoma japonicum; Nara T et al.; Passive immunization of mice with a monoclonal IgE antibody to Schistosoma japonicum (SJ18 epsilon . 1) induces significant protection to a challenge infection and the target molecule of SJ18 epsilon . 1 is paramyosin . In the present study, we demonstrate the B cell epitope of paramyosin recognized by SJ18 epsilon . 1 by using a series of deletion mutants expressed in Escherichia coli . SJ18 epsilon 1 reacted with the recombinant paramyosin containing 113 amino acids (Glu301 . Ala413) but not with a shorter peptide (Glu301-Asp343) . Further epitope mapping carried out by a multi-pin system using heptameric peptides synthesized sequentially from 71 amino acids of paramyosin (Asp343-Ala413) demonstrated significant binding of SJ18 epsilon . 1 to the sequence, 359Ile-Arg-Arg-Ala362 . Replacement set analysis of the pentameric peptide, 358Leu-Ile-Arg-Arg-Ala362, revealed that replacement of each residue with a hydrophobic or hydrophilic amino acid did not inhibit binding of SJ18 epsilon . 1, whereas replacement of positively charged Arg . or hydrophobic Ala with a negatively charged amino acid, Glu, showed reduction in binding of the antibody . Moreover, replacement of each amino acid including Arg with a positively charged amino acid, Lys, resulted in a drastic loss of the binding, indicating that binding of the antibody was markedly affected by the change of charges of the peptide as well as by the conformational alteration . The target epitope of SJ18 epsilon . 1 was common among paramyosins of S . mansoni, Taenia solium and Echinococcus granulosus but not among nematode paramyosins, suggesting that the epitope is specific for platyhelminthes.

Vaccine, 1997 Jan, 15(1), 25 - 35
Characterization of the gene encoding Mhp1 from Mycoplasma hyopneumoniae and examination of Mhp1's vaccine potential; King KW et al.; The gene encoding Mhp1, a 124 kDa protein from Mycoplasma hyopneumoniae, has been cloned, sequenced, and its product characterized . No significant homology to the gene or encoded polypeptide was found in the Genbank, NBRF, or PIR databases, though this protein appears similar to p97, a putative adhesin of M . hyopneumoniae described by Zhang et al . (Infect . Immun . 63, 1013-1019, 1995) . Two repeated motifs were identified within the 3' end of the gene and encoded polypeptide . The mhp1 gene was fused to the glutathione S-transferase (GST) gene from Schistosoma japonicum, enabling high-level expression and purification of the protein . Both the authentic and recombinant proteins were recognized by sera from pigs infected with M . hyopneumoniae . In an induced-disease model in pigs, coughing was reduced in animals vaccinated with recombinant GST-Mhp1, although differences were not significant . Only minimal protection against lung lesion formation was provided, and again differences between the Mhpl-vaccinated and nonvaccinated groups were not significant.

Mol Biochem Parasitol, 1997 Jan, 84(1), 101 - 10
A region containing repeated elements is associated with transcriptional termination of Leishmania infantum ribosomal RNA genes; Requena JM et al.; A novel repetitive DNA element has been isolated from the Leishmania infantum genome . The 348 bp long element, designated LiR3, was found to be located downstream from the 3'-end of the ribosomal RNA (rRNA) genes . This LiR3 element has short sequences with potential to form stem-loop structures similar to those of the bacterial rho-independent transcriptional terminators . Given both the structural features and the genomic location of this element we searched for a possible functional implication of these structures in the termination of rRNA transcription . Nuclear run-on assays indicated that indeed there is a transcriptional blockage associated with the LiR3 element . Several chi-like elements, resembling the recombination-promoting sites of Escherichia coli, were identified within the sequences associated with the stem-loop structures . A possible implication of these chi-like elements in rRNA gene conversion events is discussed.

Cell Transplant, 1997 Jan-Feb, 6(1), 1 - 8
Transfer and expression of the interferon gamma gene in human endothelial cells inhibits vascular smooth muscle cell growth in vitro; Stopeck AT et al.; Intimal hyperplasia in blood vessels is primarily caused by the migration and proliferation of vascular smooth muscle cells . Excessive intimal thickening characterizes atherosclerosis as well as bypass graft and angioplasty failures . Endothelial cell-smooth muscle cell interactions and local cytokine production are important regulators of smooth muscle cell growth . Interferon gamma (gamma-IFN), a product of T lymphocytes found in atherosclerotic lesions, inhibits smooth muscle cell proliferation in vitro . To determine if local delivery of gamma-IFN may be useful in the treatment or prevention of vascular proliferative diseases, we transferred the human gamma-IFN gene into endothelial cells isolated from human arteries and microvessels using a retroviral vector . Biologically active gamma-IFN was produced and secreted by gamma-IFN transduced endothelial cells, but not by control, nontransduced cells, or cells identically transduced with E . coli beta galactosidase (beta-gal) . To more closely approximate the microenvironment of blood vessels, subconfluent smooth muscle cells were plated in coculture with control, nontransduced endothelial cells, gamma-IFN transduced endothelial cells, or beta-gal transduced endothelial cells . Smooth muscle cell growth was inhibited 30-70% by coculture with gamma-IFN transduced endothelial cells compared to coculture with beta-gal transduced or control endothelial cells (p < 0.05) . Our results suggest endothelial cells modified to produce gamma-IFN may be a useful therapy in proliferative vascular diseases.

Am J Physiol, 1997 Jan, 272(1 Pt 1), C350 - 4
Reinterpretation of the RACTK1 K+ channel; Shmukler B et al.; The RACTK1 cDNA cloned from rabbit kidney cortical collecting duct cells was associated with inwardly rectifying pH-regulated K+ channel activity (M . Suzuki, K . Takahashi, M . {keda, H Hayakawa, A . Ogawa, Y . Kawaguchi, and O . Sakai . Nature Lond . 367: 642-645, 1994) . The deduced amino acid sequence of the encoded novel polypeptide lacked the signature sequence of a K(+)-selective pore region but predicted a topography suggestive of the inward rectifier K+ channel family . In subsequent articles a RACTK1 epitope was immunolocalized to the apical surface of kidney collecting duct and to arteriolar smooth muscle {M . Suzuki, T . Takigawa, K . Kimura, C . Koseki, and M . Imai . Am . J . Physiol . 269 (Cell Physiol, 38): C496-C503, 1995}, and apamin-sensitive K+ currents displaying Ca(2+)-dependent and voltage-independent activation accompanied stable heterologous overexpression of RACTK1 {M . Suzuki, M . Murata, M . Ikeda, T . Miyoshi, and M . Imai . Am . J . Physiol . 270 (Cell Physiol, 39): C964-C968, 1996} . We now report that the "RACTK1" open reading frame is a frame-shifted translation of the antisense strand of an Escherichia coli gene member of a coenzyme A transferase gene family . "RACTK1" mRNA was absent from tissues free of E . coli contamination, and the "RACTK1" gene was undetectable in Southern blots of human and rabbit genomic DNA . We conclude that the immunostaining patterns and Ca(2+)-activated K+ channel activity heretofore attributed to RACTK1 must be otherwise explained.

Virus Res, 1997 Jan, 47(1), 51 - 7
Characterization of the 3' proximal gene of the citrus tristeza closterovirus genome; Pappu SS et al.; The 3' proximal open reading frame (ORF 11) in the citrus tristeza virus (CTV) genome potentially encodes a protein of 209 amino acids with an estimated molecular weight of 23 kDa (p23) . The p23 ORF from the severe Florida strain T36 of CTV was expressed in Escherichia coli, and the expressed protein was used to raise polyclonal antibodies in a rabbit . Using these antisera on a Western blot, a protein of expected size (23 kDa) was detected in tissue extracts from CTV-infected citrus but not from uninfected citrus . Most of the p23 protein was found in the soluble, cytoplasmic fraction . Comparison of the sequence of p23 genes from several biologically and geographically diverse CTV isolates indicated a high degree of conservation for this gene and for the RNA binding motif in particular . A cluster dendrogram of the deduced amino acid sequences correlated with the biological properties of the isolates, forming distinct groups of mild, quick decline on stem pitting-inducing isolates . Therefore it is possible that, in addition to the capsid protein gene, the p23 gene also may serve as an indicator for disease severity.

Proteins, 1997 Jan, 27(1), 131 - 43
Structural homology of spinach acyl carrier protein and Escherichia coli acyl carrier protein based on NMR data; Oswood MC et al.; Acyl carrier proteins (ACPs) from spinach and from Escherichia coli have been used to demonstrate the utility of proton NMR for comparison of homologous structures . The structure of E . coli ACP had been previously determined and modeled as a rapid equilibrium among multiple conformational forms (Kim and Prestegard, Biochemistry 28:8792-8797, 1989) . Spinach ACP showed two slowly exchanging forms and could be manipulated into one form for structural study . Here we compare this single form to postulated multiple forms of E . coli ACP using the limited amount of NOE data available for the spinach protein . A number of long-range NOE contacts were present between homologous residues in both spinach and E . coli ACP, suggesting tertiary structural homology . To allow a more definitive structural comparison, a method was developed to use spinach ACP NOE constraints to search for regions of structural divergence from two postulated forms of E . coli ACP . The homologous regions of the two protein sequences were aligned, additional distance constraints were extracted from the E . coli structure, and these were mapped onto the spinach sequence . These distance constraints were combined with experimental NOE constraints and a distance geometry simulated annealing protocol was used to test for compatibility of the constraints . All of the experimental spinach NOE constraints could be successfully combined with the E . coli data, confirming the general hypothesis of structural homology . A better fit was obtained with one form, suggesting a preferential stabilization of that form in the spinach case.

Proteins, 1997 Jan, 27(1), 110 - 7
Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state; Svergun DI et al.; Solution scattering curves evaluated from the crystal structures of the T and R states of the allosteric enzyme aspartate transcarbamylase from Escherichia coli were compared with the experimental x-ray scattering patterns . Whereas the scattering from the crystal structure of the T state agrees with the experiment, large deviations reflecting a significant difference between the quaternary structures in the crystal and in solution are observed for the R state . The experimental curve of the R state was fitted by rigid body movements of the subunits in the crystal R structure which displace the latter further away from the T structure along the reaction coordinates of the T-->R transition observed in the crystals . Taking the crystal R structure as a-reference, it was found that in solution the distance between the catalytic trimers along the threefold axis is 0.34 nm larger and the trimers are rotated by 11 degrees in opposite directions around the same axis; each of the three regulatory dimers is rotated by 9 degrees around the corresponding twofold axis and displaced by 0.14 nm away from the molecular center along this axis.

Proteins, 1997 Jan, 27(1), 1 - 8
Crystal structure of a recombinant form of the maltodextrin-binding protein carrying an inserted sequence of a B-cell epitope from the preS2 region of hepatitis B virus; Saul FA et al.; We report the crystal structure of MalE-B133, a recombinant form of the maltodextrin-binding protein (MBP) of Escherichia coli carrying an inserted amino-acid sequence of a B-cell epitope from the preS2 region of the hepatitis B virus (HBV) . The structure was determined by molecular replacement methods and refined to 2.7 A resolution . MalE-B133 is an insertion/deletion mutant of MBP in which residues from positions 134 to 142, an external alpha helix in the wild-type structure, are replaced by a foreign peptide segment of 19 amino acids . The inserted residues correspond to the preS2 sequence from positions 132 to 145 and five flanking residues that arise from the creation of restriction sites . The conformation of the recombinant protein, excluding the inserted segment, closely resembles that of wild-type MBP in the closed maltose-bound form . MalE-B133 was shown by previous studies to display certain immunogenic and antigenic properties of the hepatitis B surface antigen (HBsAg), which contains the preS2 region . The crystal structure reveals the conformation of the first nine epitope residues (preS2 positions 132 to 140) exposed on the surface of the molecule . The remaining five epitope residues (preS2 positions 141 to 145) are not visible in electron density maps . The path of the polypeptide chain in the visible portion of the insert differs from that of the deleted segment in the structure of wild-type MBP, displaying a helical conformation at positions 134 to 140 (preS2 sequence numbering) . A tripeptide (Asp-Pro-Arg) at the N terminus of the helix forms a stable structural motif that may be implicated in the cross-reactivity of anti-HBsAg antibodies with the hybrid protein.

Poult Sci, 1997 Jan, 76(1), 165 - 78
Applications in in ovo technology; Johnston PA et al.; By mid-August 1995, 55% of broiler embryos in North America were vaccinated for Marek's disease using the INOVOJECT system, with 201 INOVOJECT machines placed with 16 of the top 25 poultry producers, providing the industry with the capacity to inject in excess of 400 million eggs per month or about 5 billion eggs per annum . In ovo administration of a bursal disease antibody-infectious bursal disease virus (BDA-IBDV) complexed vaccine to specific-pathogen-free (SPF) embryos was safer and more potent than conventional IBDV vaccine alone because it delayed the appearance of bursal lesions, produced no early mortality, produced higher geometric mean antibody titers against IBDV, and generated protective immunity against challenge . In ovo administration of a BDA-IBDV complexed vaccine to broiler embryos generated antibody titers against IBDV sooner than conventional virus vaccinates, and generated protective immunity against challenge Direct DNA injection of plasmid DNA encoding beta-galactosidase into breast muscle in ovo and posthatch was an effective means to achieve both gene transfer and expression, with potential for the development of gene vaccines using plasmids encoding protective antigens from poultry pathogens . In ovo administration of 800 U chicken myelomonocytic growth factor (cMGF), a chicken hematopoietic cytokine for cells of the monocytic-granulocytic lineages, significantly reduced mortality associated with Escherichia coli exposure within the hatcher when compared to PBS controls (6.1 vs 12.4, P < or = 0.05), but not when compared to a yeast expression control . A procedure was developed enabling injection prior to the onset of incubation without compromising embryo viability . This in ovo injection process has opened up the window of embryo development during incubation for intervention, as illustrated by the 100% male phenotype produced in chicks hatching from eggs injected with aromatase inhibitor prior to incubation . These data illustrate some of the in ovo applications presently in use by the poultry industry, and under development or in research at EMBREX.

Plant Mol Biol, 1997 Jan, 33(2), 301 - 11
Molecular cloning and expression of two cDNAs encoding asparagine synthetase in soybean; Hughes CA et al.; Two cDNA clones (SAS1 and SAS2) encoding different isoforms of asparagine synthetase (AS; EC 6.3.5.4) were isolated . Their DNA sequences were determined and compared . The amino-terminal residues of the predicted SAS1 and SAS2 proteins were identical to those of the glutamine binding domain of AS from pea, asparagus, Arabidopsis and human, suggesting that SAS1 and SAS2 cDNAs encode the glutamine-dependent form of AS . The open reading frames of SAS1 and SAS2 encode a protein of 579 and 581 amino acids with predicted molecular weights of 65182 and 65608 Da respectively . Similarity of the deduced amino acid sequences of SAS1 and SAS2 with other known AS sequences were 92% and 93% for pea AS1; 91% and 96% for pea AS2; 88% and 91% for asparagus; 88% and 90.5% for Arabidopsis; 70.5% and 72.5% for E . coli asnB and 61% and 63% for man . A plasmid, pSAS2E, was constructed to express the soybean AS protein in Escherichia coli . Complementation experiments revealed that the soybean AS protein was functional in E . coli . Southern blot analysis indicated that the soybean AS is part of a small gene family . AS transcript was expressed in all tissues examined, but higher levels were seen in stem and root of light-grown tissue and leaves of dark-treated tissue.

Plant Mol Biol, 1997 Jan, 33(2), 291 - 300
Cloning and characterization of a pollen-specific cDNA encoding a glutamic-acid-rich protein (GARP) from potato Solanum berthaultii; Liu J et al.; A pollen-specific cDNA was isolated from a cDNA library of in vitro germinated pollen of the diploid potato species Solanum berthaultii . The cDNA clone, designated SB401, hybridizes to a messenger RNA of 1.2 kb length in mature and germinated pollen . SB401 messenger RNA is absent from other parts of the plant, including other flower tissues . SB401 cDNA, which possesses a long stretch of AT-rich 5'-untranslated leader sequence, encodes a glutamic acid-rich protein (GARP) which is hydrophilic throughout and contains six imperfect repeated motifs of the sequence V-V-E-K-K-N/E-E with the di-basic amino acid residue pair (K-K) as the core within the repeats . These repeats are spaced at irregular intervals and predicted to form an alpha-helical structure . The SB401 protein was over-expressed in Escherichia coli and the purified protein was used for raising antiserum . Both E . coli-expressed and the endogenous SB401 proteins in pollen and pollen tubes appear much larger on SDS-polyacrylamide gels than their calculated molecular masses . Immunoblotting revealed the protein to be most abundant in germinated pollen . The structural features of SB401 protein and a possible role for the protein in pollen development, pollen germination, and pollen tube growth are discussed.

Plant Mol Biol, 1997 Jan, 33(2), 211 - 22
Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization; Schroder G et al.; We describe the molecular and functional characterization of three closely related S-adenosyl-L-methionine synthetase (SAMS) isoenzymes from Catharanthus roseus (Madagascar periwinkle) . The genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased NaCl) . Seedlings revealed organ-specific expression and differential gene regulation after salt stress . A relationship analysis indicated that plant SAMS group in two main clusters distinguished by characteristic amino acid exchanges at specific positions, and this suggested differences in the enzyme properties or the regulation . SAMS1 and SAMS2 are of type I and SAMS3 is of type II . The properties of the isoenzymes were compared after heterologous expression of the individual enzymes, but no significant differences were detected in a) optima for temperature (37 to 45 degrees C) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+, Co2+; monovalent: K+, NH4+, Na+); c) K(m)s for ATP and L-methionine; d) inhibition by reaction products (S-adenosyl-L-methionine, PPi, Pi), by the reaction intermediate tripolyphosphate, and by the substrate analogues ethionine and cycloleucine; e) response to metabolites from the methyl cycle (L-homocysteine) or from related pathways (L-ornithine, putrescine, spermidine, spermine); f) native protein size (gel permeation chromatography) . The results represent the first characterization of plant SAMS isoenzyme properties with individually expressed proteins . The possibility is discussed that the isoenzyme differences reflect specificities in the association with enzymes that use S-adenosyl-L-methionine.

Appl Microbiol Biotechnol, 1997 Jan, 47(1), 23 - 32
Synthetic spider dragline silk proteins and their production in Escherichia coli; Fahnestock SR et al.; Synthetic genes were designed to encode analogs of the two proteins of Nephila clavipes dragline silk, spidroins 1 and 2 . The genes were constructed of tandem repeats of relatively long (more than 300 bp) DNA sequences assembled from synthetic oligonucleotides, and encoded proteins of high molecular mass (65-163 kDa) . Both analogs were produced efficiently in Escherichia coli . The yield and homogeneity of the products of longer genes were limited by premature termination of synthesis, probably as a result of processivity errors in protein synthesis . Average termination rates were determined to be 1 in 1100 codons to 1 in 300 codons, depending on the length and synonymous codon choices of the gene . Both analog proteins could be induced to form stable aqueous solutions without denaturants . Circular dichroism spectra of the purified proteins in dilute solution resembled spectra of redissolved natural dragline silk in reflecting a largely disordered structure in water and more ordered structures in mixed solvents with methanol and trifluoroethanol.

Nat Biotechnol, 1997 Jan, 15(1), 79 - 84
Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones; Ryabova LA et al.; To create a rapid system to test the effect of sequence changes on recombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translation system . Functional antibodies with antigen-binding activity are obtained only if disulfide formation and rearrangement is allowed to take place during the translation reaction . The inclusion of protein disulfide isomerase (PDI) leads to a threefold increase in yield over that obtained in the presence of glutathione redox systems . DsbA had no such effect, indicating that disulfide shuffing, and not net formation, is the crucial yield-limiting step . The addition of the molecular chaperones DnaK and DnaJ increased the amount of soluble protein but not the amount of functional scFv, which appears to be limited entirely by correct disulfide formation . None of these factors significantly influenced total protein synthesis . In the presence of PDI, chaperones, reduced glutathione and oxidized glutathione, 50% of the scFv produced (about 8 micrograms/ml in only 15 min) could be recovered from immobilized antigen.

Nat Biotechnol, 1997 Jan, 15(1), 57 - 62
Nerve growth factor somatic mosaicism produced by herpes virus-directed expression of cre recombinase; Brooks AI et al.; Focal molecular genetic alteration of the intact mammalian brain will be required to elucidate gene product function in cells comprising synaptic networks . To this end, a somatic mosaic approach has been developed for the mouse whereby a dormant germline transgene is activated by the somatic delivery and expression of cre recombinase . Transgenic mice harboring a recombinational substrate, the germline-transmitted nerve growth factor excision activation transgene (NGF-XAT) were generated . Somatic delivery of virus vectors expressing cre recombinase into the brain of NGF-XAT mice resulted in regional recombination and activation of the transgene as demonstrated at the DNA level by PCR and at the protein level by both immunocytochemistry and ELISA . This approach has been used to evaluate a behavioral correlate of unilateral NGF mosaicism within the dorsal hippocampal formation . NGF-XAT mice activated by expression of cre recombinase manifest increased locomotor activity compared with NGF-XAT mice transduced by a control virus expressing Escherichia coli beta-galactosidase . These data indicate that focally increased expression of NGF in one part of a synaptic network can elicit changes in behavior presumably by altering the overall function of NGF-responsive neural circuitry . This approach should have broad application to other gene products and promises to provide the unprecedented ability to create and study discrete genetic modifications in the context of an intact adult mammal.

J Vet Med Sci, 1997 Jan, 59(1), 35 - 8
Purification of the infectious bovine rhinotracheitis virus alkaline deoxyribonuclease expressed in Escherichia coli; Chung YT et al.; Nucleotide sequence analysis within the unique long segment of the infectious bovine rhinotracheitis virus (IBRV) genome identified an open reading frame of 1461 base pairs whose deduced polypeptide of 487 amino acids exhibited homology to alkaline deoxyribonucleases of other herpesviruses . To determine whether this IBRV gene product has nuclease activity, the gene designated UL12 was inserted into the vector pET-28a(+) and expressed in Escherichia coli as an oligohistidine-tagged protein . Upon induction with isopropyl beta-D-thiogalactopyranoside E . coli BL21 (DE3){pLysS} cells harboring this recombinant plasmid produced a 57-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence . A one-step purification procedure using metal affinity chromatography resulted in a homogeneous preparation of this recombinant protein . The purified protein exhibited both exonuclease and endonuclease activities, each with an alkaline pH optimum.

Free Radic Biol Med, 1997, 22(6), 939 - 46
Antioxidant functions of inositol 1,2,3-trisphosphate and inositol 1,2,3,6-tetrakisphosphate; Phillippy BQ et al.; Iron chelates of inositol 1,2,3-trisphosphate and inositol 1,2,3,6-tetrakisphosphate lacked free coordination sites and prevented the iron-catalyzed oxidation of ascorbic acid and peroxidation of arachidonic acid . In contrast, iron chelates of inositol 1,2,6-trisphosphate and inositol 1,2,5,6-tetrakisphosphate contained available coordination sites, permitted iron-catalyzed ascorbic acid oxidation, and enhanced arachidonic acid peroxidation . It was concluded that the 1,2,3-trisphosphate grouping of inositol hexakisphosphate was responsible for the inhibition of iron-catalyzed hydroxyl radical formation . The structure of the chelate with the phosphates in an axial-equatorial-axial configuration appeared to be the only possible inositol trisphosphate that could form bonds between six oxygen atoms and the six coordination sites on iron . Km values for cleavage by Escherichia coli alkaline phosphatase were as follows: inositol 1,2,3-trisphosphate, 56 microM; inositol 1,2,6-trisphosphate, 35 microM; inositol 1,2,3,6-tetrakisphosphate, 139 microM; and inositol 1,2,5,6-tetrakisphosphate, 100 microM . The initial hydrolysis rates of 200 microM solutions of the latter three isomers by E . coli alkaline phosphatase were not affected by an equimolar concentration of iron, whereas the rate for inositol 1,2,3-trisphosphate decreased in the presence of iron to 50% of the control . Therefore, the antioxidant potential of inositol 1,2,3-trisphosphate and inositol 1,2,3,6-tetrakisphosphate in cells and other biological systems may be fortified by the resistance of their iron chelates to enzymatic hydrolysis of the functional 1,2,3-trisphosphate array.

Public Health, 1997 Jan, 111(1), 11 - 7
Enterotoxigenic Escherichia coli (ETEC) in hospitalised Arab infants from Judea area--west bank, Israel; Wolk M et al.; Enterotoxigenic Escherichia coli and other related enterotoxigenic species were isolated from 176 (44%) of 399 infants hospitalised in 'Caritas Baby Hospital' in Bethlehem, during April-December 1993 . Ninety four of the patients infected by ETEC, were clinically evaluated . Most of them suffered from diarrhoea, quite often with fever and vomiting . Dehydration occurred in 58.3% of the patients and failure to thrive (FTT) in 28.5% of them . Severe illness resulted in marasmus in five patients and in the death of two others . Most of the ETEC strains (84%) were of ST toxin type . Correlation was found between the degree of toxigenity and the severity of the gastroenteritis . The most prevalent ETEC "O' serogroups were 0-6, 0-20, 0-8, 0-86, 0-126, 0-128 and 0167 . Colonization Factors Antigens (CFAs) were identified in 36% of the isolates, CFAI was characteristic of group 0-126 and 0-128 . In the principal O-groups there were high percentages of sensitivity to the antibiotics ceftriaxone, nalidixic-acid, gentamicin and norfloxacin, with resistance to anoxycillin, tetracycline and cotrimoxazole.

Transgenic Res, 1997 Jan, 6(1), 75 - 84
Bovine alpha s1-casein gene sequences direct high level expression of human granulocyte-macrophage colony-stimulating factor in the milk of transgenic mice; Uusi-Oukari M et al.; The generation is reported of transgenic mice expressing human granulocyte-macrophage colony-stimulating factor (GM-CSF) or human erythropoietin (EPO) under the control of bovine alpha s1-casein regulatory sequences . GM-CSF expression was specific to the mammary gland, and levels of human GM-CSF in transgenic mouse milk were in the range of mg ml-1 . The specific activity of the milk GM-CSF was similar to that of the recombinant protein produced in Escherichia coli, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein . In spite of the identical bovine regulatory sequences of the fusion genes, the levels of human EPO in transgenic mouse milk were 10(3)-10(6) times lower than those of GM-CSF, ranging from 0.003 to 3 micrograms ml-1 . There appeared to be a positive correlation between the amount of EPO in the milk of lactating females and blood haematocrit values . In view of this, other type of constructs should be used to achieve more efficient EPO expression and to circumvent concomitantly-occurring adverse effects . In contrast, the high-level production of recombinant GM-CSF, its resemblance to the native mammalian protein, and mild adverse consequences of transgene expression imply that the current construct could be used for generation of larger GM-CSF transgenic animals to produce this protein in quantities sufficient for therapeutic purposes.

Transgenic Res, 1997 Jan, 6(1), 37 - 40
An improved method to detect beta-galactosidase activity in transgenic mice: a post-staining procedure on paraffin embedded tissue sections; Ave P et al.; The Escherichia coli beta-galactosidase gene is frequently used as a reporter gene in transgenic studies because its activity can be easily detected at the cellular level . Here we report a procedure for monitoring beta-galactosidase activity directly in tissue sections, which involves the use of a mixture of ethanol and poly-ethylene-glycol as a fixative (Kryofix) and a special paraffin characterized by a lower fusion point of 42 degrees C . After embedding and cutting, the sections are stained by the chromogenic substrate 5-bromo-4-chloro-3-indoyl-beta-D galactopyranoside (X-Gal) . This procedure allows both the retention of a high level of beta-galactosidase activity and the preservation of good tissue morphology . Furthermore, it can be combined with immunohistochemical methods to detect other cellular components without compromising reporter gene detection.

J Cancer Res Clin Oncol, 1997, 123(2), 91 - 9
A ribozyme specifically suppresses transformation and tumorigenicity of Ha-ras-oncogene-transformed NIH/3T3 cell lines; Chang MY et al.; In this study, the efficacy of an anti-ras ribozyme in reversing a transformed phenotype was investigated . A murine NIH/3T3-derived cell line, designated 2-12, contains an inducible Ha-ras oncogene, which is regulated by the Escherichia coli (E . coli) lac operator/repressor system, and displays a transformed phenotype after isopropyl-beta-D-thiogalactoside induction . To reverse the transformed characteristics, the ribozyme, which specifically targets the Ha-ras oncogene at the codon 12 mutation site (GGC to GUC), was transfected into 2-12 cells . Two (ribZ4 and ribZ7) clones were subsequently selected and analyzed for their transforming features . Our results show that, in the transfectants, ribozyme gene expression was detected, and the target Ha-ras transgene was expressed at basal levels . Their phenotypic responses, including morphology, cell growth rate, colony-formation efficiency and tumorigenicity in mice with severe combined immunodeficiency were more similar to those of NIH/3T3 than 2-12 transformed cells . Directly injecting the ribozyme DNA into tumors induced by transformed 2-12 cells in BALB/c mice also caused tumor regression . The enzymatic cleavage products of the ribozyme acting on mutant Ha-ras mRNA in vivo were detected by primer-extension analysis . These results indicate that the ribozyme were designed exhibits a site-specific ribonuclease function that effectively abrogates Ha-ras-oncogene-induced transformation, and this unique anti-Ha-ras property should shed light on the development of strategies against the Ha-ras-oncogene-initiated malignancy.

Steroids, 1997 Jan, 62(1), 124 - 7
Molecular recognition and electron transfer in mitochondrial steroid hydroxylase systems; Vickery LE; Mitochondrial monooxygenase systems are involved in the biosynthesis of glucocorticoids, mineralocorticoids, bile acids, and 1,25-dihydroxyvitamin D . The reactions are catalyzed by specific P450 enzymes that receive reducing equivalents via NADPH-ferredoxin oxidoreductase (adrenodoxin reductase) and ferredoxin (adrenodoxin) . Although the three-dimensional structures of the individual components have not yet been solved, methods of expressing recombinant forms of these enzymes in Escherichia coli have allowed the use of site-directed mutagenesis to investigate the roles of specific amino acids in protein binding interactions, electron transfer, and catalysis . These studies have identified key charged residues in NADPH-ferredoxin oxidoreductase, ferredoxin, and P450scc, which are involved in electrostatic interactions critical for recognition, high-affinity binding, and electron transfer . The finding that the binding sites on ferredoxin for NADPH-ferredoxin oxidoreductase and P450 show significant overlap supports the proposed function for ferredoxin as a mobile electron shuttle between the reductase and P450 enzymes and is consistent with ferredoxin's role in serving multiple P450 isoforms.

J Prosthet Dent, 1997 Jan, 77(1), 76 - 82
Lipopolysaccharide affinity for titanium implant biomaterials; Nelson SK et al.; STATEMENT OF PROBLEM: Lipopolysaccharide (LPS) affinity for titanium implant biomaterials could affect crevicular LPS concentrations and thereby influence periimplant inflammation . PURPOSE OF STUDY: The purpose of this study was to evaluate Porphyromonas gingivalis and Escherichia coli LPS affinity for titanium biomaterials groups that differed in surface oxide composition and surface roughness . MATERIAL AND METHOD: Polished and abraded grade 1 commercially pure titanium and grade 5 alloyed extra low interstitial titanium specimens were treated with 10 EU/mm2 and radiolabeled LPS . RESULTS: The resultant mean +/- SD LPS adherence values ranged from 4.17 +/- 0.29 to 4.79 +/- 0.40 EU/ mm2 . No difference in adherence and elution was indicated on the basis of LPS type, surface oxide composition, or surface roughness . Moreover, P . gingivalis and F . coli LPS desorption was below detection . CONCLUSION: Clinically, the high affinity of both LPS types for titanium biomaterials may adversely influence the periimplant tissue response.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 87 - 92
Molecular cloning of an inulin fructotransferase (depolymerizing) gene from Arthrobacter sp . H65-7 and its expression in Escherichia coli; Sakurai H et al.; The gene encoding an extracellular inulin fructotransferase (depolymerizing) (inulase II) (EC 2.4.1.93), designated ift gene, was cloned from the genomic DNA of Arthrobacter sp . H65-7, and expressed in Escherichia coli for the first time . Sequence analysis showed a single open reading frame consisting of 1314 base pairs that encoded a signal peptide of 32 amino acids and a mature protein of 405 amino acids . The primary structure showed a homology of 49.8% with that of the inulin fructotransferase (DFA I-producing) (EC 2.4.1.200) from Arthrobacter globiformis S14-3 . E . coli cells carrying the ift gene produced the active enzyme under control of the lac promoter . The expression of the ift gene was improved by a plasmid, pIFT-B, in which the ift gene was immediately downstream from the lac promoter . An E . coli transformant carrying pIFT-B expressed twice as much activity of inulase II as that of the original strain, Arthrobacter sp . H65-7 . Most of the enzyme activity existed within the cells.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 34 - 9
Analysis of low temperature inducible mechanism of gamma-glutamyltranspeptidase of Escherichia coli K-12; Hashimoto W et al.; Escherichia coli K-12 cultured at 20 degrees C has more gamma-glutamyltranspeptidase (GGT: EC 2.3.2.2) activity than that cultured at 37 or 42 degrees C . On Western blot analysis, E . coli K-12 cells cultured at 20 degrees C produced more GGT protein than those cultured at 37 degrees C . mRNA of the GGT gene (ggt) in the cells was also measured and it was found that the level of ggt mRNA at 20 degrees C was 10-fold higher than that at 37 degrees C . When the ggt promoter was replaced by a tac promoter, GGT activity at 37 degrees C from the tac promoter was 5-fold higher than that at 37 degrees C from the ggt promoter, though there was less difference in GGT activity between both grown at 20 degrees C . The ggt mRNA at 20 degrees C was found to be more stable than that at 37 degrees C . These results suggested that the higher GGT activity in E . coli K-12 cells grown at 20 degrees C was due to a higher level of GGT protein at 20 degrees C caused by higher level of ggt mRNA at 20 degrees C because of a low-temperature dependent ggt promoter as well as the stability of ggt mRNA at 20 degrees C.

Toxicon, 1997 Jan, 35(1), 27 - 37
Cloning and characterization of cDNAs encoding three isoforms of phospholipase A2 in Malayan spitting cobra (Naja naja sputatrix) venom; Armugam A et al.; cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized . One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2) . The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA . Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively . The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

Bioconjug Chem, 1997 Jan-Feb, 8(1), 44 - 8
Synthesis of the protein cutting reagent iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate and conjugation to cysteine side chains; Greiner DP et al.; Convenient methodology for preparation and conjugation of the protein-cutting iron chelate iron (S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) is given . This formulation of the reagent can be handled in a manner analogous to many other protein-labeling reagents, such as fluorescent probes or cross-linkers . By taking advantage of the recently discovered peptide hydrolysis reaction, the chelate may be tethered to a single site (e.g., a cysteine side chain) and used to map its proximity to individual peptide bonds by automated Edman sequencing of the protein fragments produced . The method is illustrated by conjugation of Fe-BABE to the carboxy terminal domain (amino acid residues 234-329) of the Escherichia coli RNA polymerase alpha subunit . The molecular mass of the protein conjugate was confirmed by electrospray ionization mass spectrometry.

Anal Biochem, 1997 Jan 1, 244(1), 62 - 6
Cationic liposomes enhanced firefly bioluminescent assay of adenosine 5'-triphosphate disodium salt; Kamidate T et al.; Cationic liposomes containing stearylamine enhanced the intensity of maximum light emission from the firefly luciferin-luciferase reaction with ATP in the range of 1.0 pM up to 5.5 nM . In contrast, the bioluminescent intensities in the presence of anionic and zwitterionic liposomes were about the same as those in water alone . The detection limit for ATP in the presence of cationic liposomes was 1.0 pM in aqueous ATP standard solutions . The sensitivity for ATP was improved by a factor of five times compared to that in water alone . The method was applied to the determination of ATP in Escherichia coli extracts . The sensitivity threshold was presumed to be 340 cells ml-1.

Plant J, 1997 Jan, 11(1), 157 - 65
Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue; McElroy D et al.; The development of a barley (Hordeum vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation (Ac/Ds) transposable elements for gene tagging in transgenic barley plants . However, barley transformation is time-consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues was developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants . In this assay, barley scutellar tissue is co-transformed with constructs containing the maize Ac transposase gene and an Escherichia coli uidA reporter gene (Gus), the expression of which is interrupted by a maize Ds element . In transformed barley scutellar cells, Ac transposase-mediated excision of the Ds element generates a functional Gus gene, leading to histochemically detectable GUS activity . Characterization of the excision products showed that they had a pattern of nucleotide deletions and/or transversions similar to that found in maize and other heterologous plant systems . In addition, although contrary to the situation observed in heterologous dicot systems, efficient Ds excision in barley, a heterologous monocot system, appears to be inversely associated with Ac copy number, a finding similar to the Ac dosage effects observed in maize . The transient assay was used to demonstrate functional transposase activity in barley callus lines stably transformed with an Ac transposase gene.

Plant J, 1997 Jan, 11(1), 73 - 82
Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L; Neuhaus HE et al.; Recently, we have sequenced a cDNA clone from Arabidopsis thaliana L . encoding a novel putative ATP/ADP translocator (AATP1) . Here, we demonstrate that the radioactively labeled AATP1 precursor protein, synthesized in vitro, is targeted to envelope membranes of isolated spinach chloroplasts . Antibodies raised against a synthetic peptide of AATP1 recognized a single polypeptide of about 62 kDa in chloroplast inner envelope preparations . The cDNA coding for the AATP1 protein was functionally expressed in Saccharomyces cerevisiae and Escherichia coli . In both expression systems, increased rates of ATP transport were observed after reconstitution of the extracted protein into proteoliposomes . To our knowledge, this is the first report on the functional expression of an intrinsic plant membrane protein in E . coli . To yield high rates of ATP transport, proteoliposomes had to be preloaded with ADP, indicating a counter-exchange mode of transport . Carboxyatractyloside did not substantially interfere with ATP transport into proteoliposomes containing the plastidic ATP/ADP translocator . An apparent KM for ATP of 28 microM was determined which is similar to values reported for isolated plastids . The data presented here strongly support the conclusion that AATP1 represents a novel eukaryotic adenylate carrier and that it is identical with the so far unknown plastidic ATP/ADP translocator.

Microbiology, 1997 Jan, 143 ( Pt 1), 147 - 56
Enterobactin synthase polypeptides of Escherichia coli are present in an osmotic-shock-sensitive cytoplasmic locality; Hantash FM et al.; The terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD, E, F and B/G polypeptides . The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated . In vitro results provided no evidence in support of the proposal: (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents, and (ii) immunoprecipitation experiments conducted on extracts engaged in Ent synthesis failed to detect any association among the Ent polypeptides . However, Western blot analyses showed that EntE, F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts . These results demonstrated that EntE, F and B/G belong to the Beacham group D class of proteins . The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and, for EntB/G, the N-terminus was sufficient for release by osmotic shock . The behaviour of group D proteins is generally attributed to their association (partial, loose or transient) with cytoplasmic membrane; therefore, the results are indirect evidence that Ent synthase interacts with membrane in vivo . At the very least, the data indicate that EntE, F and B/G are compartmentalized in E . coli and, because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins, suggest that this entire protein class may be sequestered in vivo.

Microbiology, 1997 Jan, 143 ( Pt 1), 117 - 26
ScCypB is a novel second cytosolic cyclophilin from Streptomyces chrysomallus which is phylogenetically distant from ScCypA; Pahl A et al.; A novel second streptomycete cyclophilin gene-designated sccypB-was isolated from a cosmid gene library of Streptomyces chrysomallus by using as gene probe a fragment of the previously isolated cyclophilin gene sccypA of the same organism . From its sequence the gene sccypB should encode a protein of M(r) 18868 . Expression of sccypB in Escherichia coli as a hexaHis-tagged fusion protein (H6ScCypB) and enzymic characterization of the purified protein showed that, like ScCypA, ScCypB is a peptidyl-prolyl cis-trans isomerase (PPIase) . The specific activity and substrate specificity of the enzyme were comparable to that of ScCypA, but it was threefold less sensitive to inhibition by cyclosporin A (CsA) . In contrast to ScCypA, which is abundant and exists in free and liganded form, ScCypB was 50- to 100-fold less abundant in cytosol-derived protein fractions of S . chrysomallus or Streptomyces lividans, as revealed by Western blot analyses, suggesting a specialized function for this enzyme in the streptomycete cell . Both sccypB and sccypA were found to be present as single copies in the genome of S . chrysomallus and hybridized to a single band in chromosomal DNAs of other streptomycetes . High-level expression of sccypB as well as of sccypA cloned into the expression vector pIJ702 did not produce detectable changes in growth and morphology of S . chrysomallus and S . lividans . Calculations of similarities to known cyclophilin sequences and construction of phylogenetic trees indicated that ScCypB and ScCypA are phylogenetically distant from each other . While ScCypA is clearly related to the eukaryotic cyclophilins, the analyses show the sequence of ScCypB to be the most divergent of all cyclophilin sequences, indicating that it possibly constitutes a cluster by itself.

Microbiology, 1997 Jan, 143 ( Pt 1), 45 - 53
TNF-alpha, IL-1 alpha, IL-6 and ICAM-1 expression in human keratinocytes stimulated in vitro with Escherichia coli heat-shock proteins; Marcatili A et al.; Bacterial heat-shock proteins (HSPs) from Escherichia coli (GroES, GroEL and DnaK) were studied for their ability to induce by themselves the expression and release of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) by cultured human keratinocytes . The surface expression of ICAM-1 was also investigated . In the supernatants of untreated cells none or a minimal amount of these molecules was found . After 48 h of stimulation with GroEL significant amounts of TNF-alpha, IL-1 alpha, IL-6 and soluble ICAM-1 were detected, reaching maximum concentrations at 1 microgram ml-1 . The same effect was elicited by DnaK but to a lesser extent . Treatment of keratinocytes with GroEL and DnaK also increased TNF-alpha, IL-1 alpha, IL-6 and ICAM-1 mRNA levels . GroES showed significant activity only on the expression and release of IL-6 . GroEL and DnaK were also able to up-regulate the surface expression of ICAM-1 on keratinocytes . The effects on ICAM-1 expression seemed to be directly due to HSPs and not mediated via cytokines . Furthermore, these effects were due to the properties of HSPs because they were inhibited by specific monoclonal antibodies . These findings support the potential role of HSPs in modulating cell interactions during immunological and inflammatory responses in the skin.

Mutagenesis, 1997 Jan, 12(1), 45 - 7
Mutagenicity of the potent rat hepatocarcinogen 6BT to the liver of transgenic (lacI) rats: consideration of a reduced mutation assay protocol; Lefevre PA et al.; 6-(p-dimethylaminophenylazo)benzothiazole (6BT) is an unusually potent rat hepatocarcinogen, producing large malignant liver tumours after only 2-3 months of dietary administration in a riboflavin-deficient diet . This azocarcinogen has been evaluated in a Big Blue F344 transgenic rat (lacI) gene mutation assay . In a reproduction of the early stages of the carcinogenesis bioassay of this agent, rats were maintained on a riboflavin-deficient diet and were given 10 consecutive daily doses of 6BT (10 mg/kg) by oral gavage . The animals were killed and the livers examined 11 days after the final dose . The livers of 6BT-treated rats showed evidence of hepatocellular hypertrophy in centrolobular areas, with some indication of an increased incidence of mitotic figures . An approximately 10-fold increase in the mutation frequency of DNA isolated from an aliquot of the combined liver homogenates of 6BT-treated rats was observed over that obtained from an equivalent aliquot from control animals . Examination of DNA samples isolated from the livers of individual animals confirmed that 6BT was mutagenic in Big Blue rat livers . These data extend the sensitivity of this transgenic assay to include azo hepatocarcinogens . The determination of mutation frequencies using pooled tissue samples represented a major resource-saving adaptation of the assay protocol in the present study; the general advantages and disadvantages of this practice are discussed.

Mutagenesis, 1997 Jan, 12(1), 23 - 8
Effects of nitrous acid treatment on the survival and mutagenesis of Escherichia coli cells lacking base excision repair (hypoxanthine-DNA glycosylase-ALK A protein) and/or nucleotide excision repair; Sidorkina O et al.; Deoxyinosine occurs in DNA by spontaneous deamination of adenine or by incorporation of dITP during replication . Hypoxanthine residues (HX) are mutagenic and give rise to A-T-->G-C transition . They are substrates for the Escherichia coli product of the alkA gene, the 3-methyl-adenine-DNA glycosylase II (ALK A protein) . In mammalian cells and in yeast, HX is excised by the counterpart of ALK A protein, the ANPG or the MAG proteins respectively . We have investigated in vivo the contribution of the alkA gene to counteract the lethal and/or mutagenic effects of HX residues induced by nitrous acid treatment . Using an E.coli strain allowing the detection of A-T-->G-C transition, we show that the alkA mutant has a slightly increased spontaneous rate of mutation and about the same sensitivity when treated with HNO2 as compared with the wild-type strain . Using the E.coli alkA mutant carrying a multicopy plasmid expressing the ALK A protein or the ANPG protein, we barely observe any effect of HNO2 treatment on sensitivity and mutation rate of the bacteria . In contrast, the same experiment performed with a uvrA- strain, deficient in nucleotide excision repair (NER), shows that this mutant is extremely sensitive to HNO2 treatment . Furthermore, the sensitivity and the spontaneous mutation rate observed in the double mutant alkA- uvrA- are almost identical to those of the uvrA- mutant . Hence, NER has the major role in vivo for the repair of lethal and mutagenic lesions induced by HNO2.

Rinsho Byori, 1997 Jan, 45(1), 19 - 26
{Autoimmune diseases and stress proteins}; Minota S; The heat shock protein(hsp), one of the most conserved and ubiquitous proteins in a wide range of species from bacteria to mammals, is strongly immunogenic . High conservation and potent immunogenicity, taken along with the fact that hsp is the target molecule of some gamma/delta T cells place it at the interface between immunity and tolerance . The role of hsp on autoimmune and inflammatory disorders has been vigorously investigated . There are accumulated lines of evidence suggestive of the possible involvement of hsp in clinical disorders but they still remain circumstantial . Two questions must be addressed before the exact role of hsp in these settings is established: 1) Why a conserved protein such as hsp is so immunogenic? 2) Why a ubiquitous protein can be a target of an organ-specific autoimmunity? Our data indicated that antibodies against highly conserved hsp60 in the sera from patients with autoimmune diseases, highlighted by rheumatoid arthritis, were directed mainly against epitopes specific for bacteria such as E . coli . The role of intestinal flora on the pathogenesis of rheumatoid arthritis has long been pursued and our data might support these lines.

DNA Cell Biol, 1997 Jan, 16(1), 17 - 22
The inducible lactose operator-repressor system is functional in the whole animal; Wu JD et al.; Mouse liver cell lines that bear a stably integrated lactose operon repressor (lacI) gene and a Ha-ras gene linked to a lactose operator-containing SV40 early promoter were generated . When grown in medium containing more than 0.1 mM isopropyl beta-D-thiogalactoside (IPTG), the Ha-ras gene was induced up to 20-fold . Maximum induction of Ha-ras gene expression occurred after 12 h of exposure . The tumorigenicity of these cell lines in syngeneic mice was enhanced when the mice were maintained on drinking water containing 12.5 mM IPTG . Ha-ras gene expression in tumors was strongly induced in the presence of IPTG in vivo . Induction of Ha-ras gene expression in mice was consistently observed after 48 hr of exposure to drinking water containing IPTG . This system provides an approach for studying the function of oncogene in vivo as well as other genes of interest.

J Exp Zool, 1997 Jan 1, 277(1), 38 - 48
Pheromones of the ciliate Euplotes octocarinatus not only induce conjugation but also function as chemoattractants; Kuhlmann HW et al.; Cells of the ten mating types of the ciliate Euplotes octocarinatus communicate by pheromones before they enter conjugation . The pheromones induce homotypic pairing when applied to mating types that do not secrete the same pheromone(s) . Heterotypic pairs (i.e., those between cells of different mating types) are formed only when both mating types in a mixture secrete a pheromone that the other does not . The genetics of mating types is based on four codominant mating type alleles, each allele determining production of a different pheromone . Here we report that the pheromones not only induce pair formation but also attract cells . This was shown by placing cells of various mating types in neighboring agar wells so that the pheromones could diffuse from one well to the next . We found that the cells accumulated on the side of the well where a pheromone entered by diffusion . This response was observed only if the pheromone had the capacity to induce the cells to conjugate . That the pheromones and not some other substances attract the cells was shown by placing pheromone 3, expressed in Escherichia coli, in wells next to tester strains . Mating types known to respond to pheromone 3 by pair formation also showed accumulation on the side of the well at which the pheromone entered by diffusion . Since the pattern of cell attraction corresponds with the pattern of conjugation induction, we suggest that not only conjugation induction but also cell attraction is governed by pheromone-specific receptors . In addition, we describe a succession of changes in the behavior of cells affected by the pheromones.

Mol Biol Cell, 1997 Jan, 8(1), 33 - 46
In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p; Schlaich NL et al.; The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport . To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli . When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry . In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind . For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length . Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo . When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores . We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.

Genetics, 1997 Jan, 145(1), 39 - 44
On the specificity of adaptive mutations; Hall BG; Adaptive mutations are mutations that occur in nondividing or slowly dividing cells during prolonged nonlethal selection, and that appear to be specific to the challenge of the selection in the sense that the only mutations that arise are those that provide a growth advantage to the cell . The issue of the specificity has been controversial because it violates our most basic assumptions about the randomness of mutations with respect to their effect on the cell . Although a variety of experiments in several systems in both bacteria and yeast have claimed to demonstrate that specificity, those experiments have been subjected to a variety of technical criticisms suggesting that the specificity may not be real . Here I use the ebg system to provide evidence that when selection is applied to one specific nucleotide site within a gene, mutation occurs at that site but not at an alternative and equally mutable site within the same gene.

Genetics, 1997 Jan, 145(1), 29 - 38
Highly mismatched molecules resembling recombination intermediates efficiently transform mismatch repair proficient Escherichia coli; Westmoreland J et al.; The ability of related DNAs to undergo recombination decreases with increased sequence divergence . Mismatch repair has been proposed to be a key factor in preventing homeologous recombination; however, the contribution of mismatch repair is not universal . Although mismatch repair has been proposed to act by preventing strand exchange and/or inactivating multiply mismatched heteroduplexes, there has been no systematic study to determine at what step(s) in recombination mismatch repair acts in vivo . Since heteroduplex is a commonly proposed intermediate in many models of recombination, we have investigated the consequences of mismatch repair on plasmids that are multiply mismatched in heteroduplex structures that are similar to those that might arise during recombination . Plasmids containing multiply mismatched regions were transformed into wild-type and Mut+ Escherichia coli mutants . There was only a 30-40% reduction in transformation of Mut+ as compared to mutS and mutL strains for DNAs containing an 18% mismatched heteroduplex . The products obtained from mutS hosts differed from those obtained from Mut+ hosts in that there were many more colonies containing mixtures of two plasmids, due to survival of both strands of the heteroduplex . There were nearly 10 times more recombinants obtained from the mutS as compared to the wild-type host . Based on these results and those from other studies with E . coli and yeast, we propose that the prevention of recombination between highly diverged DNAs may be at a step earlier than heteroduplex formation.

Nucleic Acids Res, 1997 Jan 1, 25(1), 188 - 91
Expansion of the 16S and 23S ribosomal RNA mutation databases (16SMDB and 23SMDB); Triman KL et al.; The Ribosomal RNA Mutation Databases (16SMDB and 23SMDB) provide lists of mutated positions in 16S and 23S ribosomal RNA from Escherichia coli and the identity of each alteration . Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation; (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods; and (iii) relevant literature citations . The databases are available via ftp and on the World Wide Web . Expansion of the databases to include information about mutations isolated in organisms other than E.coli is currently in progress.

Nucleic Acids Res, 1997 Jan 1, 25(1), 136 - 7
Databases and software for the analysis of mutations in the human p53 gene, the human hprt gene and both the lacI and lacZ gene in transgenic rodents; Cariello NF et al.; We have created databases and software applications for the analysis of DNA mutations at the humanp53gene, the humanhprtgene and both the rodent transgeniclacIandlacZlocus . The databases themselves are stand-alone dBASE files and the software for analysis of the databases runs on IBM-compatible computers . Each database has a separate software analysis program . The software created for these databases permit the filtering, ordering, report generation and display of information in the database . In addition, a significant number of routines have been developed for the analysis of single base substitutions . One method of obtaining the databases and software is via the World Wide Web (WWW) . Open the following home page with a Web Browser: ml . Alternatively, the databases and programs are available via public FTP from: anonymous@sunsite.unc.edu . There is no password required to enter the system . The databases and software are found beneath the subdirectory: pub/academic/biology/dna-mutations . Two other programs are available at the site-a program for comparison of mutational spectra and a program for entry of mutational data into a relational database.

Nucleic Acids Res, 1997 Jan 1, 25(1), 51 - 2
Genes and proteins of Escherichia coli K-12 (GenProtEC); Riley M; GenProtEC is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each . Also present are data on sequence similarities amongE.coliproteins with PAM values, percent identity of amino acids, length of alignment and percent aligned . GenProtEC can also be accessed through the World Wide Web at URL .

Nucleic Acids Res, 1997 Jan 1, 25(1), 43 - 51
EcoCyc: Enyclopedia of Escherichia coli Genes and Metabolism; Karp PD et al.; The Encyclopedia of Genes and Metabolism (EcoCyc) is a database that combines information about the genome and the intermediary metabolism of Escherichia coli . It describes 2970 genes of E.coli, 547 enzymes encoded by these genes, 702 metabolic reactions that occur in E.coli and the organization of these reactions into 107 metabolic pathways . The EcoCyc graphical user interface allows scientists to query and explore the EcoCyc database using visualization tools such as genomic-map browsers and automatic layouts of metabolic pathways . EcoCyc spans the space from sequence to function to allow scientists to investigate an unusually broad range of questions . EcoCyc can be thought of as both an electronic review article because of its copious references to the primary literature, and as an in silicio model of E.coli metabolism that can be probed and analyzed through computational means.

Nucleic Acids Res, 1997 Jan 1, 25(1), 39 - 42
Compilation of DNA sequences of Escherichia coli K12: description of the interactive databases ECD and ECDC (update 1996); Kroger M et al.; We have compiled the DNA sequence data forEscherichia coliavailable from the GenBank and EMBL data libraries and independently from the literature . We provide the most definitive version of the ECDEscherichia colidatabase now exclusively via the World Wide Web System: html/ecdc.html . Our database encloses an assembled set of contiguous sequences . Each of these contigs compiles all available sequence information, including those derived from a variety of elder sequences . The organisation of the database allows precise physical location of each individual gene or regulatory region, even taking into consideration discrepancies in nomenclature . The WWW program allows to branch into the original EMBL and SWISSPROT datafiles . A number of links to other WWW servers is provided . A FASTA and BLAST search may be performed online . Besides the WWW format a flat file version may be obtained via ftp . The ftp version may also be obtained from the EMBL data library as part of the CD-ROM issue of the EMBL sequence database, which is released and updated every 3 months . After deletion of all detected overlaps a total of 3 588 706 individual bp has been determined up to the end of September 1996 . This corresponds to a total of 77.09% of the entire E.coli chromosome consisting of approximately 4655 kb . About 479 kb (10.3%) are additionally available from Kyoto (Japan) . Another 94 kb (2%) are available, but mapping has not been confirmed . Thus the total may have reached 89.4%.

Mol Pharmacol, 1997 Jan, 51(1), 139 - 46
Aminoacyl and peptidyl analogs of chloramphenicol as slow-binding inhibitors of ribosomal peptidyltransferase: a new approach for evaluating their potency; Michelinaki M et al.; In a model system derived from Escherichia coli, acetylphenylalanyl-puromycin is produced in a pseudo-first-order reaction between the preformed acetylphenylalanyl/tRNA/poly(U)/ribosome complex (complex C) and excess puromycin . Two aminoacyl analogs {3, Gly-chloramphenicol (CAM): 4, L-Phe-CAM} and two peptidyl analogs (2, L-Phe-Gly-CAM: 5, Gly-Phe-CAM) of CAM (1) were tested as inhibitors in this reaction . Detailed kinetic analysis suggests that these analogs (I) react competitively with complex C and form the complex C*l, which is inactive toward puromycin . C*l is formed via a two-step mechanism in which C*l is the product of a slow conformational change of the initial encounter complex Cl according to the equation C + l reversible Cl reversible C*l . Furthermore, we provide evidence that analog 5 may react further with C*l forming the species C*l2 . The values of the apparent association rate constant (K(assoc)) are 1.42 x microM-1 min-1 for 2, 0.55 x microM-1 min-1 for 3, and 0.18 x microM-1 min-1 for 4 and 0.038 x microM-1 min-1 for 5 {corrected} . In the case of analog 5, K(assoc) is a linear function of the inhibitor concentration; when {I} approaches zero, the K(assoc) value is equal to 3.8 x 10(2) M-1 sec-1 . Such values allow the classification of CAM analogs as slow-binding inhibitors . According to K(assoc) values, we could surmise that analog 2 is 2.5-fold more potent than 3 and 8-fold more potent than 4 . The relative potency of analog 5 is the lowest among the analogs and is dependent on its concentration . The results are compared with previous data and discussed on the basis of a possible retro-inverso relationship between CAM analogs and puromycin.

J Virol Methods, 1997 Jan, 63(1-2), 237 - 42
A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA; Harper K et al.; A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV) . Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family . DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) . The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants . Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants . The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.

J Virol Methods, 1997 Jan, 63(1-2), 47 - 56
Detection of Maedi-Visna virus antibodies using a single fusion transmembrane-core p25 recombinant protein ELISA and a modified receiver-operating characteristic analysis to determine cut-off values; Boshoff CH et al.; The core p25 and transmembrane (TM) genes of Maedi-Visna virus (MVV) were cloned individually into the pGEX-2T expression vector . Both proteins were expressed as a combined fusion protein in frame with glutathione S-transferase (GST) . The purified recombinant antigens (GST-TM and GST-TM-p25) were used to develop a MVV ELISA . A preliminary assessment of the diagnostic potential of the recombinant antigens (GST-TM and GST-TM-p25) was made by testing the antigens against 46 seropositive and 46 seronegative sheep and comparing the results with a commercial p25 ELISA kit . A two-graph receiver operating characteristic (TG-ROC) analysis program was used to interpret the data . The GST-TM-p25 ELISA was more sensitive than the commercial assay which is based on the p25 antigen alone and more specific than the GST-TM ELISA.

Alcohol, 1997 Jan-Feb, 14(1), 99 - 105
Alcohol-induced thymocyte apoptosis is accompanied by impaired mitochondrial function; Wang JF et al.; This study examines the effects of chronic alcohol consumption on thymic apoptosis with or without pretreatment with E . coli lipopolysaccharide (LPS) . Apoptotic cell death of thymocytes was monitored by DNA fragments in gel electrophoresis and the appearance of apoptotic cells by flow cytometry . Changes in mitochondrial membrane potential (MMP), as indicated by 3,3'-dihexyloxacarbocyanine iodide {DiOC6(3)} uptake, and hydrogen peroxide (H2O2) production as indicated by oxidation of 2',7'-dichlorofluresin diacetate (DCFH-DA), were used to assess altered mitochondrial function . Glutathione levels were also determined to obtain information concerning alterations in the antioxidant potential in the cells . Male Sprague-Dawley rats, fed a nutritionally adequate liquid diet for 8-9 weeks, were divided in four groups: 1) saline-injected, diet controls; 2) LPS-injected, diet controls; 3) saline-injected, alcohol-consuming; and 4) LPS-injected, alcohol-consuming animals . LPS (0.5 mg/kg in 4 ml saline) or saline (4 ml) was continuously infused i.v . for 12 h before the experiments . The results showed that the weight and cell numbers of thymus from the chronic alcoholic rats were significantly less than values found in diet controls . Administration of LPS aggravated thymic apoptosis, as indicated by the presence of significant DNA fragments in gel electrophoresis and increased rate of apoptotic cells in flow cytometry . The alcohol-induced apoptotic changes were also accompanied by decreased MMP, indicating impaired mitochondrial function . Although H2O2 production by the total thymocyte population did not show marked changes among the experimental groups, the subpopulation of thymocytes exhibiting low H2O2 production was increased markedly in the LPS-treated groups . Ethanol consumption or LPS treatment decreased total glutathione concentration in the thymocytes . In summary, 1) chronic administration of alcohol induces atrophy of the thymus gland; 2) apoptosis is a major factor in thymic atrophy under these conditions; 3) chronic alcohol consumption is accompanied by alterations in mitochondrial function of the thymocytes, as indicated by decreased MMP and an increase in the low H2O2-producing cell subpopulation; 4) chronic alcohol abuse may impair intracellular defense mechanisms as reflected by the depletion of the intracellular antioxidant, glutathione; and 5) administration of LPS further enhances thymic apoptosis in chronic alcohol-consuming rats, suggesting that the dual insults of infection and chronic alcoholism exaggerate in vivo immunosuppression.

Cancer Gene Ther, 1997 Jan-Feb, 4(1), 59 - 65
Differential transfection efficiency rates of the GM-CSF gene into human renal cell carcinoma lines by lipofection; Hernandez A et al.; One of the major questions in any gene therapy approach is the selection of the appropriate vector system . Here, the optimization of a gene transfer protocol for renal cell carcinoma using lipofection as a nonviral gene transduction system was evaluated . To select the promoter which gives the highest expression, different plasmids which are able to express Escherichia coli beta-galactosidase gene as a reporter gene under the control of different promoters were tested: human cytomegalovirus promoter (pCMVbeta), simian virus 40 promoter (pSVbeta), adenovirus promoter (ADbeta), and herpes simplex virus thymidine kinase promoter (TKbeta) . The pCMVbeta revealed the highest expression of the beta-gal gene in the renal cell carcinoma (RCC) lines . Thus this CMV promoter was selected for the expression of the granulocyte-macrophage colony stimulator factor (GM-CSF) gene . Three different lipids (LipofectAmine, LipofectAce, and Lipofectin) were compared for their transduction efficiency, and the optimal conditions for quantitatively high lipofection rates were established . The consistently best results regarding gene expression as well as viability of the RCC lines were obtained when Lipofectin was used . Gene expression was monitored by a specific enzyme-linked immunosorbent assay and functionally validated by a cell proliferation test . The GM-CSF expression profile showed a peak at 48 hours after transfection and was still detectable after 5 days . Here the feasibility of efficient lipofection of the GM-CSF gene into RCC lines is demonstrated . Most importantly, considerable differences in the relative quantity of GM-CSF gene transfer into the different RCC lines was observed here . This may be of critical relevance for the design of any clinical gene transduction protocol in tumor cell vaccination attempts.

FEMS Immunol Med Microbiol, 1997 Jan, 17(1), 27 - 36
Effect of Escherichia coli L-form cytoplasmic membranes on the interaction between macrophages and Lewis lung carcinoma cells: scanning electron microscopy; Ivanova EH et al.; Scanning electron microscopy (SEM) investigations on the interactions between peritoneal macrophages from Lewis lung carcinoma (LLC)-bearing mice and LLC tumour cells during 21 days after tumour implantation were carried out . The action of lipopolysaccharide (LPS)-containing cytoplasmic membranes (CM), from the stable protoplast type L-form of Escherichia coli, on the activity of in vitro phagocytosis was studied; CM induced a continuous increase in macrophage numbers . Activation of macrophage surfaces in healthy and tumour-bearing mice was established . Lamelipods, pseudopods and migration fringes 14 days after CM application were seen . Crater-like cavities deeply in the macrophage cells as well as adherent or prominent engulfed tumour cells within macrophages were observed during in vitro interaction with LLC cells . Macrophages from tumour-bearing mice without CM treatment showed less activation evaluated by SEM during earlier stages of tumour growth . The SEM investigation proved the temporary stimulating effect of E . coli L-form CM on the cell surface activation of peritoneal macrophages in healthy and LLC-bearing mice.

J Gen Virol, 1997 Jan, 78 ( Pt 1), 171 - 7
Casein kinase II phosphorylates bovine papillomavirus type 1 E1 in vitro at a conserved motif; McShan GD et al.; The E1 protein of bovine papillomavirus type 1 (BPV-1) is a phosphoprotein which specifically binds and unwinds the virus replication origin by ATP-dependent helicase activity . The El protein has been shown to be multiply phosphorylated in vivo, although the sites of modification are incompletely mapped . Examination of the predicted amino acid sequence of all available E1 proteins revealed strong conservation between amino acids 25 and 60 of a motif consisting of a serine residue followed by a stretch of acidic residues . This conserved motif resembled a phosphorylation consensus site for the ubiquitous cellular kinase casein kinase II (CKII) . Biochemical and mutational analysis demonstrated that the BPV- 1 E1 protein is an in vitro substrate for CKII at the serine within this conserved motif.

J Gen Virol, 1997 Jan, 78 ( Pt 1), 119 - 24
Mapping of monoclonal antibody epitopes of the rabies virus P protein; Raux H et al.; Thirty-six monoclonal antibodies (MAbs) specific for the rabies virus P phosphoprotein were obtained from mice immunized with recombinant P (PV strain) produced in E . coli . All MAbs reacted against the corresponding rabies virus protein by ELISA and by Western blot analysis and revealed the presence of cytoplasmic inclusions in rabies virus infected cells . The epitopes of seven MAbs were mapped by testing their reactivity with protein fragments expressed from deletion mutants in transfected cells . Western blotting, immunoprecipitation and immunofluorescence assays were performed . These MAbs recognized epitopes in different domains of the P protein: 60% were directed against a region lying between residues 83-172 suggesting a major antigenic determinant of the rabies virus P protein in this region . Most of the antigenic sites appeared to be composed of linear epitopes . These MAbs will be useful as tools to dissect structural and functional properties of the rabies virus P protein.

Neuron, 1997 Jan, 18(1), 107 - 19
unc-8, a DEG/ENaC family member, encodes a subunit of a candidate mechanically gated channel that modulates C . elegans locomotion; Tavernarakis N et al.; Mechanically gated ion channels are important modulators of coordinated movement, yet little is known of their molecular properties . We report that C . elegans unc-8, originally identified by gain-of-function mutations that induce neuronal swelling and severe uncoordination, encodes a DEG/ENaC family member homologous to subunits of a candidate mechanically gated ion channel . unc-8 is expressed in several sensory neurons, interneurons, and motor neurons . unc-8 null mutants exhibit previously unrecognized but striking defects in the amplitude and wavelength of sinusoidal tracks inscribed as they move through an E . coli lawn . We hypothesize that UNC-8 channels could modulate coordinated movement in response to body stretch . del-1, a second DEG/ENaC family member coexpressed with unc-8 in a subset of motor neurons, might also participate in a channel that contributes to nematode proprioception.

Plant Physiol, 1997 Jan, 113(1), 181 - 90
Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants; Goddijn OJ et al.; As a first step toward the exploitation of the disaccharide trehalose as a stress-protective and preservative agent in plants, we engineered trehalose biosynthesis in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) by introducing the otsA and otsB genes from Escherichia coli, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively . In leaves of transgenic tobacco plants, very low levels of trehalose accumulation were obtained (0.11 mg g-1 fresh weight), whereas in transgenic potato tubers, no trehalose accumulated at all . Plant trehalase activity was shown to affect the accumulation of trehalose in these plants . An increase in trehalose accumulation, up to 0.41 and 4.04 mg g-1 fresh weight in tobacco leaves and potato micro-tubers, respectively, was noted when the potent trehalase inhibitor validamycin A was added to in vitro plants and to hydroponically grown greenhouse plants . Stunted growth and the formation of lancet-shaped leaves by trehalose-accumulating tobacco plants suggest a negative effect of trehalose biosynthesis on N . tabacum development . It is surprising that experiments with wild-type plants cultured in the presence of validamycin A indicate that, despite current belief, the capacity to synthesize trehalose may not be restricted to primitive phyla of vascular plants and certain "resurrection plants," but may exist throughout the angiosperms.

Plant Physiol, 1997 Jan, 113(1), 175 - 9
Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity; Rosler J et al.; A full-length cDNA encoding phenylalanine ammonia-lyase (PAL) from Zea mays L . was isolated and the coding region was expressed in Escherichia coli as a C-terminal fusion to glutathione S-transferase . After purification by glutathione-Sepharose chromatography, the glutathione S-transferase moiety was cleaved off and the resulting PAL enzyme analyzed . In contrast to PAL from dicots, this maize PAL isozyme catalyzed the deamination of both L-phenylalanine (PAL activity) and L-tyrosine (tyrosine ammonia-lyase activity) . These results provide unequivocal proof that PAL and tyrosine ammonia-lyase activities reside in the same polypeptide . In spite of large differences in the Michaelis constant and turnover number of the two activities, their catalytic efficiencies are very similar . Also, both activities have the same pH and temperature optima . These results imply that maize can produce p-coumaric acid from both phenylalanine and tyrosine.

Protein Sci, 1997 Jan, 6(1), 242 - 5
Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase; Pav S et al.; The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant . Both crystal forms belong to space group P2(1)2(1)2(1) . The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A . Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A . Diffraction data to 2.0 A resolution have been collected on both forms.

Protein Sci, 1997 Jan, 6(1), 141 - 6
Thermodynamics of maltose binding protein unfolding; Novokhatny V et al.; The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides . It is used widely as a carrier protein for the production of recombinant fusion proteins . The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions . MBP exhibits a single peak of heat absorption with a delta(Hcal)/delta(HvH) ratio in the range of 1.3-1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains . Binding of maltose resulted in elevation of the Tm by 8-15 degrees C, depending of pH . The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative . The delta(Hcal)/delta(HvH) ratio decreased to unity, indicating that the two domains melt together in a single two-state transition . This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the delta(Hcal)/delta(HvH) ratio . Binding of maltose to MBP is characterized by very low enthalpy changes, approximately -1 kcal/mol . The melting of MBP is accompanied by an exceptionally large change in heat capacity . 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface--0.72 A2/g--that becomes accessible to solvent in the unfolded state . The high value of delta Cp determines a very steep delta G versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures . Evidence for this was provided by changes in fluorescence intensity upon cooling the protein . A sigmoidal cooperative transition with a midpoint near 5 degrees C was observed when MBP was cooled at low pH . Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.

Protein Sci, 1997 Jan, 6(1), 119 - 24
Crystal structure of the reduced Schiff-base intermediate complex of transaldolase B from Escherichia coli: mechanistic implications for class I aldolases; Jia J et al.; Transaldolase catalyzes transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor . During catalysis, a Schiff-base intermediate between dihydroxyacetone and the epsilon-amino group of a lysine residue at the active site of the enzyme is formed . This Schiff-base intermediate has been trapped by reduction with potassium borohydride, and the crystal structure of this complex has been determined at 2.2 A resolution . The overall structures of the complex and the native enzyme are very similar; formation of the intermediate induces no large conformational changes . The dihydroxyacetone moiety is covalently linked to the side chain of Lys 132 at the active site of the enzyme . The Cl hydroxyl group of the dihydroxyacetone moiety forms hydrogen bonds to the side chains of residues Asn 154 and Ser 176 . The C3 hydroxyl group interacts with the side chain of Asp 17 and Asn 35 . Based on the crystal structure of this complex a reaction mechanism for transaldolase is proposed.

Mol Microbiol, 1997 Jan, 23(1), 151 - 9
Characterization of a mitogen-activated protein (MAP) kinase from Plasmodium falciparum; Graeser R et al.; A mitogen-activated protein (MAP) kinase gene, PfMAP, from Plasmodium falciparum was recently identified . We expressed this gene in Escherichia coli to test whether it encodes a functional MAP kinase . Recombinant PfMAP kinase autophosphorylates on both the tyrosine and threonine residues within the TXY motif, and readily phosphorylates myelin basic protein as exogenous substrate . This identifies the PfMAP gene product as a true member of the growing family of MAP kinases . Wild-type PfMAP kinase expressed in COS-7 (SV40 transformed African green monkey kidney) cells seemed to induce apoptosis in these cells . Western blots and immunoprecipitations indicated that the kinase is expressed during the growth of the parasite in the red blood cell as three major forms: truncated forms with apparent molecular masses of 40 kDa and 80 kDa, and as a protein of approximately 150 kDa . The 40 kDa form is present throughout the intraerythrocytic development, whereas the two larger forms are only detected in mature parasites . The 40 kDa and 80 kDa forms are tyrosine phosphorylated, indicating that they represent the active forms of the PfMAP kinase . The total PfMAP kinase activity constantly increases with the maturation of the parasite.

J Med Microbiol, 1997 Jan, 46(1), 67 - 74
Surface properties of diarrhoeagenic Escherichia coli isolates; Fletcher JN et al.; The surface properties of various Escherichia coli isolates associated with diarrhoeal illness were compared by aqueous partitioning between polyethylene glycol (PEG) and Dextran phases . Two well characterised strains of enteropathogenic E . coli (EPEC) were found to be very hydrophobic, based on the critical polymer concentration . EPEC strain E2348 cured of the EPEC adherence factor (EAF) plasmid had much reduced surface hydrophobicity . Partitioning of a series of diarrhoeagenic E . coli strains demonstrated that the majority of EAF+ EPEC strains were significantly more hydrophobic than EAF- EPEC strains . E . coli strains defined as enteroaggregative on the basis of hybridisation with a specific DNA probe showed much greater heterogeneity in their partitioning behaviour, possibly indicating that the AAF/I pili were not expressed in all strains . The E . coli K-12 strain used as a transformation host for adhesion studies had very low surface hydrophobicity but had a detectable negative charge . No alteration in these properties was observed when transformed with EPEC and recombinant plasmids known to specify adherence to tissue culture cells.

Biochem J, 1997 Jan 1, 321 ( Pt 1), 59 - 64
EF-hand motifs of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes; Yamada K et al.; The three diacylglycerol kinase isoenzymes (DGK alpha, DGK beta and DGK gamma) cloned so far contain in common a tandem repeat of EF-hand motifs . However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca(2+)-binding activities of these motifs have not been tested except for those present in DGK alpha . We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes . For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs . Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx . 2 mol of Ca2+ per mol . However, the apparent dissociation constant (Kd) for calcium binding to alpha-DKE (9.9 microM) was an order of magnitude greater than those estimated for beta-DKE (0.89 microM) and gamma-DKE (0.40 microM) . Experiments with 2-p-toluidinyl-naphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to beta-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of alpha-DKE and gamma-DKE were masked by the addition of Ca2+ . Taken together, these results indicate that DGK alpha, DGK beta and DGK gamma possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca(2+)-induced conformational changes.

Biochem J, 1997 Jan 1, 321 ( Pt 1), 49 - 58
The transmembrane domain of diphtheria toxin improves molecular conjugate gene transfer; Fisher KJ et al.; Vectors based on the formation of a soluble DNA-polycation complex are being developed for the treatment of human diseases . These complexes are rapidly taken up by receptor-mediated endocytosis, but are inefficiently delivered to the nucleus owing to entrapment in membrane-bound vesicles . In this study we introduced the transmembrane domain of diphtheria toxin into a DNA-polycation conjugate complex in an effort to increase gene transfer by membrane perturbation . The transmembrane domain of diphtheria toxin was expressed in Escherichia coli as a maltose-binding protein fusion and chemically coupled to high-molecular-mass poly-L-lysine . Incorporation of this conjugate into a traditional complex formed with a luciferase-containing plasmid with an asialo-orosomucoid-polycation conjugate significantly increased transfection efficiency in vitro in a manner proportional to the amount of diphtheria toxin incorporated . The delivery of luciferase RNA transcript was similarly increased when complexed with similar polycation conjugates . This study uses the structural biology of a bacterial protein to improve polycation-based gene delivery.

EXS, 1997, 80, 133 - 51
Genetically modified Escherichia coli for colorimetric detection of inorganic and organic Hg compounds; Klein J et al.; A sensitive colorimetric bacterial system was developed for the detection of Hg(II) and organomercury compounds . The bioactive species, a recombinant Escherichia coli, produces proportionally elevated levels of the enzyme beta-galactosidase with increasing amounts of Hg . This is due to a reporter plasmid which carriers a Hg(II)-inducible promoter (mer promoter) from the Hg resistance transposon Tn501 regulating the transcription of a promoterless lacZ gene . Additionally, a pMB1 origin of replication without the natural RNA polymerase start site is fused downstream of the mer promoter leading to a Hg(II)-inducible plasmid replication, which results in an improved signal-to-noise ratio . To enhance the sensitivity of this cellular biosensor, the transport proteins for Hg(II) uptake are constitutively produced by a helper plasmid . To enable the detection of organically bound Hg, the Streptomyces lividans organomercurical lyase, an enzyme which catalyses the cleavage of C-Hg-bonds of organomercurial compounds, is also provided by the helper plasmid . Hg(II) and phenylmercuric acetate (PMA) concentrations as low as 5 x 10(-10) M (0.1 ppb) may be detected within a few minutes.

Am J Respir Crit Care Med, 1997 Jan, 155(1), 343 - 50
Sensitization to Blomia tropicalis in patients with asthma and identification of allergen Blo t 5; Arruda LK et al.; In tropical and subtropical regions of the world, allergens produced by Blomia tropicalis are an important cause of IgE-mediated sensitization among patients with asthma . We compared the relative importance of sensitization to the two mite species among asthma patients from Florida, Puerto Rico, and Brazil (n = 83), who were concurrently exposed to B . tropicalis and D . pteronyssinus, with patients from the United States and from the United Kingdom (n = 56) exposed to D . pteronyssinus . In addition, molecular cloning techniques were used to clone and express a major B . tropicalis allergen . There were significant differences between IgE antibody responses to B . tropicalis and D . pteronyssinus that were related to exposure: only 22% of patients exposed to both species had a high ratio (> 10) of IgE D . pteronyssinus:B . tropicalis, whereas 68% of patients exposed only to D . pteronyssinus had a ratio of > 10 (p < 0.001) . A major 14-kD allergen (Blo t 5), cloned from a B . tropicalis cDNA library, showed 43% sequence homology to D . pteronyssinus Der p 5 . Recombinant Blo t 5 produced in E . coli reacted with 45 to 69% of sera from B . tropicalis-allergic asthmatics and induced positive immediate skin tests at 10(-3) to 1 microg/ml . In vivo and in vitro comparisons of IgE responses to B . tropicalis, D . pteronyssinus, rBlo t 5, and rDer p 5, showed that B . tropicalis has unique allergens that cause specific IgE responses . The results suggest that B . tropicalis is an independent cause of sensitization and that use of recombinant Blo t 5 should lead to a better understanding of the role of B . tropicalis in causing asthma in tropical environments.

Am J Respir Crit Care Med, 1997 Jan, 155(1), 268 - 73
Human lung mononuclear cells induce nitric oxide synthase in murine airway epithelial cells in vitro: role of TNFalpha and IL-1beta; Robbins RA et al.; Nitric oxide (NO) is a gas released by the airway epithelium, but the mechanism regulating NO release is unclear . We hypothesized that lung mononuclear cell release of tumor necrosis factor alpha (TNF) and interleukin-1beta (IL-1) would induce epithelial cells to release NO . Lung mononuclear cells were obtained from seven normal volunteers by bronchoalveolar lavage and cultured with Escherichia coli lipopolysaccharide for 24 h . The mononuclear cell culture-conditioned media (M-CM) were then applied to cultures of the murine lung epithelial cell line, LA-4 . Nitrite and nitrite + nitrate concentrations were 0.9 +/- 0.1 and 11.8 +/- 2.4 microM in the M-CM . Culturing LA-4 cells line with the M-CM (1:10 dilution) resulted in a marked and time-dependent increase in nitrite or nitrite + nitrate compared with LA-4 cells cultured in media alone (2.4 +/- 0.5 versus 0.9 +/- 0.1 microm and 16.6 +/- 0.6 versus 11.8 +/- 2.4 microM after 24 h) . Antibodies to TNF and/or IL-1 significantly reduced the nitrite or nitrite + nitrate concentrations and the concentrations of TNF and IL-1 in the M-CM correlated with nitrite concentrations in the LA-4 culture supernatant fluids (r2 = 0.848 and 0.956) . Inducible nitric oxide synthase (iNOS) protein and mRNA examined by immunohistochemistry and Northern blot analysis revealed a marked elevation in the cells cultured with the M-CM which was significantly reduced by TNF and IL-1 antibodies . These data demonstrate that mononuclear cells can stimulate LA-4 cells to express iNOS by releasing TNF and IL-1.

Br J Cancer, 1997, 75(1), 69 - 75
Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DT-diaphorase); Traver RD et al.; NAD(P)H:quinone oxidoreductase (NQO1, EC 1.6.99.2) is an obligate two-electron reductase that can either bioactivate or detoxify quinones and has been proposed to play an important role in chemoprevention . We have previously characterized a homozygous point mutation in the BE human colon carcinoma cell line that leads to a loss of NQO1 activity . Sequence analysis showed that this mutation was at position 609 of the NQO1 cDNA, conferring a proline to serine substitution at position 187 of the NQO1 enzyme . Using polymerase chain reaction (PCR) analysis, we have found that the H596 human non-small-cell lung cancer (NSCLC) cell line has elevated NQO1 mRNA, but no detectable enzyme activity . Sequencing of the coding region of NQO1 from the H596 cells showed the presence of the identical homozygous point mutation present in the BE cell line . Expression and purification of recombinant wild-type and mutant protein from E . coli showed that mutant protein could be detected using immunoblot analysis and had 2% of the enzymatic activity of the wild-type protein . PCR and Northern blot analysis showed moderate to low levels of expression of the correctly sized transcript in the mutant cells . Immunoblot analysis also revealed that recombinant mutant protein was immunoreactive; however, the mutant protein was not detected in the cytosol of either BE or H596 cells, suggesting that the mutant proteins were either not translated or were rapidly degraded . The absence of any detectable, active protein, therefore, appears to be responsible for the lack of NQO1 activity in cells homozygous for the mutation . A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for the mutation at position 609 conducted on 90 human lung tissue samples (45 matched sets of tumour and uninvolved tissue) revealed a 7% incidence of individuals homozygous for the mutation, and 42% heterozygous for the mutation . These data suggest that the mutation at position 609 represents a polymorphism in an important xenobiotic metabolizing enzyme, which has implications for cancer therapy, chemoprevention and chemoprotection.

Gen Comp Endocrinol, 1997 Jan, 105(1), 79 - 90
Evolution of insulin-like growth factor (IGF) function: production and characterization of recombinant hagfish IGF; Upton Z et al.; While there is considerable structural evidence that insulin-like growth factors (IGFs) share a long evolutionary history, little is known about the conservation of IGF function . In order to address this, we have made recombinant hagfish IGF, hence allowing characterization of an IGF from a representative of the primitive vertebrate class, Agnatha . The production of recombinant hagfish IGF has been complicated by a number of factors including the requirement of a longer leader peptide for fusion protein expression, reduced solubility of the protein, as well as problems in the refolding procedure . However, we were able to produce a small quantity of hagfish IGF with an N-terminal glycine addition which is biologically active . Furthermore, N-terminal amino acid sequencing and mass spectrometry confirm that we have produced hagfish IGF . In vitro assessment of recombinant hagfish IGF in cultured cells indicates that hagfish IGF indeed shares functional properties with mammalian IGFs . Thus, hagfish IGF stimulates protein synthesis in rat myoblasts, but 20- and 5-fold more peptide, respectively, is required to achieve the same half-maximal responses as with human IGF-I (hIGF-I) or IGF-II (hIGF-II) . Hagfish IGF also competes for binding to the type-1 IGF receptor present both on rat myoblasts and on salmon embryo fibroblasts, though with somewhat lower affinity than either hIGF-I or hIGF-II . However, studies investigating binding to the IGF-II-specific type-2 receptor suggest that hagfish IGF may in fact be more closely related to IGF-I than to IGF-II . These results indicate that motifs important for functions associated with mammalian IGFs appear to have evolved prior to the Agnathans diverging from the main line of vertebrate evolution 550 million years ago . Accordingly, we now have functional as well as structural evidence that the IGFs have a long evolutionary history.

Curr Genet, 1997 Jan, 31(1), 55 - 62
A mitochondrial sub-stoichiometric orf87-nad3-nad1 exonA co-transcription unit present in solanaceae was amplified in the genus Nicotiana; Gutierres S et al.; Unlike other plant species, two copies of nad3 are present in Nicotiana sylvestris mitochondria . Both are localized downstream from an open reading frame (orf87 ), and are associated with either rps12 or the first exon of the nad1 gene . The orf87-nad3-nad1/A cluster is present in normal stoichiometry in Nicotiana tomentosiformis and is sub-stoichiometric in other Solanaceae, revealing recent amplification in the genus Nicotiana . It is suggested from sequence analysis that this cluster originated in an homologous recombination event that involved the nad3-rps12 intergenic region and the upstream region of an ancestral nad1 gene . Transcription patterns and RT-PCR showed that orf87-nad3-rps12 and orf87-nad3-nad1/A clusters are both co-transcription units.

Parasitol Res, 1997, 83(1), 96 - 8
Identification of Entamoeba histolytica and E . dispar cysts in stool by polymerase chain reaction; Sanuki J et al.; An attempt to identify cysts of Entamoeba histolytica and E . dispar in human stool was conducted by polymerase chain reaction (PCR) using two sets of primers (p11 plus p12 and p13 plus p14) specific for either species of ameba . The cysts in stool specimens obtained from 12 infected individuals were concentrated, freeze-thawed, and treated with Triton X-100 before their examination by PCR . The results of PCR on the cysts were generally consistent with data obtained by PCR on ameba trophozoites hatched from the cysts, by zymodeme analysis, and by enzyme-linked immunosorbent assay (ELISA) and with clinical findings . This PCR was negative for the stool containing large numbers of cysts of either E . coli, E . hartmanni, or Giardia lamblia as well as for the stool specimens obtained from uninfected individuals . The ameba cyst in stool processed using the present method was effective for the PCR analysis even after 1 month of storage at 4 degrees C . The present PCR was sensitive enough to detect ten cysts of either of the amebae.

Life Sci, 1997, 60(2), PL31 - 8
A study on rat platelet responsiveness following intravenous endotoxin administration; Cicala C et al.; The aim of the present study was to evaluate platelet responsiveness in rats following E.coli endotoxin administration . Injection of E.coli to rats caused a reduction in ADP-induced pulmonary 111In labelled platelet accumulation four hours later . Similarly, when platelet aggregation was evaluated on PRP obtained from rats four hours after endotoxin administration, we found that platelet response to both ADP and collagen was significantly reduced . When platelets obtained from endotoxemic rats were suspended in normal plasma, the aggregating response to ADP and collagen was not different from that obtained with control platelets . Similarly, platelets from control rats suspended in plasma from endotoxemic rats showed hyporesponsiveness to ADP and collagen . There was no difference in the aggregatory response to collagen or to thrombin of washed platelet suspension (WPS) obtained from endotoxemic and normal rats . In conclusion, by using an in vivo minimally invasive technique and an ex vivo platelet aggregation test we demonstrate that during endotoxemia platelet are functionally unaltered and the platelet hyporesponsiveness is only observed in presence of plasma.

FEMS Microbiol Lett, 1997 Jan 1, 146(1), 143 - 8
Expression of the heme biosynthetic pathway genes hemCD, hemH, hemM, and hemA of Escherichia coli; McNicholas PM et al.; Little is known about the control of latter steps of heme biosynthesis in Escherichia coli . In this study we examined the transcriptional regulation of genes that encode two intermediate heme pathway enzymes, porphobilinogen deaminase (hemC) and uroporphyrinogen III cosynthase (hemD), and the final enzyme of the pathway, ferrochelatase (hemH) . We also reexamined the regulation of hemA and the gene located immediately upstream of hemA, hemM . The regulatory regions of hemC, hemH, hemA and hemM were fused to lacZ . The resultant operon fusions were inserted into the E . coli chromosome in single copy and expression monitored under conditions of oxygen and heme limitation . Expression of hemM appeared constitutive under the conditions tested here . In contrast, expression of hemCD, hemH and hemA were shown to be mildly regulated in response to heme availability . Thus, transcription of four of the nine genes of the E . coli heme pathway appears to be only mildly regulated in response to heme limitation.

FEMS Microbiol Lett, 1997 Jan 1, 146(1), 123 - 8
Virulence properties of Escherichia coli O111:H12 strains; Monteiro-Neto V et al.; Seventeen Escherichia coli O111:H12 strains isolated from the feces of children with acute diarrhea were studied with regard to their adherence properties and other virulence characteristics . All strains showed an aggregative adherence pattern to HEp-2 cells and agglutinated bovine and sheep red cells in the presence of mannose . These strains did not have gene sequences homologous to the aggregative adherence fimbria I gene and did not react with any of the DNA probes used to detect other virulence genes in enteropathogens . With one exception, the O111:H12 strains did not induce fluid accumulation in the rabbit ileal loop assay, although 16 of the strains had the enteroaggregative E . coli heat-stable enterotoxin 1 (EAST) gene sequences . A 60-70 MDa plasmid was present in 16 of the strains studied . We conclude that the O111:H12 serotype, one of the first E . coli identified in infantile diarrhea, belongs to the enteroaggregative E . coli category but the genes encoding its adherence phenotype are distinct from those previously described.

FEMS Microbiol Lett, 1997 Jan 1, 146(1), 103 - 8
Involvement of iclR and rpoS in the induction of acs, the gene for acetyl coenzyme A synthetase of Escherichia coli K-12; Shin S et al.; Two independent pathways in Escherichia coli convert acetate to acetyl CoA: reversal of acetate production by phosphotransacetylase and acetate kinase, and the acetyl-CoA synthetase (Acs) pathway that scavenges acetate . We investigated acs gene expression by using a cat transcriptional fusion . It was observed that acs expression varies depending on the carbon sources used and occurs in the stationary phase of growth even in the absence of acetate . Mutations in iclR for the repressor of the glyoxylate shunt and in rpoS for the stationary phase sigma factor reduced the consumption of acetate mediated by Acs, indicating that both are involved in acs regulation.

Cancer Chemother Pharmacol, 1997, 39(3), 259 - 66
Comparison of the potency of glycosylated and nonglycosylated recombinant human granulocyte colony-stimulating factors in neutropenic and nonneutropenic CD rats; Nohynek GJ et al.; Recent studies in human bone-marrow culture and healthy human volunteers suggest that lenograstim {glycosylated, recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Chinese hamster ovary (CHO) cells} has greater in vivo potency than filgrastim {nonglycosylated, methionine-extended recombinant human granulocyte colony-stimulating factor (rmetHuG-CSF) produced in Escherichia coli} . To confirm and extend these results we investigated the in vivo potency of both products in normal rats and neutropenic CD rats as an animal model of chemotherapy-induced neutropenia . In normal rats, groups of eight normal male CD rats received four subcutaneous doses of 10, 30, or 100 micrograms/kg filgrastim or lenograstim on days 1-4 of the study, whereas a control group received the vehicle . Blood samples were collected from each animal before treatment (day -5) and on days 2, 3, 5, 8, and 12 of the study for determination of red blood cell (RBC), platelet, white blood cell (WBC), and differential counts . rHuG-CSF and r-metHuG-CSF produced increased WBC counts, principally due to elevated absolute neutrophil counts (ANCs); on days 2, 3, and 5, all groups receiving rG-CSF had ANCs that increased in a progressive and dose-related manner . With the exception of a single value, mean ANCs obtained on days 2, 3, and 5 in lenograstim-treated groups were higher (statistically significant on day 3 at 30 and 100 micrograms/kg and on day 5 at 10, 30, and 100 micrograms/kg) than the respective values obtained in filgrastim-treated groups . No compound-related effect was noted in RBC or platelet parameters . Neutropenia was induced in male CD rats (12 animals/group) with a single intraperitoneal dose of 50 mg/kg cyclophosphamide (CPA) on day 0 . On days 1-4, CPA-treated groups were treated with the vehicle (control) or with filgrastim or lenograstim at 30 or 100 micrograms/kg per day . An additional group was not treated with CPA and served as the absolute control group . Blood was collected from alternating subgroups on study day -5 (pretest) and on days 2, 3, 4, 5, 6, 8, 9, and 12 for determination of RBC, platelet, WBC, and differential counts . No major adverse in-life effect was noted in neutropenic rats . Maximal depression of WBCs and ANCs occurred on day 5, followed by recovery to normal values by days 9 (ANC) and 12 (WBC) . On day 3 and days 5-9, rHuG-CSF- and metHuG-CSF-treated groups had marked and dose-related increases in WBCs as compared with CPA-treated controls, principally due to elevated ANCs . With the exception of a few values, mean ANC values obtained in lenograstim-treated groups were consistently higher than the respective values obtained in filgrastim-treated groups; the difference was statistically significant on day 3 (30-microgram/kg groups) and on days 6 and 8 (100-microgram/kg groups) . In conclusion, treatment of normal and neutropenic CD rats with lenograstim resulted in a dose-related elevation of ANCs that was consistently and significantly higher than the response to identical doses of filgrastim . These results suggest that lenograstim, the glycosylated form of rG-CSF, has superior in vivo potency in normal and neutropenic animals as compared with filgrastim, the nonglycosylated form of rG-CSF.

Biotechniques, 1997 Jan, 22(1), 140 - 9
Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions; Lindner P et al.; Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins . From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage . The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore method, Western blots and enzyme-linked immunosorbent assay (ELISA) . Western blots and ELISAs can be developed directly by using crude extracts of E.coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent . Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose . The dissociation constants, KD of the peptide KGGHHHHH (KD = 4 x 10(-7) M) and of imidazole (KD = 4 x 10(-4) M) were determined . Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.

Biotechniques, 1997 Jan, 22(1), 105 - 8, 110
Deoxyribonuclease treatment improves the homogeneity of single-stranded DNA preparations; Aliev TK et al.; The isolation of single-stranded (ss) phagemid DNA using standard protocols often results in impure preparations, which contain undesirable quantities of chromosomal and/or double-stranded (ds) phagemid DNA . Here we report a simple and efficient method for elimination of virtually all dsDNA by incubation of phagemid viral particles with deoxyribonuclease I . In addition to analyzing the ratio of linear-to-circular topological forms of ssDNA after deoxyribonuclease I treatment, we verified that no decrease in transformation efficiency occurred and demonstrated that ssDNA molecules covered by capsid proteins remained intact following such treatment.

RNA, 1997 Jan, 3(1), 89 - 103
Probing the structure of the Escherichia coli 10Sa RNA (tmRNA); Felden B et al.; The conformation of the Escherichia coli 10Sa RNA (tmRNA) in solution was investigated using chemical and enzymatic probes . Single- and double-stranded domains were identified by hydrolysis of tmRNA in imidazole buffer and by lead(II)-induced cleavages . Ribonucleases T1 and S1 were used to map unpaired nucleotides and ribonuclease V1 was used to identify paired bases or stacked nucleotides . Specific atomic positions of bases were probed with dimethylsulfate, a carbodiimide, and diethylpyrocarbonate . Covariations, identified by sequence alignment with nine other tmRNA sequences, suggest the presence of several tertiary interactions, including pseudoknots . Temperature-gradient gel electrophoresis experiments showed structural transitions of tmRNA starting around 40 degrees C, and enzymatic probing performed at selected temperatures revealed the progressive melting of several predicted interactions . Based on these data, a secondary structure is proposed, containing two stems, four stem-loops, four pseudoknots, and an unstable structural domain, some connected by single-stranded A-rich sequence stretches . A tRNA-like domain, including an already reported acceptor branch, is supported by the probing data . A second structural domain encompasses the coding sequence, which extends from the top of one stem-loop to the top of another, with a 7-nt single-stranded stretch between . A third structural module containing pseudoknots connects and probably orients the tRNA-like domain and the coding sequence . Several discrepancies between the probing data and the phylogeny suggest that E . coli tmRNA undergoes a conformational change.

RNA, 1997 Jan, 3(1), 49 - 56
Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome; von Ahsen U et al.; Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) bind efficiently in an mRNA-dependent manner to ribosomes, whereas those made from DNA do not . In order to identify the positions where ribose is required, the anticodon stem-loop region of tRNA(Phe) (Escherichia coli) was synthesized chemically using a mixture of 2'-hydroxyl- and 2'-deoxynucleotide phosphoramidites . Oligonucleotides whose ribose composition allowed binding were retained selectively on nitrocellulose filters via binding to 30S ribosomal subunits . The binding-competent oligonucleotides were submitted to partial alkaline hydrolysis to identify the positions that were enriched for ribose . Quantification revealed a strong preference for a 2'-hydroxyl group at position U33 . This was shown directly by the 50-fold lower binding affinity of a stem loop containing a single deoxyribose at position U33 . Similarly, defective binding of the corresponding U33-2'-O-methyl-substituted stem-loop RNA suggests that absence of the 2'-hydroxyl group, rather than an altered sugar pucker, is responsible . Stem-loop oligoribonucleotides from different tRNAs with U33-deoxy substitutions showed similar, although quantitatively different effects, suggesting that intramolecular rather than tRNA-ribosome interactions are affected . Because the 2'-hydroxyl group of U33 was shown to be a major determinant of the U-turn of the anticodon loop in the crystal structure of tRNA(Phe) in yeast, our finding might indicate that the U-turn conformation in the anticodon loop is required and/or maintained when the tRNA is bound to the ribosomal P site.

J Bacteriol, 1997 Jan, 179(2), 538 - 40
Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli; Dehio C et al.; The first demonstration of conjugal plasmid transfer from Escherichia coli to Bartonella henselae is reported . Transconjugants bearing plasmids of incompatibility groups P (IncP) and Q (IncQ), expressing various resistance markers, were generated . Tn5 transposons delivered on suicide plasmids by conjugation showed transpositional insertion into random chromosomal sites.

J Bacteriol, 1997 Jan, 179(2), 514 - 21
The Caulobacter heat shock sigma factor gene rpoH is positively autoregulated from a sigma32-dependent promoter; Wu J et al.; Sigma factor sigma32, encoded by rpoH, is required for the recognition of heat shock genes during normal growth conditions and in response to heat shock and other stresses . Unlike the well-studied Escherichia coli rpoH gene, which is transcribed from four promoters recognized by either a sigma70 (sigmaD)- or sigma24 (sigmaE)-containing RNA polymerase, the Caulobacter crescentus rpoH gene is transcribed from two promoters, P1 and P2 . In this study, we have examined the structure and expression of these promoters and shown that the rpoH P2 promoter is sigma32 dependent . We present evidence here that P2 is specifically recognized and transcribed by the reconstituted C . crescentus Esigma32 RNA polymerase holoenzyme . We show that site-directed mutations within either the -10 or the -35 regions of P2 have substantial effects on the levels of transcription by the Esigma32 polymerase predicted from the sigma32 promoter consensus sequence . The mutations have similar effects in vivo as assayed with rpoH-lacZ transcription fusions . Analysis of the rpoH P1 promoter provided evidence that it is sigma70 dependent . S1 nuclease protection assays of rpoH P1- and P2-specific expression after heat shock at 42 or 50 degrees C and during synchronous cell division cycles under normal growth conditions showed that the two promoters are differentially regulated . Mutations within the rpoH P2 promoter consensus sequences abolished the response to heat shock induction in C . crescentus . We conclude from these results that, unlike rpoH genes studied previously in other bacteria, the major transcriptional response of the C . crescentus rpoH gene to heat shock depends on positive autoregulation of the sigma32-dependent promoter.

J Bacteriol, 1997 Jan, 179(2), 470 - 6
Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016; Hong ST et al.; The genes for the complete pathways for two polycyclic aromatic polyketides of the angucyclinone class have been cloned and heterologously expressed . Genomic DNAs of Streptomyces rimosus NRRL 3016 and Streptomyces strain WP 4669 were partially digested with MboI, and libraries (ca . 40-kb fragments) in Escherichia coli XL1-Blue MR were prepared with the cosmid vector pOJ446 . Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes from each organism . After transfer of the four clusters to Streptomyces lividans TK24, expression of one cluster from each organism was established through the identification of pathway-specific products by high-performance liquid chromatography with photodiode array detection . Peaks were identified from the S . rimosus cluster (pksRIM-1) for tetrangulol, tetrangomycin, and fridamycin E . Peaks were identified from the WP 4669 cluster (pksWP-2) for tetrangulol, 19-hydroxytetrangulol, 8-O-methyltetrangulol, 19-hydroxy-8-O-methyltetrangulol, and PD 116740 . Structures were confirmed by 1H nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.

J Bacteriol, 1997 Jan, 179(2), 445 - 52
Heat shock regulation of sigmaS turnover: a role for DnaK and relationship between stress responses mediated by sigmaS and sigma32 in Escherichia coli; Muffler A et al.; The cellular level of the rpoS-encoded sigmaS subunit of RNA polymerase increases in response to various stress situations that include starvation, high osmolarity, and shift to acid pH, and these different stress signals differentially affect rpoS translation and/or sigmaS stability . Here we demonstrate that sigmaS is also induced by heat shock and that this induction is exclusively due to an interference with sigmaS turnover . Some sigmaS-dependent genes exhibit similar heat shock induction, whereas others are not induced probably because they need additional regulatory factors that might not be present under conditions of heat shock or exponential growth . Despite its induction, sigmaS does not seem to contribute to heat adaptation but may induce cross-protection against different stresses . While sigmaS is not involved in the regulation of the heat shock sigma factor sigma32, the heat shock protein DnaK has a positive role in the posttranscriptional control of sigmaS . The present evidence suggests that DnaK is involved in the transduction of two of the signals that result in reduced sigmaS turnover, i.e., heat shock and carbon starvation . Heat shock induction of sigmaS also clearly indicates that a cessation of growth or even a reduction of the growth rate is not a prerequisite for the induction of sigmaS and sigmaS-dependent genes and underscores the importance of sigmaS as a general stress sigma factor.

J Bacteriol, 1997 Jan, 179(2), 423 - 9
The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription; Czarniecki D et al.; The lactose (lac) operon promoter is positively regulated by the catabolite gene activator-cyclic AMP complex (CAP) that binds to the DNA located 61.5 bp upstream of the transcription start site . Between the CAP binding site and the core promoter sequence is a 13-bp sequence (from -38 to -50 {the -45 region}) . The possible roles of the -45 region in determining the CAP-independent level of lac expression and in the CAP activation process were studied by isolating and characterizing random multisite mutations . Only a small percentage of mutants have dramatic effects on lac promoter activity . Among the mutations that did affect expression, a 26-fold range in lac promoter activity in vivo was observed in the CAP-independent activity . The highest level of CAP-independent lac expression (13-fold the level of the wild-type lac promoter) correlated with changes in the -40 to -45 sequence and required an intact RNA polymerase alpha subunit for in vitro expression, as expected for an upstream DNA recognition element . Mutant promoters varied in their ability to be stimulated by CAP in vivo, with levels ranging from 2-fold to the wild-type level of 22-fold . Only a change of twofold in responsiveness to CAP could be attributed to direct DNA sequence effects . The -40 to -45 sequence-dependent enhancement of promoter activity and CAP stimulation of promoter activity did not act additively . The mutant promoters also displayed other characteristics, such as the activation of nascent promoter-like activities overlapping lac P1 and, in one case, replicon-dependent changes in promoter activity.

J Bacteriol, 1997 Jan, 179(2), 417 - 22
Proliferation of mutators in A cell population; Mao EF et al.; A Lac- strain of Escherichia coli that reverts by the addition of a G to a G-G-G-G-G-G sequence was used to study the proliferation of mutators in a bacterial culture . Selection for the Lac+ phenotype, which is greatly stimulated in mismatch repair-deficient strains, results in an increase in the percentage of mutators in the selected population from less than 1 per 100,000 cells to 1 per 200 cells . All the mutators detected were deficient in the mismatch repair system . Mutagenesis results in a similar increase in the percentage of mutators . Mutagenesis combined with a single selection can result in a population of more than 50% mutators when a sample of several thousand cells is grown out and selected . Mutagenesis combined with two or more successive selections can generate a population that is 100% mutator . These experiments are discussed in relation to ideas that an early step in carcinogenesis is the creation of a mutator phenotype.

J Bacteriol, 1997 Jan, 179(2), 382 - 8
Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli; Gonzalez-Flecha B et al.; The exponential phase of aerobic growth is associated with risk of endogenous oxidative stress in which cells need to cope with an approximately 10-fold increase in the rate of H2O2 generation . We addressed this issue by studying the regulation of the intracellular concentration of H2O2 in aerobically growing Escherichia coli . Intracellular H2O2 was kept at an almost constant steady-state value of approximately 0.2 microM (variation, less than twofold) over a broad range of cell densities in rich medium . This regulation was achieved in part by a transient increase in the OxyR-dependent transcription of the catalase gene katG (monitored by using a katG::lacZ operon fusion) during exponential growth, directly correlated with the increased rate of H2O2 generation . The OxyR-regulated alkyl hydroperoxide reductase encoded by ahpFC did not detectably affect H2O2 or catalase activity levels . Induction of katG, ahpFC, and perhaps other genes prevented the accumulation of oxidatively modified lipids but may not have protected DNA: the spontaneous mutation rate was significantly increased in both wild-type and delta(oxy)R strains during exponential growth compared to that in these strains during lag or stationary phases . Strains lacking oxyR showed throughout growth an 8- to 10-fold-higher frequency of spontaneous mutation than was seen for wild-type bacteria . The ahpdelta5 allele also had a mutator effect half of that of delta(oxy)R in exponential and stationary phases and equal to that of deltaoxyR in lag phase, perhaps by affecting organic peroxide levels . These results show that oxyR-regulated catalase expression is not solely an emergency response of E . coli to environmental oxidative stress, but also that it mediates a homeostatic regulation of the H2O2 produced by normal aerobic metabolism . The activation of the oxyR regulon in this process occurs at much lower levels of H2O2 (approximately 10(-7)M) than those reported for oxyR activation by exogenous H2O2 (approximately 10(-5) M).

J Bacteriol, 1997 Jan, 179(2), 330 - 8
Identification and characterization of hydrogen peroxide-sensitive mutants of Escherichia coli: genes that require OxyR for expression; Mukhopadhyay S et al.; Escherichia coli produces an inducible set of proteins that protect the cell from exogenous peroxide stress . A subset of these genes is induced by hydrogen peroxide and is controlled at the transcriptional level by the OxyR protein . To identify additional genes involved in protection from hydrogen peroxide, a library of random transcriptional fusions of lambda(plac)Mu53 was screened for hydrogen peroxide sensitivity and 27 such mutants were identified . These fusions were transduced into nonlysogenic strains to ensure that the phenotypes observed were the result of a single mutation . The mutants were grouped into three classes based on the expression of the lacZ fusion during growth in oxyR+ and deltaoxyR backgrounds . The expression of the lacZ fusion in 8 mutants was independent of OxyR, 10 mutants required OxyR for expression, and 6 mutants showed reduced levels of expression in the presence of OxyR . OxyR dependence varied from 2- to 50-fold in these mutants . The OxyR-dependent phenotype was complemented by a plasmid-borne copy of oxyR gene in all mutants . Three mutants exhibited dual regulation by OxyR and RpoS . We sequenced the fusion junctions of several of these mutants and identified the genetic loci responsible for the hydrogen peroxide-sensitive (hps) phenotype . In this study, we report the identification of several genes that require OxyR for expression, including hemF (encoding coproporphyrinogen III oxidase), rcsC (encoding a sensor-regulator protein of capsular polysaccharide synthesis genes), and an open reading frame, f497, that is similar to arylsulfatase-encoding genes.

J Bacteriol, 1997 Jan, 179(2), 310 - 6
nfi, the gene for endonuclease V in Escherichia coli K-12; Guo G et al.; Endonuclease V is specific for single-stranded DNA or for duplex DNA that contains uracil or that is damaged by a variety of agents (B . Demple and S . Linn, J . Biol . Chem . 257:2848-2855, 1982) . Thus, it may be a versatile DNA repair enzyme . The protein was purified to apparent homogeneity, and from its N-terminal sequence, its gene, nfi, was identified . nfi is immediately downstream of hemE, at kb 4208 (90.4 min) on the current chromosomal map of Escherichia coli K-12 . This region was cloned, and plasmid insertion and deletion mutants were used to study its molecular organization . Although nfi is the third of four closely spaced, codirectional genes, it is expressed independently.

Arch Biochem Biophys, 1997 Jan 1, 337(1), 8 - 16
Structural interactions of the oligomycin sensitivity-conferring protein in the yeast ATP synthase; Mao Y et al.; The structure/function relationship of oligomycin sensitivity-conferring protein (OSCP), subunit 5 of the mitochondrial ATP synthase, from yeast Saccharomyces cerevisiae has been studied by a combination of genetic and biochemical methods . OSCP was studied by deletion mutagenesis of the N- and C-terminal regions by modifying the gene coding for OSCP . Two deletion mutations were made immediately downstream of the leader peptide of OSCP and five were made at the C-terminus . OSCP was functional with deletions of amino acids 3 to 17 (ND15) and of the last 8 amino acids (CD8), while deletion of amino acids 3 to 31 (ND29) and the last 9 amino acids (CD9) inactivated the ATP synthase, as determined by in vivo analysis . The deletion mutants were expressed in Escherichia coli, purified, and studied by in vitro reconstitution studies . Circular dichroism studies suggested that the mutant proteins, with the possible exception of ND29, were folded in a similar fashion as wild-type OSCP . Mutants ND15 and CD8 were able to reconstitute an oligomycin-sensitive ATPase complex, although not as well as wild-type OSCP, while ND29 and CD9 were completely ineffective . Binding studies of ND29 and CD9 indicate that these mutants in OSCP were unable to bind to the membrane portion of the ATP synthase, F0, and these results were supported by competition binding studies . These results support the hypothesis that the N- and C-terminal regions of subunit 5 interact with F0 and suggest that the central region interacts with F1.

Hum Gene Ther, 1997 Jan 1, 8(1), 73 - 85
Glioma cells transduced with an Escherichia coli CD/HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity; Rogulski KR et al.; To ascertain whether concomitant expression of Escherichia coli deaminase (CD) and herpes simplex virus type-1 thymidine kinase (HSV-1 TK) could mediate greater levels of cytotoxicity beyond that observed with either suicide gene alone, 9L gliosarcoma cells were transduced with a retrovirus encoding a CD/HSV-1 TK fusion gene . The resultant CD/HSV-1 TK fusion protein (CDglyTK) was found to be bifunctional via CD and HSV-1 TK enzymatic assays, and conferred upon cells prodrug sensitivities equivalent to or better than that observed for each enzyme independently (ganciclovir {GCV} and bromovinyldeoxyuridine {BVdU} for HSV-1 TK and 5-fluorocytosine {5-FC} for CD) . Simultaneous treatment of CDglyTK-expressing cells with prodrugs specific for HSV-1 TK and CD (GCV/5-FC or BVdU/5-FC) resulted in slight synergistic toxicity, two- to three-fold greater than that expected if the cytotoxic effects of each prodrug were purely additive . More importantly, co-treatment with HSV-1 TK- and CD-specific prodrugs was found to increase greatly the radiosensitivity of CDglyTK-expressing cells . Sensitivity enhancement ratios of 2.44 (GCV/5-FC) and 3.90 (BVdU/5-FC) were achieved . The results suggest that double suicide gene therapy, using a bifunctional CD/HSV-1 TK fusion gene, coupled with radiotherapy may provide a highly efficient means of selectively treating cancer.

Shock, 1997 Jan, 7(1), 55 - 9
Dehydroepiandrosterone, an endogenous immune modulator, after traumatic shock; Schurr MJ et al.; Dehydroepiandrosterone (DHEA), an endogenous immune modulator, reduces mortality after endotoxin (lipopolysaccharide (LPS)) administration in rodents . However, there have been no studies in clinically relevant large-animal models . A unique experimental model is used to study the effects of DHEA in resuscitated trauma and to evaluate the protective effect of DHEA on the systemic inflammatory response induced by a delayed LPS challenge . Anesthetized, ventilated pigs were instrumented and then subjected to local hind-limb trauma and 35% hemorrhage . After 1 h, animals were resuscitated with shed blood, supplemental Ringers solution, and in a randomized, blinded fashion, 4 mg/kg of DHEA or vehicle . Two additional groups received 10 mg/kg or 20 mg/kg of DHEA . Animals were dosed again at 24, 48, and 72 h . After 75 h, Escherichia coli LPS was administered . LPS caused a fall in DHEA levels (0.23 +/- .05 ng/mL (60 min post-LPS) versus .94 +/- 35 ng/mL (72 h), p = .01) . DHEA levels 60 min post-LPS were significantly higher in treated animals (p < .002) . After LPS, all groups manifested progressive septic symptoms with a hyperdynamic state and pulmonary failure . These symptoms were not blunted by the administration of DHEA . DHEA levels are suppressed by LPS in this two-stage model of trauma and delayed sepsis; however, exogenous DHEA administration fails to blunt the associated systemic inflammatory response and pulmonary failure.

J Neurobiol, 1997 Jan, 32(1), 22 - 32
Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: comparison with the effects of the neurotrophins; Matheson CR et al.; We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) . All of these factors were retrogradely transported to subpopulations of sensory neuron cell bodies in the L4/ L5 DRG of neonatal rats . The size distribution of 125I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 microns2) . 125I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 microns2 . Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 microns2, respectively . A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo . GDNF and NGF rescued nearly 100% of the sensory neurons . BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport . We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats . GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not . The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model . The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons.

Nat Struct Biol, 1997 Jan, 4(1), 28 - 31
Escherichia coli positive regulator OmpR has a large loop structure at the putative RNA polymerase interaction site; Kondo H et al.; The C-terminal DNA-binding domain of OmpR, a positive regulator involved in osmoregulation expression of the ompF and ompC genes in Escherichia coli, has a helix-turn-helix variant motif . The 'turn' region, consisting of 11 residues, forms an RNA polymerase contact site.

Nat Genet, 1997 Jan, 15(1), 91 - 4
Mutations in SOD1 associated with amyotrophic lateral sclerosis cause novel protein interactions; Kunst CB et al.; A subset of familial and sporadic amyotrophic lateral sclerosis (ALS-a fatal disorder characterised by progressive motor neuron degeneration) cases are due to mutations in the gene encoding Cu,Zn superoxide dismutase (SOD1) . Two mutations which have been successfully used to generate transgenic mice that develop an ALS-like syndrome are glycine 85 to arginine (G85R) and glycine 93 to alanine (G93A) with the mutant SOD1 allele overexpressed in a normal mouse genetic background . No ALS-like phenotype is observed in mice overexpressing wild-type SOD1 or mice without any SOD1 activity . These dominant mutations, which do not necessarily decrease SOD1 activity, may confer a gain of function that is selectively lethal to motor neurons . The yeast interaction trap system allowed us to determine whether these mutations in SOD1 caused novel protein interactions not observed with wild-type SOD1 and which might participate in the generation of the ALS phenotype . Two proteins, lysyl-tRNA synthetase and translocon-associated protein delta, interact with mutant forms of SOD1 but not with wild-type SOD1 . The specificity of the interactions was confirmed by the coimmunoprecipitation of mutant SOD1 and the expressed proteins . These proteins are expressed in ventral cord, lending support to the relevance of this interaction to motor neuron disease.

Ann Surg Oncol, 1997 Jan, 4(1), 70 - 9
Transduction of murine and human tumors using recombinant adenovirus vectors; Toloza EM et al.; BACKGROUND: Most cytokine-based cancer gene therapy clinical trials have used labor-intensive, retrovirus-mediated strategies resulting in unpredictable gene expression . Recombinant AdV vectors were evaluated for easier, more reproducible gene transfer into 12 human melanoma, 2 murine fibrosarcomas, and 8 other tumor cell lines . METHODS: AdV vectors contained a reporter (Escherichia coli beta-galactosidase or firefly luciferase) or cytokine gene (human interleukin-2 {IL-2} or IL-7) . Transduction efficiencies and expression levels were assessed by histochemical staining, flow cytometry, polymerase chain reaction, fluorometry, and enzyme-linked immunosorbent assay . Tumorigenicity was determined by subcutaneous injection of cells into syngeneic mice . RESULTS: All cell lines studied were transduced with AdV . Most cell lines exhibited 100% transduction efficiencies (by flow cytometry) at multiplicities of infection (MOI) epsilon 10 . Gene expression correlated linearly with MOI, but a cytopathic effect was observed at MOI > 100 with all vectors . Nanogram gene expression levels were routinely achieved . Irradiation (30 Gy) minimally affected expression levels . Tumorigenicity of AdV-IL-2-transduced fibrosarcoma cells in mice was inversely related to IL-2 production . A majority of mice that rejected their tumor challenge were immune to tumor rechallenge . CONCLUSIONS: E1-deleted AdV vectors may prove useful in generating tumor vaccines ex vivo with high, transient cytokine expression levels.

J Virol, 1997 Jan, 71(1), 569 - 77
In vitro interactions of the aphid endosymbiotic SymL chaperonin with barley yellow dwarf virus; Filichkin SA et al.; Barley yellow dwarf virus (BYDV)-vector relationships suggest that there are specific interactions between BYDV virions and the aphid's cellular components . However, little is known about vector factors that mediate virion recognition, cellular trafficking, and accumulation within the aphid . Symbionins are molecular chaperonins produced by intracellular endosymbiotic bacteria and are the most abundant proteins found in aphids . To elucidate the potential role of symbionins in BYDV transmission, we have isolated and characterized two new symbionin symL genes encoded by the endosymbionts which are harbored by the BYDV aphid vectors Rhopalosiphum padi and Sitobion avenae . Endosymbiont symL-encoded proteins have extensive homology with the pea aphid SymL and Escherichia coli GroEL chaperonin . Recombinant and native SymL proteins can be assembled into oligomeric complexes which are similar to the GroEL oligomer . R . padi SymL protein demonstrates an in vitro binding affinity for BYDV and its recombinant readthrough polypeptide . In contrast to the R . padi SymL, the closely related GroEL does not exhibit a significant binding affinity either for BYDV or for its recombinant readthrough polypeptide . Comparative sequence analysis between SymL and GroEL was used to identify potential SymL-BYDV binding sites . Affinity binding of SymL to BYDV in vitro suggests a potential involvement of endosymbiotic chaperonins in interactions with virions during their trafficking through the aphid.

J Virol, 1997 Jan, 71(1), 138 - 44
Serine protein kinase activity associated with rotavirus phosphoprotein NSP5; Blackhall J et al.; The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine . We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11 . Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity . After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated . Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products . Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified . Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa . Moreover, there were polypeptides migrating between 28 and 35 kDa . Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation . Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A . The immunoprecipitated NSP5 possessed protein kinase activity . Incubation with {gamma-32P}ATP resulted in 32P labeling of 28- to 35-kDa NSP5 . The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo . Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.

J Virol, 1997 Jan, 71(1), 34 - 41
In vivo and in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms; Poncet D et al.; NSP5 (NS26), the product of rotavirus gene 11, is a phosphoprotein whose role in the virus replication cycle is unknown . To gain further insight into its function, we obtained monoclonal antibodies against the baculovirus-expressed protein . By immunoprecipitation and immunoblotting experiments, we showed that (i) NSP5 appears in many different phosphorylated forms in rotavirus-infected cells; (ii) immunoprecipitated NSP5 from rotavirus-infected cells can be phosphorylated in vitro by incubation with ATP; (iii) NSP5, produced either by transient transfection of rotavirus gene 11 or by infection by gene 11 recombinant vaccinia virus or baculovirus, can be phosphorylated in vivo and in vitro; (iv) NSP5 expressed in Escherichia coli is phosphorylated in vitro, and thus NSP5 is a potential protein kinase; and (v) NSP5 forms dimers and interacts with NSP2 . The intracellular localization of NSP5 in the course of rotavirus infection and after transient expression in COS7 cells has also been investigated . In rotavirus-infected cells, NSP5 is localized in viroplasms, but it is widespread throughout the cytoplasm of transfected COS7 cells . NSP5 produced by transfected COS7 cells did not acquire the multiphosphorylated forms observed in rotavirus-infected COS7 cells . Thus, there is a tight correlation between the localization of NSP5 in the viroplasms and its protein kinase activity in vivo or in vitro . Our results suggest that cellular or viral cofactors are indispensable to fully phosphorylate NSP5 and to reach its intracellular localization.

Am Surg, 1997 Jan, 63(1), 20 - 3
Nitric oxide synthase inhibition negates septic-induced alterations in cytoplasmic calcium homeostasis and membrane dynamics; Ismail NH et al.; This study was undertaken to evaluate the role of nitric oxide (NO) in the sepsis-induced disruption of intracellular calcium homeostasis and membrane dynamics . Anticoagulated whole blood was obtained from 10 healthy volunteers . Equal aliquots were incubated with saline (control), 2 microg/mL Escherichia coli endotoxin (lipopolysaccharide), 8 microg/mL NO inhibitor, N-monomethyl arginine (NMA), and endotoxin plus NO inhibitor (lipopolysaccharide/NMA) . Erythrocytes were harvested, washed, and loaded with the calcium chelator, FURA-2AM, and the fluorescent membrane probe TMA-DPH . Cells were evaluated for both intracellular calcium concentration and membrane viscosity (anisotropy) by fluorescent spectrophotometry . Endotoxin induced a significant increase in both intracellular calcium concentration and anisotropy . NMA had no intrinsic affect on either of these cellular characteristics . NMA was, however, effective in preventing the endotoxin-induced changes . These results suggest that NO may play a role in the disruption of intracellular calcium homeostasis and erythrocyte membrane deformability noted in sepsis.

J Bacteriol, 1997 Jan, 179(1), 297 - 300
The RNA-binding protein HF-I plays a global regulatory role which is largely, but not exclusively, due to its role in expression of the sigmaS subunit of RNA polymerase in Escherichia coli; Muffler A et al.; The hfq-encoded RNA-binding protein HF-I has long been known as a host factor for phage Qbeta RNA replication and has recently been shown to be essential for translation of rpoS, which encodes the sigmaS subunit of RNA polymerase . Here we demonstrate that an hfq null mutant does not synthesize glycogen, is starvation and multiple stress sensitive, and exhibits strongly reduced expression of representative sigmaS-regulated genes . These phenotypes are consistent with strongly reduced sigmaS levels in the hfq mutant . However, the analysis of global protein synthesis patterns on two-dimensional O'Farrell gels indicates that approximately 40% of the more than 30 proteins whose syntheses are altered in the hfq null mutant are not affected by an rpoS mutation . We conclude that HF-I is a global regulator involved in the regulation of expression of sigmaS and sigmaS-independent genes.

J Bacteriol, 1997 Jan, 179(1), 228 - 34
Functional characterization of roles of GalR and GalS as regulators of the gal regulon; Geanacopoulos M et al.; An isorepressor of the gal regulon in Escherichia coli, GalS, has been purified to homogeneity . In vitro DNase I protection experiments indicated that among operators of the gal regulon, GalS binds most strongly to the external operator of the mgl operon, which encodes the high-affinity beta-methylgalactoside galactose transport system, and with less affinity to the operators controlling expression of the gal operon, which codes for enzymes of galactose metabolism . GalS has even less affinity for the external operator of galP, which codes for galactose permease, the major low-affinity galactose transporter in the cell . This order of affinities is the reverse of that of GalR, which binds most strongly to the operator of galP and most weakly to that of mgl . Our results also show that GalS, like its homolog, GalR, is a dimeric protein which in binding to the bipartite operators of the gal operon selectively represses its P1 promoter . Consistent with the fact that GalR is the exclusive regulator of the low-affinity galactose transporter, galactose permease, and that the major role of GalS is in regulating expression of the high-affinity galactose transporter encoded by the mgl operon, we found that the DNA binding of GalS is 15-fold more sensitive than that of GalR to galactose.

J Bacteriol, 1997 Jan, 179(1), 41 - 5
Characterization of fhlA mutations resulting in ligand-independent transcriptional activation and ATP hydrolysis; Korsa I et al.; The FhlA protein belongs to the NtrC family of transcriptional regulators . It induces transcription from the -12/-24 promoters of the genes of the formate regulon by sigma54 RNA polymerase . FhlA is activated by binding of the ligand formate and does not require phosphorylation . A mutational analysis of the fhLA gene portion coding for the A and C domains was conducted with the aim of gaining information on the interaction between formate binding and ATP hydrolysis plus transcription activation . Four mutations were identified, all located in the A domain; one of them rendered transcription completely independent from the presence of formate, and the others conferred a semiconstitutive phenotype . The FhlA protein of one of the semiconstitutive variants was purified . Catalytic efficiency of ATP hydrolysis of the mutant FhlA was increased in the absence of formate in the same manner as formate influences the activity of wild-type FhlA . Moreover, in vitro transcription occurred at much lower threshold concentrations of the mutant protein and of nucleoside triphosphates than with the wild-type FhlA.

Mol Reprod Dev, 1997 Jan, 46(1), 31 - 7; discussion 37-8
Structure-function studies on human macrophage colony-stimulating factor (M-CSF); Koths K; M-CSF (CSF-1) can be produced in a variety of structural forms that may affect function in vivo . Truncated, nonglycosylated forms of recombinant M-CSF (rM-CSF) from E . coli have been refolded in vitro in high yield and shown to be functionally equivalent in vitro to glycosylated rM-CSF secreted from mammalian cells . An N-terminal domain of 149 amino acids is produced by all of the known M-CSF mRNA splice variants and is the region responsible for bioactivity observed in vitro . Heterodimeric rM-CSFs from different splice variants containing this domain were produced in pure form by refolding in vitro, and are fully active, but have yet to be observed in vivo . The circulating half-life of truncated M-CSF forms injected intravenously into rats increased with the MW of the M-CSF used . Large increases in half-life in vivo were observed following chemical addition of a single molecule of 10 kD polyethylene glycol to rM-CSF in vitro . The crystal structure of rM-CSF revealed that M-CSF is a member of a family of molecules related by having a distinctive four-helical-bundle structural core . Site-directed mutagenesis showed that residues in or near helix A and helix C are involved in receptor binding, as reflected by decreased bioactivity and receptor binding of certain mutants . A soluble form of the M-CSF receptor, c-fms, was produced in a baculovirus/Sf9 expression system and purified to homogeneity . The MW of rM-CSF saturated with this soluble receptor was determined by molecular sieve chromatography and light scattering . Each dimeric M-CSF molecule appears to bind two soluble receptor molecules in vitro, supporting the observation that M-CSF signaling is linked to receptor dimerization.

Antimicrob Agents Chemother, 1997 Jan, 41(1), 193 - 5
Induction of DnaK and GroEL heat shock proteins by fluoroquinolones in Escherichia coli; Mizushima T et al.; Various fluoroquinolones (norfloxacin, enoxacin, ofloxacin, levofloxacin, and sparfloxacin) induce DnaK and GroEL heat shock proteins in Escherichia coli . The induction is transient, consistent with the kinetics of cellular DNA relaxation . The concentrations of fluoroquinolones required for induction are similar to those required for DNA relaxation and much higher than those required for cell death.

Int Arch Allergy Immunol, 1997 Jan, 112(1), 44 - 8
Genomic organization and polymorphisms of the major house dust mite allergen Der f2; Yuuki T et al.; We amplified genomic DNA encoding the major house dust mite allergen, Der f2, from Dermatophagoides farinae by means of the polymerase chain reaction and cloned it into Escherichia coli . The nucleotide sequences of the amplified fragments were determined and compared with those of the cDNA previously reported . Four Der f2 genomic clones were obtained, suggesting that the genomic Der f2 gene had sequence polymorphisms like the cDNA clones . Each of the genomic clones had a single small intron . With respect to the exon sequences, two of the four genomic clones were identical with two cDNA clones, respectively . The others were combinations of the two clones . Genomic Southern blotting suggested that the Der f2 gene is located at one locus in the mite genome and that sequence substitutions were due to polymorphisms among individual mite genes.

Appl Environ Microbiol, 1997 Jan, 63(1), 263 - 9
Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone; Yuan Q et al.; The heavy-chain and kappa light-chain variable region genes of an antizearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a single-chain Fv (scFv) DNA fragment . The scFv DNA fragment was cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 in Escherichia coli TG1 . In the presence of helper phage M13K07, the scFv fusion protein was displayed on the surfaces of recombinant phages . High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate . The selected recombinant phages were used to infect E . coli HB2151 for the production of soluble scFv antibodies . One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibition of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay . However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the parent monoclonal antibody did . Nucleotide sequence analyses revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse germ line gene VK23.32 in mouse kappa light-chain variable region subgroup V, whereas the heavy-chain nucleotide sequence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity-determining region sequences . The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts.

Endocrinology, 1997 Jan, 138(1), 307 - 13
Human insulin-like growth factor I (IGF-I) produced in the mammary glands of transgenic rabbits: yield, receptor binding, mitogenic activity, and effects on IGF-binding proteins; Wolf E et al.; Insulin-like growth factor I (IGF-I) has acute insulin-like metabolic effects and long-term anabolic actions offering a range of important therapeutic applications . To evaluate a system for large-scale production of this peptide in the mammary glands of transgenic livestock, we generated transgenic rabbits carrying fusion genes in which a synthetic DNA coding for human IGF-I (hIGF-I) was placed under the transcriptional control of regulatory elements isolated from the bovine alpha S1-casein (alpha S1-cas) gene . Western blot analysis of milk from alpha S1-cas-hIGF-I transgenic rabbits demonstrated production of high amounts of mature hIGF-I peptide (7.6 kDa) . Quantitative analysis by RIA revealed hIGF-I levels between 50 and 300 micrograms/ml milk . Recombinant hIGF-I purified from the milk of alpha S1-cas-hIGF-I transgenic rabbits bound to IGF-I receptors on human IM-9 lymphoblasts and stimulated DNA synthesis by growth-arrested MG-63 human osteosarcoma cells as efficiently as hIGF-I produced in Escherichia coli . Ligand blot analysis of milk serum revealed the presence of 45-kDa, 30-kDa, and 23-kDa IGF-binding proteins (IGFBPs) . The 30-kDa IGFBP was shown to be IGFBP-2 by immunoprecipitation using an antiserum raised against human IGFBP-2 . Secretion of IGFBP-2 was markedly stimulated by hIGF-I overproduction in alpha S1-cas-hIGF-I transgenic rabbits . The latter displayed slightly increased milk yield, but no significant changes in total protein content or overall milk protein composition, and reared their offspring without any problems or clinical signs of impaired welfare, even after multiple lactations . Our results indicate that high amounts of biologically active hIGF-I can be produced in the mammary glands of alpha S1-cas-hIGF-I transgenic rabbits . Local production of hIGF-I in mammary tissue is associated with increased secretion of IGFBP-2, which may prevent major biological effects by high levels of hIGF-I on the mammary gland.

Endocrinology, 1997 Jan, 138(1), 182 - 90
Expression, purification and characterization of the rat luteal 20 alpha-hydroxysteroid dehydrogenase; Mao J et al.; The enzyme, rat ovarian 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD), plays a central role in luteolysis and parturition . It catalyzes the reduction of progesterone, leading to the formation of progestationally inactive steroid, 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-hydroxyprogesterone) . Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20 alpha HSD . To further investigate whether phosphorylation and/or glycosylation affect the activity of 20 alpha HSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression systems for obtaining large quantities of enzyme . The recombinant (rec) 20 alpha HSD expressed as glutathione-S-transferase-20 alpha HSD fusion protein was purified from bacterial lysates by affinity binding to glutathione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to apparent homogeneity by ion exchange chromatography, followed by dye affinity chromatographies . Both rec preparations of 20 alpha HSD demonstrated a single polypeptide chain of 37 kDa with similar K(m) values for 20 alpha-hydroxyprogesterone and NADP, although the corresponding maximum velocity values were slightly lower for the rec 20 alpha HSD expressed in the insect cells . The rec 20 alpha-HSD showed preference for progesterone/20 alpha-hydroxyprogesterone . 17 alpha-Hydroxyprogesterone was only 30% as effective . The enzyme also used various substrates specific for aldo-keto reductases, although with much less efficiency . The rec enzyme preparations showed an absolute requirement for NADP(H) . In vitro phosphorylation of rec bacterial enzyme with either protein kinase A or protein kinase C had no demonstrable effect on its activity . Finally, no differences in enzyme activity were noted between glycosylated (expressed in insect cells) and nonglycosylated (expressed in bacteria) forms of the enzyme . In conclusion, these studies demonstrate that rat luteal 20 alpha HSD can be prepared in large amounts from either bacterial or insect expression systems in a catalytically active form . Indirect evidence also suggests that the catalytic activity of 20 alpha HSD may be independent of phosphorylation and glycosylation states of the enzyme protein, i.e . posttranslational modification of 20 alpha HSD may not be required for the maximal expression of enzyme activity.

J Urol, 1997 Jan, 157(1), 346 - 50
Molecular evidence for pap-G specific adhesion of Escherichia coli to human renal cells; Soderhall M et al.; PURPOSE: To study the interaction between class II G-adhesin of Escherichia coli and human urogenital cells . MATERIAL AND METHODS: The adherence of two wild type P-fimbriated E . coli strains, both carrying a class II G-adhesion, and two constructed mutants (one class II G-adhesion knock-out mutant and one class switch mutant in which the papG gene was exchanged with a prsJ96 allele which is a representative of the class III G-adhesin) to human urogenital cells were examined by light microscopy and flow cytometry . RESULTS: The wild type E . coli strains adhered avidly to proximal tubular cells, but the isogenic mutant strains did only adhere in one of the experiments . A soluble receptor analogue inhibited bacterial attachment . CONCLUSIONS: These experiments strongly suggest that the papG class II tip adhesin of P-fimbriae is essential in the pathogenesis of human kidney infection.

Infect Immun, 1997 Jan, 65(1), 320 - 6
Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine; Agin TS et al.; Intimin, an outer membrane protein encoded by eaeA that mediates close attachment of enteropathogenic bacteria to apical surfaces of epithelial cells, is required for formation of the attaching-effacing lesions and for full pathogenesis of the bacteria . Analysis of the eaeA sequence indicates that there is a high degree of homology at the N termini but less at the C termini of intimins . Antisera specific for the C-terminal third of RDEC-1 intimin, used to screen outer membrane proteins from 50 rabbit enteropathogenic Escherichia coli (EPEC), human EPEC, and human enterohemorrhagic E . coli (EHEC) strains, identified cross-reactive intimins from 24 isolates . Sequence analysis of the eaeA genes from human EPEC O111 and EHEC O26 isolates indicates that their intimins have C termini nearly identical to that of RDEC-1 intimin . Our results suggest that there are at least three families of related intimins and that the presence of intimin similar to that of RDEC-1 is not restricted by serogroup or host specificity.

Infect Immun, 1997 Jan, 65(1), 267 - 71
Delayed-type hypersensitivity activity of the Brucella L7/L12 ribosomal protein depends on posttranslational modification; Bachrach G et al.; The ribosomal protein L7/L12 isolated from Brucella melitensis induces a delayed-type hypersensitivity (DTH) reaction in brucella-sensitized guinea pigs . Surprisingly, the recombinant brucella L7/L12 protein expressed in Escherichia coli as a fusion protein with a six-histidine tag cannot elicit such a reaction . The six histidines tagged to the recombinant L7/L12 protein were removed enzymatically, but the resulting protein did not induce a DTH reaction in sensitized animals . Incubation of the recombinant L7/L12 fusion protein in a B . melitensis lysate endowed the recombinant protein with a DTH activity, suggesting that the recombinant protein was modified by this treatment . Glycosylation or phosphorylation of the recombinant L7/L12 protein could not be detected . On the other hand, radiolabeled palmitic acid was found to be incorporated to the recombinant protein during its incubation in the brucella lysate . This incorporation was specific for the brucella L7/L12 protein and was inhibited when the brucella lysate was frozen and thawed before the incubation . The data reported here indicate that posttranslational modification of L7/L12 protein comprising at least an acylation step is required for the brucella L7/L12 DTH activity.

Infect Immun, 1997 Jan, 65(1), 156 - 63
Anaplasma marginale major surface protein 3 is encoded by a polymorphic, multigene family; Alleman AR et al.; The immunodominant surface protein, MSP3, is structurally and antigenically polymorphic among strains of Anaplasma marginale . In this study we show that a polymorphic multigene family is at least partially responsible for the variation seen in MSP3 . The A . marginale msp3 gene msp3-12 was cloned and expressed in Escherichia coli . With msp3-12 as a probe, multiple, partially homologous gene copies were identified in the genomes of three A . marginale strains . These copies were widely distributed throughout the chromosome . Sequence analysis of three unique msp3 genes, msp3-12, msp3-11, and msp3-19, revealed both conserved and variant regions within the open reading frames . Importantly, msp3 contains amino acid blocks related to another polymorphic multigene family product, MSP2 . These data, in conjunction with data presented in previous studies, suggest that multigene families are used to vary important antigenic surface proteins of A . marginale . These findings may provide a basis for studying antigenic variation of the organism in persistently infected carrier cattle.

Infect Immun, 1997 Jan, 65(1), 16 - 23
Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis; Manca C et al.; Proteins that are actively secreted by Mycobacterium tuberculosis generate immune responses in the infected host . This has prompted the characterization of protein components of mycobacterial culture filtrates to develop subunit vaccines and immunodiagnostic reagents . Fractionation of filtrates of M . tuberculosis cultures has yielded an abundant protein called MPT63, which has an apparent molecular mass of 18 kDa . We report the molecular cloning and nucleotide sequence of the mpt63 gene, purification of recombinant MPT63 antigen from Escherichia coli cells, and serological characterization of MPT63 . Nucleotide sequence analysis of mpt63 identified an open reading frame encoding a protein of 159 amino acids (aa) consisting of a 29-aa secretion signal peptide and a 130-aa mature MPT63 protein . Recombinant MPT63 protein, purified from E . coli cells, and native MPT63, purified from M . tuberculosis culture filtrates, were indistinguishable in serological assays . Thus, the recombinant protein constitutes a valuable reagent for immunological studies . MPT63 evoked humoral immune responses in guinea pigs infected with virulent M . tuberculosis by the aerosol route . The mpt63 gene is found only in species of the M . tuberculosis complex, as shown by DNA hybridization experiments . Moreover, polyclonal antibody against MPT63 does not cross-react with proteins of a common environmental mycobacterial species, Mycobacterium avium . The absence of cross-reactive epitopes makes MPT63 an attractive candidate as an M . tuberculosis complex-specific diagnostic reagent . In particular, evaluation of MPT63 as an M . tuberculosis complex-specific reagent for diagnostic skin testing is under way.

J Clin Microbiol, 1997 Jan, 35(1), 86 - 91
Molecular characterization of a 35-kilodalton protein of Borrelia burgdorferi, an antigen of diagnostic importance in early Lyme disease; Gilmore RD Jr et al.; Antibodies against a 35-kDa antigen of Borrelia burgdorferi are detectable in the serum of about half of patients with early Lyme disease . The gene encoding this antigen was isolated from a genomic library of B . burgdorferi B31 (low passage), and full-length expression of the recombinant gene product was achieved in Escherichia coli . Antiserum raised against the recombinant protein was reactive with a B . burgdorferi protein of the same molecular size as the diagnostic 35-kDa antigen cited in an earlier study of criteria for the sero-diagnosis of early Lyme disease . Also, the recombinant protein was reactive with serum from patients with early Lyme disease who were seropositive for the 35-kDa antigen . DNA sequence analysis of the gene indicated an open reading frame of 909 bp encoding a protein with a calculated molecular mass of 34.3 kDa . This gene did not possess the usual initiation codon ATG but rather probably used a TTG codon . The deduced amino acid sequence of the N terminus exhibited a motif similar to that for signal peptides of lipoproteins . Southern blotting revealed a chromosomal location for this gene; and it was specific for B . burgdorferi, B . afzellii, and B . garinii but not for B . hermsii, B . coriaciae, or B . turicatae.

J Clin Microbiol, 1997 Jan, 35(1), 20 - 4
Molecular characterization of Escherichia coli strains isolated from pigs with edema disease; Aarestrup FM et al.; The present study was conducted to investigate the epidemiological relationship of isolates of Escherichia coli causing edema disease . Classical edema disease has not previously been described in Denmark, but between February 1994 and November 1995 cases appeared in 51 pig herds, among which direct or indirect trading contacts were confirmed for 36 of the herds . A total of 213 isolates from pigs with edema disease in Denmark and other countries and 23 E . coli O139 isolates from pigs with diarrhea or healthy pigs were analyzed to characterize their O serogroups, HindIII ribotypes, and pulsed-field gel electrophoresis (PFGE) types, and 183 of the isolates were also analyzed for their plasmid profiles . The resulting PFGE types of the isolates from pigs with edema disease were examined by cluster analysis . Ten isolates from three herds could not be typed with the available O antisera, whereas all other isolates were of serotype O139 . However, all isolates from pigs with edema disease belonged to the same HindIII ribotype, which was not observed among the isolates from pigs with diarrhea or healthy pigs . All isolates from Danish pigs with edema disease except for three isolates originating from two herds belonged to the same or closely related XbaI PFGE types; the other three isolates were assigned to possibly related types . Isolates from pigs with edema disease in different countries belonged to different PFGE types . All isolates from Danish pigs with edema disease grouped together in one cluster, in contrast to isolates from other countries, which did not form any clusters . E . coli strains of serogroup O139 from pigs with diarrhea or isolated from the feces of healthy Danish pigs were very different . Plasmid profiles differed largely among isolates . However, among the isolates from Danish pigs with edema disease, one type predominated within herds . The present study indicated that most, if not all, of the observed cases of edema disease in Denmark were part of the same outbreak . The combination of PFGE typing and ribotyping was useful for studying the possible clonal relationship among strains, whereas plasmid profiling was less informative.

Curr Microbiol, 1997 Jan, 34(1), 27 - 32
Isolation and characterization of the Azospirillum brasilense trpE(G) gene, encoding anthranilate synthase; De Troch P et al.; The Azospirillum brasilense trpE gene has been isolated by DNA hybridization and by genetic complementation of an Escherichia coli trpE deletion mutant . DNA sequence analysis of a 3.1-kb PstI restriction fragment of A . brasilense revealed the presence of an open reading frame encoding a putative TrpE(G) fusion protein . Previously an A . brasilense clone containing trpGDC was identified (Zimmer et al . Mol Gen Genet 229:41-51, 1991) . It can, therefore, be concluded that A . brasilense contains two trpG genes . A putative leader peptide is found upstream of trpE(G), containing three consecutive tryptophan residues . Putative terminator and anti-terminator loops have also been identified . The LLESX10S motif, which is responsible for feedback inhibition by tryptophan in other TrpE proteins, is absent in the A . brasilense TrpE(G) fused protein.

Adv Biochem Eng Biotechnol, 1997, 56, 61 - 109
Inclusion bodies and purification of proteins in biologically active forms; Mukhopadhyay A; Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins . In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies . Most of these remain only as inventions and very few are commercially exploited . This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications . The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties . In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively . The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules . Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection . Lastly, two case studies are discussed to demonstrate the sequence of process steps involved.

FEMS Immunol Med Microbiol, 1996 Dec 31, 16(3-4), 183 - 92
Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein; Tarar MR et al.; Hepatitis B virus core antigen (HBcAg) has been used as a carrier for expression and presentation of a variety of heterologous viral epitopes in particulate form . The aim of this study was to produce hybrid antigens comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV) . A direct comparison was made of amino and carboxyl terminal fusions in order to investigate the influence of position of the foreign epitope on hybrid core particle formation, antigenicity and immunogenicity . HCMV DNA encoding a neutralising epitope of the surface glycoprotein gp58 was either inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg and expressed in Escherichia coli . The carboxyl terminal fusion (HBc3-144-HCMV) was expressed at high levels and assembled into core like particles resembling native HBcAg . Protein with a similar fusion at the amino terminus (HCMV-HBc1-183) could not be purified or characterised immunologically, although it formed core like particles . HBc3-144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected by radioimmunoassay or western blotting using anti-HCMV monoclonal antibody 7-17 or an anti-HCMV human polyclonal antiserum . Following immunisation of rabbits with HBc3-144-HCMV, a high titre of anti-HBc specific antibody was produced along with lower titres of HCMV/gp58 specific antibody.

DNA Res, 1996 Dec 31, 3(6), 431 - 3
Nested deletions from a fixed site as an aid to nucleotide sequencing: an in vitro system using Tn3 transposase; Morita M et al.; We have previously constructed a cloning/sequencing vector, with an in vivo system capable of creating nested deletions from the end of transposon Tn3, which is useful for sequencing large DNAs . Here we report an in vitro system which uses an ammonium sulfate fraction of extract from E . coli cells harboring a Tn3 transposase overproducer plasmid to generate nested deletions . A key feature of the procedure is exhaustive digestion of the reaction products with a restriction enzyme that cleaves only between the Tn3 "right" terminus and the cloned fragment . This step reduces the noise level due to mechanisms other than deletions from the Tn3 terminus, and facilitates detection and isolation of the desired deletion products . This system enables us to save at least 2 days' time when obtaining the necessary deletions compared with the in vivo system.

DNA Res, 1996 Dec 31, 3(6), 379 - 92
A 460-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 40.1-50.0 min region on the linkage map; Itoh T et al.; The 465,813 base pair sequence corresponding to the 40.1-50.0 min region on the genetic map of Escherichia coli K-12 (W3110) was determined . Analysis of the sequence revealed that this region contained at least 466 potential open reading frames, of which 187 (40%) were previously reported, 105 (23%) were homologous to other known genes, 103 (22%) were identical or similar to hypothetical genes registered in databases, and the remaining 71 (15%) did not show a significant similarity to any other gene . At the 45.2-46.0 min region, we found a very large cluster of about 30 genes, whose functions are involved in the biosynthesis of polysaccharides as the components of outer membranes . In addition, we identified a new asn-tRNA gene, designated asnW, between the asnT and asnU genes and a new lysogenic phage attachment site as the cis-element.

DNA Res, 1996 Dec 31, 3(6), 363 - 77
A 570-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 28.0-40.1 min region on the linkage map; Aiba H et al.; The 569,750 base pair sequence corresponding to the 28.0-40.1 min region on the genetic map of Escherichia coli K-12 (W3110) was determined . This region includes the replication terminus region and contained at least 549 potential open reading frames . Among them, 160 (29%) were previously reported, 174 (32%) were homologous to other known genes, 102 (18%) were identical or similar to hypothetical genes registered in databases, and the remaining 113 (21%) did not show a significant similarity to any other gene . Of interest was the finding of a large number of genes and gene clusters in and near the replication termination region which had been thought to be genetically silent . Those included a cluster of genes for fatty acid beta-oxidation, the third copy of the pot (spermidine/putrescine transport system) gene cluster, the second dpp (dipeptide transport system) operon, the second dsm (anaerobic dimethyl sulfoxide reductase) operon, a cluster of fim (fimbrial) genes and a DNA helicase-like gene with a high molecular weight . In addition, we found the dnaC- and dnaT-like genes in the cryptic prophage, Rac, and a number of genes originated probably from plasmids.

J Biol Chem, 1996 Dec 27, 271(52), 33664 - 9
Facilitated protein aggregation . Effects of calcium on the chaperone and anti-chaperone activity of protein disulfide-isomerase; Primm TP et al.; Protein disulfide-isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding in the eukaryotic endoplasmic reticulum . At high concentrations, it also serves as a chaperone and inhibits aggregation . However, at lower concentrations, PDI can display the unusual ability to facilitate aggregation, termed anti-chaperone activity (Puig, A., and Gilbert, H . F . (1994) J . Biol . Chem . 269, 7764-7771) . Under reducing conditions (10 mM dithiothreitol) and at a low concentration (0.1-0 . 3 microM) relative to the unfolded protein substrate, PDI facilitates aggregation of alcohol dehydrogenase (11 microM) that has been denatured thermally or chemically . But at higher concentrations (>0.8 microM), PDI inhibits aggregation under the same conditions . With denatured citrate synthase, PDI does not facilitate aggregation, but higher concentrations do inhibit aggregation . Anti-chaperone behavior is associated with the appearance of both PDI and substrate proteins in insoluble complexes, while chaperone behavior results in the formation of large (>500 kDa) but soluble complexes that contain both proteins . Physiological concentrations of calcium and magnesium specifically increase the apparent rate of PDI-dependent aggregation and shift the chaperone activity to higher PDI concentrations . However, calcium has no effect on the Km or Vmax for PDI-catalyzed oxidative folding, suggesting that the interactions that lead to chaperone/anti-chaperone behavior are distinct from those required for catalytic activity . To account for this unusual behavior of a folding catalyst, a model with analogy to classic immunoprecipitation is proposed; multivalent interactions between PDI and a partially aggregated protein stimulate further aggregate formation by noncovalently cross-linking smaller aggregates . However, at high ratios of PDI to substrate, cross-linking may be inhibited by saturation of the sites with PDI . The effects of PDI concentration on substrate aggregation and the modulation of the behavior by physiological levels of calcium may have implications for the involvement of PDI in protein folding, aggregation, and retention in the endoplasmic reticulum.

J Biol Chem, 1996 Dec 27, 271(52), 33446 - 56
Structures of active site histidine mutants of IIIGlc, a major signal-transducing protein in Escherichia coli . Effects on the mechanisms of regulation and phosphoryl transfer; Pelton JG et al.; IIIGlc (also called IIAGlc), a major signal-transducing protein in Escherichia coli, is also a phosphorylcarrier in glucose uptake . The crystal and NMR structures of IIIGlc show that His90, the phosphoryl acceptor, adjoins His75 in the active site . Glutamine was substituted for His-, giving H75QIIIGlc and H90QIIIGlc, respectively (Presper, K . A., Wong, C.-Y., Liu, L., Meadow, N . D., and Roseman, S . (1989) Proc . Natl . Acad . Sci . U . S . A . 86, 4052-4055), but the mutants showed unexpected properties . H90QIIIGlc loses regulatory functions of IIIGlc, and the phosphoryltransfer rates between HPr/H75QIIIGlc are 200-fold less than HPr/IIIGlc (Meadow, N . D., and Roseman, S . (1996) J . Biol . Chem . 271, 33440-33445) . X-ray crystallography, differential scanning calorimetry, and NMR have now been used to determine the structures of the mutants (phospho-H75QIIIGlc was studied by NMR) . The three methods gave completely consistent results . Except for the His to Gln substitutions, the only significant structural changes were in a few hydrogen bonds . H90QIIIGlc contains two structured water molecules (to Gln90), which could explain its inability to regulate glycerol kinase . Phospho-IIIGlc contains a chymotrypsin-like, hydrogen bond network (Thr73-His75-O--phosphoryl), whereas phospho-H75QIIIGlc contains only one bond (Gln75-O--phosphoryl) . Hydrogen bonds play an essential role in a proposed mechanism for the phosphoryltransfer reaction.

J Biol Chem, 1996 Dec 27, 271(52), 33440 - 5
Rate and equilibrium constants for phosphoryltransfer between active site histidines of Escherichia coli HPr and the signal transducing protein IIIGlc; Meadow ND et al.; The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) plays a central role in catabolizing many sugars; regulation is effected by phosphorylation of PTS proteins . In Escherichia coli, the phosphoryltransfer sequence for glucose uptake is: PEP --> Enzyme I(His191) --> HPr(His15) --> IIIGlc(His90) --> IIGlc(Cys421) --> glucose . A rapid quench method has now been developed for determining the rate and equilibrium constants of these reactions . The method was validated by control experiments, and gave the following results for phosphoryltransfer between the following protein pairs . For phospho-HPr/IIIGlc (and HPr/phospho-IIIGlc), k1 = 6.1 x 10(7) M-1 s-1, k-1 = 4.7 x 10(7); for the mutant H75QIIIGlc in place of IIIGlc, k1 = 2.8 x 10(5) M-1 s-1, k-1 = 2.3 x 10(5) . The derived Keq values agreed with the Keq obtained without use of the rapid quench apparatus . Keq for both reactions is 1-1.5 . The rate of phosphoryltransfer between HPr and wild type IIIGlc is close to a diffusion-controlled process, while the reactions involving the mutant H75QIIIGlc are 200-fold slower . These rate differences are explained by an hypothesis for the mechanism of phosphoryltransfer between HPr and IIIGlc based on the structures of mutant and wild type proteins (see Pelton et al . (Pelton, J . G., Torchia, D . A., Remington, S . J., Murphy, K . P., Meadow, N . D., and Roseman, S . (1996) J . Biol . Chem . 271, 33446-33456)).

J Biol Chem, 1996 Dec 27, 271(52), 33268 - 76
In vitro studies of the Prp9.Prp11.Prp21 complex indicate a pathway for U2 small nuclear ribonucleoprotein activation; Wiest DK et al.; Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6 . In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly . This paper describes a study of interactions between these proteins and their role in spliceosome assembly . The proteins were expressed in Escherichia coli . Prp9 and Prp11 were purified by metal affinity chromatography . Prp21 was purified using a solubilization/renaturation protocol . We have combined these separately purified proteins and present direct evidence of a Prp9.Prp11.Prp21 protein complex that is functional in in vitro splicing assays . Characteristics of this Prp9.Prp11.Prp21 complex were further investigated using proteins synthesized in vitro . In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage . We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome.

J Biol Chem, 1996 Dec 27, 271(52), 33192 - 200
Effect of substitutions in the thiamin diphosphate-magnesium fold on the activation of the pyruvate dehydrogenase complex from Escherichia coli by cofactors and substrate; Yi J et al.; The homotropic regulation of the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) by its coenzyme thiamin diphosphate and its substrate pyruvate was re-examined with complexes containing three and one lipoyl domains per E2 chain, and several variants of the latter, containing substitutions in the putative thiamin diphosphate fold of E1 (G231A, G231S, C259S, C259N, and N258Q) . It was found that all of the E1 variants had significantly reduced specific activities, as reported elsewhere (Russell, G . C., Machado, R . S., and Guest, J . R . (1992) Biochem . J . 287, 611-619) . In addition, extensive kinetic studies were performed in an attempt to determine the effects of the amino acid substitutions on the Hill coefficients with respect to thiamin diphosphate and pyruvate . All but one of the variants were incapable of being saturated with thiamin diphosphate, even at concentrations > 5 mM . Most importantly, the striking activation lag phase lasting for many seconds in the parental complexes containing three and one lipoyl domains per E2 chain was totally eliminated in the variants . Furthermore, activation by the coenzyme was localized to the E1 subunit, because resolved E1 exhibits virtually the same behavior during the activation lag phase as does the complex . In the parental complexes two distinct lag phases could be resolved, the duration of both decreases with increasing ThDP concentration . A mechanism that is consistent with all of the kinetic data on the parental complexes involves rapid equilibration of the first ThDP with the E1 dimer, followed by a slow conformational equilibration, that in turn is followed by slow addition of the second ThDP to form the fully activated dimer . When the diphosphate site is badly impaired, the binding affinity is very much reduced, this perhaps eliminates the slow step leading to the activated dimer form of the E1.

J Biol Chem, 1996 Dec 27, 271(52), 33176 - 80
Central role of the BvgS receiver as a phosphorylated intermediate in a complex two-component phosphorelay; Uhl MA et al.; Two-component systems use phosphorylation reactions to regulate stimulus/response pathways . In Bordetella pertussis, a human respiratory pathogen, the infectious cycle of the organism is controlled by the BvgAS two-component system . BvgS has similarities to sensor and response regulator components and is an autophosphorylating kinase that phosphorylates BvgA . BvgA, a response regulator, is a DNA-binding protein that activates virulence gene transcription . Three phosphorylated BvgS domains, the transmitter, receiver, and C terminus, are essential for signal transduction . We now demonstrate that the BvgS transmitter is sufficient for autophosphorylation but is unable to phosphorylate the C terminus or BvgA . The BvgS receiver regulates several phenotypes: dephosphorylation of both the BvgS transmitter and C terminus as well as transfer of a phosphoryl group from the transmitter to the C terminus . Our results indicate that BvgAS signal transduction initiates with autophosphorylation of the transmitter followed by phosphotransfer to the receiver . The phosphorylated receiver can donate to the C terminus or to water . The phosphorylated C terminus is then able to transfer the phosphoryl group to BvgA.

Biochemistry, 1996 Dec 24, 35(51), 16843 - 51
Real-time refolding studies of 6-19F-tryptophan labeled Escherichia coli dihydrofolate reductase using stopped-flow NMR spectroscopy; Hoeltzli SD et al.; Escherichia coli dihydrofolate reductase (ecDHFR, EC1.5.1.3) contains 5 tryptophan residues that have been replaced with 6-19F-tryptophan . Five native and four of the five unfolded tryptophan resonances can be resolved in the 1D 19F NMR spectra and have been assigned {Hoeltzli, S . D., & Frieden, C . (1994) Biochemistry 33, 5502-5509} . This resolution allows the behavior of the native and the unfolded resonances assigned to each individual tryptophan to be monitored during the unfolding or refolding process . We now use these assignments and stopped-flow NMR to investigate the real-time behavior of specific regions of the protein during refolding of DHFR after dilution from 4.6 to 2.3 M urea (midpoint of the transition = 3.8 M) at 5 degrees C . Approximately half of the intensity of each of the four unfolded resonances is present at the first measurable time point (1.5 s) . Little native resonance intensity is detectable at this time . The remaining unfolded resonance intensities present then disappear in two phases, with rates similar to the two slowest phases observed by either stopped-flow fluorescence or circular dichroism spectroscopy upon refolding under the same conditions . Substantial total resonance intensity is missing during the first 20 s of the refolding process . The appearance of the majority of native resonance intensity (as assessed by the height of each of the five native tryptophan resonances) is slow and similar for all five tryptophans . In contrast, the largest amplitude changes observed by either stopped-flow far-UV circular dichroism spectroscopy or fluorescence spectroscopy, and the greatest loss of unfolded resonance intensity, occur much more rapidly . We conclude from these studies: (1) that, under these conditions, the unfolded state remains substantially populated after initiation of refolding; (2) that the early steps in refolding involve a solvent protected intermediate containing substantial secondary structure, but (3) that the stable native side chain interactions form slowly and are associated with the final rate-limiting phase of the refolding process . Preliminary analysis of the area of broadened native resonances suggests that these resonances may appear at different rates, indicating that some regions of the protein begin to sample a native-like side chain environment while side chain environment in other regions of the protein remains less ordered . The results of this study are consistent with the earlier studies demonstrating that mobility of side chains is an early step in unfolding {Hoeltzli, S . D., & Frieden, C . (1995) Proc . Natl . Acad . Sci . U.S.A . 92, 9318-9322} and that recovery of enzymatic activity occurs as a late step in the folding process {Frieden, C . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 4413-4416}.

Biochemistry, 1996 Dec 24, 35(51), 16680 - 6
Dimer/monomer equilibrium and domain separations of Escherichia coli ribosomal protein L7/L12; Hamman BD et al.; The dimer to monomer equilibrium and interdomain separations of cysteine variants of L7/L12 have been investigated using fluorescence spectroscopy . Steady-state polarization measurements on cysteine containing variants of L7/L12, labeled with 5-(iodoacetamido)fluorescein, demonstrated dimer to monomer dissociation constants near 30 nM for variants labeled at position 33, in the N-terminal domain, and positions 63 and 89, in the C-terminal domain . A dissociation constant near 300 nM was determined for a variant labeled at position 12, in the N-terminal domain . The polarization of a labeled C-terminal fragment did not change over the range of 200 microM to 1 nM, indicating that this construct remains monomeric at these concentrations, whereas a dimer to monomer dissociation constant near 300 nM was observed for an FITC labeled N-terminal fragment . Intersubunit fluorescence resonance energy self-transfer was observed when appropriate probes were attached to cysteines at residues 12 or 33, located in the N-terminal domain . Probes attached to cysteines at positions 63 or 89 in the C-terminal domain, however, did not exhibit intersubunit self-transfer . These results indicate that these residues in the C-terminal domains are, on average, separated by greater than 85 A . Intersubunit self-transfer does occur in a C-89 double mutation variant lacking 11 residues in the putative hinge region, indicating that the loss of the hinge region brings the two C-terminal domains closer together . Rapid subunit exchange between unlabeled wild-type L7/L12 and L7/L12 variants labeled in the N-terminal domain was also demonstrated by the loss of self-transfer upon mixing of the two proteins.

Biochemistry, 1996 Dec 24, 35(51), 16672 - 9
Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12; Hamman BD et al.; Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12 . Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues . In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied . Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule . Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain . Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains . The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime . This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein . Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment . Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.

Biochemistry, 1996 Dec 24, 35(51), 16665 - 71
Specific recognition of A/G and A/7,8-dihydro-8-oxoguanine (8-oxoG) mismatches by Escherichia coli MutY: removal of the C-terminal domain preferentially affects A/8-oxoG recognition; Gogos A et al.; Escherichia coli MutY is a 39 kDa adenine DNA glycosylase and 3' apurinic/apyrimidinic (AP) lyase that is active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches . 8-oxoG (7,8-dihydro-8-oxoguanine or GO) is a major stable product of oxidative damage, and A/GO mismatches may be particularly important biological substrates for MutY . Proteolytic digestion of MutY using thermolysin was found to produce two relatively stable fragments of 25 and 12 kDa . The 25 kDa fragment begins at the N terminus of MutY and spans the region homologous with E . coli endonuclease III, a DNA glycosylase/AP lyase that repairs oxidatively damaged pyrimidines . The 12 kDa fragment, which consists of much of the rest of MutY, had no detectable activity . The purified 25 kDa fragment (M25) had nearly wild-type binding and cleavage activities with A/G-mismatched substrates . Binding to A/GO-mismatched DNA, however, was dramatically reduced in M25 compared to that in intact protein . Borohydride-dependent enzyme-DNA cross-linking, which is a hallmark of the reaction of several DNA glycosylases that possess concomitant AP lyase activity, was also substantially reduced when M25 was allowed to react with A/GO-mismatched DNA . The significant differences in M25 recognition and reactivity with A/G and A/GO mismatches suggest that the C-terminal region of MutY, a region with no homologous counterpart in E . coli endonuclease III, plays an important role in the repair of mismatched DNA arising from oxidation damage.

Biochemistry, 1996 Dec 24, 35(51), 16646 - 51
Fidelity of translesional synthesis past benzo{a}pyrene diol epoxide-2'-deoxyguanosine DNA adducts: marked effects of host cell, sequence context, and chirality; Moriya M et al.; We have used a site-specific approach to investigate the mutagenic potential of (+)- and (-)-trans-anti-benzo{a}pyrene diol epoxide (BPDE) DNA adducts . Oligodeoxyribonucleotides (5'TCCTCCTG1G2-CCTCTC), modified at the exocyclic amino groups of G1 or G2, were incorporated into a single-stranded shuttle vector and introduced into Escherichia coli or simian kidney (COS) cells . This experimental system permits translesional synthesis to proceed in the absence of DNA repair . The presence of (+)- or (-)-BPDE-N2-dG adducts strongly inhibited translesional synthesis in E . coli; induction of cellular SOS functions reduced this blocking effect . Vectors containing (+)-BPDE adducts at G1 or G2 generated mutation frequencies of 19% and 3%, respectively; these values were not altered significantly by induction of SOS functions . In COS cells, (+)-BPDE-modified vectors generated mutation frequencies of 13% at G1 and 45% at G2 . In E . coli, the (-)-BPDE adduct generated mutation frequencies of < or = 2% at G1 and G2 and, in COS cells, 13% at G1 and 21% at G2 . The predominant mutations in E . coli and COS cells were G-->T transversions targeted to the site of the lesion; however, when G2 was modified, a significant number of targeted G-->A and G-->C mutations were observed in COS cells . We conclude from this study that (+)-and (-)-BPDE-N2-dG adducts pair preferentially to dCMP and dAMP during translesional synthesis in a process that is strongly influenced by the stereochemistry of the adduct, by the bases flanking the lesion, and by host cell factors.

Biochemistry, 1996 Dec 24, 35(51), 16630 - 7
The phosphodiester bond 3' to a deoxyuridine residue is crucial for substrate binding for uracil DNA N-glycosylase; Purmal AA et al.; Using the method of water-soluble carbodiimide-induced chemical ligation, four 27-member oligodeoxyribonucleotides containing a pyrophosphate internucleotide bond near or adjacent to a deoxyuridine residue were prepared . Escherichia coli uracil DNA N-glycosylase (UDG) activity was found to be sensitive to the presence of an internucleotide pyrophosphate bond in both single- and double-stranded DNA . The rate of uracil excision from single-stranded DNA containing a pyrophosphate bond adjacent to the uracil residue, either 3' or 5', was 0.01% and 0.1% of the rate of uracil removal from control DNA without a pyrophosphate bond, respectively . The rate of uracil excision from duplex DNA containing a pyrophosphate bond 3' or 5' to the uracil residue was also reduced, being 0.1% and 1% the rate of uracil removal from the corresponding duplex DNA control . Placing the pyrophosphate bond one nucleotide 5' or 3' away from the deoxyuridine in both single- and double-stranded oligodeoxyribonucleotides provided much better substrates for UDG . Kinetic measurements showed that the pyrophosphate bond placed adjacent to the deoxyuridine residue drastically reduced the affinity of UDG toward the modified DNA substrate, with the greatest effect occurring when the pyrophosphate bond was 3' adjacent to the deoxyuridine . The enzyme was able to excise a 3'-terminal uracil at the nicked site of a nicked duplex, DNA, provided that the terminal deoxyuridine was 3'-phosphorylated . The effect of the pyrophosphate bond on the substrate susceptibility of oligonucleotides containing deoxyuridine is discussed with respect to the mechanism of action of UDG.

Biochemistry, 1996 Dec 24, 35(51), 16581 - 90
Expression, zinc-affinity purification, and characterization of a novel metal-binding cluster in troponin T: metal-stabilized alpha-helical structure and effects of the NH2-terminal variable region on the conformation of intact troponin T and its association with tropomyosin; Ogut O et al.; A repeating metal-binding (Cu2+ > Ni2+ > Zn2+ approximately Co2+) sequence {HE/AEAH}4 (Tx) has been recently identified in the NH2-terminal variable region of troponin T (TnT) isoforms specifically expressed in the breast but not leg muscles of the avian orders of Galliformes and Craciformes {Jin, J.-P., & Smillie, L . B . (1994) FEBS Lett . 341, 135-140} . In the present study, two expression plasmids were constructed to produce chicken TnT1 NH2-terminal fragments of 47 (N47) or 165 (N165) amino acids containing the Tx metal-binding cluster . The recombinant protein/peptide was expressed in Escherichia coli BL21(DE3)pLysS and purified by a highly effective Zn(2+)-affinity chromatography method . Amino acid analyses, NH2-terminal peptide sequencing, mass spectrometry and immunological identification confirmed the authenticity of the genetically engineered TnT fragments . In the presence of 2,2,2-trifluoroethanol, transition metals had significant effects on the secondary structure of TnT fragment N47, as shown by circular dichroism . N165 in non-denaturing buffer demonstrated alpha-helical content comparable to previous data from rabbit fast skeletal TnT fragment T1 . Zn(2+)-binding avidity of the metal-binding TnT and its fragments demonstrated tertiary relationships between the NH2-terminal variable region and the COOH-terminal segment of the intact TnT protein . Solid-phase protein-binding assays established that Zn(2+)-binding to the Tx cluster induces epitopic structure changes in this NH2-terminal segment, further affecting other epitopic structures of intact TnT as well as the function of TnT's tropomyosin binding-sites . The results demonstrate that metal ion-binding to the Tx cluster reconfigures the overall conformation of TnT through structural relationships between the NH2-terminal variable region and other domains of the intact TnT molecule . Accordingly, the developmental and/or muscle type specific NH2-terminal structure of TnT isoforms may modulate the Ca(2+)-activation of muscle contraction.

Biochemistry, 1996 Dec 24, 35(51), 16502 - 9
A 19F-NMR study of the equilibrium unfolding of membrane-associated D-lactate dehydrogenase of Escherichia coli; Sun ZY et al.; Partially folded protein intermediates have been observed by 19F-NMR spectroscopy during the equilibrium unfolding of the membrane-associated D-lactate dehydrogenase (D-LDH) of Escherichia coli by a denaturant, guanidine hydrochloride (Gdn.HCl) . The results from 19F-NMR and circular dichroism spectroscopic studies suggest that the intermediates observed at low Gdn.HCl concentrations (< 3.5 M) exhibit features similar to "molten globules" that contain considerable amounts of secondary and tertiary structure . The results of 19F-NMR studies on 5F-Trp-labeled D-LDH, such as the chemical shift changes, nuclear Overhauser effect, and solvent-induced isotopic shift effect, show that different regions of D-LDH unfold nonuniformly in Gdn.HCl in the presence of lysophosphatidylcholine . The polypeptide appears to unfold in a general order from the carboxyl end to the amino end, in agreement with previous findings from our laboratory that the carboxyl-terminal region of D-LDH is largely exposed to the solvent while the amino-terminal region is buried in the protein core . The structure of the partially unfolded intermediate forms of D-LDH is stabilized in the presence of lipid-like detergents, such as lysophosphatidylcholine.

Biochemistry, 1996 Dec 24, 35(51), 16412 - 20
Biosynthesis of 3,6-dideoxyhexoses: in vivo and in vitro evidence for protein-protein interaction between CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1) and its reductase (E3); Chen XM et al.; CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1), together with its reductase (E3), catalyzes a novel deoxygenation reaction essential for the biosynthesis of 3,6-dideoxyhexoses . In an attempt to gain evidence substantiating the E1.E3 complex formation as a prerequisite for the C-3 deoxygenation activity, we have carried out experiments to study the interaction between these two proteins . The detection of a new species when a mixture of E1 and E3 was analyzed by size-exclusion chromatography was the initial indication supporting the proposed complex formation . Additional evidence for the expected complex formation was provided by the change of the CD spectrum of E1 upon its coupling with E3 . The fact that the catalytic efficiency of this system is limited by the quantity of one enzyme, which becomes catalytically competent only after coupling with the second enzyme, further illustrated the importance of such a complex formation to the deoxygenation activity . By using the two-hybrid system which scores for interactions between two proteins coexpressed in yeast, the E1.E3 complex formation in vivo was also firmly established . These results, when considered with the incompatibility of other electron transfer proteins as replacements for E3 in this electron relay, nicely demonstrated the specificity of the E1-E3 recognition . The apparent dissociation constant of the E1.E3 complex formed in rapid equilibrium was estimated to be 288 +/- 22 nM from the correlation between the initial rate of the overall reaction and the concentration of one protein component, and the stoichiometry between E3 and E1 of this complex was deduced as 1.7 . Interestingly, while the conformation of the E1.E3 complex was sensitive to the salt concentration in the buffer, the decrease in the catalytic activity at high ionic strength was most likely due to the retardation of the electron transfer mediated by E3 . In conjunction with early mechanistic studies, the present data establish the significance of the E1.E3 complex formation for catalysis and, consequently, corroborate the mechanism proposed for the overall deoxygenation process.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15051 - 6
Inhibiting transthyretin amyloid fibril formation via protein stabilization; Miroy GJ et al.; Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases . Herein, we demonstrate conclusively that thyroxine (10.8 microM) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation . In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism . Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine . These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases . This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis--i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15047 - 50
Amphipols: polymers that keep membrane proteins soluble in aqueous solutions; Tribet C et al.; Amphipols are a new class of surfactants that make it possible to handle membrane proteins in detergent-free aqueous solution as though they were soluble proteins . The strongly hydrophilic backbone of these polymers is grafted with hydrophobic chains, making them amphiphilic . Amphipols are able to stabilize in aqueous solution under their native state four well-characterized integral membrane proteins: (i) bacteriorhodopsin, (ii) a bacterial photosynthetic reaction center, (iii) cytochrome b6f, and (iv) matrix porin.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15036 - 40
Methionine residues as endogenous antioxidants in proteins; Levine RL et al.; Cysteine and methionine are the two sulfur-containing residues normally found in proteins . Cysteine residues function in the catalytic cycle of many enzymes, and they can form disulfide bonds that contribute to protein structure . In contrast, the specific functions of methionine residues are not known . We propose that methionine residues constitute an important antioxidant defense mechanism . A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, available as an efficient oxidant scavenger . Reduction back to methionine by methionine sulfoxide reductases would allow the antioxidant system to function catalytically . The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system . Eight of the 16 methionine residues could be oxidized with little effect on catalytic activity of the enzyme . The oxidizable methionine residues were found to be relatively surface exposed, whereas the intact residues were generally buried within the core of the protein . Furthermore, the susceptible residues were physically arranged in an array that guarded the entrance to the active site.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15024 - 9
Chaperone activity and structure of monomeric polypeptide binding domains of GroEL; Zahn R et al.; The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities . We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase . Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL . We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties . The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions.

Biochem Biophys Res Commun, 1996 Dec 24, 229(3), 869 - 75
Structural and functional characterization of a His-tagged PhoE pore protein of Escherichia coli; Van Gelder P et al.; The recent elucidation of the 3-D structure of the outer membrane protein PhoE of Escherichia coli provides an excellent tool for a detailed analysis of the structure-function relationship of this pore-forming protein . For this purpose, a fast and efficient method for the purification of mutant porins is needed . A histidine-tag was engineered between the signal sequence and the N terminus of mature PhoE . The recombinant PhoE protein was normally assembled into the outer membrane and could be purified by immobilized metal affinity chromatography . Part of the total amount of the trimers dissociated into folded monomers during purification . The histidine-tag did not change the electrophysical characteristics of the protein in lipid bilayers . Hence, the method is useful for the fast purification of mutant porins for functional and structural characterization.

Biochem Biophys Res Commun, 1996 Dec 24, 229(3), 845 - 51
Casein kinase 2 phosphorylates recombinant human spermidine/spermine N1-acetyltransferase on both serine and threonine residues; Bordin L et al.; Casein kinase 2 purified from human erythrocyte cytosol has been found to phosphorylate human spermidine/spermine N1-acetyltransferase (SSAT) expressed as a fusion protein in E . coli and purified to homogeneity with a specific activity similar to that reported for pure human SSAT . The amino acid sequence of the protein revealed not less than four phosphorylable residues, optimal target for protein kinase 2 phosphorylation being flanked by acid residues in position +1 and +3 . Our results indicate that most 32P-phosphate is taken up by Ser residues, as evidenced by HCl hydrolysis and electrophoresis and that the phosphorylation extent is modulated by the physiological polyamine concentration . Partial digestion with trypsin at a low concentration for less than one hour preferentially hydrolyzes Lys-Arg-Arg in position 141-143 of the SSAT suggesting that the Ser-phosphorylated residues are located in the C-terminus of the protein, probably Ser 146 and 149.

Mol Biochem Parasitol, 1996 Dec 20, 83(2), 153 - 61
Cloning, sequencing and expression of a cDNA encoding an antigen from the Myxosporean parasite causing the proliferative kidney disease of salmonid fish; Saulnier D et al.; A pool of monoclonal antibodies (MAbs) raised against the unknown organism causing the proliferative kidney disease of salmonid fish (PKX) has been used to screen a cDNA expression library constructed from poly (A+) RNA extracted from PKX infected kidney . Four immunopositive lambda ZapII recombinant phages were selected . Sequencing of the cDNAs revealed identical 3' ends . The longest cDNA clone (2652 nucleotides) had an open reading frame (ORF) of 872 amino acids and encoded a protein with a predicted size of 101 kDa (PKX101) . Sequence analysis of PKX101 revealed two leucine zipper motifs, a putative transmembrane region and a microbody targeting signal at its C-terminal end . Three cDNA fragments were subcloned in pET-14b expression vector and the ORF verified by an in vitro transcription/translation procedure . Recombinant clones were expressed in Escherichia coli and the antigenicity of fusion proteins was studied by Western blotting using monoclonal antibodies directed against PKX cells and a pool of serum from preimmune or PKX-infected rainbow trout . Western blotting of enriched PKX cell antigen probed with one MAb or with sera from infected trout revealed a single protein with relative mobility of 13 kDa (PKX13) . This discrepancy between PKX101 and PKX13 observed in Western blot suggests post-translational modifications of the full-length PKX antigen.

J Mol Biol, 1996 Dec 20, 264(5), 1132 - 44
Thermal unfolding of the DNA-binding protein Sso7d from the hyperthermophile Sulfolobus solfataricus; Knapp S et al.; Thermal unfolding of the small hyperthermophilic DNA-binding protein Sso7d was studied by circular dichroism spectroscopy and differential scanning calorimetry . The unfolding transition can be described by a reversible two state process . Maximum stability was observed in the region between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperature between 370.8 to 371.9 K and an unfolding enthalpy between 62.9 and 65.4 kcal/mol . The heat capacity differences between the native and the heat denatured states obtained by differential scanning calorimetry (620 cal/(molK)) and circular dichroism spectroscopy (580 cal/(mol K)) resulted in comparable values . The thermodynamic reason for the high melting temperature of Sso7d is the shallow stability curve with a broad free energy maximum, corresponding to the relatively small heat capacity change which was obtained . The calculated stability curve shows that Sso7d has, despite of its high melting temperature, an only moderate intrinsic stability, which reaches its maximum (approximately 7 kcal/mol) at 282 K . Sso7d is particularly poorly stabilized (approximately 1 kcal/mol) at the maximum physiological growth temperature of Sulfolobus solfataricus . Sso7d has furthermore untypically low specific enthalpy (0.99 kcal/(mol residue)) and entropy (2.99 cal/(mol K)) values at convergence temperatures . No significant differences in thermal stability of the partially methylated Sso7d from Sulfolobus solfataricus and the cloned non-methylated form of the protein expressed in Escherichia coli were observed.

J Mol Biol, 1996 Dec 20, 264(5), 1117 - 31
Three-dimensional structure of the HTLV-II matrix protein and comparative analysis of matrix proteins from the different classes of pathogenic human retroviruses; Christensen AM et al.; The matrix protein performs similar roles in all retroviruses, initially directing membrane localization of the assembling viral particle and subsequently forming a stable structural shell associated with the inner surface of the mature viral membrane . Although conserved structural elements are likely to perform these functions in all retroviral matrix proteins, invariant motifs are not evident at the primary sequence level and three-dimensional structures have been available for only the primate lentiviral matrix proteins . We have therefore used NMR spectroscopy to determine the structure of the matrix protein from human T-cell leukemia virus type II (HTLV-II), a member of the human oncovirus subclass of retroviruses . A total of 577 distance restraints were used to build 20 refined models that superimpose with an rmsd of 0.71 A for the backbone atoms of the structured regions . The globular HTLV-II matrix structure is composed of four alpha-helices and a 3(10) helix . Exposed basic residues near the C terminus of helix II form a putative membrane binding surface which could act in concert with the N-terminal myristoyl group to anchor the protein on the viral membrane surface . Clear structural similarities between the HTLV-II and HIV-1 matrix proteins suggest that the topology and exposed cationic membrane binding surface are likely to be conserved features of retroviral matrix proteins.

J Mol Biol, 1996 Dec 20, 264(5), 1101 - 16
Characterizing the use of perdeuteration in NMR studies of large proteins: 13C, 15N and 1H assignments of human carbonic anhydrase II; Venters RA et al.; Perdeuteration of all non-exchangeable proton sites can significantly increase the size of proteins and protein complexes for which NMR resonance assignments and structural studies are possible . Backbone 1H, 15N, 13CO, 13C alpha and 13C beta chemical shifts and aliphatic side-chain 13C and 1H(N)/15N chemical shifts for human carbonic anhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been determined using a strategy based on 2D, 3D and 4D heteronuclear NMR experiments, and on perdeuterated 13C/15N-labeled protein . To date, HCA II is one of the largest monomeric proteins studied in detail by high-resolution NMR . Of the backbone resonances, 85% have been assigned using fully protonated 15N and 3C/15N-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR experiments . HCA II has been perdeuterated both to complete the backbone resonance assignment and to assign the aliphatic side-chain 13C and 1H(N)/15N resonances . The incorporation of 2H into HCA II dramatically decreases the rate of 13C and 1H(N)T2 relaxation . This, in turn, increases the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments . Many otherwise marginal heteronuclear 3D and 4D correlation experiments, which are important to the assignment strategy detailed herein, can now be executed successfully on HCA II . Further analysis suggests that, from the perspective of sensitivity, perdeuteration should allow other proteins with rotational correlation times significantly longer than HCA II (tau c = 11.4 ns) to be studied successfully with these experiments . Two different protocols have been used to characterize the secondary structure of HCA II from backbone chemical-shift data . Secondary structural elements determined in this manner compare favorably with those elements determined from a consensus analysis of the HCA II crystal structure . Finally, having outlined a general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this information can be used to glean more detailed structural information from the partially or fully protonated protein equivalent.

J Mol Biol, 1996 Dec 20, 264(5), 1044 - 57
Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxin reductase at 2.5 A resolution; Dai S et al.; Thioredoxin exists in all organisms and is responsible for the hydrogen transfer to important enzymes for ribonucleotide reduction and the reduction of methionine sulphoxide and sulphate . Thioredoxins have also been shown to regulate enzyme activity in plants and are also involved in the regulation of transcription factors and several other regulatory activities . Thioredoxin is reduced by the flavoenzyme thioredoxin reductase using NADPH . We have now determined the first structure of a eukaryotic thioredoxin reductase, from the plant Arabidopsis thaliana, at 2.5 A resolution . The dimeric A . thaliana thioredoxin reductase is structurally similar to that of the Escherichia coli enzyme, and most differences occur in the loops . Because the plant and E . coli enzymes have the same architecture, with the same dimeric structure and the same position of the redox active disulphide bond, a similar mechanism that involves very large domain rotations is likely for the two enzymes . The subunit is divided into two domains, one that binds FAD and one that binds NADPH . The relative positions of the domains in A . thaliana thioredoxin reductase differ from those of the E . coli reductase . When the FAD domains are superimposed, the NADPH domain of A . thaliana thioredoxin reductase must be rotated by 8 degrees to superimpose on the corresponding domain of the E . coli enzyme . The domain rotation we now observe is much smaller than necessary for the thioredoxin reduction cycle.

J Mol Biol, 1996 Dec 20, 264(5), 1013 - 27
Crystal structures of adenylosuccinate synthetase from Escherichia coli complexed with GDP, IMP hadacidin, NO3-, and Mg2+; Poland BW et al.; Crystal structures of adenylosuccinate synthetase from Esherichia coli complexed with Mg2+, IMP, GDP, NO3- and hadacidin at 298 and 100 K have been refined to R-factors of 0.188 and 0.206 against data to 2.8 A and 2.5 A resolution, respectively . Conformational changes of up to 9 A relative to the unligated enzyme occur in loops that bind to Mg2+, GDP, IMP and hadacidin . Mg2+ binds directly to GDP, NO3-, hadacidin and the protein, but is only five-coordinated . Asp13, which approaches, but does not occupy the sixth coordination site of Mg2+, hydrogen bonds to N1 of IMP . The nitrogen atom of NO3- is approximately 2.7 A from O6 of IMP, reflecting a strong electrostatic interaction between the electron-deficient nitrogen atom and the electron-rich O6 . The spatial relationships between GDP, NO3- and Mg2+ suggest an interaction between the beta,gamma-bridging oxygen atom of GTP and Mg2+ in the enzyme-substrate complex . His41 hydrogen bonds to the beta-phosphate group of GDP and approaches bound NO3- . The aldehyde group of hadacidin coordinates to the Mg2+, while its carboxyl group interacts with backbone amide groups 299 to 303 and the side-chain of Arg303 . The 5'-phosphate group of IMP interacts with Asn38, Thr129, Thr239 and Arg143 (from a monomer related by 2-fold symmetry) . A mechanism is proposed for the two-step reaction governed by the synthetase, in which His41 and Asp13 are essential catalytic side-chains.

J Mol Biol, 1996 Dec 20, 264(5), 891 - 906
Regulation of the activity of the type IC EcoR124I restriction enzyme; Kulik EM et al.; Restriction-modification (R-M) systems must regulate the expression of their genes so that the chromosomal genome is modified at all times by the methyltransferase to protect the host cell from the potential lethal action of the cognate restriction endonuclease . Since type I R-M systems can be transferred to non-modified Escherichia coli cells by conjugation or transformation without killing the recipient, they must have some means to regulate their restriction activity upon entering a new host cell to avoid restriction of unprotected host DNA and cell death . This is especially true for EcoR124I, a type IC family member, which is coded for by a conjugative plasmid . Control of EcoR124I restriction activity is most likely at the post-translational level as the transfer of the EcoR124I system into a recipient cell that already expressed the HsdR subunit of this system was not a lethal event . Additionally, the kinetics of restriction activity upon transfer of the genes coding for the EcoR124I RM system to a recipient cell are the same, irrespective of the modification state of the recipient cell or the presence or absence of the EcoR124I HsdR subunit in the new host cells . The mechanism controlling the restriction activity of a type IC R-M system upon transfer to a new host cell is different from that controlling the chromosomally coded type IA and IB R-M systems . The previously discovered hsdC mutant, which affects the establishment of the type IA system EcoKI, was shown to affect the establishment of the type IB system EcoAI, but to have no influence on EcoR124I.

J Mol Biol, 1996 Dec 20, 264(5), 878 - 90
Interactions between RuvA and RuvC at Holliday junctions: inhibition of junction cleavage and formation of a RuvA-RuvC-DNA complex; Whitby MC et al.; The RuvAB and RuvC enzymes of Escherichia coli define a molecular pathway for the resolution of Holliday intermediates in recombination and DNA repair . They bind specifically to Holliday junctions, and catalyse their branch migration and cleavage, respectively . In a RuvA(B)-junction complex, the Holliday structure is held in an open (square planar) configuration on the concave surface of a 4-fold symmetrical tetramer of RuvA, whereas in a RuvC-junction complex it is folded in an alternative arrangement as part of the cleavage reaction . Genetic studies have shown that the activity of RuvC in vivo depends on RuvAB, which suggests that the two enzymes act in concert, with junction cleavage by RuvC following from branch migration by RuvAB . We have investigated how RuvC can take over a junction from RuvAB to cleave the DNA . We show that RuvA inhibits junction cleavage by RuvC, probably by sandwiching the junction between two tetramers . The extent of inhibition depends on the reaction kinetics of RuvA binding relative to RuvC binding and cleavage . The presence of RuvB and the concentration of Mg2+ both have a significant effect on cleavage in the presence of RuvA . However, a novel protein-DNA complex can be formed when junction DNA is incubated with both RuvA and RuvC . Its mobility is consistent with a RuvC dimer binding to a junction held in an open configuration on the surface of a RuvA tetramer . We suggest that this arrangement provides RuvC with the means to scan the junction during the RuvAB-mediated branch migration reaction for DNA sequences that it can cleave . We further suggest that recognition of the target may provide a trigger for dissociating RuvA, allowing the junction to be folded and cleaved by RuvC.

J Mol Biol, 1996 Dec 20, 264(5), 852 - 62
On the role of the multiple regulatory elements involved in the activation of the Escherichia coli malEp promoter; Richet E; Activation of malEp and malKp, two divergent promoters from Escherichia coli, depends on the synergistic action of MalT and CRP . The reaction involves a common regulatory region located in between and comprising multiple binding elements for both regulatory proteins . The binding of MalT and CRP to this region is known to result in the formation of a higher-order structure that is responsible for malKp activation . This paper presents genetic data which together with previous results, provide compelling evidence that this higher-order structure is also responsible for malEp activation . The role(s) that this structure or elements thereof play in the activation of malEp is analysed by monitoring both the occupancy of the proximal MalT sites (sites 1 and 2) and the activity of different malEp variants in strains containing increasing amounts of active MalT . A truncated malEp promoter comprising only MalT sites 1 and 2 forms a minimal MalT-dependent promoter whose activity is limited by the occupancy of these sites . One role of the higher-order structure formed by MalT and CRP when bound to the entire regulatory region is to ensure high occupation of MalT sites 1 and 2, but it is not its only function . Some elements of this structure, namely the CRP site 1, located at -76.5, and the distal MalT sites, seem to play a direct role in malEp activation besides their participation in the assembly of the higher-order structure.

Mol Cell Biochem, 1996 Dec 20, 165(2), 111 - 20
Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C; Crane JK et al.; The heat-stable enterotoxin STa of E . coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP . Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion . We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro . Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with {32P}-phosphate using polyclonal anti-STa receptor antibody . The STa receptor was phosphorylated in its basal state, and 32P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h . In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC . Phosphorylation was inhibited 40% by 5 microM of a synthetic peptide corresponding to the sequence around Ser1029 of the STa receptor, a site previously proposed as a potential PKC phosphorylation site . Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold . We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser1029 of the STaR/GC remains a candidate phosphorylation site by PKC.

J Biol Chem, 1996 Dec 20, 271(51), 33009 - 17
Role of the EBNA-1 protein in pausing of replication forks in the Epstein-Barr virus genome; Ermakova OV et al.; We have previously shown that replication forks stall at a family of repeated sequences (FR) within the Epstein-Barr virus latent origin of replication oriP, both in a small plasmid and in the intact Epstein-Barr virus genome . Each of the 20 repeated sequences within the FR contains a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the only viral protein required for latent replication . We showed that the EBNA-1 protein enhances the accumulation of paused replication forks at the FR . In this study, we have investigated a series of truncated EBNA-1 proteins to determine the portion of the EBNA-1 protein that is responsible for pausing of forks at the FR . Two-dimensional agarose gel electrophoresis was performed on the products of in vitro replication reactions in the presence of full-length EBNA-1 or proteins with various deletions to assess the extent of fork pausing at the FR . We conclude that a portion of the DNA binding domain is important for fork pausing . We also present evidence indicating that phosphorylation of the EBNA-1 protein or EBNA-1-truncated derivatives is not essential for pausing . To investigate the mechanism of EBNA-1-mediated pausing of replication forks, we asked whether EBNA-1 could inhibit the DNA unwinding activity of replicative helicases . We found that EBNA-1, when bound to the FR, inhibits DNA unwinding in vitro by SV40 T antigen and Escherichia coli dnaB helicases in an orientation-independent manner.

J Biol Chem, 1996 Dec 20, 271(51), 32907 - 15
In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent; Pinnaduwage P et al.; Interaction of artificial lipid bilayers (liposomes) with the purified transit peptide (SS-tp) of the precursor form of the small subunit for ribulose-2,5-bisphosphate carboxylase/oxygenase (prSSU) has been studied using a vesicle-disruption assay (calcein dye release) and electron microscopy . Employing purified forms of Escherichia coli-expressed prSSU, mature small subunit, glutathione S-transferase-transit peptide fusion protein, and SS-tp in dye release studies demonstrated that lipid interaction is mediated primarily through the transit peptide . Using chemically synthesized peptides (20-mers), the lipid-interacting domain of the transit peptide was partially mapped to the C-terminal 20 amino acids of the transit peptide . Peptides corresponding to other regions of the transit peptide and control peptides promoted significantly less calcein release . Interaction between the transit peptide and the bilayer was very rapid and could not be resolved by stopped-flow fluorometry with a mixing time of <50 ms . Interaction between the peptides and bilayer was also lipid class-dependent . Disruption occurred only when the bilayer contained the galactolipid monogalactosyldiacylglycerol (MGDG) . The extent of bilayer disruption directly correlated with the relative concentration of MGDG in the liposome, with maximum calcein release occurring in 20 mol % MGDG liposomes . Lipid bilayers with greater than 20 mol % MGDG could not be achieved as determined by calcein entrapment . Electron microscopy of the liposomes before and after addition of the transit peptide suggested that the transit peptide induced a dramatic reorganization of lipids . These results are discussed in light of a possible mechanism for the early steps in protein transport that may involve polymorphic changes in the envelope membrane organization to include localized non-bilayer HII structures.

J Biol Chem, 1996 Dec 20, 271(51), 32900 - 6
Maize chromosomal HMGc . Two closely related structure-specific DNA-binding proteins specify a second type of plant high mobility group box protein; Grasser KD et al.; The chromosomal high mobility group (HMG) proteins are small and abundant non-histone proteins common to eukaryotes . We have purified the maize HMGc protein from immature kernels and characterized it by mass spectrometry and amino acid sequence analysis . HMGc could be resolved into two similar proteins by reversed phase chromatography . Cloning and characterization of the corresponding cDNAs revealed that they encode two closely related maize HMGc proteins, now termed HMGc1 and HMGc2 . Their theoretical masses of 15,316 and 15,007 Da are >300 Da lower than the masses determined for the proteins purified from maize, indicating post-translational modifications of the proteins . Despite sequence similarity to maize HMGa (and previously described homologous proteins of other species) amino acid sequence alignments reveal that HMGc is in several conserved regions distinct from these proteins . Consequently, we have identified a novel type of plant protein containing an HMG box DNA binding domain and belonging to the HMG1 protein family . HMGc1 and HMGc2 were expressed in Escherichia coli, purified to homogeneity, and analyzed for their DNA binding properties . They proved to bind to DNA structure-specifically since they formed complexes with DNA minicircles at concentrations approximately 100-fold lower than the concentrations required to form complexes with linear fragments of identical sequence . Furthermore, HMGc1 and HMGc2 can constrain negative superhelical turns in plasmid DNA.

J Biol Chem, 1996 Dec 20, 271(51), 32886 - 93
Nucleoside-diphosphate kinase-mediated signal transduction via histidyl-aspartyl phosphorelay systems in Escherichia coli; Lu Q et al.; Nucleoside-diphosphate kinase (NDP kinase), a key enzyme in nucleotide metabolism, is also known to be involved in growth and developmental control and tumor metastasis suppression . Interestingly, we find that coexpression of NDP kinase with Taz1, a Tar/EnvZ chimera, in the absence of its native signal, can activate a porin gene ompC-lacZ expression in Escherichia coli . Further studies show that NDP kinase can act as a protein kinase to phosphorylate histidine protein kinases such as EnvZ and CheA which are members of the His-Asp phosphorelay signal transduction systems in E . coli . Instead of ATP, the exclusive phosphodonor for histidine kinases, GTP can be utilized in vitro in the presence of NDP kinase to phosphorylate EnvZ and CheA, which then transfer the phosphoryl group to OmpR and CheY, the respective response regulators . The direct involvement of GTP for the phosphorylation of EnvZ through NDP kinase was further demonstrated by the use of a mutant EnvZ, which lost ability to be autophosphorylated with ATP . Phospho-OmpR thus formed can bind specifically to an ompF promoter sequence . These results suggest that NDP kinase may play a physiological role in signal transduction.

J Biol Chem, 1996 Dec 20, 271(51), 32857 - 62
Conserved nucleotides of 23 S rRNA located at the ribosomal peptidyltransferase center; Spahn CM et al.; Two nucleotides of the 23 S rRNA gene were mutated; the nucleotides correspond to the first two positions of the universally conserved sequence PsiGG2582 at the peptidyltransferase ring of 23 S rRNA . The ribosomes containing the altered 23 S rRNA were analyzed . Previously, it was shown that ribosomal assembly was indistinguishable from that in wild-type cells, that the flow of the corresponding 50 S subunit into the polysome fraction was not restricted, but that the ribosomes were strongly impaired in poly(Phe) synthesis (C . M . T . Spahn, J . Remme, M . A . Schafer, and K . H . Nierhaus (1996) J . Biol . Chem . 271, 32849-32856) . Here we apply assay systems exclusively testing the puromycin reaction of ribosomes carrying plasmid-born rRNA, a dipeptide assay using the minimal P site donor pA(fMet) and a translocation system not depending on the puromycin reaction . The mutations in helix 90 exclusively abolish or severely impair the ribosome capability to catalyze AcPhe-puromycin formation . A possible explanation of these observations is that G2581 and Psi2580 (and possibly also G2582) are part of the binding site of C75 of peptidyl-tRNA in the P site . The results suggest that in this case, however, such an interaction would disobey canonical base pairing.

J Biol Chem, 1996 Dec 20, 271(51), 32849 - 56
Mutational analysis of two highly conserved UGG sequences of 23 S rRNA from Escherichia coli; Spahn CM et al.; The 23 S-type rRNA contains two phylogenetically conserved UGG sequences, which have the potential to bind the universal CCA-3'-ends of tRNAs at the ribosomal peptidyltransferase center by base pairing . The first two positions, UG, of these sequences at the helix-loop 80 (U2249G2250) and helix-loop 90 (Psi2580G2581) and some related nucleotides were tested by site-directed mutagenesis for their involvement in ribosomal function, i.e . peptidyltransferase . The plasmid-derived mutated 23 S rRNA comprised about 50% of the total 23 S rRNA . None of the single mutations caused an assembly defect, and all 50 S subunits carrying an altered 23 S rRNA could freely exchange with the pools of 70S ribosomes and polysomes . The mutations at the helix-loop 80 region hardly affected bacterial growth . However, mutations at the helix 90 caused severe growth effects and severely impaired the in vitro protein synthesis, showing that this 23 S rRNA region is of high importance for ribosomal function.

J Biol Chem, 1996 Dec 20, 271(51), 32737 - 42
Cross-linking of engineered subunit delta to (alphabeta)3 in chloroplast F-ATPase; Lill H et al.; Ser --> Cys mutations were introduced into subunit delta of spinach chloroplast F0F1-ATPase (CF0CF1) by site-directed mutagenesis . The engineered delta subunits were overexpressed in Escherichia coli, purified, and reassembled with spinach chloroplast F1-ATPase (CF1) lacking the delta subunit (CF1(-delta)) . By modification with eosin-5-maleimide, it was shown that residues 10, 57, 82, 160, and 166 were solvent-accessible in isolated CF1 and all but residue 166 also in membrane-bound CF0CF1 . Modification of the engineered delta subunit with photolabile cross-linkers, binding of delta to CF1(-delta), and photolysis yielded the same SDS gel pattern of cross-link products in the presence or absence of ADP, phosphate, and ATP and both in soluble CF1 and in CF0CF1 . By chemical hydrolysis of cross-linked CF1, it was shown that deltaS10C was cross-linked within the N-terminal 62 residues of subunit beta . deltaS57C, deltaS82C, and deltaS166C were cross-linked within the N-terminal 192 residues of subunit alpha . Cross-linking affected neither ATP hydrolysis by soluble CF1 nor its ability to reassemble with CF0 and to structurally reconstitute ATP synthesis . Functional reconstitution, however, seemed to be impaired.

J Biol Chem, 1996 Dec 20, 271(51), 32729 - 36
Thermal stability of Escherichia coli ribonuclease HI and its active site mutants in the presence and absence of the Mg2+ ion . Proposal of a novel catalytic role for Glu48; Kanaya S et al.; Escherichia coli ribonuclease HI, which requires divalent cations (Mg2+ or Mn2+) for activity, was thermostabilized by 2.6-3.0 kcal/mol in the presence of the Mg2+, Mn2+, or Ca2+ ion, probably because the negative charge repulsion around the active site was canceled upon the binding of these metal ions . The dissociation constants were determined to be 0.71 mM for Mg2+, 0.035 mM for Mn2+, and 0.16 mM for Ca2+ . Likewise, various active site mutants at Asp10, Glu48, Asp70, or Asp134 were thermostabilized by 0.4-3.0 kcal/mol in the presence of the Mg2+ ion, suggesting that this ion binds to these mutant proteins as well . The dissociation constants of Mg2+ were determined to be 9.8 mM for D10N, 1.1 mM for E48Q, 18.8 mM for D70N, and 1.8 mM for D134N . Thus, the mutation of Asp10 or Asp70 to Asn considerably impairs the Mg2+ binding, whereas the mutation of Glu48 to Gln or Asp134 to Asn does not . Comparison of the thermal stability of the mutant proteins with that of the wild-type protein in the absence of the Mg2+ ion suggests that the negative charge repulsion between Asp10 and Asp70 is responsible for the binding of the metal cofactor . Glu48 may be required to anchor a water molecule, which functions as a general acid.

J Biol Chem, 1996 Dec 20, 271(51), 32623 - 8
The trapping of different conformations of the Escherichia coli F1 ATPase by disulfide bond formation . Effect on nucleotide binding affinities of the catalytic sites; Gruber G et al.; Two mutants of the Escherichia coli F1 ATPase, betaY331W:E381C/epsilonS108C and alphaS411C/betaY331W/epsilonS108C, have been used to relate nucleotide binding in catalytic sites with different interactions of the stalk-forming subunits gamma and epsilon at the alpha3beta3 subunit domain . Essentially full yield cross-linking between beta + gamma and beta + epsilon, or between alpha + gamma and alpha + epsilon, was obtained in these mutants by Cu2+-induced disulfide bond formation, thereby trapping the enzyme in states with the small subunits interacting either with beta or alpha subunits . The presence of the Trp for beta Tyr-331 in both mutants allowed direct measurement of nucleotide occupancy of catalytic sites . Before cross-linking, Mg2+ATP could be bound in all three catalytic sites in both mutants with a Kd of around 0.1 microM for the highest affinity site and Kd values of approximately 2 microM and 30-40 microM for the second and third sites, respectively . In the absence of Mg2+, ATP also bound in all three catalytic sites but with a single low affinity (above 100 microM) in both mutants . Cu2+-induced cross-linking of ECF1 from the mutant betaY331W:E381C/epsilonS108C had very little effect on nucleotide binding . The binding affinities of the three catalytic sites for Mg2+ATP were not significantly altered from those obtained before cross-linking, and the enzyme still switched between cooperative binding and equal binding affinities of the three catalytic sites (when Mg2+ was absent) . When the gamma and epsilon subunits were cross-linked to alpha subunits, ATP binding in the highest affinity catalytic site was dramatically altered . This site became closed so that nucleotide (ATP or ADP) that had been bound into it prior to cross-linking was trapped and could not exchange out . Also, ATP or ADP could not enter this site, although empty, once the enzyme had been cross-linked . Finally, cross-linking of the gamma and epsilon to the alpha subunits prevented the switching between cooperative binding and the state where the three catalytic sites are equivalent . We argue that the conformation of the enzyme in which the small subunits are at alpha subunits occurs during functioning of the enzyme in the course of the rotation of gamma and epsilon subunits within the alpha3beta3 hexamer and that this may be the activated state for ATP synthesis.

J Biol Chem, 1996 Dec 20, 271(51), 32617 - 22
Expression, purification, and characterization of an active RNase H domain of the hepatitis B viral polymerase; Wei X et al.; The replication of the hepatitis B viral DNA genome proceeds through a pregenomic RNA intermediate . This pregenomic RNA subsequently serves as the template for the formation of the viral DNA by the reverse transcriptase activity of the viral P gene product . The P gene product is believed to be a multifunctional enzyme with DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, and RNase H activities . Detailed biochemical studies of this protein have not been performed because of the inability to obtain sufficient amounts of the enzyme from the virus and by the inability to produce the enzyme in heterologous expression systems . The RNase H activity is essential for viral replication and is believed to be responsible for the degradation of the RNA pregenomic intermediate as well as for generating the short RNA primer that is required for DNA second strand synthesis . We have assembled an expression vector which directs the synthesis of a protein that corresponds to the putative RNase H domain of the P gene product and having a carboxyl-terminal polyhistidine tag to facilitate purification . The protein has been expressed in Escherichia coli and purified to yield 1-2 mg of protein/liter of culture . This protein has RNase H activity as defined by its ability to degrade the RNA component of RNA-DNA hybrids but not the DNA component . The RNase H has a basic optimum pH, is active only in the presence of reducing agents, and is dependent on the presence of divalent cations, with magnesium being preferred over manganese.

J Biol Chem, 1996 Dec 20, 271(51), 32507 - 10
Human coproporphyrinogen oxidase is not a metalloprotein; Medlock AE et al.; Coproporphyrinogen oxidase (CPO) (EC 1.3.3.3), the antepenultimate enzyme in the heme biosynthetic pathway, catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen IX . Previously, based upon metal analysis and site-directed mutagenesis of purified recombinant enzyme, it has been suggested that CPO contains and requires copper for activity (Kohno, H., Furukawa, T., Tokunaga, R., Taketani, S., and Yoshinaga, T . (1996) Biochim . Biophys . Acta 1292, 156-162) . To examine this putative metal site in human CPO, the cDNA encoding human CPO was engineered into an expression vector with a His6 tag at its amino terminus, and the protein was expressed in Escherichia coli and purified to apparent homogeneity using nickel-nitroliotriacetic acid resin . Activity of the purified protein was monitored by a coupled fluorometric assay that employed purified protoporphyrinogen oxidase to convert protoporphyrinogen to protoporphyrin, thereby allowing the direct fluorescent determination of protoporphyrin IX produced . CPO has an apparent Km of 0.6 microM and an apparent Kcat of 16 min-1 with coproporphyrinogen III as substrate . Metal analysis of the enzyme was carried out via ultraviolet and visible spectroscopy, inductively coupled plasma atomic emission spectroscopy metal analysis, and electron paramagnetic resonance spectroscopy . The data presented demonstrate that human CPO contains no metal center, that it is not stimulated in vitro by iron or copper, and that addition of these metals to cultures expressing the protein has no effect.

Science, 1996 Dec 20, 274(5295), 2082 - 6
Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II; Pfeifer A et al.; Cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinases (cGKs) mediate cellular signaling induced by nitric oxide and cGMP . Mice deficient in the type II cGK were resistant to Escherichia coli STa, an enterotoxin that stimulates cGMP accumulation and intestinal fluid secretion . The cGKII-deficient mice also developed dwarfism that was caused by a severe defect in endochondral ossification at the growth plates . These results indicate that cGKII plays a central role in diverse physiological processes.

Nature, 1996 Dec 19-26, 384(6610), 641 - 3
The CBP co-activator is a histone acetyltransferase; Bannister AJ et al.; The CBP protein acts as a transcriptional adaptor for many different transcription factors by directly contacting DNA-bound activators . One mechanism by which CBP is thought to stimulate transcription is by recruiting the histone acetyltransferase (HAT) P/CAF to the promoter . Here we show that CBP has intrinsic HAT activity . The HAT domain of CBP is adjacent to the binding site for the transcriptional activator E1A . Although E1A displaces P/CAF from CBP, it does not disrupt the CBP-associated HAT activity . Thus E1A carries HAT activity when complexed with CBP . Targeting CBP-associated HAT activity to specific promoters may therefore be a mechanism by which E1A acts as a transcriptional activator.

Nature, 1996 Dec 19-26, 384(6610), 638 - 41
NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain; Huang B et al.; Programmed cell death (apoptosis) mediated by the cytokine receptor Fas is critical for the removal of autoreactive T cells, the mechanism of immune privilege, and for maintenance of immune-system homeostasis . Signalling of programmed cell death involves the self-association of a conserved cytoplasmic region of Fas called the death domain and interaction with another death-domain-containing protein, FADD (also known as MORT1) . Although death domains are found in several proteins, their three-dimensional structure and the manner in which they interact is unknown . Here we describe the solution structure of the Fas death domain, as determined by NMR spectroscopy . The structure consists of six antiparallel, amphipathic alpha-helices arranged in a novel fold . From the structure and from site-directed mutagenesis, we have identified the region of the death domain involved in self-association and binding to the downstream signalling partner FADD.

J Natl Cancer Inst, 1996 Dec 18, 88(24), 1857 - 63
Phase I study of a recombinant adenovirus-mediated gene transfer in lung cancer patients; Tursz T et al.; BACKGROUND: Despite vigorous efforts at curbing tobacco consumption and aggressive combined-modality treatment programs, both the incidence of and the mortality from lung cancer have remained virtually unchanged in the last 10 years . More effective innovative therapies are clearly needed . The direct transfer into tumor cells of tumor suppressor genes or toxic gene products that specifically promote tumor cell death and spare nonmalignant cells is a potentially novel anticancer treatment approach that should be investigated . PURPOSE: On the basis of compelling preclinical data, we initiated a phase I study involving six patients with inoperable lung cancer and an endobronchial lesion accessible by bronchoscopy . Our purpose was to evaluate the feasibility, tolerance, and clinical, biologic, and immunologic effects of the intratumoral administration of a recombinant, replication-deficient adenovirus (rAd.RSV beta-gal), using the Rous sarcoma virus promoter to drive transcription of the Escherichia coli lacZ marker gene that encodes for the bacterial enzyme beta-galactosidase (beta-gal) . METHODS: From June 1994 through April 1995, six patients (five males and one female) were enrolled in the trial . A single dose of recombinant virus suspension containing 10(7) or 10(8) plaque-forming units (PFU) was injected intratumorally into two successive cohorts of three patients . Eligible patients received concomitant chemotherapy . Patients were kept under isolation conditions from 3 days before the injection was given until virus excretion was undetectable . Biopsy specimens of the tumor and surrounding mucosa were collected on the 8th day and at 1, 2, and 3 months after injection . They were analyzed by cell culture, polymerase chain reaction (PCR), and beta-gal expression for the presence of recombinant adenovirus . So that the risk of virus recombination or complementation could be minimized, wildtype adenovirus carriers among the hospital staff (identified by PCR) were excluded from contact with patients who were potentially excreting recombinant virus . RESULTS: beta-gal was expressed in tumor biopsy specimens of three patients (one who received the 10(7) PFU dose level and two who received 10(8)) . Bronchoalveolar lavage specimens collected immediately after injection were positive for recombinant adenovirus when analyzed in culture and by PCR . All biologic fluids were negative for recombinant virus as judged by PCR after day 12, with the exception of bronchoalveolar lavage specimens (positive PCR up to 90 days in two of three patients treated with 10(8) PFU) . The blood samples obtained from the three patients treated with 10(8) PFU showed positive PCR results immediately after virus injection . Patients were kept in isolation for a median of 17 days . The most common toxic effects were moderate bleeding (occurring in two patients) during bronchoscopy and fever (seen in four patients) . Endoscopic and clinically objective antitumor responses were seen in four patients, including one patient who showed a complete response by pathologic evaluation . The median survival for the patients was 12.5 months (range, 3-16+ months) . Throughout the study, hospital staff remained negative for recombinant adenovirus infection . CONCLUSIONS: This ongoing phase I study has demonstrated that a recombinant adenovirus-mediated marker gene, such as rAd.RSV beta-gal, can be safely introduced into humans and that the gene product is expressed by lung tumor cells of the host.

Biochemistry, 1996 Dec 17, 35(50), 16378 - 90
13C NMR spectroscopic and X-ray crystallographic study of the role played by mitochondrial cytochrome b5 heme propionates in the electrostatic binding to cytochrome c; Rodriguez-Maranon MJ et al.; The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme propionate groups in the electrostatic binding between OM cytochrome b5 and horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray crystallography . To achieve these aims, 13C-labeled heme OM cytochrome b5 was expressed in Escherichia coli as previously described {Rivera M., Walker, F.A . (1995) Anal . Biochem . 230, 295-302} . Assignment of the resonances arising from the heme propionate carbons in ferricytochrome b5 was carried out by a combination of one- and two-dimensional NMR experiments . Titrations of {13C}heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b5 . The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b5 participates in the electrostatic stabilization of the complex between OM cytochrome b5 and horse heart cytochrome c . Similar experiments carried out monitoring 13C resonances arising from several other heme substituents demonstrated that the stoichiometry of the complex is 1:1 . A conditional binding constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained for the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry . The X-ray crystal structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that the structures of bovine liver microsomal cytochrome b5 and rat liver OM cytochrome b5 are almost identical when compared at medium resolution . The similarity between the two structures, combined with the findings that only the heme propionate located on the exposed heme edge of OM cytochrome b5 participates in the electrostatic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b5 is almost identical to the one elucidated for microsomal cytochrome b5 . It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b5 and cytochrome c (recently reviewed) {Mauk, A . G . Mauk, M . R., Moore, G . R., & Northrup, S . H . (1995) Bioenerg . Biomembr . 27, 311-330} has indeed biological as well as pedagogical validity.

Biochemistry, 1996 Dec 17, 35(50), 16336 - 45
Oligomers of the cytoplasmic fragment from the Escherichia coli aspartate receptor dissociate through an unfolded transition state; Seeley SK et al.; The kinetic and equilibrium properties of a clustering process were studied as a function of temperature for two point mutants of a 31 kDa fragment derived from the cytoplasmic region of the Escherichia coli aspartate receptor (C-fragment), which were shown previously to have a greater tendency to form clusters relative to the wild-type C-fragment {Long, D . G., & Weis, R . M . (1992) Biochemistry 31, 9904-9911} . The clustering equilibria were different for the two C-fragments . Monomers of a serine-461 to leucine (S461L) mutant C-fragment were in equilibrium with dimers, while monomers of a S325L C-fragment were in equilibrium with trimers . The positive values for delta H degree, delta S degree, and delta Cp degree of dissociation estimated from a van't Hoff analysis, and the differences in the CD spectra of isolated monomers and oligomers, demonstrated that the monomers were less well-folded than the clustered forms . The oligomer dissociation rate exhibited a marked temperature dependence over the range from 4 to 30 degrees C and was remarkably slow at low temperatures; e.g . t1/2 of dimer dissociation for the S461L C-fragment was 85 h at 4 degrees C . The values for delta H degree +2, delta S degree +2, and delta Cp degree +2 derived from the temperature dependence of the dissociation rate were comparable to the corresponding parameters determined in a DSC study of C-fragment denaturation {Wu, J., Long, D . G., & Weis, R . M . (1995) Biochemistry 34, 3056-3065}, which indicated that the transition state resembled thermally denatured C-fragment . Octyl glucoside accelerated the dissociation rate by 3-5-fold presumably by lowering the barrier to dissociation . This acceleration and the positive value of delta Cp degree +2 were interpreted as evidence for an increase in solvent accessible hydrophobic groups in the transition state . The molecular basis for the slow rate of dissociation is proposed to result from the conversion of intermolecular coiled coils in the oligomers to an intramolecular coiled coil in the monomer.

Biochemistry, 1996 Dec 17, 35(50), 16270 - 81
An essential role for water in an enzyme reaction mechanism: the crystal structure of the thymidylate synthase mutant E58Q; Sage CR et al.; A water-mediated hydrogen bond network coordinated by glutamate 60(58) appears to play an important role in the thymidylate synthase (TS) reaction mechanism . We have addressed the role of glutamate 60(58) in the TS reaction by cocrystalizing the Escherichia coli TS mutant E60(58)Q with dUMP and the cofactor analog CB3717 and have determined the X-ray crystal structure to 2.5 A resolution with a final R factor of 15.2% (Rfree = 24.0%) . Using difference Fourier analysis, we analyzed directly the changes that occur between wild-type and mutant structures . The structure of the mutant enzyme suggests that E60(58) is not required to properly position the ligands in the active site and that the coordinated hydrogen bond network has been disrupted in the mutant, providing an atomic resolution explanation for the impairment of the TS reaction by the E60(58)Q mutant and confirming the proposal that E60(58) coordinates this conserved hydrogen bond network . The structure also provides insight into the role of specific waters in the active site which have been suggested to be important in the TS reaction . Finally, the structure shows a unique conformation for the cofactor analog, CB3717, which has implications for structure-based drug design and sheds light on the controversy surrounding the previously observed enzymatic nonidentity between the chemically identical monomers of the TS dimer.

Biochemistry, 1996 Dec 17, 35(50), 16241 - 6
The "ferrous-oxy" intermediate in the reaction of dioxygen with fully reduced cytochromes aa3 and bo3; Verkhovsky MI et al.; We have studied the reactions with oxygen of two terminal oxidases, cytochrome c oxidase from mitochondria and cytochrome bo3 from Escherichia coli . In each case, flow-flash methodology was used to react the fully reduced enzyme with a high concentration of oxygen (1 mM), and absorbance changes were recorded for a number of separate wavelengths in the alpha-band (visible) region . In both enzymes, an early kinetic phase could be resolved, corresponding to the binding of oxygen to produce a ferrous-oxy heme intermediate . In cytochrome c oxidase, this intermediate appears with a time constant of 10 microseconds; its spectrum has a peak at 595 nm (relative to the unliganded reduced enzyme) . In cytochrome bo3, the ferrous-oxy intermediate, resolved by optical absorbance spectroscopy for the first time, appears with a time constant of 11 microseconds and has a broad maximum near 570 nm.

Biochemistry, 1996 Dec 17, 35(50), 16222 - 9
Site-directed mutagenesis and spectroscopic characterization of human ferrochelatase: identification of residues coordinating the {2Fe-2S} cluster; Crouse BR et al.; The five cysteines closest to the carboxyl terminus of human ferrochelatase have been individually mutated to serine, histidine, or aspartate residues in an attempt to identify the protein ligands to the {2Fe-2S} cluster . Mutations of cysteines at positions 403, 406, and 411 (C403D, C403H, C406D, C406H, C406S, C411H, and C411S mutants) all resulted in inactive enzyme that failed to assemble the {2Fe-2S} cluster as judged by whole-cell EPR studies . In contrast, mutation of the cysteines at positions 360 and 395 to serines (C360S and C395S mutants) did not affect the enzymatic activity, and the resulting enzyme assembled a {2Fe-2S} cluster that was spectroscopically indistinguishable from the wild-type enzyme . The results indicate that three of the conserved cysteines in the 30-residue C-terminal extension of mammalian ferrochelatase are involved in ligating the {2Fe-2S} cluster . Resonance Raman and variable-temperature magnetic circular dichroism studies of heme-free preparations of human ferrochelatase are reported, and the spectra are best interpreted in terms of one non-cysteinyl, oxygenic ligand for the {2Fe-2S} cluster . Such anomalous coordination could account for the cluster lability compared to similar clusters with complete cysteinyl ligation and hence may be intrinsic to the proposed regulatory role for this cluster in mammalian ferrochelatases.

Biochemistry, 1996 Dec 17, 35(50), 16180 - 5
Substrate specificity and identification of functional groups of homoserine kinase from Escherichia coli; Huo X et al.; Homoserine kinase, an enzyme in the aspartate pathway of amino acid biosynthesis in Escherichia coli, catalyzes the conversion of L-homoserine to L-homoserine phosphate . This enzyme has been found to have broad substrate specificity, including the phosphorylation of L-homoserine analogs where the carboxyl functional group at the alpha-position has been replaced by an ester or by a hydroxymethyl group . Previous pH profile studies {Huo . X., & Viola, R . E . (1996) Arch . Biochem . Biophys . 330, 373-379} and chemical modification studies have suggested the involvement of histidinyl, lysyl, and argininyl residues in the catalytic activity of the enzyme . With the assistance of sequence alignments, several potential amino acids have been targeted for examination . Site-directed mutagenesis studies have confirmed a role for arginine-234 in the binding of the carboxyl group of L-homoserine, and the involvement of two histidine at the homoserine binding site . Mutations at these sites have led to the decoupling of the kinase activity from an inherent ATPase activity in the enzyme, and suggest the presence of independent domains for the binding of each substrate in homoserine kinase.

Biochemistry, 1996 Dec 17, 35(50), 16174 - 9
Chemical rescue by guanidine derivatives of an arginine-substituted site-directed mutant of Escherichia coli ornithine transcarbamylase; Rynkiewicz MJ et al.; Escherichia coli ornithine transcarbamylase (OTCase) catalyzes the production of L-citrulline and phosphate from carbamyl phosphate and L-ornithine in L-arginine biosynthesis . We show that exogenous guanidines can restore activity to (chemically rescue) a catalytically-impaired site-directed mutant OTCase, R57G, in which glycine replaces an an active site arginine . The best rescue agent is guanidine hydrochloride, which enhances the rate of the mutant 2000-fold . The turnover number for the guanidine-rescued R57G mutant is 10% that of wild-type . The addition of guanidine to the R57G mutant has little effect on KMCP values, and the rescue effect is therefore attributed principally to an increase in kcat . Other compounds were screened as potential rescue agents, but rate enhancement is highly selective for guanidines . Not all guanidines show large increases in kcat . For a comparative series that includes guanidine and alkylguanidines, substituent size is inversely related to kcat . Bronsted analysis of guanidines with varying pKa values indicates that a partial positive charge is implicated in rescue, consistent with the proposed role of arginine 57 in catalysis . In UV difference and 31P-NMR spectra, carbamyl phosphate-induced effects associated with wild-type OTCase are observed in the R57G mutant only in the presence of guanidine . The kinetic mechanism of the mutant is random in the presence or absence of guanidine, in contrast to the sequential ordered mechanism of the wild-type enzyme . Thus, chemical rescue of R57G by guanidine hydrochloride restores many but not all wild-type properties to the mutant enzyme.

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