J Virol, 1992 Jun, 66(6), 3860 - 8 Amino acid sequence and structural properties of protein p12, an African swine fever virus attachment protein; Alcami A et al.; The gene encoding the African swine fever virus protein p12, which is involved in virus attachment to the host cell, has been mapped and sequenced in the genome of the Vero-adapted virus strain BA71V . The determination of the N-terminal amino acid sequence and the hybridization of oligonucleotide probes derived from this sequence to cloned restriction fragments allowed the mapping of the gene in fragment EcoRI-O, located in the central region of the viral genome . The DNA sequence of an EcoRI-XbaI fragment showed an open reading frame which is predicted to encode a polypeptide of 61 amino acids . The expression of this open reading frame in rabbit reticulocyte lysates and in Escherichia coli gave rise to a 12-kDa polypeptide that was immunoprecipitated with a monoclonal antibody specific for protein p12 . The hydrophilicity profile indicated the existence of a stretch of 22 hydrophobic residues in the central part that may anchor the protein in the virus envelope . Three forms of the protein with apparent molecular masses of 17, 12, and 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been observed, depending on the presence of 2-mercaptoethanol and alkylation with 4-vinylpyridine, indicating that disulfide bonds are responsible for the multimerization of the protein . This result was in agreement with the existence of a cysteine-rich domain in the C-terminal region of the predicted amino acid sequence . The protein was synthesized at late times of infection, and no posttranslational modifications such as glycosylation, phosphorylation, or fatty acid acylation were detected. J Cell Biol, 1992 Jun, 117(5), 1023 - 9 Effects of profilin and profilactin on actin structure and function in living cells; Cao LG et al.; Previous studies have yielded conflicting results concerning the physiological role of profilin, a 12-15-kD actin- and phosphoinositide-binding protein, as a regulator of actin polymerization . We have addressed this question by directly microinjecting mammalian profilins, prepared either from an E . coli expression system or from bovine brain, into living normal rat kidney (NRK) cells . The microinjection causes a dose-dependent decrease in F-actin content, as indicated by staining with fluorescent phalloidin, and a dramatic reduction of actin and alpha-actinin along stress fibers . In addition, it has a strong inhibitory effect toward the extension of lamellipodia . However, the injection of profilin causes no detectable perturbation to the cell-substrate focal contact and no apparent depolymerization of filaments in either the nonlamellipodial circumferential band or the contractile ring of dividing cells . Furthermore, cytokinesis of injected cells occurs normally as in control cells . In contrast to pure profilin, high-affinity profilin-actin complexes from brain induce an increase in total cellular F-actin content and an enhanced ruffling activity, suggesting that the complex may dissociate readily in the cell and that there may be multiple states of profilin that differ in their ability to bind or release actin molecules . Our results indicate that profilin and profilactin can function as effective regulators for at least a subset of actin filaments in living cells. Exp Cell Res, 1992 Jun, 200(2), 263 - 71 Establishment of a human cell line for the detection of demethylating agents; Biard DS et al.; To detect xenobiotics affecting the DNA methylation pattern we have established a human clonal cell line (A4/4) derived from the 293 epithelial cells . This cell line is stable after more than 1 year in culture and we report here its characteristics . The A4/4 cells carry the Escherichia coli lacI gene and the symmetric lacO sequence upstream of the beta-galactosidase coding gene (lacZ) . Both sequences have been independently integrated into the genomic DNA . The lacZ gene is under the control of the metal-inducible mouse metallothionein-I (mMT-I) promoter . Vector integration followed by cell ageing in culture gave rise to the methylation of the 5'CpG3' sites in the mMT-I promoter and the 5' part of the lacZ gene . The reactivation of the lacZ gene by 5-azacytidine (5-Aza-CR) treatment allowed a tremendous lacZ expression which is correlated with demethylation in the mMT-I promoter and its neighboring regions . This enhanced transcription is easily quantified by measuring the beta-galactosidase activity in cell extracts . 5-Aza-CR, the specific isopropyl beta-D-thiogalactoside inducer, and heavy metal ions added together trigger an increase of the beta-galactosidase activity up to 600-fold over the basal level . These cells can be used for a rapid assessment of the demethylating potential of environmental chemicals. J Bacteriol, 1992 Jun, 174(11), 3541 - 8 RNase E-dependent cleavages in the 5' and 3' regions of the Escherichia coli unc mRNA; Patel AM et al.; The endonucleolytic processing of the unc mRNA encoding the eight subunits of the Escherichia coli F1F0-ATPase was studied . Northern (RNA) blots of mRNA expressed from a plasmid which contained the 3'-terminal portion of the operon including the uncDC sequences revealed, in addition to the expected 2-kb mRNA, a 0.5-kb RNA species which hybridized to an uncC antisense RNA probe . An uncD antisense RNA probe hybridized to only the 2-kb mRNA, implying that the upstream 1.5-kb fragment is rapidly degraded . The 5' end of the 0.5-kb fragment was determined by primer extension analysis to be 11 bases into the coding region of the uncC gene . In RNase E-deficient strains, the amount of the 0.5-kb product was strongly reduced while the levels of the precursor uncDC transcript remained high . Similar RNase E-dependent processing was found in the chromosomally encoded unc mRNA . As this RNase E-dependent cleavage directly inactivates uncC and appears to leave uncD susceptible to degradation, it seems unlikely to play a role in differential expression of the gene products but may be an important event in unc mRNA degradation . RNase E mutants also showed altered processing of the chromosomally encoded unc mRNA in the uncB region near the 5' end . The expected full-length (7-kb) transcript was recognized when RNA from the RNase E-deficient strain was subjected to Northern blot analysis with uncB- and uncC-specific probes . RNA from strains with functional RNase E lacked the 7-kb transcript but had a 6.2-kb mRNA detectable with the uncC but not the uncB probe . RNase E is therefore implicated in multiple cleavages of the unc mRNA. Mol Cell Biol, 1992 Jun, 12(6), 2837 - 46 E1A-responsive elements for repression of rat fibronectin gene transcription; Nakajima T et al.; The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of E1A expression . To study the mechanism of this E1A-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with E1A expression plasmids . The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary E1A-responsive elements for repression of the FN gene . Two GC boxes also contain a G10 stretch that is interrupted by the presence of an internal C residue . These sequences overlap with the Sp1 motif GGGCGG . Substitution of the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, completely abolished the E1A sensitivity of the promoter . Analysis of the E1A domains by using various E1A deletion mutants indicated that the domain for binding to the retinoblastoma susceptibility gene product (RB) is essential for efficient repression . These results suggest that the gene encoding a negative factor(s) binding to the three G-rich sequences in the FN promoter is repressed by RB in resting 3Y1 cells and derepressed by expression of E1A. J Surg Res, 1992 Jun, 52(6), 555 - 9 Interleukin-1 alpha prevention of the lethality of Escherichia coli peritonitis; Lange JR et al.; Interleukin-1 (IL-1) is an inflammatory mediator with a variety of described physiologic functions . IL-1 alpha has been shown to confer a survival advantage to experimental animals when administered before a lethal bacterial challenge . The experiments reported here were performed to define the effective pretreatment interval of a single intravenous dose of IL-1 alpha in a murine model of bacterial peritonitis, to examine the differential induction of cytokines in animals with and without IL-1 alpha pretreatment, and to assess differences in histologic evidence of end organ damage . IL-1 alpha (27 micrograms/kg iv) conferred a survival advantage to mice given a lethal challenge of live Escherichia coli (2 x 10(8) CFU/mouse ip) when the pretreatment was given 2 to 24 hr before the bacterial inoculum . Longer pretreatment intervals were not significantly protective . Treatment with IL-1 alpha at 1 hr after bacterial inoculum also did not improve survival . Mice pretreated with IL-1 alpha developed significantly lower peak serum levels of TNF-alpha after E . coli injection than did control mice . Pretreated and control mice had similar peak serum levels of IL-6 after bacterial challenge; however, IL-1 alpha-pretreated mice had a less prolonged elevation of serum levels of IL-6 . IL-1 alpha-pretreated animals were protected from the histologic evidence of end organ damage seen in control animals . Thus, in this model of E . coli peritonitis pretreatment with a single intravenous dose of IL-1 alpha confers a significant protective effect when given within a limited time range . Treatment outside this interval has no apparent beneficial effect.(ABSTRACT TRUNCATED AT 250 WORDS) J Surg Res, 1992 Jun, 52(6), 549 - 54 Modulation of macrophage procoagulant activity by arachidonic acid metabolites; Kucey DS et al.; Macrophage (M phi)-mediated fibrin deposition via induction of procoagulant activity (PCA) is an important component of the host response during various infections . While endotoxin (LPS) is a well-known stimulus of PCA, the factors modulating its activity within the inflammatory microenvironment are unknown . The purpose of these studies was to determine the relative roles of two pathways of arachidonic acid metabolism, i.e., the cyclooxygenase (CO) and 5-lipoxygenase (5-LO) pathways, in modulating M phi PCA induction by LPS . Thioglycolate-elicited murine peritoneal M phi were treated with the CO inhibitor indomethacin (INDO), the 5-LO inhibitor nordihydroguaiaretic acid (NDGA), or control vehicle for 15 min prior to a 4-hr exposure to LPS (10 micrograms/ml) . The ability of M phi to shorten the clotting time of plasma (i.e., PCA) was measured and clotting times were converted to PCA units via a thromboplastin standard . While CO blockade had no effect on PCA induction by LPS (without INDO 30 microM 446 +/- 131, with INDO 30 microM 546 +/- 193, mU/2 x 10(6) cells, n = 4), NDGA caused a dose-dependent inhibition (IC50 = 3 microM) without affecting cell viability (without NDGA 3 microM 446 +/- 131, with NDGA 3 microM 191 +/- 67, mU/2 x 10(6) cells, n = 6, P less than 0.05) . Induction of PCA by Escherichia coli was similarly inhibited (E . coli 10(6) alone = 518 +/- 130; with NDGA 3 microM = 234 +/- 100, n = 2) . Combined NDGA/INDO reduced PCA comparable to NDGA alone, ruling out the possibility that NDGA acted through generation of inhibitory prostanoids like PGE2.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Microbiol, 1992 Jun, 138 ( Pt 6), 1265 - 70 Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form; Onoda T et al.; The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8 . The cessation of growth at alkaline pH was not due to cell death . In complex media containing K+ or Na+, the L-form grew ove a wide pH range . Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH . The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2 . The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions. J Gen Microbiol, 1992 Jun, 138 ( Pt 6), 1123 - 31 Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762; Pfeifer O et al.; A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tu24, was used as a probe in Southern blot hybridization of total DNA from S . aureofaciens ATCC 10762 . A single SstI fragment of 5.4 kb was detected, which was cloned via an enriched gene library into Escherichia coli . The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S . lividans TK64, using the multicopy plasmid pIJ486 . The enzyme was overproduced in S . lividans TK64 (up to 30,000 times compared to S . aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S . aureofaciens ATCC 10762 . DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M(r) and N-terminal amino acid sequence as the purified subunit of BPO-A2. Biol Chem Hoppe Seyler, 1992 Jun, 373(6), 333 - 5 Incorporation of the enantiomers of lipoic acid into the pyruvate dehydrogenase complex from Escherichia coli in vivo; Oehring R et al.; The uptake of 35S-labelled enantiomers of lipoic acid into cells from Escherichia coli was studied . The R-enantiomer was taken up by a factor of two more efficiently than the S-form . Autoradiography of polyacrylamide gels of partially purified pyruvate dehydrogenase complex from these cells showed that only the R-lipoic acid was covalently incorporated as a cofactor into the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. Indian J Biochem Biophys, 1992 Jun, 29(3), 227 - 30 In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate; Sibghat-Ullah et al.; The size of the repair patch produced by E . coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined . A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E . coli endonuclease IV . Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase . Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E . coli endonuclease IV . Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated. Thromb Haemost, 1992 Jun 1, 67(6), 697 - 701 On the fibrinolytic system in aged rats, and its reactivity to endotoxin and cytokines; Emeis JJ et al.; Aged rats are more susceptible to endotoxin-induced effects, including microthrombosis and platelet aggregation, than are young rats . To investigate whether changes in the fibrinolytic system might be involved, we investigated the fibrinolytic activity in plasma euglobulin fractions and tissues (lung and heart) of young (6-months old) and aged (24-months old) rats under baseline conditions and after challenge with endotoxin . Aged rats had lower plasma levels of tissue-type plasminogen activator (t-PA) and of urokinase-type PA (u-PA) activity . PA inhibitor (PAI) activity was higher in the plasma of aged rats, as was t-PA activity in lung and heart . Rats were treated with either a low dose (1 microgram/kg) or a high dose (10 mg/kg) of endotoxin . Both treatments induced a transient phase of increased blood fibrinolytic activity, as evidenced by higher levels of tissue-type plasminogen activator (t-PA) activity and decreased levels of PA inhibitor (PAI) activity . Over time, the fibrinolytic activity decreased, probably due to increased levels of PA inhibitor . Both the early increase in t-PA activity, and the subsequent increase in PAI activity, were more pronounced in the aged rats, as compared with the younger rats, after the high dose of endotoxin . The aged rats also responded to an injection of interleukin-1 beta or tumor necrosis factor-alpha with a larger increase of PAI activity than did the younger rats . Together the data suggest that, compared to young rats, aged rats have a decreased base-line plasma fibrinolytic activity, while their fibrinolytic system is more responsive to challenge by endotoxin and cytokines. Arch Ital Urol Nefrol Androl, 1992 Jun, 64(2), 123 - 6 {Percutaneous drainage in renal emphysema . Clinical case}; Mancini R et al.; Renal emphysema is a rare and severe infection, that constitutes two clinical entities: emphysematous pyelonephritis and emphysematous pyelitis . We report a case in which the gas is localized both in the collecting system and in the parenchyma of the kidney . In our experience the percutaneous renal drainage has revealed to be able to dominate the infection but not to preserve renal function. FEMS Microbiol Lett, 1992 Jun 1, 72(2), 139 - 46 Incorporation of S-{3H}methyl-D-cysteine into the peptidoglycan of ether-treated cells of Escherichia coli; Caparros M et al.; Certain D-amino acids can be incorporated into the murein sacculus of Escherichia coli apparently through a direct transpeptidation reaction independent of the normal biosynthetic pathway . Investigation of this process is important because it could lead to the identification of hitherto unknown enzymes involved in murein metabolism . However, a serious drawback is the lack of an appropriate in vitro assay . We have analysed the suitability of a system based on the incorporation of a radioactive substrate (S-{3H}methyl-D-cysteine) by ether-treated cells, a method successfully applied before to the study of murein biosynthesis . The results reported here indicate that ether-treated cells are indeed proficient in the incorporation of D-amino acids, matching closely the properties of the reaction in growing cells. Cell Calcium, 1992 Jun-Jul, 13(6-7), 391 - 400 Solution structure of calmodulin and its complex with a myosin light chain kinase fragment; Ikura M et al.; The solution structure of Ca2+ ligated calmodulin and of its complex with a 26-residue peptide fragment of skeletal muscle myosin light chain kinase (skMLCK) have been investigated by multi-dimensional NMR . In the absence of peptide, the two globular domains of calmodulin adopt the same structure as observed in the crystalline form . The so-called 'central helix' which is observed in the crystalline state is disrupted in solution . 15N relaxation studies show that residues Asp78 through Ser81, located near the middle of this 'central helix', form a very flexible link between the two globular domains . In the presence of skMLCK target peptide, the peptide-protein complex adopts a globular ellipsoidal shape . The helical peptide is located in a hydrophobic channel that goes through the center of the complex and makes an angle of approximately 45 degrees with the long axis of the ellipsoid. Hybridoma, 1992 Jun, 11(3), 385 - 90 Monoclonal antibodies to Escherichia coli beta-galactosidase and their use for detection and purification of recombinant expression products; Draber P et al.; A panel of seven mouse monoclonal antibodies (BG-01-BG-07) was prepared against beta-galactosidase derived from E . coli . The antibodies are beta-galactosidase specific, show no cross-reactivity with other E . coli proteins and can be used for identification and characterization of beta-galactosidase fusion proteins expressed in lambda expression vectors . One of the antibodies allows a simple, one-step isolation of the fusion proteins directly from the crude bacterial lysates using immunoaffinity chromatography. Differentiation, 1992 Jun, 50(2), 67 - 74 Characterization of bep1 and bep4 antigens involved in cell interactions during Paracentrotus lividus development; Romancino DP et al.; We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins) . The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli . These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated . Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins . Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers . Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface . Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos . This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development. Mol Microbiol, 1992 Jun, 6(12), 1655 - 61 In vivo characterization of tus gene expression in Escherichia coli; Roecklein BA et al.; The tus gene encodes a DNA-binding protein (Tus) that inhibits replication forks at specific block-sites within the terminus region of the Escherichia coli chromosome . One of these block-sites, TerB, is adjacent to the tus gene . Using primer extension and a promoter fusion to characterize in vivo expression, we have demonstrated that the tus transcription start site is within TerB, and that Tus protein autoregulates expression at this weak promoter . We have also demonstrated that a minority of tus transcripts are initiated from an upstream region that contains two additional open reading frames . This readthrough transcription into tus is reduced in the presence of Tus protein. Leuk Lymphoma, 1992 Jun, 7(3), 217 - 24 A phase I/II study of dose and administration of non-glycosylated bacterially synthesized G-M CSF in chemotherapy-induced neutropenia in patients with non-Hodgkin's lymphomas; Hovgaard D et al.; Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) derived from E . coli was administered to 24 previously untreated patients with non-Hodgkin's lymphoma following the first cycle of CHOP chemotherapy . Four dose levels were examined, 1.5, 3.0, 5.5 and 11 micrograms/kg and patients were randomized to receive the drug either once or twice daily subcutaneously (s.c.) . During rhGM-CSF treatment, the leucocyte counts increased up to 3-4 fold in 20/24 patients, reaching a peak 24-48 (mean 35) hours after initiation of rhGM-CSF . The leukopenic period in cycle one of the CHOP chemotherapy with rhGM-CSF, was shorter than after the course of chemotherapy without rhGM-CSF and also shorter when compared to cycle one of CHOP in the 127 historical controls (p < 0.05 and p < 0.001 respectively) . Similar results were observed for neutrophil counts . No effect was seen on platelet counts at nadir but a significant, although moderate increase occurred in the recovery period on days 15 and 22 when compared to control cycles and historical controls . When dose levels were compared, there was only a trend to higher WBC counts at the higher dose groups (5.5 and 11 micrograms/kg) when compared to the two lower dose groups (1.5 and 3.0 micrograms/kg) . In the overall evaluation there was no statistical significant difference in results between patients treated s.c . once daily versus twice daily . However when only the two highest dose levels (5.5 + 11 micrograms/kg) were compared, s.c . administration of rhGM-CSF twice daily led to higher leucocyte counts than once daily in the recovery period on day 15 (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) Genet Anal Tech Appl, 1992 Jun, 9(3), 91 - 5 A plasmid that improves the efficiency of foreign gene expression by intracellular T7 RNA polymerase; Takahashi T et al.; To facilitate the construction of recombinant plasmids for expressing cloned genes with T7 RNA polymerase supplied by recombinant vaccinia virus, a plasmid expression vector was designed by combining parts of plasmids pTZ18R, pBluescript II KS+, and pAR2529 . The 3043-bp plasmid pTF1 has a T7 RNA polymerase promoter, multiple cloning site for insertion of foreign genes, and a T7-specific transcription termination signal . Plasmid pTF1 had several advantages compared with the reference plasmid pAR2529, including more efficient replication in bacteria, greater flexibility in the insertion and subcloning of foreign genes, and increased efficiency of liposome-mediated introduction into cultured cells for expression of the foreign gene. Kobe J Med Sci, 1992 Jun, 38(3), 175 - 89 Phosphorylation of CRE-BP1, a cyclic AMP response element binding protein, by protein kinase C and cyclic AMP-dependent protein kinase; Sakurai A; CRE-BP1 is a transcriptional activator binding to the cyclic AMP response element, which has a putative metal finger structure and the leucine zipper motif linked to a cluster of basic amino acids in the amino and carboxyl-terminal regions, respectively . The activities of a number of transcription factors are known to be controlled through phosphorylation and dephosphorylation . At the first step for understanding of the regulation of CRE-BP1, phosphorylation of CRE-BP1 was studied in vitro . The human recombinant CRE-BP1 was phosphorylated by protein kinase C and cyclic AMP-dependent protein kinase . These two protein kinases recognized distinct seryl residues of CRE-BP1 . Amino acid sequence analysis after phosphopeptide map indicated that two seryl residues, Ser-340 and Ser-367, located in the basic region of CRE-BP1 were identified as the major protein kinase C phosphorylation sites, whereas Ser-62 downstream of the metal finger structure was determined as the phosphorylation site by cyclic AMP-dependent protein kinase . The phosphorylation of CRE-BP1 by these two protein kinases may regulate the function of this transcriptional activator protein. Biochem Cell Biol, 1992 Jun, 70(6), 460 - 5 Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids; Schutzbach JS et al.; The activity of purified recombinant yeast dolichyl-phosphomannose synthase (EC 2.4.1.83) was assessed following reconstitution of the enzyme with phospholipids . The yeast synthase, similar to the mammalian enzyme, was active when reconstituted with phosphatidylethanolamine dispersions but had little (less than 5%) activity in stable phosphatidylcholine bilayers . The enzyme was activated by adding increasing amounts of diacylglycerol to phospholipid bilayers, suggesting that activity of the yeast enzyme was dependent on lipid phase properties rather than specific phospholipids . The synthase could also be reconstituted as an active enzyme in bilayers prepared with a commercial crude lipid preparation containing 40% phosphatidylcholine, but at a rate 10% of that occurring in phosphatidylethanolamine . Vesicles composed of the 40% phosphatidylcholine lipid mixture, dolichyl phosphate, and enzyme were leaky in the presence of divalent cations, and dolichyl-phosphomannose synthase did not appear to catalyze the translocation of dolichyl phosphomannose across membranes at a catalytically significant rate under the assay conditions employed. Yakugaku Zasshi, 1992 Jun, 112(6), 349 - 57 {Translocation mechanism of presecretory protein across the cytoplasmic membrane of Escherichia coli}; Tokuda H; Translocation of presecretory proteins across the cytoplasmic membrane of Escherichia coli requires several Sec proteins and two kinds of energies, adenosine triphosphate (ATP) and a proton motive force . In vitro assay system established for the analysis of protein translocation revealed that ATP and the proton motive force play different roles in protein translocation . SecA, a peripheral membrane protein, was shown to be essential for in vitro protein translocation . SecE and SecY, both of which are integral membrane proteins, were purified from E . coli cells overproducing these proteins, and reconstituted into proteoliposomes . The reconstituted proteoliposomes exhibited protein translocation activity in the presence of ATP and SecA, indicating that SecE and SecY as well as SecA are indispensable components of protein translocation machinery . In this paper, the molecular mechanism of protein translocation across the membrane of E . coli is discussed based on the recent data obtained by both in vitro and reconstitution systems. Tuber Lung Dis, 1992 Jun, 73(3), 129 - 33 Skin testing with recombinant Mycobacterium intracellulare antigens; Morris SL et al.; The immunoreactivity of four recombinant Mycobacterium intracellulare beta-galactosidase fusion proteins, which correspond to 22, 40, 43 and 85 kDa M . intracellulare antigens, was assessed . Lymphoproliferative assays demonstrated that Escherichia coli lysates containing each of the fusion proteins stimulated T cells in vitro . Purified preparations of three of these recombinant M . intracellulare antigens (22, 43 and 85 kDa) also induced delayed-type hypersensitivity (DTH) reactions in sensitized guinea pigs . However, the skin test responses evoked by each of these antigens was not species-specific . Given these results, the potential utility as skin test reagents of the purified antigens or peptides derived from these proteins is discussed. Bioorg Khim, 1992 Jun, 18(6), 766 - 76 {Secretion of recombinant human epidermal growth factor into the periplasm in Escherichia coli cells}; Batchikova NV et al.; Secretion vectors were constructed in which a synthetic gene of human epidermal growth factor (hEGF) joined with a gene coding for the leader peptide to one of the E . coli outer membrane major proteins (OmpF) is controlled by tac promoter . The increase of the hEGF yield was achieved by the multiplication of the gene copies . The hEGF in bacterial cells was secreted into periplasm . The recombinant protein was isolated by means of reverse phase chromatography as almost homogenous preparation (greater than 98%), the yield being 7 mg/l bacterial culture . The sequence of twenty-five N-terminal amino acid residues of the isolated hEGF coincided with that of the natural protein . The preparation proved to be biologically active. Anal Biochem, 1992 Jun, 203(2), 274 - 80 A solid-phase screen for protein kinase substrate selectivity; Carmel G et al.; Cassette mutagenesis was used to synthesize an Escherichia coli expression library of unique phosphorylation sites . The cassette encodes a central serine residue surrounded by every combination of Ala, Arg, Gln, Glu, Gly, and Pro residues over a 7-residue segment (a total of 6(7) approximately 2.8 x 10(5) sequences) . The cassette was inserted into the gene of a suitable carrier protein and expressed in E . coli with the T7 expression system, and the resultant library was subjected to solid-phase protein phosphorylation assays on nitrocellulose filters . When the library was screened with TPK1 delta, the modified catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase, individual colonies that expressed substrates for this kinase were identified . By DNA sequencing through the cassette region of positive clones, the consensus recognition sequence for TPK1 delta was deduced and found to conform with the well-established substrate selectivity of its mammalian homolog (Arg-Arg-Xaa-Ser) . Because a large number of clones can be sequenced rapidly, and the positions of invariant residues composing a recognition site identified, this approach may be useful as a general screen of protein kinase substrate selectivity. Protein Eng, 1992 Jun, 5(4), 351 - 9 Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer; Peter W et al.; The progesterone-binding protein uteroglobin has been expressed in Escherichia coli in an unfused, soluble form . Like mature uteroglobin from rabbit endometrium (UG), the E.coli produced uteroglobin (UG1) dimerizes in vitro, forms an antiparallel dimer with Cys3-Cys69' and Cys69-Cys3' disulfide bonds and binds progesterone under reducing conditions . In order to analyze the dimerization and the reduction dependence of progesterone binding in more detail, we separately replaced cysteine 3 and cysteine 69 by serines . Under reducing conditions, both uteroglobin variants (UG1-3Ser and UG1-69Ser) bind progesterone with the same affinity as the wild-type suggesting that both cysteine residues are not directly involved in progesterone binding . In contrast to the wild-type protein, both cysteine variants also bind progesterone with high affinity in the absence of reducing agents . In addition, UG1-3Ser and UG1-69Ser both form covalently linked homodimers . Thus, unnatural Cys69-69' and Cys3-3' disulfide bonds exist in UG1-3Ser and UG1-69Ser, respectively . These data together with computer models based on X-ray diffraction data strongly support the idea that progesterone reaches its binding site located in an internal hydrophobic cavity via a hydrophobic tunnel along helices 1 and 4 . Under non-reducing conditions the tunnel is closed by two disulfide bridges (Cys3-Cys69' and Cys69-Cys3') that lie in the most flexible region of the dimer . Reduction or replacement of a cysteine residue enables conformational changes that open the channel allowing progesterone to enter. Mol Cell Probes, 1992 Jun, 6(3), 209 - 14 Identification of Shiga-like toxin type II producing Escherichia coli using the polymerase chain reaction and a digoxigenin labelled DNA probe; Jackson MP; Epidemiological studies have demonstrated that enterohaemorrhagic strains of Escherichia coli which cause the haemolytic uremic syndrome in humans and the oedema disease in pigs more frequently produce Shiga-like toxin type II (SLT-II) than any other member of the Shiga-like toxin family . A technique has been developed for the identification of SLT-II producing E . coli using the polymerase chain reaction (PCR) and a digoxigenin (DIG)-labelled DNA probe to facilitate the early detection and epidemiological analysis of these pathogens . Whole cell DNA liberated from isolated colonies during the denaturation step of PCR was amplified using a primer pair which is homologous to the slt-II gene sequences . The amplification products were transferred directly to a nitrocellulose membrane or following agarose gel electrophoresis and DNA denaturation . A chemically labelled DNA probe, prepared using PCR with the incorporation of DIG, was used to identify the PCR products of strains which produced SLT-II or a variant of SLT-II. Mol Cell Probes, 1992 Jun, 6(3), 201 - 8 Colorimetric detection of Plasmodium falciparum and direct sequencing of amplified gene fragments using a solid phase method; Holmberg M et al.; A rapid colorimetric assay for the detection of DNA from Plasmodium falciparum malaria is described, allowing direct sequencing of amplified fragments in the positive samples . The method is based on amplification by the polymerase chain reaction (PCR), with incorporation of biotin and a lac operator sequence in the amplified target DNA . The PCR product was immobilized on streptavidin-coupled magnetic beads, and detected by the specific binding of an Escherichia coli lac repressor beta-galactosidase fusion protein . Positive samples were subsequently treated with alkali to generate single stranded templates, which were used for solid phase genomic sequencing . As targets for amplification and sequencing we selected a region of the gene for the antigen Pf155/RESA and a region of the parasite dihydrofolate reductase gene (PfDHFR/TS) . We show here that both of these gene targets can be used for specific detection of P . falciparum in patient blood samples . Genomic sequencing of five patient isolates revealed no variation in the Pf155/RESA gene fragment . In a comparison of this sequence with conserved protein domains, a marked similarity to the src homology region 3 was detected . A point mutation was found in the PfDHFR/TS gene fragment of one of the clinical samples, replacing Ser108 with Asn . This mutation has earlier been described in pyrimethamine and cycloguanile-resistant strains of P . falciparum. Immunobiology, 1992 Jun, 185(1), 41 - 52 Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli; Bautsch W et al.; A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli . A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene . The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor . After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets . The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1992 Jun, 14(3), 173 - 8 {Synthesis and cloning of the whole human erythropoietin (EPO) gene}; Cai Y; A 600 bp synthetic erythropoietin (EPO) gene encoding all 166 amino acids of the EPO protein and 27 amino acids of the signal peptide has been constructed . The whole gene was divided into three large fragments consisting of a total of 32 oligonucleotides . These oligonucleotides were synthesized by the solid-phase phosphor-amidite method and ligated into three large fragments . These latter three were separately cloned into vector M13mp19 and then transformed into E . coli JM 103 . Positive clones were screened with 32P-labeled probes . The sequences of the fragments were confirmed by DNA sequencing, and the sequence of the whole synthetic EPO gene was confirmed by enzymatic digestion and sequencing . The results indicated that the nucleotide sequence of the synthetic EPO gene is identical to that of the original. Chem Pharm Bull (Tokyo), 1992 Jun, 40(6), 1644 - 6 Bacteriostatic effect of 4,7-dicyanobenzofurazan due to inactivation of 2,3-dihydroxyisovalerate dehydratase; Takabatake T et al.; As part of our research on benzofurazans (BZs), we have reported the bacterioses of BZs in Escherichia coli, which may be due to O2- . produced within E . coli in the presence of dioxygen (O2) . Incubation of E . coli with 4,7-dicyanobenzofurazan (1) lowered the 2,3-dihydroxyisovalerate dehydratase activity detectable in extracts from these cells . Addition of branched chain amino acids such as valine and leucine protected E . coli from growth inhibition by compound 1, though it could not protect E . coli from the damage by paraquat (PQ) . Addition of Fe(III)-tris{N-(2-pyridylmethyl)-2-aminoethyl}amine (Fe-TPAA), a novel superoxide dismutase mimic, protected the dehydratase in a dose-dependent manner, which confirms that inactivation of the dehydratase is largely due to production of O2-. . The possibility was discussed that the bacteriostatic effect of compound 1 is due to the inactivation of 2,3-dihydroxyisovalerate dehydratase. Protein Expr Purif, 1992 Jun, 3(3), 256 - 62 Expression of human NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in Escherichia coli: purification and partial characterization; Yang XM et al.; NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is a bifunctional enzyme synthesized as a 37-kDa precursor that is imported into the mitochondria of embryonic and transformed mammalian cells . The cDNA encoding the human bifunctional enzyme was modified to remove nucleotides corresponding to the mitochondrial targeting sequence and was subcloned into a procaryotic expression vector under the control of the T7 RNA polymerase promoter . The soluble dehydrogenase-cyclohydrolase was expressed in Escherichia coli at levels up to 150-fold higher than those found in transformed mammalian cells . Forms of the recombinant enzyme with one, three, or seven additional amino-terminal residues were purified to homogeneity and shown to have similar kinetic properties . Investigation of the absolute requirement of the enzyme for Mg2+ using fluorescence quenching indicates that this ion binds in the absence of substrates. Protein Expr Purif, 1992 Jun, 3(3), 246 - 55 Expression of the adenovirus E1B 175R protein and its association with membranes of Escherichia coli; Dalie B et al.; The E1B 175-amino-acid (175R) protein of adenovirus 2 is required for cellular transformation of primary cells and establishing cell morphology in lytically infected cells . To investigate the biochemical function of this protein, we constructed a bacterial expression vector (pKHB1-T) to produce the 175R protein in sufficient amounts for purification and biochemical analysis . On the basis of DNA sequencing, gel electrophoresis, and immunoblot analysis, the pKHB1-T-encoded 175R protein appears to be identical to that expressed transiently in mammalian or adenovirus-transformed cells . The bacterially produced viral protein was also found to be quite stable and without any modifications . Partial purification of the pKHB1-T-encoded protein revealed that the majority of its associates with the inner membrane of the bacterial cell . This, together with the possibility of the 175R protein containing an N-terminal amphipathic alpha-helix as a potential translocation signal, suggests that there may be a common mechanism of protein transport operating in both eucaryotic and procaryotic systems. Protein Expr Purif, 1992 Jun, 3(3), 204 - 11 Isolation and purification of a biologically active human platelet-derived growth factor BB expressed in Escherichia coli; Alexander DM et al.; Human platelet-derived growth factor (PDGF) was expressed in Escherichia coli from a high-level cytoplasmic expression vector . A cDNA fragment encoding the mature form of the human PDGF B chain (hPDGF-B) was cloned into a plasmid under transcriptional control of the inducible E . coli Tac promoter . Expression of hPDGF-B from the final construct, pTacBIq, is regulated by the lactose repressor (LacIq) . Upon induction, a polypeptide of approximately 14 kDa that had the same molecular mass and immunoreactivity as authentic hPDGF-B was produced . The production of recombinant hPDGF-B was significantly increased in an E . coli strain (CAG629) defective in expression of the lon protease . Expression of hPDGF-B in the CAG629 strain accounted for approximately 1% of total cell protein . In this system, hPDGF-B is expressed as an insoluble, intracellular protein and can readily be obtained in a partially purified form after differential centrifugation . Amino acid sequence determination of the purified protein has verified that the amino-terminal portion of the recombinant PDGF is correct . After renaturation into dimers, the purified recombinant hPDGF is fully functional in assays for receptor binding and mitogenesis. Protein Expr Purif, 1992 Jun, 3(3), 178 - 84 An import-competent precursor of small subunit of ribulose-1,5-bisphosphate carboxylase generated by factor Xa cleavage from a beta-galactosidase fusion expressed in Escherichia coli; Evans A et al.; A plasmid-encoding fusion protein interlinked by factor Xa recognition sequence between beta-galactosidase and a precursor of the small subunit of wheat ribulose-1,5-bisphosphate carboxylase has been constructed . The plasmid directed abundant synthesis of the fusion protein in Escherichia coli . The recombinant protein was accumulated in an aggregated form that was associated with the bacterial membranes . A procedure was developed to isolate the fusion protein in a relatively pure and soluble form . Bovine factor Xa cleaved the isolated chimera to generate the complete chloroplast precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase from the fused beta-galactosidase . The cleaved precursor protein was imported into the isolated chloroplasts and processed to yield its mature counterpart. Anal Biochem, 1992 Jun, 203(2), 269 - 73 A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs; Lakshman DK et al.; We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs . A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants . First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT) . Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E . coli DNA polymerase I, E . coli DNA ligase, and beta-NAD+ . The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme . The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E . coli strain DH5 alpha . This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence. J Protein Chem, 1992 Jun, 11(3), 225 - 30 Both ends of Escherichia coli ribosomal protein S13 are immunochemically accessible in situ; Syu WJ et al.; To investigate the structure of Escherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence . The availability of these S13 epitopes in situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation . We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera . However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers . When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed . Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation . Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region . The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits . These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits . Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13 in situ. Cell Struct Funct, 1992 Jun, 17(3), 157 - 60 Chemical modification of recombinant human granulocyte colony-stimulating factor by polyethylene glycol increases its biological activity in vivo; Satake-Ishikawa R et al.; Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Escherichia coli was chemically modified by polyethylene glycol (PEG) of molecular weights 4,500 or 10,000 . The neutrophils observed at 32 hours after intravenous injection of the rHuG-CSF modified with PEG (4,500) or PEG (10,000) to mice were, respectively, 2.5 times and 5 times more than that observed after the injection of the unmodified rHuG-CSF . These results show that the covalent attachment of PEG to rHuG-CSF enhanced its pharmacological activity in vivo and that the modification with the larger PEG molecule is more effective to enhance the in vivo activity of rHuG-CSF. J Interferon Res, 1992 Jun, 12(3), 177 - 83 Differential inactivation of interferons by a protease from human granulocytes; Cantell K et al.; Human leukocyte suspensions produced interferon-alpha (IFN-alpha) without induction during incubation at 37 degrees C . The highest titers were obtained at about 30 million cells/ml . The best yields, approximately 2 IU per 10(6) cells, were achieved in medium with or without albumin; serum inhibited the production . The uninduced IFN-alpha peaked at 24 h . The titers dropped on further incubation due to release of a protease from polymorphonuclear cells . The protease inactivated all tested human class I IFNs, but recombinant IFN-alpha 1 was clearly more resistant to the enzyme than rIFN-alpha 2 . Human IFNs-gamma from different sources exhibited striking differences in their sensitivity to the protease . Glycosylated natural IFN-gamma from human leukocytes and glycosylated rIFN-gamma from CHO cells were relatively resistant, whereas unglycosylated rIFN-gamma from Escherichia coli was rapidly degraded by the protease . The protease was inhibited by PMSF and by greater than or equal to 1% human or fetal bovine serum but not by EDTA or less than or equal to 1% human albumin . Its optimum pH was between 7 and 8 . It was resistant to treatment for 30 min at 56 degrees C. Clin Biochem, 1992 Jun, 25(3), 209 - 12 Molecular basis for the association between HLA DR4 and rheumatoid arthritis . From the shared epitope hypothesis to a peptidic model of rheumatoid arthritis; Albani S et al.; Susceptibility to rheumatoid arthritis (RA) maps to residues QKRAA/QRRAA in the third hypervariable region of the HLA DR beta 1 chain . Peptides from the same area of MHC class II molecules are able to modulate the T-cell repertoire by deleting self-reactive T-cells . The Epstein Barr virus glycoprotein gp110 and the dna J heat-shock protein from E . coli mimic the third hypervariable region of HLA-Dw4DR beta 1 . Thus, the same area of HLA DR beta 1 carries susceptibility to RA, modulates the T-cell repertoire and is mimicked by human pathogens . RA may originate from a particular shape imposed on the T-cell repertoire by the QKRAA/QRRAA sequence in the third hypervariable region of HLA DR beta 1. Comput Appl Biosci, 1992 Jun, 8(3), 215 - 25 An algorithm for comparing RNA secondary structures and searching for similar substructures; Chevalet C et al.; To access the functional informations carried by RNA molecules at the level of their secondary structure interactions, we propose a comparison method based on a tree edit algorithm which takes into account the tree structure of RNA foldings . Any secondary structure is translated into a tree involving all its elementary substructures; then a shorter condensed tree is built in which any unbranched helix interspersed with bulges and interior loops is taken as a single node . This method includes several parameters: a comparison matrix between structural units, gap penalties, and the scoring between nodes of the condensed trees . Their effects have been analysed using as a model a rapidly divergent domain of the large ribosomal RNA, for which structural variation during evolution is well known . This method allows one to recognize precisely, in large target molecules, definite substructures that present with the query molecules only a limited set of closely related secondary structure features; it is still efficient if intervening features, which can correspond to insertion/deletion of entire stem regions, separate such structural elements . When coupled with a hierarchical clustering algorithm, this method is suitable for classifying RNA molecules according to their secondary structure homologies. J Mol Endocrinol, 1992 Jun, 8(3), 213 - 23 Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency; Francis GL et al.; An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described . These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action . The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include {Met1}-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, {Met1}-pGH(1-11)-Val-Asn-{Gly3}-IGF-I and {Met1}-pGH(1-11)-Val-Asn-{Arg3}-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively . The three peptides are referred to as Long IGF-I, Long {Gly3}-IGF-I or Long {Arg3}-IGF-I depending on the IGF-I sequence present . Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product . Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs . The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I . In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown . In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts . The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long {Arg3}-IGF-I-des(1-3)IGF-I greater than Long {Gly3}-IGF-I greater than Long IGF-I greater than IGF-I . In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long {Arg3}-IGF-I, was less potent than IGF-I . Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions. Int Immunol, 1992 Jun, 4(6), 627 - 36 Kinetic immunodominance: functionally competing antibodies against exposed and cryptic epitopes of Escherichia coli beta-galactosidase are produced in time sequence; Kunkl A et al.; The murine antibody response to Escherichia coli beta-galactosidase (GZ) was analyzed in vivo and in vitro by focusing on two families of antibodies that exert distinct conformational/functional activity on the antigen . Activating antibodies--defined by their capacity to increase the enzymatic activity of defective GZ produced by mutant strains of E . coli--are detected early after secondary challenge . Inhibiting antibodies, which interfere with antibody-mediated enzyme activation, appear later and cause the abrupt fall of activating titer, a scenario suggesting either idiotype/anti-idiotype interaction or opposite pulsions exerted on the antigen molecule . Supporting the latter mechanism, the confrontation of mAbs of the two families produced classical competitive inhibition curves when the readout was enzyme activation, although they recognize two different epitopes of the same molecule: the activating mAb a quaternary conformation-dependent site of wild-type GZ, the inhibiting mAb a sequential determinant exposed only in denatured or in defective enzyme . The different timing of generation of these antibodies during the response may depend on a processing step necessary for unfolding of native antigen and consequent display of certain cryptic epitopes before they can trigger specific B cells . A picture emerges where the response to the various epitopes of a complex antigen is sequentially connected and where the uptake by antigen-presenting cells of antigen complexed with antibodies specific for the exposed epitopes may favor revelation of the cryptic ones. Mutat Res, 1992 Jun, 282(2), 93 - 8 Glycyrrhiza glabra extract as an effector of interception in Escherichia coli K12+; Kuo S et al.; Glycyrrhiza glabra polar lipid extract contains a number of flavonoids and related chemical compounds . Studies on the effectiveness of Glycyrrhiza glabra polar lipid extract in intercepting reactive molecules generated from the illumination of the photosensitizers rose bengal and phenosafranin indicate that it is effective in preventing cytotoxicity against E . coli K12+ in a dose-related fashion using illuminated rose bengal . Since only a modest scavenging of singlet oxygen generated from phenosafranin is observed, the effects of the extracts are less related to singlet oxygen-mediated oxidation of substrate (type II reactions) than non-singlet oxygen-mediated oxidation of substrate (type I reactions) . Elevated levels of glutathione observed in exponentially growing cells of E . coli K12 were also observed. J Cell Biol, 1992 Jun, 117(6), 1343 - 50 Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility; Hardwick C et al.; A cDNA encoding a unique hyaluronan receptor has been molecularly cloned from a lambda GT11 3T3 cDNA expression library . Immunoblot analyses of cell lysates, using antibodies to peptides encoded in the cDNA, specifically react with a 58-kD protein . This protein is regulated by the mutant H-ras gene in cells containing a metallothionein promoter H-ras hybrid gene . Further, antibodies to peptide sequences encoded in the cDNA block the increase in locomotion resulting from induction of the mutant H-ras gene in this cell line . In a transblot assay, the bacterially expressed protein binds to biotinylated hyaluronan . Antibodies to peptides encoded in the cDNA react in immunoblot assays with the 58- and 52-kD proteins of a novel hyaluronan receptor complex previously implicated in cell locomotion . Furthermore, antibodies specific to the 58- and 52-kD proteins, which block ras-induced locomotion, also cross-react with the expressed, encoded protein . The gene product described here appears to be a new type of hyaluronan receptor that is involved in cell locomotion . It is named RHAMM, an acronym for receptor for hyaluronan-mediated motility. EMBO J, 1992 Jun, 11(6), 2345 - 55 Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope; Denecke J et al.; We studied protein sorting signals which are responsible for the retention of reticuloplasmins in the lumen of the plant endoplasmic reticulum (ER) . A non-specific passenger protein, previously shown to be secreted by default, was used as a carrier for such signals . Tagging with C-terminal tetrapeptide sequences of mammalian (KDEL) and yeast (HDEL) reticuloplasmins led to effective accumulation of the protein chimeras in the lumen of the plant ER . Some single amino acid substitutions within the tetrapeptide tag (-SDEL, -KDDL, -KDEI and -KDEV) can cause a complete loss of its function as a retention signal, demonstrating the high specificity of the retention machinery . However, other modifications confer efficient (-RDEL) or partial (-KEEL) retention . It is also shown that the efficiency of protein retention is not significantly impaired by an increased ligand concentration in plants . The efficiently retained chimeras (-KDEL, -HDEL and -RDEL) were shown to be recognized by a monoclonal antibody directed against the C-terminus of the mammalian reticuloplasmin protein disulfide isomerase (PDI) . The recognized epitope is also present in several putative reticuloplasmins in microsomal fractions of plant and mammalian cells, suggesting that the antibodies recognize an important structural determinant of the retention signal . In addition, data are discussed which support the view that upstream sequences beyond the C-terminal tetrapeptide can influence or may be part of the structure of reticuloplasmin retention signals. Am J Hum Genet, 1992 Jun, 50(6), 1203 - 10 A molecular defect in human protoporphyria; Brenner DA et al.; Protoporphyria is generally an autosomal dominant disease that is characterized clinically by photosensitivity and hepatobiliary disease and that is characterized biochemically by elevated protoporphyrin levels . The enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is deficient in all tissues of patients with protoporphyria . In this study, sequencing of ferrochelatase cDNAs from a patient with protoporphyria revealed a single point mutation in the cDNAs resulting in the conversion of a Phe(TTC) to a Ser(TCC) in the carboxy-terminal end of the protein, F417S . Further, the human ferrochelatase gene was mapped to chromosome 18q21.3 by chromosomal in situ suppression hybridization . Finally, expression of recombinant ferrochelatase in Escherichia coli demonstrated a marked deficiency in activity of the mutant ferrochelatase protein and of mouse-human mutant ferrochelatase chimeric proteins . Therefore, a point mutation in the coding region of the ferrochelatase gene is the genetic defect in some patients with protoporphyria. Eur J Biochem, 1992 Jun 1, 206(2), 381 - 90 Attenuation in the rph-pyrE operon of Escherichia coli and processing of the dicistronic mRNA; Andersen JT et al.; We have substituted on a plasmid the native promoter of the Escherichia coli rph-pyrE operon with an inducible transcription-initiation signal . The plasmid was used to study the mRNA chains derived from the operon at different intracellular concentrations of UTP and as a function of time following induction of transcription . The results showed that dicistronic rph-pyrE mRNA was formed when the UTP pool was low, and that a monocistronic rph mRNa was the major transcription product in high-UTP pools, thus supporting an UTP-controlled attenuation mechanism for regulation of pyrE gene expression . However, the dicistronic rph-pyrE transcript was rapidly processed into two monocistronic mRNA units, and a cleavage site was mapped near the attenuator in the intercistronic region, close to the site where transcription was terminated in high-UTP pools . Furthermore, the major 3' end of the pyrE mRNA was mapped near a palindromic structure of similarity to the family of repetitive extragenic palindromic sequences, 35 nucleotide residues after stop codon of the pryE gene. Eur J Biochem, 1992 Jun 1, 206(2), 315 - 21 Small RNA helices as substrates for aminoacylation and their relationship to charging of transfer RNAs; Francklyn C et al.; RNA microhelices that reconstruct the acceptor stems of transfer RNAs can be aminoacylated . The anticodon-independent aminoacylation is sequence-specific and suggests a relationship between amino acids and nucleotide sequences which is different from that of the classical genetic code . The specific aminoacylation of RNA microhelices also suggests a highly differentiated adaptation of the structures of aminoacyl-tRNA synthetases to sequences in the acceptor stems of transfer RNAs. Mutat Res, 1992 Jun, 274(1), 65 - 71 Differences in DNA damage induced by mutagenic and nonmutagenic 4-aminoazobenzene derivatives in Escherichia coli; Kojima M et al.; DNA lesions produced in Escherichia coli AB2500 (uvrA) exposed to the carcinogen N-hydroxy-3-methoxy-4-aminoazobenzene (N-OH-3-MeO-AAB) or the noncarcinogen N-hydroxy-2-methoxy-4-aminoazobenzene (N-OH-2-MeO-AAB) were investigated by alkaline sucrose gradient sedimentation and 32P-postlabeling analysis . Alkali-labile sites appeared to be formed equally in cells treated with both aminoazobenzene derivatives . 32P-Postlabeling analysis revealed that the 3-MeO-AAB-DNA adduct level was 25-fold higher than that for 2-MeO-AAB-DNA adducts . In addition to major adducts, 4 minor spots were detected in N-OH-3-MeO-AAB-treated cells, while only one major adduct was found in N-OH-2-MeO-AAB-treated cells . The mutagenicities and cytotoxicities were also determined with E . coli with different repair capacities; we found that repair of 3-MeO-AAB damages is strongly dependent on the UVR repair system . Moreover, N-OH-3-MeO-AAB, but not N-OH-2-MeO-AAB, could induce recA and umuC gene expression, which was higher in uvrA strains than in the wild type. Mutat Res, 1992 Jun, 274(1), 29 - 43 HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro; Carty MP et al.; To determine whether cellular replication factors can influence the fidelity of DNA replication, the effect of HeLa cell single-stranded DNA-binding protein (SSB) on the accuracy of DNA replication by HeLa cell DNA polymerase alpha has been examined . An in vitro gap-filling assay, in which the single-stranded gap contains the supF target gene, was used to measure mutagenesis . Addition of SSB to the in vitro DNA synthesis reaction increased the accuracy of DNA polymerase alpha by 2- to 8-fold . Analysis of the products of DNA synthesis indicated that SSB reduces pausing by the polymerase at specific sites in the single-stranded supF template . Sequence analysis of the types of errors resulting from synthesis in the absence or presence of SSB reveals that, while the errors are primarily base substitutions under both conditions, SSB reduces the number of errors found at 3 hotspots in the supF gene . Thus, a cellular replication factor (SSB) can influence the fidelity of a mammalian DNA polymerase in vitro, suggesting that the high accuracy of cellular DNA replication may be determined in part by the interaction between replication factors, DNA polymerase and the DNA template in the replication complex. Infect Immun, 1992 Jun, 60(6), 2201 - 10 Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A; Kuhn HM et al.; A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures . These compounds vary in phosphate substitution, acylation pattern (type, number, and distribution of fatty acids), and, in the case of monosaccharides, in their backbone glycosyl residue . The monoclonal antibodies tested could be subdivided into five groups according to their reactivity patterns . One group reacted exclusively with 1,4'-bisphosphoryl lipid A, and a second also reacted with 4'-monophosphoryl lipid A . Two further groups recognized either 4-phosphoryl or 1-phosphoryl monosaccharide partial structures of lipid A . The fifth group reacted with 4-phosphoryl monosaccharide structures and with phosphate-free compounds . Antibodies reactive with monosaccharide structures also recognized their epitopes in corresponding phosphorylated disaccharide compounds . Both groups of monosaccharide and monophosphoryl lipid A-recognizing antibodies have access to their epitopes in bisphosphoryl compounds as well . Because of this unidirectional reactivity with more complex structures, the various specificities cannot be distinguished by using bisphosphoryl lipid A (e.g., Escherichia coli lipid A) as a test antigen . The epitopes recognized by the various monoclonal antibodies all reside in the hydrophilic backbone of lipid A, and there was no indication that fatty acids were part of the epitopes recognized . Nevertheless, the reactivities of compounds in the different test systems are strongly influenced by their acylation patterns; i.e., acyl groups may modulate the exposure of lipid A epitopes. Infect Immun, 1992 Jun, 60(6), 2174 - 81 Analysis of Escherichia coli colonization factor antigen I linear B-cell epitopes, as determined by primate responses, following protein sequence verification; Cassels FJ et al.; Colonization factor antigen I (CFA/I)-bearing strains of enterotoxigenic Escherichia coli (ETEC) are responsible for a significant percentage of ETEC diarrheal disease worldwide whether the disease presents as infant diarrhea with high mortality or as traveler's diarrhea . CFA/I pili (fimbriae) are virulence determinants that consist of repeating protein subunits (pilin), are found in several ETEC serogroups, and promote attachment to human intestinal mucosa . While CFA/I pili are highly immunogenic, the antigenic determinants of CFA/I have not been defined . We wished to identify the linear B-cell epitopes within the CFA/I molecule as determined by primate response to the immunizing protein . To do this, we (i) resolved the discrepancies in the literature on the complete amino acid sequence of CFA/I by N-terminal and internal protein sequencing of purified and selected proteolytic fragments of CFA/I, (ii) utilized this sequence to synthesize 140 overlapping octapeptides covalently attached to polyethylene pins which represented the entire CFA/I protein, (iii) immunized three rhesus monkeys with multiple intramuscular injections of purified CFA/I subunit in Freund's adjuvant, and (iv) tested serum from each monkey for its ability to recognize the octapeptides in a capture enzyme-linked immunosorbent assay . Eight linear B-cell epitopes were identified; the region containing an epitope at amino acids 11 to 21 was strongly recognized by all three individual rhesus monkeys, while the amino acid stretches 22 to 29, 66 to 74, 93 to 101, and 124 to 136 each contained an epitope that was recognized by two of the three rhesus monkeys . The three other regions containing epitopes were recognized by one of the three individuals . The monkey antiserum to pilus subunits recognized native intact pili by immunogold labeling of CFA/I pili present on whole H10407 cells . Therefore, immunization with pilus subunits induces antibody that clearly recognizes both synthetic linear epitopes and intact pili . We are currently studying the importance of these defined epitope-containing regions as vaccine candidates. J Virol, 1992 Jun, 66(6), 3593 - 601 Human immunodeficiency virus type 1 integration protein: DNA sequence requirements for cleaving and joining reactions; Sherman PA et al.; Using purified integration protein (IN) from human immunodeficiency virus (HIV) type 1 and oligonucleotide mimics of viral and target DNA, we have investigated the DNA sequence specificity of the cleaving and joining reactions that take place during retroviral integration . The first reaction in this process is selective endonucleolytic cleaving of the viral DNA terminus that generates a recessed 3' OH group . This 3' OH group is then joined to a 5' phosphoryl group located at a break in the target DNA . We found that the conserved CA located close to the 3' end of the plus strand of the U5 viral terminus (also present on the minus strand of the U3 terminus) was required for both cleaving and joining reactions . Six bases of HIV U5 or U3 DNA at the ends of model substrates were sufficient for nearly maximal levels of selective endonucleolytic cleaving and joining . However, viral sequence elements upstream of the terminal 6 bases could also affect the efficiencies of the cleaving and joining reactions . The penultimate base (C) on the minus strand of HIV U5 was required for optimal joining activity . A synthetic oligonucleotide mimic of the putative in vivo viral "DNA" substrate for HIV IN, a molecule that contained a terminal adenosine 5'-phosphate (rA) on the minus strand, was indistinguishable in the cleaving and joining reactions from the DNA substrate containing deoxyadenosine instead of adenosine 5'-phosphate at the terminal position . Single-stranded DNA served as an in vitro integration target for HIV IN . The DNA sequence specificity of the joining reaction catalyzed in the reverse direction was also investigated. Biotechnology (N Y), 1992 Jun, 10(6), 682 - 5 Renaturation of a single-chain immunotoxin facilitated by chaperones and protein disulfide isomerase; Buchner J et al.; B3(Fv)-PE38KDEL, a recombinant immunotoxin, forms inclusion bodies when produced in Escherichia coli . In renaturation experiments, nonspecific aggregation of non-native polypeptide chains, and the formation of incorrect disulfide linkages lead to inactive molecules . To prevent these side reactions, we added molecular chaperones and protein disulfide isomerase (PDI) to the refolding buffer . Both DnaK and GroEL/S influenced the reactivation process . GroEL alone inhibited reactivation, but in the presence of ATP, GroEL and GroES significantly increased the yield of active protein . DnaK also increased the yield of properly folded protein and the stimulating effect of DnaK was also observed using immobilized DnaK, which can be used repeatedly without significant loss of activity . PDI, which catalyzes disulfide bridging of proteins, also stimulated reactivation of the immunotoxin . Under optimum conditions, reactivation yields in the presence of PDI were about twice that obtained with nonenzymatic disulfide bond formation . Furthermore, DnaK and PDI were additive when renaturation was performed in the presence of both proteins. Appl Microbiol Biotechnol, 1992 Jun, 37(3), 352 - 7 Secretion of a functional Fab fragment in Escherichia coli and the influence of culture conditions; Shibui T et al.; Genes encoding a light chain and an Fd region (a variable region and a CH1 domain of a heavy chain) of a mouse-human chimeric antibody with specificity for human carcinoembryonic antigen (CEA) were fused to a DNA segment coding for the signal peptide of Escherichia coli ompF . E . coli cells harbouring an expression vector containing these genes downstream of a tac promoter were able to secrete a Fab fragment of the antibody efficiently . When the cells were cultured at 37 degrees C and the inducer (isopropyl-beta-D-thiogalactopyranoside, IPTG) concentration was 1 mM (standard conditions), production of functional Fab was very low (medium; 200 ng/l culture and periplasm; less than 90 ng/l culture) . In order to optimize functional Fab production, we examined the influence of culture conditions (i.e . temperature and the inducer concentration) on secretion of the product . It was found that a 12.7-fold higher amount of Fab fragment could be produced at 30 degrees C using 0.1 mM IPTG, as compared with standard conditions . Under these optimal conditions, functional Fab accumulated in the periplasm and culture medium for 10 h after induction and the total production level was found to reach approximately 4.5 mg/l culture. Appl Microbiol Biotechnol, 1992 Jun, 37(3), 335 - 41 Proteolytic response to the expression of an abnormal beta-galactosidase in Escherichia coli; Kosinski MJ et al.; Because induction of proteolytic activity and stress-response proteins can significantly affect expression levels in recombinant Escherichia coli, the influence of low-level expression of a mutant beta-galactosidase was investigated . A single copy of the well-characterized CSH11 mutant of the lacZ gene was integrated into the chromosome . Induction of expression of the mutant beta-galactosidase caused a measurable increase in ATP-dependent intracellular proteolytic activity but resulted in no significant change in ATP-independent proteolytic activity . Growth at temperatures above 40 degrees C resulted in a significant decrease in the level of ATP-independent proteolytic activity compared to growth at 37 degrees C, and the ATP-dependent activity increased 2.5-fold from 30 to 42 degrees C . Synthesis of stress-response proteins was evident in two-dimensional gel electrophoresis analysis of proteins in the strain expressing the abnormal beta-galactosidase at 37 degrees C, but no such response was evident when mutant beta-galactosidase expression was induced at 30 degrees C . In separate experiments, stress proteins were overexpressed by inducing expression of the htpR gene on a plasmid . Resulting increases in stress-protein levels correlated with an increase in ATP-dependent proteolytic activity with no significant change in the intracellular ATP-independent proteolytic activity . These data suggest that even very low levels of abnormal protein can substantially influence protease levels and stress response in E . coli . These responses were reduced by induction at lower temperatures. Lett Appl Microbiol, 1992 Jun, 14(6), 250 - 4 An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range; Azad AK et al.; An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4.3 to 73 kb . This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods . The principal modification is the replacement of NaCl by MgCl2 in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA . Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments. Biosci Biotechnol Biochem, 1992 Jun, 56(6), 906 - 12 Cloning and sequencing of the xynA gene encoding xylanase A of Aspergillus kawachii; Ito K et al.; We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii . The cDNA was isolated from an A . kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein . Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues . The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid . The transformed yeast with a cloned cDNA produced xylanase . The genomic DNA was arranged as ten exons and nine introns. Br J Pharmacol, 1992 Jun, 106(2), 287 - 94 The role of CD18 in IL-8 induced dermal and synovial inflammation; Forrest MJ et al.; 1 . The intradermal administration of endothelial IL-8 (IL-8(1-77) or monocyte derived IL-8 (IL-8(1-72) to rabbits produced a concentration-dependent increase in plasma extravasation and an accumulation of polymorphonuclear leukocytes (PMNs) when measured over a 3 h time period . When plasma extravasation and PMN accumulation were measured over a 30 min time period no significant increases in PMN accumulation or plasma extravasation were observed in response to IL-8 alone . However, under these conditions, the addition of prostaglandin E2 (100 pmol) produced a significant potentiation of IL-8-induced plasma extravasation . There was no significant difference between the biological activities of IL-8(1-77) and IL-8(1-72) . 2 . Plasma extravasation and PMN accumulation induced by IL-8 were inhibited in rabbits pretreated with the monoclonal antibody designated IB4 (1 mg kg-1, i.v.) directed against the common beta chain (CD18) of the leukocyte integrins . 3 . The intra-articular administration to rabbits of IL-8(1-77) (1 nmol) resulted 24 h later in the appearance of a mixed population of leukocytes (PMNs and mononuclear cells) in synovial lavage fluid . Biochemical analyses revealed the presence of an increased level of sulphated proteoglycans (sPG) and of the metalloproteinase stromelysin . Pretreatment of rabbits with IB4 (3 mg kg-1, i.v.) inhibited the accumulation of PMNs but had no effect on the mononuclear infiltrate nor on the levels of sPG or stromelysin . 4 . The intradermal or intra-articular injection of E . coli-derived endotoxin induced similar inflammatory changes to those observed with IL-8.(ABSTRACT TRUNCATED AT 250 WORDS) Acta Trop, 1992 Jun, 51(2), 135 - 42 The antibody response to well-defined malaria antigens after acute malaria in individuals living under continuous malaria transmission; Petersen E et al.; The IgG and IgM antibody responses to the C-terminal 783 amino acids of the P . falciparum glutamate-rich protein, GLURP489-1271, expressed as an E . coli fusion protein, the IgG response to a 18-mer synthetic peptide EDKNEKGQHEIVEVEEIL (GLURP899-916) representing the C-terminal repeats of GLURP, and a synthetic peptide (EENV)6 representing the C-terminal repeats from Pf155/RESA, were investigated longitudinally in 13 children and 7 adults living under conditions of continuous, intense malaria transmission . Some subjects did not recognize the antigens after malaria infection, and in subjects recognizing the antigens, the responses were often short-lived . In adults, the antibody responses to the GLURP489-1271 fusion protein and the (EENV)6 peptide peaked after 2 weeks, and not all individuals responded to all antigens . The antibody response, even against large fragments of conserved antigens, is not uniformly elicited by natural malaria infection in previously primed donors. Electrophoresis, 1992 Jun, 13(6), 346 - 53 Deletion studies to reveal the basis for size discrepancy in proliferating cell nuclear antigen; Liang CP et al.; Proliferating cell nuclear antigen (PCNA), an essential component for DNA replication in eukaryotes, is a highly conserved nonhistone nuclear protein of 261 amino acids . The molecular weight of mammalian PCNA, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), differs notably from that predicted by the cDNA sequences, that is, 36,000 in comparison with 29,261 and 28,748 for human and rat PCNA, respectively . To investigate if this discrepancy is due to posttranslational modifications, we studied the PCNA protein synthesized by an in vitro transcription/translation system as well as the protein overproduced in bacteria . We found that both PCNA protein samples were indistinguishable from the authentic protein from the protein mobility in SDS-PAGE . The finding indicates that the size discrepancy is not due to the posttranslational modifications . Hence, the size discrepancy may be due to the protein sequence per se, namely a sequence-related anomaly in SDS-PAGE . Results from the analyses of a series of PCNA derivatives with various lengths of C- or N-terminal deletion indicate that the putative sequence is in the region of residues 128-150. J Biochem (Tokyo), 1992 Jun, 111(6), 793 - 7 Characterization of the bacterially expressed Drosophila engrailed homeodomain; Yamamoto K et al.; To investigate the mechanism of DNA recognition by the homeodomain, truncated proteins containing the entire homeodomain encoded by the Drosophila engrailed gene were expressed in Escherichia coli . Each protein was accumulated to an amount representing more than 40% of the total bacterial protein and recovered in the soluble fraction . Of the three truncated proteins, the shortest one (71 amino acid residues) was further purified by conventional chromatography . The purified engrailed homeodomain (En-HD) protected a DNA sequence, TTAATT, the core element of consensus sequences recognized by many other homeodomain proteins, from DNase I digestion . UV-CD spectra of the En-HD showed that it mainly consisted of alpha-helix . Based on one-dimensional 1H-NMR spectra, the tertiary structure of the En-HD was shown to be stable against temperature up to 50 degrees C and low pH . The low pH resistance of the protein was also demonstrated by UV-CD measurement . Thus, the current over-production system provides an active and stable homeodomain, which is suitable for structure-function analysis. Mol Pharmacol, 1992 Jun, 41(6), 1073 - 80 Identification of two affinity states of low affinity receptors for Escherichia coli heat-stable enterotoxin: correlation of occupation of lower affinity state with guanylate cyclase activation; Crane MR et al.; Two distinct affinity states of low affinity Escherichia coli heat-stable enterotoxin (ST) receptors in rat intestinal membranes, with dissociation constants of 0.12 and 2.5 nM, were identified . Kinetic binding studies demonstrated biphasic association kinetics, whereas dissociation was unimodal . These studies also confirmed that ligand bound to each receptor state in an independent bimolecular reaction . In contrast, equilibrium binding studies yielded linear Scatchard plots, indicative of a single class of noninteractive binding sites, with a Kd = 2.3 nM . Close agreement of the dissociation constants determined by kinetic and equilibrium methods suggested that receptors were in the lower affinity state at equilibrium . Several models, including binding site heterogeneity, cooperativity, and ligand-induced alterations in receptor conformation were inconsistent with these observations . Indeed, these data were most consistent with a two-step binding process involving a third component . Comparison of the ligand dependence of enzyme activation (EC50 = 124 nM) and the calculated fractional receptor occupancy of the lower affinity component at 5 min (EC50 = 40 nM) demonstrated that occupation of the lower affinity state of low affinity ST receptors correlated with guanylate cyclase activation . The close correlation between receptor occupation and enzyme activation suggests that there are no spare receptors for ST in intestinal membranes . These data resolve the previously observed discrepancy between the affinity of receptors for ST and the potency of this ligand for activating guanylate cyclase . Receptor affinity state alterations may represent a common mechanism for receptor-effector coupling of particulate guanylate cyclases. Int J Radiat Biol, 1992 Jun, 61(6), 759 - 65 Induction of lacI- mutations in Escherichia coli cells after single and split-dose irradiation; Kozubek S et al.; In the lacI system of Escherichia coli, X-ray mutagenesis follows a linear-quadratic curve with suppression; the survival curve is exponential . Dose fractionation leads to nearly complete repair of premutational lesions during an incubation interval of 3.5 h . Repair starts with a delay of 1.5-2 h, suggesting the involvement of an inducible repair/mutation fixation system . The dose-dependence of mutagenesis is described by a simple model assuming two hits being required . A probable explanation might be that the premutagenic lesions consist of two closely spaced lesions on the opposite strands of the DNA molecule. Proc Natl Acad Sci U S A, 1992 Jun 1, 89(11), 5088 - 92 Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription; Weiss V et al.; We studied the effect of phosphorylation of nitrogen regulator I (NRI) on its binding properties . Both phosphorylated and unphosphorylated NRI bind linearly to a single binding site but cooperatively to two adjacent binding sites . Cooperative binding of NRI is severely affected by phosphorylation: half-maximal binding of NRI-phosphate is at 20-fold lower concentrations than that of unphosphorylated NRI . This is more due to a huge increase in the cooperativity constant--which is the strength of interaction between two NRI dimers--than to an increase in the microscopic binding constant which is the binding affinity to a single binding site . In vitro transcription and DNA footprinting experiments showed that occupation of a single binding site by NRI is not enough for efficient activation and that activation only occurs at a higher NRI concentration . We propose an activation mechanism for NRI in which the phosphorylation of NRI induces a conformational change in the N-terminal domains of the NRI-phosphate dimers, which now interact strongly with each other, leading to a tetramerization of NRI upon binding to two adjacent binding sites . We propose that not the phosphorylation of NRI itself but rather the tetramerization of NRI-phosphate on DNA binding induces the conformational change of the central domain to the active conformation. Infect Immun, 1992 Jun, 60(6), 2425 - 31 Characterization of the heat shock response in Brucella abortus and isolation of the genes encoding the GroE heat shock proteins; Lin J et al.; In an effort to define the heat shock response in the bovine intracellular pathogen Brucella abortus, a rough variant lacking extensive lipopolysaccharide was pulse-labeled with {35S}methionine following exposure to elevated temperatures . The major heat shock proteins observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography migrate at 70, 62, 18, and 10 kDa . The maximum response was observed between 42 and 46 degrees C and within 2 to 3 h of the shif in temperature and varied slightly for the different proteins . Accumulation of the 62-kDa heat shock protein (62-kDa Hsp) was observed to continue for up to 5 h following the shift in temperature . In an effort to better define the heat shock response and its potential relationship with protective immunity, genes encoding the major heat shock proteins were isolated from recombinant libraries constructed from B . abortus S19 and S2308 and sequenced . The 62-kDa Hsp shares more than 60% amino acid homology with members of the GroEL family and is immunoprecipitated with polyclonal antibodies to Escherichia coli GroEL and monoclonal antibodies to mycobacterial Hsp 65 . Western blot (immunoblot) analysis with pooled sera from vaccinated and infected cattle revealed that the 62-kDa Hsp is a predominantly recognized antigen . The roles of these gene products during environmental stress and in protective immunity against brucellosis are under investigation. Infect Immun, 1992 Jun, 60(6), 2297 - 304 Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes; Nataro JP et al.; Strains of enteroaggregative Escherichia coli (EAggEC) have been implicated in several studies as important agents of persistent diarrhea among infants in the developing world . We have previously shown that the aggregative adherence (AA) property of EAggEC is associated with the presence of a 60-MDa plasmid which confers AA when introduced into E . coli HB101 . Here, we report the cloning of the AA determinant from EAggEC strain 17-2 into the 21.5-kb cosmid vector pCVD301 . TnphoA mutagenesis of the AA cosmid clone pJPN31 implicated an AA region of approximately 12 kb . Transmission electron microscopy of HB101 (pJPN31) revealed the presence of bundle-forming fimbriae, which were absent in AA- TnphoA insertion mutants . The presence of these fimbriae, AA, and hemagglutination (HA) of human erythrocytes were all concurrently lost by single-insertion mutations . A 14-kDa protein was seen on polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of surface shear preparations from fimbriated clones . Twelve of nineteen volunteers fed EAggEC 17-2 developed rises in antibodies to the 14-kDa protein as determined by Western blot . We have termed the cloned bundle-forming fimbriae aggregative adherence fimbriae I (AAF/I); positivity with a previously described EAggEC probe and human erythrocyte HA appear to correlate with the presence of AAF/I. Microb Releases, 1992 Jun, 1(1), 47 - 50 OmpC and OmpF porins influence viability and culturability of Escherichia coli cells incubated in seawater; Gauthier MJ et al.; The contribution of the major outer membrane porins OmpF and OmpC to the maintenance of viability and culturability of Escherichia coli cells in seawater was analyzed using isogenic mutant strains lacking one or both porins . Cells that possessed OmpF and OmpC survived better than those lacking one or both of them . However, the results differed, depending on whether the cells were adapted to high osmolarity or not before transfer to seawater . When cells were grown at low osmolarity, survival was largely influenced by porins, the OmpF+ strains surviving better than those lacking this porin . Addition of an OmpF plasmid to OmpF- OmpC- cells also improved their viability . When grown at high osmolarity, the role of porins was less critical since both the viability and culturability of the cells increased . However, cells that expressed only OmpC showed the most dramatic loss of viability . Cells lacking both OmpF and OmpC exhibited a higher loss of viability and culturability in seawater . Regarding the influence of porins on survival, these results show that the conditions that prevail during the growth of cells before their transfer to seawater are highly influential: cells that express the porin corresponding to the growth conditions they are in at the time of transfer survive better. J Gen Microbiol, 1992 Jun, 138 ( Pt 6), 1109 - 16 Nucleotide sequence of the rhaR-sodA interval specifying rhaT in Escherichia coli; Garcia-Martin C et al.; The precise location of the rhaT gene, encoding rhamnose permease, has been established between sodA and rhaC at 3605-3607 kb of Kohara's physical map, which corresponds to 88.4 min on the Escherichia coli chromosomal map . The dependence of the activity of the rhaT product on the function of rhaC, the rhamnose operon regulatory gene, was established by measuring rhamnose transport in wild-type and rhaC-deficient strains . The sequence of the sodA-rhaC interval displayed a single ORF corresponding to rhaT, which is transcribed counterclockwise on the E . coli chromosome . The ORF was shown to be preceded by a ribosome binding consensus sequence and a catabolite repression protein consensus sequence . The derived amino acid sequence displayed very low homology with any other permease and was clearly dissimilar to the homologous group formed by the xylose, arabinose, galactose and several glucose transporters . Analysis of the rhaT primary sequence identified potential membrane-spanning regions, possibly defining a protein structure model different from the one corresponding to the above-mentioned homologous group. Chin Med Sci J, 1992 Jun, 7(2), 72 - 4 Construction of an Rb gene expression plasmid; Huang Q et al.; We obtained an 844 bp Bg1II fragment from an Rb cDNA clone and inserted it into the expression vector pWR-13 to construct an Rb gene expression plasmid . When the Rb Bg1II fragment was fused in-frame into pWR-13, it was operated by a Lac Z promoter and produced a fusion protein which consisted of expressed Rb protein and a small peptide from Lac Z . The recombinants were transformed into E . coli with the CaCl2 method, screened by in situ hybridization, and restriction mapped . Total cellular protein of transformed clones was analyzed by SDS-PAGE and Commassie blue staining . The sense clones showed a unique band at 28,000 . On Western blot, this band specifically reacted with 125I-labelled antibody against synthetic Rb peptide . This protein comprised more than 5% of total bacterial protein. Br J Pharmacol, 1992 Jun, 106(2), 489 - 92 Peripheral analgesic activities of peptides related to alpha-melanocyte stimulating hormone and interleukin-1 beta 193-195; Poole S et al.; 1 . The hyperalgesic effects of interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE2) were measured in rats . 2 . Hyperalgesic responses to IL-1 beta were inhibited in a dose-dependent manner by alpha-melanocyte stimulating hormone (alpha-MSH)-related peptides with the following order of potency: {N1(4),D-Phe7}alpha-MSH greater than alpha-MSH greater than Lys-D-Pro-Val greater than Lys-Pro-Val greater than Lys-D-Pro-Thr greater than D-Lys-Pro-Thr . 3 . Hyperalgesic responses to PGE2 were not inhibited by Lys-D-Pro-Thr and D-Lys-Pro-Thr but were inhibited in a dose-dependent manner by the other peptides with the same order of potency as against IL-1 beta . 4 . The potencies of {N1(4), D-Phe7}alpha-MSH and alpha-MSH were greatly diminished by deletion of their C-terminal tripeptide, Lys11-Pro-Val13 . 5 . Nor-binaltorphimine (Nor-BNI) largely reversed the analgesic effects of alpha-MSH, {N1(4), D-Phe7}alpha-MSH, Lys-Pro-Val and Lys-D-Pro-Val indicating that kappa-opioid receptors mediated the analgesic activity of these peptides . 6 . Nor-BNI did not antagonize the inhibition by Lys-D-Pro-Thr and D-Lys-Pro-Thr of IL-1 beta evoked hyperalgesia indicating that these peptides were not acting via kappa-opioid receptors. Protein Expr Purif, 1992 Jun, 3(3), 212 - 22 Expression and purification of kringle 4-type 2 of human apolipoprotein (a) in Escherichia coli; Li Z et al.; The most frequently occurring kringle 4 domain of human apolipoprotein (a), Kringle 4-subtype 2 (K4(2)), was expressed as a fusion protein with the maltose binding protein in Escherichia coli using the "tac" promoter . Although the fusion protein was expressed without a signal sequence, 25% was secreted into the periplasmic space; the remainder was found associated with the soluble cytosolic fraction . The fusion protein was readily isolated from whole cell lysate by amylose agarose affinity chromatography . Although a factor Xa cleavage site was engineered into the fusion protein, it was found that release of the K4(2) protein was most conveniently achieved by proteolysis with subtilisin A . The cleavage product produced in this way was shown to be intact K4(2) with only the first three amino acid residues of the leading flanking peptide missing, as judged by N-terminal sequence analysis . K4(2) was isolated from the hydrolysate by FPLC on a Mono-Q column with a yield of 170 +/- 30 micrograms/g wet cells . The resulting protein was monomeric in phosphate-buffered saline as judged by size-exclusion chromatography and appeared to be folded as shown by spectroscopic and immunological assays . The recombinant K4(2) did not bind to either lysine- or proline-Sepharose, suggesting that the ligand binding activities of lipoprotein (a) may reside in the other kringle domains of apolipoprotein (a). Hum Cell, 1992 Jun, 5(2), 150 - 9 {Gene expression of hepatitis viruses in the liver and hepatocarcinogenesis}; Koike K; Human hepatitis B virus (HBV) carriers run an increased risk of hepatocellular carcinoma (HCC), where the expression of HBV genes play the most important role in the initial stage of hepatocarcinogenesis . As the integration of HBV DNA into the cellular DNA of HCC as well as chronic hepatitis was demonstrated very frequently, the virus-cell fusion gene was considered to be most essential for hepatocarcinogenesis . Among the virus-cell fusion genes, the X gene is known to function as a transactivator for viral and cellular genes at the time of chronic infection . One mechanism for hepatocarcinogenesis that appears particularly reasonable is transactivation of cellular oncogenes by the X-cell fusion protein . In 1990, we found a part of the amino acid sequences in the X protein to be highly homologous to functionally essential sequences in the Kunitz domain, characteristic of Kunitz-type serine protease inhibitors . It has been recently demonstrated that X protein expressed in E . coli or from the in vitro translation system binds to a specific serine protease from the liver cells . These results indicate that transactivation function of X protein may be exerted by acting as a protease inhibitor analogue to control the proteolytic pathway of cellular transcription factor(s) . On the other hand, viral hepatitis resulting from viruses other than hepatitis A virus and HBV has been referred to as non-A, non-B hepatitis . In 1989, the viral genome was molecularly cloned as a positive-strand RNA having about 10 kb in size and named as hepatitis C virus (HCV) . Details of genetic structure and mechanism of expression are currently under investigation at molecular level. FEMS Microbiol Lett, 1992 Jun 1, 72(2), 115 - 20 A pSC101-par sequence-mediated study on the intracellular state of supercoiling of the pBR322 genome in Escherichia coli DNA topoisomerase I deletion mutant; Ishii S et al.; In Escherichia coli DNA topoisomerase I deletion mutant DM800, transcription of the tetracycline-resistance gene (tet) in the pBR322 genome is thought to create and maintain two domains of positive supercoils ahead, and negative supercoils behind, the transcription complex . To assess the actual intracellular state of twin-supercoiled domains, par sequence (365 bp) of plasmid pSC101, which shows a high affinity for DNA gyrase, was inserted into the EcoRI site upstream, or the AvaI site downstream, of the tet gene on the pBR322 genome . Analysis of the oxolinic acid-induced sites of cleavage by gyrase in DM800 revealed that the pBR322 derivatives are highly preferentially cleaved at the par sequence of the EcoRI site as well as the AvaI site and efficiently linearized when compared with pBR322 . Assessment of the state of negative supercoiling of the pBR322 derivatives isolated suggested that the DNA (containing the AvaI site) ahead of the tet transcripts, is not so positively supercoiled and preferential interaction of gyrase with the EcoRI-par sequence does not result in removing negative superhelical turns so effectively as DNA topoisomerase I does on pBR322 DNA in the isogenic wild-type cells. J Biochem (Tokyo), 1992 Jun, 111(6), 707 - 13 Transmembrane signal transduction and osmoregulation in Escherichia coli: functional importance of the transmembrane regions of membrane-located protein kinase, EnvZ; Tokishita S et al.; EnvZ is a membrane-located protein kinase which modulates expression of the ompF and ompC genes through phosphotransfer signal transduction in Escherichia coli . Previously, we developed an in vitro method for analyzing the intact form of EnvZ in isolated cytoplasmic membranes, and demonstrated that this particular form of EnvZ exhibits the ability not only of OmpR phosphorylation but also OmpR dephosphorylation . Taking advantage of this in vitro system, in this study, to assess the structural and functional importance of the membrane-spanning (transmembrane) regions of EnvZ, a set of mutant envZ genes, each of which specifies a mutant EnvZ protein with a single amino acid replacement within or very near the transmembrane regions, were isolated and characterized in terms of their in vivo osmoregulatory phenotypes and in vitro EnvZ-OmpR phosphotransfer activities . On the basis of the results, it was suggested that the transmembrane regions of EnvZ play roles in transmembrane signaling and consequent modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an osmotic stimulus. Mol Microbiol, 1992 Jun, 6(12), 1617 - 24 gyrB mutations which confer coumarin resistance also affect DNA supercoiling and ATP hydrolysis by Escherichia coli DNA gyrase; Contreras A et al.; Coumarins are inhibitors of the ATP hydrolysis and DNA supercoiling reactions catalysed by DNA gyrase . Their target is the B subunit of gyrase (GyrB), encoded by the gyrB gene . The exact mode and site of action of the drugs is unknown . We have identified four mutations conferring coumarin resistance to Escherichia coli: Arg-136 to Cys, His or Ser and Gly-164 to Val . In vitro, the ATPase and supercoiling activities of the mutant GyrB proteins are reduced relative to the wild-type enzyme and show resistance to the coumarin antibiotics . Significant differences in the susceptibility of mutant GyrB proteins to inhibition by either chlorobiocin and novobiocin or coumermycin have been found, suggesting wider contacts between coumermycin and GyrB . We discuss the significance of Arg-136 and Gly-164 in relation to the notion that coumarin drugs act as competitive inhibitors of the ATPase reaction. Biotechniques, 1992 Jun, 12(6), 798 - 800, 802, 804 Enzymatic purification of plasmid DNA; Isfort RJ; A novel method for plasmid DNA purification using enzymes that degrade all major types of contaminating nucleic acids present in crude plasmid DNA mixtures (but not plasmid DNA) has been devised . This method is quick (can be accomplished in two hours), requires no expensive laboratory equipment (an ultracentrifuge is not necessary) and is inexpensive . Plasmid DNA purified by this methodology can be used in a variety of molecular biological techniques including restriction enzyme digestions, subcloning, sequencing, nick translation and end labeling . This plasmid purification technique will be very useful for the molecular biologist performing cloning experiments. Mol Gen Genet, 1992 Jun, 233(3), 443 - 8 Escherichia coli umuDC mutants: DNA sequence alterations and UmuD cleavage; Koch WH et al.; The products of the chromosomally encoded umuDC genes are directly required for mutagenesis in Escherichia coli . Strains with either umuD or umuC mutations are rendered phenotypically non-mutable . To ascertain the molecular basis of this non-mutability, we determined the DNA sequence alterations of seven chromosomal umuDC mutants . Six mutants (umuD1, umuD44, umuD77, umuC36, umuC25, and umuC104) were found to be single base-pair substitutions that resulted in missense mutations . The Tn5 transposon insertion mutation (umuC122) resulted in a missense mutation followed immediately by a termination codon, producing a truncated UmuC protein lacking 102 carboxyl-terminal amino acids . All of the mutations were found to reside in regions of the UmuD and UmuC proteins that share high homology with analogous proteins . Chemiluminescent immunoassays revealed that the umuD1, umuD44, and umuD77 mutations all resulted in a non-cleavable UmuD protein . Because UmuD cleavage is a prerequisite for mutagenesis, the lack of UmuD processing appears to be the molecular basis for the non-mutable phenotype in these strains . These studies re-emphasize the critical nature of the RecA-mediated cleavage of UmuD for inducible mutagenesis and provide insights into the functional domains of the UmuC protein. Int J Biochem, 1992 Jun, 24(6), 945 - 50 Gene cloning of Porphyromonas gingivalis specific antigens recognized by serum of adult periodontitis patient; Hayakawa M et al.; 1 . Porphyromonas gingivalis is believed an important pathogen of adult periodontitis . A gene library of P . gingivalis 381 was constructed in lambda phage vector L47.1 . The library was probed with serum obtained from patients of severe adult periodontitis . Two clones, lambda MDBG101 and lambda MDBG103 which were expressed, 200 and 160 kDa respectively, were selected and further studied . 2 . The expressed antigens in these two clones were also reacted with rabbit antiserum against whole cells, capsular fraction and cell surface fraction of P . gingivalis . 3 . Genes coding protein antigens in lambda MDBG101 and lambda MDBG103 were subcloned into high-copy-number plasmid vector pACYC184 and subclones obtained were designated as MD101 and MD103 . Recombinant plasmids, pMD101 and pMD103, differed in their restriction endonuclease digestion . 4 . Immunodiffusion analysis showed that cloned proteins from MD101 and MD103 reacted with antiserum against P . gingivalis but did not react with antiserum against Prevotella intermedia, Prevotella loescheii and Prevotella asaccharolyticus . 5 . These data suggest that P . gingivalis species-specific antigens has been successfully cloned and expressed in Escherichia coli . Since these cloned specific antigens were recognized by adult periodontitis patient sera, the recombinant antigen will be useful material for the development of serodiagnosis of P . gingivalis infection in adults periodontitis. J Bacteriol, 1992 Jun, 174(12), 4175 - 8 An Escherichia coli DNA topoisomerase I mutant has a compensatory mutation that alters two residues between functional domains of the DNA gyrase A protein; Oram M et al.; Nucleotide sequence analysis revealed that the compensatory gyrA mutation in Escherichia coli DM750 affects DNA supercoiling by interchanging the identities of Ala-569 and Thr-586 in the DNA gyrase A subunit . These residues flank Arg-571, a site for trypsin cleavage that splits gyrase A protein between DNA breakage-reunion and DNA-binding domains . The putative interdomain locations of the DM750 mutation and that of E . coli DM800 (in gyrase B protein) suggests that these compensatory mutations may reduce DNA supercoiling activity by altering allosteric interactions in the gyrase complex. J Bacteriol, 1992 Jun, 174(12), 3915 - 20 Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli; Wu J et al.; soxR and soxS are adjacent genes that govern a superoxide response regulon . Previous studies revealed that induction of the regulon is accompanied by increased transcription of soxS, which can activate the target genes . Therefore, induction may occur in two stages: the soxR-dependent activation of soxS, followed by the soxS-dependent induction of other genes . However, the requirement for soxR was unproven because the only existing soxR mutations either were of the regulon-constitutive type or also involved soxS . Therefore, we produced an insertion mutation that was shown by complementation to inactivate only soxR . In confirmation of the two-stage model, soxR was required for the induction by paraquat of the target genes studied (nfo, zwf, and sodA), for paraquat resistance, and for the 47- to 76-fold induction of soxS-lacZ gene fusions . Paraquat did not affect the expression of soxR-lacZ gene fusions . In a soxRS deletion mutant, the regulon was constitutively activated by a runaway soxS+ plasmid . However, a lower-copy-number plasmid failed to activate nfo, zwf, or sodA but did increase the paraquat resistance of a soxRS mutant . Therefore, there is a differential response of the regulon genes to soxS overproduction . A soxR regulon-constitutive mutation was suppressed by a soxR+ plasmid, suggesting a competition between native and activated forms of SoxR . It is proposed that to enhance the sensitivity of the response, the cell minimizes such potential competition by manufacturing only a small amount of this sensor protein, thereby necessitating signal amplification via induction of soxS. Eur J Biochem, 1992 Jun 1, 206(2), 337 - 43 Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI; Kimura S et al.; The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme . Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed . Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein . The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves . Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme . The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C . Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, {D}1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively . The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine . However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability . In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins. J Bacteriol, 1992 Jun, 174(11), 3637 - 44 osmY, a new hyperosmotically inducible gene, encodes a periplasmic protein in Escherichia coli; Yim HH et al.; A new osmotically inducible gene in Escherichia coli, osmY, was induced 8- to 10-fold by hyperosmotic stress and 2- to 3-fold by growth in complex medium . The osmY gene product is a periplasmic protein which migrates with an apparent molecular mass of 22 kDa on sodium dodecyl sulfate-polyacrylamide gels . A genetic fusion to osmY was mapped to 99.3 min on the E . coli chromosome . The gene was cloned and sequenced, and an open reading frame was identified . The open reading frame encoded a precursor protein with a calculated molecular weight of 21,090 and a mature protein of 18,150 following signal peptide cleavage . Sequencing of the periplasmic OsmY prot