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 p