Escherichia coli

Infection, 1993 May-Jun, 21(3), 179 - 86
Cross-reactivity of monoclonal antibodies and sera directed against lipid A and lipopolysaccharides; Kuhn HM; The cross-reactive capacity of monoclonal and polyclonal lipid A antibodies was tested with rough and smooth lipopolysaccharides (LPS) . The antibodies represented different specificities recognizing epitopes in the hydrophilic lipid A backbone . In none of the various assay systems applied did the antibodies react with complete rough or smooth-form LPS . Cross-reactions, in general, were only detected with the most rudimentary rough LPS tested, i.e . Re-LPS . A variety of reactivities with other LPS was shown not to be related to lipid A antibodies; such reactivities were present in rabbit sera as well as in crude ascites . These results underline the need for careful checks on the origin of reactivities observed . In addition, rabbit antisera raised with R- and S-LPS were screened for lipid A reactivity using synthetic lipid A and partial structures as antigens . No cross-reactivity of LPS antibodies with lipid A was detected in these sera.

Biol Pharm Bull, 1993 May, 16(5), 437 - 43
Proton NMR study on a histone-like protein, HU alpha, from Escherichia coli and its complex with oligo DNAs; Shindo H et al.; It was confirmed that the flexible arm region of HU alpha forms an antiparallel beta-sheet and that all of the residues of phenylalanines, together with some of leucines and/or valines, form a hydrophobic core within the dimer of HU alpha . HU alpha protein alone is thermally labile and melts at 38 degrees C, but it becomes remarkably stabilized and melts at 59 degrees C in the presence of DNA . Several resonances from both HU alpha and DNA perturbed by their complex formation, notably those of His C-2 and C-4 protons, downfield shifted C alpha protons in the antiparallel beta-sheet, as well as Arg C delta and Lys C epsilon protons . The results indicated that a beta-sheet region of HU alpha binds to DNA, and also showed that rapid equilibrium occurs on the NMR time scale between bound and unbound states of HU alpha . A few intermolecular nuclear Overhauser effects (NOEs) were also observed between the protein and H1' protons of DNA in the complex, suggesting that HU alpha binds primarily to the minor groove of DNA.

Vet Microbiol, 1993 May, 35(1-2), 179 - 85
Species-specific recombinant DNA probes for Mycoplasma meleagridis; Zhao S et al.; Two recombinant DNA probes (pMM-2 and pMM-13) were isolated from a Mycoplasma meleagridis strain 17529 genomic library prepared in plasmid pUC8, and Escherichia coli strain JM83 . In dot blot assays, 32P-labeled pMM-13 with a DNA insert of 3.5 kbp, hybridized with 18 isolates of M . meleagridis but not with 16 other known species of avian mycoplasmas . Except for weaker signals on hybridization with the M . meleagridis cultures, pMM-2 with an DNA insert of 0.85 kbp, showed a similar reaction pattern . The minimal concentration of M . meleagridis strain 17529 chromosomal DNA that pMM-13 and pMM-2 detected were 1 and 8 ng, respectively . Neither probe hybridized with chromosomal DNA of M . gallisepticum strain S6, M . synoviae strain WVU-1853, or M . iowae strain I-695 at concentration of 256 ng.

J Surg Res, 1993 May, 54(5), 474 - 9
Effect of anti-lipid A monoclonal antibody (E5) on microcirculatory function during lipopolysaccharide shock; Schwartz RW et al.; Early septic shock is characterized by fever, increased cardiac output, decreased systemic vascular resistance, and dilation of higher-order arterioles in peripheral tissues, such as skeletal muscle . We used a rat model of low-dose lipopolysaccharide (LPS) "septic" shock to investigate the potential benefit of an anti-lipid A monoclonal antibody preparation (E5) on macro- and microcirculatory function . Twenty-five male Sprague-Dawley rats were anesthetized and instrumented for measurement of arterial pressure (AP), heart rate (HR), and cardiac output (CO) . The left cremaster muscle of each rat was prepared for in vivo video microscopic examination of changes in third-order arteriolar (A3) diameter and erythrocyte velocity . Rats were randomly assigned to two groups: Group I (n = 13) received E5 vehicle and 200 micrograms/kg Escherichia coli LPS; Group II (n = 12) received 2 mg/kg E5 iv prior to LPS administration . All variables were recorded at 15-min intervals for 30 min prior to and 150 min following LPS . Microcirculatory recordings were restricted to those rats where arteriolar diameters were 20-40 microns and vessels displayed obvious vasomotion (n = 7/group) . Infusion of LPS caused no significant change in AP, an increase in CO by 105 min, an increase in HR by 75 min, an increase in diameter by 75 min, and a decrease in velocity by 165 min (P < 0.01) . Pretreatment with E5 inhibited the A3 vasodilation but did not affect the macrocirculatory changes . These data suggest a potential therapeutic role for E5 in ameliorating LPS-induced changes in skeletal muscle microcirculation.

J Surg Res, 1993 May, 54(5), 418 - 25
In vivo hepatocyte transduction with retrovirus during in-flow occlusion; Rettinger SD et al.; Gene therapy research would be facilitated by a technically simple procedure for transducing hepatocytes in vivo . Previously reported methods have employed partial hepatectomy followed 24 hr later by asanguineous perfusion of the regenerating liver with retrovirus . We have developed a simpler method of in vivo transduction in which we deliver an intraportal bolus of retrovirus to the regenerating rodent liver during a brief period of hepatic in-flow occlusion . On Day 0, adult male Sprague-Dawley rats (N = 19) underwent 70% hepatectomy to induce hepatocyte replication . On Day 1, retroviral supernatant was harvested from an amphotropic retroviral packaging cell line that packaged an LNL6-derived vector containing the cytomegalovirus promoter driving expression of the Escherichia coli beta-galactosidase (beta gal) gene . Twenty-four hours after partial hepatectomy, experimental rats (N = 17) received 6 x 10(5) colony-forming units of retrovirus by intraportal injection during a 3-min occlusion of the hepatic artery and portal vein . Control rats (N = 2) received intraportal medium (without retrovirus), also during in-flow occlusion . The procedure required 20-25 min, and the survival rate was 84% . Cryostat sections were prepared from liver biopsies obtained on Post-transduction Days 8 and 15 and stained with 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside to detect beta gal expression . Light microscopic examination of Day 8 sections from surviving experimental rats (N = 14) revealed 0.10-1.00% blue (i.e., transduced) hepatocytes per low power field, while sections from control rats (N = 2) exhibited no blue cells . Day 15 sections from experimental rats revealed a somewhat lower frequency of hepatocytes expressing beta gal.(ABSTRACT TRUNCATED AT 250 WORDS)

J Surg Res, 1993 May, 54(5), 393 - 400
Endotoxin increases hepatic glutamine transport activity; Inoue Y et al.; Glutamine uptake by the liver is accelerated during endotoxemia, but little is known regarding the influence of sepsis on the plasma membrane transport systems catalyzing hepatic glutamine uptake . We hypothesized that this augmented uptake was due to an increase in hepatocyte plasma membrane transport activity . We investigated the activities of the Na(+)-dependent transport System N (transports glutamine into the hepatocyte) and the Na(+)-independent System n (transports glutamine out of the cell) in hepatocyte plasma membrane vesicles (HPMVs) prepared from livers of rats treated with Escherichia coli endotoxin (LPS) in vivo . HPMVs were prepared by differential centrifugation and the transport of {3H}glutamine was assayed by a rapid mixing/filtration technique in the presence and absence of sodium . Vesicle integrity and functionality were confirmed by enzyme marker enrichments and classic "overshoots" in the presence of sodium . Carrier-mediated Na(+)-independent glutamine transport activity was not altered by LPS administration . In contrast, endotoxemia resulted in a time- and dose-dependent two- to threefold increase in Na(+)-dependent glutamine transport activity in HPMVs secondary to an increase in the transport Vmax, consistent with the appearance of increased numbers of corresponding transporter proteins in the hepatocyte plasma membrane . The Km (affinity for glutamine) of the System N transporter was not affected by LPS treatment . Maximal increases in transport were observed 4 hr after exposure to endotoxin . System N transport activity returned to basal levels by 12 hr . This increase in transport activity represents an important mechanism regulating the accelerated hepatic glutamine uptake that occurs during severe infection.

Plasmid, 1993 May, 29(3), 236 - 40
Overproduction and purification of the colicin E1 immunity protein; Shirabe K et al.; Colicin E1 immunity protein encoded by the plasmid colicin E1 was overproduced in Escherichia coli from an expression plasmid which was constructed by placing the immunity protein-encoding sequence downstream of the tac promoter and by properly positioning the ribosome binding site on a run-away replication vector . The immunity protein was solubilized with 0.5% Brij 58 from the membrane fraction and purified to homogeneity by a simple batch procedure with hydroxyapatite gel and reverse-phase chromatography . A 15-residue NH2-terminal amino acid sequence was determined to be the same as that deduced from the DNA sequence . The effect of the purified immunity protein on membranes was tested in vitro using solute-loaded liposomes . The immunity protein added to the liposomes showed a small but significant channel or lytic activity that is an indicator of its hydrophobic nature.

Plasmid, 1993 May, 29(3), 222 - 32
A natural A/T-rich sequence from the yeast FBP1 gene exists as a cruciform in Escherichia coli cells; del Olmo M et al.; Palindromic or semipalindromic sequences can adopt cruciform structures in DNA in vitro . It has been demonstrated in some cases that A/T-rich cruciforms exist also in vivo in Escherichia coli . The biological function of those structures is not understood although putative cruciforms have been found in interesting locations on replication origins, operators, or transcriptional termination regions . Here we show by means of the use of structure-dependent nucleases that the 3' end of the yeast FBP1 gene contains a stable cruciform both in vitro and in E . coli cells and that in both cases, its extrusion depends on the DNA supercoiling state.

Mol Microbiol, 1993 May, 8(5), 903 - 14
Sequence, genetic, and lacZ fusion analyses of a nifR3-ntrB-ntrC operon in Rhodobacter capsulatus; Foster-Hartnett D et al.; Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen . Regulatory genes required to sense and relay the nitrogen status of the cell are glnB, ntrB (nifR2), and ntrC (nifR1) . R . capsulatus nifA1 and nifA2 require ntrC for activation when fixed nitrogen is limiting . The polypeptides encoded by nifA1 and nifA2 along with the alternate sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen . In this study we report the sequence and genetic analysis of the previously identified nifR3-ntrB-ntrC regulatory locus . nifR3 is predicted to encode a 324-amino-acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli regulatory gene, fis . Analysis of ntrC-lacZ fusions and complementation data indicate that nifR3 ntrBC constitute a single operon . nifR3-lacZ fusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product . Tn5, a kanamycin-resistance cassette, and miniMu insertions in nifR3 are polar on ntrBC (required for nif transcription) . This gene organization suggests that the nifR3 gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid . In addition, a R . capsulatus strain with a 22-nucleotide insert in the chromosomal nifR3 gene was constructed . This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for ntrC-dependent gene activation in R . capsulatus.

Mol Microbiol, 1993 May, 8(5), 875 - 89
Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation; Dersch P et al.; Mutations in the structural gene (hns) for the Escherichia coli nucleoid-associated DNA-binding protein H-NS cause highly pleiotropic effects on gene expression, site-specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA . We have investigated the regulation of hns expression and found that hns transcription is subjected to stationary phase induction and negative autoregulation . A set of hns-lacZ protein and operon fusions was constructed in vitro and integrated in single copy into the attB site of the bacterial genome . Quantification of beta-galactosidase activity along the bacterial growth curve showed that hns expression increases approximately 10-fold in stationary phase compared with exponentially growing cells . Immunological detection of the H-NS protein in growing and stationary phase cells supported the genetic data and showed that H-NS synthesis varies with growth phase . In addition, primer extension experiments demonstrated that the amount of hns mRNA is elevated in stationary phase cultures and that hns transcription is directed by a unique promoter functioning in both log and stationary phase . Disruption of the hns gene by an insertion mutation led to the derepression (approximately fourfold) of the expression of an hns-lacZ operon fusion integrated at the attB site, showing that hns transcription is subjected to negative regulation by its own gene product . Autoregulation of hns expression is particularly pronounced in log phase . Both stationary phase control and autoregulation of hns transcription are associated with a 130 bp fragment that contains the hns promoter . In order to study the interaction of H-NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H-NS . DNA gel retardation assays showed that the H-NS protein can preferentially interact with a restriction fragment carrying the hns promoter . This restriction fragment showed features of curved DNA as judged by two-dimensional polyacrylamide gel electrophoresis performed at 4 degrees C and 60 degrees C.

Mol Gen Mikrobiol Virusol, 1993 May-Jun, (3), 26 - 9
A method for isolation of sequences missing in one of two related genomes; Lisitsyn NA et al.; We describe a novel technique for isolation of sequences that are present in one genome (tracer), but absent in another (driver) . Tracer DNA, cleaved with Sau 3A and capped with a single stranded PCR adapter, is allowed to hybridize with an excess of sheared biotinylated driver; biotinylated DNA and its hybrids with the tracer are removed by phenol/chloroform extraction after incubation with streptavidin . After several rounds of subtraction the ends of self-annealed tracer molecules from the nonextractable fraction are filled-in with Tag polymerase and amplified, using the single stranded PCR adapter as a primer . The method has been applied to purification of fragments from a 2.9 kb plasmid added to E . coli DNA at equimolar quantity . Plasmid derived fragments (250-1000 bp), initially comprising 1/1400th part of tracer DNA, were purified to homogeneity after two rounds of subtraction followed by PCR.

J Photochem Photobiol B, 1993 May, 18(2-3), 205 - 10
Inhibition of dimer excision in repeatedly UV-irradiated Escherichia coli: its requirement for RecA protein and de novo protein synthesis; Fridrichova I et al.; In UV-irradiated Escherichia coli dimer excision was found to be inhibited by predamage (M . Sedliakova, F . Masek and J . Brozmanova, FEBS Lett., 23 (1972) 325-326) or overproduction of RecA protein, which suggests that the coating of the dimers by this protein may make them inaccessible to the excision nuclease (M . Sedliakova, K . Kleibl and F . Masek, Mutat . Res., 191 (1987) 13-16) . We measured the levels of RecA protein and dimer excision in cells irradiated with (i) a single dose of 50 J m-2, (ii) two separate doses of 30 and 50 J m-2, post-incubated with chloramphenicol; (iii) two separate doses of 30 and 50 J m-2, post-incubated without chloramphenicol . Dimer excision was complete in the first two cases, but in the latter it was inhibited by 40% . At the time of active dimer excision, there were marked differences in RecA protein content between the cells irradiated with a single dose and cells irradiated with two separate doses (both post-incubated without chloramphenicol), which might account for the differences in dimer excision . However, relatively small differences in RecA protein content were found in cells irradiated with two doses and post-incubated with or without chloramphenicol, which could therefore not account for the differences in dimer excision . The data suggest that the inhibition of dimer excision involves some short-lived component(s) other than RecA protein.

J Photochem Photobiol B, 1993 May, 18(2-3), 191 - 6
Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 632.8 nm light; Bertoloni G et al.; Irradiation of Escherichia coli cells with either coherent or non-coherent 632.8 nm light (4 J cm-2) causes a transient acceleration of cell proliferation, which is maximal about 60 min after the end of the phototreatment . The stimulatory effect is dose dependent and is especially evident in the case of defective E . coli strains which are in the logarithmic phase of growth, while it becomes less important when cells are exposed to non-coherent 600-700 nm light . Stimulated cells exhibit biochemical and morphological changes, such as an intensified synthesis of cytoplasmic membrane proteins, increased cell volume and ribosomal content, which are suggestive of an enhanced cell metabolism.

J Biochem (Tokyo), 1993 May, 113(5), 568 - 72
Properties of DNA-binding of HU heterotypic and homotypic dimers from Escherichia coli; Tanaka H et al.; The interactions of three forms of HU dimer from Escherichia coli with DNA were compared . The complexes formed between HU and short DNA fragments (35 to 132 bp), uncurved oligodeoxyribonucleotide (oligo) with and without 5-bp deoxyriboadenosine (dA) stretches and curved oligo with 5-bp dA stretches, were analyzed by the gel retardation technique . The binding of HU homodimers to the DNA was less efficient than that of HU heterodimer, and the HU-1 homodimer had lower DNA-binding capacity than the HU-2 homodimer . The equilibrium dissociation constant (KD) for DNA of the HU-1 homodimer was 3 times that of the HU heterodimer . The HU dimers had two- to fourfold higher affinity for DNA duplexes containing 5-bp dA stretches, particularly for curved DNA . The binding of HU heterodimer to the DNA was inhibited more by the curved DNA competitor than the uncurved DNA competitor without dA stretches.

Biotech Histochem, 1993 May, 68(3), 169 - 74
In situ hybridization at the electron microscopic level using a bromodeoxyuridine labeled DNA probe; Yao CH et al.; A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level . A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells . After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM), and others were embedded in Quetol for electron microscopy (EM) . The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid . In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum . Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level . Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here . This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.

Res Vet Sci, 1993 May, 54(3), 290 - 8
Experimental infection of neonatal pigs with CNF toxin-producing strains of Escherichia coli; Wray C et al.; To examine and compare the pathogenicity of cytotoxic necrotising factor (CNF)-producing Escherichia coli, two litters of piglets were infected orally with 10(10) E coli O88 or 10(10) E coli O32 strains . Of the six piglets infected with E coli O88, two died within 24 hours and three developed blood-stained diarrhoea . The other piglets were killed one, five, six and eight days after infection, when bacterial cultures indicated an overwhelming bacteraemic infection with E coli O88 in the early stages followed by clearance through the large intestine . The pathological changes consisted of an early enteritis, progressing to enterocolitis and a bacteraemic spread to the lungs . The histopathological changes were characteristic of toxaemic effects in brain, heart, liver and kidney, and characterised by congestion, oedema and exudation . Infection with E coli O32 produced a milder but similar enterocolitis, also with bacterial colonisation in the lungs . The histopathological findings again reflected a toxaemia . The enteritis was more persistent after E coli O32 infection and the strain persisted in large numbers in the intestine . No evidence of bacterial adherence to the intestinal mucosa was found with either strain . Enteroinvasion was only evident in one E coli O88-infected piglet, but the consistent occurrence of interstitial pneumonia showed the predilection of these organisms for the lung . The results confirm the toxigenic properties of CNF+ E coli and suggest an important role for this organism in enteric infection of young pigs.

Photochem Photobiol, 1993 May, 57(5), 895 - 904
Photochemistry, photophysics, and mechanism of pyrimidine dimer repair by DNA photolyase; Kim ST et al.; DNA photolyases photorepair pyrimidine dimers (Pyr < > Pyr) in DNA as well as RNA and thus reverse the harmful effects of UV-A (320-400 nm) and UV-B (280-320 nm) radiations . Photolyases from various organisms have been found to contain two noncovalently bound cofactors; one is a fully reduced flavin adenine dinucleotide (FADH-) and the other, commonly known as second chromophore, is either methenyltetrahydrofolate (MTHF) or 8-hydroxydeazaflavin (8-HDF) . The second chromophore in photolyase is a light-harvesting molecule that absorbs mostly in the near-UV and visible wavelengths (300-500 nm) with its high extinction coefficient . The second chromophore then transfers its excitation energy to the FADH- . Subsequently, the photoexcited FADH- transfers an electron to the Pyr < > Pyr generating a dimer radical anion (Pyr < > Pyr.-) and a neutral flavin radical (FADH.) . The Pyr < > Pyr.- is very unstable and undergoes spontaneous splitting followed by a back electron transfer to the FADH. . In addition to the main catalytic cofactor FADH-, a Trp (Trp277 in Escherichia coli) in apophotolyase, independent of other chromophores, also functions as a sensitizer to repair Pyr < > Pyr by direct electron transfer.

J Gen Microbiol, 1993 May, 139 ( Pt 5), 1083 - 92
Expression of a plasmid gene of Chlamydia trachomatis encoding a novel 28 kDa antigen; Comanducci M et al.; Plasmid pCT is present in essentially all isolates of Chlamydia trachomatis and may encode factors important for survival in the natural environment . However, no pCT-associated phenotype has been described so far . With the purpose of investigating the possibility of a role of pCT in C . trachomatis pathogenicity we examined the expression of an ORF (ORF3), potentially encoding a 28 kDa polypeptide (pgp3) . Analysis of RNA extracted from chlamydia-infected Vero cells detected ORF3-specific transcripts, from 20 h post-infection onwards, mainly as discrete RNA species of 1390 nucleotides comprising the downstream ORF4 sequence . ORF3 DNA was cloned and expressed in Escherichia coli as a 39 kDa fusion protein (MS2/pgp3) . Antibodies raised against purified MS2/pgp3, specifically recognized a 28 kDa protein on Western blots of protein from purified chlamydial elementary bodies (EBs) . The same antibodies detected chlamydial inclusions in methanol-fixed infected cells by immunofluorescence . Western blot analysis of EBs extracted with 2% Sarkosyl, showed that a large proportion of the 28 kDa antigen is associated with the detergent-insoluble ('membrane') fraction . Antibodies recognizing pgp3 epitopes were detected in sera from patients with chlamydial infections, but not in sero-negative control sera . The finding support the hypothesis that pCT may provide a function related to chlamydial cell physiology.

J Appl Physiol, 1993 May, 74(5), 2155 - 60
Attenuation of acute lung injury and oxygen radical production by the 21-aminosteroid, U-78518F; Tanigaki T et al.; Oxygen radicals play an important role in the mechanism of acute lung injury . The 21-aminosteroid lazaroid, U-78518F, is a potent antioxidant . We examined the effect of intravenous U-78518F on acute lung injury in septic guinea pigs over 8 h . The experimental groups (n = 6) were 1) saline control, 2) Escherichia coli (2 x 10(9)/kg i.v.), 3) pretreatment (U-78518F 5 mg/kg bolus + 1 mg.kg-1 x h-1, 15 min before E . coli injection), and 4) posttreatment (U-78518F 30 min after E . coli injection) . We measured wet-to-dry weight ratio (W/D) as an index of pulmonary edema and concentration ratios of 125I-labeled albumin in lung tissue and bronchoalveolar lavage fluid compared with plasma (L/P and BAL/P, respectively) as indexes of lung protein fluxes . In septic guinea pigs, pretreatment with U-78518F attenuated W/D, L/P, and BAL/P and posttreatment attenuated W/D and BAL/P (P < 0.05 for each) . Furthermore, we studied the effect of U-78518F on human neutrophil oxygen radical production (ORP) by using flow cytometry to assess intracellular ORP and lucigenin-dependent chemiluminescence to assess extracellular ORP . Neutrophils (5 x 10(5) were stimulated with 0.5 micrograms/ml of phorbol myristate acetate . With flow cytometry, we measured intracellular ORP, cross-sectional cell area, and degranulation in neutrophils . U-78518F (minimum concn 1.0 microM) decreased intracellular ORP (n = 4; P < 0.05) when the dihydrorhodamine 123 assay was used . U-78518F (minimum concn 1.0 microM) inhibited phorbol myristate acetate-induced neutrophil chemiluminescence (n = 4; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Genetika, 1993 May, 29(5), 748 - 59
{Increased frequency of unequal crossing over in recBC mutant cells in conjugational crosses in Escherichia coli K-12}; Sukhodolets VV; The formation of heterozygous tandem duplications was studied in conjugational crosses of Escherichia coli using mutations for deo-operon genes . The frequency of tandem duplications in recF::Tn3 and recBC sbcB mutants was not substantially changed from that of the wild type strain . In contrast, the frequency of duplications was increased about ten-fold when using recBC (sbcB+) mutants as recipients . This result can be explained by a participation of the RecBCD protein in the pairing of chromosomes during recombination . We also found that HfrH thyA recBC (sbcB+) bacteria grow very poorly on rich medium and that good growth is restored by conversion from the Hfr to the F+ state, as well as by mutations suppressing the RecBC phenotype; such F+ strains retain sensitivity to UV light and to mitomycin C, which are characteristic of the recBC phenotype.

Toxicon, 1993 May, 31(5), 615 - 26
Immunization against Echis ocellatus (carpet viper) venom using liposomes incorporating immunostimulants: role of lipopolysaccharide in conferring protection in a mouse model; Laing GD et al.; Varying doses of whole West African Echis ocellatus venom were incorporated, with or without immunostimulants, into membrane-stabilized reverse phase evaporation (REV) liposomes . Preparations were given either subcutaneously (s.c.) or intravenously (i.v.) to mice and the immune responses compared . Addition of lipopolysaccharide (LPS) significantly increased the venom antibody response . Lipid A produced a less pronounced and less sustained effect and the addition of muramyl dipeptide analogues made no significant contribution to the antibody response . The protective ability of circulating venom antibodies was assessed by challenging the immunized mice with a minimum lethal dose of whole venom after 175 days . A dose of 250 micrograms E . ocellatus venom + 300 micrograms LPS in REVs injected s.c . conferred the highest protection against lethal venom effects . Orally administered venom/liposomes incorporated with the mucosal adjuvant avridine primed the antibody response and produced a classic secondary response following a sublethal boost of whole venom . Single injections of venom or venom fraction/liposome preparations which produce a high and sustained immune response have potential in commercial antivenom production and in active immunization of man in areas of high snakebite incidence and mortality.

Nippon Geka Gakkai Zasshi, 1993 May, 94(5), 466 - 74
{Experimental studies on the relation of platelet activating factor (PAF) to high mortality in sublethal endotoxemia after massive hepatectomy and effects of its antagonist}; Onuki Y et al.; The effects of platelet activating factor antagonist (PAF-A) for sublethal endotoxemia after 70% or 84% hepatectomy in rats were studied . Endotoxin of 1mg/kg b.w . was intravenously given once on the first, third or fifth day hepatectomy . One week survival rates of 84%-hepatectomized rats with endotoxin injection on the third postoperative day was the lowest at 20%, and the lowest survival rate improved to 80% with PAF-A administration . Pathophysiology of PAF-A treated and nontreated rats with endotoxemia was investigated on the third day after 84% hepatectomy . There was no significant difference in SGOT, SGPT and blood glucose level between the two groups . PAF-A nontreated rats' prothrombin time was prolonged (p < 0.05) and serum fibrinogen level significantly decreased (p < 0.01), compared to treated rats . Platelet count distinctly decreased in both groups . Fibrin was pathologically proved in the glomeruli of the kidney in both groups . However, single cell necrosis of hepatocytes was significantly reduced by PAF-A administration . Rats without PAF-A had bloody ascites in 75% of cases and showed shock and pathologically pulmonary congestion . PAF may be related to DIC, shock, bloody ascites and high mortality rate in sublethal endotoxemia after massive hepatectomy . PAF-A was significantly effective for endotoxemia after hepatectomy.

Mol Microbiol, 1993 May, 8(4), 697 - 706
Phosphate starvation and low temperature as well as ultraviolet irradiation transcriptionally induce the Escherichia coli LexA-controlled gene sfiA; Dri AM et al.; The LexA repressor controls the expression of several SOS genes, such as lexA, recA and sfiA, which are induced by DNA damage . Induction results from the activation of the RecA protein that favours the cleavage and thus the inactivation of LexA . It has been shown that the activation of RecA results from its binding to damaged DNA . It is therefore believed that in growing bacteria, in the absence of any DNA-damaging treatment, the intracellular level of LexA remains stable at a high basal level and, hence, SOS genes are expressed at relatively low basal levels . In contrast, we show here that the intracellular level of LexA and the rate of transcription of the sfiA gene may vary markedly throughout the growth cycle of wild-type Escherichia coli . We provide evidence that such changes result from two superimposed processes: proteolytic cleavage of LexA upon dilution of stationary phase bacteria, and increase in strength of the promoters of the lexA and sfiA genes when bacteria approach the stationary phase . We show that a signal which strongly increases the strength of the sfiA gene promoter is starvation for phosphate . Such induction was not significantly affected by mutations either in phoB (encoding the transcriptional regulator for the phosphate regulon) or rpoS (encoding a putative stationary phase-specific sigma factor) . However, sfiA induction by phosphate starvation appeared to be markedly inhibited by the presence of the osmZ205 mutation which alters the histone-like protein H-NS, suggesting that changes in the DNA structure may play a role in signal transduction during phosphate starvation . As previously shown for several processes which are controlled by H-NS, induction of sfiA was modulated by growth temperature.

J Am Soc Nephrol, 1993 May, 3(11), 1783 - 91
Cytokine formation within rat glomeruli during experimental endotoxemia; Fouqueray B et al.; Increasing evidence supports a role of cytokines, tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and IL-6 in the development of endotoxin-induced acute renal failure . Several activities of these cytokines require a local rather than a systemic production and function . Thus, this study investigates the chronology of cytokine expression in glomeruli isolated from normal rats or rats given iv lipopolysaccharide injections . Detectable levels of TNF alpha could be found in glomeruli isolated from normal rats as assessed by L-929 fibroblast lytic assay and ELISA . Glomeruli isolated from rats given lipopolysaccharide transiently released increased amounts of TNF alpha in relation to the dose of lipopolysaccharide (10 to 500 micrograms/kg body wt) and the lag period between lipopolysaccharide injection and glomerular isolation (20 to 120 min) . TNF alpha was released in similar amounts by glomeruli from normal rats that were exposed in vitro to lipopolysaccharide challenge (0.01 to 10 micrograms/mL), indicating that lipopolysaccharide had direct effects on the release of TNF alpha from glomerular cells . These cells consisted mainly of resident cells because reduction of glomerular infiltration by bone marrow-derived cells after the irradiation of normal rats did not affect TNF alpha release . Glomerular IL-1 and IL-6 production was evaluated by specific bioassays under identical conditions . No IL-1 activity could be detected in the medium or within the glomerular cells at any time within 120 min after lipopolysaccharide injection . By contrast, glomerular IL-6 production was induced after lipopolysaccharide challenge both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

J Am Soc Nephrol, 1993 May, 3(11), 1753 - 64
Dysfunction of glomerular fibrinolysis in experimental antiglomerular basement membrane antibody glomerulonephritis; Feng L et al.; Glomerular plasminogen activator inhibitor-1 (PAI-1) steady-state mRNA and bioactivity were increased after the induction of an augmented form of antiglomerular basement membrane (GBM) antibody glomerulonephritis . PAI-1 mRNA expression was noted at 6 h, peaking at 1 day, and although falling thereafter, remained higher than that of the control group through Day 17 . PAI-1 mRNA expression correlated with glomerular PAI-1 bioactivity as determined by a functional tissue type plasminogen activator (t-PA) binding assay . Glomerular PAI-1 bioactivity, not detected in controls, increased to 1.4 +/- 0.3 ng/mg of glomerular lysate at 6 h and then decreased to 0.7 +/- 0.1 ng/mg of glomerular lysate by Day 6 . The mRNA of the plasminogen activators (urokinase plasminogen activator), t-PA) either remained unchanged or declined through Day 1, with a slight increase in t-PA mRNA at Day 6 . Interleukin-1 beta mRNA expression was maximal at 6 h, declining by Day 3 . Transforming growth factor beta 1 (TGF-beta 1) mRNA began to increase at Day 1, was maximal at Day 6, and fell only slightly by Day 17 . Epidermal growth factor mRNA decreased . The increase in PAI-1 mRNA and bioactivity, possibly induced early by the interleukin-1 beta response and perhaps later by the TGF-beta 1 response, was associated with striking glomerular capillary lumen fibrin accumulations on Day 1, which decreased and appeared to recanalize as the PAI-1 mRNA and bioactivity fell . The glomerular lesion continued to have some fibrin deposits even on Day 17 and, in addition, had changes of thickened GBM, suggestive of the early stages of diffuse glomerulosclerosis . The latter had a temporal relationship with the persisting increase in TGF-beta 1 and PAI-1 mRNA levels . These observations suggest the possibility that inhibition of enzymes capable of remodeling excessive extracellular matrix production may have contributed to the thickened GBM.

Br J Nutr, 1993 May, 69(3), 757 - 66
The effect of Escherichia coli heat-stable (STA) enterotoxin on in vitro uptake of amino acids by small intestine of sucking rats during fluid accumulation; Tin-Aung et al.; In vitro uptake of 14C-labelled amino acids by segments of small intestine was determined in sucking (2-4-d-old) Wistar rats . Intragastric injections of heat-stable (ST) toxin of enterotoxigenic Escherichia coli (ETEC) were given to produce fluid accumulation, defined as a gut weight: carcass weight value of > 0.085 . Continued active uptake of the prototypic amino acids, leucine (by active transport system 1 for monoamino monocarboxylic (neutral) amino acids), lysine (by active transport system 2 for dibasic amino acids), and proline (by active transport system 3 for N-substituted amino acids), persisted during the active fluid accumulation response to ETEC ST toxin . The mean Kt and mean Vmax of the amino acid transport systems were similar in control (non-injected) and ST toxin-injected rats . The present study provides a scientific basis for the use of amino acids in oral rehydration solutions utilizing amino acid transport systems which are linked to absorption of Na (and water) so that reduction in diarrhoeal stools can be achieved, and emphasizes the importance of maintaining feeding during acute diarrhoea to prevent development of malnutrition.

Mol Microbiol, 1993 May, 8(3), 525 - 33
Charged amino-terminal amino acids affect the lethal capacity of Lambda lysis proteins S107 and S105; Steiner M et al.; The lysis inhibitor protein S107 and the lysis effector protein S105 start at Met codons 1 and 3 of the Lambda S gene, respectively . The antagonistic action of both proteins precisely schedules lysis by formation of a non-specific lesion in the inner membrane through which the Lambda-encoded murein transglycosylase can pass . Here, we show that the main difference between lysis-effector and lysis-inhibitor is the degree by which an energized membrane inhibits either protein from hole formation . To dissect the structural parameters responsible for intrinsic inhibition of both proteins, charged amino acids were replaced proximal to the first putative membrane-spanning region in both S proteins . Our results show that the distribution of amino-terminal charged amino acids as well as the total amino-terminal net charge of S107 and S105 influence their lethal potential . The data are interpreted in terms of a model in which the electrostatic status of the amino-terminus of both S107 and S105 is an important feature affecting their conformational change required for formation of the S-dependent hole.

Mol Microbiol, 1993 May, 8(3), 435 - 41
Intermolecular complementation of the kinase activity of CheA; Swanson RV et al.; CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis in Escherichia coli . The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions . Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase-defective mutant, CheA470GK . The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans-phosphorylation within a dimer . The carboxy-terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW . Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphorylation requires the presence of both carboxy-terminal portions in a CheA dimer . These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.

Dev Comp Immunol, 1993 May-Jun, 17(3), 195 - 200
Specific immune recognition of insect hemolin; Schmidt O et al.; Bacteria entering the body cavities of insects are recognized by hemolymph components and subsequently inactivated by phagocytosis and nodule formation . A hemolymph component called hemolin is apparently involved in the recognition process . It binds to a bacterial surface molecule and forms a stable complex with other hemolymph proteins . Hemolin binding is independent of bacterial lipopolysaccharide (LPS) structure, whereas the complex formation is dependent on the presence of the carbohydrate moiety of LPS molecules . The specificity of immune recognition in insects is discussed.

PCR Methods Appl, 1993 May, 2(4), 305 - 12
Importance of different variables for enhancing in situ detection of PCR-amplified DNA; Nuovo GJ et al.; This study determined the effects of several variables on the in situ signal after PCR amplification in fixed cells . A signal was evident in all human peripheral blood monocytes fixed in buffered formalin using primers for the human proto-oncogene bcl-2 with in situ PCR only after prolonged fixation and protease digestion . A much lower detection rate was noted after ethanol or acetone fixation due to loss of amplified product out of the nucleus into amplifying solution . This observation demonstrates the importance of cross-linking fixatives for retention of amplified DNA at the site of origin . The increased amount of target-specific DNA synthesis evident with the manual hot start modification to in situ PCR was also seen with chemical hot start mediated by the Escherichia coli single-stranded DNA-binding protein . The manual hot start method strongly suppressed in situ unwanted DNA synthesis dictated by nonsense primers; residual nonspecific synthesis was influenced by annealing temperatures and post-fixation protease digestion conditions.

Acta Anaesthesiol Scand, 1993 May, 37(4), 396 - 403
Effects of pentoxifylline on hemodynamics, gas exchange and multiple organ platelet sequestration in experimental endotoxic shock; Sigurdsson GH et al.; In an intensive care setting we studied the effects of pentoxifylline on hemodynamics, gas-exchange and platelet sequestration in multiple organs in three groups of sheep exposed to endotoxin shock (n = 7 in each) . Group P-E was given pentoxifylline before and group E-P after E . coli endotoxin infusion, while group E received normal saline (controls) . The endotoxin infusion caused a three-fold increase in pulmonary artery pressure (PAP) and a significant decrease in mean arterial pressure (25-30%; MAP), respiratory compliance (CT; 60%) and arterial oxygen tension (65-70%; Pao2) in all groups after 30 min . After 4 h MAP had improved significantly in the pretreated animals (group P-E) and arterial pH, Pao2 and CT improved in both pentoxifylline-treated groups compared with the controls . On the other hand, the effects of endotoxin on PAP and cardiac index were not significantly influenced by pentoxifylline treatment . In addition, there was a pronounced platelet sequestration in the lungs and in the liver in groups E and E-P during the 4 h study, but in the pretreated group (group P-E) the changes were significantly less marked (P < 0.01) . The wet-to-dry weight ratios of the lungs were significantly lower in both pentoxifylline-treated groups compared with the controls (P < 0.01) . It was concluded that pentoxifylline modified the effects of endotoxin on hemodynamics, gas exchange and platelet sequestration in the lungs and liver in sheep when it was given prior to endotoxin . However, when it was given after hemodynamic and respiratory signs of shock had appeared, the effects were more moderate.

J Cell Biochem, 1993 May, 52(1), 78 - 83
Unphosphorylated alpha-PKC exhibits phorbol ester binding but lacks protein kinase activity in vitro; Filipuzzi I et al.; Expression of the alpha-isoform of protein kinase C (alpha-PKC) in E . coli yielded the unphosphorylated 74 kD precursor molecule . This precursor form exhibited phospholipid- and calcium-dependent phorbol ester binding but lacked, in contrast to the phosphorylated enzyme, protein kinase activity . In addition, the precursor molecule was found to interact with both threonine and an ATP analogon, which demonstrates that phosphorylation of alpha-PKC is not required for binding of substrates, cofactors, or activators . These results, therefore, suggest that posttranslational phosphorylation of alpha-PKC is not needed for the formation of a functional enzyme-substrate complex but is necessary for the catalytic transfer of phosphate residues from ATP to protein substrates.

Virus Res, 1993 May, 28(2), 153 - 70
Non-homologous recombination between adenovirus and AcNPV DNA fragments in cell-free extracts from insect Spodoptera frugiperda nuclei; Schorr J et al.; In previous work, we have developed a cell-free system from nuclear extracts of hamster cells to study the mechanism of integrative recombination between adenovirus type 12 (Ad12) DNA and hamster cell DNA (Jessberger et al., 1989; Tatzelt et al., 1992) . We have also demonstrated that in insect cells the left terminal fragment of Ad2 DNA can insert by non-homologous recombination into the 32.6 to 34.5 map unit (EcoRI-O) fragment and into other segments of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA (Xiong et al., 1991) . We have now imitated this recombination event in vitro by incubating the E1 fragment of Ad2 DNA and the EcoRI-O fragment of AcNPV DNA, both in the plasmid-cloned circular forms, with partly purified nuclear extracts from Spodoptera frugiperda insect cells . Proteins from these extracts have been fractionated by gel filtration . After the reextraction of DNA from the incubation mixture, recombinants generated in this cell-free system have been identified directly with the polymerase chain reaction (PCR) by using Taq polymerase and appropriate primers which are unique to either of the two reaction partners . The recombinants identified are all different . The results of control experiments argue against the possibility that unspecific reaction products might have been generated during PCR . Nucleotide sequence determinations in some of the recombinants localize the sites of genetic exchange between the two partners and assess the non-homologous nature of the reaction . The recombinants are characterized by the presence of short patch homologies at or close to the sites of linkage between the reaction partners, as described earlier in the hamster cell system (Tatzelt et al., 1992) . The occurrence of recombinants in the cell-free system can also be demonstrated by a biological test in which potential recombinants are isolated by transfection into recA- strains of Escherichia coli.

Mol Biol (Mosk), 1993 May-Jun, 27(3), 589 - 607
{Stable maintenance of dicentric mini-chromosomes in CHL4 mutants in yeast}; Kuprina NIu et al.; Earlier we have identified the chl4-1 mutation in a screen for yeast mutants with increased loss of chromosome III and circular artificial minichromosome in mitosis . Mutation in the CHL4 gene leads to a 50-100-fold promotion in the rate of chromosome loss per cell division compared to the isogenic wild type strain . Detailed analysis of behaviour of the circular minichromosome marked by the CUP1 gene has shown that minichromosome nondisjunction (2:0 segregation) leading to an increase in the copy number of minichromosome in part of a cell population is the main reason of minichromosome instability in the mutant . The unique peculiarity of chl4-1 mutation is the ability of the strains carrying this mutation to stably maintain circular dicentric minichromosomes without any rearrangement during many generations . (In the wild type strains dicentric minichromosomes are extremely unstable . As a consequence of that there is a strong selection for cells harboring monocentric derivatives in a population of cells derived from a cell containing a dicentric plasmid) . Introduction of the second centromere into one of the natural chromosomes (chromosomes II or III) in the chl4-1 mutant leads to the same dramatic consequences as that in the wild type strain (mitotic lag of cells harboring dicentric chromosomes and, as a result of that, selective pressure for cells harboring monocentric derivatives of dicentric chromosome) . A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation . Nucleotide sequence analysis of CHL4 revealed a 1.4-kb open reading frame with a predicted 53-kDa protein sequence . Analyzing the sequence of the CHL4 protein we have found a region meeting the necessary requirements for the helix-turn-helix (HTH) structure . This region of the CHL4 protein has about 40% homology with the repressor of tryptophane operon (TrpR) of E . coli . A strain containing a null allele of CHL4 was viable under standard growth conditions, but had temperature-sensitive phenotype (conditional lethality at 34 degrees C) . We suggest that the CHL4 gene product is one of the components of the segregation cell machinery.

Zhonghua Yu Fang Yi Xue Za Zhi, 1993 May, 27(3), 141 - 3
{The genotoxicity of air particles tested by SOS chromotest}; Lei XF et al.; Samples of variously-sized total suspended particles in the air of one sampling site of TaiYuan city were collected . The samples were extracted with acid and a simulated lung fluid (SLF) respectively . Both extracts mainly consisted of metallic elements . Using SOS chromotest as a means to test the gentoxity of the extracts and five metallic compounds, namely Cr6+, Ni2+, Pb2+, Mn2+ and Cd2+ were tested first . All the compounds tested could induce the SOS response to various extent, showing that the method was sensitive to metallic compounds . For the extracts of air particles, both the extracts of acid and of SLF of the smaller-sized particles could induce SOS response . This indicated the existence of metaklic genotoxicants in the smaller-sized particles . Being convenient to use fast and precise, the SOS Chromotest has its unique advantage for detecting carcinogenic metallic compounds.

Zhonghua Yu Fang Yi Xue Za Zhi, 1993 May, 27(3), 135 - 8
{Inhibitory effects of garlicin and cinnamaldehyde on SOS response and their mechanisms in Escherichia coli}; Jin ZC; Inhibitory effects of garlicin and cinnamaldehyde on SOS response and their mechanisms in Escherichia coli were investigated . Garlicin and cinnamaldehyde suppressed the lambda cI depended SOS response induced by 4-nitroquinoline-N-oxide (4NQO), mytomycin (MMC) and methyl methane-sulfonate (MMS), and the lexA-depended SOS response induced by 4NQO and UV to various extent, respectively . They also diminished the rexA 441-depended SOS response induced by temperature (at 42 degrees C) However, they did not show any effect on the constitution of SOS functions . It suggested that this inhibitory effect could be via RecA protease which regulates the cleavage of lexA repressor, and perhaps, lambda cI repressor.

J Hered, 1993 May-Jun, 84(3), 157 - 65
Studies in swine on inheritance and variation in expression of small intestinal receptors mediating adhesion of the K88 enteropathogenic Escherichia coli variants; Hu ZL et al.; Small intestinal enterocyte preparations from 368 pigs were phenotyped by an in vitro adhesion test using six strains of K88 Escherichia (E.) coli, each expressing one of three K88 fimbrial antigenic variants: K88ab, K88ac, or K88ad . All pigs tested were classified into one of four adhesion brush border phenotypes: I (K88ab-, K88ac-, K88ad-); II (K88ab-, K88ac-, K88ad+); III (K88ab+, K88ac+, K88ad-); or IV (K88ab+, K88ac+, K88ad+) . The segregation and adhesion affinity data suggest that there are two adhesion affinity receptors for K88ad+ E . coli: a high affinity (adH) and a low affinity (adL) receptor . The high affinity receptor cosegregates with receptors for K88ab and K88ac fimbrial antigens forming together the phenotype IV; the low affinity receptor is associated with the adhesion phenotype II, and its physiological expression is terminated by 16 weeks of age . In contrast, the K88adH receptor is expressed during the entire life cycle . The presence of a mixed adhesion phenotype, K88adM, assumed to be determined by K88ab(-),ac(-),adL(+)/K88ab(+),ac(+),adH(+) heterozygous genotype, is interpreted as an indication that each of the two types of brush border adhesion for the K88ad antigen is expressed on independent enterocytes.

Bull Cancer, 1993 May, 80(5), 418 - 30
{Phase I trial of recombinant human granulocyte-macrophage colony stimulating factor . Results in patients with advanced tumors}; Berthaud P et al.; Fourteen patients with advanced solid tumors were included in a phase I trial of recombinant human E coli derived granulocyte-macrophage colony-stimulating factor (GM-CSF) given daily subcutaneously for 10 consecutive days . Dose levels were increased from 250 micrograms/m2 to 500, 750 and 1,000 micrograms/m2 . Adverse effects were mainly fever, local irritation, lethargia, arthalgia . Three patients did not complete the 10-day cycle: one patient died due to progressive disease without toxic effects related to GM-CSF, one was withdrawn because of suspicion of pulmonary embolism (not confirmed), one patient had hypotension, not recurring after treatment with GM-CSF . Although the maximum tolerated dose was not reached, the trial was stopped at 1,000 micrograms/m2, considering the satisfactory response and the high white blood cell counts observed with lower dose levels . N-fold increases of leucocyte count ranged between 4.2 and 8.2 for the first dose level (250 micrograms/m2), 4 and 10.1 for 500 micrograms/m2, 8.5 and 12.3 for 750 micrograms/m2 and 5.6 and 8.3 for 1,000 micrograms/m2 . Increases of granulocyte, neutrophil and eosinophil counts had a similar pattern with a weaker response at 1,000 micrograms/m2 (two patients who completed the cycle) . In contrast, even for the first three levels, no dose response relationship was shown for increases of monocytes (between 2.8 and 12 n-fold whatever the dose), or lymphocytes (between 1.7 and 10.7 n-fold whatever the dose) . Decreases of platelets (between 6 and 55%) were observed, followed by a rebound after stopping treatment . No modifications of erythrocyte count were observed . Subcutaneous GM-CSF was well-tolerated up to 1,000 micrograms/m2 during a 10-day course . Hematological effects were observed from the first dose level of 250 micrograms/m2.

Tsitol Genet, 1993 May-Jun, 27(3), 68 - 71
{The ultrastructural characteristics of the changes in the rat liver in the dynamics of experimental endotoxemia}; Kharlanova NG et al.; Electron microscopic study was performed in the experiments on the rat liver while intravenously administering E . coli endotoxin . The dynamics of ultrastructural disorders during endotoxemia has been established and the role of "Kupffer cell-hepatocyte" microsystem in the liver detoxification function is shown.

Gen Pharmacol, 1993 May, 24(3), 681 - 5
Changes in pre- or postsynaptic adrenergic mechanisms modify the thermoregulatory responses produced by pyrogen in rabbits; Gagalo IT et al.; 1 . Thermoregulatory responses to BHT 920, prazosin (PRA) and 6-hydroxydopamine (6-OHDA) were investigated in pyrogen (lipopolysaccharide Escherichia coli, LPS) treated rabbits . 2 . All the compounds in question, despite their different selectivity for pre- or postsynaptic adrenergic structures, significantly reduced pyrogen fever . Antipyresis was associated with inhibition of the metabolic rate . 3 . The role of adrenergic mechanisms in fever, with particular respect to those of postsynaptic alpha-2, is discussed.

Mol Microbiol, 1993 May, 8(5), 865 - 73
Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors; Almenoff JS et al.; The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors . Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology . To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester . The derivatized toxin (BST) has an apparent Kd of 11.7 +/- 10 nM for rat brush border receptors . We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells . Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity . This system provides a novel approach to screening cells for the presence of unique ST-binding proteins . BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level . Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine . Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes . Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes . Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts . Thus, ST receptors are concentrated in villus enterocytes, while guanylin appears to be produced at the base of the crypts.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Radiat Biol, 1993 May, 63(5), 597 - 607
Background and radiation-induced 8-hydroxy-2'-deoxyguanosine in gamma-irradiated Escherichia coli; Claycamp HG et al.; The DNA base damage product, 8-hydroxy-2'-deoxyguanosine (7-hydro-8-oxo-2'-deoxyguanosine, or 8-OHdG) was measured in 2'-deoxyribonucleoside digests of DNA from irradiated E . coli or model DNA using HPLC separation and electrochemical detection (LCED) . The LCED technique enabled the specific quantitation of 25 fmol of 8-OHdG (signal: noise ratio = 4:1) among a variety of DNA base lesions within the sample . A radiation dose-yield relationship for 8-OHdG was observable in cells that were rapidly lysed after irradiation using a chloroform-saturated, sarkosyl and EDTA solution at 4 degrees C (Tilby and Loverock 1983) . The observed yield of 8-OHdG was 7.05 +/- 0.27 pmol J-1 and was against a background of 120 8-OHdG per E . coli genome . This yield translates to an average of 12 additional 8-OHdG lesions per genome at the mean lethal dose . The low radiochemical yield and high background suggests that factors other than the toxicity of 8-OHdG per se--such as its participation in clusters of base lesions (e.g . Ward 1985)--must contribute significantly to cell killing . Although exposure of DNA to phenol mixtures has often been reported to increase 8-OHdG, we found that a more significant effect of phenol mixtures was the potentiation of the radiochemical yields of 8-OHdG in model DNA from 5.26 +/- 0.48 to 158 +/- 12.2 nmol J-1 . Other factors, such as the exposure of dried 2'-deoxyribonucleoside digests to ambient humidity in the presence of buffer components, were shown to contribute more significantly to the background of 8-OHdG.

J Clin Microbiol, 1993 May, 31(5), 1194 - 9
A novel mu-capture enzyme-linked immunosorbent assay based on recombinant proteins for sensitive and specific diagnosis of hemorrhagic fever with renal syndrome; Zoller LG et al.; Hantavirus nucleocapsid protein has recently been identified as a major antigen inducing an early and long-lasting humoral immune response in patients with hemorrhagic fever with renal syndrome . A mu-capture enzyme-linked immunosorbent assay utilizing recombinant nucleocapsid proteins of Hantavirus strains Hantaan 76-118 (Hantaan serotype) and CG 18-20 (Puumala serotype) as diagnostic antigens and specific monoclonal antibodies as the detection system has been developed . Histidine-tailed recombinant proteins were expressed in Escherichia coli and purified in a single step by affinity chromatography on a nickel-chelate resin . The assay was evaluated with a panel of sera from patients with hemorrhagic fever with renal syndrome originating from various geographic regions . The overall sensitivity of the mu-capture enzyme-linked immunosorbent assay (both recombinant antigens) was 100%, and its specificity was also found to be 100% . Immunoglobulin M antibodies were detected as early as on day 3, and maximum titers were obtained between days 8 and 25 after onset of the disease . The assay was regularly found to be positive within 3 to 4 months but in some cases up to 2 years after the acute phase of the disease.

J Clin Microbiol, 1993 May, 31(5), 1185 - 8
Clonal analysis of Escherichia coli of serogroups O9, O20, and O101 isolated from Australian pigs with neonatal diarrhea; Woodward JM et al.; The genetic diversity of 87 isolates of Escherichia coli recovered from Australian pigs with neonatal diarrhea was examined by multilocus enzyme electrophoresis . The isolates were of serogroups O9, O20, and O101, and although most isolates lacked K88(F4), K99(F5), 987P(F6), and F41 fimbriae, they were considered to be involved in the etiology of the diarrhea . The isolates were extremely diverse, considering their origin from a single pathological condition in one country . There were estimated to be 18, 16, and 12 clones of the three respective serogroups in the collection, with serogroup diversities of 0.387, 0.448, and 0.275, respectively . Comparison with the results previously obtained for isolates from piglets with postweaning diarrhea suggested that bacteria from piglets with these two conditions did not come from any particular common genetic background . The overall genetic diversity for the combined collection was the same as that reported by others for representative isolates selected from throughout the species (0.47) . The current results indicate that if isolates of these O groups are involved in porcine diarrhea, their pathogenicity is directly linked to their O somatic antigen type and is not simply due to the wide distribution of a small number of virulent clones.

Protein Sci, 1993 May, 2(5), 800 - 13
Time-resolved fluorescence and computational studies of adenylylated glutamine synthetase: analysis of intersubunit interactions; Atkins WM et al.; Adenylylation of Tyr-397 of each subunit of Escherichia coli glutamine synthetase (GS) down-regulates enzymatic activity in vivo . The overall structure of the enzyme consists of 12 subunits arranged as two hexamers, face to face . Research reported in this paper addresses the question of whether the covalently attached adenylyl group interacts with neighboring amino acid residues to produce the regulatory phenomenon . Wild-type GS has two Trp residues (positions 57 and 158) and the adenylylation site lies within 7-8 A of the Trp-57 loop in the adjacent subunit of the same hexameric ring; Trp-158 is about 35 A from the site of adenylylation . Fluorescence lifetimes and quantum yields have been determined for two fluorophores with wild-type and mutant GS . One fluorophore is epsilon-AMP adenylylated GS (at Tyr-397), and the other fluorophore is the intrinsic protein residue Trp-57 . These experiments were conducted in order to detect possible intersubunit interactions between adenylyl groups and the neighboring Trp-57 to search for a role for the Trp-57 loop in the regulation of GS . The fluorescence due to epsilon-AMP of two adenylylated enzymes, wild-type GS and the W158F mutant, exhibits heterogeneous decay kinetics; the data adequately fit to a double exponential decay model with recovered average lifetime values of 18.2 and 2.1 ns, respectively . The pre-exponential factors range from 0.66 to 0.73 for the long lifetime component, at five emission wavelengths . The W57L-epsilon-AMP enzyme yields longer average lifetime values of 19.5 and 2.4 ns, and the pre-exponential factors range from 0.82 to 0.85 for the long lifetime component . An additional residue in the Trp-57 loop, Lys-58, has been altered and the K58C mutant enzyme has been adenylylated with epsilon-AMP on Tyr-397 . Lys-58 is near the ATP binding site and may represent a link by which the adenylyl group controls the activity of GS . The fluorescence of epsilon-AMP-adenylylated K58C mutant GS is best described by a triple exponential decay with average recovered lifetime values of 19.9, 4.6, and 0.58 ns, with the largest fraction being the median lifetime component . Relative quantum yields of epsilon-AMP-Tyr-397 were measured in order to determine if static quenching occurs from adenine-indole stacking in the wild-type GS . The relative quantum yield of the epsilon-AMP-adenylylated W57L mutant is larger than the wild-type protein by the amount predicted from the difference in lifetime values: thus, no static quenching is evident.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1993 May, 175(10), 3236 - 9
Change in direction of flagellar rotation in Escherichia coli mediated by acetate kinase; Dailey FE et al.; Strains of Escherichia coli lacking all cytoplasmic chemotaxis proteins except CheY swim smoothly under most conditions . However, they tumble when exposed to acetate . Acetate coenzyme A synthetase (EC 6.2.1.1) was thought to be essential for this response . New evidence suggests that the tumbling is mediated instead by acetate kinase (EC 2.7.2.1), which might phosphorylate CheY via acetyl phosphate . In strains that were wild type for chemotaxis, neither acetate coenzyme A synthetase, acetate kinase, nor phosphotransacetylase (EC 2.3.1.8) (and thus acetyl phosphate) was required for responses to aspartate, serine, or sugars sensed by the phosphotransferase system . Thus, acetate-induced tumbling does not appear to play an essential role in chemotaxis in wild-type cells.

J Bacteriol, 1993 May, 175(10), 2970 - 9
The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity; Doublet P et al.; The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E . coli B/r (P . Doublet, J . van Heijenoort, and D . Mengin-Lecreulx, J . Bacteriol . 174:5772-5779, 1992) . We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan . A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E . coli strains grown in rich medium to internalize D-glutamic acid . The murI gene product was overproduced and identified as a glutamate racemase activity . UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.

Biochem J, 1993 May 1, 291 ( Pt 3), 927 - 32
{13C}propionate oxidation in wild-type and citrate synthase mutant Escherichia coli: evidence for multiple pathways of propionate utilization; Evans CT et al.; The metabolism of propionate was examined in wild-type Escherichia coli and cells lacking citrate synthase by high-resolution 13C n.m.r . Spectra of cell extracts from wild-type E . coli show that glutamate becomes highly enriched in 13C when 13C-enriched propionate is the sole carbon source . No glutamate labelling was detected when the tricarboxylic acid cycle was blocked either by deletion of citrate synthase or by inhibition of succinate dehydrogenase by malonate . The 13C fractional enrichment in glutamate C-2, C-3 and C-4 in wild-type cells was quantitatively and qualitatively different when {2-13C}propionate as opposed to {3-13C}propionate was supplied . Approximately equal labelling occurred in the C-2, C-3 and C-4 positions of glutamate when {3-13C}propionate was available, and multiplets due to carbon-carbon spin-spin coupling were observed . However, in cells supplied with {2-13C}propionate, very little 13C appeared in the glutamate C-4 position, and the remaining glutamate resonances all appeared as singlets . The unequal and non-identical labelling of glutamate in cells supplied with {2-13C}- as opposed to {3-13C}propionate is consistent with the utilization of propionate by E . coli via two pathways, oxidation of propionate to pyruvate and carboxylation of propionate to succinate . These intermediates are further metabolized to glutamate by the action of the tricarboxylic acid cycle . The existence of an organized tricarboxylic acid cycle is discussed as a consequence of the ability to block utilization of propionate in tricarboxylic acid-cycle-defective E . coli.

Arch Biochem Biophys, 1993 May, 302(2), 455 - 60
Lysine241 of tyrosine hydroxylase is not required for binding of tetrahydrobiopterin substrate; Daubner SC et al.; The lysine residues at positions 194 and 198 in phenylalanine hydroxylase have been shown to react with a photoaffinity label which is an analog of phenyltetrahydropterin (Gibbs, B . S., and Benkovic, S . J . (1991) Biochemistry 30, 6795-6802), in a manner suggesting that these lysine residues are involved in tetrahydrobiopterin binding . The related enzyme tyrosine hydroxylase has a lysine at position 241 which, given the 75% identity between its C-terminal 330 amino acids and those of phenylalanine hydroxylase, corresponds to lysine194 of phenylalanine hydroxylase . Site-directed mutagenesis was used to alter lysine241 of tyrosine hydroxylase to alanine . Steady-state kinetic parameters were measured for wild-type and K241A tyrosine hydroxylase . No kinetic parameter differed between the wild-type and K241A enzymes, including Vmax values, Michaelis constants for tetrahydrobiopterin, 6-methyl-tetrahydropterin, and tyrosine, and the inhibition constants for norepinephrine . These results show that lysine241 is not required for tetrahydrobiopterin binding to tyrosine hydroxylase.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 3978 - 82
A polypeptide bound by the chaperonin groEL is localized within a central cavity; Braig K et al.; Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments . They appear to bind early folding intermediates, preventing their aggregation; in the presence of MgATP and a cochaperonin, bound polypeptides are released in a stepwise manner, associated with folding to the native state . Chaperonin complexes appear in the electron microscope as cylindrical structures, usually composed of two stacked rings, each containing, by negative staining, an electron dense central "hole" approximately 6.0 nm in diameter . We sought to identify the site on the Escherichia coli chaperonin groEL, where the "molten globule"-like intermediate of dihydrofolate reductase (DHFR) becomes bound, by examining in the scanning transmission electron microscope complexes formed between groEL and DHFR molecules bearing covalently crosslinked 1.4-nm gold clusters . In top views of the groEL complexes, gold densities were observed in the central region; in side views, the densities were seen at the end portions of the cylinders, corresponding to positions within the individual rings . In some cases, two gold densities were observed in the same groEL complex . We conclude that folding intermediates are bound inside central cavities within individual chaperonin rings . In this potentially sequestered location, folding intermediates with a compact conformation can be bound at multiple sites by surrounding monomeric members of the ring; localization of folding within the cavity could also facilitate rebinding of structures that initially fail to incorporate properly into the folding protein.

J Bacteriol, 1993 May, 175(9), 2692 - 701
Alterations of highly conserved residues in the regulatory domain of nitrogen regulator I (NtrC) of Escherichia coli; Moore JB et al.; Transcription of many nitrogen-regulated (Ntr) genes requires the phosphorylated form of nitrogen regulator I (NRI, or NtrC), which binds to sites that are analogous to eukaryotic enhancers . A highly conserved regulatory domain contains the site of phosphorylation and controls the function of NRI . We analyzed the effects of substitutions in highly conserved residues that are part of the active site of phosphorylation of NRI in Escherichia coli . Fourteen substitutions of aspartate 54, the site of phosphorylation, impaired the response to nitrogen deprivation . Only one of these variants, NRI D-54-->E (NRI-D54E), could significantly stimulate transcription from glnAp2, the major promoter of the glnALG operon . Cells with this variant grew with arginine as a nitrogen source . Experiments with purified components showed that unphosphorylated NRI-D54E stimulated transcription . In contrast, substitutions at aspartate 11 were not as deleterious as those at aspartate 54 . Finally, we showed that NRI-K103R, in which arginine replaces the absolutely conserved lysine, is functionally active and efficiently phosphorylated . This substitution appears to stabilize the phosphoaspartate of NRI . The differences between our results and those from study of homologous proteins suggest that there may be significant differences in the way highly conserved residues participate in the transition to the activated state.

Infect Immun, 1993 May, 61(5), 2249 - 52
Identification of plasmid and chromosomal copies of 987P pilus genes in enterotoxigenic Escherichia coli 987; Casey TA et al.; A DNA probe derived from the subunit gene of a cloned 987P determinant was used to characterize the locations of the 987P genes in several Escherichia coli strains . We examined E . coli 987, a nonpiliated mutant of strain 987 (I36) that does not express 987P in vitro, and a variant of I36 that expressed 987P following growth in vivo . We found that plasmid and chromosomal copies of the 987P subunit gene could be differentiated in strain 987 by restriction endonuclease analysis and Southern blot hybridization . The nonpiliated mutant I36 had lost the plasmid copy but retained the chromosomal copy of the 987P gene . A 987P-piliated variant of I36, which did not contain the 987P plasmid, colonized and caused diarrhea in neonatal pigs similarly to wild-type 987 . The plasmid that hybridized with the 987P probe was transferred from strain 987 to an E . coli K-12 strain by conjugation . We were unable to demonstrate expression of 987P by these transconjugants . The data suggest that the chromosomal and plasmid copies of the 987P genes may interact to influence 987P expression.

Infect Immun, 1993 May, 61(5), 2108 - 15
Specificity of S fimbriae on recombinant Escherichia coli: preferential binding to gangliosides expressing NeuGc alpha (2-3)Gal and NeuAc alpha (2-8)NeuAc; Hanisch FG et al.; The adhesins of Escherichia coli strains HB101(pANN801-13) and HB101(pAZZ50), which express S fimbriae encoded by a recombinant plasmid containing the sfaI and sfaII gene clusters, respectively, were characterized with regard to the detailed structural requirements of their binding to sialyloligosaccharides on (neo)glycoproteins and (neo)glycolipids . From binding and binding inhibition studies in solid-phase enzyme immunoassays with isolated S fimbriae, several major conclusions can be drawn . S fimbriae bind specifically to sialic acid on gangliosides . The most active structural variant of sialic acid on GM3 ganglioside is N-glycolylneuraminic acid (NeuGc) . In contrast to previous reports, high binding activities were measured also for b-series gangliosides expressing NeuAc alpha (2-8)NeuAc . In agreement with earlier studies, the site of sialic acid substitution to subterminal sugars strongly influences the binding to sialyloligosaccharides, i.e., alpha-6-linked sialic acid is only poorly recognized by the adhesin compared with alpha-3-linked sialic acid . C-8 and C-9 hydroxyl groups form essential structural elements of sialic acid in the binding event.

Urol Nefrol (Mosk), 1993 May-Jun, (3), 13 - 6
{Fibronectin and its significance in pyelonephritis}; Rudenko AV et al.; Fibronectin levels were measured by enzyme immunoassay in 68 patients with pyelonephritis and 10 patients with chronic cystitis . The patients with chronic cystitis, acute and chronic non-obstructive pyelonephritis exhibited significantly high average blood fibronectin levels, whereas those with chronic obstructive pyelonephritis displayed the levels slightly different from those observed in healthy individuals . Hypofibronectinemia was detected in 23.3% of patients with chronic pyelonephritis . Preincubation of the neutrophils isolated from pyelonephritis patients with fibronectin increased the initially low phagocytic capacity without enhancing their metabolic activity . In experimental pyelonephritis, administration of exogenous fibronectin was demonstrated to contribute to a rapid bacterial elimination from the kidneys, to decrease the intensity of an inflammatory response, to prevent renal histostructural lesions, to enhance the functional activity of immunocompetent cells and to stabilize their membranes . The findings may serve the basis for using fibronectin in clinical practice as a promising agent to treat pyelonephritis.

Biosci Biotechnol Biochem, 1993 May, 57(5), 760 - 5
Sequencing analysis of mutation points in the biotin operon of biotin-overproducing Escherichia coli mutants; Ifuku O et al.; We analyzed mutation points of the biotin operon from biotin-overproducing mutants of Escherichia coli resistant to two biotin analogs, actithiazic acid and 5-(2-thienyl)-valeric acid, by DNA sequencing . The biotin operons cloned from these mutants were classified into three groups . One point mutation, which was a GC-->AT change within the operator overlapping the -10 region of the rightward (bioB) promoter, was considered to result in disruption of operator structure and enhancement of promoter activity . Two other point mutations, which were both GC-->AT changes just before and after the initiation codon of the bioB gene, were considered to activate the translation efficiency . These mutations significantly accelerated the biotin-forming activity from dethiobiotin in cell-free extracts.

Biosci Biotechnol Biochem, 1993 May, 57(5), 710 - 4
Cleavage of potato virus Y polyprotein in Escherichia coli depending on the proteolytic activity of viral protease; Hidaka M et al.; We have cloned a 5-kb cDNA corresponding to the 3'-half of genomic RNA of potato virus Y (PVY), the type member of plant potyvirus . Based on its nucleotide sequence, the cDNA was presumed to encode PVY protease responsible for the self-processing of polyprotein precursor expressed from PVY genome . To test its proteolytic activity, the cDNA was modified so as to express the protease-polymerase-coat protein (CP) portion of PVY polyprotein in Escherichia coli . By Western blotting analysis of the cell extract of E . coli expressing the polyprotein, mature CP was detected . A large deletion in the polymerase-coding region did not affect the emergence of mature CP, but a small deletion in the putative protease region resulted in disappearance of mature CP and appearance of the intact polyprotein . These results indicated that the polyprotein expressed in E . coli was cleaved depending on the proteolytic activity of PVY protease.

Appl Microbiol Biotechnol, 1993 May, 39(2), 227 - 34
Biosafety investigations in an r-DNA production plant; Weibel EK et al.; Employees of a biotechnological production plant for recombinant interferon alpha-2a have been participating in a 1-year study on occupational hygiene and the resulting biosafety aspects . Most of the employees have been employed in the plant for more than 6 years . Weekly stool samples were analysed for tetracycline (used as selection marker)-resistant coliforms as well as for rDNA (IFN gene) (interferon gene) and for the production organism . Various analytical methods, including the polymerase chain reaction, were applied to show that neither rDNA nor the production organism could be found in any of the stool samples and that there was no change or trend in the gut flora with respect to tetracycline resistance . In addition it could be shown that the tetracycline-resistance gene, as well as the rDNA, are completely inactivated in the course of the production process and thus no further recombination can take place . Blood samples were taken to show that none of the employees had anti-product antibodies.

Appl Microbiol Biotechnol, 1993 May, 39(2), 221 - 6
Cloning, expression, and characterization of a synthetic analog to the bioadhesive precursor protein of the sea mussel Mytilus edulis; Salerno AJ et al.; Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were designed, constructed, and expressed in Escherichia coli . The bioadhesive precursor (BP) with a relative molecular mass of 25,000 was expressed from one 600-bp gene at levels approaching 60% of total cell protein in strains employing T7 RNA polymerase for induction and carrying a repetitious gene comprised of a 30-bp unit repeat that accounts for E . coli codon bias . BP forms intracellular inclusions and yet methionine was processed from the N-terminus of the purified protein, as shown by amino acid composition and N-terminal sequencing, to give an authentic consensus precursor protein.

Biotechnology (N Y), 1993 May, 11(5), 601 - 5
Recombinant colorimetric antibodies: construction and characterization of a bifunctional F(ab)2/alkaline phosphatase conjugate produced in Escherichia coli; Ducancel F et al.; We have designed a vector which allows the synthesis in Escherichia coli of bifunctional F(ab)2-alkaline phosphatase conjugates . The vector contains a di-cistronic operon encoding truncated heavy chain (Fd or VH-CH1) of an IgG inserted between residues +6 and +7 of bacterial alkaline phosphatase (PhoA), and the light chain of the same IgG . We demonstrate the utility of this approach with the heavy and light chain domains of a snake toxin-specific monoclonal antibody, M alpha 2-3 . We show that the VH-CH1-PhoA hybrid and VL-CL are concomitantly expressed and exported to the periplasm of E . coli where they form a disulfide-linked chimeric protein . The hybrid has the same affinity as M alpha 2-3 for the snake toxin antigen and possesses PhoA enzymatic activity.

Gastroenterol Jpn, 1993 May, 28 Suppl 5, 52 - 4
High detection rate of hepatitis C virus E2 antibody in patients with type C hepatitis; Yokosuka O et al.; Putative hepatitis C virus E2 protein was applied for detection of anti-E2 antibody in patients with type C hepatitis . The Putative E2 sequence was expressed in E . coli and approximately 38 KDa hepatitis C E2 protein fused with 42 KDa maltose binding protein was obtained . The HCV E2 antibody was detected in 2 of 7 acute hepatitis cases, in 8 of 12 chronic persistent hepatitis, in 17 of 25 chronic active hepatitis, and in 2 of 4 cirrhosis . It was not detected in 10 normal subjects . Anti-E2 antibody became undetectable after successful interferon treatment in patients with type C hepatitis . The High detection rate of E2 antibody in chronic hepatitis C and disappearance of the antibody after the interferon treatment indicate that the E2 antibody is related to viral replication and is unlikely to be a neutralizing antibody.

Plasmid, 1993 May, 29(3), 241 - 4
Development of a native plasmid as a cloning vector in Clavibacter xyli subsp . cynodontis; Taylor J et al.; A 50-kilobase (kb) cryptic plasmid was found in three geographic isolates of Clavibacter xyli subsp . cynodontis (Cxc) . The DNA region essential for replication of the plasmid was cloned in an Escherichia coli vector . The resulting vector, which functions as a shuttle vector between E . coli and Cxc, was characterized further with respect to its stability and copy number.

Parasitology, 1993 May, 106 ( Pt 4), 413 - 20
Cloning, over-expression and evaluation of a recombinant fusion protein of Wuchereria bancrofti towards its application as a diagnostic agent for bancroftian filariasis; Theodore JG et al.; A low molecular weight (15 kDa) surface antigen of the cattle filarial nematode, Setaria digitata, was earlier shown to be specifically recognized by the antibodies from human bancroftian filarial (Mf positive) patients' sera (Theodore & Kaliraj, 1990) . The filarial specific antibodies bound to a 15 kDa peptide in preparative Western blots were eluted and employed in screening of candidate antigens expressed in the genomic library of Wuchereria bancrofti at the IgG4 subclass antibody level . A recombinant clone (lambda WbG7) reacting strongly with filarial sera but poorly with sera from patients infected with non-filarial helminths was selected for further studies . The 2 kb DNA insert of the clone lambda WbG7 was recloned into a pMAL vector and the recombinant clone pGT7 thus obtained was over-expressed and affinity purified . The purified 105 kDa fusion protein of the clone pGT7 was specific and was not recognized by the non-filarial sera at the IgG4 level . All microfilaraemic individuals were positive by IgG4 assay . However, similar attempts to diagnose by filarial-specific IgE assays failed to recognize microfilaraemic individuals . Moreover, by filarial-specific IgG4 assays, the endemic normals were distinctly divided into two groups, showing higher and lower recognition for this antigen indicating current and past/no infection . Among the filarial-IgG4 (assay)-positive 'endemic normals', 14% showed 'microfilariae' during repeated peripheral night blood examination, confirming the validity of the recombinant antigen, pGT7 based assay.

Mol Biol (Mosk), 1993 May-Jun, 27(3), 618 - 30
{Reverse transcriptase of the human immunodeficiency virus: isolation and substrate specificity}; Rozovskaia TA et al.; Human immunodeficiency virus (HIV-I) reverse transcriptase was expressed in E . coli and purified to homogeneity (E . coli strain RRI (pRC-RT, pRK 248cIts)) . We have investigated the substrate properties toward to DNA synthesis, catalyzed by this enzyme, of some nucleoside-5'-triphosphate analogues, previously studied in the same reactions, catalyzed by AMV and M-MLV reverse transcriptases . We have investigated substrate properties of new analogues of 2',3'-dideoxy-2',3'-didehydro- and 2',3'-dideoxytubercidin-5'-triphosphates . We have compared the relative efficiency of incorporation of different analogues tested in the DNA chain . It has been shown that expressed and purified HIV reverse transcriptase had the same specificity to analogues of 2'-deoxyribonucleoside-5'-triphosphates as was described for reverse transcriptases and natural HIV reverse transcriptase as well . These properties allow to apply the expressed HIV reverse transcriptase in different model systems.

Mol Biol (Mosk), 1993 May-Jun, 27(3), 538 - 51
{Design of four-helix protein--a possible vaccine against human immunodeficiency virus (HIV-1)}; Eroshkin AM et al.; Successful approach to the development of safe and effective synthetic vaccines requires that different B- and T-cell epitopes of the infectious agent be included into the vaccine construction . It is suggested that vaccines should be constructed as proteins with both optimal epitope composition and predetermined tertiary structure . Based on analysis of B-cell and T-cell epitope properties, a possibility to use one well-known protein spatial motif--four-alpha-helix bundle--for vaccine construction is substantiated . Antigenic determinants of cellular immunity (amphipathic alpha-helices) and humoral immunity (flexible hydrophilic loop regions) can be used as blocks for vaccine design . Nonloop B-epitopes and nonhelical T-epitopes may be introduced in the protein N- and C-terminal regions . General principles of PTS-vaccine construction have been applied to anti-HIV-1 vaccine design . Experimentally studied T- and neutralizing B-cell epitopes from HIV-1 proteins were analyzed . The sequence of one possible four-alpha-helix protein vaccine has been constructed . Predicted secondary structure and T- and B-cell epitopes of this protein coincided with the planned ones . The amino acid composition of the protein was found to be consistent with the composition of globular water-soluble proteins . The gene of the protein with codon composition optimal for expression in E . coli has been synthesized . The advantages and limitations of this approach to vaccine design are discussed.

J Neuroimmunol, 1993 May, 44(2), 157 - 62
Biological activity of recombinant human myelin basic protein; Oettinger HF et al.; Using an inducible expression vector in Escherichia coli, we have expressed, purified, and biologically characterized recombinant human myelin basic protein (r-MBP) . The recombinant protein binds in cation-exchange chromatography with similar affinity to purified, human MBP, and elutes as a single, 18.5-kDa species as judged by SDS-PAGE . The recombinant protein exhibits similar conformation to native MBP, as demonstrated by ELISA reactivity with a panel of six monoclonal antibodies directed against at least three different epitopes on human MBP . Additionally, recombinant MBP can trigger the proliferation of human T cell clones recognizing MBP, can induce experimental autoimmune encephalomyelitis (EAE) in the SJL mouse, and is capable of suppressing EAE following tolerization via oral administration . Most important, by using a novel method of purification, the recombinant protein preparation contains no detectable proteolytically derived fragments of MBP, a significant advantage over MBP purified from protease-rich central nervous system tissue . Purified recombinant MBP produced in E . coli will be useful as a biological reagent where highly purified protein devoid of degradation products is needed . Relevance to the study of antigen processing, interactions between MHC and the TCR, as well as for the investigation of antigen-driven immune tolerance are discussed.

J Bacteriol, 1993 May, 175(10), 2895 - 906
Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication; Siemering KR et al.; Physical analysis of RNA I, the small antisense RNA which regulates the replication of IncB miniplasmid pMU720, showed that it is a highly structured molecule containing an imperfectly paired stem closed by a 6-base hairpin loop . Mutational studies revealed that a 3-base sequence in the hairpin loop is critical to the interaction between RNA I and its complementary target in the RepA mRNA (RNA II) . Furthermore, a 2-base interior loop in the upper stem was found to play an important role in facilitating effective binding between RNA I and RNA II . From these analyses, a model describing the molecular mechanism of binding between RNA I and RNA II is proposed.

Genes Dev, 1993 May, 7(5), 904 - 10
Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase; Citovsky V et al.; In host plants, cell-to-cell spread of tobacco mosaic virus (TMV) presumably occurs through intercellular connections, the plasmodesmata . TMV movement is mediated by a specific virus-encoded single-strand nucleic acid-binding protein, P30 . The mechanism by which P30 operates is largely unknown . Here, we demonstrate that P30 expressed in transgenic plants is a phosphoprotein . We have developed an assay for in vitro phosphorylation of purified P30 by plant cell wall fractions and have localized the phosphorylation sites to amino acid residues Ser-258, Thr-261, and Ser-265 . Interestingly, the P30 phosphorylation sites do not correspond to any known consensus phosphorylation sites for protein kinases . While P30 binding to single-stranded DNA (ssDNA) was shown to involve Thr-261, phosphorylation of this residue does not appear to play a role in binding activity . The protein kinase activity contained in the cell wall fractions was developmentally regulated, expressed predominantly in leaves . Within a leaf, this protein kinase activity increased with leaf maturation and correlated with the reported development of secondary plasmodesmata, sites of P30 accumulation . We suggest that phosphorylation may represent a mechanism for the host plant to sequester P30 following its localization to cell walls.

Biochem J, 1993 May 1, 291 ( Pt 3), 757 - 63
Identification of the domain recognized by anti-(ryanodine receptor) antibodies which affect Ca(2+)-induced Ca2+ release; Treves S et al.; In the present paper we have defined putative functional domains of the ryanodine receptor Ca2+ channel . cDNA fragments of the skeletal muscle ryanodine receptor were fused in-frame with the Escherichia coli trpe protein and the resulting fusion proteins were evaluated for their ability to react with anti-(ryanodine receptor) antibodies, which are known to block Ca(2+)-dependent activation of the Ca(2+)-release channel . Anti-(ryanodine receptor) antibodies react with epitopes lying within a 245-amino-acid-long polypeptide which is located in a region (residues 4380-4625) encompassing most of myoplasmic loop 2, the predicted transmembrane segment M5 and part of the next lumenal loop (45 residues) . Purification of the anti-(ryanodine receptor) antibodies by affinity chromatography led to the isolation of a population of antibodies which was capable of decreasing (by > 30%) the doxorubicin-induced Ca2+ release from isolated terminal cisternae . Polyclonal antibodies raised against a ryanodine receptor fusion encompassing part (198 out of 245 residues) of the immunopositive polypeptide decreased by 2-fold the first-order rate constant of Ca(2+)-induced 45Ca2+ efflux from isolated terminal cisternae . These results suggest strongly that the Ca(2+)-activating domain of the skeletal muscle Ca(2+)-release channel is close to, or associated with, myoplasmic loop 2.

Mol Immunol, 1993 May, 30(7), 613 - 25
Identification of helper T cell epitopes of dengue virus E-protein; Leclerc C et al.; The T cell proliferative response to dengue 2 (Jamaica) E-glycoprotein (495 amino acids) was analyzed in vitro using either killed virus or E-protein fragments or synthetic peptides . Inactivated dengue virus stimulated dengue-specific lymph node (LN) CD4+T cell proliferation in BALB/c (H-2d), C3H (H-2k) and DBA/1 (H-2q) but not in C57BL/6 (H-2b) mice . Moreover, LN cells from dengue-virus primed BALB/c mice proliferated in vitro in response to three purified non-overlapping E-protein fragments expressed in E . coli as polypeptides fused to trpE (f22-205, f267-354, f366-424) . To further determine T cell epitopes in the E-protein, synthetic peptides were selected using prediction algorithms for T cell epitopes . Highest proliferative responses were obtained after in vitro exposure of virus-primed LN cells to peptides p135-157, p270-298, p295-307 and p337-359 . Peptide p59-78 was able to induce specific B and T cell responses in peptide-primed mice of H-2d, H-2q and H-2k haplotypes . Two peptides p59-78 corresponding to two dengue (Jamaica and Sri Lanka) isolates and differing only at position 71 cross-reacted at the B but not at the T cell level in H-2b mice . This analysis of murine T helper cell response to dengue E-protein may be of use in dengue subunit vaccine design.

Clin Exp Immunol, 1993 May, 92(2), 308 - 16
Sera from patients with tuberculosis recognize the M2a-epitope (E2-subunit of pyruvate dehydrogenase) specific for primary biliary cirrhosis; Klein R et al.; Anti-M2 antibodies in primary biliary cirrhosis (PBC) have been shown to react with the alpha-ketoacid dehydrogenase complex of the inner mitochondrial membrane consisting of six epitopes (E2 subunit of the pyruvate dehydrogenase complex (PDC), 70 kD; protein X of the PDC, 56 kD; alpha-ketoglutarate dehydrogenase complex, 52 kD; branched-chain alpha-ketoacid dehydrogenase, 52 kD; E1 alpha subunit of PDC, 45 kD; and E1 beta-subunit of PDC, 36 kD) . These epitopes are also present in the M2 fraction which is a chloroform extract from beef heart mitochondria . The E2 subunit of the PDC at 70 kD (M2a), especially, is a major target epitope which is recognized by about 85% of all PBC sera . However, analysing sera from 28 patients with active pulmonary tuberculosis it became evident that 12 (43%) also recognized the PDC-E2 subunit (M2a), as shown by Western blotting using the M2 fraction, the purified PDC, and the recombinant PDC-E2 . In contrast, only two of 82 patients with other bacterial and viral infections including 25 patients with Escherichia coli infections reacted with the PBC-specific epitope at 70 kD . Naturally occurring mitochondrial antibodies (NOMA) were present in 54% of the patients with tuberculosis and in 50% of patients with other infectious disorders . They recognized either a determinant at 65 kD (epsilon) or at 60/55 kD (zeta/eta) . None of the sera from 100 blood donors had anti-M2 but 14 had NOMA . Testing anti-M2 and NOMA-positive marker sera by Western blotting against membrane fractions derived from mycobacteria and E . coli it could be shown that--like mammalian mitochondria--they contain both the PBC-specific M2 antigen as well as the non-PBC-specific naturally occurring mitochondrial antigen system (NOMAg) . The observation that PBC-specific antibodies were preferentially induced in patients suffering from a mycobacterial infection may provide some new clues to the still unknown etiology of PBC.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 4092 - 6
Evasion of protective immunity by Borrelia burgdorferi by truncation of outer surface protein B; Fikrig E et al.; We analyzed variability in outer surface protein B (OspB) from Borrelia burgdorferi (Bb), the causative agent of Lyme disease, to determine how Bb escapes immune destruction . We have shown that vaccination with OspB from Bb strain B31 protected mice from infection with Bb B31 but not against Bb N40 . The present study demonstrates that Bb N40 spirochetes which evade vaccination immunity to OspB have a truncated form of OspB, due to a TAA stop codon at nucleotide 577 . In contrast, Bb N40 spirochetes that express full-length OspB are unable to infect mice immunized with OspB, analogous to our previous studies with Bb B31 . Mapping of the OspB antibody response shows that epitopes in the C terminus of OspB are surface-exposed and bind protective monoclonal and polyclonal antibodies . This suggests that the C terminus of OspB is important for eliciting a protective immune response to OspB . Truncation or modification of outer surface proteins that do not bind protective antibody may be a means by which Bb evades host defenses.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 3938 - 42
Molecular cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger (NHE-2); Collins JF et al.; The present studies demonstrate cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger which was isolated from a rat intestinal cDNA library . The cloned cDNA recognizes two transcripts in poly(A)+ RNA from the stomach, jejunum, ileum, liver, large intestine, and uterus . Based on deduced amino acid sequences, this clone shares sequence homology with the other known Na+/H+ exchanger isoforms (NHE-1, NHE-3, and NHE-4) except for its 5' end . Overall, the protein exhibits 47.8%, 41.2%, and 56.2% amino acid sequence identity to NHE-1, NHE-3, and NHE-4, respectively . The hydropathy profile of the predicted protein shows 10 transmembrane domains, suggesting a protein with transport characteristics . The tissue distribution differs from that of the other Na+/H+ exchanger isoforms . The cDNA hybridizes to two closely related transcripts in the mRNA of these tissues, which suggests that the predominant transcript of this clone is alternatively spliced . Transfection of this cDNA into Na+/H+ exchanger-deficient mutant fibroblasts (PS120 cells) results in functional Na+/H+ exchange activity . These data suggest that we have cloned a member of the Na+/H+ exchanger family with tissue-specific expression . We suggest the designation of NHE-2 for this Na+/H+ exchanger.

Dev Biol, 1993 May, 157(1), 214 - 23
Cloning and analysis of expression of a ubiquitin carboxyl terminal hydrolase expressed during oogenesis in Drosophila melanogaster; Zhang N et al.; Using the enhancer trap approach we have searched for genes with important functions in oogenesis . We selected a line of flies with a P insertion, carrying the Escherichia coli lac Z gene, which showed beta-galactosidase expression in the nurse cell nuclei during oogenesis . Surrounding the P insertion we discovered a cluster of transcription units with enriched expression in the ovary . One of these encodes a protein with extensive sequence similarity to the human and yeast ubiquitin carboxyl terminal hydrolase . Analysis of a fusion protein including the putative ubiquitin carboxyl terminal hydrolase indicated that this protein does have the appropriate enzyme activity, and the gene was assigned the name ubiquitin carboxyl terminal hydrolase uch-D . The expression of this gene is enriched in the nurse cells and transcripts are transported to the embryo . Transcripts are abundant for the first few hours of development . The transcripts are found to be enriched on the ventral side of the oocyte and nurse cells . Rather little is known about the ubiquitin pathway in Drosophila and the discovery of this gene enables us to make predictions as to the roles it may play during early embryogenesis.

Oncogene, 1993 May, 8(5), 1241 - 7
Recombinant Csk expressed in Escherichia coli is autophosphorylated on tyrosine residue(s); Bougeret C et al.; The C-terminal src kinase (Csk) is responsible for the phosphorylation of the carboxy-terminal tyrosine of several tyrosine kinases of the Src family . This phosphorylation site has a negative regulatory function . Csk is unique among the members of the protein tyrosine kinase family because it lacks the conserved tyrosine autophosphorylation site and has been thought to be devoid of autophosphorylation activity . Using the glutathione S-transferase (GST) bacterial expression system, we have produced large amounts of a chimeric rat Csk protein . We have determined that the GST-Csk fusion protein isolated from bacteria is autophosphorylated on tyrosine residue(s) . GST-Csk and purified Csk are capable of undergoing autophosphorylation on tyrosine residue(s) in vitro . The GST-Csk fusion protein also phosphorylates exogenous substrates, including the heteropolymer poly-Glu/Tyr and enolase . This is the first indication that Csk is autophosphorylated on tyrosine residue(s) both in vivo in bacteria expressing Csk cDNA and in vitro . These findings suggest that the autophosphorylation of Csk might play a role in the regulation of its kinase activity as well as its binding to other cellular proteins.

Mutat Res, 1993 May, 302(1), 53 - 9
The effect of non-mutagenic stress on liquid holding recovery in Escherichia coli AB2463; Fitt PS et al.; Previous work has shown that exposure to many non-mutagenic stresses causes greater UV resistance in Escherichia coli K12 via an error-free, excision repair-dependent process . Induction of the latter should enhance liquid holding recovery in the bacteria . The results in this paper show that this is the case and that the increased UV-resistance is due entirely to an increase in the capacity of the cells for DNA excision repair . The latter arises wholly or in part from an increase in the intracellular level of the key enzyme of the pathway, UvrABC endonuclease.

Mutat Res, 1993 May, 302(1), 13 - 8
Mutation enhancing effect of o-vanillin in the lacZ gene of Escherichia coli: characterization of mutational spectrum; Watanabe K et al.; The enhancing effect of o-vanillin (2-hydroxy-3-methoxybenz-aldehyde) on mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was characterized with mutational specificity . The mutational spectrum of MNNG-induced mutations in the presence of o-vanillin was compared with that in the absence of o-vanillin by means of a series of mutant lacZ genes in E . coli that can detect each of the six types of base substitutions and five kinds of frameshift events . In the absence of o-vanillin, the mutational spectrum induced by MNNG consisted mainly of G.C-->A.T transitions and, to a lesser extent, -1(G.C) frameshift mutations . By adding o-vanillin at 75 micrograms/plate, a marked enhancement was observed in two transitions, G.C-->A.T and A.T-->G.C, and in two frameshift mutations, +1(G.C) and -1(G.C) . Only a small change was observed in the -2(C.G-G.C) fraction . Regarding the MNNG-induced frameshifts at the A.T base pair, the +1(A.T) frameshift was much more enhanced by o-vanillin than the -1(A.T) frameshift.

Mutat Res, 1993 May, 299(3-4), 233 - 50
Double-strand breaks in plasmid DNA and the induction of deletions; Schulte-Frohlinde D et al.; Double-strand breaks (dsbs) have been produced in plasmid DNA by various restriction endonucleases and the survival and the deletion mutation incidence have been measured in E . coli . The deletion formation is known to depend upon the occurrence of short direct repeats within the DNA molecule . In order to study the role of these repeats we constructed plasmid molecules with repeats of various lengths or with a 10-base pair repeat at different distances from each other . Furthermore the influence of the location and the structure of the dsb was studied . Repair and deletion frequencies of the linearized plasmids were measured after transformation of E . coli . The yield of the specific deletion mutation (the one which occurs between the introduced repeats) increases nearly linearly with the square of the length of the repeat, while the yield of the correctly repaired DNA and the yield of all other deletion mutants remained constant . The slope of the linear increase of the yield of the specific deletion depends on the location and the structure of the dsb . The yield of the specific deletion mutation decreases with increasing distance between the repeats . A proposal for the rate-determining step of the deletion formation is made.

Mutat Res, 1993 May, 299(3-4), 193 - 209
Multiple pathways of deletion formation in Escherichia coli; Balbinder E; We are investigating the mechanisms for deletion formation through the use of mutants which alter deletion frequency together with well characterized systems for deletion detection . We report here on three mutations which were isolated for their ability to stimulate deletions in plasmid pMC874 (dli mutations) . The mutation rec-2251 (formerly known as dli1) is a new allele of recBCD, a group of genes coding for the polypeptide components of the major recombination enzyme complex in E . coli; the second one, dli2 may be a new allele of uvrD, which codes for DNA helicase II; and the third one, dli3, has the phenotype of a mismatch repair mutation . Here we compare the effects of mutations in SOS-repair genes to those of the dli mutations on three different deletion events: (a) the deletion of short (60-100-bp) palindromic and non-palindromic inserts in derivatives of plasmid pBR325; (b) larger (600-800-bp) deletions in plasmid pMC874; and (c) the excision of the Tn10 transposon from chromosomal sites . Our results indicate that some form of SOS processing stimulates the loss of palindromes but not non-palindromes in plasmid pBR325 derivatives, and that RecA is necessary for UV-induced excision of Tn10 but this event is inhibited by UmuCD or its homolog MucAB . Each of the dli mutations showed unique effects on different classes of deletions . Mutation rec-2251 stimulated specifically deletions in pMC874 but had no effect on the deletion of non-palindromes in pBR325, and reduced the incidence of the other deletion events tested including loss of palindromic inserts in pBR325 as well as Tn10 excision . Mutation dli2, on the other hand, stimulated all deletions tested to varying extents, while dli3 did not affect markedly deletion formation in pBR325 plasmids but had a large stimulatory effect on both deletions in plasmid pMC874 and Tn10 excision . These results reveal that (a) some SOS-repair functions participate in deletion formation, (b) mutations selected for altering the incidence of one class of deletions may have totally different effects on other deletion events, and (c) the differences in mutant behavior may result in part from the ability of some pathways to discriminate among different deletion intermediates such as hairpins or cruciforms formed by palindromic sequences vs . transient secondary structures stabilized by direct repeats flanking non-palindromic sequences.

Mutat Res, 1993 May, 299(3-4), 147 - 56
Thymine ring saturation and fragmentation products: lesion bypass, misinsertion and implications for mutagenesis; Evans J et al.; We have used thymine glycol and dihydrothymine as representative ring saturation products resulting from free-radical interaction with DNA pyrimidines, and urea glycosides and beta-ureidoisobutyric acid (UBA) as models for pyrimidine-ring fragmentation products . We have shown that thymine glycol and the ring-fragmentation products urea and beta-ureidoisobutyric acid, as well as abasic sites, are strong blocks to DNA polymerases in vitro . In contrast, dihydrothymine is not a block to any of the polymerases tested . For thymine glycol, termination sites were observed opposite the putative lesions, whereas for the ring-fragmentation products, the termination sites were primarily one base prior to the lesion . These and other data have suggested that thymine glycol codes for an A, and that a base is stably inserted opposite the damage, whereas when a base is inserted opposite the non-coding lesions, it is removed by the 3-->5 exonuclease activity of DNA polymerase I . Despite their efficiency as blocking lesions, thymine glycol, urea and UBA can be bypassed at low frequency in certain specific sequence contexts . When the model lesions were introduced individually into single-stranded biologically active DNA, we found that thymine glycol, urea, beta-ureidoisobutyric acid, and abasic sites were all lethal lesions having an activation efficiency of 1, whereas dihydrothymine was not . Thus the in vitro studies predicted the in vivo results . When the survival of biologically active single-stranded DNA was examined in UV-induced Escherichia coli cells where the block to replication was released, no increase in survival was observed for DNA containing urea or abasic sites, suggesting inefficient bypass of these lesions . In contrast, beta-ureidoisobutyric acid survival was slightly enhanced, and transfecting DNA containing thymine glycols was significantly reactivated . When mutation induction by unique lesions was measured using f1-K12 hybrid DNA containing an E . coli target gene, thymine glycols and dihydrothymine were found to be inefficient as premutagenic lesions, suggesting that in vivo, as in vitro, they primarily code for A . In contrast, urea and beta-ureidoisobutyric acid were efficient premutagenic lesions, with beta-ureidoisobutyric acid being about 4-5-fold more effective than urea glycosides, which have approximately the same rate of mutation induction as abasic sites from purines . Sequence analysis of the mutations resulting from these ring-fragmentation products shows that the mutations produced are both lesion and sequence context dependent . The possible roles that bypass efficiency and lesion-directed misinsertion might play in mutagenesis are discussed.

Mutat Res, 1993 May, 299(3-4), 123 - 33
Site-directed mutagenesis in single cells: transitions produced by DNA carrying a single O6-alkylguanine residue; Chambers RW; Using a single burst assay based on a Poisson Distribution, I have determined the mutant virus frequency in single spheroplasts transfected with phi X174 form I' DNA carrying an O6-methyl-, ethyl-, n-propyl- or n-butylguanine residue at position 2401 of the minus strand . One set of experiments was performed with spheroplasts derived from Escherichia coli AB1157, which has normal DNA-repair systems . Of the cells examined after transfection with DNA carrying a methylguanine moiety, 30% produced mutant virus and 12% contained only mutants; with ethylguanine, 55% of the cells had mutants and 41% produced only mutants; with butylguanine, 6% of the cells had mutants and 3% contained only mutants; with propylguanine no mutants were detected in the 33 cells examined . In similar experiments carried out with spheroplasts defective in excision repair (E . coli AB1157 uvrA6) the percentage of cells producing mutant phage after transfection with DNA carrying an O6-butylguanine residue increased from 6 to 21%, and the percentage of cells producing only mutants increased from 3 to 8%; with DNA carrying an O6-methylguanine moiety, the percentage of cells producing mutants decreased from 30 to 6% and the percentage of cells producing only mutants fell from 12% to 0 . In order for an individual uvrA cell to produce exclusively mutant phage from a single O6-alkylguanine residue some form of selection must occur during replication because one strand of the transfecting DNA is wild-type, and excision repair, which could lead to a homoduplex with the transition in both strands, is defective in these cells . This selection must also occur in cells with normal DNA repair . The first event in the selection process is critical; if replication of the alkyl-DNA occurs first and if a mutation is produced, then there is a significant probability that the cell will produce only mutant virus regardless of whether or not repair occurs in subsequent events, but the frequency one observes is influenced strongly by the status of the repair systems in the cell.

J Bacteriol, 1993 May, 175(9), 2754 - 7
Specific regions of Escherichia coli OmpF protein involved in antigenic and colicin receptor sites and in stable trimerization; Fourel D et al.; Four different mutations were obtained by selecting for resistance to colicin N and screening for continued production of the OmpF protein of Escherichia coli . Two of them also conferred resistance to colicin A . The substitutions C for R-168 (R168C) and E284K caused the loss of the E21 epitope, while the transition G285D altered the E18, E19, and E20 antigenic sites . The substitution G119D drastically affected the stability of the trimeric conformation.

Infect Immun, 1993 May, 61(5), 2200 - 2
Membrane translocation and channel-forming activities of diphtheria toxin are blocked by replacing isoleucine 364 with lysine; Cabiaux V et al.; A mutant of diphtheria toxin in which Ile-364 was replaced by Lys was at least 500-fold less toxic to Vero cells than the parental toxin . Its ability to undergo low-pH-triggered translocation across the plasma membrane was greatly diminished, as was its ability to form ion-conductive channels . In addition, the mutant toxin was inactive in the pH-dependent killing of Escherichia coli.

Infect Immun, 1993 May, 61(5), 2003 - 10
A major outer membrane protein of Moraxella catarrhalis is a target for antibodies that enhance pulmonary clearance of the pathogen in an animal model; Helminen ME et al.; A murine immunoglobulin G monoclonal antibody (MAb) raised against outer membrane vesicles of Moraxella catarrhalis 035E was shown to bind to a surface-exposed epitope of a major outer membrane protein of this organism . This outer membrane protein, which had an apparent molecular weight of approximately 80,000 in sodium dodecyl sulfate-polyacrylamide gels, was designated CopB . MAb 10F3, reactive with CopB, bound to a majority (70%) of M . catarrhalis strains tested . More importantly, mice passively immunized with MAb 10F3 exhibited an enhanced ability to clear a bolus challenge of M . catarrhalis from their lungs, a result which suggested that CopB might have potential as a vaccine candidate . The M . catarrhalis gene encoding CopB was cloned in Escherichia coli, and nucleotide sequence analysis of the copB gene indicated that the CopB protein was synthesized with a leader peptide, a finding confirmed by N-terminal amino acid sequence analysis of the mature CopB protein purified from M . catarrhalis 035E . Southern blot analysis showed that chromosomal DNA from seven different M . catarrhalis strains hybridized with a probe comprising the majority of the copB structural gene from strain 035E . Additional data emphasizing the extent of conservation of the CopB protein among M . catarrhalis strains were obtained from Western immunoblot analyses with polyclonal antisera raised against CopB proteins from different M . catarrhalis strains used to probe the recombinant form of the CopB protein from strain 035E . The ability of the CopB protein to function as a target for biologically active antibodies and its apparent conservation among M . catarrhalis strains warrant further investigation of this outer membrane protein as a potential vaccine candidate.

Infect Immun, 1993 May, 61(5), 1772 - 8
Neutralizing monoclonal antibody epitopes of the Entamoeba histolytica galactose adhesin map to the cysteine-rich extracellular domain of the 170-kilodalton subunit; Mann BJ et al.; Entamoeba histolytica adheres to human colonic mucins and colonic epithelial cells via a galactose-binding adhesin . The adhesin is a heterodimeric glycoprotein composed of 170- and 35-kDa subunits . Fragments of the hgl1 gene encoding the 170-kDa subunit were expressed as recombinant fusion proteins in Escherichia coli and reacted with anti-adhesin monoclonal antibodies (MAbs) or pooled human immune sera . The MAbs tested recognize seven distinct epitopes on the 170-kDa subunit and have distinct effects on the adherence and complement-inhibitory activities of the adhesin . All seven MAbs reacted with a fusion protein containing the cysteine-rich domain of the protein . Pooled human immune sera reacted with the same cysteine-rich domain as the MAbs and also with a construct containing the first 596 amino acids . Reactivity of three MAbs with the surface of intact trophozoites confirmed that the cysteine-rich domain was located extracellularly . The location of individual epitopes was fine mapped by constructing carboxy-terminal deletions in the cysteine-rich region of the fusion protein . The locations of adherence-enhancing and -inhibiting epitopes were partially distinguished, and the epitopes where complement-inhibitory MAbs bound were demonstrated to be near the adhesin's area of sequence identity with the human complement inhibitor CD59.

Eur J Immunol, 1993 May, 23(5), 1064 - 71
B cell epitope on the U1 snRNP-C autoantigen contains a sequence similar to that of the herpes simplex virus protein; Misaki Y et al.; The mechanism of autoantibody production in autoimmune diseases is not well understood . In the present study we performed the B cell epitope mapping of the U1 small nuclear ribonucleoprotein (snRNP)-C, one of the target molecules of anti-nRNP autoantibody to investigate how B cells respond to the autoantigen . After cloning and expression of a full length complementary DNA (cDNA) encoding the U1-C protein, several truncated mutants of the cDNA were constructed and expressed in E . coli . Although a few epitopes were distributed on the whole molecule, all anti-C protein antibody-positive patients' sera tested recognized the region between amino acid residues 102 and 125 of the coding sequence . This universal epitope region contains an amino acid sequence similar to that of the herpes simplex virus type 1 ICP4 protein . The peptides representing each molecule were cross-reactive to each other . In addition this region cross-reacted to the B/B' protein . These observations suggest that molecular mimicry might be involved in the initiation of autoantibody production, followed by cross-reactive events between the autoantigens and by antigen-driven mechanisms to generate more complicated autoantibody patterns against the U1 snRNP complexes.

Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 948 - 53
Facile cloning and sequence analysis of goose delta-crystallin gene based on polymerase chain reaction; Yu CW et al.; To facilitate the cloning of delta-crystallin gene, the product of which is a major lens protein present in the avian and reptilian lenses, polymerase chain reaction (PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of goose lenses . The PCR product was then subcloned into pUC19 vector and transformed in E . coli strain JM109 . Plasmids purified from the positive clones were prepared for nucleotide sequencing by dideoxynucleotide chain-termination method . Sequencing several clones containing 1.4 kb DNA inserts encoding delta-crystallin constructed a complete and unambiguous full-length reading frame of 1401 base pairs covering a deduced protein sequence of 465 amino acids excluding the universal translation-initiating methionine . The goose delta-crystallin shows 88, 94, 88 and 69% sequence identity to pigeon delta, duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively . It is also shown that, similar to duck delta 2 and in contrast to pigeon delta crystallin, goose delta-crystallin appears to possess high argininosuccinate lyase activity despite the fact that a highly homologous structure is shared among these homologous proteins . Structural analysis and comparison of these closely related delta-crystallin homologues with or without enzymatic activity should be of value in unraveling the intriguing evolutionary process leading to the development and evolution of active enzymatic crystallins in the lenses of certain avian species.

Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 738 - 46
Comparison of the disulfide bond arrangements of human recombinant and bovine brain heparin binding neurite-promoting factors; Hulmes JD et al.; Heparin binding neurite-promoting factor (HBNF) is a highly basic 136 amino acid protein containing 10 cysteine residues . We have determined the redox status and the disulfide arrangement of the cysteine residues in HBNF from bovine brain and refolded human recombinant protein produced in E . coli . Our data indicate that all 10 cysteines are involved in disulfide bond formation . The disulfide linkages of human recombinant and bovine brain HBNF, as determined after proteolytic digestions of the non-reduced proteins by peptide mapping and sequence analysis are: Cys15-Cys44, Cys23-Cys53, Cys30-Cys57, Cys67-Cys99 and Cys77-Cys109 . Thus, recombinant HBNF has the same disulfide arrangement as the native brain-derived protein.

Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 720 - 7
Bacterially expressed murine CSF-1 possesses agonistic activity in its monomeric form; Krautwald S et al.; CSF-1 is a dimeric peptide growth factor, stabilized by disulfide bonds . We expressed mouse CSF-1 in bacteria as a fusion protein either with glutathione S-transferase (GST) or with a six histidine tag (His-tag) . Large amounts of recombinant material were obtained and purified by a single affinity chromatography step . Purified CSF-1-His-tag monomers efficiently dimerized in vitro, but the presence of variable amounts of GST-moiety in CSF-1 preparations obtained by thrombin cleavage of GST-fusion proteins (thrombin-released CSF-1) interfered with dimerization . However, the thrombin-released CSF-1 monomers possessed agonistic activity, being capable of stimulating tyrosine phosphorylation of the CSF-1 receptor and of an array of cellular proteins in living macrophages and of supporting their growth . These results show that CSF-1 dimerization is not essential for receptor activation in vivo.

Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 373 - 80
Isolation and characterization of four basic proteins from horse eosinophilic granules; Piller K et al.; Four new basic proteins were isolated from horse eosinophils and purified . The eosinophils release these proteins after permeabilization with saponin and degranulation stimulized by guanosine 5'-O-thiotriphosphate . The proteins were separated and purified on a Superose P12- and a Mono S-column by fast protein liquid chromatography . The amino acid composition, the relative molecular mass, the isoelectric point and the partial N-terminal sequence of the four proteins were determined . Papain-activation and ribonuclease activity of the four proteins were tested for comparison with the human eosinophil basic granular proteins . The cytotoxicity of the hole granular extract and of the isolated basic proteins against Escherichia coli K12 was also studied.

Gene, 1993 Apr 30, 126(2), 251 - 5
Cloning of a cDNA encoding a DNA-binding protein TAXREB302 that is specific for the tax-responsive enhancer of HTLV-I; Nyunoya H et al.; The transcriptional activator, Tax, of human T-cell leukemia virus (HTLV-I) has been considered to interact with cellular proteins to act on target enhancer motifs . Using oligodeoxyribonucleotides containing the tax-responsive element (TAXRE) of the HTLV-I enhancer, we have cloned multiple cDNAs coding for TAXRE-binding proteins (TAXREB), and determined the cDNA and the deduced 200-amino-acid sequences for TAXREB302 . The recombinant protein binds to the enhancer DNA by specific interaction to the CRE-like sequence . A single 1.8-kb species of mRNA was detected in cultured cells, as well as in normal human tissues, especially brain and skeletal muscle . The 22-kDa native protein was detected in the cultured-cell lysate by immunoblotting analysis . TAXREB302 does not have structural features common to the CRE-binding protein or activating transcription factor (CREB/ATF) family, but has homology to chicken erythroid transcription factor (Eryf1 or GATA-1), suggesting a possible protein-protein interaction.

Neurosci Lett, 1993 Apr 30, 153(2), 157 - 60
Immunocytochemical localization of rat substance P receptor in the striatum; Shigemoto R et al.; A trp E fusion protein containing a C-terminal portion of the rat substance P receptor (SPR) was expressed in bacteria and used to produce an antibody . The antibody specifically reacted with SPR expressed in a mammalian cell line and rat striatum . Light and electron microscope analyses of the rat striatum revealed intense SPR-like immunoreactivity in neuronal somata and dendrites . These immunoreactive neurons constituted approximately 3% of the total population of striatal neurons; they were putative interneurons of large and medium-sized aspiny type.

Nature, 1993 Apr 29, 362(6423), 860 - 2
Yeast DNA repair and recombination proteins Rad1 and Rad10 constitute a single-stranded-DNA endonuclease; Tomkinson AE et al.; Damage-specific recognition and incision of DNA during nucleotide excision repair in yeast and mammalian cells requires multiple gene products . Amino-acid sequence homology between several yeast and mammalian genes suggests that the mechanism of nucleotide excision repair is conserved in eukaryotes, but very little is known about its biochemistry . In the yeast Saccharomyces cerevisiae at least 6 genes are needed for this process, including RAD1 and RAD10 (ref . 1) . Mutations in the two genes inactivate nucleotide excision repair and result in a reduced efficiency of mitotic recombinational events between repeated sequences . The Rad10 protein has a stable and specific interaction with Rad1 protein and also binds to single-stranded DNA and promotes annealing of homologous single-stranded DNA . The amino-acid sequence of the yeast Rad10 protein is homologous with that of the human excision repair gene ERCC1 (ref . 3) . Here we demonstrate that a complex of purified Rad1 and Rad10 proteins specifically degrades single-stranded DNA by an endonucleolytic mechanism . This endonuclease activity is presumably required to remove non-homologous regions of single-stranded DNA during mitotic recombination between repeated sequences as previously suggested, and may also be responsible for the specific incision of damaged DNA during nucleotide excision repair.

Nature, 1993 Apr 29, 362(6423), 844 - 6
Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo; Nabel EG et al.; The prototype members of the heparin-binding fibroblast growth factor (FGF) family, acidic FGF (FGF-1) and basic FGF (FGF-2), are among the growth factors that act directly on vascular cells to induce endothelial cell growth and angiogenesis . In vivo, the role of the FGF prototypes in vascular pathology has been difficult to determine . We report here the introduction, by direct gene transfer into porcine arteries, of a eukaryotic expression vector encoding a secreted form of FGF-1 . This somatic transgenic model defines gene function in the arterial wall in vivo . FGF-1 expression induced intimal thickening in porcine arteries 21 days after gene transfer, in contrast to control arteries transduced with an Escherichia coli beta-galactosidase gene . Where there was substantial intimal hyperplasia, neocapillary formation was detected in the expanded intima . These findings suggest that FGF-1 induces intimal hyperplasia in the arterial wall in vivo and, through its ability to stimulate angiogenesis in the neointima, FGF-1 could stimulate neovascularization of atherosclerotic plaques . Potentially, gene transfer of FGF-1 could also be used as a genetic intervention to improve blood flow to ischaemic tissues in selected clinical settings.

Biochemistry, 1993 Apr 27, 32(16), 4466 - 73
Mechanisms of mutation by oxidative DNA damage: reduced fidelity of mammalian DNA polymerase beta; Feig DI et al.; Reactive oxygen species, produced in cells by a variety of mechanisms, damage DNA and cause mutations . To characterize the types of mutations produced in mammalian cells, we copied DNA damaged by reactive oxygen species with mammalian DNA polymerase beta . Double-stranded circular M13mp2 DNA containing a 361-nucleotide single-stranded gap within the lacZ gene was damaged by aerobic incubation with Fe2+ and H2O2 . The gap then was filled by purified recombinant rat DNA polymerase beta, and the DNA was transfected into Escherichia coli . Mutations within the nonessential lacZ gene for beta-galactosidase were identified by reduced alpha-complementation . In this system, oxidative damage increased the mutation frequency within the target region by an average of 4.3-fold . At certain sites, the base substitution rate is nearly 300 times greater than would be expected to result from a random distribution of damage . The oxidatively induced mutations fall into two categories: those apparently caused by direct miscoding of modified DNA and those associated with enhanced misincorporation at prexisting polymerase-specific hot spots . The latter group may be due to a conformational change in the DNA caused by oxidative modification and could be indicative of a novel mutagenic mechanism.

Biochemistry, 1993 Apr 27, 32(16), 4455 - 60
Structure-function relationship of a recombinant human galactoside-binding protein; Ochieng J et al.; A galactoside-binding lectin (hL-31) containing a collagen-like sequence was identified in human tumor cells . It was found to be the homologue of the IgE-binding protein, the macrophage cell-surface Mac-2 antigen, and the murine CBP35, RL-29, and mL-34 lectins . Here we report on the expression in Escherichia coli and functional analysis of recombinant hL-31 (rhL-31) . The rhL-31 was purified in one step through an asialofetuin affinity column . The rhL-31 was reactive to anti-lectin antibodies and retained its lactose-dependent hemagglutination of trypsin-treated glutaraldehyde-fixed rabbit erythrocytes . The rhL-31 elutes from an affinity column as a 31-kDa monomer and undergoes homodimerization at relatively high protein concentrations, comparable to those used to mediate hemagglutination . Electron microscopy showed that the rhL-31 appears as a Y-shaped structure . Lactoperoxidase-catalyzed iodination of murine tumor cell-surface proteins followed by collagenase treatment revealed that the lectin is probably a peripheral membrane protein whereby both the amino and the carboxy termini are exposed on the outer cell membrane . These results point to the membrane disposition and orientation of the lectin and suggest a mechanism for a structure-function relationship of lectin activity.

Biochemistry, 1993 Apr 27, 32(16), 4338 - 43
Direct evidence for the exploitation of an alpha-helix in the catalytic mechanism of triosephosphate isomerase; Lodi PJ et al.; In previous work, we have shown that the first (and, presumably, the second) pKa of the active-site histidine-95 in triosephosphate isomerase has been lowered by about 2 units {Lodi, P . J., & Knowles, J . R . (1991) Biochemistry 30, 6948-6956} . One reason for the perturbed pKa of this residue appears to be its location at the N-terminus of a short alpha-helix that runs from residues 95 to 102 . Fortuitously, the residue at the C-terminus of this helix is also a histidine residue (histidine-103), and the existence of a histidine side chain at each end has allowed us directly to implicate the helix in the perturbation of the pKa value of histidine-95 . 15N NMR titration studies of the native enzyme and 13C NMR titration studies of the denatured enzyme show that while the pKa of histidine-95 is lowered by a least 2 units in the folded versus the unfolded state, the pKa of histidine-103 is raised by about 0.6 unit on protein folding . These complementary effects on the pKa values of histidine-95 and histidine-103 suggest that the alpha-helix is indeed responsible for the perturbation of the pKa values . The larger effect on the pKa of histidine-95 is readily rationalized in terms of the local structure of the enzyme . The disparity in the perturbation for the two histidine side chains illustrates how an alpha-helix can be functionally utilized by proteins, directly to affect (as in the present case) the chemistry of catalysis by an enzyme.

Biochemistry, 1993 Apr 27, 32(16), 4322 - 9
Engineered disulfide bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation; Clarke J et al.; Disulfide bridges have been introduced into barnase to act as probes of folding . One disulfide (between residues 85 and 102) links two loops known to pack together early in the folding pathway . A second disulfide bond (between residues 43 and 80) links two elements of secondary structure known to pack together only after the rate-determining step of folding . The disulfide-bridged proteins are more stable than wild-type by 4.1 and 1.2 kcal mol-1, respectively . The kinetics of unfolding and refolding of the mutant proteins has been measured, and a comparison of the disulfide proteins and their corresponding dithiol forms has been made by use of thermodynamic cycles . These data have been used to construct folding profiles of the disulfide proteins . The disulfide bond engineered into the part of the protein that folds early confers stability upon the intermediate and transition states of folding . The protein with a disulfide bond connecting parts of the protein that fold late is not stabilized until the protein reaches its final folded form . Conversely, in the unfolding pathway, the rate of unfolding of this mutant is lowered considerably . This points to a method of decreasing the rate of denaturation of proteins that are used in medical and biotechnological applications: elements of structure that unfold in or before the rate-determining step of overall unfolding may be stabilized and so slow down the overall unfolding process . The barnase mutant linked between Cys 43 and Cys 80, for example, unfolds 20 times slower than wild-type and 170 times slower than the reduced protein.

Biochemistry, 1993 Apr 27, 32(16), 4314 - 21
Three-state denaturation of DnaK induced by guanidine hydrochloride . Evidence for an expandable intermediate; Palleros DR et al.; The denaturation of the heat shock protein DnaK induced by guanidine hydrochloride (Gdn-HCl) was investigated by circular dichroism, fluorescence, size-exclusion HPLC, and dynamic light scattering . DnaK unfolding takes place in two discrete steps . The midpoint (Cm) of the first transition (0.5 M) was shifted to higher denaturant concentrations (0.8 M) in the presence of Mg/ADP or Mg/ATP, whereas the second transition (Cm = 1.6 M) was unaffected by nucleotides . An intermediate state which continuously expands with increasing Gdn.HCl concentration was observed; its relation to molten globules is discussed . In addition, a direct correlation between molecular volume and ellipticity at 222 nm was found, regardless of the conformational state (native, intermediate, unfolded); the implications of these findings are discussed . The unfolding of DnaK is best explained by a hierarchical model of unfolding.

Biochemistry, 1993 Apr 27, 32(16), 4281 - 5
Kinetic analysis of T7 RNA polymerase transcription initiation from promoters containing single-stranded regions; Maslak M et al.; T7 RNA polymerase is highly specific for the initiation of transcription from a relatively small consensus promoter sequence . Previous footprinting studies suggested that the enzyme binds specifically to a fully closed duplex form of the promoter, recognizing functional groups along one face of the helix {Muller, D . K., Martin, C . T., & Coleman, J . E . (1989) Biochemistry 28, 3306-3313} . Steady-state kinetic analysis of oligonucleotide-based promoters shows that removal of the nontemplate strand completely within the message region of the DNA (positions +1 through +5) results in no change in binding (as reflected in the parameter Km) and a 2-fold increase in kinetics (as reflected in kcat) . Further deletion of the nontemplate strand as far upstream as position -4 has no effect on binding, and although deletion upstream through position -6 weakens binding, specific initiation continues at a high rate . The temperature dependence of the initiation kinetics shows a single apparent activation energy of approximately 26 kcal/mol for the fully duplex promoter . Similar measurements on the promoter lacking the nontemplate strand in the message region show that less than 10% of this barrier is related to melting of the downstream region of the promoter . These results lead us to revise the previous model for recognition to include specific binding to a form of the promoter which is duplex upstream of about position -6 and melted downstream through the start site . Within the melted region, the polymerase interacts significantly only with the template strand of the promoter DNA.

Biochemistry, 1993 Apr 27, 32(16), 4275 - 80
Identification of specific contacts in T3 RNA polymerase-promoter interactions: kinetic analysis using small synthetic promoters; Schick C et al.; The T7, T3, and SP6 RNA polymerases recognize very similar, yet distinct, promoter sequences . The high homology among the promoter sequences suggests that differential promoter recognition must derive from relatively small changes in the protein . Steady-state kinetic analyses of transcription from the T3 consensus promoter and from promoters modified in the region critical to specific recognition reveal details concerning which functional groups contribute to this recognition . Modifications include base pair substitutions, single base substitutions (mismatches), and simple functional group modifications at unique sites in the promoter . The results show that T3 RNA polymerase recognizes the amino group on the nontemplate cytidine in the major groove at position -10, while the identity of the base on the template strand is less critical to binding . In contrast, recognition at position -11 allows a greater range of modifications and seems to have a more complex recognition . The results do not seem to be consistent with a single recognition contact at this position; however, some groups may be ruled out as simple recognition contacts . While major groove modifications weaken binding at positions -10 and -11, the removal of an exocyclic amino group from the minor groove at either position does not disrupt binding, further supporting a model for promoter recognition in which the enzyme binds to one face of closed duplex DNA in this region . The effects of these changes in the DNA structure on the kinetics of initiation are compared to complementary results from the T7 system.

Biochemistry, 1993 Apr 27, 32(16), 4225 - 30
Transforming the Escherichia coli Trp repressor into a site-specific nuclease; Sutton CL et al.; The Escherichia coli Trp repressor has been converted into an operator-specific nuclease by alkylating cysteine-49, inserted by site-directed mutagenesis, with 5-(iodoacetamido)-1,10-phenanthroline . In the presence of copper ion and thiol, high yields (> 50%) of double-stranded breaks of DNA are observed after a 20-min reaction . The high cleavage efficiency of this derivatized protein (Trp repressor E49C-OP) can be attributed to the proximity of cysteine-49 to the minor groove, the site of the C-1H of the deoxyribose and the target of the oxidative nuclease activity of (1,10-phenanthroline)copper . Since sequence position 49 is close to the protein's C2 dyad axis and adjacent to the minor groove, Trp repressor E49C-OP reacts with the operator DNA near the binding site of this symmetry locus of the protein . The patterns of scission of the trpR, aroH, and trpEDCBA operators (a) confirm the orientation of the repressor to the operator predicted from the X-ray study of a cocrystal (Otwinowski et al., 1988) and (b) support the model for tandem binding of the repressor to the trpR, aroH, and trpEDCBA operators based on DNase I footprinting and methylation interference (Kumamoto et al., 1987) . There are one, two, and three binding sites for the repressor on the trpR, aroH, and trpEDCBA operators, respectively . In addition to providing a novel approach to studying the interactions of DNA binding proteins, 1,10-phenanthroline-derivatized proteins such as Trp repressor E49C-OP may be useful as rare cutters in the analysis of high molecular weight DNAs, especially if their binding specificities can be altered.

Biochemistry, 1993 Apr 27, 32(16), 4430 - 43
Comprehensive explanation of the anomalous EPR spectra of wild-type and mutant cytochrome c peroxidase compound ES; Houseman AL et al.; Although the cytochrome c peroxidase/H2O2 reaction product, compound ES, has been a long-standing subject of research, only recently has its broad EPR signal been proven to arise from a radical at Trp-191 . Despite this advance, no model has satisfactorily explained the anomalous breadth and shape of this signal, which is conventionally interpreted as having axial symmetry with g parallel approximately 2.04 > g perpendicular approximately 2.01, contrary to expectations for a planar pi radical . Furthermore, these g values exhibit marked temperature and preparation dependencies as well as an unexplained high-field "tail" extending from the g = 2.01 peak . We have reexamined the EPR and ENDOR spectra of compound ES at 35 GHz, as well as those of compound ES in the mutant D235E . This mutation significantly alters the line shape of the Trp-191 free radical . We present a comprehensive model that successfully accounts for the properties of this unusual protein free radical . We show that the EPR spectra of both proteins can be described in terms of a weak exchange interaction between the S = 1 oxyferryl (Fe = O)2+ moiety and a radical on Trp-191; a distribution in protein conformation leads to a distribution in the coupling, which ranges from ferromagnetic to antiferromagnetic . We also derive, for the first time, explicit expressions for frozen-solution and single-crystal spectra of such spin-coupled systems and show that the model accounts for all the data that previously led to apparent anomalies in the interpretation of the frozen-solution and single-crystal {Hori, H., & Yonetani, T . (1985) J . Biol . Chem . 260, 349-355} EPR properties . Finally, we have used the CW EPR and pulsed-EPR saturation-recovery methodology to address reports that the broad signal from the spin-coupled Trp-191 radical is accompanied by a minority (approximately 10%), narrow signal that is associated with a radical site other than Trp-191 . We find no evidence for such a species and discuss the earlier reports in light of our model.

Biochemistry, 1993 Apr 27, 32(16), 4403 - 10
Characterization of mutations in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides that affect the quinone reductase site (Qc); Hacker B et al.; The cytochrome b subunit of the bc1 complex contains two heme components, cytochrome bL and cytochrome bH, and is the locus of both a quinol oxidizing site (Qo or Qz) and a quinone reducing site (Qi or Qc) . The quinone reductase site has been previously characterized as the site of interaction for a set of inhibitors including antimycin A, diuron, funiculosin, and HQNO . In this paper, four highly conserved residues in the cytochrome b subunit of Rhodobacter sphaeroides (A52, H217, K251, and D252) were targeted for site-directed mutagenesis . These residues were chosen as being likely to be at or near the quinone reductase site, on the basis of known locations of missense mutations in the homologous yeast subunit that confer resistance to Qc-directed inhibitors . The site-directed mutants all exhibit a normal rate of reduction of cytochrome bH, suggesting a fully functional quinol oxidizing site . However, each of the mutants is impaired, to varying degrees, in the rate of reoxidation of cytochrome bH . Two mutants (H217A and D252A) are unable to grow photosynthetically, indicating a severe defect in the bc1 complex . In both cases, the cause of the defect is the lack of reoxidation of cytochrome bH by ubiquinone . This is the first report of mutations that selectively impair the rate of electron transfer from cytochrome bH to the Qc-site . This set of mutations will be useful not only for modeling the structure of the quinone reducing site but also in elucidating the catalytic mechanism of this portion of the Q-cycle.

FEBS Lett, 1993 Apr 26, 321(2-3), 267 - 73
The structure of neutrophil defensin genes; Linzmeier R et al.; Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils, in rabbit alveolar macrophages, and in human and murine intestinal Paneth cells . We cloned and sequenced the genes of three neutrophil-specific defensins . Human HNP-1 and HNP-3 are nearly identical and rabbit NP-3a is closely related . The four known neutrophil-specific defensin genes are strikingly similar in the structure and organization of their three exons and two introns, but the three defensin genes expressed in macrophages (MCP-1 and -2) or Paneth cells (HD-5) are organized differently: HD-5 had only two exons, and MCP-1 and -2 have a comparatively short first intron . The diverse genomic organization of defensin genes may contribute to their cell-specific expression.

FEBS Lett, 1993 Apr 26, 321(2-3), 233 - 6
The ZNF35 human zinc finger gene encodes a sequence-specific DNA-binding protein; Pengue G et al.; We developed a rapid method to determine DNA-binding sites for putative DNA-binding proteins . This procedure has been successfully used to define a specific consensus site for the human ZNF35 zinc finger gene . ZNF35 encodes a 58-kDA polypeptide containing 11 consecutive finger motifs located at the amino terminus, and an acidic domain located at the carboxy terminus . These features suggest that ZNF35 is a site-specific DNA-binding protein involved in the regulation of gene expression . We have expressed the ZNF35 protein from E . coli and have employed a Southwestern-polymerase chain reaction method using random oligonucleotides to identify its high-affinity binding site . The core sequence for the ZNF35 protein-binding site is 5'-C/GC/GAAG/TA-3'.

FEBS Lett, 1993 Apr 26, 321(2-3), 169 - 72
Stabilization of mRNA in an Escherichia coli cell-free translation system; Hirao I et al.; It became clear that mRNA can be stabilized in a cell-free translation system of Escherichia coli by hybridization with a small DNA fragment at its 3' terminus . The stability increased when a small DNA fragment containing a stable hairpin structure with a GAAA loop was used . The enhancement of stabilization was brought about because the hairpin structure is resistant towards the nucleases contained in the translation system . The hairpin structure is effective by stabilizing the added DNA fragment itself towards the nucleases.

FEBS Lett, 1993 Apr 26, 321(2-3), 149 - 52
Divergent effects of fluoroaluminates on the peptide chain elongation factors EF-Tu and EF-G as members of the GTPase superfamily; Mesters JR et al.; Fluoraluminates are thought to mimic the gamma-phosphate of GTP and thus, together with GDP, perturb the functioning of heterotrimeric GTP-binding G-proteins . Here we show they do inhibit the ribosome-stimulated GTPase activity of EF-G from Escherichia coli via the formation of a stable complex with EF-G-GDP and ribosomes . In contrast, no perturbed interactions were observed in a similar ribosomal complex with EF-Tu . Interestingly, in the absence of ribosomes both EF-Tu an EF-G remain totally unaffected by fluoraluminates . For members of the GTPase superfamily such differential effects have not been described before.

FEBS Lett, 1993 Apr 26, 321(2-3), 107 - 10
Secondary structure of the oct-3 POU homeodomain as determined by 1H-15N NMR spectroscopy; Morita EH et al.; Most of the 1H and 15N magnetic resonances of the 66 amino acid long POU homeodomain of mouse Oct-3 have been assigned by the combined use of the two-dimensional homonuclear, and two- and three-dimensional heteronuclear NMR methods . The sequential NOE connectivities and amide proton exchange measurements indicate the presence of three helical regions within the domain . The positions of the three helices correspond well to those of other homeodomains, the three-dimensional structures of which have already been determined . The present NMR study provides the first experimental evidence for the existence of a helix-turn-helix motif in the oct-3 POU homeodomain.

FEBS Lett, 1993 Apr 26, 321(2-3), 215 - 8
Diethyldithiocarbamate inhibits induction of macrophage NO synthase; Mulsch A et al.; We investigated whether sodium diethyldithiocarbamate (DETC), an inhibitor of the nuclear transcription factor kappa B (NFkappa B), modulates induction of NO synthase (NOS) in murine bone marrow-derived macrophages . A short exposure (between 1 and 16 h) of L929-cell medium-preconditioned macrophages to E . coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite . DETC (0.1-1 mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity . These findings suggest that NFkappa B is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of the gene encoding inducible NOS.

FEBS Lett, 1993 Apr 26, 321(2-3), 111 - 5
Cloning and sequencing of rat liver cDNAs encoding the regulatory protein of glucokinase; Detheux M et al.; cDNAs encoding the rat liver regulatory protein of glucokinase were cloned and sequenced . The deduced protein contains 568 amino acids for a molecular mass of 62,867 Da . Northern blot analysis showed the presence of a major RNA species of 2.35 kb in rat liver . No signal was observed with muscle, brain, heart, testis, intestine or spleen RNA . Recombinant regulatory protein expressed in Escherichia coli was insoluble and inactive, and was presumably contained in inclusion bodies . Western blot analysis showed that the recombinant protein was recognized by antibodies raised against regulatory protein purified from rat liver.

Nucleic Acids Res, 1993 Apr 25, 21(8), 1837 - 43
Analysis of effects of tRNA:message stability on frameshift frequency at the Escherichia coli RF2 programmed frameshift site; Curran JF; The codon that is in-frame prior to +1 frameshifting at the E.coli prfB (RF2 gene) frameshift site is randomized to create thirty-two variants . These alleles vary 1000-fold in frameshift-dependent expression in fusions to lacZ . Frameshifting is more frequent at sites where the in-frame codon ends in uridine, as if third position wobble pairs to message uridine facilitate slippage into the +1 frame . Consistent with other studies of programmed frameshift sites, efficient frameshifting depends on stable message:tRNA base pairs after rephasing . For complexes with mispairs, frameshift frequency depends on the nature, number, and position of mispairs . Central purine:purine mispairs are especially inhibitory . Relative stabilities of +1 rephased complexes are estimated from published data on the stabilities of tRNA:tRNA complexes . Stability correlates with frameshifting over its entire range, which suggests that stability is an important determinant of the probability of translation of the rephased complex.

Nucleic Acids Res, 1993 Apr 25, 21(8), 1727 - 34
Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B; Matthews JR et al.; A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B . Differential labelling with {14C} iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein . To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined . Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity . Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly . Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E . coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA . Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.

J Biol Chem, 1993 Apr 25, 268(12), 9063 - 70
Fpg protein of Escherichia coli is a zinc finger protein whose cysteine residues have a structural and/or functional role; O'Connor TR et al.; The Fpg protein of Escherichia coli is a DNA repair enzyme with DNA glycosylase, abasic site nicking, and deoxyribose excising activities . Analysis of the amino acid sequence of this protein suggests that the Fpg protein is a zinc finger protein with a Cys-X2-Cys-X16-Cys-X2-Cys motif . Competition experiments show that the Fpg protein substitutes Cu(II), Cd(II), and Hg(II), metal ions classically associated with substitutions in zinc finger proteins . The Fpg protein activities are inhibited following the reaction with a Cys-specific reagent at low protein:reagent ratios, suggesting that these residues are important for the enzymatic activities . Site-directed mutagenesis was used to produce 6 mutant Fpg proteins with Cys-->Gly mutations . Substitution of the zinc in these proteins by 65Zn(II) indicates that all the proteins bind zinc, but the Zn(II) is not retained as strongly in the zinc finger mutants . The mutations in the Fpg protein outside the zinc finger consensus sequence do not eliminate the Fapy-DNA glycosylase and abasic site nicking . One of the Fpg mutant proteins outside the zinc finger has a reduced capacity to release deoxyribose from abasic sites . Cys-->Gly mutations in the zinc finger consensus sequence reduce all three aforementioned activities substantially . The purified Fpg proteins with Cys-->Gly mutations in the zinc finger consensus sequence do not incise DNA at abasic sites with the same efficiency nor mechanism as the native Fpg protein . The wild type Fpg protein and the Fpg proteins mutated outside the zinc finger sequence bind an oligonucleotide with a unique chemically reduced abasic site in a defined sequence as assayed by retention on nitrocellulose filters, whereas the mutant Fpg proteins within the zinc finger sequence do not bind to the same oligonucleotide . Therefore, the disruption of zinc coordination in the zinc finger of the Fpg protein is associated with decreased binding capacity to DNA as well as decreased enzymatic activities.

J Biol Chem, 1993 Apr 25, 268(12), 8972 - 9
Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor by proline; Brown ED et al.; The PutA protein is both the put repressor and a membrane-bound enzyme with proline and delta 1-pyrroline-5-carboxylate dehydrogenase activities . The conditions required for association of purified PutA protein with membrane vesicles suggested that a redox switching mechanism might determine the proportion of PutA protein functioning as a dehydrogenase (Wood, J . M . (1987) Proc . Natl . Acad . Sci . USA 84, 373-377) . The FAD cofactor was released from the PutA protein with 1 M KBr at neutral pH . The apoprotein retained delta 1-pyrroline-5-carboxylate dehydrogenase and DNA binding but not proline dehydrogenase activity . Reconstitution with FAD fully restored proline dehydrogenase activity . Proline at a concentration of 0.11 mM caused half-maximal bleaching of the FAD in PutA . Chymotryptic digestion of the PutA protein in the presence and absence of proline demonstrated that the persistence of a 119-kDa protein fragment was characteristic of the reduced protein . Identical digestion patterns were obtained from the apoprotein in the presence and absence of proline . The quantity of the 119-kDa fragment produced varied with proline concentration, yielding a midpoint of 0.056 mM proline . The fraction of PutA protein associated with membrane vesicles was also a function of proline concentration, yielding a titration midpoint of 0.10 mM proline . Membrane binding was thus coincident with both flavin reduction and a change in protein conformation.

J Biol Chem, 1993 Apr 25, 268(12), 8769 - 76
Properties and regulation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans; Lu KP et al.; NIMA is the protein product of the nimA gene of the filamentous fungus Aspergillus nidulans, required for progression of cells from G2 into mitosis . The protein kinase activity of NIMA, assayed by phosphorylation of beta-casein, varies during the nuclear division cycle, reaching a maximum in late G2 and M . To investigate the biochemical properties of this cell cycle-regulated protein kinase, we have expressed nimA cDNA that encodes full-length NIMA in Escherichia coli as a fusion product with glutathione S-transferase . Purified NIMA phosphorylated beta-casein, with a Km of 38 microM and Vmax of 156 nmol/min/mg . NIMA also demonstrated a Km of 69 microM for ATP . Both recombinant and cellular NIMA kinases behaved as oligomers on gel filtration chromatography, and their kinase activities were strongly inhibited by various salts . By using both protein and peptide substrates, NIMA demonstrated a serine/threonine-specific protein kinase activity . Cellular NIMA exists as a phosphoprotein, and bacterially expressed NIMA was also phosphorylated on multiple serine/threonine residues . Some of these phosphorylations appeared essential for NIMA activity as the enzyme could be dephosphorylated and inactivated in vitro by protein serine/threonine phosphatases . Use of a kinase-negative mutant of NIMA revealed that the NIMA enzyme undergoes autophosphorylation when expressed at high concentrations in bacteria . Taken together, these data suggest that cellular mechanisms may exist to regulate the phosphorylation state and activity of the NIMA protein kinase during the nuclear division cycle in A . nidulans.

J Biol Chem, 1993 Apr 25, 268(12), 8717 - 26
Mutagenic analysis of AMP nucleosidase from Escherichia coli . Deletion of a region similar to AMP deaminase and peptide characterization by mass spectrometry; Kvalnes-Krick K et al.; AMP nucleosidase (EC 3.2.2.4) from Escherichia coli and AMP deaminase (EC 3.5.4.6) from bakers' yeast are proposed to regulate cellular AMP levels under allosteric control of the activator ATP and the inhibitor, PO4 . Both enzymes contain catalytic sites which bind AMP and regulatory sites which bind ATP . The deduced amino acid sequences of the proteins revealed only one region of homology in which six of eight amino acids are identical . A similar sequence is found in glyceraldehyde-3-phosphate dehydrogenase, phoE, ras proteins, RNA polymerase, K(+)-ATPase, nucleolin, and other proteins expected to have nucleotide or phosphate binding properties . In the crystal structure of glyceraldehyde-3-phosphate dehydrogenase, this sequence is part of the NAD(+)-binding site . The function of these amino acids was explored with a deletion mutant of AMP nucleosidase . The protein was over-produced in a pTZ construct using the AMP nucleosidase promoter which resulted in approximately 30% of the total protein as the desired enzyme . The mutation was characterized by DNA sequence analysis and by direct analysis of the peptides using high performance liquid chromatography-mass spectrometry . Deletion of amino acids 128-135, corresponding to DGSELTLD, produced an enzyme with a 20-fold decrease in Vmax but with smaller changes in substrate saturation kinetics, activation by MgATP, inhibition by inorganic phosphate, and inhibition by the tight-binding inhibitor, formycin 5-phosphate . The deletion mutant of AMP nucleosidase exhibits hysteresis in establishing a steady-state rate of product formation which is most pronounced in the absence of MgATP . These results establish that the sequence DGSELTLD in E . coli AMP nucleosidase is not required for binding of AMP, MgATP, or inorganic phosphate . However, the mutant enzyme has a structural defect related to the polymerization state which delays the onset of catalysis and decreases the catalytic efficiency.

J Biol Chem, 1993 Apr 25, 268(12), 8590 - 5
Isolation, sequence analysis, and cloning of haemadin . An anticoagulant peptide from the Indian leech; Strube KH et al.; A slow, tight-binding inhibitor of thrombin with an apparent molecular mass of about 5 kDa has been isolated from Haemadipsa sylvestris, an Indian leech of the family of Haemadipsidae . The inhibitory activity, called haemadin, is thrombin specific since it does not inhibit other proteases like trypsin, chymotrypsin, factor Xa, or plasmin . NH2-terminal amino acid sequence analysis (residues 1-45) does not reveal any homology to known serine protease inhibitors, including the thrombin-specific inhibitor hirudin . The haemadin cDNA cloned by polymerase chain reaction techniques codes for a polypeptide of 57 amino acid residues preceded by 20 residues of a signal peptide sequence . A synthetic gene coding for the mature haemadin was expressed in Escherichia coli . Recombinant haemadin displays a similar inhibition constant and specific activity as its natural counterpart . Although there is no obvious sequence identity between haemadin and hirudin, both proteins seem to share common mechanisms for thrombin inhibition.

J Biol Chem, 1993 Apr 25, 268(12), 8429 - 35
His865 is the catalytically important histidyl residue of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase; Darnay BG et al.; Involvement in catalysis of a histidyl residue of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was suggested by the ability of diethyl pyrocarbonate to abolish catalytic activity, accompanying spectral changes, and reactivation by hydroxylamine . The 7 histidines present in the catalytic domain of the hamster enzyme were changed to glutamine (His474, His487, His634, His751, His860, and His865), lysine (His865), or tyrosine (His868) . Overexpression in Escherichia coli yielded six soluble mutant proteins, one insoluble protein (H634Q), and one which was degraded in vivo (H487Q) . Following purification to homogeneity, mutant enzymes H474Q, H751Q, H860Q, and H868Y had essentially wild-type catalytic activity, while mutant enzymes H865K and H865Q had less than 0.6% wild-type activity . The low activity of mutant enzymes H865K and H865Q is unlikely to reflect altered structural integrity since both chromatographed on affinity supports like wild-type enzyme and had Km values for (S)-HMG-CoA (31 and 16 microM) and for NADPH (60 and 24 microM) close to those for wild-type enzyme (31 and 52 microM for (S)-HMG-CoA and NADPH, respectively) . His865 of hamster HMG-CoA reductase, the histidine of the consensus Leu-Val-Xaa-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif and the only histidine conserved among the catalytic domains of all HMG-CoA reductases, thus appears to be a general acid/base functional in catalysis.

J Biol Chem, 1993 Apr 25, 268(12), 8402 - 5
Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type IV collagen; Neilson EG et al.; Type IV collagen has recently emerged as a family composed of five known chains (alpha 1-alpha 5), each of which contains a carboxyl-terminal noncollagenous domain (NC1) of approximately 230 amino acids . The NC1 domain of the alpha 3(IV) chain is the probable target for autoantibodies in patients with Goodpasture syndrome (GP), as evidenced from studies employing bovine type IV collagen . In the present experiments, the specificity of GP antibodies for the five NC1 domains of human type IV collagen was determined by using recombinant NC1 domains as the antigen . cDNAs encoding each NC1 domain were expressed in E . coli as fusion proteins with a 6-histidine amino-terminal leader . The recombinant NC1 monomers r alpha 1(IV), r alpha 2(IV), r alpha 3(IV), r alpha 4(IV), and r alpha 5(IV) were purified by affinity chromatography to the fusion protein using a nickel resin column, and then characterized by electrophoresis and immunoblot analysis using chain-specific peptide antibodies . The specificity of GP antibodies from four patients to these recombinant proteins was then further evaluated by immunoblot analysis and enzyme-linked immunosorbent assay measurements . The GP antibodies reacted strongly with the r alpha 3(IV) NC1 domain but were not reactive when tested against the other four recombinant monomers . In contrast, neither antisera from patients with two other forms of autoimmune disease (anti-tubular basement membrane disease and Wegener's syndrome) nor normal control sera bound to any of the recombinant NC1 moieties . These results unambiguously establish that GP antibodies are specifically targeted to the NC1 domain of the alpha 3(IV) chain of human type IV collagen . The findings also establish a methodology for large scale preparation of r alpha 3(IV) NC1 domain for use in diagnostic tests and development of therapeutic procedures and offer a strategy for the elucidation of a more complete GP epitope by site-directed mutagenesis.

J Biol Chem, 1993 Apr 25, 268(12), 8391 - 3
Phosphorylation and dephosphorylation catalyzed in vitro by purified components of the nitrate sensing system, NarX and NarL; Walker MS et al.; The regulation of specific gene expression by nitrate in Escherichia coli is mediated by the NarX/NarQ-NarL system . Based on sequence homologies with a family of two-component regulatory systems in bacteria, NarL has been identified as a putative response regulator while NarX and NarQ were proposed to be alternative membrane-associated sensors that activate NarL in the presence of nitrate . To investigate the interaction of NarX and NarL in vitro, both proteins were purified from overproducing strains . Purified NarX was rapidly labeled when incubated with {gamma-32P} ATP but not with {alpha-32P}ATP in a reaction that required Mg2+ but was unaffected by nitrate . Incubation of the labeled NarX with purified NarL resulted in the transient phosphorylation of NarL . Both the phosphorylation and dephosphorylation of NarL required Mg2+, and neither reaction was affected by the presence of nitrate . NarL-phosphate, stabilized by the addition of EDTA, ran as a monomer on gel filtration . Dephosphorylation of the isolated NarL-phosphate required the addition of both Mg2+ and the NarX protein . The relative stabilities of the phosphorylated forms of the two proteins at different pH values were consistent with the proposal that, in analogy to other related two-component regulatory systems, NarX and NarL were phosphorylated on specific histidine and aspartate residues, respectively.

Nucleic Acids Res, 1993 Apr 25, 21(8), 1827 - 35
Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket; Belduz AO et al.; The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82 . Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure . Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity . This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP . Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide . Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP . CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket . Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP.

Nucleic Acids Res, 1993 Apr 25, 21(8), 1719 - 25
Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli; Lloyd RG et al.; The RuvAB, RuvC and RecG proteins of Escherichia coli process intermediates in recombination and DNA repair into mature products . RuvAB and RecG catalyse branch migration of Holliday junctions, while RuvC resolves these structures by nuclease cleavage around the point of strand exchange . The overlap between RuvAB and RecG was investigated using synthetic X- and Y-junctions . RuvAB is a complex of RuvA and RuvB, with RuvA providing the DNA binding subunit and RuvB the ATPase activity that drives branch migration . Both RuvA and RecG form defined complexes with each of the junctions . The gel mobilities of these complexes suggests that the X-junction attracts two tetramers of RuvA, but mainly monomers of RecG . Dissociation of the junction in the presence of ATP requires high levels of RuvAB . RecG is shown to have a much higher specific activity to the extent that very little of this protein would be required to match RuvAB in vivo . Both proteins also dissociate a Y-junction, which is consistent with helicase activity . However, RecG shows no ability to unwind more conventional substrates and the suggestion is made that its helicase activity is directed towards specific DNA structures such as junctions.

J Biol Chem, 1993 Apr 25, 268(12), 9105 - 9
Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts . II . Defective DNA repair synthesis by xeroderma pigmentosum cell extracts; Masutani C et al.; We have constructed a cell-free DNA repair system with UV-irradiated SV40 minichromosomes, as described in the accompanying paper (Sugasawa, K, Masutani, C., and Hanaoka, F . (1993) J . Biol . Chem 268, 9098-9104) . In this study, we examined DNA repair synthesis by cell extracts from seven xeroderma pigmentosum (XP) complementation groups, A through G . DNA repair synthesis by XP cell extracts was lower than that with repair-proficient human 293 cell extract and did not increase to the level with the latter on increase in the amount of cell extract or the incubation time . The defects of XP cell extracts were complemented by addition of extracts from cells of different complementation groups, indicating that defective proteins in XP-A through G cells are directly involved in DNA repair . Addition of T4 endonuclease V, which is reported to complement defects of XP cells, stimulated DNA repair synthesis by the 293 cell extract, and also complemented the defects of all XP cell extracts . The XPAC gene product was shown to be involved in DNA repair synthesis using anti-xpac serum and xpac protein produced in Escherichia coli . Anti-xpac serum inhibited DNA repair synthesis by the 293 cell extract and xpac protein reversed the inhibition . Furthermore, xpac protein complemented the defects of extracts of two lines of XP-A cells (XP2OSSV and XP12ROSV) but had no effect on the reactions of extracts from cells of other complementation groups . These findings are consistent with previous results obtained in experiments with cells, indicating that our system is useful for analyzing the mechanisms of DNA excision repair in mammalian cells.

J Biol Chem, 1993 Apr 25, 268(12), 8777 - 80
Formation of a stable adduct between ubiquitin and the Arabidopsis ubiquitin-conjugating enzyme, AtUBC1+; Sullivan ML et al.; Ubiquitin conjugating enzymes (E2s) are an integral part of a multienzyme pathway that ligates ubiquitin to intracellular target proteins . This ligation has been implicated in a number of fundamental processes including protein degradation, cell cycle progression, DNA repair, and organelle biogenesis . To function, E2s form a labile thiol-ester intermediate between a specific cysteine within the E2 and the carboxyl terminus of ubiquitin; this high energy intermediate then serves as the donor for ubiquitin ligation . To aid in the characterization of E2s, we have created a stable ubiquitin-E2 intermediate using a mutant form of the 16-kDa E2 encoded by the Arabidopsis thaliana AtUBC1 gene in which the active-site cysteine at residue 88 was replaced with serine . The mutant protein synthesized in Escherichia coli formed an adduct with ubiquitin in vitro, but in this case the E2 and ubiquitin were linked via a more stable ester bond . The ester-linked ubiquitin could not be transferred subsequently to substrate proteins in an E3 alpha-dependent conjugation reaction . The ester adduct was sufficiently stable to survive purification by anion exchange high performance liquid chromatography . As a result, this adduct may prove useful for the structural analysis of ubiquitin-E2 intermediates and in the study of E2s interacting with other ubiquitin pathway enzymes.

Nucleic Acids Res, 1993 Apr 25, 21(8), 1935 - 9
The 3'-5' exonuclease of human DNA polymerase delta (pol delta) is regulated by pol delta accessory factors and deoxyribonucleoside triphosphates; Lee SH; Human DNA polymerase delta (pol delta) is required for the synthesis of leading strand of simian virus 40 (SV40) DNA replication in vitro . Pol delta requires the accessory factors, proliferating cell nuclear antigen (PCNA), activator 1 (A1; also known as replication factor C {RF-C}), human single-stranded DNA binding protein (HSSB; also known as replication protein A {RP-A}) for the elongation of primed template DNA . Since pol delta has an associated 3'-5' exonuclease activity, the effect of pol delta accessory factors on the exonuclease activity was examined . The 3'-5' exonuclease activity was stimulated 8-10 fold by the addition of HSSB, and this stimulatory effect was preferential to HSSB since other SSBs from E . coli, T4 or adenovirus, had a little or no effect . The stimulatory effect of HSSB was markedly inhibited by the combined action of A1 and PCNA . Furthermore, the addition of deoxyribonucleoside triphosphates (dNTPs) completely abolished the effect of HSSB on the 3'-5' exonuclease activity even in the absence of pol delta accessory factors . These results suggest that accessory factors and dNTPs regulate both the polymerase and the 3'-5' exonuclease activities.

Nucleic Acids Res, 1993 Apr 25, 21(8), 1941 - 7
Mosaic tile model for tRNA-enzyme recognition; Steinberg SV et al.; An improved algorithm was elaborated to analyse tRNA interaction with aminoacyl-tRNA synthetase based on analysis of tRNA sequences . The fundamental element defining the interaction between the tRNA and the synthetase is not a single nucleotide but a nucleotide combination named a tile which comprises of a given nucleotide and its neighbours as they are defined by the tertiary structure of the molecule . Informational content of each tile is calculated as its probability to occur exclusively in a set of cognate tRNAs . Based on this algorithm the identity sites of E . coli tRNA(Ala) and tRNA(Gln) were determined . The results are in a good agreement with the biochemical data and provide new information about identity sites of these tRNAs.

J Biol Chem, 1993 Apr 25, 268(12), 8617 - 23
Identification of two hyaluronan-binding domains in the hyaluronan receptor RHAMM; Yang B et al.; We have identified two discrete hyaluronan- (HA) binding domains in the HA receptor RHAMM (Receptor for HA-Mediated Motility) that mediates the locomotion of H-ras transformed fibroblasts . A complete RHAMM cDNA (1.43 kilobases (kb)) was expressed as a fusion protein with pGEX-2T in Escherichia coli HB101 and was shown to bind specifically to both biotin-labeled HA in a transblot assay and to HA-Sepharose . The complete cDNA was truncated with restriction endonucleases from the 3' end resulting in 1.30-, 1.02-, 0.71-, and 0.41-kb cDNAs which were then expressed in HB101 . Only the fusion peptide expressed from the complete cDNA and the 1.30-kb cDNA bound to HA indicating that the region located between 1.02-1.30 kb of RHAMM cDNA was critical for recognition of this glycosaminoglycan . Deletion of 114 bases in this region virtually eliminated HA binding activity thus defining the major glycosaminoglycan binding region to amino acids 400-434 located near the carboxyl terminus of RHAMM . Two domains containing clusters of basic amino acids were identified within this region . Synthetic peptides mimicking these two domains both inhibited HA binding to the complete 1.43-kb expressed glutathione s-transferase-RHAMM fusion protein, and also directly bound to HA-Sepharose . Random peptides and peptides mimicking other regions in RHAMM did not inhibit HA-RHAMM interactions and bound weakly to HA-Sepharose . Oligonucleotides encoding either of these two peptides were linked to the NH2-terminal 0.71 kb of RHAMM which encoded a peptide that did not contain HA binding activity . Fusion proteins containing either of these recombinant peptides acquired HA binding activity as assessed with a transblot assay . Thus, we have identified two domains within RHAMM that are responsible for its HA binding activity.

Biochim Biophys Acta, 1993 Apr 23, 1167(3), 257 - 63
In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) to apolipoprotein AI in rabbits; Saku K et al.; In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) from E . coli to apolipoprotein (apo) AI was investigated . rh-Met-proapo AI was labeled with 125I, and then administered intravenously to rabbits . Blood was sampled periodically for 6 days . The plasma decay curves of radioiodinated rt-Met-proapo AI were similar to those of human mature apo AI (fractional catabolic rate (FCR); 1.018 +/- 0.090/day vs . 0.976 1 0.031/day, respectively) . In vivo conversion of rh-Met-proapo AI to mature apo AI was examined by autoradiography of the isoelectric focusing (IEF) slab gel, i.e., the HDL fraction from each sampling point was semiquantitatively applied to IEF . It was found that the radioactivity of rh-Met-proapo AI migrated to more acidic isoproteins, the conversion was complete within 24 h, and the FCR of rh-Met-proapo AI was 9.20 +/- 1.34/day . Although the plasma decay curves of both human pro (rh-Met-proapo AI) and mature apo AI were significantly steeper than those of rabbit mature apo AI4 and apo AI5 (FCR; 0.703 +/- 0.027/day and 0.795 +/- 0.031/day, respectively), the conversion rate of human rt-Met-proapo AI to mature apo AI in rabbit was assumed to be 1:1 . In vitro incubation of rh-Met-proapo AI with rabbit serum produced mature apo AI isoproteins, as determined by the apo AI immunoblotting method . Prediction of the amino acid sequence at the NH2 terminus of rabbit proapo AI showed that the prosegment consisted of an alpha helix with a high probability of a beta turn at Pro9, which is close to that in humans . Thus, (1) the proteolytic cleavage of proapo AI is an extracellular event, (2) the converting enzyme in rabbits can also process human proapo AI, (3) this converting enzyme does not specifically and directly attack the Gln6-Asp7 bond which links the carboxyl-terminal residue of the hexapeptide to the amino-terminal residue of human mature apo AI . The conformation of proapo AI at the NH2 terminus (alpha helix of the prosegment and a beta turn at Pro9) may have a key role in this cleavage, and (4) the examination of rh-Met-proapo AI in rabbits helps to explain the early events of HDL biogenesis.

Cell, 1993 Apr 23, 73(2), 375 - 9
Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers; Zhou Y et al.; We have constructed heterodimers consisting of two subunits: one CAP subunit that has a nonfunctional activating region but wild-type DNA binding specificity, and one CAP subunit that has a functional activating region but non-wild-type DNA binding specificity . We have oriented the heterodimers on lac promoter DNA by use of promoter derivatives that have DNA sites for CAP consisting of one wild-type half site and one non-wild-type half site, and we have analyzed the abilities of the oriented heterodimers to activate transcription . Our results indicate that transcription . Our results indicate that transcription activation requires the activating region of only one subunit of CAP: the promoter-proximal subunit . The oriented heterodimers method of this report should be generalizable to other dimeric transcription activator proteins.

Eur J Pharmacol, 1993 Apr 22, 235(1), 45 - 50
Recombinant human type II phospholipase A2 lacks edema producing activity in rat; Morgan DW et al.; The rat paw edema-inducing, acute inflammatory activity of four snake venom phospholipase A2S (PLA2) (Naja naja, Naja mocambique mocambique, Crotalus atrox and recombinant Naja naja naja) and of recombinant human type II PLA2 (rh-PLA2) found in rheumatoid synovial fluid, were compared after a bolus subplantar injection . The snake venom-derived PLA2s, including the recombinant Naja naja naja, were potent inducers of paw edema . On the other hand, when given in similar amounts (protein and/or enzymatic activity), the rh-PLA2 did not produce paw edema . Furthermore, the addition of Naja naja PLA2, blood plasma from rats with adjuvant arthritis or the E . coli-based enzymatic incubation mixture used to measure PLA2 activity to the injection mixture containing rh-PLA2, did not result in paw edema-inducing activity . These results suggest that the lack of paw edema-inducing activity of rh-PLA2 may be due to significant structural differences between snake venom PLA2s and human PLA2 which allow snake venom PLA2s, but not the human group II PLA2, to express inflammatory activity as measured by paw edema in rat induced by a bolus injection.

Biochim Biophys Acta, 1993 Apr 21, 1163(1), 75 - 80
Characterization of yeast EF-1 alpha: non-conservation of post-translational modifications; Cavallius J et al.; Elongation factor 1 alpha (EF-1 alpha) is an abundant cellular protein and its amino-acid sequence has been inferred from numerous organisms, including bacteria, archaebacteria, plants and animals . In large measure, it would appear that the overall structure has probably been maintained given the 33% identity and 56% similarity of Escherichia coli EF-Tu with human EF-1 alpha . Chemical sequencing of EF-Tu and EF-1 alpha has revealed that these proteins are post-translationally modified . In order to assess the possible function of these modifications, we have chemically sequenced the EF-1 alpha from the lower eukaryote Saccharomyces cerevisiae (yeast) . To our surprise, the methylation pattern of yeast EF-1 alpha was quite different from either rabbit or brine shrimp EF-1 alpha with only the trimethyllysine at position 79 conserved although the yeast protein is 81% identical to rabbit EF-1 alpha . A dimethyllysine was observed at position 316 which corresponds to a trimethyllysine in brine shrimp and rabbit EF-1 alpha . The other positions in yeast EF-1 alpha which were methylated were unrelated to the other six possible positions for modification observed in brine shrimp or rabbit EF-1 alpha . In addition, the unique glyceryl-phosphorylethanolamine observed in mammalian EF-1 alpha and suspected in brine shrimp EF-1 alpha was not found in yeast EF-1 alpha.

Biochim Biophys Acta, 1993 Apr 21, 1163(1), 67 - 74
Ligand affinities in mutant metmyoglobins; Biram D et al.; Ligand binding to the wild-type and a series of mutant porcine myoglobins, expressed and purified from Escherichia coli cells, has been studied using UV-VIS absorption spectroscopy . The proximal pocket mutation, F7 Ser-->Leu (F7), causes an increased affinity for OH- and N3- binding to metmyoglobin . A hydrogen bond between the F7 serine residue and the imidazole side-chain of the proximal histidine has been removed by this mutation . It is suggested that this allows the imidazole group to reorientate, reducing the steric clash between itself and the haem pyrrole nitrogen atoms and leading to a shortening of the bond between the proximal histidine and the haem iron . Other conformational changes further away from the haem pocket have also been induced, but the mutant still crystallizes under the same conditions as for the wild-type protein . A series of distal pocket mutants, E11 Val-->Thr (VT), E7 His-->Val (HV) and a mutant with both of these substitutions (M2) all have greatly reduced the OH- and N3- binding affinity . These effects have been interpreted by considering several factors: the changed stability of the aquometmyoglobin form, hydrogen-bond formation between the ligand and the E7 residue, and electrostatic repulsion between the ligand and the E11 threonine residue.

Biochim Biophys Acta, 1993 Apr 21, 1163(1), 54 - 60
Comparison of several new chromogenic galactosides as substrates for various beta-D-galactosidases; Pocsi I et al.; The kinetic characteristics of beta-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates . All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum . Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used . The substrates attached particularly tightly to the active centre of E . coli beta-D-galactosidase resulting in low Km values . The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed to interactions not involving the catalytic site . When the product of the maximum observed velocity (Vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed . The beta-D-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.

Biochim Biophys Acta, 1993 Apr 21, 1163(1), 49 - 53
Expression in Escherichia coli of the 36 kDa domain of poly(ADP-ribose) polymerase and investigation of its DNA binding properties; Thibodeau J et al.; We have expressed in Escherichia coli the 36 kDa domain of the human poly(ADP-ribose) polymerase . This polypeptide comprises the C-terminal part of the DNA binding domain, as well as the automodification region of the enzyme, but lacks the zinc-finger motifs of the N-terminal region and the C-terminal catalytic domain . By probing the crude E . coli protein extracts with radioactive DNA probes (South-Western blots), we have shown that the 36 kDa domain binds a DNA probe of 222 bp but does not bind a shorter probe of 66 bp . This interaction is stronger when the polypeptide is fused to the 55 kDa catalytic domain of the enzyme.

J Mol Biol, 1993 Apr 20, 230(4), 1291 - 6
A mutagenic study of the allosteric linkage of His(HC3)146 beta in haemoglobin; Shih DT et al.; We have examined the contribution of His(HC3)146 beta to the alkaline Bohr effect of human haemoglobin (HbA) by replacing it with Gln, using site-directed mutagenesis, and studying the structural and functional consequences . Oxygen equilibrium curves of the mutant show that the effect of pH on the oxygen affinity, the alkaline Bohr effect, is half that of HbA in the presence of chloride ion and less than 10% in its absence . Crystallographic analysis shows that the mutation introduced only small structural changes localized to the site of substitution, proving that the replacement of the hydrogen bond between the ionizable side-chain of His146 beta and Asp94 beta by a hydrogen bond between the unionizable side-chain of Gln146 beta and the same aspartate is solely responsible for the reduction of the alkaline Bohr effect . Our data confirm that His(HC3)146 beta is predominantly responsible for the chloride-independent component of the alkaline Bohr effect which results from the breaking of the hydrogen bond between His(HC3)146 beta and Asp(FG1)94 beta accompanying the transition from the quaternary deoxy to oxy-structure.

Biochemistry, 1993 Apr 20, 32(15), 4128 - 38
Kinetics of Escherichia coli helicase II-catalyzed unwinding of fully duplex and nicked circular DNA; Runyon GT et al.; Escherichia coli helicase II (UvrD) protein can initiate unwinding of duplex DNA at blunt ends or nicks, although these reactions require excess protein . We have undertaken kinetic studies of these reactions in order to probe the mechanism of initiation of unwinding . DNA unwinding was monitored directly by using agarose gel electrophoresis and indirectly through the rate of ATP hydrolysis by helicase II in the presence of an ATP-regenerating system . In the presence of fully duplex DNA and excess helicase II, the rate of ATP hydrolysis displays a distinct lag phase before the final steady-state rate of hydrolysis is reached . This reflects the fact that ATP hydrolysis under these conditions results from helicase II binding to the ssDNA products of the unwinding reaction, rather than from an intrinsic duplex DNA-dependent ATPase activity . Unwinding of short blunt-ended duplex DNA (341 and 849 base pairs) occurs in an "all-or-none" reaction, indicating that initiation of unwinding by helicase II is rate-limiting . We propose a minimal mechanism for the initiation of DNA unwinding by helicase II which includes a binding step followed by the rate-limiting formation of an initiation complex, possibly involving protein dimerization, and we have determined the phenomenological kinetic parameters describing this mechanism . Unwinding of a series of DNA substrates containing different initiation sites (e.g., blunt ends, internal nicks, and four-nucleotide 3' vs 5' ssDNA flanking regions) indicates that the rate of initiation is slowest at nicks and, surprisingly, at ends possessing a four-nucleotide 3' ssDNA flanking region.

Biochemistry, 1993 Apr 20, 32(15), 4112 - 20
UV irradiation of Escherichia coli modulates mutagenesis at a site-specific ethenocytosine residue on M13 DNA . Evidence for an inducible recA-independent effect; Palejwala VA et al.; Mutagenic action of chemical and physical mutagens is mediated through DNA damage and subsequent misreplication at sites of unrepaired damage . Most DNA damage is noninstructive in the sense that the causative chemical modification either destroys the template information or renders it inaccessible to the DNA polymerase . Noninstructive adducts possess high genotoxicity because they stop DNA replication . Replication past noninstructive adducts is thought to depend on induced functions in addition to the regular replication machinery . In Escherichia coli, noninstructive DNA damage leads to induction of the SOS regulon, which in turn is thought to provide the inducible functions required for replicative bypass of the lesion . Because of the absence of accessible template instruction, base incorporation opposite noninstructive lesions is inherently error-prone and results in mutagenesis . Ethenocytosine (epsilon C), an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly mutagenic, noninstructive lesion on the basis of its template characteristics in vivo and in vitro . However, mutagenesis at epsilon C does not require SOS functions, as evidenced by efficient mutagenesis in recA-deleted E . coli . Even though efficient mutagenesis in recA-deleted cells shows a lack of SOS dependence, the question remains whether SOS induction can modulate mutagenesis opposite epsilon C . To examine the possible contribution of SOS functions to mutagenesis at epsilon C, we constructed an M13 duplex circular DNA molecule containing an epsilon C residue at a unique site . The construct was transfected into nonirradiated or UV-irradiated E . coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Apr 20, 32(15), 4105 - 11
Quantitative multiplex sequence analysis of mutational hot spots . Frequency and specificity of mutations induced by a site-specific ethenocytosine in M13 viral DNA; Palejwala VA et al.; We describe an assay for determining the frequency and specificity of mutations occurring at hot spots within a population of DNA molecules . The procedure consists of (a) annealing the DNA population with a labeled oligonucleotide designed to prime DNA synthesis at the mutational hot spot; (b) DNA elongation in the presence of a single dideoxynucleoside triphosphate together with 1-3 deoxynucleoside triphosphates, and (c) quantitation of all limit elongation products by high-resolution gel electrophoresis followed by autoradiography and computing densitometry . Derivation of mutational frequency and specificity over a wide range of values is demonstrated for M13 viral DNA mixtures containing defined proportions of wild-type and mutant DNAs, as well as for M13 viral DNA populations obtained by transfection of DNA bearing a defined site-specific ethenocytosine lesion . The assay is shown to yield results similar to those obtained by laborious clone-by-clone sequencing of viral progeny . The method is not affected significantly by several tested variables and appears to be suitable for use as a quantitative assay for sequence microheterogeneity at defined positions within DNA populations . Application of the methodology demonstrates that ethenocytosine, an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly efficient mutagenic lesion with a mutational specificity expected for noninstructive lesions.

Biochemistry, 1993 Apr 20, 32(15), 3907 - 12
Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli: a new type of proteolytic enzyme; Roderick SL et al.; The X-ray structure of Escherichia coli methionine aminopeptidase (MAP) has been determined to 2.4-A resolution and refined to a crystallographic R-factor of 18.2% . The fold is novel and displays internal pseudo-2-fold symmetry which structurally relates the first and second halves of the polypeptide chain . The topology consists of a central antiparallel beta-sheet covered on one side by two pairs of alpha-helices and by a C-terminal loop . The other face of the beta-sheet, together with some irregular loops, forms the active site, which contains two cobalt ions 2.9 A apart . These metal ions are liganded by the side chains of Asp 97, Asp 108, Glu 204, Glu 235, and His 171 with approximate octahedral coordination . In terms of both the novel backbone fold and the constitution of the active site, MAP appears to represent a new class of proteolytic enzyme.

J Mol Biol, 1993 Apr 20, 230(4), 1145 - 50
RecQ DNA helicase of Escherichia coli . Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein; Umezu K et al.; RecQ protein of Escherichia coli is a DNA helicase implicated in the RecF pathway of genetic recombination . To gain insight into the mode of its action, the effect of single-stranded DNA-binding proteins (SSBs) on the RecQ-mediated unwinding reaction was investigated . When the unwinding of M13-based, circular partially duplex substrates was measured as a function of the enzyme dose, a markedly sigmoidal relation was revealed, with relatively large amounts of the enzyme being necessary for substantial unwinding to occur . For instance, unwinding 50% of a 71 base-pair (bp) partial duplex substrate in ten minutes required an enzyme-to-substrate molar ratio of about 60 . However, these features, indicating the enzyme's "inefficiency", were reversed by SSBs: in the presence of a saturating amount of E . coli SSB the sigmoidal relation was converted to a typically hyperbolic one, and the enzyme-to-substrate molar ratio at 50% unwinding of the 71 bp substrate was reduced to as low as 0.5 . Phage T4 gene 32 protein also showed similar stimulatory activity . Further, the single-stranded DNA-dependent ATPase activity of RecQ was found to be relatively insensitive to E . coli SSB; its large excess brought about only a 60% inhibition . It is postulated that RecQ helicase is highly adapted to an SSB-rich environment, where the strand exchange reaction mediated by RecA protein, perhaps coupled closely with the RecQ reaction, should also take place.

Biochemistry, 1993 Apr 20, 32(15), 4083 - 9
Frameshift fidelity during replication of double-stranded DNA in HeLa cell extracts; Roberts JD et al.; The processes by which minus-one frameshifts arise during replication of double-stranded DNA by a human replication apparatus were examined . Using M13mp2 DNA containing the simian virus 40 (SV40) origin of replication and a plus-one frameshift mutation in the lacZ alpha reporter gene, we performed replication reactions using a HeLa cell extract and the SV40 large T antigen . Frameshifts that restore the reading frame to give a blue-plaque phenotype include the loss of one of five consecutive A.T base pairs or any one of 36 non-reiterated base pairs . Although both types of deletions were generated at rates substantially above the background mutant frequency of unreplicated DNA, the rate was highest at the A.T run, suggesting the involvement of a misaligned replication intermediate at this homopolymeric sequence . The error rate for both types of deletions increased as the concentration of dNTPs was increased . A small increase in error rate at the run of A.T base pairs was also observed when a dNMP was added to the replication reaction . These results are consistent with the correction of frameshift intermediates during replication by exonucleolytic proofreading . To examine frameshift error rates on the leading and lagging strands, we compared reversion frequencies for two vectors containing the origin of replication close to, but on opposite sides of, the mutational target . To generate strand-specific errors, nucleotide substrate imbalances were used in replication reactions with these vectors . The results suggest that there is less than a 2-fold difference in the fidelity of leading- and lagging-strand synthesis for deletions at the run of A.T base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Apr 20, 32(15), 3836 - 41
A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase; Sylvers LA et al.; Early investigations into the interaction between Escherichia coli glutamyl-tRNA synthetase (GluRS) and tRNAGlu have implicated the modified nucleoside 5-{(methylamino)methyl}-2-thiouridine in the first position of the anticodon as an important contact for efficient aminoacylation . However, the experimental methods employed were not sufficient to determine whether the interaction was dependent on the presence of the modification or simply involved other anticodon loop-nucleotides, now occluded from interaction with the synthetase . Unmodified E . coli tRNA(Glu), derived by in vitro transcription of the corresponding gene, is a poor substrate for GluRS, exhibiting a 100-fold reduction in its specificity constant (kcat/KM) compared to that of tRNA(Glu) prepared from an overproducing strain . Through the use of recombinant RNA technology, we created several hybrid tRNAs which combined sequences from the in vitro transcript with that of the native tRNA, resulting in tRNA molecules differing in modified base content . By in vitro aminoacylation of these hybrid tRNA molecules and of tRNAs with base substitutions at positions of nucleotide modification, we show conclusively that the modified uridine at position 34 in tRNA(Glu) is required for efficient aminoacylation by E . coli GluRS . This is only the second example of a tRNA modification acting as a positive determinant for interaction with its cognate aminoacyl-tRNA synthetase.

Biochemistry, 1993 Apr 20, 32(15), 4060 - 6
Folding of the four domains and dimerization are impaired by the Gly446-->Glu exchange in human glutathione reductase . Implications for the design of antiparasitic drugs; Nordhoff A et al.; Glutathione reductase (NADPH+GSSG+H+-->NADP(+) + 2GSH) is a homodimeric flavoenzyme of known geometry . Each subunit contains four well-defined domains and contributes essential residues to the active sites; consequently, the monomer is expected to be inactive . As part of our program to develop dimerization inhibitors of human glutathione reductase (hGR) as antimalarial agents, we mutagenized the residues 446 and 447 which, together with their counterparts on the other subunit, represent the tightest contact between the subunits {Karplus, P . A., & Schulz, G . E . (1987) J . Mol . Biol . 195, 701-729} . Wild-type human glutathione reductase and mutants of this protein were produced in plasmid-transformed Escherichia coli SG5 cells . Active enzyme species, namely, wild-type hGR, N-terminally truncated delta(1-15)hGR, and the point mutant F447P-hGR, were purified by 2',5'-ADP-Sepharose chromatography and crystallization . Inactive mutants such as G446E-hGR or the double mutants G446E/F447P-hGR and G446P/F447P-hGR were isolated by immunoadsorption chromatography . G446E/F447P-hGR was studied in detail . This mutant behaved like a poorly folded monomeric protein, as indicated by the following properties: absence of the intersubunit disulfide bridge, Cys90-Cys90'; failure to bind FAD; failure to bind NADPH and analogues thereof; a short half-life (< 4 min) in E . coli cells; and high susceptibility to trypsin in vitro . The results suggest that the sequence around G446 can control dimerization as well as domain folding . This is unexpected since the FAD-binding domain and the NADPH-binding domain occur in many different enzymes and have been regarded as autonomous folding units.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Apr 20, 32(15), 4007 - 13
Comparison of the conformation of the epitope of alpha(2-->8) polysialic acid with its reduced and N-acyl derivatives; Baumann H et al.; The immunological properties of alpha(2-->8) polysialic acid have been rationalized in terms of the presence of an epitope situated on a unique extended helical segment (n approximately 9) of the polymer . The critical importance of the carboxylate group to the stability of the extended helical epitope can be ascertained from NMR spectrocopic studies and potential energy calculations on the carboxyl reduced alpha(2-->8) polysialic acid . These studies indicate that the extended helix (n approximately 9) is not stabilized in the reduced polymer and that the majority of conformers can only have helical parameters with n = 2 and 3 . This result is also consistent with the fact that the reduced alpha(2-->8) polysialic acid, contrary to its acidic counterpart, exhibits conventional immunological properties . Only five to six reduced oligomers are required to inhibit the binding of the reduced polysialic acid to its homologous antiserum . NMR spectroscopic analysis and potential energy calculations on the N-propionyl, N-butanoyl, N-isobutanoyl, N-pentanoyl, N-hexanoyl, and N-glycolyl derivatives of alpha(2-->8) polysialic acid indicate that, despite the bulk of some of these substituents, they did not disrupt the extended helical conformer . The presence of the extended helical epitope in some of these N-acyl derivatives has also been confirmed from immunological data.

FEBS Lett, 1993 Apr 19, 321(1), 93 - 7
Cloning, expression and characterization of human kininogen domain 3; Auerswald EA et al.; The internal domain 3 of the heavy chain of human kininogen, a cysteine proteinase inhibitor, was amplified by a polymerase chain reaction from the kininogen cDNA clone phKG36 . The DNA fragment was expressed in Escherichia coli using the ompA expression vector pASK40 and the resulting protein was isolated from periplasm, purified by S-carboxymethylpapain affinity- and ion-exchange chromatography . The recombinant human kininogen domain 3 is 92% pure, reacts with anti-kininogen antibodies and is actively inhibitory . The expected amino acid sequence of ANSM-{G253-S377} kininogen was confirmed; the inhibitor has a molecular mass of 14,396 Da and an isoelectric point of 6.0 (pH) . The determined Ki values of the complexes with papain and cathepsin L are similar to those measured previously with proteolytically liberated kininogen domain 3, and those of single-domain cystatins, like chicken egg white cystatin . However, recombinant kininogen domain 3 is a weak inhibitor of cathepsin B (Ki = 63 nM) as it has been found for native L-kininogen (Ki = 340 nM).

FEBS Lett, 1993 Apr 19, 321(1), 63 - 8
Molecular cloning of a cDNA encoding calmodulin from Neurospora crassa; Capelli N et al.; A full-length cDNA encoding Neurospora crassa calmodulin was isolated from a lambda ZAP II cDNA expression library . The open reading frame encodes a protein of 148 amino acid residues with a calculated M(r) of 16,865 Da . Using site-directed mutagenesis, the complete cDNA was ligated into a trc promoter-regulated bacterial expression vector to allow expression of N . crassa calmodulin in E . coli . The expressed protein was found to be identical to the native protein on the basis of some of its biochemical properties . Finally, Southern analysis of restriction digests of genomic DNA indicates that calmodulin is encoded by a single-copy gene.

FEBS Lett, 1993 Apr 19, 321(1), 55 - 8
A thioredoxin-independent fully active NADP-malate dehydrogenase obtained by site-directed mutagenesis; Issakidis E et al.; A triple cysteine mutant of sorghum leaf NADP-malate dehydrogenase has been constructed by site-directed mutagenesis, combining the previously obtained mutation of the two N-terminal cysteines with the mutation of the most internal of the two C-terminal cysteines . The construct, over-expressed in E . coli, yielded an always active, dithiol-insensitive enzyme . It can be concluded that the dithiol activation of the unmodified enzyme involves a maximum of two different disulfides per subunit, and that none of the mutated cysteines is implicated in catalysis.

FEBS Lett, 1993 Apr 19, 321(1), 89 - 92
Neopterin modulates toxicity mediated by reactive oxygen and chloride species; Weiss G et al.; Neopterin, a pyrazino-pyrimidine derivative, is synthesized in excess by human monocytes/macrophages upon stimulation with interferon-gamma, a cytokine derived from activated I cells . Neopterin is furthermore produced constitutively . A relatively constant ratio between neopterin and its reduced form . 7,8-dihydroneopterin, has been described in human serum . In the study presented here we tested the ability of neopterin and its reduced form to modulate the effects of cytotoxic substances like hydrogen peroxide or hypochlorous acid and N-chloramine derivatives . We show that 7,8-dihydroneopterin potently reduces biological and chemical effects of these substances independently from the pH value . In contrast, at slightly alkaline pH (pH 7.5) neopterin enhances hydrogen peroxide and chloramine-T activity . This is demonstrated by increase of signal intensity in a luminol assay and also by enhancement of toxicity towards bacteria . Thus, the macrophage derived substance neopterin is able both to enhance and to reduce cytotoxicity in dependence of pH value and its oxidation state, and it may have a pivotal role in modulation of macrophage mediated effector mechanism.

FEBS Lett, 1993 Apr 19, 321(1), 41 - 5
Mouse monoclonal antibodies that specifically recognize an amino-terminal epitope of p56lck protein tyrosine kinase; Stieger M et al.; p56lck is a T cell-specific protein tyrosine kinase of the src family of proto-oncogenes which has been implicated in T cell signal transduction . Here we describe the production of mouse monoclonal antibodies directed against recombinant human p56lck purified from an E . coli expression system . The antibodies were characterized, by ELISA . RIA and immunoprecipitation of p56lck from T cell lysates . A specific epitope was revealed at the aminoterminus of the p56lck molecule by using Western blotting of deletion mutants and distinct domains of p56lck expressed in E . coli . Potential applications of the results obtained are discussed.

Neurosci Lett, 1993 Apr 16, 153(1), 88 - 92
Preparation of an antiserum using a fusion protein produced by a cDNA for rat aromatic L-amino acid decarboxylase; Krieger M et al.; Aromatic L-amino acid decarboxylase (AADC) decarboxylates L-DOPA and 5-hydroxytryptophan into dopamine and serotonin, respectively . Starting from a rat AADC cDNA clone isolated in our laboratory, we produced a beta-galactosidase-AADC fusion protein in E . coli . It was purified from inclusion bodies and injected into a rabbit . The antiserum identified AADC on a Western blot of extracts from rat organs as a unique 50 kDa band; it also strongly reacted by immunohistochemistry with dopaminergic and serotonergic neurons . This new beta-galactosidase-AADC fusion protein then constitutes a useful tool for producing AADC as an antigen free of contamination by mammalian proteins.

Science, 1993 Apr 16, 260(5106), 352 - 5
Structure of DNA polymerase I Klenow fragment bound to duplex DNA; Beese LS et al.; Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain . When the fragment bound DNA, a region previously referred to as the "disordered domain" became more ordered and moved along with two helices toward the 3' to 5' exonuclease domain to form the binding groove . A single-stranded, 3' extension of three nucleotides bound to the 3' to 5' exonuclease active site . Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polymerase catalytic site.

Cell Immunol, 1993 Apr 15, 148(1), 157 - 65
Transfection of human immunodeficiency virus type 1 proviral DNA into primary human monocytes; Weir JP et al.; To investigate the expression of human immunodeficiency virus (HIV) genes in human monocytes, a DNA transfection system was developed and characterized using cultured primary monocytes . Monocytes that were cultured 6-7 days in an adherent monolayer were efficiently recovered and transfected by electroporation with an expression vector containing the Escherichia coli lacZ gene under control of the cytomegalovirus immediate-early promoter . Successful transfection was detected by expression of beta-galactosidase activity and by histochemical staining for beta-galactosidase in cells that were allowed to readhere to plastic following transfection . Over 30% of the surviving adherent monocytes expressed the transfected beta-galactosidase gene . In the same manner, monocytes were transfected with HIV provirus clones pIIIB and pIIB/PB . The provirus pIIIB/PB differs from pIIIB only in that it contains a small sequence from the env gene of a macrophage tropic HIV-1 . Virus derived from pIIIB will not replicate in monocytes whereas virus derived from pIIIB/PB will . Monocytes transfected with either provirus DNA expressed high levels of p24 antigen within 1 day of transfection, and cell-free supernatants contained virus that was infectious for T cells . In contrast, only supernatants from pIIIB/PB transfections contained virus capable of infecting monocytes . Thus, proviral DNA of T cell tropic HIV efficiently completes the retroviral life cycle in monocytes in a manner indistinguishable from that of macrophage tropic HIV, and progeny virus retain their T cell tropism.

Biochem J, 1993 Apr 15, 291 ( Pt 2), 493 - 504
Determination of the order of substrate addition to MspI DNA methyltransferase using a novel mechanism-based inhibitor; Taylor C et al.; The cloning and overexpression of the MspI DNA methyltransferase as a functional fusion with glutathione S-transferase is described . The fusion enzyme retains full biological activity and has been used to investigate the interaction of substrates and inhibitors with MspI DNA methyltransferase . The fusion enzyme has been purified to homogeneity in a single step on GSH-agarose and is free from contaminating exonuclease activity . The enzyme can be photolabelled with S-adenosyl-L-methionine and the level of incorporation of label is enhanced by the presence of a nonspecific DNA duplex . In the presence of a cognate oligodeoxynucleotide, no photolabelling was observed since methyl transfer occurs instead . The inclusion of a mechanism-based inhibitor of C-5 deoxycytidine DNA methylation (an oligodeoxynucleotide containing the base 2-pyrimidinone-1-beta-D-2'-deoxyribofuranoside in the position of the deoxycytidine to which methyl addition occurs), which is thought to form a covalent interaction with the reactive cysteine of such enzymes, led to an enhancement of S-adenosyl-L-methionine photolabelling which suggests that, in contrast with results obtained with EcoRII DNA methyltransferase {Som and Friedman (1991) J . Biol . Chem . 266, 2937-2945}, methylcysteine is not the photolabelled product . The implications of the results obtained with this mechanism-based inhibitor are discussed with respect to other C-5-specific DNA methyltransferases . Gel-retardation assays in the presence of cognate oligodeoxynucleotides that contain the reactive pyrimidinone base in place of the deoxycytidine target base are described . These demonstrate that most probably a stable covalent bond is formed between the methyltransferase and this oligodeoxynucleotide . However, the alternative of extremely tight non-covalent binding cannot be rigorously excluded . Furthermore, the results from these experiments indicate that the reaction mechanism proceeds in a manner similar to that of HhaI DNA methyltransferase with sequence-specific DNA binding being followed by addition of S-adenosyl-L-methionine and concomitant isomerization of the ternary complex leading to methyl transfer . S-Adenosyl-L-homocysteine appears to inhibit the reaction pathway as a result of either competition with the methyl donor and potentiation of a high-affinity interaction between the enzyme and DNA in an abortive ternary complex or through an allosteric interaction.

Eur J Biochem, 1993 Apr 15, 213(2), 749 - 56
Functional domains in the Escherichia coli release factors . Activities of hybrids between RF-1 and RF-2; Moffat JG et al.; Chimeras between Escherichia coli release factors RF-1 and RF-2 have been constructed to study the role of the release factors in termination, in particular whether each possesses specific domains for recognition of the stop codon, and for facilitating peptidyl-tRNA hydrolysis . One hybrid factor showed normal codon-recognition activity but was defective in its ability to facilitate hydrolysis . Overexpression of this protein was toxic to the cell . Conversely, another hybrid factor showed complete loss of codon recognition but retained some hydrolysis activity . These two functional activities of the release factors were not localised in domains within either the amino-terminal or carboxy-terminal halves of the primary sequence as previously predicted . Evidence from the activities of the hybrid proteins and from earlier studies suggests that a combination of residues from the beginning and middle of the sequence, including a region of very high sequence conservation, contribute to the hydrolysis domain, whereas residues from both the amino-terminal and carboxy-terminal halves of the molecule are important for the codon recognition domain.

Biochem Biophys Res Commun, 1993 Apr 15, 192(1), 15 - 21
Enhancement of the stability and activity of aspartase by random and site-directed mutagenesis; Zhang HY et al.; Enzymatic generation of mutant libraries for random mutagenesis of aspartase gene from E . coli J2 was made . A mutant enzyme with 4-fold increase in aspartase activity was found . It is stable at pH7.5-9.0 (wild-type: pH7.0-8.0); heat stability and alpha-helicity are higher than those of the wild-type . By using site directed mutagenesis, the aspartase was activated by replacement of Lys-126 with an arginine residue . The mutation produced functional alterations without appreciable structure changes . The optimum pH for the mutant enzyme is 8.5 . The stable pH range is 7.0-9.0 . Heat stability is higher than that of the wild-type one . Activity of the mutant enzyme is about 5-fold as much as that of wild-type one.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3358 - 62
Signal sequence region of mitochondrial precursor proteins binds to mitochondrial import receptor; Murakami H et al.; An integral mitochondrial membrane protein (p32) of yeast has previously been molecularly cloned and sequenced and suggested to function as a mitochondrial import receptor . However, this protein has also been proposed to function as phosphate translocator {Guerin, B., Bukusoglu, C., Rakotomanana, F . & Wohlrab, H . (1990) J . Biol . Chem . 265, 19736-19741; Phelps, A., Schobert, C.T . & Wohlrab, H . (1991) Biochemistry 30, 248-252} . Here we have purified p32 after expression of its gene in Escherichia coli and assayed its ability to bind to various preproteins containing signal sequences for protein translocation into mitochondria, chloroplasts, or the endoplasmic reticulum . Our data suggest that p32 contains a binding site specific for the signal sequence region of mitochondrial preproteins . These data are consistent with the previous assignment of p32 as an import receptor and are discussed with regard to the apparently conflicting assignment of this protein as phosphate translocator.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3343 - 7
The nucleotide in position 32 of the tRNA anticodon loop determines ability of anticodon UCC to discriminate among glycine codons; Lustig F et al.; We have investigated the influence of structures in the tRNA anticodon loop and stem on the ability of the anticodon to discriminate among codons . We had previously shown that anticodon UCC, when placed in the structural context of tRNA(Gly1) from Escherichia coli, discriminated efficiently between the glycine codons, as required by the wobble rules . Thus, this anticodon read GGA and GGG but did not read GGU and GGC, whereas in mycoplasma tRNA(Gly), the same anticodon did not discriminate among the glycine codons . We have now determined the reading properties of three constructions based on tRNA(Gly1) containing the anticodon UCC in different structural contexts . In one of these constructs, tRNA(Gly1-ASL), the anticodon loop and stem are the same as in mycoplasma tRNA(Gly) . The second construct, tRNA(Gly1-AS), has an anticodon stem identical with the mycoplasma tRNA(Gly), whereas in the last construct, tRNA(Gly1-C32), the only difference from tRNA(Gly1)(UCC) is that the uridine in position 32 of the anticodon loop has been replaced by cytidine . These constructs were tested for ability to read glycine codons in an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each codon . We found that the anticodon UCC, when present in tRNA(Gly1-AS), discriminated among the glycine codons, whereas in the constructs tRNA(Gly1-ASL) and tRNA(Gly1-C32), the same anticodon had lost its ability to discriminate--i.e., it behaved as in mycoplasma tRNA(Gly) . These results strongly suggest that nt 32 of the anticodon loop of tRNA(Gly1)(UCC) decisively influences the reading properties of the anticodon UCC.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3202 - 6
Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB); Dreyfus LA et al.; The heat-stable enterotoxin B (STB) of Escherichia coli is a 48-amino acid extracellular peptide that induces rapid fluid accumulation in animal intestinal models . Unlike other E . coli enterotoxins that elicit cAMP or cGMP responses in the gut {heat-labile toxin (LT) and heat-stable toxin A (STA), respectively}, STB induces fluid loss by an undefined mechanism that is independent of cyclic nucleotide elevation . Here we studied the effects of STB on intracellular calcium concentration ({Ca2+}i), another known mediator of intestinal ion and fluid movement . Ca2+ and pH measurements were performed on different cell types including Madin-Darby canine kidney (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat pituitary cells . Ca2+ and pH determinations were performed by simultaneous real-time fluorescence imaging at four emission wavelengths . This allowed dual imaging of the Ca(2+)- and pH-specific ratio dyes (indo-1 and SNARF-1, respectively) . STB treatment induced a dose-dependent increase in {Ca2+}i with virtually no effect on internal pH in all of the cell types tested . STB-mediated {Ca2+}i elevation was not inhibited by drugs that block voltage-gated Ca2+ channels including nitrendipine, verapamil (L-type), omega-conotoxin (N-type), and Ni2+ (T-type) . The increase in {Ca2+}i was dependent on a source of extracellular Ca2+ and was not affected by prior treatment of MDCK cells with thapsigargin or cyclopiazonic acid, agents that deplete and block internal Ca2+ stores . In contrast to these results, somatostatin and pertussis toxin pretreatment of MDCK cells completely blocked the STB-induced increase in {Ca2+}i . Taken together, these data suggest that STB opens a GTP-binding regulatory protein-linked receptor-operated Ca2+ channel in the plasma membrane . The nature of the STB-sensitive Ca2+ channel is presently under investigation.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3147 - 51
T7 RNA polymerase mutants with altered promoter specificities; Raskin CA et al.; The amino acid at position 748 in T7 RNA polymerase (RNAP) functions to discriminate base pairs at positions -10 and -11 in the promoter . We have constructed a series of T7 RNAP mutants having all possible amino acid substitutions at this position . Surprisingly, most (13/19) substitutions result in active RNAPs, and many of these exhibit altered promoter specificities . Identification of mutant RNAPs with altered specificities expands the repertoire of highly specific phage RNAPs that are available for use in phage RNAP-based transcription systems and highlights the complexity of sequence-specific DNA recognition.

Gene, 1993 Apr 15, 126(1), 93 - 7
Propagation of phage Mu in IHF-deficient Escherichia coli in the absence of the H-NS histone-like protein; Kano Y et al.; Integration host factor (IHF) is known to be required for the expression of early genes and formation of the transpososome of mutator phage Mu . Prophage Mucts62 was stably maintained at 30 degrees C and proliferated effectively after thermal induction at 42 degrees C in an Escherichia coli mutant defective in the histone-like H-NS and IHF proteins . No IHF activity was detected in cells lacking H-NS and IHF; cells could not be transformed with plasmid pCL1920, which is based on the pSC101 replicon whose replication requires IHF . No difference in the superhelical densities of the reporter plasmid was detected in the H-NS, IHF null mutant and parental cells . From these results it is concluded that IHF is not essential for Mu development . These results also suggest that H-NS may function as a silencer for Pe operon expression and that IHF overcomes the inhibitory effect of H-NS.

Gene, 1993 Apr 15, 126(1), 85 - 92
Overproduction of Anabaena 7120 ribulose-bisphosphate carboxylase/oxygenase in Escherichia coli; Larimer FW et al.; As a prerequisite to protein engineering, we have overexpressed the rbcLS operon of the cyanobacterium Anabaena 7120, in Escherichia coli . The operon encodes the large and small subunits of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) . Levels of active enzyme exceed 6% of soluble protein . We noted an apparent third gene, an unidentified open reading frame (URF) referred to here as rbcX, in the 558-bp intergenic space between the large and small subunit encoding genes . The URF, rbcX, has no known function . High-level production of Rubisco activity from the rbc operon in E . coli required simultaneous overproduction of the GroESL chaperonins under a regimen of limited growth, in contrast to more modest conditions which suffice for efficient production of the Anacystis nidulans cyanobacterial Rubisco . Deletion of rbcX or inversion of the rbcL-rbcS order did not enhance expression levels . The recombinant Rubisco, purified to near homogeneity, exhibited functional properties {Km(ribulose-P2), kcat, transition-state-analogue binding stoichiometry/exchange, and specificity factor} essentially identical to those of the enzyme obtained from Anabaena.

Gene, 1993 Apr 15, 126(1), 35 - 41
A method for the site-directed mono- and multi-mutagenesis of double-stranded DNA; Weiner MP et al.; A general solid-phase method for the site-directed mutagenesis of double-stranded DNA (dsDNA) is described . Plasmid DNA is linearized using either a restriction endonuclease (ENase) or the RecA-assisted ENase or RecA-AC cleavage method . Alternatively, PCR may be used to generate linear dsDNA . One or both strands of the DNA is biotinylated and attached to a solid support, and the DNA strands are separated using 0.2 M NaOH . An extension oligodeoxyribonucleotide (oligo) and a single or multiple oligo(s) containing the desired mutation(s) are annealed to one of the bound DNA strands and used to initiate the synthesis of a complementary strand by a nonstrand-displacing DNA polymerase . The in vitro synthesized strand incorporating the desired alteration(s) is melted off of the support and recircularized using one of several types of bridging oligos, DNA ligase, and a DNA polymerase and transformed into the host . Greater than 90% mutagenic efficiency has been obtained using this method.

Gene, 1993 Apr 15, 126(1), 129 - 33
Organization of the genes encoding p-aminobenzoic acid synthetase from Streptomyces lividans 1326; Arhin FF et al.; Genes involved in the biosynthesis of p-aminobenzoic acid (PABA) in Streptomyces lividans 1326 were cloned in pBR322 by complementing a pabB mutant of Escherichia coli . A 2.7-kb BamHI-SstI fragment of the cloned DNA complemented pabA and pabB mutations in both E . coli and S . lividans; complementation in S . lividans was accompanied by integration of the recombinant plasmid into the host chromosome . The nucleotide (nt) sequence of the 2.7-kb fragment contained two open reading frames, the deduced amino acid sequences of which were similar to those of pabA and pabB products from other bacteria . The nt sequences indicated that pabA and pabB are closely linked in S . lividans and supported cloning evidence that the genes are expressed from a promoter with features resembling those of most E . coli promoters.

Gene, 1993 Apr 15, 126(1), 115 - 7
Deletion mutations in the speED operon: spermidine is not essential for the growth of Escherichia coli; Xie QW et al.; Null mutants of Escherichia coli were constructed that cannot synthesize spermidine, because of deletions in the gene encoding S-adenosylmethionine decarboxylase . These mutants are still able to grow at near normal rates in purified media deficient in polyamines . These results in E . coli differ from recent findings that null mutants of Saccharomyces cerevisiae and of Neurospora crassa have an absolute growth requirement for spermidine.

Gene, 1993 Apr 15, 126(1), 105 - 7
Overproduction and single-step purification of GAL4 fusion proteins from Escherichia coli; Reece RJ et al.; A DNA fragment encoding the yeast GAL4 transcriptional activator DNA-binding domain (amino acids 1-93) was cloned into an Escherichia coli expression vector such that the overproduced protein is tagged with six histidine residues and a factor Xa protease cleavage site . The vector also contains unique restriction sites at the 3' end of the gene to allow the construction of fusion proteins . These fusion proteins can easily be purified to homogeneity and their activity tested in vitro.

Nature, 1993 Apr 15, 362(6421), 652 - 4
Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage; Branch P et al.; Acquired resistance to alkylating agents such as N-methyl-N-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine results from the ability to tolerate the potentially cytotoxic methylated base O6-methylguanine (m6-G) in DNA . In the absence of repair by demethylation in situ, m6-G is probably lethal through its inappropriate processing by the cell . DNA mismatch correction is an attractive candidate for the processing function because although it is replicated, m6-G has no perfect complementary base . Thus, m6-G in DNA might provoke abortive mismatch repair and tolerance could subsequently arise through loss of a mismatch repair pathway . Mismatch correction helps maintain genomic fidelity by removing misincorporated bases and deaminated 5-methylcytosine from DNA, and its loss by mutation confers a mutator phenotype on Escherichia coli . Here we describe human and hamster cell lines that are tolerant to N-methyl-N-nitrosourea and are defective in a DNA mismatch binding activity . The loss of this activity, which acts on G.T mispairs, confers a mutator phenotype.

J Biol Chem, 1993 Apr 15, 268(11), 8284 - 9
On the mechanism of the activation of human plasminogen by recombinant staphylokinase; Collen D et al.; The mechanism of activation of human plasminogen by recombinant staphylokinase (STAR) was studied using the active site titrant p-nitrophenyl-p'-guanidinobenzoate (NPGB) . NPGB prevented active site exposure in equimolar mixtures of plasminogen and STAR but reacted stoichiometrically with mixtures preincubated in the absence of titrant . Active site generation occurred progressively, with a marked initial lag phase followed by an exponential growth phase, and was associated with the conversion of single-chain plasminogen to two-chain plasmin . Incubation of mixtures of plasminogen and STAR with catalytic amounts (< 0.2% molar ratio) of preformed plasmin.STAR complex or of urokinase shortened the lag hase, whereas catalytic amounts (5% molar ratio) of the plasmin inhibitor alpha 2-antiplasmin delayed active site generation . The following kinetic model for the activation of plasminogen (P) by STAR (S) fits the experimental data, {formula: see text} and is described by {formula: see text} or {formula: see text} In this model, plasminogen and STAR produce an inactive complex (P.S), in which active plasmin.STAR (p.S) is generated in a rate limiting step, which is accelerated by plasminogen activators and delayed by plasmin inhibitors . At room temperature in a 0.1 M Veronal buffer, pH 8.3, containing 0.1 M arginine, the data are adequately fitted by the integrated equation with k1 = 4.0 x 10(-7) s-1 and k2 = 1.3 x 10(-2) microM-1 s-1 . The k1 value could be explained by contamination of the plasminogen preparation with 3 ppm plasmin, converted by S to p.S . It is concluded that STAR activates plasminogen via a mechanism which differs in several essential aspects from that of streptokinase.

J Biol Chem, 1993 Apr 15, 268(11), 8256 - 60
The binding site for the beta gamma subunits of heterotrimeric G proteins on the beta-adrenergic receptor kinase; Koch WJ et al.; The beta gamma subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes . Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T . (1992) J . Biol . Chem . 267, 2222-2227) and beta-adrenergic (Pitcher, J . A., Inglese, J., Higgins, J . B., Arriza, J . L., Casey, P . J., Kim, C., Benovic, J . L., Kwatra, M . M., Caron, M . G., and Lefkowitz, R . J . (1992) Science 257, 1264-1267) receptors . Beta gamma mediates the membrane targeting of the beta-adrenergic receptor kinase (beta ARK), in response to receptor activation, through a specific beta ARK-beta gamma interaction . This process utilizes the membrane-anchoring properties of the isoprenylated gamma subunit of beta gamma . In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of beta ARK which binds beta gamma and thereby results in membrane translocation . We studied the ability of beta gamma to enhance the enzymatic activity of a series of truncated mutants of bovine beta ARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of beta ARK to bind beta gamma subunits, and the ability of synthetic peptides comprised of beta ARK sequences to inhibit beta gamma activation of beta ARK1 . We find that the minimal beta gamma binding domain of beta ARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus . A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited beta gamma activation of beta ARK1, with an IC50 of 76 microM . The identification of this "beta gamma binding domain" on beta ARK and the development of peptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of beta gamma activation of other G protein-effector systems.

J Biol Chem, 1993 Apr 15, 268(11), 8213 - 20
Partial revertants of tryptophan synthetase alpha chain active site mutant Asp60-->Asn; Yanofsky C et al.; Residue Asp60 of the tryptophan synthetase alpha chain of Escherichia coli is though to interact with the pyrrole NH of substrate indole-3-glycerol phosphate and facilitate its cleavage to indole and glyceraldehyde 3-phosphate . Two distinguishable partial revertants of DN60 tryptophan synthetase alpha mutant trpA34 were analyzed . The slower growing partial revertant, PR1, had the second-site change, YD102 . The other partial revertant, PR2, lacked three consecutive base pairs, resulting in replacement of Ala59 and Asn60 of the DN60 mutant alpha polypeptide by Asp . Inspection of the three-dimensional structure of the enzyme-substrate analog complex revealed that Tyr102 is in the vicinity of the pyrrole NH of the substrate . The PR1 alpha chain has a near normal Km for substrate, whereas the PR2 polypeptide has greatly reduced substrate affinity . The PR2 polypeptide is more active than the PR1 polypeptide in the alpha beta reaction in vitro and appears to be more active than the PR1 polypeptide in vivo . Attempts to obtain repeat occurrences of the PR2 deletion mutation were unsuccessful . A third type of trpA34 partial revertant, PR3, that grows very poorly in minimal medium, also has a Tyr102 replacement: YF102 . These findings demonstrate that each of the second-site mutations affects a residue located in the vicinity of the active site residue altered by the primary mutation . Slightly leaky mutant trpA89, genetically altered near the site of the trpA34 mutation, was found to have a GS61 substitution.

J Biol Chem, 1993 Apr 15, 268(11), 8193 - 8
Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and countermovement of protons in a SecA- and presecretory protein-dependent manner; Kawasaki S et al.; Everted membrane vesicles were prepared from Escherichia coli cells containing either overproduced amounts (OP-membrane vesicles) or normal amounts (normal membrane vesicles) of SecY and SecE, both of which are essential components of the protein translocation apparatus . The rates of translocation of pro-OmpA were similar in the two types of membrane vesicles, whereas translocation ATPase activity, which requires SecA, a precursor protein (pro-OmpA), and membrane vesicles, was appreciably higher with OP-membrane vesicles than with normal membrane vesicles . Since ATP hydrolysis has been shown to take place at an earlier part of the translocation reaction, these results suggest that the overproduction of SecY and SecE enhanced the activity of the earlier process, but not the entire process, of the translocation reaction . The addition of pro-OmpA in the presence of SecA caused the partial collapse of delta pH (inside acidic) generated on OP-membrane vesicles, suggesting that protons come out from the inside of the membrane vesicles in a pro-OmpA-dependent manner . The collapse of delta pH caused by pro-OmpA required SecA, ATP, and SecY and was not detected when normal membrane vesicles were used . These results indicate that the early event of protein translocation, which requires the functioning of SecA, SecY, and SecE, causes the countermovement of protons.

J Biol Chem, 1993 Apr 15, 268(11), 7990 - 8002
Repair of aflatoxin B1 DNA adducts by the UvrABC endonuclease of Escherichia coli; Oleykowski CA et al.; The repair by UvrABC endonuclease of two major adducts formed by aflatoxin B1 in DNA was found to be similar . Aflatoxin epoxide was used to generate the aflatoxin B1.N7-guanine adduct which can convert to aflatoxin B1-formamidopyrimidine adduct . The reaction of the aflatoxin B1 epoxide with DNA follows pseudo-first order kinetics . The DNA sequence-specific relative reactivity of the epoxide is the same as previously observed for aflatoxin B1 activated by liver microsomes, therefore strongly reinforcing the notion that aflatoxin B1 reacts with DNA through the epoxide intermediate . For the majority of lesion sites, a high affinity protein-DNA complex was formed from the UvrA and the UvrB proteins with similar efficiency to both adducts, and to pyrimidine dimers, and then nicks the DNA when UvrC was added . The two incisions are at the eighth phosphodiester moiety 5' and the sixth phosphodiester moiety 3' of a modified guanine nucleotide . Both incisions appeared to be concerted . For some sites, the DNA sequence can alter the relative incision efficiency up to 15-fold . However, the majority of these AFB1 lesion structures in most DNA sequences are similar with respect to recognition by this nucleotide excision repair enzyme . Therefore the observation that the aflatoxin B1.N7-guanine lesion is removed rapidly, while the aflatoxin B1-formamidopyrimidine lesion persists in the mammalian cell may have other mechanistic explanations.

J Biol Chem, 1993 Apr 15, 268(11), 7885 - 92
Modulation of ligand binding affinity of the adipocyte lipid-binding protein by selective mutation . Analysis in vitro and in situ; Sha RS et al.; The crystal structure of the adipocyte lipid-binding protein (ALBP) with coordinated fatty acid shows the hydrophobic ligand bound within a water-filled central cavity with its carboxyl group engaged in a hydrogen bonding network involving, at least in part, the functional groups of residues R126 and Y128 . We produced mutant forms of ALBP which altered these amino acids, expressed these in Escherichia coli as glutathione S-transferase (GST) fusion proteins, and examined their ligand-binding properties using the fluorescent fatty acids cis-parinaric acid (c-PA) and 12-(9-anthroyloxy)-oleate (12-AO) . The wild-type and all mutated forms of GST-ALBP displayed similar binding affinities for 12-AO, with Kd,app values ranging from 0.5 to 2.4 microM . The binding affinity of ALBP forms R126Q and Y128W for c-PA were reduced about 30-50-fold in comparison to GST-ALBP, while that for the double mutation R126L + Y128F was below the limits of detection . To determine if the hydrogen bonding system functioned in situ, Chinese hamster ovary (CHO) cell transfectants expressing wild-type ALBP demonstrated a moderate (1.5-2-fold) increase in the total rate of {3H}oleate uptake and trafficking into the esterified lipid pools over that of untransfected cells, while the rate of {3H}oleate uptake of the transfected CHOs expressing the R126L + Y128F mutation was identical to that of the control CHOs . In summary, these results suggest that the primary factor contributing to binding affinity of ALBP for fatty acids such as c-PA or oleic acid both in vitro and in situ is the hydrogen bonding network involving at least R126, Y128, and the lipid carboxyl group . However, a ligand with sufficiently large hydrophobic character such as 12-AO can bind in the absence of a functional carboxylate hydrogen bonding network, presumably due to stabilizing entropic interactions with other cavity atoms.

J Biol Chem, 1993 Apr 15, 268(11), 7818 - 24
Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning; Gutteridge S et al.; Amino acids composing a flexible loop (loop 6) of the eight-stranded barrel domain of the L-subunit of Synechococcus ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) involved in reaction intermediate stabilization have been modified by site-specific mutagenesis . Changes at positions both distant and within the active site affect overall catalysis and substrate partitioning . Most significantly, replacement of the active site Lys (Lys-334) with Arg at the apex of the loop almost completely suppressed the carboxylase activity of the enzyme relative to oxygenation, with only a modest reduction in overall catalysis . Val-331 and Thr-342, more distant from the active site but with interacting side chains, were changed to larger and smaller residues with differential effects on both turnover and substrate partitioning . Substitution of the loop with the sequence found in more efficient carboxylases only increased partitioning marginally when accompanied by alterations in the C-terminal tail of the L-subunit that interacts with the loop . Generally, modifications to the loop composition also affected enediol formation, the first step of catalysis, suggesting that the geometry and hence flexibility of this segment affect more than just stabilization of the intermediates immediately following reaction with CO2 or O2.

J Biol Chem, 1993 Apr 15, 268(11), 7660 - 7
Primary structure of the gene for glycyl-tRNA synthetase from Bombyx mori; Nada S et al.; The primary structure of the gene encoding Bombyx mori glycyl-tRNA synthetase was determined by sequence analysis of one cDNA and two genomic clones . The sequence of the protein deduced from the nucleotide sequence was verified by sequence analysis of eight peptides . The M(r) 77,667 protein is encoded in a single open reading frame of 2061 nucleotides . There are no introns in the gene . The deduced protein sequence has no obvious similarity to Escherichia coli glycyl-tRNA synthetase but contains a sequence in its amino terminus that is similar to a sequence found in the Drosophila melanogaster and human glutamyl-tRNA synthetases, the hamster and human histidyl-tRNA synthetases, bovine tryptophanyl-tRNA synthetase, and the mammalian peptide chain release factor . The B . mori glycyl-tRNA synthetase also has sequence similarity with the Saccharomyces cerevisiae (cytoplasmic and mitochondrial), E . coli, and human threonyl-tRNA synthetases . This sequence similarity occurs in a sequence motif that is characteristic of other class II aminoacyl-tRNA synthetases . Two transcription start sites approximately 100 nucleotides apart were identified by ribonuclease mapping . One of the transcription start sites is used preferentially in the posterior silk gland . The peak in mRNA accumulation occurs 80-100 h prior to the peak in glycyl-tRNA synthetase activity and enzyme protein.

Eur J Biochem, 1993 Apr 15, 213(2), 797 - 804
Human beta 2-adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP-binding protein to adenylyl cyclase; Kleymann G et al.; Spodoptera frugiperda insect cells (Sf9) containing the stably integrated human beta 2-adrenergic receptor gene under the control of the baculovirus IE1 promoter expressed up to 350,000 human receptors/cell . The number of receptors did not change with cell density or age of culture . The adrenergic receptors overexpressed in the insect cells were functional with respect to their ligand binding and signalling properties . Coupling of the receptors to endogenous GTP-binding proteins is demonstrated by hormone-dependent stimulation of GTPase and adenylyl cyclase activity in the transformed insect cells . Western-blot analysis revealed that the endogenous GTP-binding protein appears to be of the heterotrimeric type . Antibodies raised against the mammalian alpha subunit of stimulatory GTP-binding proteins cross-react with the insect alpha subunit of GTP-binding proteins, which also exhibits the same apparent molecular mass as its mammalian counterpart . The beta subunit of GTP-binding proteins from insect cells reacts with anti-peptide serum directed against the C-terminal amino acids of the mammalian beta subunit of GTP-binding proteins, but is about approximately 2 kDa larger than that of the beta subunit of GTP-binding proteins from bovine brain . Exposure of the transformed insect cells to L-isoproterenol rapidly induces uncoupling and internalization of 30% of the heterologously expressed receptors . In contrast to the situation in mammalian cells, prolonged exposure of the agonist (24 h) does not result in down regulation of the remaining 70% of the receptors.

Eur J Biochem, 1993 Apr 15, 213(2), 781 - 8
Nucleotide binding and GTP hydrolysis by the 21-kDa product of the c-H-ras gene as monitored by proton-NMR spectroscopy; Low A et al.; Proton-NMR signals in the downfield region (below approximately 10 ppm) have been shown to provide a useful spectroscopic window to monitor the binding of guanine nucleotides to the active site of GTP/GDP-binding proteins via H-bonds, as specified here by the 21-kDa product of the c-H-ras gene (p21) . The time course of the intensity change of certain peaks upon addition of GTP to nucleotide-free p21 corresponds to the GTP hydrolysis rate as determined by HPLC . Though there are fewer potential H-bond acceptors in the GDP-bound protein than in the GTP complex, more downfield peaks are found in the former complex, suggesting tighter binding of GDP . Moreover, inspection of the downfield proton-NMR spectra permits rapid detection of subtle changes of the active site induced by complexation with slowly hydrolyzing GTP analogues resulting from mutations of the amino acid sequence, especially in the phosphate binding loop . Our studies strongly suggest that no major conformational change of the phosphate-binding region occurs upon nucleotide complexation that precedes the catalytic step . Besides, it is suspected that the Ser17 hydroxyl group is involved in nucleotide binding and GTP hydrolysis.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3428 - 32
Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding; Bushman FD et al.; The integrase protein of human immunodeficiency virus type 1 carries out a set of polynucleotidyl transfer reactions that result in the covalent attachment of the retroviral cDNA to host DNA . We have analyzed the activities of a set of deletion derivatives of the integrase protein . The analysis reveals that a central domain of only 137 amino acids is sufficient in vitro to catalyze a subset of the reactions carried out by the complete protein . This polypeptide contains an amino acid sequence motif, Asp-Xaa39-58-Asp-Xaa35-Glu (DX39-58DX35E, where X and the subscript indicate the intervening amino acids between the invariant acidic residues), that is found in the integrases of retroviruses and retrotransposons and also the transposase proteins of some bacterial transposable elements . We also find that the integrase protein can bind Zn2+, and the histidine and cysteine residues of another conserved motif (HX3-7HX23-32CX2C) are required for efficient Zn2+ binding . The activities displayed by deletion mutants suggest to us possible functions for the various parts of integrase.

Blood, 1993 Apr 15, 81(8), 2076 - 84
Interleukin-6 inhibits the proliferation of B-chronic lymphocytic leukemia cells that is induced by tumor necrosis factor-alpha or -beta; Aderka D et al.; Tumor necrosis factor (TNF-alpha) acts as a growth stimulatory factor on leukemic B lymphocytes from many patients with chronic lymphocytic leukemia (CLL) . Because TNF induces production of interleukin-6 (IL-6), which has been shown to be a growth factor for myeloma and other transformed B cells, we examined the possibility that IL-6 mediates the growth-stimulatory effect of TNF on B-CLL cells . In fact, we found that IL-6 is an inhibitor of B-CLL growth . The addition of recombinant human IL-6 markedly decreased the TNF-induced B-CLL growth, and this decrease was even greater when soluble IL-6 receptor, known to act as IL-6 agonist, was added with recombinant IL-6 . Conversely, neutralizing monoclonal antibodies to IL-6 and to the IL-6 receptor potentiated the growth stimulation of TNF on B-CLL cells, in line with the possibility that IL-6 functions as a negative feedback regulator of an autocrine TNF action on these B-leukemic cells . Evidence is presented that production of IL-6 by monocytes and B cells of CLL patients is low, suggesting that administration of IL-6 may be beneficial in CLL to reduce the eventual growth stimulation by TNF and, possibly, also the deficiency in platelets and Ig production in this disease.

J Biol Chem, 1993 Apr 15, 268(11), 8105 - 10
Poliovirus protein 2C has ATPase and GTPase activities; Rodriguez PL et al.; Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence . We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA . Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP) . The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region . Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C . Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted . The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity . Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein . Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255 . None of the fusion proteins had detectable helicase activity . We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes . These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids.

J Biol Chem, 1993 Apr 15, 268(11), 8078 - 84
Expression of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli . Role of N-2 proline in degradation of the protein; Lange AJ et al.; Human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is 96% identical to the rat and bovine liver enzymes, and all of the critical catalytic and substrate binding residues in both the kinase and bisphosphatase domains are conserved in the three enzymes . However, in contrast to rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which is readily expressed in an Escherichia coli T-7 RNA polymerase-based expression system, the human liver bifunctional enzyme could not be expressed in this system . Western blot and slot blot analysis revealed that although both the bifunctional enzyme protein and its mRNA were rapidly induced by the addition of isopropyl-1-thio-beta-D-galactopyranoside, the protein underwent rapid degradation . Deletion of the N-2 proline residue or its mutation to arginine, the corresponding residue in the rat liver enzyme, revealed that this proline residue was responsible for its rapid degradation . The Pro-2-->Arg mutant could be expressed with a high yield (20 mg/liter) in E . coli . The results support the hypothesis that a proline residue at N-2 facilitates bifunctional enzyme degradation in E . coli . The E . coli expressed mutant form was purified to homogeneity by phosphocellulose chromatography, and its kinetic properties were compared with those of the rat liver enzyme . The kinetic properties of the two enzymes were identical except for the presence of substrate (fructose 6-phosphate) inhibition of the human liver enzyme but not of the rat liver enzyme . The ability to express and purify large amounts of human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase will permit structure/function and x-ray crystal structure studies of the enzyme and ultimately its targeting for drug therapy.

J Biol Chem, 1993 Apr 15, 268(11), 8046 - 52
Expression of rat liver phenylalanine hydroxylase in insect cells and site-directed mutagenesis of putative non-heme iron-binding sites; Gibbs BS et al.; Rat liver phenylalanine hydroxylase was expressed in both Escherichia coli and the Spodoptera frugiperda insect cell line, Sf9 . Recombinant enzyme from E . coli was inactive and contained less than 0.1 iron atom/subunit . In contrast, recombinant enzyme expressed in Sf9 cells using a baculovirus vector was active and identical in several properties to phenylalanine hydroxylase from rat liver: the Km for 6-methyltetrahydropterin was 39 microM (compared with 35 microM for the rat liver enzyme), 1 atom of iron was "associated" per enzyme subunit, and electron paramagnetic resonance spectra showed that iron was distributed within two distinct environments . Putative iron-binding sites of phenylalanine hydroxylase were studied by mutating either histidine 284 or 289 to serine and expressing these mutant enzymes (PAH-H284S and PAH-H289S) in Sf9 cells . Mutants were expressed at levels similar to wild-type PAH, but contained < or = 0.1 iron/subunit and were inactive . Thus, both His284 and His289 apparently are required for iron binding and hydroxylation activity.

J Biol Chem, 1993 Apr 15, 268(11), 7636 - 9
Isolation and characterization of recombinant mitochondrial 4-aminobutyrate aminotransferase; Park J et al.; 4-Aminobutyrate aminotransferase, which catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde, is a key enzyme of the 4-aminobutyrate shunt . The amino acid sequence predicted from the cDNA sequence shows that the precursor protein consists of the mature enzyme of 473 amino acid residues and an amino-terminal segment of 27 amino acids (Kwon, O . S., Park, J., and Churchich, J . E . (1992) J . Biol . Chem . 267, 7215-7216) . A recombinant 4-aminobutyrate aminotransferase has been expressed in Escherichia coli using pETIId as expression vector . The protein has been purified and characterized as a dimer (2 x 55 kDa) . NH2-terminal sequence analysis has revealed the presence of an extra amino-terminal segment (signal peptide) predicted from the cDNA sequence . The isolated precursor of 4-aminobutyrate aminotransferase contains pyridoxal 5-phosphate and exhibits catalytic activity (18 units/mg) comparable to that of the mature enzyme (20 units/mg) . The presequence peptide of the precursor of mitochondrial 4-aminobutyrate aminotransferase does not interfere with the folding and functional properties of the mature moiety of the aminotransferase.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3670 - 4
Outer-membrane PapC molecular usher discriminately recognizes periplasmic chaperone-pilus subunit complexes; Dodson KW et al.; P pili are highly ordered composite structures consisting of thin fibrillar tips joined end-to-end to rigid helical rods . The production of these virulence-associated structures requires a periplasmic chaperone (PapD) and an outer membrane protein (PapC) that is the prototype member of a newly recognized class of proteins that we have named "molecular ushers." Two in vitro assays showed that the preassembly complexes that PapD forms with the three most distal tip fibrillar proteins (PapG, PapF, and PapE) bound to PapC . The relative affinity of each complex for PapC was found to correlate with the final position of the subunit type in the tip fibrillum . In contrast, the complexes PapD forms with the major component of the pilus rod, PapA, or the pilus rod initiating protein, PapK, did not recognize PapC . The in vitro data argue that differential targeting of chaperone-subunit complexes to PapC may be part of a mechanism to ensure the correctly ordered assembly of adhesive composite pili.

Eur J Biochem, 1993 Apr 15, 213(2), 859 - 64
Structural studies of the O-antigenic polysaccharide of an enteroaggregative Escherichia coli strain; Weintraub A et al.; The polysaccharide part of the lipopolysaccharide obtained from an enteroaggregative Escherichia coli strain isolated from a young child with diarrhoea in Santiago, Chile (strain 73-1) was investigated . Sugar and methylation analyses of native and partially degraded polysaccharide together with 1H-NMR and 13C-NMR spectroscopy revealed that the polysaccharide is built of pentasaccharide repeating units . The structure of the repeating unit of E . coli strain 73-1 O-polysaccharide is (formula: see text)

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3641 - 4
Molecular characterization of a structural epitope that is largely conserved among severe isolates of a plant virus; Pappu HR et al.; Direct molecular evidence was obtained for the critical role of a single amino acid residue in a structural epitope distinguished by the monoclonal antibody MCA-13, which reacts selectively with severe isolates of citrus tristeza virus (CTV) . Different CTV isolates cause a wide range of symptoms in the diverse citrus species they affect . Severe symptoms include decline, stem pitting, and seedling yellows . Plants infected by mild isolates are essentially symptomless . The monoclonal antibody MCA-13, which discriminates severe isolates from mild isolates of the virus, was used to map its epitope on the coat protein of CTV . A diverse group of coat protein genes of geographically and biologically distinct CTV isolates which are either MCA-13-reactive or MCA-13-nonreactive was cloned and sequenced . A series of mutant coat protein genes was constructed through oligonucleotide-directed, site-specific mutagenesis . The reactivity of the wild-type and mutant coat proteins expressed in Escherichia coli was evaluated by Western blotting using MCA-13 and polyclonal antibody prepared to CTV-coat protein . A single nucleotide alteration resulting in a Phe-->Tyr mutation at position 124 of the coat protein abolished the MCA-13 reactivity of a severe isolate, whereas a Tyr-->Phe mutation at the same site conferred MCA-13 reactivity on the coat protein of a previously nonreactive mild isolate of CTV.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3569 - 73
Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm; Gmachl M et al.; The venom of honeybees, Apis mellifera, contains several biologically active peptides and two enzymes, one of which is a hyaluronidase . By using degenerate oligonucleotides derived from the amino-terminal sequence of this hyaluronidase reported by others, clones encoding the precursor for this enzyme could be isolated from a cDNA library prepared from venom glands of worker bees . The deduced amino acid sequence showed that bee venom hyaluronidase is a polypeptide composed of 349 amino acids containing four cysteines and three potential sites for N-glycosylation . The sequence of the precursor also indicated that the conversion of the pro-enzyme to the end product must involve cleavage of a Thr-Pro bond, a most unusual processing reaction . The mRNA encoding hyaluronidase could also be detected in testes from drones . Expression of the cloned cDNA in Escherichia coli yielded a 41-kDa polypeptide that had hyaluronidase activity . Interestingly, the hyaluronidase from bee venom glands exhibited significant homology to PH-20, a membrane protein of guinea pig sperm involved in sperm-egg adhesion . These structural data support the long-held view that hyaluronidases play a role in fertilization.

Gene, 1993 Apr 15, 126(1), 109 - 13
An Escherichia coli plasmid vector system for high-level production and purification of heterologous peptides fused to active chloramphenicol acetyltransferase; Robben J et al.; A very small plasmid vector system is described for construction and high-level production of C-terminal chloramphenicol acetyltransferase (CAT) fusion proteins in Escherichia coli . The only functional elements of the plasmid are a minimal region of the ColE1 origin of DNA replication and the Tn9 cat gene, both under control of a tac promoter . Since C-terminal fusion to CAT does not interfere with chloramphenicol (Cm) resistance, plasmids are maintained under Cm selection . Because of its small size (1392 bp), the system is especially convenient for building and expression of synthetic genes and gene fragments . This concept was utilized to generate a fusion with a synthetic gene encoding the multiple-epitope fragment from the rubella virus E1 membrane protein . Affinity-purified fusion proteins were obtained in mg amounts from 100-ml batches of culture fluid, and incorporated as a specific antigen in a rubella immunoglobulin G enzyme-linked immunosorbent assay.

J Immunol, 1993 Apr 15, 150(8 Pt 1), 3389 - 96
Requirement for both H and L chain V regions, VH and VK joining amino acids, and the unique H chain D region for the high affinity binding of an anti-phosphotyrosine antibody; Ruff-Jamison S et al.; Sequence analysis of a panel of antibodies to phosphotyrosine revealed predominant H and L chain V regions in the immune response and a unique D segment in the Py20 mAb, which exhibits a high affinity for phosphotyrosine . In order to determine the influence of somatic diversity on the high affinity binding of Py20, H and L chain V regions were expressed in Escherichia coli as an Fv dimer . Whereas the H or L chain V regions of Py20 alone were unable to bind phosphotyrosine, the Fv binds phosphotyrosine with an affinity comparable with the intact IgG as determined by fluorescence quenching experiments (1.55 x 10(-7) M vs 1.25 x 10(-7) M, respectively) . Substitution of the Py20 V regions with other IgG V regions that differed greatly in sequence abolished binding . A high affinity Py20-combining site was dependent on the presence of the unique D-D segment . Replacement of the Py20 D-D region with a single homologous D region resulted in a decrease in affinity (5.9 x 10(-7) M) . Substitution of this D-D region for the D region of another anti-phosphotyrosine antibody that is known to bind phosphotyrosine weakly (1 x 10(-3) M) conferred high affinity binding . Removal of three tyrosines from the first of the two D regions was accompanied by a fivefold reduction in affinity for phosphotyrosine . In addition, changing the VK and VH junctional amino acids resulted in a complete loss of binding . Therefore, the formation of the high affinity Py20 combining site requires both a H and L chain that are similar in sequence to those of Py20 including the unique D region and the junctional amino acids.

Biochemistry, 1993 Apr 13, 32(14), 3783 - 9
A reexamination of the folding mechanism of dihydrofolate reductase from Escherichia coli: verification and refinement of a four-channel model; Jennings PA et al.; The mechanism of folding of dihydrofolate reductase from Escherichia coli was reinvestigated by studying the unfolding and refolding kinetics using absorbance and fluorescence spectroscopies . The original kinetic model proposed that folding involved a series of native, intermediate, and unfolded forms which interconverted through four independent channels linked by slow cis/trans isomerization reactions at Xaa-Pro peptide bonds {Touchette, N . A., Perry, K . M., & Matthews, C . R . (1986) Biochemistry 25, 5445} . Recently, alternative sequential models have been proposed {Frieden, C . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 4413; Kuwajima et al . (1991) Biochemistry 30, 7693} which challenge the original proposal . Stopped-flow studies of the intrinsic tryptophan fluorescence demonstrated the presence of three (and tentatively four) kinetic phases in unfolding which correlated well with four phases previously observed in refolding experiments . By monitoring the binding of the inhibitor methotrexate during folding at varying relative concentrations of inhibitor to protein, it was found that the selective loss of the slow-folding phases at substoichiometric levels could only be explained by a four-channel folding model . Double-jump experiments (native-->unfolded-->native) showed that the four refolding channels are populated within 20 s at 15 degrees C and are not likely to be due to proline isomerization . Reverse double-jump experiments (unfolded-->native-->unfolded) demonstrated that interconversions between native conformers are more rapid than originally proposed . Interestingly, the majority of the protein folds through a channel to a native conformer that is minimally populated at equilibrium . This implies that although the folding of dihydrofolate reductase is ultimately under thermodynamic control, kinetic factors contribute to the transient populations of native species during folding.

Biochemistry, 1993 Apr 13, 32(14), 3778 - 82
Biosynthesis of lipoic acid: characterization of the lipoic acid auxotrophs Escherichia coli W1485-lip2 and JRG33-lip9; Hayden MA et al.; The abilities of the Escherichia coli lipoic acid auxotrophs W1485-lip2 and JRG33-lip9 to grow on succinate medium in the presence of octanoate, 8-mercaptooctanoate, or 6-mercaptooctanoate have been determined . Both organisms are mutated in lipA . Neither organism can use octanoate or 6-mercaptooctanoate for production of lipoate, but the lip2 allele can use 8-mercaptooctanoate . Chromosomal DNA from the auxotrophs was amplified by PCR using primers derived from the DNA sequence of wild-type lipA and then sequenced . Both mutants contain single G/C to A/T mutations in lipA, resulting in conversion of Ser307 into Phe in W1485-lip2 and Glu195 into Lys in JRG33-lip9 . These results support the hypothesis that lipA is involved in the sulfur insertion step(s) of lipoate biosynthesis and indicate that it is possible to selectively block formation of the C8-S bond through suitable mutation in lipA.

Biochemistry, 1993 Apr 13, 32(14), 3644 - 8
Differential scanning calorimetry of the irreversible denaturation of Escherichia coli glucosamine-6-phosphate deaminase; Hernandez-Arana A et al.; The thermal denaturation of Escherichia coli glucosamine-6-phosphate deaminase (G6PD) at neutral pH was studied by means of differential scanning calorimetry (DSC) . In the concentration range 0.6-7.3 mg mL-1, the denaturation of this hexameric enzyme was completely irreversible as judged by the absence of any endotherm on rescanning of previously scanned samples . However, the study of the effect of scanning rate on DSC curves indicated that the denaturation of G6PD is, most likely, a complex process which includes transitions in equilibrium as well as an irreversible step; in addition, it was found that application of the equilibrium formalism to the analysis of calorimetric data seems to be justified in this case, provided that scanning rates used are above 0.75 K min-1 . The calorimetric and van't Hoff enthalpies for G6PD were 1260 +/- 118 and 160 +/- 27 kcal mol-1, respectively, indicating the presence of intermediates in the process . Accordingly, the DSC curves were adequately fitted to a model including six two-state sequential transitions . The observed protein-concentration dependence of the temperature at the maximum heat capacity, Tm, for each of the individual transitions suggests that G6PD dissociates to dimers in two consecutive steps . Using a model that includes dissociation explicitly, we calculated the thermodynamic parameters for each step . From this data, the enthalpy and free energy for the disruption of one dimer-dimer contact were roughly estimated, at pH 7.1 and 51 degrees C, as 57 and 2.1 kcal mol-1, respectively.

Biochemistry, 1993 Apr 13, 32(14), 3557 - 63
Inhibitor binding to the Phe53Trp mutant of HIV-1 protease promotes conformational changes detectable by spectrofluorometry; Rodriguez EJ et al.; HIV-1 protease contains two identical, conformationally mobile loops, known as flaps, which form in part the binding pockets for substrates and inhibitors . We have constructed a site-specific mutant of the protease in which residues Phe-53 and Phe-153 at the end of the flaps have been mutated to Trp residues, in order to incorporate a specific fluorescent probe to monitor conformational changes upon the binding of an inhibitor . The Phe53Trp (F53W) mutant of HIV-1 protease was expressed in Escherichia coli and purified from bacterial lysates . Analysis of the purified mutant protease demonstrated that its kinetic properties were highly similar to those of the wild-type protease . While binding of a potent peptide-analogue inhibitor (Ki = 9 nM) to the wild-type enzyme led to no change in protein fluorescence, a 5-8% increase in fluorescence was observed with the F53W mutant, indicating an enhancement of the Trp fluorescence due to flap movement upon inhibitor binding . Investigation of the kinetics of the F53W protease-inhibitor binding by stopped-flow spectrofluorometry revealed a rapid increase in protein fluorescence upon formation of the enzyme-inhibitor complex . These data were consistent with a one-step mechanistic model of inhibitor binding in which flap movement was concomitant with inhibitor binding, from which respective rate constants of association and dissociation of 2.5 x 10(6) M-1 s-1 and 0.023 s-1 were obtained.

Biochemistry, 1993 Apr 13, 32(14), 3638 - 43
Effects of topoisomerase II-targeted drugs on enzyme-mediated DNA cleavage and ATP hydrolysis: evidence for distinct drug interaction domains on topoisomerase II; Robinson MJ et al.; Topoisomerase II is the target for two broad groups of clinically relevant drugs . Members of these groups are classically defined by their ability to enhance enzyme-mediated DNA cleavage (such as etoposide and m-AMSA) or to inhibit enzyme-catalyzed ATP hydrolysis (such as novobiocin) . The above notwithstanding, little is known concerning the interactions of drugs in either mechanistic class with the topoisomerase II-DNA complex . In order to further delineate the mechanism of drug action, the effects of several topoisomerase II-targeted agents on the DNA cleavage and ATP hydrolysis steps of the enzyme's catalytic cycle were determined . Of the drugs examined (genistein, quercetin, quercitrin, etoposide, m-AMSA, CP-115,953, and novobiocin), only novobiocin was unable to enhance topoisomerase II-mediated DNA cleavage . Moreover, with the exception of etoposide, all of the drugs were found to inhibit enzyme-catalyzed ATP hydrolysis . This latter finding undercuts the common assumption that DNA cleavage-enhancing drugs are specific for the cleavage/religation activity of topoisomerase II . Finally, by utilizing a series of competition experiments that took advantage of mechanistic differences between drug classes, it was possible to functionally define drug interaction domains on the eukaryotic type II enzyme . Results of this novel approach indicate that the interaction domain for novobiocin on topoisomerase II is distinct from those of the DNA cleavage-enhancing drugs.

Biochemistry, 1993 Apr 13, 32(14), 3540 - 8
Specific anionic residues of the recombinant kringle 2 domain of tissue-type plasminogen activator that are responsible for stabilization of its interaction with omega-amino acid ligands; De Serrano VS et al.; The involvement of specific aspartic acid (D) and glutamic acid (E) residues of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) in stabilizing its interaction with omega-amino acid ligands has been assessed by examination of these binding events subsequent to site-directed mutagenesis of the relevant amino acid residues . We have expressed and purified nonconservative alanine (A) replacement mutants at the following amino acid sequence locations in r-K2tPA:E17 (r-{K2tPA/E17A}), E75 (r-{K2tPA/E75A}), and D78 (r-{K2tPA/D78A}) . More conservative E for D replacements were generated at the only other anionic (at neutral pH) amino acids of r-{K2tPA}, viz., D57 (r-{K2tPA/D57E}) and D59 (r-{K2tPA/D59E}) . Each of these variant polypeptides was then utilized for binding investigations with a series of omega-amino acids . No substantial differences were found in the binding constants (pH 8.0, 25 degrees C) for the ligands, 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine, and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), among wild-type (wt) r-K2tPA, r-{K2tPA/E17A}, r-{K2tPA/E75A}, and r-{K2tPA/D78A} . On the other hand, dramatic effects on this same binding were observed in recombinant mutants with alterations at D57 and D59 . In these cases, even with the most conservative replacements, i.e., r-{K2tPA/D57E} and r-{K2tPA/D59E}, the Kd values for these ligands were increased approximately 3-6-fold and 18-85-fold, respectively . NMR analysis of these variants suggested that no substantial gross conformational changes occurred as a result of the mutations made, but some localized alterations in amino acid microenvironments did take place.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Apr 13, 32(14), 3763 - 8
p-Aminobenzoate synthesis in Escherichia coli: mutational analysis of three conserved amino acid residues of the amidotransferase PabA; Roux B et al.; p-Aminobenzoate synthesis in Escherichia coli requires three enzymes, PabA, PabB, and PabC, acting respectively as glutaminase, chorismate aminase, and 4-amino-4-deoxychorismate aromatase . PabA requires stoichiometric amounts of PabB to display glutaminase activity . PabA has conserved cysteine (C79), histidine (H168), and glutamate (E170) residues that have been suggested in the analogous anthranilate synthase to form a type of catalytic triad in an acylenzyme mechanism . Mutations at each of these residues of PabA lead to the following observations . C79S PabA has 40-fold lower kcat and 10(4) lower kcat/Km with no detectable acylenzyme accumulation in steady-state turnover (vs wild-type PabA at 0.56 mol fraction of gamma-glutamyl-enzyme) . H168Q has no catalytic activity and does not compete with wild-type PabA for PabB (this may indicate a folding defect) . Four E170 mutants give three outcomes . E170D and E170A yield active PabA species, down 4-fold and 150-fold, respectively, in kcat/Km ratios from wild-type PabA . E170Q has no detectable glutaminase activity but does bind to PabB in competition with wild-typoe PabA while E170K has neither detectable catalytic activity nor the ability to be recognized by PabB.

FEBS Lett, 1993 Apr 12, 320(3), 224 - 8
Nicking of the tryptophan synthase beta 2-subunit at Glu-296 prevents the conformational change undergone on binding the alpha-subunit; Linkens HJ et al.; Using a monoclonal antibody as conformational probe it has been shown that the weakly active nicked-beta 2 dimer of tryptophan synthase generated by proteolytic cleavage at Glu-296, does not undergo on association with alpha subunit a conformational change known to occur in intact beta 2 subunit . This alpha induced conformational change is also prevented in intact beta 2 by the coenzyme pyridoxal-5'-phosphate when the substrate L-serine is absent.

FEBS Lett, 1993 Apr 12, 320(3), 256 - 60
Characterization of reactions catalysed by yeast phosphatidylinositol synthase; Klezovitch O et al.; The nature of reactions catalysed by yeast phosphatidylinositol synthase expressed in E . coli has been investigated . The single enzyme is shown to carry both CDP-diacylglycerol-dependent incorporation of inositol into phosphatidylinositol (Km for inositol of 0.090 mM) and a CDP-diacylglycerol-independent exchange reaction between phosphatidylinositol and inositol (Km for inositol of 0.066 mM) . The exchange reaction and reversal of phosphatidylinositol synthase were both stimulated by CMP, but had different optimum pH and requirements for substrates . These results suggest that CMP-stimulated exchange and CMP-dependent reverse reactions are distinct processes catalysed by the same enzyme, phosphatidylinositol synthase.

FEBS Lett, 1993 Apr 12, 320(3), 198 - 202
Interaction of homologues of Hsp70 and Cpn60 with ferredoxin-NADP+ reductase upon its import into chloroplasts; Tsugeki R et al.; A homologue of the 70-kDa heat-shock protein (Hsp70) was purified from pumpkin chloroplasts . The molecular mass of the purified protein was approximately 75 kDa and its N-terminal amino acid sequence was very similar to those of homologues of Hsp70 from bacterial cells and from the mitochondrial matrix and stroma of pea chloroplasts . The purified homologue of Hsp70 was found in the stroma of chloroplasts . To investigate the role(s) of the homologue of Hsp70 in the chloroplast stroma, we examined the possibility that the homologue of Hsp70 might interact with newly imported proteins to assist in their maturation (for example, in their folding and assembly) . Ferredoxin NADP+ reductase (FNR) imported into chloroplasts in vitro could be immunoprecipitated with antisera raised against the homologue of Hsp70 from pumpkin chloroplasts and against GroEL from Escherichia coli, which is a bacterial homologue of chaperonin 60 (Cpn60), in an ATP-dependent manner, an indication that newly imported FNR interacts physically with homologues of Hsp70 and Cpn60 in chloroplasts . Time-course analysis of the import of FNR showed that imported FNR interacts transiently with the homologue of Hsp70 and that the association of FNR with the homologue of Hsp70 precedes that with the homologue of Cpn60 . These results suggest that homologues of Hsp70 and Cpn60 in chloroplasts might sequentially assist in the maturation of newly imported FNR in an ATP-dependent manner.

Nucleic Acids Res, 1993 Apr 11, 21(7), 1527 - 32
cDNA cloning and transcriptional properties of a novel GC box-binding protein, BTEB2; Sogawa K et al.; We have cloned a cDNA for a novel GC box-binding protein designated BTEB2 from a human placenta cDNA library using rat BTEB cDNA (Imataka et al . (1992) . EMBO J . 11,3663-3671 . as a hybridization probe . BTEB2 consists of 219 amino acids and contains three contiguous zinc finger motifs at its C-terminus . The zinc finger domains showed 59% and 64% sequence similarity to those of Sp1 and BTEB, respectively . Adjacent to the N-terminal of the zinc finger motifs, a short sequence rich in basic amino acids is conserved between BTEB2 and Sp1 . Furthermore, This basic sequence concurs with the N-terminal half of the consensus sequence for basic domains of the proteins containing both helix-loop-helix and leucine zipper motifs . The other region of BTEB2 is notably rich in proline, serine, threonine, and alanine residues . BTEB2 expressed in Escherichia coli showed DNA-binding activity whose specificity was closely similar to that of Sp1 . Cotransfection experiments using Hepa-1 cells (a mouse hepatoma cell line) with a BTEB2 expression plasmid and GC box-containing reporter plasmids revealed that BTEB2 apparently activated the expression of the CAT activity . Moreover, when BTEB2 was fused to GAL4 DNA-binding domain, the chimeric protein could enhance the transcription through promoters containing GAL4-binding sites . Analysis of the BTEB2 mRNA by RNA blot analysis demonstrated that the mRNA was expressed specifically in testis and placenta with different sizes, 20S and 28S, respectively, among various organs examined.

Mol Cell Biochem, 1993 Apr 7, 121(1), 67 - 74
Biochemical and electron microscopy analysis of the endotoxin binding to microtubules in vitro; Risco C et al.; The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined . In particular, there is little information about possible intracellular targets of endotoxins . Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested . In the present work, we have studied the effect of E . coli lipopolysaccharide on the polymerization of microtubular protein in vitro . Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations . At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules . Electron microscopy showed that LPS binds to microtubules of tubulin + MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol . Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS-MAP-2 was detected . The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.

Carbohydr Res, 1993 Apr 7, 242, 91 - 107
The stepwise synthesis of oligo(glycosyl phosphates) via glycosyl hydrogenphosphonates . The chemical synthesis of oligomeric fragments from Hansenula capsulata Y-1842 exophosphomannan and from Escherichia coli K51 capsular antigen; Nikolaev AV et al.; A stepwise approach has been used in the syntheses of pentamannosyl tetraphosphate HO-{-6Man(alpha)-PO4-}4-6Man(alpha)-OMe and tetra(N-acetylglucosaminyl) triphosphate HO-{-3GlcNAc(alpha)-PO4}3- 3GlcNAc(beta)-OC6H4NO2, which are fragments of the yeast and bacteria extracellular phosphoglycans . Elongation of the chain was performed with the use of suitably protected glycosyl hydrogenphosphonate derivatives for successive introduction of glycosyl phosphate residues . Partially protected monosaccharide derivatives and oligomeric blocks served as hydroxylic components.

Biochemistry, 1993 Apr 6, 32(13), 3479 - 87
How do mutations at phenylalanine-153 and isoleucine-155 partially suppress the effects of the aspartate-27-->serine mutation in Escherichia coli dihydrofolate reductase?
Dion A, Linn CE, Bradrick TD, Georghiou S, Howell EE.
Several second-site suppressors of the D27S lesion in Escherichia coli dihydrofolate reductase (DHFR) have been identified . The activity of the primary mutant, D27S DHRF, was found to be greatly decreased at pH 7.0, consistent with aspartic acid-27 being critically involved in proton donation during catalysis . Partial suppressors of the D27S mutation have been selected by their ability to confer an increased resistance to trimethoprim upon host E . coli; the suppressors have been identified as F153S or I155N substitutions . D27S+F153S and D27S+I155N DHFRs display 2-3-fold increases in kcat over D27S DHFR values, but only the F153S mutation decreases the Km for dihydrofolate by a factor of 2 . Neither double mutant approaches wild-type DHFR activity . Unexpectedly, Phe153 and Ile155 occur on the surface of the protein and are approximately 8 and 14 A distant from the active site . Ile155 is a member of a beta-bulge . A previously identified suppressing mutation, F137S, occurs nearby and is also a member of the same beta-bulge {Howell et al . (1990) Biochemistry 29, 8561-8569} . Clustering of these three second-site mutations indicates this area of the structure may be important in protein function . Conformational changes due to the presence of these suppressing mutations are likely as the F153S and I155N mutations do not affect hydride-transfer rates upon introduction in wild-type DHFR and alterations in circular dichroism spectra are associated with the double-mutant DHFRs.

Biochemistry, 1993 Apr 6, 32(13), 3363 - 7
Novel ion specificity of a carboxylate cluster Mg(II) binding site: strong charge selectivity and weak size selectivity; Needham JV et al.; Carboxylate cluster Mg(II) binding sites consist of a cluster of side-chain carboxylates, typically 3-4 in number, partially buried in a shallow cleft on the surface of a Mg(II) binding protein . Such clusters are often found in the active sites of enzymes catalyzing phosphochemistry . An example is the phospho-signaling protein CheY of the Escherichia coli chemotaxis pathway, which binds Mg(II) via a cluster of three carboxylates at its phosphorylation site . The present study quantitates both the ion charge and size specificity of the CheY site by measuring the dissociation constants of metal ions from groups Ia, IIa, IIIa, and the lanthanides; these spherical cations provide a range of substrates with incrementally varying charge and radius . The site binds divalent and trivalent cations, but it effectively excludes monovalent cations, including the physiological ions Na(I) and K(I) . This charge specificity is in contrast to the site's remarkable lack of size specificity: divalent and trivalent cations exhibit affinities which are essentially independent of radius . It is revealing to compare the ion specificity of the Mg(II) site with the previously characterized specificity of the EF-hand class of Ca(II) sites commonly found in Ca(II) signaling proteins . The Mg(II) and Ca(II) sites exhibit similar charge selectivity, but the Ca(II) site is highly size-selective, preferring divalent and trivalent ions with radii similar to that of Ca(II).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Apr 6, 32(13), 3377 - 80
GroEL and GroES increase the specific enzymatic activity of newly-synthesized rhodanese if present during in vitro transcription/translation; Tsalkova T et al.; Enzymatically active mammalian rhodanese, a mitochondrial matrix enzyme, which has been found to require assistants for efficient refolding in vitro, has been synthesized from a plasmid in a cell-free, fractionated, coupled transcription/translation system derived from Escherichia coli . The bacterial chaperonins, GroEL and GroES, along with the rhodanese substrate thiosulfate greatly enhance the specific enzymatic activity of the rhodanese polypeptide that is formed . Indirect evidence suggests that the effect of the GroEL/ES chaperonins is on ribosome-bound nascent peptides . The in vitro transcription/translation system produces sufficient amounts of rhodanese to provide a system for studying factors that control the initial steps in folding of nascent proteins.

Biochemistry, 1993 Apr 6, 32(13), 3282 - 90
Calcium and lipid regulation of an Arabidopsis protein kinase expressed in Escherichia coli; Harper JF et al.; Calcium-dependent protein kinases (CDPKs) represent a new family of protein kinases which are proposed to contain, in a single polypeptide, both a kinase domain and an adjoining calmodulin-like domain with four calcium-binding EF-hand motifs {Harper, J.F., Sussman, M.R., Schaller, G.E., Putnam-Evans, C., Charbonneau, H., & Harmon, A.C . (1991) Science 252, 951-954} . DNA cloning and Western blot analysis indicate that multiple CDPK isoforms are present in the model plant system Arabidopsis thaliana . One CDPK gene called AK1 was isolated from Arabidopsis as a full-length cDNA . The predicted AK1 protein has a M(r) of 72,645 and is 116 amino acid residues longer at the amino terminus than the prototype CDPK alpha gene previously identified in soybean . The most highly conserved region between these two CDPKs is a region of 31 amino acids that joins the kinase and calmodulin-like domains . To verify the kinase activity of the enzyme encoded by AK1, a fusion of an amino-terminally truncated AK1 to the C-terminus of glutathione S-transferase was expressed in Escherichia coli . The fusion protein was purified and displayed a maximum kinase activity of 40 nmol of phosphate/(min.mg), using histone IIIs as a substrate . The enzyme activity was stimulated 3-6-fold by calcium and 2-5-fold by crude lipid . However, a synergistic stimulation of 16-30-fold was observed by the addition of both calcium and crude lipid . Lipid stimulation was specific for lysophosphatidylcholine and phosphatidylinositol and did not occur with the addition of phosphatidylserine or phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1993 Apr 5, 230(3), 966 - 78
Characterization of the zinc-induced structural transition to *H-DNA at a d(GA.CT)22 sequence; Beltran R et al.; Zinc induces transition of a d(GA.CT)22 sequence to a novel DNA conformation, called *H-DNA . In this paper, the structural characteristics of this altered DNA conformation are determined . Formation of *H-DNA is induced at zinc concentrations higher than 70 microM (Zn/P = 2) and it is favoured by negative supercoiling and low ionic strength . Two different structural conformations of the d(GA.CT)22 sequence are observed upon increasing the zinc concentration . At low zinc concentration (Zn/P < 15), half of the purine strand falls back upon itself giving rise to the formation of an RRY intramolecular triplex (H*-triplex) . At higher zinc concentration, the complete pyrimidine strand is single-stranded and an RR hairpin is formed (*H-hairpin) . Protection towards dimethylsulphate modification suggests that zinc binds to the N-7 group of guanine residues in the *H-hairpin with a higher affinity than in B-DNA . The dissociation constant of the *H-zinc complex is estimated to be in the range of 10(-3) M to 10(-4) M.

J Mol Biol, 1993 Apr 5, 230(3), 739 - 49
Undiscriminating codon reading with adenosine in the wobble position; Boren T et al.; To investigate the reading properties of adenosine in the wobble position we have used site-directed mutagenesis of the Escherichia coli glycine tRNA1(CCC) gene to substitute the nucleotide A in the wobble position of the corresponding tRNA . The effect of this change on the ability of the tRNA to discriminate between the nucleotides in the third position of the glycine codons has been investigated . We have compared the ability of the mutant glycine tRNA1(UCC) and glycine tRNA1(ACC) as well as the mycoplasma glycine tRNA(UCC) to read the glycine codons . The results showed that glycine tRNA1(ACC) unlike glycine tRNA1(UCC) did not fully discriminate between the glycine codons . These experiments were carried out using a new in vitro protein synthesizing system that allows us to monitor the reading of all four glycine codons . In the present paper we give a detailed description of this new in vitro system.

J Mol Biol, 1993 Apr 5, 230(3), 725 - 32
Functional expression of the uncomplexed serum retinol-binding protein in Escherichia coli . Ligand binding and reversible unfolding characteristics; Muller HN et al.; The serum retinol-binding protein solubilizes the lipophilic vitamin A alcohol and plays an important physiological role in the transport of this compound . The monomeric single-domain protein, the three-dimensional structure of which is known, constitutes a well-characterized member of the lipocalin family of proteins . We report here the functional expression of the apo-protein in Escherichia coli by secretion to the periplasm . The recombinant protein, purified in a single step by metal chelate affinity chromatography, exhibits the same ligand binding characteristics as described for the natural protein . Guanidinium chloride-induced unfolding and refolding experiments suggest that the recombinant retinol-binding protein adopts a stable conformation despite being expressed and purified in the absence of the large hydrophobic ligand . The expression system described here should also be useful for the recombinant production of other lipocalin proteins, thus permitting the elucidation of the structure-function relationships of ligand binding by protein engineering.

J Mol Biol, 1993 Apr 5, 230(3), 1097 - 100
Crystallization of DsbA, an Escherichia coli protein required for disulphide bond formation in vivo; Martin JL et al.; DsbA is a 21 kDa protein that facilitates disulphide bond formation and is required for the correct folding and stability of a number of exported proteins in Escherichia coli . Crystals of oxidized DsbA have been obtained from polyethylene glycol 8000 (20 to 25%), 0.1 M-cacodylate buffer (pH 6.5) and 1% 2-methyl-2,4-pentanediol . Oxidation of the protein is critical for reproducibly obtaining high quality crystals . The resulting crystals diffract to 2 A and belong to the monoclinic space group C2 with cell dimensions a = 117.5 A, b = 65.0 A, c76.3 A, beta = 126.3 degrees with two molecules in the asymmetric unit.

J Mol Biol, 1993 Apr 5, 230(3), 1094 - 6
Diffraction grade crystals of Escherichia coli serine hydroxymethyltransferase; Stover P et al.; Diffraction grade crystals of serine hydroxymethyltransferase were obtained after extensive screening of source of enzyme, purification procedures, ligand binding and crystallization conditions . Serine hydroxymethyltransferase purified from Escherichia coli crystallizes in two forms that are suitable for crystal structure determination.

J Mol Biol, 1993 Apr 5, 230(3), 1015 - 24
Complement-stabilized D-loop . RecA-catalyzed stable pairing of linear DNA molecules at internal sites; Jayasena VK et al.; The RecA protein (RecA) of Escherichia coli has the ability to pair a single-stranded DNA to a homologous sequence in a duplex DNA without requiring denaturation of the duplex . This ability has stimulated interest in the use of RecA for targeting probes to genomic DNA . However, because pairing generally requires that the double-stranded DNA either have a homologous end or be negatively supercoiled, the application of RecA to targeting has been very limited . Here, we show that if the sequence complementary to the probe is also included in the reaction, RecA can pair the two single strands to sites distant from any ends on linear DNA . The resulting structure, termed a complement-stabilized D-loop (csD-loop), is cleavable by restriction endonucleases and, upon removal of RecA, remains stable to temperatures up to the tm of the double-stranded probe . These results indicate that the csD-loop probably consists of two side-by-side Watson-Crick duplexes, much like a replication bubble . This novel reaction of RecA may be useful in gene mapping and isolation, as well as in sequence-specific cleavage of genomic DNA, and might have functions in vivo.

J Biol Chem, 1993 Apr 5, 268(10), 6989 - 94
Lysine 155 in beta-subunit is a catalytic residue of Escherichia coli F1 ATPase; Senior AE et al.; The "homology A" ("glycine-rich" or "P-loop") consensus sequence occurs in the catalytic sites of F1F0 ATP synthase enzymes . The conserved lysine of this motif is beta-subunit Lys-155 in Escherichia coli F1 . The role of this lysine in binding and catalysis at the high affinity ATP binding site was studied with the mutants beta K155Q and beta K155E by measuring the rates of ATP binding/release, ATP hydrolysis/synthesis, and Pi release as a function of pH varied from 5.5 to 9.5 . In wild type, protonated beta Lys-155 appears to contribute significantly to high affinity binding of ATP, probably through hydrogen bonding to the gamma-phosphate . ATP hydrolysis and synthesis were impaired strongly in the mutants, and the reaction equilibrium constant, which was pH-independent in wild type, was highly pH-dependent in beta K155Q and beta K155E . Studies of steady-state ATPase turnover showed that positive catalytic cooperativity was virtually absent and the pH-dependent component of positive catalytic cooperativity was abolished or reversed in the mutants . The data demonstrate that residue beta K155 is a critical catalytic residue in F1 ATPase.

J Biol Chem, 1993 Apr 5, 268(10), 6978 - 84
The cysteine introduced into the alpha subunit of the Escherichia coli F1-ATPase by the mutation alpha R376C is near the alpha-beta subunit interface and close to a noncatalytic nucleotide binding site; Turina P et al.; Mutation of the alpha subunit of the Escherichia coli F1-ATPase to convert Arg-376 to a Cys (alpha R376C) lowers multisite ATPase activity 400-1,000-fold while affecting unisite catalysis only around 6-fold, suggesting that the mutation is in a region important for transmission of conformational changes between catalytic sites (Soga, S., Noumi, T., Takeyama, M., Maeda, M., and Futai, M . (1989) Arch . Biochem . Biophys . 268, 643-648; this study) . To learn more of the structural features of the segment of the alpha subunit around Arg-376, mutant enzyme with a Cys at this position was modified with several maleimides . N-{14C}Ethylmaleimide reacted rapidly with this Cys in one of the three alpha subunits/F1 (2,500 M-1 s-1); more slowly with a second alpha subunit (390 M-1 s-1); and the same Cys in the third copy of the alpha subunit was completely unreactive to the reagent, indicating asymmetry of alpha subunits in the ECF1 complex . The photoactivatable cross-linker N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide++ +, when reacted via its maleimide to alpha Cys-376 of the mutant, covalently linked alpha to beta subunits upon photolysis, indicating that Cys-376 of alpha is close to an interface between the alpha and beta subunits . The EDTA-induced exchangeable noncatalytic site could be filled by TNP-ATP in both wild type and alpha R376C mutant ECF1 . Occupancy of this site in the alpha R376C mutant altered the rate of reaction of the second-fastest reacting Cys-376 from 390 M-1 s-1 to below 130 M-1 s-1, suggesting that the two sites are on the same alpha subunit . TNP-ATP in the EDTA-induced exchangeable noncatalytic site was quenched by reacting Cys-376 with 4-maleimido-(2,2,6,6-tetramethylpiperidine-N(oxyl), indicating that the region around Cys-376, which is involved in transmission of conformational changes between alpha and beta, and noncatalytic sites are maximally 10-12 A from each other.

J Biol Chem, 1993 Apr 5, 268(10), 6939 - 44
A single amino acid substitution confers progesterone 6 beta-hydroxylase activity to rabbit cytochrome P450 2C3; Hsu MH et al.; A cDNA encoding a naturally occurring variant of cytochrome P450 (P450) 2C3 that catalyzes the 6 beta- and 16 alpha-hydroxylation of progesterone exhibits six differences of nucleotide sequence leading to five amino acid substitutions from that encoding 2C3, a progesterone 16 alpha-hydroxylase that does not catalyze 6 beta-hydroxylation . Analysis of chimeric and mutant enzymes indicates that a Ser/Thr difference at position 364 underlies the difference between the two enzymes in 6 beta-hydroxylase activity as well as sensitivity to the inhibitor, 16 alpha-methylprogesterone . In addition, an Ile/Met difference at position 178 influences the apparent Km for progesterone . The two mutations, S364T and 1178M, together convert 2C3 to a form that exhibits kinetic properties which are similar to the 2C3v enzyme, and the reciprocal mutations in 2C3v convert it to an enzyme that resembles 2C3 . Interestingly, position 364 of 2C3 maps to a substrate-contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450cam . Ile178 is highly conserved among mammalian microsomal P450s with the exception of CYP4A and CYP19 enzymes which exhibit a Met at this alignment position.

J Biol Chem, 1993 Apr 5, 268(10), 6925 - 31
Biochemical analysis of rab9, a ras-like GTPase involved in protein transport from late endosomes to the trans Golgi network; Shapiro AD et al.; rab9 is a ras-like GTPase which has been implicated in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network . We have expressed recombinant rab9 in Escherichia coli, purified the protein to homogeneity, and initiated a biochemical analysis of this enzyme . rab9 hydrolyzed GTP with a rate constant of 0.0052 min-1 at 37 degrees C . rab7, a highly homologous endosomal GTPase, hydrolyzed GTP with a rate constant of 0.0023 min-1 at 37 degrees C . At this temperature, GDP and GTP each dissociated from rab9 with first-order rate constants of 0.017 min-1 . GDP and GTP dissociated from rab7 at 37 degrees C with first-order rate constants of 0.0054 and 0.0024 min-1, respectively . We modified the procedure of John et al . (John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., and Goody, R . (1990) Biochemistry 29, 6058-6065) for the preparation of nucleotide-free ras such that the procedure can now be applied to 1000-fold smaller quantities of protein . Using this method, we prepared microgram quantities of nucleotide-free rab9 in a form which is heat-stable, free of exogenous nucleotide-degrading enzymes and which can be stored at -80 degrees C . At 37 degrees C for GDP and GTP, the second-order rate constants for association with nucleotide-free rab9 were 1.7 x 10(6) M-1 s-1 and 1.2 x 10(5) M-1 s-1, respectively, and equilibrium binding constants were 170 pM and 2.4 nM, respectively.

FEBS Lett, 1993 Apr 5, 320(2), 107 - 10
Kinetic mechanism of ketoreductase activity of prostaglandin F synthase from bovine lung; Barski OA et al.; The kinetic mechanism of ketoreductase activity of bovine lung prostaglandin F synthase, expressed in E . coli, was investigated . Data on initial velocity and radioisotope exchange between {3H}prostaglandin D2 and 9 alpha,11 beta-prostaglandin F2 suggest that the enzyme obeys the ping-pong mechanism . Using a fluorescence technique we obtained a binding constant of 3 microM for NADPH . This is in close correlation with the kinetically determined intrinsic Michaelis constant for NADPH . Activation energy of the redox process was determined from the temperature dependence of maximal velocities for nitrobenzaldehyde and menadione and was found to be 119 and 96 kJ/mol, respectively.

J Biol Chem, 1993 Apr 5, 268(10), 7107 - 17
The alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase is phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor; Hayashi H et al.; We have shown previously that insulin stimulated the tyrosine phosphorylation of the alpha-type 85-kDa subunit (p85) of phosphatidylinositol (PI) 3-kinase in vitro and in vivo . In the present work, we identified the major tyrosine phosphorylation sites of the alpha-type p85 by the insulin receptor . {32P}Phosphopeptides obtained from lysylendopeptidase digestion of phosphorylated alpha-type p85 in intact cells after insulin treatment were analyzed using reverse-phase high performance liquid chromatography and thin layer electrophoresis . The tyrosine phosphorylation sites of alpha-type p85 in vivo were assigned to three major phosphopeptides, designated p1, p2, and p3 . Highly purified insulin receptor also phosphorylated the purified p85 of PI 3-kinase from the bovine thymus at p1 . The purified glutathione S-transferase (GST)-p85 (alpha-type) fusion protein and its truncated proteins from Escherichia coli were also phosphorylated by the purified insulin receptor at p1, p2, and p3 in vitro . Analysis of {32P}phosphopeptide of the truncated GST-p85 (alpha-type) fusion proteins and radiosequence analysis revealed that the p1, p2, and p3 phosphopeptides were phosphorylated at tyrosines 607, 580, and 368, respectively . In addition, phenylalanine substitutions at tyrosine 607 and 580 reduced the p1 and p2 phosphopeptides in vivo, respectively . We conclude that the alpha-type p85 of PI 3-kinase was phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor in vivo.

FEBS Lett, 1993 Apr 5, 320(2), 160 - 4
Crystallization and preliminary X-ray investigation of the Escherichia coli molecular chaperone cpn60 (GroEL); Spangfort MD et al.; The Escherichia coli molecular chaperone, cpn60 (GroEL), has been purified from an overproducing E . coli strain and crystallized . Of the two crystal forms that were obtained, one was found to be suitable for crystallographic and structural studies at low resolution . Preliminary X-ray investigation of the crystals show unit cell dimensions: a = 143.3, b = 154.6 and c = 265 A, with alpha = 82 degrees, beta = 95 degrees and gamma = 107 degrees . The space group is P1 and the crystals diffract to a maximum of 7 A when using CuK alpha X-rays from a rotating anode . Both electron microscopy and non-denaturing electrophoretic analysis of redissolved cpn60 crystals show that cpn60 crystallizes in the native oligomeric form . Comparison between the dimensions of oligomeric cpn60 and the crystallographic unit cell volume suggests that the unit cell contains two oligomeric cpn60 molecules . The VM value for two cpn60 molecules per unit cell is 3.5 A3/Da, corresponding to a water content of 65% . Electrophoretic analysis under denaturing conditions shows that the cpn60 in crystals is heterogeneous, and this probably explains the limited resolution of the diffraction data.

J Mol Biol, 1993 Apr 5, 230(3), 750 - 6
Pathway of activation by magnesium ions of substrates for the catalytic subunit of RNase P from Escherichia coli; Perreault JP et al.; The pathway is described for activation by Mg2+ of substrates for M1 RNA, the catalytic subunit of the RNase P from Escherichia coli . The dissociation constants are reported for binding of Mg2+ to the substrate and for the binding of the metal ion-substrate complex to the enzyme . The enzyme binds the substrate with the same affinity whether or not Mg2+ is already bound to the substate . However, only substrates with bound Mg2+ can make a productive ternary complex when combined with the enzyme . The presence of certain 2'-hydroxyl groups in the substrate is required to stabilize the binding of Mg2+ and, thereby, to increase the lifetime of the ternary complex . An energy profile for the reaction of M1 RNA with a small model substrate is presented and the role of Mg2+ bound to the substrate is discussed.

J Mol Biol, 1993 Apr 5, 230(3), 704 - 8
Symmetrical interactions between the translational operator of the thrS gene and dimeric threonyl transfer RNA synthetase; Bedouelle H; Threonyl-tRNA synthetase from Escherichia coli represses the translation of its coding gene, thrS, by binding an operator located in the leader region of its messenger RNA . Published data on the structure of this leader region and on its interaction with threonyl-tRNA synthetase support a model in which each of two stem-and-loop structures mimics the anticodon arm of tRNA(Thr) and binds a different subunit of one synthetase dimer.

Neurosci Lett, 1993 Apr 2, 152(1-2), 155 - 7
The protein VAT-1 from Torpedo electric organ exhibits an ATPase activity; Linial M et al.; VAT-1 is an abundant protein in Torpedo electric organ which copurifies with a major ATPase activity from synaptic vesicles . VAT-1 was expressed in E . coli and the product was purified and analyzed . The protein binds specifically to an ATP column and displays an ATPase activity as measured by the kinetics of {32P}phosphate release . The activity is dependent on divalent ions, with both Mg2+ and Ca2+ supporting the reaction . The apparent Km for ATP is 18 microM . This ATPase activity is not affected by known inhibitors of the vesicular V- and P-type ATPases such as vanadate and N-ethylmaleimide . We suggest that VAT-1 activity may affect ATP-dependent reactions in Torpedo nerve terminals, such as phosphorylation and dephosphorylation of proteins.

Science, 1993 Apr 2, 260(5104), 53 - 8
Molecular mechanism of transcription-repair coupling; Selby CP et al.; Lesions in the transcribed strand block transcription and are repaired more rapidly than lesions in the nontranscribed (coding) strand which do not block RNA polymerase (RNAP) . It has been shown previously that in Escherichia coli the mfd (mutation frequency decline) gene is necessary for strand-specific repair . The mfd gene was cloned and sequenced and the Mfd protein was purified and used to reconstitute strand-specific repair in a completely defined system . The mfd gene encodes a protein of 130 kilodaltons and contains the so-called "helicase motifs," a leucine zipper motif, and regions of sequence similarity to UvrB and RecG proteins . The Mfd protein was shown to (i) displace RNAP stalled at a lesion in an adenosine triphosphate-dependent reaction, (ii) bind to the damage recognition subunit (UvrA) of the excision nuclease, and (iii) stimulate the repair of the transcribed strand only when transcription is taking place . Thus, Mfd appears to target the transcribed strand for repair by recognizing a stalled RNAP and actively recruiting the repair enzyme to the transcription blocking lesion as it dissociates the stalled RNAP.

J Immunol Methods, 1993 Apr 2, 160(2), 155 - 61
Improvement of antisera raised against complex antigen mixtures by the use of heterologous sources of antigen for immunization; Hammerl P et al.; Polyspecific antisera against antigen mixtures are important tools for many experimental purposes . However, following immunization with extracts containing a great number of different proteins, many antigens remain non-immunogenic . Therefore, the improvement of the antibody spectrum of antisera is an essential goal in maximising the power and applicability of many serological analyses . In the present report we demonstrate that the additional use of heterologous (i.e., antigenically related, but not identical) antigen mixtures for immunization increases the variety of antibodies in polyspecific antisera as well as the antibody titer against weak immunogens.

Protein Sci, 1993 Apr, 2(4), 543 - 58
Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques; Pelton JG et al.; IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli . The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment . The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples . The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations . The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein . Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety . The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0 . These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10 . The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.

Protein Sci, 1993 Apr, 2(4), 498 - 505
The Escherichia coli adenylyl cyclase complex: stimulation by GTP and other nucleotides; Peterkofsky A et al.; Escherichia coli cells permeabilized by treatment with low concentrations of toluene contain an adenylyl cyclase activity that can be stimulated 3.6-7.6-fold by GTP . The stimulatory effect of GTP is maximal at concentrations of the nucleotide in the physiological range (above 0.7 mM) . Studies of the dependence of velocity on substrate (ATP) concentration indicate that the velocity vs . substrate plots are sigmoid in the absence of GTP but hyperbolic in the presence of GTP, suggesting an allosteric regulatory site that can be occupied by either ATP or GTP . Replacement of ATP by AMPPNP as substrate results in velocity vs . substrate plots that are hyperbolic in the absence or presence of GTP, although GTP increases the Vmax by a factor of 2.2; these findings indicate that AMPPNP specifically occupies the substrate site and GTP exclusively occupies the regulatory site . A test of the capacity of other guanine nucleotides to stimulate adenylyl cyclase activity showed that 2'-deoxy-GTP was almost as effective as GTP, but that GDP, GMP, ppGpp, and 3',5'-cGMP were not stimulatory effectors; GTP-gamma-S and GMPPNP stimulated adenylyl cyclase activity but to a lesser degree than did GTP . In addition to the previous indication that ATP can occupy the regulatory site on adenylyl cyclase, it was found that CTP and UTP were potent stimulators . Thus, all the naturally occurring RNA precursor nucleoside triphosphates are capable of stimulating adenylyl cyclase activity . In contrast, PPPi inhibits adenylyl cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioessays, 1993 Apr, 15(4), 249 - 58
Genes controlling nucleotide excision repair in eukaryotic cells; Weeda G et al.; The maintenance of genetic integrity is of vital importance to all living organisms . However, DNA--the carrier of genetic information--is continuously subject to damage induced by numerous agents from the environment and endogenous cellular metabolites . To prevent the deleterious consequences of DNA injury, an intricate network of repair systems has evolved . The biological impact of these repair mechanisms is illustrated by a number of genetic diseases that are characterized by a defect in one of the repair machineries and in general predispose individuals to cancer . This article intends to review our current understanding of the complex nucleotide excision repair pathway, a universal repair system with a broad lesion specificity . Emphasis will be on the recent advances in the genetic analysis of this process in mammalian cells.

J Biochem (Tokyo), 1993 Apr, 113(4), 488 - 92
Postnatal change of pig intestinal ganglioside bound by Escherichia coli with K99 fimbriae; Yuyama Y et al.; Enterotoxigenic Escherichia coli possessing K99 fimbriae (E . coli K99) causes diarrhea in piglets of less than 1 week old . The first stage of the bacterial infection is adhesion by the fimbriae on the small intestinal mucosa and the adhesion is followed by colony formation . K99 fimbriae bind specifically to N-glycolylneuraminyl-lactosyl-ceramide, GM3(NeuGc) {Ono, E . et al . (1989) Infect . Immun . 57,907-911} . We examined the postnatal change of the content and the molecular species of GM3(NeuGc) in the small intestinal mucosa of 0- to 14-day-old piglets and adult pigs . GM3(NeuGc) was a major ganglioside of piglet intestinal mucosa . GM3(NeuGc) content was maximal at birth and gradually decreased to 1/16 in adult animals (5 months old) . The ceramide moiety of piglet intestinal GM3(NeuGc) was characterized by the presence of 2-hydroxylated palmitic acid . 125I-labeled bacteria strongly bound to GM3(NeuGc) containing 2-hydroxylated palmitic acid and phytosphingosine compared with GM3(NeuGc) containing any other ceramide moiety . The time when this particular GM3(NeuGc) appears coincides with the time that the infection occurs, and it may explain the susceptibility of newborn piglets to E . coli K99 infection.

J Appl Physiol, 1993 Apr, 74(4), 1528 - 33
Left ventricular performance during continuous endotoxin-induced hyperdynamic endotoxemia in sheep; Noshima S et al.; Cardiac function was studied in an unanesthetized ovine model of hyperdynamic endotoxemia . Sixteen sheep were instrumented with ultrasonic crystals on the left ventricle to measure changes in its external diameter, a pressure transducer in the left ventricle, and aortic and Swan-Ganz catheters . The animals received either Escherichia coli endotoxin {lipopolysaccharide (LPS), 10 ng.kg-1.min-1; LPS group, n = 10} or an equivalent amount of 0.9% NaCl (sham group, n = 6) . Between 1 and 8 h after LPS, a hypodynamic state with low cardiac output ensued (LPS 5.0 +/- 0.2 vs . sham 6.3 +/- 0.4 l.min-1.m-2) . During this period, the end-systolic pressure-diameter relationship, a sensitive index of myocardial contractility, was reduced (LPS 10.4 +/- 1.2 vs . sham 17.2 +/- 0.8 mmHg/mm) . After this first phase, the sheep developed a persistent hyperdynamic state characterized by a significant increase in cardiac output . By 24 h after LPS administration, the cardiac output was 10.1 +/- 0.5 l.min-1.m-2 (sham 6.3 +/- 0.3) . Despite the marked elevation of cardiac output, the end-systolic pressure-diameter relationship had fallen to 8.5 +/- 0.9 mmHg/mm (sham 16.0 +/- 1.2) . In a model of hyperdynamic state, an increased cardiac output occurs despite a significant depression in myocardial contractility.

Genome, 1993 Apr, 36(2), 244 - 54
Identification of short tandemly repeated sequences in extrachromosomal circular DNAs from Drosophila melanogaster embryos; Renault S et al.; A sequence (scl) belonging to the recently identified dodeca satellite family was found to be a major family of extrachromosomal circular DNA molecules from Drosophila melanogaster embryos . The basic unit consists of the 11-bp repeat 5' ACTGGTCCCGT 3', is 63% G + C rich, and shares some similarity with the Escherichia coli chi sequence . This family accounts for only about 0.06% of the genome but very likely for a higher proportion of the circular DNA molecules . It is organized in the genome into at least five main clusters contained in DNA fragments larger than 20 kb and several minor clusters . These clusters are located in the heterochromatic pericentromeric regions . Two other families of simple repeated sequences, the 1.686 g/cm3 (5' AATAACATAG 3') and the 1.705 g/cm3 (5' AAGAG 3') satellite DNAs, were also found in circular DNAs, while another family, the 1.672 g/cm3 (5' AATAT 3'), was not detected . The representation of the simple repeated sequences in circular molecules is not correlated to their genomic representation . Among the seven families of sequences identified to date in extrachromosomal circular DNAs from embryos, the dodeca satellite, the 240-bp repeat of the rDNA intergenic spacer, and the 1.688 and 1.705 g/cm3 satellite DNAs are the most represented families, while the 5S genes, the histone genes, and the 1.686 g/cm3 satellite DNA are present in a lower amount.

J Interferon Res, 1993 Apr, 13(2), 91 - 7
Production and characterization of recombinant canine interferon-gamma from Escherichia coli; Zucker K et al.; We have used the recently cloned cDNA for canine interferon-gamma (IFN-gamma) to engineer bacteria to produce large amounts of the recombinant cytokine . The resulting protein can be recognized by monoclonal and polyclonal antibodies largely species specific for canine IFN-gamma . The purified recombinant IFN-gamma (rIFN-gamma) also had biological activity in vitro in three assay systems: (i) vesicular stomatitis virus plaque inhibition, (ii) class II major histocompatibility complex antigen upregulation on canine kidney parenchymal cells, and (iii) amplification of in vitro tissue-associated lymphoproliferation, all known to be effected by native IFN-gamma (nIFN-gamma) . The availability of large amounts of active canine rIFN-gamma will be an important tool in studies of the role of this cytokine in the widely used experimental canine organ transplant model and also will be of diagnostic and therapeutic veterinary interest.

Vet Immunol Immunopathol, 1993 Apr, 36(3), 281 - 91
Detection of mucosal immune responses in chickens after immunization or infection; Zigterman GJ et al.; In order to measure mucosal antibody responses in the chicken intestine an ELISA-based assay was developed that was able to detect antigen-specific antibodies in an isotype-specific way . Locally produced antibodies could be detected after overnight culture at 37 degrees C . In particular the production of IgA, more than IgM and IgG, was significantly increased by immunization of the animals with K99 pilus antigen or by infection with Eimeria tenella parasites . Data presented here indicate that the assay can be used to estimate the magnitude of the mucosal antibody response in experimental conditions where antibody levels in bile or intestinal contents were not significantly changed.

Photochem Photobiol, 1993 Apr, 57(4), 648 - 54
Relaxation of supercoiled DNA by aminomethyl trimethylpsoralen and UV photons: action spectrum; Oroskar AA et al.; An action spectrum for the relaxation of supercoiled plasmid DNA (induction of the first single-strand break) by photoactivated 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) has been determined using monochromatic UV photons from 254 to 405 nm . The spectrum of AMT-induced plasmid DNA relaxation fits closely with the absorbance spectrum of AMT in the spectral region between 313 nm and 405 nm but deviates at wavelengths shorter than 313 nm . This assay also reveals that the psoralen photosensitization reaction with DNA also produces piperidine-labile sites . Addition of mannitol and azide partially quenches the supercoil relaxation reaction, evidence for a role of Type II photosensitization pathway.

FEMS Immunol Med Microbiol, 1993 Apr, 6(4), 325 - 30
Antibodies to Bordetella pertussis adenylate cyclase toxin in neonatal and maternal sera; Arciniega JL et al.; To investigate the high prevalence among infants of antibodies to Bordetella pertussis adenylate cyclase toxin (ACT), cord-blood sera were examined for antibodies to ACT, filamentous hemagglutinin (FHA) and pertussis toxin (PT) using immunoblot analysis . Antibodies reactive with ACT were the most prevalent in neonatal sera . Similar reactivity of IgG with ACT was found in each sample of a given neonatal-maternal pair, yet IgM reactive with ACT was virtually absent in neonatal sera, suggesting that antibodies to ACT are maternally derived . Antibodies to ACT might come from infection or childhood vaccination of the mothers since pertussis vaccines from all US manufacturers elicited antibodies to ACT in mice . Alternatively, these antibodies may have been elicited by a cross-reactive antigen such as Escherichia coli alpha-hemolysin, since all of the neonatal and maternal sera contained antibodies reactive with alpha-hemolysin.

Plant Mol Biol, 1993 Apr, 22(1), 171 - 6
Nucleotide sequence of a genomic gene encoding tritin, a ribosome-inactivating protein from Triticum aestivum; Habuka N et al.; A genomic gene of tritin, a ribosome-inactivating protein (RIP) from Triticum aestivum, was cloned using a barley RIP gene as a probe . The 5'-non-coding region has potential TATA boxes and three sequences homologous to the binding sequence of the transcriptional activator protein Opaque-2 which activates maize RIP gene expression . The cloned DNA encoded tritin consists of 275 amino acids with no secretion signal sequence . The coding region of tritin was expressed in Escherichia coli using lac promoter and yielded a protein similar to the native one, as determined by SDS-polyacrylamide gel electrophoresis and immunological analysis.

Mol Microbiol, 1993 Apr, 8(1), 61 - 8
Properties of FNR proteins substituted at each of the five cysteine residues; Green J et al.; FNR is a transcriptional regulator controlling the expression of a number of Escherichia coli genes in response to anoxia . It is structurally-related to CRP (the cyclic AMP receptor protein) except for the presence of a cysteine-rich N-terminal extension, which may form part of an iron-binding, redox-sensing domain in FNR . Site-directed substitution has previously shown that four of the cysteine residues (C20, C23, C29 and C122) are essential for FNR function, whereas the fifth (C16) is not . The FNR protein exists in two forms separable by non-reducing SDS-PAGE, and in studies with altered FNR proteins containing single substitutions at each of the five cysteine residues it was concluded that the faster-migrating form (FNR(27)), possesses an intramolecular disulphide bond linking C122 to one of the cysteines near the N-terminus . FNR(27) was more abundant in aerobic cells but the physiological significance of this was not established . Footprint studies indicated that FNR proteins lacking essential cysteine residues are impaired in their ability to protect FNR sites in the ndh promoter . The non-essential cysteine residue (C16) was identified as the source of the most reactive sulphydryl group and all of the inactive proteins exhibited different sulphydryl reactivities . The iron content of the C122A-substituted protein was much reduced but those of the other proteins were less affected . Electrospray mass spectrometry confirmed the accuracy of the gene-derived amino acid composition of FNR with a mutant protein and it showed that a fraction of the wild-type protein may carry a 78 Da substituent which could not be removed with dithiothreitol or beta-mercaptoethanol.

Mol Microbiol, 1993 Apr, 8(1), 43 - 51
DNA sequencing and expression of the gene rnb encoding Escherichia coli ribonuclease II; Zilhao R et al.; The Escherichia coli ribonuclease II (RNase II) is an exonuclease involved in mRNA degradation that hydrolyses single-stranded polyribonucleotides processively in the 3' to 5' direction . Sequencing of a 2.2 kb MseI-RsaI fragment containing the rnb gene revealed an open reading frame of 1794 nucleotides that encodes a protein of 598 amino acid residues, whose calculated molecular mass is 67,583 Da . This value is in good agreement with that obtained by sodium dodecyl sulphate/polyacrylamide gel electrophoresis of polypeptides synthesized by expression with the T7 RNA polymerase/promoter system . This system was also used to confirm the correct orientation of rnb . Translation initiation was confirmed by rnb-lacZ fusions . The mRNA start site was determined by S1 nuclease mapping . Two E . coli mutants harbouring different rnb alleles deficient in RNase II activity were complemented with the expressed fragment carrying the rnb gene.

Biophys J, 1993 Apr, 64(4), 1361 - 5
The effect of hydration on the dynamics of trimethoprim bound to dihydrofolate reductase . A deuterium NMR study; Yang QX et al.; To determine the effect of hydration on the dynamics of a protein complex, we used deuterium nuclear magnetic resonance (NMR) techniques to examine a trimethoprim (TMP)/E . coli dihydrofolate reductase (DHFR) complex in its lyophilized, partially hydrated, polycrystalline, and ammonium sulfate-precipitated states . The results indicate that TMP is rigid in the lyophilized powder state . The dynamic behavior could be restored by partial rehydration . At 30 wt% hydration the deuterium spectrum of the partially hydrated sample was indistinguishable from that of the polycrystalline and ammonium sulfate-precipitated samples, suggesting that the structure of the protein/TMP complex is similar in the three physical states . Furthermore, we found that the para- and meta-methoxyl groups have very different dynamical behavior.

Hum Gene Ther, 1993 Apr, 4(2), 171 - 8
Retrovirus-mediated gene transfer into canine thyroid using an ex vivo strategy; O'Malley BW Jr et al.; We describe studies in a canine model aimed at establishing methods for ex vivo gene delivery to thyroid follicular cells . Canine follicular cells were harvested from tissue obtained by unilateral lobectomy, grown in thyrotropin-containing media, and transduced with amphotropic retroviral vectors carrying Escherichia coli beta-galactosidase or Tn7 neomycin-resistance genes . Up to 30% of cells were transduced with retroviral vectors containing the neomycin resistance gene, and transduced cells could be selected with G418 . Significantly, transduced and selected cells exhibited the morphology of thyroid follicular cells and continued to express thyroglobulin . To assess the viability of cultivated and transduced cells for transplantation, cells were stained with the vital fluorescent dye DiI, recovered by trypsinization, and transplanted into the contralateral thyroid lobe of autologous animals . Engraftment was demonstrated by fluorescence microscopy and identification of proviral sequences 7-10 days after transplantation . Proviral transcripts were evident using coupled reverse transcription and the polymerase chain reaction using total RNA from transplanted glands . Thyroid follicular cells may represent an attractive target for gene therapy due to their proliferative potential, their large protein synthetic and secretory capacity, and their susceptibility to regulation . The thyroid might be a target for therapy of congenital or acquired thyroid diseases as well as disorders requiring regulated expression of proteins in the circulation . This work demonstrates the feasibility of ex vivo gene delivery to thyroid follicular cells that may be used in future investigations.

Virus Res, 1993 Apr, 28(1), 29 - 35
Expression and assembly of the potato virus Y (PVY) coat protein (CP) in Escherichia coli cells; Stram Y et al.; A clone harboring the full-length cDNA of potato virus Y in a lambda-DASH vector under the control of a T7 promoter was introduced into Escherichia coli carrying the T7-RNA-polymerase gene on a plasmid . The viral coat protein was expressed and the product was of the same size as the corresponding mature protein in infected plants . Immunoelectronmicroscopy of transfected cell extracts revealed virus-like particles, indicating that the proteins involved in its processing and the viral coat protein retained their native activity.

Mol Gen Genet, 1993 Apr, 238(3), 333 - 8
pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo; Gigliani F et al.; Evidence is presented that the pR bat gene is essential for plasmid replication and for spontaneous induction of the SOS response in Escherichia coli . Mutations preventing single-stranded DNA production, needed for pR plasmid replication, also prevent the induction of the SOS system . The following experimental design was used . Firstly, we identified the minima rep region, defined as the minimal DNA sequence necessary for pR plasmid replication and, secondly, analyzed the nucleotide sequence of this region . This identified structures and functions (ori-plus, ori-minus and Rep protein) homologous to those found in phages and plasmids replicating by the rolling-circle mechanism . Finally, mutations were introduced either in the replication protein catalytic site or in the nick site consensus sequence, which caused the pR plasmid to lose its ability to induce the SOS system . We conclude that, in this system, the in vivo SOS-inducing signal appears to be the single-stranded DNA produced during pR replication.

Inflammation, 1993 Apr, 17(2), 107 - 19
Antiinflammatory action of thielocin A1 beta, a group II phospholipase A2 specific inhibitor, in rat carrageenan-induced pleurisy; Tanaka K et al.; Extracellular phospholipase A2 (PLA2) activity was detected in exudate from rat carrageenan-induced pleurisy using {3H}oleic acid-labeled Escherichia coli as substrate . Both exudate volume and PLA2 activity increased up to 24 h after carrageenin injection . Specific absorption of this activity by anti-group II PLA2 (PLA2-II) antibody indicated that the PLA2 activity in the pleural exudate was PLA2-II . Thielocin A1 beta, a novel type of PLA2 inhibitor from fungi, inhibited this PLA2-II activity in a dose-dependent manner (IC50 = 0.32 microM) . Thielocin A1 beta correspondingly reduced both exudate volume and PLA2-II activity in the exudate in a dose-dependent manner when coinjected with carrageenan . The exudate volume was also significantly decreased when indomethacin, a cyclooxygenase inhibitor, or dexamethasone, a steroidal antiinflammatory drug, was coinjected with carrageenan . However, neither indomethacin nor dexamethasone could significantly attenuate the PLA2-II activity in exudate . In addition, indomethacin and dexamethasone significantly reduced the levels of PGE2 in the exudate . However, thielocin A1 beta had no effect on the PGE2 content in the exudate . These results suggest that thielocin A1 beta shows antiinflammatory activity due to inhibition of PLA2-II and offer evidence for the significance of PLA2-II in the propagation of inflammatory processes.

J Protein Chem, 1993 Apr, 12(2), 121 - 31
Refolding of cytochrome b562 and its structural stabilization by introducing a disulfide bond; Kobayashi Y et al.; The packing mechanism of the secondary structures (4-alpha-helices and 3(10)-helix) of cytochrome b562 is simulated by the "island model," where the formation of protein structure is accomplished by the growth-type mechanism with the driving force of packing of the long-range and specific hydrophobic interactions . Packing proceeds through the formation of the structure at the nonhelical part, where a lot of hydrophobic pairs are distributed . Consequently, conformation, nearly similar to the native one, is successfully obtained . With the help of this result, the theoretical prediction of the possibility of forming this disulfide mutant (N22C/G82C) of b562 can be performed prior to the experiments by our geometrical criterion ("lampshade") . This criterion is expected to be a significant principle for introducing possible disulfide bonds into a protein to be engineered.

Mol Immunol, 1993 Apr, 30(6), 593 - 602
Extensive somatic mutation in the Ig heavy chain V genes in a late primary anti-hapten immune response; Tao W et al.; Somatic mutations and cell lineage relationships were examined in a large panel of hybridomas derived from a single mouse 21 days after a primary immunization with NP-CGG . Among 21 lambda-bearing anti-NP hybridomas 18 distinct cell lineages were observed . Ten of the hybridomas used the V186-2 gene which is the most frequently utilized VH gene in the anti-NP response . Analysis of DNA sequence of the entire VH region of these antibodies revealed extensive somatic mutations . The selection for certain codon changes and the level of mutation observed is comparable to that observed in an early secondary anti-NP response . An unexpected observation was that one-third of the hybridomas produced IgM antibodies . Two IgM antibodies expressing the V186-2 gene contained extensive mutations in the VH region . These results indicate that once the somatic mutation process is initiated, it progresses rapidly and continues for at least two weeks during the development of the response . A highly mutated repertoire of memory B cells is formed by three weeks post-immunization that can be rapidly utilized to generate the secondary immune response.

Mol Immunol, 1993 Apr, 30(6), 559 - 68
Recombinant Fel d.I: Expression, purification, IgE binding and reaction with cat-allergic human T cells; Rogers BL et al.; This study describes the properties of the two recombinantly expressed polypeptide chains of Fel d I, the major allergen produced by the domestic cat (Felis domesticus) . An inframe linker encoding polyhistidine has been added to the 5' ends of the Fel d I chains 1 and 2 cDNAs to facilitate purification using Ni2+ ion affinity chromatography . This method provides high yields in a single step of rchain 1 and rchain 2 of Fel d I with a > 90% level of purity . Polymerase chain reaction (PCR) methods were used to introduce a thrombin cleavage site (LVPR decreases GS) at the N-terminus of both chains . Thrombin cleavage of rchain 1 and rchain 2 followed by HPLC purification of the cleavage products allowed the isolation of each recombinant chain with only two additional residuals (GS) at the N-terminus of the native sequence . Amino acid sequencing analysis of the N-terminus and mass spectrometry of these polypeptides demonstrated that they are highly pure and full-length . Direct ELISA assays showed that IgE from cat-allergic patients binds to both rchain 1 and rchain 2 of Fel d I, demonstrating that both these chains contribute to the allergenicity of this heterodimeric protein . An examination of the reactivity of T cells derived from cat-allergic patients revealed that both polypeptide chains contribute to the T cell response to this allergen . Consequently, it is concluded that the immunological response to Fel d I is composed of a reaction at both the B and T cell level to each of the two chains that constitute the native allergen.

J Appl Bacteriol, 1993 Apr, 74(4), 402 - 5
Enumeration of Escherichia coli in cooked and raw meats by ion mobility spectrometry; Ogden ID et al.; A novel method for enumerating Escherichia coli in foods is described . It is based on the production of o-nitrophenol (ONP) from o-nitrophenyl-beta-D-glucuronide as detected by an ion mobility spectrometer . The time taken for the detection of ONP in a food sample was inversely proportional to the logarithm of the initial E . coli population . Escherichia coli could be detected at a level of 10 g-1 within 9 h.

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 237 - 42
Identification of the transcriptional start site of the cyd operon from Escherichia coli; Fang H et al.; The cydAB operon encodes the two subunits of the cytochrome bd ubiquinol oxidase from Escherichia coli . This enzyme is one of two terminal oxidases in the aerobic respiratory chain of E . coli . It has been demonstrated that expression of the cyd operon is transcriptionally regulated by oxygen via both the fnr and arcA gene products . Whereas arcA clearly serves as a positive regulator at low oxygen tension, there is no consensus concerning the role of the fnr gene product (FNR) . In this paper, the transcriptional start site of the cyd operon is identified at position -287 with respect to the translational start . This is located 53 basepairs downstream from the center of dyad symmetry of the putative FNR recognition sequence.

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 231 - 6
Expression of human alpha 1 interferon genes in vectors containing tandemly located promoters recognized by two different RNA polymerases (Escherichia coli and T7); Ivanov IG et al.; An expression vector containing two tandemly located promoters (T7 and P1) and two transcription terminators recognized by two different RNA polymerases (T7 RNA polymerase and Escherichia coli RNA polymerase) was constructed . Human alpha 1 interferon gene variants were cloned in this vector and their expression was studied in E . coli strains containing {E . coli BL2I (DE3)} or devoid (E . coli BL21) of the gene for the T7 RNA polymerase . We report that simultaneous activity of the two promoters reduces the level of gene expression when compared with the levels of expression corresponding to either P1 or T7 promoter alone.

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 157 - 61
Amino acid sequence of heat-labile enterotoxin from chicken enterotoxigenic Escherichia coli is identical to that of human strain H 10407; Inoue T et al.; The DNA sequence of heat-labile enterotoxin from the chicken enterotoxigenic Escherichia coli 21d strain was determined by direct dideoxy sequencing of polymerase chain reaction (PCR)-amplified DNA and was compared with those of heat-labile enterotoxins from porcine and human enterotoxigenic E . coli strains EWD 299 and H 10407 . The structural genes of the A and B subunits of chicken heat-labile enterotoxin were identical to those of human heat-labile enterotoxin from the human H 10407 strain . Moreover, 67 base pairs of the upstream and 60 base pairs of the downstream region of the chicken heat-labile enterotoxin gene were also identical to that of the human heat-labile enterotoxin from strain H 10407 . However, the patterns of plasmids from the 21d and H 10407 strains were different . The 21d strain had no band corresponding to the 42-MDa plasmid of the H 10407 strain encoding the heat-labile enterotoxin gene but it had a smaller plasmid . These data suggest that although the DNA sequence of chicken heat-labile enterotoxin is identical to that of human heat-labile enterotoxin, the plasmid encoding the chicken heat-labile enterotoxin gene in the chicken might be different from that encoding the human heat-labile enterotoxin gene in the H 10407 strain.

Circ Shock, 1993 Apr, 39(4), 285 - 92
Analysis of endotoxin effects on pulmonary circulation in terms of pressure-flow characteristics; D'Orio V et al.; The purpose of the present work was to explore the hypothesis that pulmonary vasoconstriction secondary to endotoxin insult results mainly from an increase in the critical closing pressure of the pulmonary vessels . Specifically, we reasoned that in the face of a Starling resistor located between pulmonary arteries and left atrium, upstream transmission of increased left atrial pressure (Pla) would be inversely related to the level of the pressure intercept (Pi) obtained by extrapolation from the linear pulmonary arterial pressure (Ppa)--flow (Q degrees) plot . Six dogs (group E) were infused with Escherichia coli endotoxin (0.25 microgram/kg/min) for 2 hr, whereas six additional dogs (group C) served as control . During baseline conditions, Pi approximated LAP in both groups . In group C dogs, increasing LAP at constant Q degrees led to a proportional augmentation of Ppa . In group E dogs, endotoxin resulted in a shift of the Ppa-Q relationships to higher pressures due to both increases in Pi and slope . In addition, changes in Pla over the same range as in control dogs affected Ppa only at the highest levels of Pla . We conclude that endotoxin insult increases the critical closing pressure that exceeds Pla and induces the occurrence of a Starling resistor responsible for the production of an effective vascular waterfall.

Biochem Med Metab Biol, 1993 Apr, 49(2), 164 - 72
Sandwich enzyme immunoassay for rat retinol-binding protein using antibody against recombinant antigen and its application; Matsumoto T et al.; The development of a sandwich enzyme immunoassay for rat retinol-binding protein using molecular biological techniques was described . Rat retinol-binding protein gene cloned by the PCR method was expressed by a fusion vector pEZZ18 in Escherichia coli strain HB101 . A recombinant retinol-binding protein fused with IgG-binding domain ZZ of protein A was purified with IgG-Sepharose . Antibody against the recombinant protein was found to be specific to rat retinol-binding protein in plasma by immunoblot analysis . Affinity-purified anti-recombinant protein IgG was biotinylated and used for the sandwich enzyme immunoassay . In this assay, the measurable range is 1.9-60 ng/ml and the coefficients of variation within and between the assay series (assay range: 4-30 ng/ml) are 4.30 +/- 4.33 and 5.32 +/- 1.45%, respectively . Cross-reactivity of the immunoassay was examined using bovine, human, and mouse serum . There was a cross-reaction only with mouse serum . In an in vitro experiment, retinol-binding protein produced by rat hepatocytes could be measured by the sandwich enzyme immunoassay.

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