Escherichia coli

Rheumatol Int, 1986, 6(3), 139 - 44
Measurement of serum DNA binding in chronic active hepatitis and systemic lupus erythematosus using the Farr assay; Pollard KM et al.; Antibodies to double-stranded DNA (dsDNA), generally regarded as highly specific for systemic lupus erythematosus (SLE), have also been reported in chronic active hepatitis (CAH) . Using the Farr assay and E . coli DNA, fractionated by benzoylated-naphthoylated-DEAE cellulose chromatography into dsDNA and dsDNA containing single-stranded regions, we compared the serum DNA binding of CAH and SLE patients . Although CAH sera were found to have dsDNA binding significantly above the normal control group such binding was of low level and we could find no evidence of markedly elevated dsDNA binding in CAH . However 12 of the 20 CAH sera studied did bind preferentially to dsDNA containing single-stranded regions suggesting the presence of anti-single-stranded DNA antibodies . We conclude that the description of elevated anti-dsDNA antibodies, as measured by the Farr assay, in CAH is due to the interaction of anti-single-stranded DNA antibodies or other serum components with single-stranded DNA contaminating dsDNA preparations.

Gene, 1986, 43(3), 311 - 7
Synthesis of fused glycoprotein D of herpes simplex virus type 1 but not type 2 inhibits Escherichia coli hosts; Steinberg DA et al.; Glycoprotein D from either Herpes simplex virus type 1 (gD-1) or type 2 (gD-2) has been expressed in Escherichia coli as a series of chimeric proteins . The expression vector used in this study, pJS413, was derived from pBR322 and contains several cloning sites between the lacZ promoter-operator and the phage lambda cro gene . Plasmids containing fusions between the cro gene, gD-related sequences and lacZ was constructed and shown to direct the synthesis of 160-kDa proteins . The accumulation of fusion protein could be visualized as inclusion bodies when the cells were examined by dark phase-contrast or transmission electron microscopy . None of the plasmids that encoded cro::gD gene fusions yielded significant amounts of material upon induction with isopropyl-beta-D-thiogalactopyranoside . In addition, certain plasmids produced a form of Cro-gD-1 fusion protein which resulted in severe growth inhibition of E . coli . These inhibitory effects were attributed to the presence of specific gD-1 sequences, i.e., the transmembrane and cytoplasmic anchor region of the protein.

Gene, 1986, 43(3), 287 - 93
Nucleotide sequence and deduced amino acid sequence of Escherichia coli adenine phosphoribosyltransferase and comparison with other analogous enzymes; Hershey HV et al.; The Escherichia coli apt gene has been analyzed and its nucleotide (nt) sequence and the deduced amino acid (aa) sequence compared to those of other phosphoribosyltransferases (PRTs) . The apt mRNA has a 102-nt leader sequence which may form alternate secondary structures . The RNA transcript may also form several 3' hairpin structures, which, however, do not appear to act as Rho-independent terminators . All PRTs, including E . coli adenine PRT (APRT), have a strongly conserved 13-aa sequence, as well as other regions of aa sequence or structural similarity . E . coli APRT is remarkably similar to the mouse enzyme.

Gene, 1986, 43(3), 183 - 96
Overlapping arrangement of the recF and dnaN operons of Escherichia coli; positive and negative control sequences; Armengod ME et al.; The recF gene of Escherichia coli controls one of the recombination pathways and UV sensitivity, but its precise function and expression pattern are still largely unknown . We have characterized the promoter region of the recF gene by mapping for E . coli RNA polymerase binding sites, in vitro transcription experiments, cloning, and S1 mapping of in vivo mRNAs . It contains three overlapping promoters, two initiating transcription towards recF and one in the opposite direction . The recF promoter region is located about 600 bp upstream from the start codon of the recF structural gene and resides entirely within the translated region of the preceding gene, dnaN, which encodes for the beta subunit of DNA polymerase III . This unusual arrangement might provide discoordinate regulation of the recF and dnaN genes, thus controlling the level of DNA polymerase III holoenzyme . Expression of recF is also negatively controlled by sequences located upstream as well as inside the recF coding frame . Such negative regulation may serve to prevent toxic effects due to accumulation of an excessive number of copies of the recF gene product.

Circ Shock, 1986, 19(4), 409 - 22
The pulmonary microvascular response to infusion of live Escherichia coli in sheep with acutely or chronically prepared lung lymph fistula; Smith L et al.; The effects of infusion of live Escherichia coli bacteria in awake sheep with a chronic lung lymph fistula (n = 15) were compared to anesthetized animals (n = 7) receiving the same septic insult after surgical trauma including bilateral thoracotomies for lung lymph cannulation (acute group) . During preseptic baseline conditions, pulmonary arterial pressure (Ppa) and central venous pressure (Pcv) were increased and leukocytes decreased in the newly operated animals compared to the sheep with a chronic lung lymph fistula . After i.v . infusion of live E . coli 10(9) X kg-1 b.w . over 20 min, arterial pressure (Psa), cardiac output (Qt), leukocytes, and partial pressure of arterial oxygen (PaO2) decreased in both groups . Ppa peaked after 15 min at 37.2 +/- 2.5 in the chronic and 33.4 +/- 3.3 mm Hg in the acute group . In the chronic group, Ppa remained elevated but not in the acute group during the rest of experiment . Lung lymph flow (QL) increased significantly in both groups during the initial high Ppa, but it increased to a higher level in the chronic group . After 150 min, QL did not differ between the groups but remained elevated over baseline . Lymph-to-plasma concentration ratio (L/P) for total protein decreased in the chronic group during the initial high QL . This decrease was not seen in the acute group that had a significantly higher L/P between 30 and 120 min after sepsis . The high QL with unchanged L/P compared to baseline indicated increased permeability in the pulmonary microvessels in both groups but the changes in permeability, hemodynamics, or respiratory parameters after sepsis were not aggravated by the surgical trauma.

Basic Life Sci, 1986, 38, 439 - 52
Use of gradient denaturing gels to determine mutational spectrum in human cells; Cariello NF et al.; Based on the fact that mutagens induce specific patterns of gene mutations, this paper outlines a method to allow discrimination among mutagen-treated populations . The technique should allow direct screening of human tissue for genetic change, using human peripheral blood lymphocytes deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase . The method is based on gradient denaturing gel electrophoresis, which separates short DNA molecules according to their melting properties . The melting behavior of DNA fragments is extremely sequence-dependent, and DNAs with single basepair substitutions often migrate differently . Even DNA fragments with the same basepair substitutions at different locations in the molecule have been resolved . Gradient-denaturing gel electrophoresis has the capacity to separate mutant DNA on the basis of the nature and position of the mutation.

Basic Life Sci, 1986, 38, 311 - 8
Molecular approaches to the study of nucleotide excision repair in eukaryotes; Friedberg EC et al.; Very little is known about the molecular mechanism of nucleotide excision repair in eukaryotes . Studies on human cells have been stimulated by the availability of excision repair-defective cell lines from patients suffering from the autosomal recessive disease xeroderma pigmentosum (XP) . Such studies have contributed significantly to an understanding of the genetic complexity of excision repair in human cells . However, to date, no human excision repair genes or gene products known to complement the repair defect in XP cells have been isolated . The yeast Saccharomyces cerevisiae is an interesting model for exploring the molecular mechanism of nucleotide excision repair in eukaryotic cells . As is true in human cells, multiple yeast genes are involved and at least five genes are required for the specific incision of UV-irradiated DNA in vivo . These five genes have been isolated by molecular cloning and the nucleotide sequences of four of them have been determined . Each of these cloned genes is being used for overexpression of protein.

Am J Pediatr Hematol Oncol, 1986 Summer, 8(2), 99 - 104
Antibody response to Escherichia coli L-asparaginase . Prognostic significance and clinical utility of antibody measurement; Cheung NK et al.; By using a modification of the microtiter solid-phase radioimmunoassay, we have measured Escherichia coli L-asparaginase (L-ASP) specific IgG, IgG4, and IgE antibodies in children who received L-ASP as part of their chemotherapy for leukemia and lymphoma . In 13 children with acute lymphoblastic leukemia induced with vincristine, prednisone, and L-ASP (10,000 IU/M2 i.v . each week for 3 weeks), seven developed high titer specific IgG antibodies . Four of the seven relapsed at the time of their peaking IgG response (6-10 months) . None of the six with low or absent L-ASP antibody response have relapsed (followed for 20-35 months) . In six children with allergic reactions to L-ASP reinduction, all had high titers of L-ASP specific IgG4 (greater than or equal to 20 U/ml) at the time of their reaction . In 16 other children with low L-ASP IgG4 (less than 13 U/ml), none demonstrated allergic reactions to rechallenge . Specific IgE was not consistently detectable in either group . In 21 patients with leukemia or lymphoma on L-ASP with cyclophosphamide-containing regimens, none developed significant IgG antibody response, compared with seven of 13 not receiving cyclophosphamide (p less than 0.001) . We conclude: (a) development of L-ASP antibodies may have prognostic significance; (b) the detection of specific IgG4 can predict L-ASP allergy; and (c) cyclophosphamide-containing regimens reduce antibody formation to L-ASP and may allow repetitive (without anaphylaxis) and more effective (avoiding neutralizing antibodies) use of L-ASP.

Vet Med Nauki, 1986, 23(4), 7 - 11
{Isolation of Escherichia coli O 157 from pigs with diarrhea}; Petkov A et al.; Biochemical and serologic studies were carried out with Escherichia coli strains isolated from pigs affected with diarrhea . A total of 161 were investigated, 13 (8.01 per cent) of which were found to belong to serogroup O 157 . Almost all strains proved beta-hemolytic, enterotoxigenic, and K 88-positive . The strains of this serogroup were found for the first time in this country in swine colibacteriosis.

Med Microbiol Immunol (Berl), 1986, 175(4), 251 - 60
Indirect haemagglutination test for the detection of thermolabile (LT) enterotoxin from Escherichia coli; Ricci LC et al.; An indirect haemagglutination test (IH) for the detection of enterotoxigenic E . coli (LT) was developed . Twenty-five enterotoxigenic E . coli (ETEC) from human and porcine diarrhoea and from river water were examined . The described IH test was more specific and sensitive than the passive immune haemolysis test (PIH).

Med Microbiol Immunol (Berl), 1986, 175(4), 221 - 7
Treatment of LD100 Escherichia coli septic shock with netilmicin and methylprednisolone in baboons; Flournoy DJ et al.; Treatment efficacy with netilmicin sulphate/methylprednisolone sodium succinate in a severe septic shock baboon model, using an LD100 of live Escherichia coli, was evaluated . All the animals treated with both netilmicin and methylprednisolone were permanent (greater than or equal to 7 days) survivors, whereas none of the untreated baboons lived more than 24 hours . These results indicate that, in a baboon model, netilmicin is an effective alternative to gentamicin (with methylprednisolone) in the treatment of severe septic shock.

Int J Immunopharmacol, 1986, 8(3), 357 - 68
Recombinant human tumor necrosis factor--II . Antitumor effect on murine and human tumors transplanted in mice; Sohmura Y et al.; Recombinant human tumor necrosis factor (rHu-TNF) was found to exhibit potent antitumor activities not only against murine tumors, i.e . Meth A sarcoma, B 16 melanoma, colon 26 adenocarcinoma, Lewis lung carcinoma and MH134 hepatoma, transplanted in syngeneic mice but also against human tumors, i.e . HMV-2 melanoma, PC-10 lung carcinoma and GOTO neuroblastoma, heterotransplanted in nude mice . rHu-TNF caused necrosis of all tumors tested and inhibited their growth in a dose dependent manner . Complete regression of tumors was observed in mice bearing Meth A, B16, colon 26, MH134, HMV-2 and PC-10 but not in mice bearing Lewis lung carcinoma and GOTO neuroblastoma . The prolongation of survival time was also observed in syngeneic mice transplanted with murine tumors except Lewis lung carcinoma . The antitumor effect of rHu-TNF was more evident when it was given intratumorally than when given intravenously . The feasibility of rHu-TNF as a drug for cancer therapy is discussed.

Int J Immunopharmacol, 1986, 8(3), 347 - 55
Recombinant human tumor necrosis factor--I . Cytotoxic activity in vitro; Nakano K et al.; Cytotoxic activity of recombinant human TNF (rHu-TNF) on various human cell lines was examined in vitro . rHu-TNF exerted a cytostatic effect on various types of human tumor cells such as carcinoma, sarcoma, leukemia, melanoma and other types . When the cytocidal effect was examined on the tumor cells which were cytostatically susceptible to rHu-TNF, the cytocidal effect of rHu-TNF was also noticed on many of these tumor cells . However, some tumor cells were affected cytostatically only . Human diploid cells were not affected cytostatically or cytocidally by rHu-TNF . WI-38 VA13 cells which are an SV-40-transformed derivative of WI-38 diploid cells, were affected both cytostatically and cytocidally by rHu-TNF . These results suggest that rHu-TNF exerts cytostatic and cytocidal effects against a broad spectrum of human tumor cells, and its cytotoxic activity is tumor-specific.

Int J Immunopharmacol, 1986, 8(3), 313 - 21
A comparative evaluation of particulate and soluble glucan in an endotoxin model; Bowers GJ et al.; Particulate glucan (P) but not soluble glucan (F) has been shown to sensitize rats to endotoxins . This phenomenon is believed to be mediated by the reticuloendothelial system (RES) . The effect of glucan-P and -F on the RES, and the response of glucan-treated rats to nonlethal doses of endotoxin were investigated . Rats were injected for 5 days with 10 mg/kg of glucan-P, -F or saline . Three days later rats were either (1) injected with colloidal carbon for clearance studies, (2) sacrificed for organ histology and determination of serum glucose, plasma thromboxane (Tx) B2, and plasma 6-keto-prostaglandin (PG) F1 alpha concentrations, or (3) challenged with a nonlethal dose of endotoxin . The latter were further subdivided into groups for either 30-day survival or for sacrifice at 30 min or 4 h post-endotoxin infusion to obtain blood samples for glucose, TxB2, and 6-keto-PGE1 alpha determinations . Glucan-P induced hepatosplenomegaly and granulomatous changes within the liver and spleen . The carbon clearance halftime was markedly decreased in these animals . In glucan-P-treated rats challenged with endotoxin, elevated concentrations of both plasma prostanoids were observed as well as alterations in serum glucose levels . These changes were less pronounced in glucan-F- or saline- treated rats . Following endotoxin challenge, only 40% of glucan-P-treated rats survived 30 days whereas 100% of both the glucan-F and saline-treated rats survived . We conclude that glucan-P, in contrast to glucan-F, significantly heightens RES function and that this effect likely accounts for the endotoxin sensitivity.

Environ Mutagen, 1986, 8(4), 571 - 7
Induction of SOS genes of Escherichia coli by chromium compounds; Llagostera M et al.; The induction of several SOS genes of Escherichia coli such as recA, umuC, and sfiA by hexavalent (K2Cr2O7, K2CrO4, and CrO3) and trivalent (CrCl3, Cr(NO3)3, and (CH3COO)3Cr) compounds of chromium was studied . Induction was measured as beta-galactosidase activity, using lacZ gene fusions under the control region of different SOS genes . The hexavalent chromium forms induced the genes responsible for massive synthesis of RecA protein, error-prone repair, and inhibition of cell division . On the other hand, the trivalent chromium compounds were unable to induce any of the SOS genes tested . Individual assay of hexavalent chromium compounds showed that K2Cr2O7 was a stronger inducing agent of those three SOS genes tested than K2CrO4, which, in turn, was stronger than CrO3 . All this data led to the conclusion that hexavalent chromium compounds, but not trivalent, are proficient agents of induction of the SOS system and can produce indirect mutagenesis in Escherichia coli.

CRC Crit Rev Biochem, 1986, 21(1), 27 - 52
Molecular biology of terminal transferase; Chang LM et al.; Terminal transferase is an unusual deoxynucleotide polymerizing enzyme found only in prelymphocytes . The protein was purified to homogeneity from calf thymus glands in 1971 as a 32 kDa protein with a two peptide structure . Subsequent biochemical and immunological analyses of terminal transferase protein in crude extracts from a number of animal species showed a single peptide with a molecular weight of about 58,000 . The two peptide structure found earlier was caused by proteolysis . Homogeneous 58 kDa terminal transferase has now been produced from human lymphoblastoid cells and calf thymus glands by immunoaffinity chromatography . In vitro phosphorylation studies showed that the terminal transferase protein contains one phosphorylation site near one end of the polypeptide chain, and the phosphorylation of the enzyme has been confirmed by in vivo labeling experiments . Unambiguous demonstration of the molecular weight of the human terminal transferase was obtained by translation of the cloned human terminal transferase DNA sequence to a 58,308 Da protein . The translated amino acid sequence also provided a possible phosphorylation site near the amino-terminus of the protein . Preliminary analysis of the genomic structure shows a simple intron/exon pattern with the total human terminal transferase gene spanning at least 65 Kb.

Ann N Y Acad Sci, 1986, 469, 97 - 103
Model for the dynamics of colicin plasmids in continuous culture; Lauffenburger DA; Bacteriocin plasmids may be useful in preventing plasmid instability because the bacteriocin they produce selectively kills otherwise identical bacterial cells lacking the plasmid . This capability may allow plasmid-bearing cells to persist despite the problems of growth-rate depression and segregational plasmid loss that often lead to displacement by plasmid-free cells . In order to determine the conditions under which bacteriocin plasmids can prevent instability, we have developed and analyzed a mathematical model for the growth of colicin plasmid-bearing E . coli in continuous culture . Model behavior compares successfully with experimental results of Adams et al . Dependence of the system dynamic behavior on key parameters has been elucidated and a simple parameter criterion for prevention of plasmid instability has been derived . The possibility of instability prevention was found to be extremely sensitive to dilution rate.

Vet Med Nauki, 1986, 23(1), 3 - 9
{Therapeutic effect and residues of a Gentavet preparation in chicks, piglets and calves}; Vangelov S et al.; Studied were the therapeutic and prophylactic effects of the preparation Gentavet (containing gentamycin complex II) with pigs, birds and calves affected with colibacteriosis . It was found that in conc . 80,000 IU (0.08 g or 0.8 g) with the drinking water (1 lit.) given 14 days to birds lowered the mortality rate to a higher extent than Avimycin (containing oxitetracycline and neomycin) . At the rate of 10,000 IU (0.01 g or 0.1 g) per kg body mass, orally, split in two for 24 h with newborn pigs Gentavet produced a very good prophylactic effect, and when used at a higher rate--16,000 IU (0.016 g or 0.16 g) per kg under the same conditions up to 90 per cent of the pigs with polyenteritis recovered . At 10,000 IU (0.01 g or 0.1 g) per kg body mass, orally, split in two for 24 h with newborn calves with colibacteriosis produced therapeutic and prophylactic effects . Gentamycin complex II residual amounts remained longest in the parenchymal tissue of the kidneys and liver . The withdrawal times in slaughtering birds, pigs, and calves treated with Gentavet should last from 30 to 46 days (31 days for birds, 46 days for pigs and calves of up to 100 kg, and 30 days for heifers of up to 400-500 kg) . This period could be shortened 4 to 5 days if both kidneys and liver are to be eliminated after slaughter.

Gene, 1986, 42(1), 49 - 57
High-level expression vectors to synthesize unfused proteins in Escherichia coli; Seth A et al.; A new class of plasmid vectors (pANK-12, pANH-1, and pPL2) for synthesizing unfused proteins was constructed by inserting synthetic linkers at the NdeI site (CATATG) of plasmid pJL6, which contains the lambda cII gene initiator codon . These expression vectors contain the lambda pL promoter, the cII ribosome-binding site, cII start codon and unique restriction sites (KpnI, Asp718, HpaI, BamHI) downstream from the initiator ATG for expression of unfused proteins . The main advantage of these vectors is that any DNA fragment with an open reading frame that does not possess a start and/or a stop codon can be directed to overproduce protein in an unfused form.

Circ Shock, 1986, 19(1), 55 - 67
Metabolic effects of sodium dichloroacetate in endotoxemic minipigs; Weingand KW et al.; Lactic acidosis is significant in the pathophysiology of endotoxicosis . By increasing the activity of pyruvate dehydrogenase, sodium dichloroacetate (DCA) decreases lactate production and may be useful as a therapy for endotoxic shock . Five (n = 5) 50-80-kg Yucatan minipigs were fitted with jugular, portal, hepatic vein, and carotid artery catheters, and hepatic artery and portal vein flow cuffs to quantitate transhepatic kinetics of glucose, lactate, and insulin . Three days later, they were placed in slings, and a primed-continuous intravenous infusion of 3-tritiated glucose was initiated to monitor whole body glucose kinetics . Following a 3-hour control period, an intravenous infusion of E . coli endotoxin (LPS) was administered at 15 micrograms/kg/hr for 6 hours . After 1 hour of LPS infusion, DCA was administered as a primed (30 mg/kg)-continuous (30 mg/kg/hr) intravenous infusion for 5 hours . These DCA-treated endotoxemic minipigs (group DE) were compared statistically to a group (n = 8) of minipigs given endotoxin only (group E) . Group DE arterial lactate concentrations eventually decreased (19.2 +/- 1.0 mg/dl) significantly (P less than or equal to 0.05) below group E (39.3 +/- 7.7 mg/dl) in the last hour of DCA infusion . The progressive hypoglycemia of group DE decreased below that of group E and became significantly (P less than or equal to 0.05) lower in the last hour of the experiment (33.6 +/- 11.5 vs 50.6 +/- 10.0 mg/dl, respectively) due to decreased hepatic gluconeogenesis as evidenced by a lower relative rate of appearance (%Ra) of glucose and decreased hepatic glucose output and lactate extraction . A large relative decrease in pancreatic output of insulin in group DE contributed to the lower serum insulin levels than group E throughout the treatment period . Lethality in group DE (60%) was unaltered from that of group E (67%) . Despite its ability to decrease arterial lactate concentrations, DCA alone does not appear to be an effective treatment for endotoxicosis.

Circ Shock, 1986, 19(1), 39 - 45
Potentiated cardiodepressant effect of serum by endotoxin in adrenalectomized rats; Carli A et al.; We have previously shown that in intact rats (IT) a sublethal and nonhypotensive dose (2 mg/kg IV) of Escherichia coli endotoxin was able to induce the early and sustained release of a lipid-soluble cardiodepressant factor, decreasing contractility of cultured rat heart cells by about 35% . As a humoral mediation may also be envisaged in cardiac dysfunction observed by other investigators in adrenal insufficiency, this study was designed to assess serum cardiodepressant effects of endotoxin in 6-10-day, saline-maintained, adrenalectomized rats (ADX) . In ADX 2 mg/kg endotoxin caused severe hypotension and 100% lethality within the first 90 min . To obtain in ADX a depressant effect of serum on cultured heart cells fairly similar to that observed in IT, it was necessary to reduce the endotoxin dose by about 200 times (0.01 mg/kg) . The latter proved sublethal and nonhypotensive in ADX and was without depressant effect of serum in IT . Serum from ADX (no endotoxin) induced a slight but significant decrease by about 9% in cultured heart cell contractility when compared to serum from IT or sham-operated controls . These data show that adrenalectomy confers a cardiodepressant effect on rat serum and potentiates the release of endotoxin-induced cardiodepressant substance(s) without relation to systemic hypotension.

Ultramicroscopy, 1986, 19(1), 43 - 56
Contrast and resolution of scanning transmission electron microscope imaging modes; Reichelt R et al.; Image blurring due to delocalization of inelastic events was studied for scanning transmission electron microscopy (STEM) of unstained thin sections . The delocalization probability was obtained from the angular distribution of inelastic scattering, which was calculated from experimental electron loss spectra of organic samples . This probability was implemented in a Monte Carlo program to simulate the effects of multiple scattering and delocalization for STEM images collected by either the annular detector or the spectrometer, and images generated by a combination of these two signals . Depending on the illumination, the detector geometry and the energy-loss range selected for imaging the annular detector image is blurred by a non-negligible fraction of inelastically scattered electrons . Simultaneous acquisition of an inelastic image using a spectrometer allows the blurring to be reduced by calculation of either the ratio or the difference of the two darkfield signals . While inherent nonlinearities reduce the interpretability of ratio-contrast images, difference-contrast improves the visibility of details submerged in a diffuse background without introducing artifacts.

Proc Natl Sci Counc Repub China B, 1986 Jan, 10(1), 35 - 42
Lung vascular injury after Escherichia coli endotoxin and phorbol myristate acetate infusion in dogs; Hsu K et al.; The effects of Escherichia coli endotoxin and phorbol myristate acetate (PMA), a potential stimulator of polymorphonuclear leukocyte (PMN), on circulating PMN counts, gas exchange, protein concentration of lavage fluid, pulmonary hemodynamics and pathology of the lung were studied in ten anesthetized dogs . Six dogs were infused with 1 microgram/kg endotoxin plus 10 micrograms/kg of PMA; four other dogs were infused with the same amount of endotoxin but 5 micrograms/kg of PMA . After administration of endotoxin plus 10 micrograms/kg PMA, the number of circulating PMN (per mm3) decreased dramatically from 4081 +/- 1041 to 303 +/- 119, arterial oxygen partial pressure (PaO2) dropped to 49.1 +/- 2.4 mmHg and the arterial alveolar oxygen partial pressure difference (A-a DO2) increased significantly above baseline . Lungs from this group appeared to be grossly damaged: edema with distinct petechial hemorrhage and areas of hemorrhagic consolidation; frothy edema fluid often emanated from the tracheas . The group infused with endotoxin plus 5 micrograms/kg PMA showed no significant decrease in the number of PMN; PaO2 and A-a DO2 maintained comparatively stable . Protein concentration of lavage fluid and lung wet/dry weight ratios in dogs of 10 micrograms/kg PMA group were significantly increased (P less than 0.05) as compared to those of 5 micrograms/kg PMA group . Our study showed that the magnitude of leukopenia after endotoxin and PMA was paralleled with the severity of lung vascular injury . These results support the potential role of PMN in the pathogenesis of acute edematous lung injury.

Physiol Behav, 1986, 36(4), 745 - 9
Effect of centrally administered interleukin-1 and endotoxin on food intake of fasted rats; McCarthy DO et al.; We have previously shown that interleukin-1 (IL-1), a polypeptide known to mediate many aspects of the acute phase response to infection, suppresses food intake when injected intraperitoneally into fasted rats . IL-1 acts at the level of the hypothalamus to induce fever . In view of the large number of peptides that have been shown to alter food intake as well as body temperature when injected intracerebroventricularly (ICV), we hypothesized that the receptor site for the anorexigenic activity of IL-1 would be located in a central nervous site bathed by the cerebrospinal fluid . In the present study, ICV injection of IL-1 or E . coli endotoxin (a stimulus for the synthesis of IL-1), significantly elevated body temperature, but did not affect food intake of fasted rats . We conclude that receptors mediating the anorexigenic actions of IL-1 or endotoxin are not located at a central nervous site bathed by the cerebrospinal fluid . Furthermore, fever per se is not responsible for the reduction in food intake seen following peripheral injection of IL-1 or endotoxin.

J Gen Microbiol, 1986 Jan, 132 ( Pt 1), 171 - 81
Effects of pho regulatory mutations and phoA gene amplification on alkaline phosphatase synthesis and release by lky mutants of Escherichia coli K12; Atlan D et al.; lky mutants of Escherichia coli K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml-1 . APase is a phosphate repressible periplasmic enzyme encoded by the gene phoA . With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic lky strains containing either a constitutive pho regulatory mutation, or a hybrid plasmid carrying the structural gene phoA+ and pho regulatory genes, or a transducing phi 80 phoA+ phage . In the presence of the phoS2333 mutation, F- lky strains lysogenized with phi 80 phoBin phoA+ phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml-1) than haploid strain 2336 (phoS+ lky) grown in low phosphate medium . Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release . Sufficient APase was released into the medium to facilitate its purification.

Int J Nucl Med Biol, 1986, 12(6), 477 - 81
{Detection of endotoxins in radiopharmaceutical preparations--III . Limulus test assessment using radiopharmaceutical preparations; correlation with the rabbit pyrogen test}; Cohen Y et al.; Experiments using 17 radiopharmaceuticals containing known amounts of added endotoxin show that none of them inhibits the pyrogenic reaction of the rabbit . Gelation of the Limulus amoebocyte lysate (LAL) is inhibited by 4 of them: colloidal erbium 169Er citrate, colloidal rhenium 186Re sulfide, colloidal technetium 99mTc (Re) sulfide for liver scintigraphy and the colloidal technetium 99mTc (Re) sulfide for lymphography . This inhibition is cancelled, either by dilution or after neutral pH adjustment . Both controls were performed on 313 batches of various radiopharmaceuticals, 95% of results were identical (93% negative, 2% positive) . The remaining 5% correspond to positive LAL tests vs negative rabbit tests on the same batches . No negative LAL test vs positive rabbit test was observed.

Int J Nucl Med Biol, 1986, 12(6), 471 - 6
{Detection of endotoxins in radiopharmaceutical preparations--II . Comparison of the sensitivity of methods using the rabbit and the Limulus amoebocyte lysate for the detection of endotoxins}; Bruneau J et al.; The rise of the rabbit internal temperature after i.v . injection of an endotoxin solution is proportional to concentration . Gelation of Limulus amoebocyte, when in presence of an endotoxin solution, is also related to concentration . We compared the sensitivity of these two methods . With our experimental procedure, the rabbit is sensitive to a 0.40 ng/mL solution and the Limulus amoebocyte lysate to a 0.14 ng/mL solution . The rabbit sensitivity increase is related to the per kilogramme injected volume, whereas sensitivity is not related to the volume to check in the case of the lysate.

Int J Nucl Med Biol, 1986, 12(6), 467 - 70
{Detection of endotoxins in radiopharmaceutical preparations--I . Comparison of rabbit hyperthermia after intravenous or intrathecal administration of reference endotoxin preparations}; Merlin L et al.; The rise of the rabbit internal temperature after endotoxin injection is related to the route of administration . A rise of 1.71 +/- 0.411 degrees C is obtained after i.v . injection of 1 ng/kg Escherichia coli 0111.B.4 endotoxin . An increase of 1.93 +/- 0.236 degrees C is obtained after suboccipital intrathecal injection of 0.1 ng/kg of the same endotoxin; with the intrathecal route, the hyperthermia is induced by E . coli endotoxin after a dose ten times lower than with i.v . injection as shown by statistical analysis.

Folia Microbiol (Praha), 1986, 31(2), 81 - 5
The control of adenylate cyclase activity in Escherichia coli; Janecek J et al.; Cell-free extract of E . coli possessed an inhibited adenylate cyclase activity after a previous anaerobic incubation of cells with glucose which is transported and metabolized . The degree of the inhibition depends on incubation conditions . Glucose analogues that are only transported but not metabolized, are not inhibitory . To restore the adenylate cyclase activity, the cells have to be cultivated aerobically prior to disintegration for a defined period of time without glucose.

Gene, 1986, 41(2-3), 225 - 31
Oligonucleotide directed mutagenesis of cauliflower mosaic virus DNA using a repair-resistant nucleoside analogue: identification of an agnogene initiation codon; Dixon L et al.; Mutation of the initiation codon of the dispensible open reading frame, ORF VII, of cauliflower mosaic virus (CaMV) delayed the appearance of disease symptoms, but the mutants reverted with high frequency . This suggests a role of this start codon in viral expression . Oligonucleotide-directed mutagenesis, utilizing a novel, repair-resistant deoxyguanosine analogue, 2'-deoxy-7-deazainosine (dDI), highly improved the yield of mutants.

Int J Biochem, 1986, 18(5), 431 - 5
Uridine phosphorylase from Escherichia coli B . Enzymatic and molecular properties; Vita A et al.; Uridine phosphorylase (EC 2.4.2.3) from Escherichia coli B is an oligomeric protein composed of four identical subunits of 29,000 mol . wt . The enzyme has four half-cystine residues per subunit titrable only in denaturing condition . No disulphide linkages either inter- or intra-chain are present . The isoelectric point is 5.25 . The enzyme shows strict specificity toward uridine and 5-methyluridine and is inhibited by thymine, deoxycytidine and heavy metal ions.

Eur Surg Res, 1986, 18(2), 112 - 21
Application of a quantitative spectrophotometric endotoxin assay on lymph and plasma from the rat; Olofsson P; A quantitative spectrophotometric assay for endotoxin, utilizing Limulus amebocyte lysate and a chromogenic peptide substrate, was standardized for the application on lymph and plasma from the rat . The influence of inhibitors and unspecific amidolytic activity, the optimal incubation times, the adhesion of endotoxin to platelets and glass surface, the variation in endotoxin standard as well as the effects of cold storage on endotoxin activity were settled . In vitro added as well as endogenously derived endotoxin was studied . The method was found to be reliable when in practical use on these media.

Res Vet Sci, 1986 Jan, 40(1), 128 - 35
Emigration of polymorphonuclear leucocytes into the intestinal lumen of the neonatal piglet in response to challenge with K88-positive Escherichia coli; Sellwood R et al.; The emigration of neutrophils from the blood of neonatal piglets into the intestinal lumen in response to a K88-positive strain of Escherichia coli was investigated . The pig herd used was of known genetic susceptibility to K88-positive E coli and had recently experienced an outbreak of neonatal diarrhoea . Neutrophil emigration depended on certain factors . Neutrophils emigrated into ligated loops in susceptible piglets sucking immune colostrum from susceptible dams but not into loops in colostrum deprived resistant piglets or piglets sucking non-immune colostrum from resistant dams . In susceptible, colostrum deprived piglets neutrophils in intestinal contents were only associated with severe lesions . Large numbers of neutrophils which appeared at several foci on the villi were observed in three of six resistant piglets that sucked immune colostrum from susceptible dams . It was concluded that neutrophil emigration into the intestinal lumen of piglets could occur in response to K88-positive E coli and resulted not from the presence or absence of the intestinal K88-receptor but from the ingestion of immune colostrum.

J Clin Microbiol, 1986 Jan, 23(1), 92 - 9
Immunologic response of patients with legionellosis against major protein-containing antigens of Legionella pneumophila serogroup 1 as shown by immunoblot analysis; Sampson JS et al.; Major protein-containing antigens of Legionella pneumophila serogroup 1 were were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with rabbit antisera to 14 different Legionella species or serogroups . Fourteen bands were observed in immunoelectropherograms of whole-cell, sonicated cell, and heated cell preparations, seven of which appeared in the supernatant fluid from the heated cells and three of which were shown in an outer membrane fraction . Immunoblots of whole-cell antigen preparations of 14 Legionella species or serogroups revealed seven major Legionella proteins: antigens with molecular weights of 58,000, 79,000, and 154,000 were present in all Legionella sp . strains, antigens with molecular weights of 44,000 and 97,000 occurred in multiple species, and antigens with molecular weights of 14,000 and 25,000 were present only in L . pneumophila strains . All sera from 15 patients with culture-confirmed L . pneumophila serogroup 1 disease and 14 of 18 (78%) sera from serologically diagnosed patients reacted with the 58-kilodalton (kDa) common antigen . In contrast, less than one-half of the sera reacted with the L . pneumophila-specific proteins (14 and 25 kDa) . Absorption of sera with Escherichia coli cells had no effect on their reactivity with the 58-kDa antigen, whereas absorption with L . pneumophila serogroup 1 cells removed reactivity . These data suggest that the 58-kDa antigen may prove useful in serodiagnostic tests for legionellosis.

Gene, 1986, 41(1), 67 - 74
Mutations that reduce expression from the P2 promoter of the Escherichia coli galactose operon; Bingham AH et al.; We describe the isolation and characterisation of twelve different mutations that reduce gene expression from the galP2 promoter, starting with a gal regulatory region with a mutation that inactivated galP1, the cAMP-CRP-dependent promoter . Seven of the new mutations reduce the initiation of transcription at P2 whereas the others reduce translation initiation of the first gal operon gene, galE . Two of the mutations affecting translation fall in the galE initiation codon and the Shine-Dalgarno sequence . Mutations that allow the formation of a stem-loop structure in the messenger including this sequence also reduce translation . A deletion of 11 bp, upstream of the Shine-Dalgarno sequence, almost totally prevents translation . Although none of the point mutations that reduced transcription initiation at P2 fall in the -35 region, we repeatedly isolated insertions in this zone . The point mutations all fell around the -10 region: the strongest effects were found with mutations that altered the sequence away from the consensus that has been established for Escherichia coli promoters . The effects of the two strongest P2 mutations were investigated in the absence of the P1 mutation used for their isolation . One mutation, a T:A to C:G transition at -12, inactivates both P2 and P1 . In contrast the other, a T:A to G:C transversion at -19, specifically inactivates P2, but leaves P1 partially active even in the absence of cAMP-CRP . The implications of this are discussed in the context of how cAMP-CRP controls the balance between transcription from P2 and P1 at the gal operon regulatory region.

Environ Mutagen, 1986, 8(2), 263 - 71
The genetic toxicology of metal compounds: II . Enhancement of ultraviolet light-induced mutagenesis in Escherichia coli WP2; Rossman TG et al.; Salts of metals which are carcinogenic, noncarcinogenic, or of unknown carcinogenicity were assayed for their abilities to modulate ultraviolet (UV)-induced mutagenesis in Escherichia coli WP2 . In addition to the previously reported comutagenic effect of arsenite, salts of three other compounds were found to enhance UV mutagenesis . CuCl2, MnCl2 (and a small effect by KMnO4), and NaMoO4 acted as comutagens in E coli WP2, which has wild-type DNA repair capability, but were much less comutagenic in the repair deficient strain WP2s (uvrA) . The survival of irradiated or unirradiated cells was not affected by these compounds . No effects on UV mutagenesis were seen for 16 other metal compounds . We suggest that the comutagenic effects might occur either via metal-induced decreases in the fidelity of repair replication or (in the case of CuCl2) via metal-induced depurination.

Circ Shock, 1986, 18(4), 289 - 300
Metabolic effects of glucagon in endotoxemic minipigs; Weingand KW et al.; A treatment group of four 50-80-kg Yucatan minipigs was fitted with jugular, portal, hepatic vein, and carotid artery catheters, and hepatic artery and portal vein flow cuffs to quantitate transhepatic kinetics of glucose, lactate, and insulin . Three days later, they were placed in slings, and a primed-continuous intravenous infusion of 3-tritiated glucose was initiated to monitor whole body glucose kinetics . Following a 3-hour control period, an intravenous infusion of E . coli endotoxin was administered at 15 micrograms/kg/hr for 6 hours . After 1 hour of endotoxin infusion, glucagon was administered as a primed (50 micrograms/kg)-continuous (50 micrograms/kg/hr) intravenous infusion for 5 hours . These results were compared statistically to a control group of eight minipigs given endotoxin only . Glucagon caused a transient (less than 1 hour) peak elevation of arterial glucose levels due to increased hepatic glycogenolysis immediately following initiation of the glucagon infusion . Despite enhanced extrahepatic (renal) gluconeogenesis, plasma glucose concentrations decreased to the hypoglycemic levels of the control group, as glucagon was unable to overcome the relative endotoxic inhibition of hepatic gluconeogenesis . Pancreatic output of insulin increased sixfold resulting in threefold increments in transhepatic uptake and arterial serum insulin levels . Arterial lactate and pyruvate concentrations were elevated due to increased peripheral production and hepatic output . Survivability in the treatment group (75%) improved over the control group (33%).

Circ Shock, 1986, 18(4), 267 - 75
Regional blood flow during continuous low-dose endotoxin infusion; Fish RE et al.; Escherichia coli endotoxin (ET) was administered to adult rats by continuous IV infusion from a subcutaneously implanted osmotic pump (Alzet) . Cardiac output and regional blood flow were determined by the radiolabeled microsphere method after 6 and 30 hr of ET or saline infusion . Cardiac output (CO) of ET rats was not different from time-matched controls, whereas arterial pressure was 13% lower after 30 hr of infusion . After both 6 and 30 hr of ET, pancreatic blood flow and percentage of cardiac output were lower than in controls . Estimated portal venous flow was decreased at each time point, and an increased hepatic arterial flow (significant after 30 hr) resulted in an unchanged total hepatic blood flow . Blood flow to most other tissues, including epididymal fat, muscle, kidneys, adrenals, and gastrointestinal tract, was similar between treatments . Maintenance of blood flow to metabolically important tissues indicates that the previously reported alterations in in vitro cellular metabolism are not due to tissue hypoperfusion . Earlier observations of in vitro myocardial dysfunction, coexistent with the significant impairment in pancreatic flow, raise the possibility that release of a myocardial depressant factor occurs not only in profound shock but also under less severe conditions of sepsis and endotoxemia.

Circ Shock, 1986, 18(3), 179 - 92
Effects of methylprednisolone upon vascular permeability changes in endotoxic shock; Hubbard JD et al.; This study was designed to examine glucocorticoid effects on increases in vascular permeability caused by endotoxic shock in dogs anesthetized with pentobarbital sodium (30 mg/kg) . Methylprednisolone sodium succinate was administered IV in two doses (30 mg/kg each) before Escherichia coli endotoxin was administered (0.5 mg/kg) . Samples of serum and lymph from the left thoracic duct were collected for measurement of total protein and separation by polyacrylamide gel electrophoresis . The same four protein electrophoretic fractions with molecular weights (M.W.) ranging from 60,000 to 1,000,000 were consistently chosen for analysis . Methylprednisolone treatment given prior to endotoxin administration resulted in an attenuation of the early increase in total protein flux and lymph to plasma protein (L/P) ratio and prevented significant increases in the permeability surface area product observed in the group given endotoxin alone . Endotoxin administration alone resulted in significant increases in the permeability-surface area product and L/P ratio for all four electrophoretic fractions . Pretreatment with glucocorticoid partially attenuated the increase in the L/P ratio for only those fractions of 100,000 M.W . or less . These results suggest MP provides partial but not complete protection from increases in vascular permeability during endotoxic shock.

Can J Microbiol, 1986 Jan, 32(1), 52 - 5
Site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide; Ferris FG et al.; The site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide was assessed by collecting high-resolution phosphorus nuclear magnetic resonance spectra in the presence of manganese, a paramagnetic divalent cation . This technique revealed high-affinity interactions between the cation and all of the lipopolysaccharide phosphoryl groups . To ascertain whether the carboxyl groups of 2-keto-3-deoxyoctonate contributed to the metal cation binding, lipopolysaccharide was chemically modified using a glycine ethyl ester - carbodiimide reaction . Of the three available carboxyl groups, only one was neutralized by the exogenously added ligand; the others appeared to be cross-linked within the molecule . By analogy, only one carboxyl group should be freely available for binding metallic ions, while the others are probably neutralized by the close proximity of endogenous amino substituents . Although high-resolution phosphorus nuclear magnetic resonance showed that an intermolecular conformational change had occurred after the carboxyl groups were neutralized, titration with manganese revealed no differences in the apparent strength of the interactions between the cation and the phosphoryl groups . Together, these data suggest that the high affinity of lipopolysaccharide for divalent metallic ions can be attributed primarily to the phosphoryl substituents and not free carboxyl groups.

Biol Neonate, 1986, 49(2), 102 - 8
Metabolic effects of endotoxin in newborn rabbits; Molnar D et al.; The metabolic and hormonal effects of Escherichia coli endotoxin injected intraperitoneally (IP) or into the cerebral ventricles (ICV) and that of passive hyperthermia were studied in rabbits aged 6-10 days . Irrespective of the route of administration, endotoxin caused a transient rise in blood glucose with a simultaneous rise in plasma insulin . In contrast, only in the IP, but not in the ICV group, the endotoxin resulted in a rise of the free fatty acid and a fall in the ketone body concentrations by the second hour . The blood level of pyruvate, lactate, alanine and glycerol was not altered by endotoxin . No parameter was affected by the injection of saline or passive hyperthermia.

Ann Inst Pasteur Immunol, 1986 Jan-Feb, 137C(1), 39 - 50
{Application of the vapor test for the detection and immunologic determination of Escherichia coli heat labile enterotoxin}; Germani Y et al.; The principle of this thin-layer immunoassay (vapour condensation technique or TVAP) is based on the ability of antibodies to absorb firmly to polystyrene surfaces and to retain their reactivity . A condensation pattern consisting of large confluent water drops is noticable when an antibody-antigen reaction takes place . We used this technique to detect and assay the Escherichia coli heat-labile enterotoxin (ETEC LT+) and compared the results of 53 strains (40 positives and 13 negatives) with single radial immune haemolysis, Gm1-ELISA and Vero cell culture tests . With the reagents used, this reaction was specific for a toxin dilution up to 1/14 . As little as 0.025 micrograms/ml of cholera toxin could be detected . The TVAP-test is simple, rapid and cost-effective . It is thus quite suitable for use in diarrhoeal endemic areas.

Scand J Infect Dis, 1986, 18(1), 65 - 70
Effective treatment of diarrhoeal dehydration with an oral rehydration solution containing citrate; Ahmed SM et al.; To compare the clinical efficacy of oral rehydration salts (ORS) from effervescent tablets containing citrate with the WHO recommended ORS for the treatment of dehydration due to acute diarrhoea, a randomized clinical trial was carried out in 57 adults and 58 children . These patients had mild or moderate degrees of dehydration and acidosis due to acute watery diarrhoea that was caused by enterotoxigenic Escherichia coli in 43-47% of the cases . Efficacies were compared by measuring oral fluid intake, stool output, gain in body weight, decrease in serum specific gravity and correction of acidosis during treatment . Successful rehydration and maintenance of hydration was achieved in 25 adults and 24 children treated with citrate containing ORS and 25 adults and 24 children treated with WHO ORS . The mean intake of ORS/kg body weight in children receiving WHO ORS was greater (p less than 0.05) and correction of acidosis was faster than the citrate group during the initial 24 h of therapy (p less than 0.05) . By 48 h, however, both groups showed satisfactory and comparable intake of ORS and correction of acidosis . Thus ORS from effervescent tablets containing sodium citrate base is effective for management of diarrhoea in both adults and children and is a convenient stable form of ORS for use in the home and for travelers.

Neuroendocrinology, 1986, 42(4), 285 - 8
Indomethacin, an antipyretic drug, prevents the endotoxin-induced and potentiates the hemorrhage-induced oxytocin release into the plasma of the male rat; Kasting NW; Oxytocin (OXY) is a nonapeptide of hypothalamic origin which has defined roles in the female reproductive functions of lactation and labor . However, OXY may have other physiological functions because of its presence in the male and its release in response to stress . Available evidence suggests prostaglandins may stimulate the release of OXY . These experiments sought to determine if the stressors, endotoxin and hemorrhage, would release OXY in the chronically catheterized, freely behaving male rat and what effect the antipyretic and prostaglandin synthesis inhibiting drug, indomethacin, would have on these responses . Endotoxin caused a marked release of OXY from mean baseline levels of 5 pg/ml to mean peak levels of 168 pg/ml . Indomethacin greatly attenuated this increase . In contrast, OXY release in response to hemorrhage of either 22 or 44% of the blood volume of the rat was enhanced by indomethacin . Indomethacin increased the hemorrhage-induced OXY levels about 2-fold over a 2-hour posthemorrhage period . Indomethacin alone had no effect on OXY levels . These data verify that stress is a potent stimulus for OXY release and strengthen the hypothesis that prostaglandins mediate OXY release . The paradoxical effects of indomethacin on OXY release suggest that the prostaglandins may have different effects on OXY release depending upon the evoking stimulus.

Med Microbiol Immunol (Berl), 1986, 175(1), 55 - 60
Stability of thermolabile (LT) enterotoxin produced from enterotoxigenic Escherichia coli strains maintained in vitro; Gatti MS et al.; The stability of thermolabile (LT) enterotoxin in 26 strains of porcine enterotoxigenic Escherichia coli (PETEC) belonging to serogroups 08 and 0149 was assayed by the passive immune hemolysis (PIH) test, over a period of 9 months at -70 degrees C . It was found that the percentage of LT+ colonies (% LT+) and the mean value of hemoglobin release (XHb), could predict a change from LT+ to LT-.

Med Microbiol Immunol (Berl), 1986, 175(1), 15 - 26
Phenotypic variations among enterotoxigenic O-groups of Escherichia coli from various human populations; Kuhn I et al.; ETEC isolates from various sources (children from Ethiopia and some Asian countries, and Swedish tourists suffering from traveller's disease) were analysed with the aid of "biochemical fingerprinting", which is a highly discriminative, computerized method designed to subdivide E . coli isolates into different phenotypes . Isolates belonging to the most common ETEC O-groups and others which had not been typeable with available O-antisera were selected . It was found that certain phenotypes of O-groups 6 and 114 could be found in materials from several continents . Phenotypes of other O-groups were usually more restricted to certain geographic areas . Among children in Addis Abeba, 19 out of 25 isolates carrying O-antigen 78 belonged to the same phenotype . Some possible explanations for the fact that certain phenotypes of enterotoxigenic E . coli could be found over the whole world are that they might represent relatively recent developed clones, or they may represent unusually stable clones . Of the isolates that had been nontypeable with available antisera, some had lost their LT-productivity after one year of storage . These isolates proved to belong to a wide variety of phenotypes, whereas nontypeable isolates which were stable LT-producers could be clustered into distinct groups . It is suggested that the non-stable LT-producers are members of the normal E . coli flora of these children, which have occasionally picked up the enterotoxin-producing plasmids, whereas most stable LT-producers represent true ETEC clones.

Mol Gen Genet, 1986 Jan, 202(1), 90 - 5
Base pair substitution and frameshift mutagenesis induced by apurinic sites and two fluorene derivatives in a recA441 lexA (Def) strain; Granger-Schnarr M; One of the consequences of the induction of the Escherichia coli SOS system is the increased ability of the cells to perform mutagenesis . Induction of the SOS system is the result of derepression of a set of genes through a regulatory mechanism controlled by LexA and RecA . In response to an inducing signal, RecA is activated in a form that facilitates the proteolytic cleavage of LexA repressor . Previous works have shown that activated RecA plays a second role, i.e . it is required for the establishment of base pair substitution mutations promoted by UV irradiation . Using a forward mutational assay and recA441 lexA (Def) host bacteria, we show that the result can be extended not only to other mutagens promoting base pair substitution mutations (Apurinic sites, Ap sites and N-hydroxy-N-2-aminofluorene, N-OH-AF) but also mutagens promoting frameshift mutations (N-Acetoxy-N-2-acetylaminofluorene, N-AcO-AAF) . In the recA441 lexA (Def) strain all the genes which are part of the lexA regulon, including recA itself, are expressed constitutively . The recA441 mutation allows RecA to acquire its activated form when the bacteria are grown at 42 degrees C . We show that in such strains Ap sites or N-OH-AF induce a high level of mutations only when the bacteria are grown at 42 degrees C . On the other hand, we show that N-AcO-AAF can promote mutations even at 30 degrees C; the number of mutations being increased when the bacteria were grown at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1986 Jan, 202(1), 162 - 8
Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator; Piechocki R et al.; The dnaQ (mutD) gene product which encodes the epsilon-subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3'-5' exonucleolytic editing capacity . It is shown in this paper that more than 95% of all dnaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A . Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E . coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites . Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background . The introduction of a lexA (Ind-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect . Both, the mutational specificity observed and the partial lexA+ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.

J Biochem (Tokyo), 1986 Jan, 99(1), 303 - 6
Differential effect of phosphatidylethanolamine molecular species on glycerophosphate acyltransferase activity of Escherichia coli; Ishinaga M; The effect of phosphatidylethanolamine (PE) molecular species on the reconstitution of partially purified glycerophosphate acyltransferase of Escherichia coli was investigated . The acyltransferase activity was abolished by 1,2-di-unsaturated (U-U) PE, but not by 1-saturated-2-unsaturated (S-U) PE or 1-saturated-2-cyclopropanoyl PE . Since both the U-U and S-U PE used in the present work are in a fluid state at temperatures above about 30 degrees C, the differential effect cannot be accounted for in terms of the membrane fluidity . Therefore, the inactivation of the reconstituted enzyme was attributed to the large amount of the 1,2-di-cis-vaccenoyl species of PE.

Infection, 1986 Jan-Feb, 14(1), 7 - 12
Colonization and persistence of Escherichia coli phenotypes in the intestines of children aged 0 to 18 months; Kuhn I et al.; The aim of the present investigation was to study the intestinal colonization of Escherichia coli in newborn children, and to determine which strains become residential within the human intestine . The E . coli flora of 89 newborn children was studied by repeated sampling during their first 11 or 18 months of life . The E . coli isolates from the samples were subdivided into phenotypes by the aid of biochemical fingerprinting, a method which measures the kinetics of 24 selected biochemical tests as a tool for discriminating bacterial strains . It was found that E . coli strains colonizing children soon after birth persisted longer than strains colonizing them later . Especially those phenotypes which were defined as hospital strains persisted longer . Certain phenotypes were commonly found among the children, and these phenotypes were more persistent and more homogeneous than other phenotypes with respect to their pattern of biochemical activities . They might be specially adapted to colonize the human intestine . It was concluded that the generally long persistence of the first E . coli strains colonizing a newborn child indicates that the first case of bacterial colonization in children may be an event too important to be allowed to happen at random.

Int J Biochem, 1986, 18(3), 257 - 62
The synthesis and use of oligodeoxynucleotides in plasmid DNA sequencing; Ner SS et al.; A convenient procedure for the synthesis and purification of oligonucleotides is described . 16-base long primers synthesised by this method were used to investigate DNA sequencing using plasmid DNA as a template . This allowed the further analysis of the E . coli glt A sequence coding for citrate synthase and enabled determination of the 5'-non-coding regulatory region of the aminoglycoside phosphotransferase gene.

EMBO J, 1986 Jan, 5(1), 175 - 9
Near ultraviolet DNA damage induces the SOS responses in Escherichia coli; Caldeira de Araujo A et al.; The influence of the growth delay induced by near u.v . radiation on the SOS response was monitored by comparing the level of sfiA expression by means of a sfiA::lacZ fusion in both a nuvA+ cell and an isogenic nuvA mutant . The mutant lacks 4-thiouridine in its tRNA and does not exhibit the near u.v.-induced growth delay . Although the two strains exhibit similar sfiA induction levels after 254 nm irradiation, their behaviour is different after illumination with near u.v . light, including solar u.v . Inducibility is 10-20 times higher in the nuvA mutant than in the parent strain . Furthermore, pre-illumination with broad band near u.v . light does not affect the 254 nm-induced sfiA response in the mutant but reduces it by a factor of 3-4 in the parent strain . The kinetics of sfiA induction in near u.v.-illuminated nuvA+ cells, whether treated with 254 nm light or not, is unusual and follows the growth curve: only after 50 min is sfiA derepression observed . It can be concluded that (i) near u.v.-induced DNA lesions are able to trigger the SOS response and (ii) the growth delay effect reduces this response, whether triggered by u.v . or near u.v . light . Hence 4-thiouridine in tRNA acts as a built-in antiphotomutagenic 'device' protecting Escherichia coli cells against mutagenesis and the induction of the SOS response by near u.v . light and sunlight.

Diagn Immunol, 1986, 4(1), 17 - 23
ELISA detection of specific functional antibodies in human serum to Escherichia coli, tetanus toxoid, and diphtheria-tetanus toxoids: normal values for IgG, IgA, and IgM; Moen RC et al.; An inexpensive, easily performed enzyme-linked immunosorbent assay (ELISA) was developed to measure specific IgG, IgA, and IgM antibodies to the common antigens Escherichia coli, diphtheria-tetanus toxoid, and tetanus toxoid . Normal values were established . Classical antibody deficiency disease states were confirmed and delineated by these assays . Additionally, several instances were discovered when functional antibody levels were abnormal when the serum immunoglobulin levels were normal . The use of ELISA assays for antibodies to common antigens provides a useful technique to measure and monitor isotype responses of the humoral immune system.

Brain Res Bull, 1986 Jan, 16(1), 99 - 105
alpha-Melanocyte-stimulating hormone infused ICV fails to affect body temperature or endotoxin fever in the cat; Rezvani AH et al.; Permanent cannulae for intracerebroventricular (ICV) infusion were implanted bilaterally in cats following stereotaxic procedures . After colonic temperature was recorded for a one-hour baseline, a 300 microliter ICV infusion was given of CSF control vehicle, 1:100 dilution of W3110 E . coli endotoxin (10(8) organisms/ml) or alpha-melanocyte-stimulating hormone (alpha-MSH) in one of seven doses ranging from 50.0 ng to 50.0 micrograms . Whereas ICV E . coli always induced an intense and prolonged fever of rapid onset, alpha-MSH infused similarly was essentially without effect on the deep body temperature of the normothermic cat . When each of the doses of alpha-MSH was infused ICV, either during the rising phase of an E . coli fever or after the febrile response had reached its asymptote, the core temperature of the cat was unaffected . Similarly, a mixture of E . coli combined with alpha-MSH given ICV failed to alter the characteristics of the rapidly developing fever produced in the cat by this endotoxin . On the other hand, either excess Ca++ ions (50 mM) given ICV or the antipyretic drug . Dipyrone, administered systematically during the course of an endotoxin fever effectively attenuated the animal's elevated body temperature . These results demonstrate that alpha-MSH is apparently neither involved in the central mechanisms underlying normal thermoregulation, nor does it act as an endogenous antipyretic in the cat as has been postulated in another species.

Biophys J, 1986 Jan, 49(1), 251 - 8
Alignment and merging of electron microscope images of frozen hydrated crystals of the T4 DNA helix destabilizing protein gp32*I; Grant RA et al.; Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I . Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space . Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.

Arch Microbiol, 1986 Jan, 143(4), 400 - 2
Role of relA mutation in the survival of amino acid-starved Escherichia coli; Hecker M et al.; Amino acid-starved cells of Escherichia coli relA+, which contain a large number of glycogen particles, are able to survive in phosphate buffer for a longer time period than their relaxed counterparts . With regard to NH4+ starvation differences in the survival of both strains were not found . NH4+ starved cells of E . coli relA are able to synthesize glycogen but amino acid-starved cells of the relA strain are not . We suggest that the synthesis of glycogen triggered by guanosine tetraphosphate during amino acid starvation is responsible for the prolonged viability of the E . coli relA+ strain.

Anal Biochem, 1986 Jan, 152(1), 89 - 93
A nitrocellulose filter binding assay for ribonucleotide reductase; Soderman K et al.; Protein B1, one of the two nonidentical subunits of Escherichia coli ribonucleotide reductase, contains two classes of binding sites for nucleoside triphosphates . One class (h-sites), with a high affinity for dATP (KD = 30 nM) regulates the substrate specificity, while the other (l-sites), with a lower affinity for dATP, regulates overall activity . These classes were defined from experiments involving equilibrium dialysis . Here we describe a sensitive alternative method to measure nucleotide binding to ribonucleotide reductases that gave the same results as equilibrium dialysis . The method involves the protein-specific binding of radioactive nucleotides to nitrocellulose filters . We believe that this method will be useful in binding studies with pure reductases from sources other than E . coli, for the characterization of mutants with changed allosteric properties, and as an assay during purification of reductases containing an h-site for dATP.

Plasmid, 1986 Jan, 15(1), 35 - 47
Directly repeated, 20-bp sequence of plasmid R1162 DNA is required for replication, expression of incompatibility, and copy-number control; Lin LS et al.; DNA required in cis for the replication of the broad-host-range plasmid R1162 is located on two contiguous HpaII fragments of 210 and 370 bp . The latter of these contains three and one-half, perfectly conserved, 20-bp directly repeated sequences . The significance of these for plasmid replication, incompatibility, and copy-number control was examined by generating deletions into these repeats and testing the properties of the remaining DNA . We conclude from the results that the direct repeats are essential for expression of incompatibility and for the decrease in copy number observed when the directly repeated DNA is cloned into R1162 . Little, if any, additional DNA is required from the ori region for these properties . Moreover, deletions of intermediate size result in an intermediate level of incompatibility, indicating the importance of the periodic structure of the direct repeats . The directly repeated DNA is also required for an active origin of replication, as are additional, nonrepeated sequences adjacent to this DNA . The properties of the direct repeats are discussed with respect to their possible role in the replication of R1162 DNA.

Mol Biol (Mosk), 1986 Jan-Feb, 20(1), 242 - 9
{Symmetry of multienzyme complexes}; Gol'dshtein BN et al.; A model for studying the symmetry of stable states arising from polyenzymic complex conformations is proposed . A formal scheme of submolecular structure self-assembly, on which the model is based, enables it not only to limit the class of conformations but in some cases to determine the structure of a complex in an unambigous manner . The model is shown in its application to polyenzymic complexes of dehydrogenases of alpha-keto acids.

Mol Biol (Mosk), 1986 Jan-Feb, 20(1), 224 - 32
{A model of an enzyme with mixed cooperation: 3 states of aspartate transcarbamylase}; Saifullin SR et al.; Allosteric enzyme models on the basis of the known properties of aspartate transcarbamylase (ATCase) from Escherichia coli are suggested . In the first model molecules are supposed to equilibrate between two states . In contrast to the classical Monod-Wyman-Changeux model the symmetry of enzyme molecules changes during the conformational transition . It is shown that the number of binding sites of the enzyme defined from the Scatchard plots is sufficiently dependent on values of parameters of enzyme reaction . This fact results from the mixed (both positive and negative) cooperative effects . However the complex kinetic of ATCase is not completely simulated by this model . Therefore the model is complicated by taking into account the inactive third state of the enzyme . Thus the complex kinetic behaviour of ATCase is explained . The models may be also used for other enzymes.

Microbiologica, 1986 Jan, 9(1), 53 - 61
Toxicity of granular activated carbon treated coal gasification water as determined by the Microtox test and Escherichia coli; Makino Y et al.; The Microtox assay and various parameters (growth, ATP concentration and electrochemical detection) of Escherichia coli were used to assess the toxicity of various levels of granular activated carbon treated coal gasification process water . The generation time of E . coli was statistically significantly slower at the level of 50 percent treatment than any other level of treatment . No differences were seen for ATP concentration per cell or in the electrochemical detection methods for any level treatment . There was a very high correlation between total organic carbon removal by GAC treatment and reduction in toxicity as measured by the Microtox system . However, even the treated water which had 91 percent of the TOC removed was still highly toxic.

J Antibiot (Tokyo), 1986 Jan, 39(1), 136 - 40
Effects of cations, polyamines and other aminoglycosides on gentamicin C2 . Binding to ribosomes from sensitive and resistant Escherichia coli strains; Moukaddem M et al.; Gentamicin C2 interacts cooperatively with ribosomes from a sensitive Escherichia coli strain in a multiphasic way with several classes of sites . It is shown that this binding is highly-dependent on Mg++ and natural endogenous polyamine concentrations . The differences observed between ribosomes from sensitive and resistant strains may be explained by the absence of specific cooperative gentamicin interactions with resistant ribosomes . The effects of other aminoglycoside antibiotics are discussed in terms of structure-activity relationships.

CRC Crit Rev Biochem, 1986, 19(3), 191 - 245
Kinetics of protein-nucleic acid interactions: use of salt effects to probe mechanisms of interaction; Lohman TM; The kinetics of protein-nucleic acid interactions are discussed with particular emphasis on the effects of salt concentration and valence on the observed rate constants . A general review is given of the use of experimentally determined salt dependences of observed kinetic parameters as a tool to probe the mechanism of interaction . Quantitative analysis of these salt dependences, through the application of polyelectrolyte theory, can be used to distinguish reactions which occur in a single step from those reactions which involve distinct intermediates . For those rate constants which display a large salt dependence, in either the association or dissociation reaction, this is due to the high concentration of counterions (e.g., Na+) in the vicinity of the nucleic acid which are subsequently released (or bound in the case of dissociation) at some point before the rate limiting step of the reaction . A general discussion of other features which affect protein-nucleic acid kinetics, such as nucleic acid length and the ratio of nonspecific to specific DNA binding sites (in the case of sequence specific binding proteins), is also given . The available data on the nucleic acid binding kinetics of small ligands (ions, dyes, oligopeptides), nonspecific binding proteins (T4 gene 32 protein, fd gene 5 and Escherichia coli SSB), and sequence specific binding proteins (lac repressor, RNA polymerase, Eco RI restriction endonuclease) are discussed with emphasis on the interpretation of the experimentally determined salt dependences.

Chem Biol Interact, 1986 Jan, 57(1), 41 - 53
Efficient breakage of DNA apurinic sites by the indoleamine related 9-amino-ellipticine; Malvy C et al.; The aromatic amine, 9-NH2-ellipticine, is a synthetic DNA intercalating derivative of the antitumor agent ellipticine, which breaks circular DNA containing apurinic sites . This breakage is inhibited when the apurinic (AP) sites are reduced . The concentration of 9-NH2-ellipticine required to get a significant effect (0.1 microM) is the lowest known among chemicals which induce the same breakage reaction . Comparison with the action of structurally related amines shows that the amino-indole structure is specific for AP sites . The ability of ellipticine derivatives to induce breakage in DNA containing apurinic sites is related to the nucleophile substituent in position 9 . Two ellipticine derivatives with known antitumor activity, BD 40 and 9-OH-ellipticine, were able to break purified DNA at apurinic sites.

Arch Biochem Biophys, 1986 Jan, 244(1), 218 - 25
Spatial relationship between the intrinsic metal in the beta subunit and cysteine-132 in the sigma subunit of Escherichia coli RNA polymerase: a resonance energy transfer study; Chatterji D et al.; Fluorescence excited-state energy transfer measurements were carried out between the N-(1-pyrene)maleimide (PM)-labeled sigma subunit and Co in the beta subunit of Co-Zn RNA polymerase (RPase) . sigma subunit with or without PM labeling was cleaved with 2-nitro-5-thiocyanobenzoic acid, and the reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . One molecule of the fluorescent probe (PM) was found to be attached to the cysteine-132 residue of the sigma subunit . When excited at 340 nm, the fluorescence emission bands from 380 to 420 nm of PM-labeled sigma overlap with the charge transfer absorption band of Co-Zn RPase around 400 nm . Based on Forster's equation, the R0 values for the donor-acceptor pair were calculated to be 21.5 and 22 A in the absence and presence of template analog (dA-dT)60, respectively . Using these R0 values and the observed energy transfer efficiencies, the distance between the cysteine-132 of the sigma subunit and Co located at the initiation site of the beta subunit was calculated to be 22 A with or without the template present, indicating that no major conformational change of the enzyme was induced upon template binding . However, a small but significant change in the above distance was observed upon the addition of ATP to RPase in the presence (dA-dT)60 but not in the absence of (dA-dT)60 template . The biological implications of these observations are discussed.

Arch Biochem Biophys, 1986 Jan, 244(1), 128 - 36
The 18O isotope effect in 13C nuclear magnetic resonance spectroscopy: mechanistic studies on asparaginase from Escherichia coli; Rohm KH et al.; The mechanism of the enzyme asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli was examined using 13C NMR spectroscopy . The pH-dependent oxygen exchange reactions between water and aspartic acid were followed by use of the 18O isotope-induced shift of the resonance positions of directly bonded 13C nuclei . Both L-1- and L-1,4-{13C}aspartic acid were used in experiments with previously 18O-labeled aspartic acid, or in experiments involving the use of 18O-labeled solvent water . Asparaginase catalyzes a relatively efficient exchange between the oxygens of water and those on one carboxyl group of aspartic acid . Exchange at C-4 occurs rapidly but, within experimental error, no exchange at C-1 could be detected . These and related experiments involving the position of 18O incorporation during hydrolysis of aspartic acid beta-methyl ester are all consistent with possible acyl-enzyme mechanisms involving C-4, but do not support a free aspartic acid anhydride mechanism.

Antibiot Med Biotekhnol, 1986 Jan, 31(1), 40 - 3
{Comparative study of the chemotherapeutic effectiveness of mecillinam, ampicillin and their combination in coli bacillary pyelonephritis in rats}; Zinchenko TA et al.; The chemotherapeutic effect of mecillinam and ampicillin was studied comparatively on rats with hematogenic obturation colibacillary pyelonephritis . The antibiotics were administered intragastrically in a dose of 100 mg/kg for 7 days . The treatment was started 24 hours after infection . When the drugs were used in combination their doses were twice as lower . When used alone mecillinam and ampicillin had a significant effect which was practically the same . A marked increase in the therapeutic effect was observed with the use of the antibiotics in combination: sterilization of the tissues of the affected kidney and prevention of development of macroscopic lesions in it in all the animals.

South Med J, 1986 Jan, 79(1), 41 - 6
Emphysematous pyelonephritis: diagnosis and treatment; Klein FA et al.; Emphysematous pyelonephritis is a rare life-threatening bacterial infection usually occurring in patients with diabetes mellitus and producing gas within the renal parenchyma and/or perirenal tissue . We add four cases to the 62 previously reported in the literature . The overall mortality is 38%, with only 29% survival for those treated medically compared to 71% for those given both medical and surgical treatment . Early diagnosis with the aid of renal ultrasonography and/or computerized axial tomography and aggressive early combined medical-surgical management are emphasized.

Nephron, 1986, 42(2), 171 - 6
Progression of chronic pyelonephritis in the rat; Mackenzie R et al.; Thirteen of 20 female Wistar rats developed perihilar kidney scars 6 weeks after ascending infection with Escherichia coli 078 . After removal of the lesser scarred kidney from 6 of the animals, all animals were followed for 54 weeks . Proteinuria (greater than 18 mg/24 h) developed at 20 weeks in the uninephrectomised infected rats and at 34 weeks in the 2-kidney infected model . In 10 uninephrectomised controls significant proteinuria did not appear until 52 weeks . In 9 2-kidney controls proteinuria did not develop at all . The speed of onset and severity of proteinuria was related to the extent of the renal parenchymal loss . Pyelonephritic scars did not show macroscopic progression over the 54-week observation period, even though the original renal infection persisted . Uninephrectomised animals with infected scars developed a highly significant rise of creatinine/body weight (p less than 0.02) and of heart weight/body weight p less than 0.02) ratios compared with the non-infected controls . Their kidneys showed focal and segmental hyalinosis and sclerosis of the glomeruli adjacent to the scars . Immunofluorescent staining for serum proteins was negative, but mesangial deposits of Tamm-Horsfall protein were found in the glomeruli between the scars in animals with renal impairment . These findings establish that progressive renal impairment in rats with infected kidney scars is associated with the development of proteinuria and a glomerulopathy . The cause of the glomerulopathy is not clear, both glomerular hyperfiltration and deposition of Tamm-Horsfall protein in glomerular mesangial cells may be involved.

Nephron, 1986, 42(2), 128 - 32
Endotoxin level of sterile injection solutions and substitution fluid for hemofiltration in Japan and Australia; Tominaga H et al.; The endotoxin contamination levels of sterile distilled water and saline solutions used for injection and hemofiltration in Japan and Australia were examined with a colorimetric limulus test using a chromogenic substrate . The endotoxin levels of injection solutions in most products from both countries measured against E . coli 0111:B4 endotoxin or USP reference standard endotoxin were less than 0.2 pg or 0.0006 endotoxin units (EU) X ml-1 . Only 2 solutions in 30 from both countries showed higher levels: 7.7 and 10.8 pg for E . coli 0111:B4 endotoxin X ml-1 (0.02 and 0.03 EU X ml-1) . Even these higher values were well below the level recommended by the draft guideline published by the United States Food and Drug Administration (FDA) . The endotoxin contamination level of a Japanese hemofiltration substitution fluid ranged from 8.2 to 9.2 pg E . coli 0111:B4 endotoxin X ml-1 (0.024-0.027 EU X ml-1).

J Pediatr Gastroenterol Nutr, 1986 Jan, 5(1), 70 - 3
Ultrastructural and biochemical changes in human jejunal mucosa associated with enteropathogenic Escherichia coli (0111) infection; Taylor CJ et al.; A case of prolonged diarrhoea following Escherichia coli 0111 gastroenteritis is reported . Electron microscopy of the jejunal biopsy revealed effacement of the brush border and attachment of bacteria by pedestal formation . Specific activities of brush border enzymes showed marked depression of disaccharidases, zinc-resistant alpha-glucosidase, and alkaline phosphatase . In contrast, marker enzymes for basolateral membranes and endoplasmic reticulum were unaffected . The biochemical changes support the pathogenic mechanism suggested by ultrastructural studies previously reported.

J Am Vet Med Assoc, 1986 Jan 1, 188(1), 57 - 9
High technology diagnostics: detection of enterotoxigenic Escherichia coli, using DNA probes; Maddox CW et al.; A relatively new technique termed colony blot hybridization appears to be a reliable replacement for the ligated porcine gut loop assay previously used to detect stable toxin-B producing Escherichia coli . The highly reproducible blot assay enables screening of large numbers of isolants for the presence of E coli stable-B toxin genes, thus avoiding variations in phenotypic expression and poor repeatability inherent in the in vivo assays . Availability of this diagnostic test can aid practitioners in identifying enterotoxigenic E coli related disease in swine and in determining the benefits of vaccination programs.

Environ Mutagen, 1986, 8(1), 161 - 9
The reproductive effects assessment group's review of the mutagenicity of vinylidene chloride; Jacobson-Kram D; A large number of studies indicate that vinylidene chloride is mutagenic to bacteria and that this activity is largely dependent on microsomal activation . Vinylidene chloride gave positive results for gene reversion and conversion in yeast that was also dependent on metabolic activation, and was positive in tradescantia . In mammalian systems, vinylidene chloride failed to induce gene mutations in V79 cells at two separate loci, failed to induce chromosomal aberrations in mouse bone marrow in vivo, and failed to induce dominant lethals in either mice or rats . Vinylidene chloride was found to alkylate DNA of mice exposed through inhalation and may have caused unscheduled DNA synthesis in kidneys of similarly exposed mice . The studies on the mutagenicity of vinylidene chloride are evaluated in this review.

Circ Shock, 1986, 18(1), 31 - 42
Regional blood flow and metabolism in canine endotoxin shock before, during, and after infusion of glucose-insulin-potassium (GIK); Bronsveld W et al.; Glucose-insulin-potassium infused (GIK) during endotoxin shock causes increased cardiac output (CO) accompanied by decreased systemic vascular resistance . We have studied the effects of GIK on the distribution of cardiac output with radioactive microspheres to see if this decrease in resistance is equally distributed over all organs . GIK resulted in increased CO and increased flow to heart, splanchnic bed, kidneys, adrenals, and skeletal muscle, but fractional flow to these organs did not change . Thirty minutes after the GIK infusion, CO and organ flow had fallen again and differences between the endotoxin and control groups were no longer significant . GIK thus increases CO during endotoxin shock but does not influence its distribution . Systemic oxygen transport increased after GIK, but oxygen extraction decreased . Myocardial and splanchnic oxygen consumption did not change significantly . Oxygen extraction also diminished in these areas after GIK . GIK did not influence serum lactate: In both groups lactate increased significantly.

Circ Shock, 1986, 18(1), 21 - 9
Lipolytic patterns in isolated adipocytes of continuously endotoxemic rats; Spitzer JA et al.; Lipolytic patterns were studied in adipocytes isolated from rats after 6 and 30 hr of continuous Escherichia coli endotoxin (ET) or saline infusion via a subcutaneously implanted osmotic pump . By 6 hr, ET cells responded to norepinephrine (NE) stimulation with significantly greater increase above basal rates of glycerol and free fatty acid (FFA) release than did control adipocytes . By 30 hr of continuous infusion, basal glycerol release was enhanced; the in vitro lipolytic response evoked by NE was significantly reduced in ET cells compared to rates on the previous day, and NE-stimulated lipolysis in ET cells was significantly below that of controls . At the same time, the in vitro antilipolytic effect of insulin was attenuated . We conclude that 1) an initial metabolic response can be observed within a few hours of a continuous, low dose ET infusion, 2) the biphasic nature of the sequential changes in lipolysis is likely to reflect alterations in the hormonal environment in vivo, and 3) these features are consonant with some aspects of the metabolic profile of septic patients.

Circ Shock, 1986, 18(1), 11 - 9
Ultrastructural studies of experimental endotoxin shock in the liver and spleen: therapeutic effects of low-dose heparin on reticuloendothelial disturbances; Onda M et al.; To determine the effects of low-dose heparin on the reticuloendothelial system in shock, rats were subjected to intraperitoneal administration of E coli endotoxin . Alterations such as degeneration and necrosis of the Kupffer cells and sinusoidal endothelial cells were observed in the livers of the non-heparin-treated animals 4 hours after the administration of the endotoxin . These changes progressed with time . Kupffer cells in the heparin-treated rats appeared undisturbed up to 8-12 hours post endotoxin challenge . Phagocytosis of cell remained slight in the heparin-treated rats . The mortality rate of the heparin-treated group was 10% with LD50 endotoxin (7.6 mg/kg) . The mortality rate of the untreated control group 24 hours after administration was 50% . However, when endotoxin was administered in a dosage of 30 mg/kg (MLD), the mortality rate of the heparin-treated rats was 60%--10% greater than that of controls (P less than 0.05).

Arch Surg, 1986 Jan, 121(1), 65 - 70
In vitro reversal of cardiac deterioration in septic shock with tetraethylammonium chloride; DeMeules JE; Overwhelming sepsis associated with cardiac failure continues to be a major clinical problem . This is commonly associated with a failure to respond to conventional pharmacologic therapy . This study was undertaken to see if manipulations of the electrophysiologic defects previously described by treatment with tetraethylammonium chloride (TEA) would be advantageous . Septic shock was induced in rabbits by a lethal dose of Escherichia coli . Peak tension and velocities of contraction and relaxation were measured in papillary muscle with and without 5mM TEA . Exposure to this compound improved peak tension and velocities of contraction and relaxation to normal values . The action of TEA is not specific to septic tissue as values in normal muscles are similarly improved . Tetraethylammonium chloride or other drugs that decrease outward potassium current and prolong the action potential duration may be helpful in treating cardiac dysfunction that accompanies sepsis.

Am Rev Respir Dis, 1986 Jan, 133(1), 62 - 7
Reduction of total hemolytic complement activity with Naja haje cobra venom factor does not prevent endotoxin-induced lung injury in sheep; Flick MR et al.; We studied the effects of reducing total hemolytic complement activity with Naja haje cobra venom factor on the lung injury caused by intravenously infused endotoxin in 5 unanesthetized sheep with lung lymph fistulas . In normal sheep, infusions of lipopolysaccharide W from Escherichia coli (1.0 micrograms/kg) intravenously over 30 min caused increases in protein-rich lung lymph flow as well as the appearance in plasma and lung lymph of complement (C5)-derived chemotactic activity for polymorphonuclear leukocytes . Reduction of total hemolytic complement activity by treatment with Naja haje cobra venom factor (12 to 17 U/kg intraperitoneally) did not prevent the lung injury caused by endotoxin and also did not prevent the appearance in plasma and lung lymph of chemotactic activity . We conclude that although complement appears to be activated following intravenously infused endotoxin in sheep, a completely intact complement system is not necessary for endotoxin-induced lung injury.

Proc Natl Acad Sci U S A, 1986 Jan, 83(2), 483 - 7
Design of a photoreactive analogue of the Escherichia coli heat-stable enterotoxin STIb: use in identifying its receptor on rat brush border membranes; Gariepy J et al.; The Escherichia coli heat-stable enterotoxin, STIb was prepared by solid-phase peptide synthesis and purified to homogeneity by high-pressure liquid chromatography . This analogue was iodinated and shown to bind specifically to rat intestinal membranes . The radiolabeled peptide was derivatized at the amino terminus with the photoreactive heterobifunctional crosslinking agent N-hydroxysuccinimidyl p-benzoylbenzoate . This photoreactive probe also exhibited binding specificity . It was mixed with rat intestinal brush border membranes and photolyzed in the presence or absence of excess unlabeled STIb . Polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate and 2-mercaptoethanol indicated that the peptide probe was crosslinked specifically to two molecular species of 57 and 75 kDa . One or both of these molecules appear to constitute the enterotoxin receptor or to be in close proximity to it.

Proc Natl Acad Sci U S A, 1986 Jan, 83(2), 303 - 7
Evidence for a contact between glutamine-18 of lac repressor and base pair 7 of lac operator; Ebright RH; Glutamine-18 of the lac repressor (lacR) has been substituted by glycine, by serine, and by leucine . The specificities of wild-type lacR and of the three substituted lacR variants have been analyzed with respect to base pairs 5, 6, 7, 8, 9, and 10 of the lac operator (lacO) . The data indicate that {Gly18}lacR, {Ser18}lacR, and {Leu18}lacR lose the ability to distinguish between the O+ base pair G . C and the Oc base pairs T . A and A . T at position 7 of lacO (KdOc/KdO+ approximately equal to 1) . In contrast, the three substituted variants retain the ability to discriminate O+ from Oc at each other position, by factors of 9 to 37 . Therefore, I propose that glutamine-18 contacts base pair 7 of lacO . These data suggest that the interaction between the helix-turn-helix motif and DNA may be very similar or identical in lacR and the catabolite gene activator protein.

Proc Natl Acad Sci U S A, 1986 Jan, 83(2), 275 - 9
DNA hybridization electron microscopy: ribosomal RNA nucleotides 1392-1407 are exposed in the cleft of the small subunit; Oakes MI et al.; The ribosomal sequence corresponding to Escherichia coli 16S rRNA nucleotides 1392-1407 (the "1400 region") is phylogenetically conserved and is in a functionally important region of the subunit . Using the technique of DNA hybridization electron microscopy, we have mapped this sequence on the surface of the small ribosomal subunit . In this procedure a synthetic oligodeoxynucleotide probe, complementary to a specific rRNA sequence and carrying an attached marker molecule, is hybridized to ribosomal subunits in order to determine the dimensional site of attachment . In the E . coli ribosome, the 1400 region is located at the level of the neck, near the cleft and most likely on the head of the small subunit . The related sequence in yeast 18S rRNA, nucleotides 1618-1633, is located in the topological equivalent of the E . coli site . The location of this region, which has been crosslinked to the anticodon of a peptidyl-site-bound tRNA, indicates that this part of the cleft of the small subunit has a similar three-dimensional organization in phylogenetically diverse organisms and suggests that it is the site of the codon-anticodon interaction.

Proc Natl Acad Sci U S A, 1986 Jan, 83(2), 231 - 5
Mechanism of the idling-turnover reaction of the large (Klenow) fragment of Escherichia coli DNA polymerase I; Mizrahi V et al.; The mechanism of the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I has been investigated . The reaction cycle involved is one of excision/incorporation, in which the 3' deoxynucleotide residue of the primer DNA strand is partitioned into its 5'-mono- and 5'-triphosphate derivatives, respectively . Mechanistic studies suggest the 5'-monophosphate product is formed in the first step by simple 3'----5' exonucleolytic cleavage . Rapid polymerization follows with the concomitant release of inorganic pyrophosphate . In the second step, the 5'-triphosphate product is generated by a pyrophosphorolysis reaction, which, despite the low concentration of pyrophosphate that has accumulated, occurs at a rate that is comparable with that of the parallel 3'----5' hydrolysis reaction.

Mutat Res, 1986 Jan, 173(1), 13 - 8
Studies on mutagenesis and repair induced by platinum analogs; Fram RJ et al.; Mutagenesis and cytotoxicity were studied in Escherichia coli by iproplatin and carboplatin, two analogs of cisplatin (CDDP) currently undergoing clinical trial . As with CDDP, mutagenesis by these agents was mediated by the umuDC gene product . In contrast to CDDP, however, mismatch repair did not substantially contribute to survival of cells after exposure to these agents since dam-3 E . coli were not more sensitive than wild type E . coli . UvrA- E . coli, however were more sensitive to these analogs demonstrating that as with CDDP, uvr endonuclease-mediated excision contributes to the repair of DNA damage induced by platinum compounds.

Mutat Res, 1986 Jan, 165(1), 39 - 44
Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells; Wang TC et al.; The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation . These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps . By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks . Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed.

Mutat Res, 1986 Jan-Feb, 159(1-2), 41 - 6
The molecular basis of the origin of complete and mosaic mutants; Dianov GL et al.; To study the molecular basis of the origin of complete and mosaic mutants, pBR322 plasmids with damage to one or both DNA strands were constructed by limited chemical modification of plasmid DNA . Damage to one strand of DNA resulted in the induction of predominantly mosaic mutants . Data were obtained indicating that complete mutations arise as a result of damage to both strands in the region of the mutated gene.

Mutat Res, 1986 Jan-Feb, 159(1-2), 31 - 9
Inducible error-prone repair in yeast . Suppression by heat shock; Mitchel RE et al.; The production of reversion mutations in wild-type, diploid Saccharomyces cerevisiae by the alkylating agents N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU) was suppressed in cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide . The same cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide . The same treatment after mutagen exposure did not lower the induced mutation frequency . In split-dose experiments, a first MNNG exposure prevented subsequent heat (or cycloheximide) treatment from blocking mutation by a second, later mutagen exposure . These data suggest that, in yeast, MNNG or MNU induces an error-prone DNA-repair system, and that this induction is blocked by protein-synthesis inhibitors . The specificity of this system for different types of DNA damage was investigated using a variety of other mutagenic agents . A prior heat shock did not suppress mutation produced by exposure to ethyl methanesulfonate, ethylnitrosourea, 8-methoxypsoralen + UVA, or gamma-radiation . Partial suppression was observed in cells exposed to methyl methanesulfonate or to 254-nm ultraviolet light . These results indicate that, unlike the SOS system of E . coli, this inducible error-prone process of yeast is responsive to only certain mutagens . Heat shock suppression of mutation produced by MNNG exposure was also demonstrated in wild-type haploid cells, as well as haploid strains mutant in representative genes of the RAD52 epistasis group (rad52, rad53, rad54), the RAD3 epistasis group (rad1, rad2, rad3) and the RAD6 epistasis group (rad9, rad18) . The rad6 mutant itself was immutable with MNNG and therefore untestable by these techniques . These data indicate that this error-prone repair system is not absolutely dependent on the integrity of the RAD52 (recombination) or the RAD3 (excision) systems, or on at least some parts of the RAD6 system.

Mutat Res, 1986 Jan-Feb, 159(1-2), 13 - 21
RecBC enzyme activity is required for far-UV induced respiration shutoff in Escherichia coli K12; Swenson PA et al.; Shutoff of respiration is one of a number of recA+ lexA+ dependent (SOS) responses caused by far ultraviolet (245 nm) radiation (UV) damage of DNA in Escherichia coli cells . Thus far no rec/lex response has been shown to require the recB recC gene product, the RecBC enzyme . We report in this paper that UV-induced respiration shutoff did not occur in either of these radiation-sensitive derivatives of K12 strain AB1157 nor in the recB recC double mutant . The sbcB gene product is exonuclease I and it has been reported that the triple mutant strain recB recC sbcB has near normal recombination efficiency and resistance to UV . The sbcB strain shut off its respiration after UV but the triple mutant did not show UV-induced respiration shutoff; the shutoff and death responses were uncoupled . We concluded that respiration shutoff requires RecBC enzyme activity . The RecBC enzyme has ATP-dependent double-strand exonuclease activity, helicase activity and several other activities . We tested a recBC+ (double dagger) mutant strain (recC 1010) that had normal recombination efficiency and resistance to UV but which possessed no ATP-dependent double-strand exonuclease activity . This strain did not shut off its respiration . The presence or absence of other RecBC enzyme activities in this mutant is not known . These results support the hypothesis that ATP-dependent double-strand exonuclease activity is necessary for UV-induced respiration shutoff.

J Virol, 1986 Jan, 57(1), 101 - 9
Hepatitis B virus polypeptide X: expression in Escherichia coli and identification of specific antibodies in sera from hepatitis B virus-infected humans; Meyers ML et al.; Sequence analysis of the hepatitis B virus (HBV) genome revealed the presence of an open reading frame (ORF X) which has the potential to encode a 154-amino acid polypeptide . A fusion protein containing 145 of the amino acids encoded by ORF X and 8 amino acids of beta-galactosidase was expressed and characterized in bacterial extracts . Immunoprecipitations with the ORF X fusion protein as a radioactively labeled antigen were performed to screen sera of humans infected with HBV for the presence of antibodies against ORF X-encoded determinants (anti-X) . Such antibodies were identified in 9 samples from a set of 26 sera characterized as positive for HBV surface antigen but were not found in 16 normal human sera . The data reported here demonstrate that sera from some patients with markers of HBV infection contain antibodies directed against the polypeptide encoded by ORF X . As such, these findings represent evidence that ORF X constitutes a gene, or a portion of a gene, which is expressed during HBV infection . Although there does not appear to be a direct relationship between anti-X and any individual markers of HBV infection, our data suggest that anti-X is more prevalent in HBV-positive sera containing antibodies to HBe3 antigen (anti-HBe3).

J Infect Dis, 1986 Jan, 153(1), 98 - 108
Relation of structure to function for the U . S . reference standard endotoxin after exposure to 60Co radiation; Csako G et al.; The structure and function of the highly purified U.S . reference standard endotoxin (RSE) were studied after exposure to ionizing radiation from a 60Co source . With increasing doses of radiation, the trilaminar ribbon-like structure of untreated endotoxin exhibited focal swelling, after which only spherical particles were seen by electron microscopy . These morphological changes were paralleled by the respective loss of O-side chain repeating units and pieces of the R-core from the lipopolysaccharide molecules, as demonstrated by electrophoresis . The biologic function of the irradiated endotoxin was assessed with a variety of tests . At higher doses of radiation, a direct relation was observed between the degradation of the molecular and supramolecular structure and the loss of biologic function . At lower doses of radiation, however, there was variability among the functional assays in their rate of change with progressive irradiation of the RSE . The results suggest that the carbohydrate moiety plays an important role both in determining the supramolecular structure and in modulating certain biologic activities of bacterial endotoxins.

J Bacteriol, 1986 Jan, 165(1), 94 - 100
Highly mobile DNA segment of IncI alpha plasmid R64: a clustered inversion region; Komano T et al.; When R64 DNA was digested with EcoRI, two DNA fragments not equimolar to the plasmid DNA were produced . A DNA region including these fragments was cloned (pKK009), and the pKK009 DNA sample was found to be a mixture of six or more DNA species with EcoRI, PstI, and AvaI cleavage sites at different positions, suggesting a complex rearrangement of DNA . When a part of the pKK009 DNA was removed by HindIII digestion, 33 different types of plasmids (pKK010-series plasmids) were obtained out of 58 clones tested, but no DNA rearrangement could be observed . On the basis of a comparison of the detailed restriction maps of these pKK010-series plasmids, we propose a model in which four DNA segments invert independently or in groups within the 1.95-kilobase region of R64, so that the arrangements of these four segments change randomly . The fixed pKK010-series plasmid DNA was again rearranged in the presence of R64, indicating that trans-acting gene function may be present to mediate the DNA rearrangement . The gene (tentatively designated as rci) was located on a 4.5-kilobase E9' fragment of R64.

J Bacteriol, 1986 Jan, 165(1), 82 - 7
Plasmid segregation into minicells is associated with membrane attachment and independent of plasmid replication; Rashtchian A et al.; The role of plasmid replication in the segregation of plasmids into Escherichia coli minicells was investigated with temperature-sensitive replication mutants derived from E . coli plasmids ColE1 and pSC101 . For as long as six generations of growth, at permissive or nonpermissive temperatures (when greater than 80% of plasmid replication was inhibited), the same amount of previously 3H-labeled plasmid DNA segregated into minicells . Density gradient separations of wild-type and temperature-sensitive plasmid DNA from both replicons segregated into the minicells showed that about 20 to 25% was stably associated with the minicell membrane at both temperatures . Electron microscopy showed this DNA to consist of circular plasmid molecules attached to the minicell membrane . These combined findings suggest that segregation of plasmids into minicells and their association with the minicell membrane are interrelated and independent of plasmid replication.

J Bacteriol, 1986 Jan, 165(1), 34 - 40
Mutations in tar suppress defects in maltose chemotaxis caused by specific malE mutations; Manson MD et al.; Maltose-binding protein (MBP), which is encoded by the malE gene, is the maltose chemoreceptor of Escherichia coli, as well as an essential component of the maltose uptake system . Maltose-loaded MBP is thought to initiate a chemotactic response by binding to the tar gene product, the signal transducer Tar, which is also the aspartate chemoreceptor . To study the interaction of MBP with Tar, we selected 14 malE mutants which had specific defects in maltose taxis . Three of these mutants were fully active in maltose transport and produced MBP in normal amounts . The isoelectric points of the MBPs from these three mutants were identical to (malE461 and malE469) or only 0.1 pH unit more basic than (malE454) the isoelectric point of the wild-type protein (pH 5.0) . Six of the mutations, including malE454, malE461, and malE469, were mapped in detail; they were located in two regions within malE . We also isolated second-site suppressor mutations in the tar gene that restored maltose taxis in combination with the closely linked malE454 and malE461 mutations but not with the malE469 mutation, which maps in a different part of the gene . This allele-specific suppression confirmed that MBP and Tar interact directly.

J Bacteriol, 1986 Jan, 165(1), 198 - 203
Posttranscriptional autoregulation of Escherichia coli threonyl tRNA synthetase expression in vivo; Butler JS et al.; Five mutations in thrS, the gene for threonyl-tRNA synthetase, have been characterized, and the sites of the mutations have been localized to different regions of the thrS gene by recombination with M13 phage carrying portions of the thrS gene . Quantitative immunoblotting shows that some of these mutations cause the overproduction of structurally altered threonyl-tRNA synthetase in vivo . The amounts of in vivo thrS mRNA as measured by quantitative hybridization are, however, the same as wild-type levels for each mutant . These results demonstrate that the expression of threonyl-tRNA synthetase is autoregulated at the posttranscriptional level in vivo.

J Bacteriol, 1986 Jan, 165(1), 161 - 6
Nucleotide sequence corresponding to five chemotaxis genes in Escherichia coli; Mutoh N et al.; The nucleotide sequence of DNA which contains five chemotaxis-related genes of Escherichia coli, cheW, cheR, cheB, cheY, and cheZ, and part of the cheA gene was determined . Molecular weights of the polypeptides encoded by these genes were calculated from translated amino acid sequences, and they were 18,100 for cheW, 32,700 for cheR, 37,500 for cheB, 14,100 for cheY, and 24,000 for cheZ . Nucleotide sequences which could act as ribosome-binding sites were found in the upstream region of each gene . After the termination codon of the cheW gene, a typical rho-independent transcription termination signal was observed . There are no other open reading frames long enough to encode polypeptides in this region except those which code for the two previously reported genes tar and tap.

Fed Proc, 1986 Jan, 45(1), 19 - 24
Endotoxin-mediated pulmonary endothelial cell injury; Meyrick BO; Infusion of endotoxin into sheep results in physiological and structural damage to the pulmonary endothelium . It is uncertain whether complement activation and granulocyte sequestration in the pulmonary microcirculation and the ensuing granulocyte migration into the interstitium seen with endotoxemia contribute to the endothelial damage . We have shown that infusion of complement-activated plasma into sheep, although causing the same degree of granulocyte sequestration in the lungs, results in only modest and transient endothelial damage . In addition, migration or chemotaxis of granulocytes across the endothelial layer of intimal explants is not accompanied by either structural evidence of endothelial damage or a detectable increase in vascular permeability . Such studies indicate that neither complement/granulocyte activation nor granulocyte migration across a vessel wall is entirely responsible for the severe endothelial damage seen with endotoxin . In vitro studies of bovine pulmonary endothelial monolayers indicate that endotoxin can cause direct damage to the endothelium; the damage is dose-dependent and more severe in the presence of serum . Structural studies show endothelial cell retraction, pyknosis, and sloughing . Prostacyclin production and lactic dehydrogenase release are increased, as are permeability to small solutes and hydraulic conductance across the endothelium . It seems that endotoxin can cause a direct injury to pulmonary endothelium but complement and granulocyte activation may enhance the damage.

FEBS Lett, 1986 Jan 1, 194(1), 12 - 5
The roles of HPr and FPr in the utilization of fructose by Escherichia coli; Kornberg H; A mutant impaired in FPr activity was isolated . The altered gene (fpr), which was located near min . 44 on the E . coli genome, was transferred by phage-mediated transduction to appropriate recipients that lack HPr (ptsH), or Enzyme IIman (ptsM), or neither . The rates of growth on fructose of such transductants indicate that phosphate from PEP is transferred predominantly via FPr to fructose that enters the cells by Enzyme IIfru, but that HPr can play a role in transferring phosphate to fructose taken up via Enzyme IIman.

Free Radic Res Commun, 1986, 2(1-2), 27 - 42
Anti-inflammatory activity of superoxide dismutases: studies on adjuvant induced polyarthritis in rats; Jadot G et al.; The anti-arthritic activities of various superoxide dismutases and of liposomal bovine Cu-SOD have been compared in the adjuvant induced Lewis Inbred Rat model . Various approaches, including plethysmometric measurements, red cell sedimentation rates, while cell counts, levels of IgA and IgG immunoglobulins and scoring by visual, radiographic and scintigraphic techniques all concord in a demonstration of different activities for different SODs . The most efficient are liposomal bovine Cu-SOD and E . coli Mn-SOD, a moderate activity being shown by free bovine Cu-SOD . Poor or zero results are obtained with human Mn-SOD, human Cu-SOD or the homologous rat Cu-SOD.

Free Radic Res Commun, 1986, 2(1-2), 19 - 26
Anti-inflammatory activity of superoxide dismutases: inhibition of adriamycin induced edema in rats; Jadot G et al.; Various superoxide dismutases from different sources, containing Cu, Mn or Fe at the active centre, have been examined with respect to anti-inflammatory activity in a model using adriamycin-induced edema in rats . Very large differences in efficiency are observed, the most active being E . coli Mn-SOD and bovine Cu-SOD . The Fe-SOD from E . coli is active whereas P . leiognathi Fe-SOD is not . Human Mn-SOD shows no significant activity and homologous rat Cu-SOD is totally inactive . Yeast Cu-SOD shows pro-inflammatory properties . Anti-inflammatory activity is not a function of molecular weight or circulation life-time.

Int J Immunopharmacol, 1986, 8(8), 1009 - 15
The effect of cadmium on antibody responses to antigens with different cellular requirements; Blakley BR et al.; Six week old BDF1 or CD-1 female mice were exposed to cadmium chloride in the drinking water at concentrations ranging from 0 to 50 ppm cadmium for 3 weeks . The in vivo antibody response against dinitrophenyl-aminoethylcarbamylmethyl-Ficoll (DNP-Ficoll), a T-lymphocyte independent, macrophage dependent response, was enhanced by cadmium . Similarly, the in vivo antibody response against Escherichia coli 0127 (LPS), a T-lymphocyte and macrophage independent response, was also enhanced by cadmium . In contrast, the in vitro antibody response against sheep red blood cells (SRBC), a T-lymphocyte and macrophage dependent response, was suppressed in spleen cell cultures that contained cadmium-exposed non-adherent cells (lymphocytes) . Cultures containing cadmium-exposed adherent cells (macrophages) were not suppressed by cadmium . These results suggest that the immunosuppressive effects of cadmium as it relates to humoral immunity involve T-lymphocyte function rather than macrophage or B-lymphocyte activity . The enhanced T-lymphocyte independent antibody responses which accompany suppressed T-lymphocyte-dependent responses following cadmium exposure are an indication of compensatory mechanisms that are associated with the immune system.

Gene, 1986, 41(2-3), 201 - 6
Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes; McCarthy JE et al.; The cDNA sequences encoding mature human interleukin 2 (IL2) and beta-interferon (INF beta), respectively, were fused with various translational initiation regions and inserted into two different types of expression vector . The relative levels of expression of the two genes and the functional stability of their respective mRNAs were examined in vivo in Escherichia coli hosts . The addition of the 30-bp sequence, found immediately upstream of the E . coli atpE gene Shine-Dalgarno (SD) sequence, to the translational initiation regions of IL2 and INF beta increased the expression of both these genes by a factor of 6-10 . Thus this sequence, which naturally acts within the E . coli atp operon to enhance the translational initiation frequency of the atpE gene, can increase the expression of other genes in E . coli . It may exemplify a specific type of recognition signal for the E . coli translational apparatus.

Int J Immunopharmacol, 1986, 8(1), 93 - 9
The effect of oral exposure to the n-butylester of 2,4-dichlorophenoxyacetic acid on the immune response in mice; Blakley BR; Six week old female BDF1 mice were administered the n-butylester of 2,4-dichlorophenoxyacetic acid (2,4-D) by gastric intubation at dosages ranging from 0 to 200 mg/kg (2,4-D content) in the acute studies and 0 to 100 mg/kg in the subacute studies . Following acute exposure, the antibody production against sheep red blood cells and the induction of DNA synthesis with Escherichia coli lipopolysaccharide, a B-lymphocyte mitogen, were enhanced . Comparable T-lymphocyte mitogen responses induced by concanavalin A, a T-lymphocyte mitogen, were not affected by 2,4-D, though 2,4-D did exhibit a weak, non-specific, dose-related mitogenic effect . Subacute exposure to the 2,4-D ester did not alter antibody production . B-lymphocyte mitogen responses were enhanced, though a linear dose-response relationship was not observed . Histopathological alterations in the brain and spinal cord and clinical symptoms of toxicity were observed in the 200 mg/kg treatment group (acute exposure) . Similar effects were not seen in the subacute study . The immunostimulatory effects of 2,4-D were observed at relatively high exposures . It is unlikely that these immune alterations will have any major toxicological significance in agricultural communities utilizing 2,4-D herbicides, though further studies with commercial grade formulations which may contain other compounds in addition to the pure 2,4-D esters must be evaluated at similar levels of exposure.

Vet Immunol Immunopathol, 1986 Jan, 11(1), 91 - 100
Response of bovine and porcine peripheral blood mononuclear cells to human recombinant interleukin 2(125); Fong S et al.; Bovine and porcine peripheral blood mononuclear cells (PBMC) were tested for their response to human recombinant interleukin 2(125) (rIL 2(125)) . The rIL 2(125) used in these experiments was purified to homogeneity from Escherichia coli, contained a site-specific modification at amino acid #125 replacing a cysteine with a serine residue and had a specific activity of 4 X 10(6) units/mg . Human rIL 2(125) was shown to be directly mitogenic for bovine and porcine PBMC and was able to maintain the long-term growth of mitogen-activated PBMC of both species . Long-term cultures were highly sensitive to low levels of rIL 2(125) and showed dose-dependent responses when used in short-term IL 2 assays . Bovine and porcine PBMC preincubated with human rIL 2(125) for 1 and 5 days demonstrated enhanced levels of cell-mediated cytotoxicity against both allogeneic and xenogeneic cell lines.

Avian Dis, 1986 Jan-Mar, 30(1), 37 - 42
Use of hybridoma antibodies and recombinant DNA technology in protozoan vaccine development; Danforth HD et al.; The use of hybridoma antibodies developed against the sporozoite stage of avian coccidia, coupled with genetic-engineering techniques, has made it possible to begin bird-immunization studies utilizing an Escherichia coli-elicited coccidial protein . The coccidia are currently controlled in the poultry industry by use of anticoccidial compounds, but it now may be possible to use the bird's own immune system for defense against the parasitic infection . Since the sporozoite stage, which initiates the infection in poultry, is quite complex and is made up of hundreds of proteins or antigens, hybridoma antibodies were produced to identify specific antigens . These antigens, once identified, were found in such minute amounts that it became necessary to utilize genetic engineering in order to produce enough protein for immunization studies . One such protein, designated 5401, has been shown to stimulate an antibody response in immunized birds and to impart partial protection against a coccidial challenge infection . The results of these studies indicate that development of a vaccine against coccidial parasites may someday be possible.

Proc Natl Acad Sci U S A, 1986 Jan, 83(1), 28 - 32
O4-Methyl, -ethyl, or -isopropyl substituents on thymidine in poly(dA-dT) all lead to transitions upon replication; Singer B et al.; In a previous paper, we reported that O4-methyl dTTP can be incorporated into poly(dA-dT) in place of thymidine without distortion of the helical structure, but on replication it could behave as deoxycytidine and misincorporate dGTP . Only weak interactions are possible for any O4-modified T X A pair . While O4-alkyl T X G pairing should be favored, experiments to detect the ability of Escherichia coli DNA polymerase I (pol I) to utilize the triphosphate as dCTP were ambiguous . dTTPs with larger alkyl groups (ethyl, isopropyl) have now been synthesized and tested for their recognition as dTTP by pol I . Enhanced steric hindrance could be expected, particularly for O4-isopropyl dTTP, which has a three-carbon branched chain . However, both compounds behaved qualitatively like O4-methyl dTTP, being incorporated into poly(dA-dT) and then directing deoxyguanosine misincorporation by pol I . Quantitative comparisons of mutagenicity were not possible because of the finding that, unlike polymers made with O4-methyl dTTP, those made with ethyl or isopropyl dTTP were resistant to hydrolysis by using a variety of nucleases . The frequent misincorporations of dGTP would be expected to produce transitions in vivo . O4-ethyldeoxythymidine is very poorly repaired in vivo, which would also be expected for repair of O4-isopropyldeoxythymidine . Therefore, under suitable conditions, these particular carcinogen products are likely to be initiators of carcinogenesis.

Am J Physiol Imaging, 1986, 1(4), 181 - 5
Assessment of cardiopulmonary alterations in endotoxic shock; Christenson JT et al.; Using radionuclide first-pass technique, seven sheep were studied prior to and during endotoxic shock . In vitro labeled 99mTC red blood cells were used to measure right and left ventricular ejection fractions (RVEF and LVEF) and cardiopulmonary transit time (CPTT) prior to and at 1, 4, 30, and 60 minutes after the intravenous injection of endotoxin Escherichia coli, 3 mg/kg bodyweight . The LVEF decreased slightly from 51 to 42% but became normal four minutes later, while the RVEF remained decreased from 49 to 26% for 60 minutes . The CPTT was prolonged from 13 to 26 cardiac beats . The decrease in RVEF was well correlated with the prolongation of the CPTT.

Curr Genet, 1986, 10(12), 903 - 7
Structural organization of the genome of the zygomycete Absidia glauca: evidence for high repetitive DNA content; Wostemeyer J et al.; Total cell DNA of Absidia glauca has a GC-content of 44.6% +/- 0.5% as determined from optical melting profiles which is in good accordance with values from equilibrium centrifugation in bisbenzimide containing CsCl gradients (46.2% +/- 1.1%), whereas mitochondrial DNA has a GC-content of only 30% . The genome size of Absidia glauca is approximately 36,000 kb, 8.6 times that of Escherichia coli . Three kinetically different fractions could be identified in reassociation experiments: a foldback-DNA fraction, comprising approximately 10% of the total DNA, repetitive DNA (25%) and single copy DNA (65%) . This relatively high amount of repetitive DNA could partly be ascribed to ribosomal DNA (13%) and a new interspersed repetitive element ("rAg1") which has been cloned in pBR325.

Microcirc Endothelium Lymphatics, 1986-87, 3(3-4), 231 - 40
Terbutaline increases survival after endotoxin shock . A comparative study of methyl prednisolone, isoprenaline and terbutaline in rats; Sigurdsson GH et al.; Since beta 2-receptor agonists and corticosteroids can counteract inflammatory mediator induced protein leakage and corticosteroids have been proposed as effective in treatment of experimental septic shock, we compared the effects of these agents on rats in endotoxin shock . Sixty adult wistar rats were anaesthetised with thiopentone sodium intra-peritoneally (i.p.), before shock was induced by E coli endotoxin (3 mg/kg bwt i.v.) . Ten minutes later the rats were divided into four groups (n = 15 in each) and received either normal saline (controls) 4 ml/kg i.p., methyl prednisolone (MP) 40 mg/kg i.p., isoprenaline (ISO) 200 micrograms/kg i.p . or terbutaline (TER) 200 micrograms/kg i.p . 25% of the initial dose was repeated after 15, 60 and 120 minutes . After 2 hours observation 3-5 animals had died in each group (n.s.), but at the end of the observation period (96 hrs), 87% of the controls, 73% of the MP-group, 53% of the ISO-group and 20% of the TER-group had died . At this time all the surviving animals were looking well and behaving normally . It was concluded that terbutaline significantly increased the survival of rats exposed to a lethal dose of endotoxin compared with controls (p less than 0.001) and compared with methyl prednisolon (p less than 0.05) . The mechanism may be that of decreased pulmonary capillary leakage with terbutaline . However, the survival rate was not significantly improved with methyl prednisolon or isoprenaline.

J Enzyme Inhib, 1986, 1(2), 151 - 61
Interaction between L-aspartic acid and L-asparaginase from Escherichia coli: binding and inhibition studies; Jayaram HN et al.; Experiments using equilibrium dialysis and fluorescence quenching provided direct evidence that approximately four moles of L-aspartic acid were bound per mole of tetrameric L-asparaginase from Escherichia coli, with a dissociation constant on the order of 60-160 microM . In addition, a set of weaker binding sites with a dissociation constant in the millimolar range were detected . Kinetic studies also revealed that L-aspartic acid inhibited L-asparaginase competitively, with an inhibition constant of 80 microM at micromolar concentrations of L-asparagine; at millimolar concentrations of the amide, an increase in maximal velocity but a decrease in affinity for L-asparagine were observed . L-Aspartic acid at millimolar levels again displayed competitive inhibition . These and other observations suggest that L-aspartic acid binds not only to the active site but also a second site with lower intrinsic affinity for it . The observed "substrate activation" is most likely attributable to the binding of a second molecule of L-asparagine rather than negative cooperativity among the tight sites of the subunits of this tetrameric enzyme . Further support for L-aspartic acid binding to the active site comes from experiments in which the enzyme, when exposed to various group-specific reagents suffered parallel loss of catalytic activity and in its ability to bind L-aspartic acid . Different commercial preparations of Escherichia coli L-asparaginase were found to contain approximately 2-4 moles of L-aspartic acid; these were incompletely removed by dialysis, but could be removed by transamination or decarboxylation . Efficiency of dialysis increased with increasing pH . Taken together, this set of results is consistent with the existence of a covalent beta-aspartyl enzyme intermediate.

Gene Amplif Anal, 1986, 4, 53 - 72
Structure and function of p21 ras proteins; Shih TY et al.; Cancer is a malfunction of cellular growth control . The discovery of oncogenes, first in transforming retroviruses, and later in human and animal tumors, may have uncovered the key to understanding one of the most elusive subjects of basic cell biology, namely, the controlling mechanisms of cell growth . The ras gene family encodes a group of closely related 21,000 dalton (p21) proteins with special affinity for guanine nucleotides . Other cellular proteins with similar biochemical properties, collectively known as G-proteins, include the regulatory G proteins of adenylate cyclase, the alpha subunit of transducin of retina rod outer segments, the recently identified rho gene proteins, and perhaps also the elongation factors, EF-Tu and EF-G, of the protein synthesis system . These G-proteins have roles in cellular signal transduction; by analogy p21 may have a similar cellular function in mediating the flow of growth control signals . Recent progress in the cloning and sequencing of these genes, overproduction of gene products in E . coli, protein engineering, detailed biochemical characterization, and the molecular structure determined by high resolution X-ray crystallography, have helped to elucidate in great detail the structure and function of p21 ras proteins . p21 appears to have a small membrane binding domain at the C-terminus, which contains a palmitylation site at cysteine-186, four amino acid residues from the end . Separated by a variable "hinge" region, most of the rest of ras amino acid sequences are highly conserved in nature . Four regions of extensive sequence homology among G-proteins constitute the GTP/GDP binding domain . In the crystal structure of EF-Tu, four peptide loops connecting beta sheets and alpha helices form the pocket for binding GDP . Studies using site-directed mutagenesis and immnochemical probes, indicate that the basic structure of the GDP binding site is conserved between p21 and EF-Tu . Furthermore, these studies also conclude that GTP binding is crucial for p21 ras cellular function . Although the precise target molecules for p21 are still unknown, the finding of the on/off switch function for ras genes have provided a better understanding of the mechanism of proto-oncogene activation, and may also provide further impetus to explore means of cancer intervention by interfering with the switch function.

Free Radic Res Commun, 1986, 1(6), 395 - 403
Anti-inflammatory activity of superoxide dismutases: inhibition of carrageenan induced edema in rats; Jadot G et al.; Eighteen different superoxide dismutases from procaryote, plant, fish, bird and mammalian species have been tested for anti-inflammatory activity in the rat paw pad carrageenan-induced inflammation model . Very large differences in activity are observed . Homologous rat Cu-SOD is not active and indeed shows slight pro-inflammatory activity . The different SODs have different iso-electric values, different metals (Cu, Mn or Fe) at the active centre, different molecular weights and different circulation lifetimes . Biological activity is a function of amino acid sequence rather than of such secondary parameters.

Free Radic Res Commun, 1986, 2(1-2), 43 - 56
Anti-inflammatory activity of superoxide dismutases: comparison of enzymes from different sources in different models in rats: mechanism of action; Michelson AM et al.; Comparison of the anti-inflammatory properties of superoxide dismutases from different sources using different models (carrageenan and adriamycin induced inflammation, adjuvant-induced arthritis) in rats shows a very wide range of activity from extremely good to zero . Neither circulating life time nor intracellular penetration are of importance . The mechanism of biological activity of the SODs is discussed in detail, and binding to an interphase situation on the outer cell surface is postulated . As a consequence of these various considerations it is predicted that clinical application of human Cu-SOD in humans may well be much less spectacular than is commonly assumed, and indeed may be somewhat disappointing.

Acta Microbiol Hung, 1986, 33(4), 311 - 5
Comparison of adjuvanticity and autoantibody inducing capacity of endotoxin and radio-detoxified endotoxin in mice; Klimovich VB et al.; Endotoxin (LPS) and radio-detoxified endotoxin (RD-LPS: 150 kGy 60Co-gamma irradiated) preparations were compared in mice (C57Bl X CBA: F1/Rapo) for capacity to enhance the immune response against sheep red blood cells and to induce the production of antibody against autologous (bromelain-treated) erythrocytes . As RD-LPS retains its capacity to stimulate immune response against heterologous antigen, it may be used as an immuno-adjuvant . The LPS preparation gave rise to a significant increase of autoreactive cells . However, RD-LPS activated the autoantibody forming cells only to a very small degree.

Acta Paediatr Scand Suppl, 1986, 325, 33 - 40
Clinical experience with somatrem in growth hormone deficiency; Vicens-Calvet E et al.; Three studies of human growth hormone (hGH) in hGH deficiency were initiated . In the first of these, adolescent patients were switched from pituitary hGH to somatrem (SI preparation) for 1 month . No significant differences were noted in any of the clinical parameters measured during treatment with either preparation . In the second study, nine patients (six of them naive) were treated with somatrem (SII preparation) for 9-12 months . The naive patients exhibited catch-up growth, and bone age developed in parallel to chronological age during the study period . Somatomedin activity increased and correlated positively with growth . Antibodies to hGH and Escherichia coli polypeptide (ECP) developed in some patients, but titres and binding capacities were low . In the third study, 21 patients are currently being treated with Somatonorm; the first 3-6 months are evaluable . Growth velocities increased to normal values . Antibodies to hGH and ECP were present in several patients, but again the titres and binding capacities were low, and Somatonorm was less antigenic than the SI and SII preparations.

Acta Paediatr Scand Suppl, 1986, 325, 29 - 32
Clinical experience with Somatonorm; Girard F et al.; A total of 21 patients with hGH deficiency was included in a double-blind, randomized, comparative multicentre trial of somatrem (Somatonorm) and pituitary hGH . Owing to the withdrawal of the latter product, only 6 months follow-up can be evaluated . Growth velocities on both preparations were similar, and patients with complete hGH deficiency responded better than those with only partial deficiency . No specific side-effects were noted, but antibodies to hGH were noted in two patients on Somatonorm . Antibodies to Escherichia coli polypeptide (ECP) were found in both groups . In an open study of 14 children treated with Somatonorm, growth velocity increased from a mean of 3.26 +/- 0.42 cm/year to 7.1 +/- 0.47 cm/year during the 1 year of follow-up; in this time, bone age increased by 10 months . No clinical or biological abnormalities were detected, though 50% of the children developed anti-hGH antibodies and 13 out of 14 developed Z scores greater than 0.1 for ECP antibodies, compared with 5 out of 14 before treatment.

Acta Paediatr Scand Suppl, 1986, 325, 13 - 8
Treatment of pituitary dwarfism with biosynthetic growth hormone; Bierich JR; Over 3 1/2 years, 54 previously untreated hypopituitary children were enrolled in three studies; 48 had idiopathic pituitary dwarfism . Three preparations of somatrem were used (SI, SII, Somatonorm) . Growth velocities on all three preparations were similar, though SI and SII tended to produce slightly higher growth velocities . Serum somatomedin and alkaline phosphatase levels increased during treatment . Antibodies to Escherichia coli polypeptide (ECP) increased to a maximum Z score of about 0.7 with SI, but this was considerably lower with SII and below 0.1 with Somatonorm . Anti-hGH antibodies had the highest titre in children treated with SI and these antibodies also had quite high binding capacities . Anti-hGH antibody formation was negligible during Somatonorm treatment.

J Free Radic Biol Med, 1986, 2(4), 245 - 8
The effects of paraquat on Escherichia coli: distinction between bacteriostasis and lethality; Kitzler J et al.; Paraquat exerted a progressively more pronounced bacteriostatic effect on Escherichia coli as its concentration was raised in the range 0-1.0 microM . In contrast, concentrations of 100 microM or greater were required before significant lethality could be observed . This bacteriostatic effect of paraquat could be eliminated by supplementation of the glucose-plus-salts medium with either yeast extract or a casein hydrolysate . This protection was seen whether the supplement was added a few minutes prior to or following the addition of paraquat and was thus not due to the inhibition of active uptake of paraquat by the cells . The lethal effect of high levels of paraquat was not influenced by supplementation of the medium with yeast extract . It follows that the bacteriostatic and lethal effects of paraquat involve attack upon distinct targets within the cell.

Curr Genet, 1986, 11(3), 211 - 5
Hyperresistance to DNA damaging agents in yeast; Ruhland A et al.; In order to study resistance to DNA damaging agents, yeast DNA segments conferring hyperresistance in this organism to such genotoxic agents were selected for among yeast cells transformed by a yeast genome library based on the multi-copy vector plasmid YEp13 . Genetic variants hyperresistant to 4-nitroquinoline-N-oxide, formaldehyde, and alkylating agents were isolated and the respective hyperresistance determinants shown to co-segregate with the vector plasmid . Phenotypical characterization indicated different degrees of resistance, few cases of cross-resistance and differing structural stability of the cloned DNA . By transfer to E . coli and subsequent retransformation of yeast a number of plasmids was shown to stably carry the genetic information for hyperresistance.

Gene, 1986, 49(1), 61 - 8
Expression in Escherichia coli of synthetic human interleukin-1 alpha genes encoding the processed active protein, mutant proteins, and beta-galactosidase fusion proteins; Zurawski SM et al.; We have synthesized, cloned, and expressed the coding region for the C-terminal 159 amino acids (aa) of the human active interleukin polypeptide hormone IL-1 alpha . The sequence was assembled in stages and includes preferred Escherichia coli codons and unique restriction sites . The coding region was cloned on a multicopy plasmid vector adjacent to signals for transcription and translation that directed synthesis of 6% of total E . coli protein as IL-1 alpha . Active IL-1 alpha mutants that have a C-terminal additional eleven aa and that have N-terminal deletions of six and fourteen aa are described . Plasmids expressing beta-galactosidase fusion proteins with various parts of IL-1 alpha at their N-termini were constructed.

Gene, 1986, 47(2-3), 193 - 9
Vaccinia virus vectors utilizing the beta-galactosidase assay for rapid selection of recombinant viruses and measurement of gene expression; Panicali D et al.; Plasmids were constructed fusing vaccinia transcriptional regulatory sequences (promoters) to the lacZ gene of Escherichia coli . These recombinant plasmids were used to compare relative promoter strengths in transient expression assays and to construct recombinant vaccinia viruses producing beta-galactosidase (beta Gal) . Viruses synthesizing beta Gal were determined by utilizing the chromogenic substrate, 5-bromo-4-chloro-3-indoyl-beta-D-galactoside to form blue plaques . A recombinant virus producing beta Gal was then used to select a second recombinant virus . This was accomplished via in vivo recombination replacing the lacZ gene with a sequence coding for the gp85 protein of Friend murine leukemia virus . The recombinant virus was selected by its inability to form blue plaques under appropriate conditions.

Microbiol Immunol, 1986, 30(11), 1129 - 39
A monoclonal antibody with broad reactivity to human interferon-alpha subtypes useful for purification of leukocyte-derived interferon; Tsukui K et al.; A monoclonal antibody to human interferon-alpha, termed HT-1 antibody, with a broad reactivity to various subtypes of interferon-alpha was prepared . It bound and neutralized all of the four subtypes of E . coli-derived human recombinant interferon-alpha (alpha 1, alpha 2, alpha 4, and alpha 6) tested; it also neutralized human natural leukocyte interferon but only partially . Human interferon-beta and -gamma were not bound . The antibody conjugated to Sepharose beads retained over 90% of human leukocyte interferon induced by Sendai virus . The bound interferon was recovered by acid elution in good yields and in almost pure form (specific activity was about 2 X 10(8) international units/mg protein) . The purified interferon showed, in SDS-polyacrylamide gel electrophoresis, an activity profile with major peaks in a mol . wt . range of 17,000-22,000, which completely agreed with the profile shown by polyclonal antibody-purified interferon . Such purified leukocyte interferon-alpha preparations containing most of the naturally occurring subtypes can be useful for clinical and other purposes.

J Mol Evol, 1986, 23(3), 259 - 66
An evolvant of Escherichia coli that employs the L-fucose pathway also for growth on L-galactose and D-arabinose; Zhu Y et al.; L-Galactose, D-arabinose, and L-fucose form six-membered rings with identical stereoconfigurations . However, only L-fucose can serve as the sole carbon and energy source of wild-type Escherichia coli K-12 . A mutant that can grow on L-galactose and D-arabinose was isolated by alternate selection on the two sugars . The L-fucose pathway became inducible by all three sugars . Transduction into the mutant of the wild-type fuc+ region containing both the regulatory and structural genes abolished the novel growth abilities on L-galactose and D-arabinose, whereas transduction into the mutant of a fuc deletion abolished the growth abilities on all three sugars . Introduction of the wild-type fucR+ (which encodes the activator protein for the fuc regulon) on a multicopy plasmid depressed the growth abilities of the mutant on L-galactose and D-arabinose, but not on L-fucose . The results suggest that the effector specificity of the activator protein in the mutant was broadened . It is proposed that an adaptive response of an activator-controlled system is more likely than that of a repressor-controlled system to achieve fixation in a population, because the first variant to emerge in response to a novel metabolic demand has a good chance of having an altered specificity of regulation . Such a change entails little or no metabolic liability during the absence of the novel substrate . In contrast, the first variant of a negatively controlled system to emerge has an overwhelming chance of being the result of a random mutation that destroys repressor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1986, 46(1), 123 - 6
A linear shuttle vector for yeast and the hypotrichous ciliate Stylonychia; Ascenzioni F et al.; A linear plasmid was constructed in vitro using the telomeres of the rDNA of Tetrahymena pyriformis . These telomeres were added to a yeast circular vector containing an ARS sequence from Dictyostelium, the LEU2 gene of yeast and the neo gene from Escherichia coli Tn5 fused with a eukaryotic promoter . The resulting plasmid was used to transform yeast . During the replication of the linear plasmid in yeast it was spontaneously modified at the extremity by the addition of 300 bp of yeast telomeric sequence for each end . Total DNA prepared from yeast transformants was used to transform the hypotrichous ciliate Stylonychia lemnae . The same plasmid isolated from Stylonychia can again be replicated in yeast.

Gene, 1986, 45(1), 113 - 6
Molecular cloning of the phosphoenolpyruvate carboxylase gene of Anabaena variabilis; Harrington TR et al.; Purified Anabaena variabilis chromosomal DNA was partially digested with restriction endonuclease Sau3A and ligated into the BamHI site of plasmid pBR322 . Escherichia coli 342-167, a mutant with a decreased level of phosphoenolpyruvate carboxylase (PEPCase) activity was transformed with plasmids from the A . variabilis genomic library . A transformant that grew on minimal media in the absence of glutamate was isolated and its plasmid, pTRH1, was shown to encode the A . variabilis PEPCase . E . coli HB101 cells transformed with plasmid pTRH1 have approx . 50 times the normal amount of PEPCase activity and also overproduce a protein with the apparent Mr (99,000) of the A . variabilis PEPCase.

Gen Pharmacol, 1986, 17(5), 611 - 4
The effect of 6-hydroxydopamine pretreatment on the metabolic response produced by endotoxin in rabbits; Gagalo IT et al.; The thermoregulatory, effector processes were investigated in rabbits after treatment with 6-hydroxydopamine (6-OHDA) and lipopolysaccharide Escherichia coli (LPS) . Pyrogen (1 microgram/kg, i.v.) produced a fever reaction resulting from stimulation of the metabolic rate and heat conservation responses . Pretreatment with 6-OHDA (3 X 500 micrograms, i.c.v.) reduced the metabolic as well as pyretic activity of pyrogen . It is suggested that stimulation of the thermoregulatory heat production which contributes to the febrile rise in body temperature is dependent on the intact adrenergic structures in the central nervous system.

Circ Shock, 1986, 20(1), 25 - 34
Release of eicosanoids from white blood cells, platelets, smooth muscle cells, and endothelial cells in response to endotoxin and A23187; Bottoms GD et al.; Endotoxin produces numerous pathophysiologic changes in animals, including vascular endothelial cell damage and hematologic changes . Direct effects of endotoxin on arachidonic acid metabolism and the release of eicosanoids from endothelial cells and neutrophils have been reported . A rapid release of these autocoids occurs when cells are incubated with endotoxin, and this appears to be one of the earliest endotoxin-induced changes . Some of these eicosanoids may result in beneficial effects, and others may result in detrimental effects . This study was to determine the release of eicosanoids from white blood cells, platelets, smooth muscle cells, and endothelial cells in response to varying amounts of endotoxin and the calcium ionophore A23187 . The results indicate that endotoxin has a major direct effect on vascular endothelial cells and smooth muscle cells as indicated by its ability to increase the synthesis of predominately i6-keto-PGF1 alpha by these cells . These effects were seen within a dose range of endotoxin that is lethal in horses . Very high concentrations of endotoxin (100 micrograms/ml) were required to stimulate a small increase in the production of i6-keto-PGF1 alpha and iLTC4 by freshly isolated neutrophils . Stimulation of cells with A23187 revealed that, of the eicosanoids measured, the one produced predominately by endothelial cells and smooth muscle cells was 6-keto-PGF1 alpha, by platelets was TxB2, and by neutrophils was LTC4 (LTB4 was not measured) . A mixture of all white blood cells including platelets when incubated with A23187 produced large amounts of TxB2, LTB4, and LTC4 with smaller amounts of 6-keto-PGF1 alpha . The results indicate that endotoxin directly affects cells and stimulates them to produce thromboxane and prostacyclin, but very high concentrations of endotoxin were required to stimulate neutrophils to produce rather small increases in iLTC4.

Gene, 1986, 43(3), 281 - 6
Plasmid vectors useful in the study of translation initiation signals; Wyckoff E et al.; The construction and characterization of plasmid vectors useful in the analysis of translation initiation signals in Escherichia coli and in the construction of lacZ gene hybrids are described . Transcription on the vectors proceeds from a cAMP-independent lac promoter through several restriction sites into a truncated lacZ structural gene lacking its first eight codons . Because this gene has no translation initiation signal, its level of expression is extremely low . A DNA fragment containing a translation start signal can be inserted between the promoter and truncated lacZ gene to produce a hybrid protein with functional beta-galactosidase activity . The vectors described here differ in sequence between the EcoRI cloning site and the lacZ gene to allow easy, in-frame joining of DNA containing a translation initiation signal to the lacZ gene . Cells containing plasmids can be screened directly for in-frame inserts by colony color on indicator plates.

Histochemistry, 1986, 85(1), 81 - 7
Controlled growth of colloidal gold particles and implications for labelling efficiency; van Bergen en Henegouwen PM et al.; A new method is reported for the preparation of colloidal gold particles with diameters ranging between 5 and 12 nm . The initial gold particle population, with an average diameter of 5.6 +/- 0.9 nm, is prepared by reduction of chloroauric acid with white phosphorous . An increase in particle diameter by growth is obtained by reduction of chloroauric acid with white phosphorous in the presence of colloidal gold particles . The labelling efficiency of these gold particles, conjugated with protein A, in indirect immunolabelling experiments is investigated by labelling of beta-galactosidase on ultrathin cryosections of Escherichia coli cells . We demonstrate that the labelling efficiency is at least dependent on particle diameter, probe concentration and preparation method . In addition it is shown, that with this new method, gold particle populations can be prepared with minor overlap in diameter spreading . Therefore these gold probes are suitable for qualitative double labelling experiments . The quantitative aspect of immunolabelling is discussed.

Gene, 1986, 42(1), 97 - 100
Expression vectors based on the rac fusion promoter; Boros I et al.; The -35 region of the rrnB P2 promoter and the -10 region of the lacZpo promoter-operator were fused to form the strong and regulatable rac promoter . Vectors were constructed that allow the attachment of protein-coding sequences to the beta-galactosidase alpha-peptide (LacZ alpha) in any reading frame . By introducing a high-copy-number mutation, the synthesis of a LacZ alpha-chloramphenicol acetyltransferase fusion protein reached more than 60% of total cell protein in Escherichia coli.

Crit Rev Immunol, 1986, 6(1), 47 - 69
Gene expression and structure of immunoglobulin epsilon chains; Liu FT; The study of immunoglobulin genes has been an active area of research in recent years . The study of IgE heavy (epsilon) chain gene, in particular, has a significant impact on our understanding of the IgE system . The localization of the epsilon gene in the cluster of mouse heavy chain constant region genes has been determined, and models for gene rearrangements leading to epsilon chain gene expression have been provided . The regulation of IgE antibody production can thus be discussed in the context of these models in combination with knowledge of the cellular basis of IgE production . Comparison of the epsilon chain sequence among human, rat, and mouse has allowed a better understanding of the evolution and structure-function relationship of the epsilon chain . Furthermore, expression of epsilon chain cDNA in Escherichia coli and demonstration of biological activities of expression products have paved the basis for identification of defined peptides representing the structures for recognition by IgE receptors . These studies are discussed in this review.

J Mol Evol, 1986, 23(1), 53 - 60
Primary structure of the Paramecium tetraurelia small-subunit rRNA coding region: phylogenetic relationships within the Ciliophora; Sogin ML et al.; We have sequenced the coding region for the small-subunit rRNA gene from Paramecium tetraurelia . Similarity comparisons between small-subunit rRNAs from representatives of the Metazoa, the Plantae, the Fungi and four other members of the Ciliophora were used to construct phylogenetic trees . In these phylogenies the Ciliophora diverged from the eukaryotic line of descent as a loose phylogenetic grouping during a radiative period that gave rise to the Fungi, the Plantae and the Metazoa.

Gene, 1986, 41(1), 113 - 20
A reconstruction of the gene for ribulose bisphosphate carboxylase from Rhodospirillum rubrum that expresses the authentic enzyme in Escherichia coli; Larimer FW et al.; Escherichia coli plasmid pRR36, which expresses Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) as a fusion protein {Nargang et al., Mol . Gen . Genet . 193 (1984) 220-224}, was used to construct a new clone of the carboxylase gene (rbc) whose expression product is the wild-type enzyme . This construction entailed removing all lacZ-coding sequences and a portion of the 5'-noncoding leader of the R . rubrum rbc gene . The highest specific activity of carboxylase was observed with an expression vector which juxtaposed the trp-lac (tac) hybrid promoter with the R . rubrum ribosome binding site and the rbc structural gene . The carboxylase expressed in E . coli JM107 was purified to near homogeneity and, based on subunit Mr and specific enzymic activity, the isolated protein appeared indistinguishable from authentic ribulose bisphosphate carboxylase from R . rubrum . N-terminal sequence analyses of the cloned enzyme verified that the cloned and wild-type enzymes are the same.

Acta Anaesthesiol Scand, 1986 Jan, 30(1), 44 - 6
Circulatory effects of carbon dioxide in experimental endotoxic shock; Larsson LE et al.; In seven anaesthetized dogs the central circulatory effects of changes in Paco2 were studied before and after an intravenous injection of E . coli endotoxin 1.0 mg kg-1 . The animals were on controlled ventilation with constant minute volumes, and Paco2 was changed by variations in the inspired gas mixture . Before endotoxin was given, cardiac output, mean aortic pressure, mean pulmonary artery pressure, total peripheral resistance and stroke volume were little affected by the changes in Paco2 from 3.9 +/- 0.4 to 7.2 +/- 0.4 kPa (mean +/- s.e.mean) . Only heart rate decreased significantly (P less than 0.05) . The intravenous endotoxin injection resulted in a decreased cardiac output (P less than 0.01), a decreased mean aortic pressure (P less than 0.01) and a decreased stroke volume (P less than 0.05) . Mean pulmonary artery pressure, total peripheral resistance and heart rate showed only minor changes . In endotoxic shock an increase in Paco2 from 4.4 +/- 0.4 to 7.8 +/- 0.3 kPa (mean +/- s.e.mean) resulted in a significant increase in cardiac output (P less than 0.05), stroke volume (P less than 0.05) and mean pulmonary artery pressure (P less than 0.05), while the other parameters remained unchanged . It can be concluded that the carbon dioxide tension is of importance for the cardiac performance in experimental endotoxic stock in a manner not seen in control animals . The mechanisms behind these findings need further investigation.

Mol Gen Genet, 1986 Jan, 202(1), 112 - 9
The regulatory region of the uxuAB operon in Escherichia coli K12; Blanco C et al.; The uxuAB operon is composed of two genes coding for enzymes involved in hexuronate degradation . This operon is negatively controlled by the uxuR and exuR regulatory gene products . Starting from uxu hybrid plasmids, a 300 bp Sau3A restriction fragment was isolated and shown to contain the entire uxu regulatory region using in vitro gene fusions that brought lac gene expression under the control of the transcriptional and translational signals of the uxuA gene . The nucleotide sequence of this fragment was established . The start of the uxu mRNA was localized by S1 mapping experiments (the presence of a CAP binding site upstream of the promoter was shown by DNAse I footprinting and in vitro titration) . The N-terminal amino acid sequence of the purified uxuA-lacZ gene product allowed the mapping of the uxuA initiation codon at 115 bp from the start of transcription.

Jpn J Cancer Res, 1986 Jan, 77(1), 45 - 51
Synthesis and expression of a synthetic gene for the activated human c-Ha-ras protein; Miura K et al.; A 576 base-pair DNA encoding human c-Ha-ras protein (p21) with a valine codon at position 12 has been synthesized by ligation of chemically synthesized oligodeoxyribonucleotides . The duplex was inserted into a plasmid with the E . coli tryptophan promoter and was expressed in E . coli efficiently.

Pharmacol Res Commun, 1986 Jan, 18(1), 17 - 30
The acid-base balance during the time course of endotoxin fever and sodium salicylate antipyresis in the rabbit; Gagalo IT et al.; Blood acid-base balance in sodium salicylate antipyresis was investigated in adult rabbits at ambient temperature of 21.5 +/- 0.5 degrees C . The experimental fever elicited by iv injection of lipopolysaccharide Escherichia coli 1 microgram/kg/ was accompanied by a slight metabolic acidosis . A decrease in pH by 0.09 and HCO3- by 4.8 mEq/1 was noticed during the rising phase of pyrogen fever . There were no concomitant changes in blood PCO2 during that period of time . Although the concentrations of HCO3- were decreasing till the end of the experiment, the parallel falls in PCO2 led to a partial compensation of the noticed acidosis . Pretreatment with 200 mg/kg of sodium salicylate /an hour's iv infusion/reduced the febrile response by 42% and completely reversed the postpyrogen changes in pH and HCO3- . The falls in PCO2 during antipyresis, however, were similar to those observed in febrile rabbits . Possible mechanisms by which sodium salicylate could affect the pyrogen-induced disturbances of acid-base balance are being considered.

Eur J Immunol, 1986 Jan, 16(1), 35 - 40
Monocyte-dependent induction of proliferation of human peripheral T cells by recombinant interleukin 2; Roosnek EE et al.; The in vitro proliferation of human peripheral lymphocytes induced by interleukin 2 (IL2), a product of cDNA, cloned in E . coli has been studied . The maximal mitogenic signal is given by concentrations greater than or equal to 2.5 U/ml . Due to growth factor consumption, at least 10 U/ml are needed to maintain a logarithmic response until day 6 . The anti-Tac antibody, directed against the IL2 receptor, effectively blocks this response, but we could not obtain a decrease of IL2-reactive cells by depletion of putative in vivo activated Tac+ cells, using this antibody and a fluorescence-activated cell sorter . Depletion of Leu7+ and Leu11b+ cells does not cause a decrease of the response, which indicates that the responding cells are not confined to the natural killer lineage . By simultaneous staining of cell-surface markers and DNA, the nature of the proliferating cell was determined . More than 90% of the dividing cells expressed HLA class II and the Tac antigen, whereas the lymphocyte populations, defined by the surface markers Leu2, Leu3, Leu4, Leu7 and Leu11b, were all represented in the dividing cells . The magnitude of the response was proportional to the number of monocytes present in the culture . Depletion of monocytes completely abrogated the response, whereas an increase in the number of monocytes to a 1:1 ratio with lymphocytes caused a 2-fold increase in proliferation . However, purified T cells do proliferate to IL2 when cultured in the presence of a supernatant that was harvested from a 2-day culture of adherent monocytes . The proliferation-inducing activity in the supernatant eluted with apparent molecular weights of 15 000 and 75 000 on an Ultrogel AcA-54 column . Therefore, we conclude that in vitro, in the presence of an IL1-like activity produced by monocytes, IL2 is mitogenic for a population of T cells.

Am J Vet Res, 1986 Jan, 47(1), 110 - 3
Modulation of arachidonic acid metabolism in endotoxic horses: comparison of flunixin meglumine, phenylbutazone, and a selective thromboxane synthetase inhibitor; Moore JN et al.; Two cyclooxygenase inhibitors (flunixin meglumine and phenylbutazone) and a selective thromboxane synthetase inhibitor were assessed in the management of experimental equine endotoxemia . Drugs or saline solution were administered to 16 horses 15 minutes before administration of a sublethal dose of endotoxin (Escherichia coli 055:B5) . Plasma concentrations of thromboxane B2 (TxB2), prostacyclin (6-keto PGF1 alpha), plasma lactate, and hematologic values and clinical appearance were monitored for 3 hours after endotoxin administration . Pretreatment with flunixin meglumine (1 mg/kg of body weight) prevented most of the endotoxin-induced changes and correlated with a significant decrease in plasma TxB2 and 6-keto PGF1 alpha concentrations, compared with concentrations in nontreated horses (ie, pretreated with saline solution) . Pretreatment with phenylbutazone (2 mg/kg) attenuated the effects of endotoxin and was associated with a brief, early, significant increase in plasma TxB2 concentrations, but not in plasma 6-keto PGF1 alpha concentrations . Pretreatment with the thromboxane synthetase inhibitor did not appear to clinically benefit the horses involved; however, arachidonic acid metabolism was redirected to prostacyclin production.

Vet Pathol, 1986 Jan, 23(1), 29 - 34
Ileal Peyer's patches in experimental infections of calves with rotaviruses and enterotoxigenic Escherichia coli: a light and electron microscopic and enzyme histochemical study; Pospischil A et al.; The effect of rotaviruses and enterotoxigenic Escherichia coli administered in various sequences to cesarean-derived, colostrum-deprived calves was studied using light and electron microscopy . The structure of the lymphoid tissue in the ileum, the number of mitoses in the crypts, number of intraepithelial lymphocytes, and enzyme histochemistry (alkaline phosphatase, acid phosphatase, succinic dehydrogenase, beta-galactosidase, and leucinaminopeptidase) of the ileal dome epithelium were evaluated . The area of lymphoid follicles in Peyer's patches of the ileum was investigated morphometrically . Monoinfections with either rotavirus or enterotoxigenic E . coli induced a significant increase in lymphoid follicle area, but did not affect dome epithelial cells . Dual infections did not consistently affect the follicle area, but the number of intraepithelial lymphocytes and the mitotic indices exceeded those of comparable monoinfections . Changes in activity of enzymes in the ileal dome epithelial area were minor.

Genetics, 1986 Jan, 112(1), 11 - 26
Escherichia coli gene induction by alkylation treatment; Volkert MR et al.; Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased beta-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation . In this report we describe gene fusions that are specifically induced by alkylation treatments . Nine new mutants are described, and their properties are compared with the five mutants described previously . The total of 14 fusion mutants map at five distinct genetic loci . They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) . alkA, aidB and aidD are induced by both agents and appear to be regulated by ada . Neither aidC nor aidI is regulated by ada . Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated . Thus, there appear to be at least three different regulatory mechanisms controlling aid genes.

Circ Shock, 1986, 18(1), 43 - 52
Lack of defective cardiac oxidative metabolism in intact dogs subjected to a prolonged low-dose infusion of Escherichia coli endotoxin; D'Orio V et al.; The aim of the present study was to determine possible direct adverse effects of a 2-hour Escherichia coli endotoxin infusion (50 ng kg-1 min-1) on myocardial oxidative carbohydrate metabolism . The experiments were performed in intact dogs to assay glucose and lactate cardiac uptake and relate them to oxygen consumption (MVO2), CO2 production, and myocardial hemodynamics . Coronary sinus blood flow (CSBF) was measured by thermodilution, and the arteriovenous differences in glucose, lactate, pyruvate, O2, and CO2 were determined by blood samples obtained simultaneously from the carotid artery and sinus coronary . The adequacy of CSBF in meeting cardiac oxygen needs was evaluated by calculating the percentage of anaerobic metabolic rate (% AMR) . During endotoxin infusion, CSBF was significantly lowered by 33% while mean aortic blood pressure was decreased by 43% . Cardiac index exhibited a minimal reduction of 14% . Mean arterial blood glucose decreased 30% and arterial lactate increased 100% . Despite the progressively developing hypoglycemia, cardiac glucose uptake increased 140% . Although MVO2 was reduced to 70% of control value, lactate uptake increased 50% . Throughout the experimental period, the % AMR remained negative . Under endotoxin infusion, up to 78% of the cardiac CO2 production was derived from carbohydrate utilization, as compared to 40% prior to endotoxin infusion . Our findings suggest the absence of any toxic action by an endotoxin-sustained infusion on cardiac oxidative metabolism.

Circ Shock, 1986, 18(1), 1 - 10
Effects of naloxone and thyrotropin-releasing hormone on plasma catecholamines, corticosterone, and arterial pressure in normal and endotoxemic rats; Long JB et al.; To investigate the possible involvement of the adrenal cortex and medulla in the cardiovascular effects of naloxone and thyrotropin-releasing hormone (TRH) in endotoxic shock, plasma epinephrine, norepinephrine, dopamine, and corticosterone were measured along with hemodynamic variables during naloxone and TRH treatment of normal and endotoxemic rats . In the absence of endotoxemia, naloxone (3 mg/kg, iv) did not significantly alter mean arterial pressure or plasma catecholamine or corticosterone levels . In contrast, following TRH administration (4 mg/kg, iv), an increase in mean arterial pressure was associated with significant increases in plasma epinephrine, norepinephrine, and corticosterone . TRH also produced a transient increase in plasma glucose levels . Endotoxic shock was associated with marked increases in plasma catecholamine levels, with epinephrine levels showing the greatest change, and significant though less pronounced increases in corticosterone . Both naloxone and TRH significantly elevated mean arterial pressures of endotoxemic rats, although neither of these compounds significantly altered the plasma catecholamine and corticosterone responses to endotoxin . Naloxone and TRH also failed to alter endotoxin-induced changes in plasma glucose levels . These results indicate that the cardiovascular effects of naloxone and TRH in endotoxic shock do not simply arise from an enhancement of adrenal catecholamine or corticosterone secretion.

Arch Dermatol, 1986 Jan, 122(1), 52 - 7
Local environment of chronic wounds under synthetic dressings; Varghese MC et al.; Local wound environment under oxygen-permeable and oxygen-nonpermeable dressings in patients with chronic ulcers was investigated . The oxygen tensions under both these dressings were very low or zero . Wound fluid was more acidic under the nonpermeable hydrocolloid dressing than under the oxygen-permeable polyurethane dressing . Bacterial growth studied in vitro was retarded at the more acidic pH similar to that found under the hydrocolloid dressing . Viable and functioning neutrophils were found under both the polyurethane and hydrocolloid dressings, with a greater percentage of viable cells under the polyurethane film . Our data suggest that these synthetic dressings create hypoxic conditions in which wound healing occurs whether or not the dressing is permeable to oxygen . Furthermore the local wound environment can be modified by use of synthetic dressings.

Am Rev Respir Dis, 1986 Jan, 133(1), 55 - 61
Influence of steroidal and nonsteroidal anti-inflammatory agents on the accumulation of arachidonic acid metabolites in plasma and lung lymph after endotoxemia in awake sheep . Measurements of prostacyclin and thromboxane metabolites and 12-HETE; Ogletree ML et al.; The influence of methylprednisolone and meclofenamate on endotoxin-induced release of 3 arachidonic acid metabolites was studied in unanesthetized sheep . Concentrations in plasma and lung lymph of prostacyclin and thromboxane (Tx) A2 metabolites (6-keto-PGF1 alpha and TxB2, respectively) were measured by radioimmunoassay . Concentrations of 12-HETE in lung lymph were measured by stable isotope dilution assay employing gas chromatography-mass spectroscopy . Thromboxane B2 concentrations increased quickly to peak values during the first hour after endotoxin infusion, then decreased to baseline by 1.5 hr . 6-keto-PGF1 alpha concentrations increased more gradually to peak values between 1 and 2 h after endotoxin infusion and remained elevated at 2.5 h . Lymph concentrations of both cyclooxygenase metabolites exceeded those in blood plasma . Methylprednisolone significantly inhibited accumulation of 6-keto-PGF1 alpha in lymph and plasma, but did not significantly inhibit accumulation of TxB2 in lymph or plasma . The combination of meclofenamate and methylprednisolone completely inhibited accumulation of TxB2 and 6-keto-PGF1 alpha in lymph and plasma . The concentration of 12-HETE in lung lymph increased significantly to peak values by 2.5 h after endotoxemia, and methylprednisolone, with or without meclofenamate, inhibited accumulation of 12-HETE in lung lymph . These data support participation of TxA2 in acute pulmonary hypertension after endotoxemia . That methylprednisolone treatment inhibited accumulation of 6-keto-PGF1 alpha and prevented the increase in lung vascular permeability suggests that prostacyclin production is a consequence of lung vascular injury . Increased lung lymph concentrations of the lipoxygenation product, 12-HETE, were coincident with physiologic evidence of increased lung vascular permeability, but whether release of lipoxygenase products after endotoxemia contributes to or results from lung vascular injury remains to be established.

J Basic Microbiol, 1986, 26(10), 621 - 5
Restriction enzyme map of cryptic plasmid accompanying pR711b and pR409; identity of pR409 and pR388; Heidorn JV et al.; A reference strain containing the IncW plasmid, pR409, was found to contain also a cryptic, conjugative plasmid, here named pJR15 . After the separation of the two plasmids into different strains of bacteria, pR409 was found to have an identical restriction enzyme map to the IncW plasmid, pR388 . Both pR409 and pR388 were isolated from the same hospital in London and confer sulphonamide and trimethoprim resistance . Originally pR409 was reported to confer resistance to tetracycline, but this was not confirmed by the Plasmid Reference Center . pR409 appears to represent another isolate of pR388 in a different host background . The restriction enzyme map of the cryptic plasmid, pJR15 (39 kb), has been determined . pJR15 was found compatible with plasmids from 15 different incompatibility groups and has been found also in the reference strain of the IncD plasmid, pR711b . The HindIII and BglII digests of pR711b are shown . The possible presence of the conjugative plasmid, pJR15, should be examined for studies on the sex pili or chromosomal mobilization properties of pR711b.

Gene, 1986, 50(1-3), 3 - 40
Plasmid vector pBR322 and its special-purpose derivatives--a review; Balbas P et al.; The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli . This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence . The widespread use of pBR322 has prompted numerous studies into its molecular structure and function . These studies revealed two features that detract from the plasmid's effectiveness as a cloning vector: plasmid instability in the absence of selection and, the lack of a direct selection scheme for recombinant DNA molecules . Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vector's versatility to accommodate special cloning purposes . The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.

Gene, 1986, 50(1-3), 215 - 24
Construction of an infectious pseudorabies virus recombinant expressing a glycoprotein gIII-beta-galactosidase fusion protein; Keeler CL Jr et al.; An infectious herpesvirus mutant has been constructed in which a major structural envelope glycoprotein gene was replaced by a hybrid gene encoding a novel fusion protein consisting of the N-terminus of the viral glycoprotein joined to Escherichia coli beta-galactosidase (beta Gal) . Specifically, we fused DNA encoding the first 157 amino acids of the structural glycoprotein gIII from pseudorabies virus strain Becker to the E . coli lacZ gene in a bacterial expression vector . The resulting hybrid gene was then used to replace the wild-type gIII gene in the virus by cotransfection of plasmid and viral DNA . The desired viral recombinants were identified by their inability to react with specific monoclonal antibodies that recognized only wild-type gIII protein . One such mutant virus, PRV-Z1, was chosen for further analysis . PRV-Z1 expressed a glycosylated gIII-beta Gal fusion protein after infection of PK15 cells . The fusion protein has no demonstrable beta Gal activity and, although glycosylated, remains sensitive to the enzyme endo-beta-N-acetylglucosaminidase H, unlike the mature gIII gene product, indicating that the fusion protein was incompletely processed.

Biomed Biochim Acta, 1986, 45(11-12), 1529 - 35
Protein products synthesized by a cloned viral protease gene; Korant BD et al.; A region of the poliovirus genome coding for a cysteine protease was expressed in E . coli . Following separation in isoelectric focussing gels, four new proteins were identified and were shown to be products of the viral cDNA . Although a majority of the protein products is aggregated, the system offers the first practical basis for production of large quantities of a viral protease.

J Basic Microbiol, 1986, 26(8), 453 - 9
{Autonomously replicating sequence activity of kinetoplast DNA in yeast cells}; Gutter HP et al.; Different fragments of the maxicircle of the kinetoplast DNA (kpDNA) from Crithidia oncopelti were cloned (Fig . 1, 2) and tested for ARS-activity (ARS, autonomously replicating sequences) . ARS-activity was expressed as number of transformed yeast cells per microgram plasmid DNA, as number of transformed cells per number of plasmid molecules (transformation efficiency, TE) and as number of transformed cells per kilobase pair of cloned kpDNA (specific transformation efficiency, TES) (Fig . 3, Table 1) . All DNA fragments studied showed ARS-activity . Large fragments exhibited higher ARS-activities than their smaller subfragments . The number of fragments showing ARS activity and their distribution within the maxicircles (Fig . 4) suggest that there was no strong correlation between sites with ARS-activity and the replication origin of kpDNA.

Gene, 1986, 49(1), 129 - 38
Identification of Paramecium mitochondrial proteins using antibodies raised against fused mitochondrial gene products; Mahalingam R et al.; Paramecium aurelia mitochondrial (mt) DNA fragments carrying the coding regions for two proteins, P1 (in the region adjacent to the origin of replication) and COII (subunit II of cytochrome oxidase), were used to study mt gene expression . The sequence for the portion of mtDNA containing P1 has already been described {Pritchard et al., Gene 44 (1986) 243-253} . The complete nucleotide sequence of the portion containing the COII gene is presented here . An 18.5-kDa protein was produced in maxicells when a fragment containing a major portion of the sequence coding for P1 was used . This fragment and a fragment carrying the COII gene were cloned into the expression vector pTRPLE', and antibodies were raised against the resulting fusion proteins in an Escherichia coli lysate . Western blots of Paramecium mt extracts identified two proteins, one 21 kDa (COII) and the other 23.5 kDa (P1) . The size of the P1 protein is in agreement with the size of the open reading frame in that region of mitochondrial DNA . Based on extensive amino acid homology to the Paramecium gene and limited homology to COII genes from other organisms, the COII gene in another ciliate, Tetrahymena pyriformis, was identified just upstream of the small subunit rDNA in previously published sequences (Schnare et al., 1986) . The size of the COII gene and the homology with the COII gene from Tetrahymena suggest that ATC, ATT, GTG and GTC could be used as translational initiators in Paramecium mitochondria.

Gene, 1986, 47(2-3), 211 - 9
Synthesis of the outer-capsid glycoprotein of the simian rotavirus SA11 in Escherichia coli; Arias CF et al.; The major outer layer protein, VP7, of the simian rotavirus SA11 has been synthesized in Escherichia coli, under the control of the lac promoter, as a fusion polypeptide with beta-galactosidase (beta Gal) . The viral protein in the hybrid polypeptide is missing its N-terminal hydrophobic region and 26 amino acids (aa) at its C-terminus; it is flanked at both ends by beta Gal sequences . We have purified the hybrid 145-kDa protein by affinity chromatography using a column specific for beta Gal . Unexpectedly, a second protein of 118-kDa was also specifically bound to the column . N-terminal aa sequence analysis of these two proteins showed that the 145-kDa protein represented the expected fusion product, whereas the 118-kDa protein was apparently the result of initiation of translation at an internal site close to the 3' end of the viral sequence, in the chimeric mRNA . Each of the two polypeptides represented about 2 to 3% of the total protein of the recombinant-plasmid-carrying bacteria . When a bacterial lysate enriched for the hybrid polypeptides was injected into mice, it induced neutralizing antibodies to SA11 rotavirus.

Adv Enzyme Regul, 1986, 25, 99 - 122
The enzymatic basis of cyclophosphamide specificity; Hohorst HJ et al.; Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP . Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP) . Protein-S-CP stores activated CP in a highly stable form . From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days . From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells . On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity . No spontaneous toxicogenation of activated CP was noted under in vivo conditions . 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products . Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo . 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E . coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate . In experiments with DNA polymerase I from E . coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme . This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase . Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated . Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target . In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.

Microbiol Immunol, 1986, 30(11), 1095 - 104
Conjugal acquisition and stable maintenance of Ent plasmids in nontoxigenic wild-type strains of Escherichia coli; Danbara H et al.; In spite of the ability of the genetic determinants for enterotoxin production to be conjugally transferred, mobilized or transposed, enterotoxigenic Escherichia coli (ETEC) isolated from diarrheal patients is restricted to certain serotypes . Four conjugative enterotoxigenic plasmids (Ent plasmids) encoding either a heat-labile enterotoxin or a heat-stable enterotoxin or both and belonging to one of three incompatibility groups IncFI, IncHl, or IncX, were examined for their transferability to and stability in 157 nonenterotoxigenic Escherichia coli strains belonging to various serotypes and 89 clinical isolates nonenterotoxigenic but belonging to those serotypes in which ETEC from diarrheal patients are usually found . The serotypes of the strains to which Ent plasmids were efficiently transferred and in which they were maintained stably were not always the serotypes in which ETEC had usually been found and vice versa . The frequencies of transfer of four Ent and two R plasmids to each of the 157 recipients were correlated with each other, indicating that the frequency of transfer of the plasmid is not determined by a resident plasmid, if there is one, but by a recipient factor which commonly affects transferability to all donors . These results have led to the conclusion that the reason why only certain serotypes are found among ETEC isolated from diarrheal patients is not the ability of these strains specifically and preferentially to acquire and maintain the Ent plasmids.

Gene, 1986, 46(1), 97 - 101
In vitro packaging into phage T4 particles and specific recircularization of phage lambda DNAs; Black LW; Concatemeric phage lambda imm434 DNA packaged in vitro into phage T4 particles produced plaques on a selective host . Moreover, lambda DNA containing a pBR322 derivative flanked by the lambda attL and attR sites could be specifically recircularized by excisive lambda recombination to yield the pBR322 derivative . A host deficient in generalized recombination and containing a defective lambda c Its prophage which provided Int and Xis proteins was the recipient for this plasmid derivative carried by T4 . Such a T4-lambda hybrid may potentially allow almost one T4 headful of donor DNA (166 kb) to be packaged and recircularized.

Gene, 1986, 46(1), 65 - 9
Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid; Finkel S et al.; The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype) . The old gene product also kills Escherichia coli recB or recC mutants . We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages . We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid . The plasmid-borne old gene kills E . coli recB mutants, providing a selection for old- mutants.

Gene, 1986, 46(1), 103 - 12
Use of a modified Escherichia coli trpR gene to obtain tight regulation of high-copy-number expression vectors; Warne SR et al.; It has been found that with high-copy-number vectors utilising the Escherichia coli trp promoter the amount of repressor protein produced from the single chromosomally located trpR gene is inadequate for tight repression to be obtained . An attempt has been made to overcome this problem by inserting the trpR gene in cis into the expression vector . This proved unsuccessful because transcription from the trp promoter of such a plasmid could not be induced with 3,beta-indole acrylic acid, probably because the trpR gene is autogenously regulated . However, it was found that when the natural trpR promoter was replaced with a relatively weak constitutive promoter a useful self-repressible vector could be formed . A modified trpR gene of this type has been used to obtain tightly controlled expression of human interferon-beta (IFN-beta) from a vector having a copy number of 400 . Tight regulation is particularly important in this case as IFN-beta is highly toxic to the E . coli cell.

Gene, 1986, 45(3), 333 - 8
An improved filamentous helper phage for generating single-stranded plasmid DNA; Russel M et al.; Gene cloning in plasmid vectors that contain a filamentous phage intergenic region presents several advantages . However, technical difficulties have been a problem, primarily low yields of packaged single stranded (ss) plasmid DNA from the rapid, small scale procedures usually employed, and ambiguities in sequencing reactions attributed to the contamination by helper phage ss DNA . We report here the construction and some properties of a new f1 helper phage . Using this phage, R408, plasmid ss DNA is packaged and exported preferentially over phage ss DNA, and the absolute yield of plasmid ss DNA is usually increased.

Gene, 1986, 45(3), 317 - 25
Synthesis of fusion and mature murine alpha interferons in Escherichia coli; Kelley KA et al.; Four murine interferon-alpha (MuIFN-alpha) genes (alpha 1, alpha 4, alpha 5, alpha 6) were previously identified and characterized . The coding regions of these IFN-alpha genes were inserted into bacterial expression vectors behind the lpp promoter under the control of the lac promoter-operator region, resulting in fusion peptides containing additional N-terminal amino acids (aa) . Plasmids coding for the expression of mature IFN-alpha 1 and alpha 5 were also constructed using the same vector system, by inserting a 30-bp synthetic oligodeoxynucleotide, which contains a stop codon for the lpp gene, a ribosome-binding sequence and an ATG start codon for the IFN peptides . The amounts of IFN polypeptides synthesized in Escherichia coli were estimated in the maxi-cell system and their biological activities were measured on mouse and other mammalian cells . The yields of mature IFN produced in this vector were 2 to 4 X 10(6) units/liter; the antiviral activity of the majority of the MuIFNs on human and bovine cells was 100- to 1000-fold lower than on mouse cells . IFN-alpha 4, which contains an internal deletion of 5 aa, showed a lower antiviral activity than other MuIFNs on mouse cells.

Gene, 1986, 45(3), 299 - 310
Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions; Myers AM et al.; We report yeast/Escherichia coli shuttle vectors suitable for fusing yeast promoter and coding sequences to the lacZ gene of E . coli . The vectors contain a region of multiple unique restriction sites including EcoRI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII . The region with the unique cloning sites has been introduced in both orientations with respect to lacZ and occurs proximal to the eighth codon of the gene . All the restriction sites have been phased to three different reading frames . Two series of vectors have been constructed . The first series (YEp) has two origins of replication (ori), i.e., of the yeast 2 mu circle and of the ColE1 plasmid of E . coli, and can therefore replicate autonomously in both organisms . These shuttle vectors also have the ApR gene of E . coli and either the yeast LEU2 or URA3 genes to allow for selection of both E . coli and yeast transformants . The second series of vectors (YIp) are identical in all respects to the YEp vectors except that they lack the 2 mu ori . The YIp vectors can be used to integrate lacZ fusions into yeast chromosomal DNA . None of the vectors express beta-galactosidase (beta Gal) in yeast or E . coli in the absence of inserted yeast promoter sequences . The 5'-nontranslated sequences and parts of the coding sequences of various yeast genes have been cloned into representative lacZ fusion vectors . In-frame gene fusions can be detected by beta Gal activity when either yeast or E . coli clones are plated on media containing XGal indicator . Quantitative determinations of promoter activity were made by colorimetric assay of beta Gal activity in whole cells . Fusion of the yeast CYC1 gene to lacZ in one of the vectors allowed detection of regulated expression of this gene when cells were grown under conditions of catabolite repression or derepression.

Gene, 1986, 45(2), 233 - 5
Insertion element IS1 can generate a 10-base pair target duplication; Iida S et al.; Transposable element IS1 is known to generate mainly 9-bp and occasionally 8-bp target duplications upon transposition . We have isolated a plasmid pBR322 derivative having IS1 inserted into a site between the promoter and the structural gene for tetracycline resistance . DNA sequence analysis revealed that integration of this IS1 resulted in a 10-bp target duplication.

Gene, 1986, 45(2), 167 - 74
Cloning and expression in biologically active form of the gene for human interferon alpha 2 in Streptomyces lividans; Pulido D et al.; A fragment of human DNA encoding the mature form of interferon alpha 2 (hIFN-alpha 2), and carrying both an in-phase ATG initiation codon and the ribosome binding site (RBS) of the Escherichia coli membrane lipoprotein gene (lpp), was fused to the aminoglycoside phosphotransferase gene (aph) promoter (aphP) from Streptomyces fradiae . When this construction was inserted, in the two possible orientations, in the Streptomyces plasmid pIJ702, plasmids pNIS19 and pNIS91 were obtained . A 20-kDa polypeptide that immunoreacted with an hIFN-alpha 2 monoclonal antibody was expressed in S . lividans clones carrying these plasmids . Moreover, these clones contained an intracellular antiviral activity similar to that of hIFN-alpha 2 . When plasmids pNIS19 and pNIS91 were deprived of the aphP no expression of activity was found . Therefore, it is concluded that the hIFN gene can be efficiently expressed in Streptomyces as directed by the aph gene promoter.

Gene, 1986, 45(2), 159 - 65
Transient expression of murine interferon-alpha genes in mouse and monkey cells; van Heuvel M et al.; The coding regions of murine interferon-alpha (IFN-alpha) genes were combined with promoter and 3'-noncoding sequences from other eukaryotic genes . Transient expression of these fusion genes was achieved in monkey COS cells and in a mouse cell line (TOP cells) expressing polyoma virus (Py) large T antigen constitutively . The efficiency of the different expression plasmids was determined by measuring the amount of IFN secreted into the medium . Replacement of the 3'-noncoding region of an IFN-alpha gene by that of the rabbit beta-globin gene resulted in a fourfold higher IFN-alpha production . The SV40 early promoter and the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR) produced similar amounts of IFN-alpha in COS cells . However, a tandem combination of the SV40 enhancer/early promoter and the mouse metallothionein-I promoter appeared fivefold more active than the SV40 early promoter . In TOP cells the MoMLV LTR was found to be threefold more active than the Py early promoter.

Circ Shock, 1986, 20(3), 181 - 91
Inhibition by naloxone of neutrophil superoxide release: a potentially useful antiinflammatory effect; Simpkins CO et al.; The working hypothesis of many studies of shock has been that naloxone acts by blocking centrally and/or peripherally located opioid receptors . At plasma concentrations used to treat experimental shock (10(-6) M and above), naloxone inhibited the in vitro release of superoxide (O2-) by human neutrophils that were stimulated by the E . coli peptide N-formyl methionyl leucyl phenylalanine (FMLP) . Superoxide release stimulated by phorbol 12,13-dibutyrate (PDB) was also inhibited by naloxone . Naloxone had no effect on the FMLP-stimulated release of beta-glucuronidase or lysozyme . Naloxone had no effect on 3H FMLP receptor binding . Studies utilizing 3H naloxone revealed the presence of a ligand-specific naloxone binding site on human neutrophils with a Kd of 1.2 X 10(-5) M, which is close to the ID50 of the inhibitory effect upon O2- release (1.8 X 10(-5) . Thyrotropin releasing factor (TRF) had no effect upon 3H naloxone binding or on O2- release . Verapamil, a calcium channel blocker, inhibited 3H naloxone binding, and O2- release while nifedipine, another calcium channel blocker had no effect on either assay except at 10(-4) M, at which concentration 3H naloxone binding as well as the release of O2- were increased . These experiments suggest that the inhibitory effect of naloxone upon O2- release is mediated via a specific binding site.

Radiat Environ Biophys, 1986, 25(3), 159 - 73
Radiation damage to phi X174 DNA and biological effects; Lafleur MV et al.; Dilute aqueous solutions of biologically active DNA can serve as a simplified model system of the cell . As a biological endpoint the survival of the DNA (after transfection to E . coli spheroplasts) is used . Damage in the DNA, irradiated in water with gamma rays, can be ascribed to reactions with primary waterradicals . By introducing additives in such solutions, which will scavenge the primary waterradicals, competition between a scavenger and DNA for such radicals can be studied . Comparison of different additives makes it possible to decide whether a compound behaves like a simple scavenger, radiosensitizer or like a radioprotector . In this context work has been done with the electron-affinic radiosensitizers metronidazole, misonidazole and nifuroxime . We have found that these wellknown cellular sensitizers do not enhance the inactivation of biologically active DNA . They act as simple competitive scavenger for waterradicals . However, if besides a sensitizer a trace of a metalloporphyrin containing compound (e.g . cyt . c) is present during irradiation an enhanced DNA inactivation, which can be interpreted as sensitization, is observed . Without sensitizer metalloporphyrins induce an enhanced protection of DNA . Apart from these effects the consequences of both chemical-(sulphydryl) and enzymatic-(excision; recombination) repair has been studied . It has been found that sulphydryl compounds are able to react with DNA radicals, modifying the radiation damage in such a way that e.g . breaks are prevented . Further in double-stranded DNA a considerable amount of OH and also H radical damage appeared to be reparable by the excision-repair mechanism . However, post-replication repair had only very small or no effect on the amount of damage.

Vet Med Nauki, 1986, 23(7), 13 - 7
{Structure and mechanism of action of restriction endonucleases}; Popovski B; The structure and the mode of action of restrictional endonucleases are dealt with in detail . Described are some more important representatives of the three types of endonucleases . Stated are their role and place in present-day molecular biology.

Gene, 1986, 44(2-3), 347 - 51
An improved Escherichia coli expression vector for the construction and identification of full-length cDNA clones; Nakamura K et al.; An Escherichia coli expression vector designed for the efficient synthesis and identification of a full-length cDNA clone is constructed . The vector allows the synthesis of double-stranded cDNAs downstream from the tandem lac control regions employing the vector-primer and linker procedure of Okayama and Berg {Mol . Cell Biol . 2 (1982) 161-170} . Full-length cDNA clones carrying the 5'-noncoding region in addition to the entire coding and 3'-noncoding regions can be expressed in E . coli cells without fusing their coding region to that of E . coli proteins; these clones are identified by colony immunoassay . The entire cDNA insert can be easily excised from the plasmid, since the multiple cloning sites in the vector are duplicated at both ends of the cDNA insert during its synthesis.

Gene, 1986, 44(2-3), 279 - 85
Identification of the thymidine kinase gene of infectious bovine rhinotracheitis virus and its function in Escherichia coli hosts; Liu KY et al.; In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase gene (tk), a well regulated viral gene has been chosen for this study . A cosmid library of IBRV has been constructed in Escherichia coli HB101 by cloning partially Sau3A-digested DNA fragments into a cosmid vector, pJB8 . Recombinant cosmids were further analyzed by restriction digestions and by Southern blot hybridization . Results showed that this cosmid library comprised all of the IBRV genome with the exception of both termini . The individual recombinant cosmid clones were then used to transform E . coli tdk- mutant strains, Ky895 or C600tdk- for the selection of the IBRV tk gene . The clones able to grow on the selection plates containing 5-fluorouracil, uridine, thymidine and ampicillin were selected and further characterized . The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and the sequences complementing the tk activity have been isolated by subcloning . The plasmid, pIBRTK, was shown to grow on selection plates and therefore, retained the ability to complement the tk gene . The E . coli mutant strain C600tdk- harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of {3H}thymidine into bacterial DNA over that of C600tdk- mutant.

Gene, 1986, 44(2-3), 271 - 8
Isolation of the Rhodopseudomonas sphaeroides form I ribulose 1,5-bisphosphate carboxylase/oxygenase large and small subunit genes and expression of the active hexadecameric enzyme in Escherichia coli; Gibson JL et al.; A library of cloned Rhodopseudomonas sphaeroides DNA was screened by colony hybridization for form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) sequences using heterologous RuBPC/O probes . A recombinant plasmid was identified that hybridized to both the Anacystis nidulans and the R . sphaeroides form II RuBPC/O genes . Subcloning of a hybridizing 4-kb SmaI fragment allowed expression of active enzyme in Escherichia coli that was identical to form I RuBPC/O based on polyacrylamide gel electrophoresis and Western immunoblot analysis.

Gene, 1986, 44(2-3), 263 - 70
Covalently linked sequencing primer linkers (splinkers) for sequence analysis of restriction fragments; Kalisch BW et al.; A new method for direct sequence analysis of DNA restriction fragments uses synthetic covalently linked complementary oligodeoxynucleotides, as universal sequencing primer linkers (splinkers) . These splinkers were designed to contain an inverted repeat sequence which forms a double-stranded hairpin structure with a known restriction site . The splinkers were characterized by their ability to be self-ligating (dimerized) and by their restriction digest product analysis of both the monomer and dimer . They can also be ligated to dephosphorylated DNA restriction fragments which contain the appropriate end . This was evidenced by mobility shifts of the splinker-ligated restriction fragments . The splinker-ligated restriction fragments, after denaturation, form a single-stranded DNA template containing an inverted repeat sequence (from splinker) at one terminus . The splinker is thus suitably oriented and serves as a primer for dideoxy nucleotide (nt) sequencing catalyzed by either Klenow fragment of Escherichia coli DNA polymerase I or avian myeloblastosis virus reverse transcriptase . As demonstrated for both pBR322 and phi X174, release of the primer extension strand by digestion at the splinker restriction site results in a ladder of labelled fragments which corresponds to a unique nt sequence.

Gene, 1986, 44(2-3), 219 - 26
Cloning and characterization of the glycine-cleavage enzyme system of Escherichia coli; Stauffer LT et al.; The glycine-cleavage enzyme system of Escherichia coli has been cloned in the cosmid vector pMF7 . The recombinant plasmid, designated pGS64, carries two 19.4-kb EcoRI insert fragments . One of these fragments, which carries the gcv system, was subcloned from plasmid pGS64 into the plasmid vectors pACYC184 and pSC101 (creating plasmids pGS96 and pGS97, respectively) . Plasmid pGS97, but not pGS96, complements a gcv mutant on glycine-supplemented plates . Enzyme assays, however, verified that both plasmids carry an inducible gcv system . The location of the gcv system in plasmid pGS97 was determined by Tn5 insertional inactivation . Subcloning experiments identified the region on the 19.4-kb fragment that inhibits growth in strains transformed with plasmid pGS96 and a region that is possibly involved in negative regulation of the gcv system.

Gene, 1986, 44(2-3), 193 - 9
The lac operator as a phenotypic label for DNA fragments cloned in Escherichia coli; Danner DB; To confer a detectable phenotype on any DNA fragment cloned in Escherichia coli, one can label the fragment by ligating it to the lac operator so that host cells can be identified as blue colonies on agar plates . This screening strategy is similar to that used for the pUC and M13 series of vectors, but does not require the vector to contain the lacZ gene . Instead, the presence of the lac operator on the multicopy vector results in the induction of the host cell beta-galactosidase by titrating out the repressor . This paper describes how pUC/M13 vectors or synthetic oligodeoxynucleotides can be used to supply the operator label, and shows how this method has been used to position unique restriction sites for initiating BAL 31 deletions . This approach may be particularly helpful when a given DNA fragment is to be cloned in many different constructs or is to pass through many sequential cloning steps.

Gene, 1986, 44(2-3), 177 - 83
Cloning of random-sequence oligodeoxynucleotides; Oliphant AR et al.; Methods are described for cloning random or highly degenerate nucleotide (nt) sequences . The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases . Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region . Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage . A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I . If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization . The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site . After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures . Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis.

Folia Microbiol (Praha), 1986, 31(4), 257 - 66
An in vivo cointegrate of two plasmids from incompatibility group X; Nesvera J et al.; Formation of a recombinant plasmid designated pNH603 was observed when two plasmids from incompatibility group X, the multicopy plasmid pNH602 (a higher-copy-number deletion derivative of R6K) and the oligocopy plasmid R485, coexisted in a single Escherichia coli cell . According to its size and its restriction endonuclease cleavage pattern, plasmid pNH603 is a true cointegrate of pNH602 and R485 . An insertion-sequence-like element coming from plasmid R485 is supposed to mediate the fusion of both replicons . The pNH603 copy number (1-2 per chromosome) indicates that the mechanism of replication of the low-copy-number plasmid is dominant in this cointegrate . No dissociation of pNH603 to parental plasmids was observed even in E . coli K-12 recA+ cells . On the other hand, deletion derivatives of four size classes originate from pNH603 in both recA+ and recA hosts . A miniplasmid designated pNH604, a representative of the most frequent 7 Mg/mol size class, was found, in a low number of copies per host chromosome.

Gene, 1986, 44(1), 55 - 62
Molecular cloning and characterization of the purE operon of Escherichia coli; Kamholz J et al.; The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment . The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions . The purE regulation region has been identified by assay of beta-galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments . Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated . The latter may be internal to the purE1 structural gene.

Gene, 1986, 44(1), 47 - 53
Molecular cloning of a recA-like gene of Methylophilus methylotrophus and identification of its product; Finch PW et al.; A recombinant plasmid, pPF5, has been constructed which contains a 5.5-kb fragment of Methylophilus methylotrophus DNA inserted into pAT153, and which confers resistance to UV and mitomycin C (MC) upon Escherichia coli recA mutants . Hybridisation analysis indicates that sequence homology exists between the cloned DNA and a fragment of E . coli chromosomal DNA that contains the recA gene . Tn1000 insertions have been isolated within pPF5 that inactivate its ability to complement recA mutations, and the site of each insertion has been determined by restriction mapping . A 36-kDa protein is synthesised by pPF5 but not by any of the Tn1000 derivatives, indicating that this protein is the product of the gene responsible for the complementation . Comparison of the size of truncated polypeptides produced by selected pPF5::Tn1000 derivatives with the position of the insertion sites of the transposon, gave the direction of transcription of the M . methylotrophus recA+ gene . SOS proteins are induced in E . coli recA mutants harbouring pPF5 following MC treatment.

Gene, 1986, 44(1), 37 - 45
Transcription of the speC (ornithine decarboxylase) gene of Escherichia coli is repressed by cyclic AMP and its receptor protein; Wright JM et al.; The speC gene encoding ornithine decarboxylase (ODC) in Escherichia coli is negatively regulated by cAMP and the cAMP receptor protein (CRP) . In minicells transformed with the plasmid pODC bearing speC, cAMP supplementation repressed ODC synthesis . In a cell-free protein synthesizing system directed by pODC, cAMP at 10(-5) M repressed ODC synthesis by 90% . This repression required a functional CRP as cAMP failed to repress ODC synthesis in vitro in an extract prepared from a crp- strain; the addition of purified CRP to the crp- extract restored the ability of cAMP to repress ODC synthesis . In a prototroph transformed with the plasmid pCOD bearing a speC::tet chimeric gene, cAMP supplementation decreased tetracycline (Tc) resistance . In contrast, in crp- strains transformed with pCOD or pTET (TcR), cAMP supplementation did not change their Tc resistance . When a cya- strain was supplemented with 2 mM cAMP, steady state levels of ODC mRNA were repressed by 80% . However, when a isogenic crp- strain was supplemented with 2 mM cAMP, no repression of ODC mRNA was observed . These results indicate that the cAMP-CRP complex exerts negative transcriptional control of ODC synthesis as a function of the speC promoter.

Gene, 1986, 43(1-2), 51 - 8
Sequence analysis of the pyr-4 (orotidine 5'-P decarboxylase) gene of Neurospora crassa; Newbury SF et al.; The pyr-4 gene of Neurospora crassa encodes orotidine-5' -phosphate decarboxylase, which catalyses the sixth step in the pyrimidine biosynthetic pathway . The complete nucleotide sequence of a 1.8-kb genomic fragment containing the pyr-4 gene has been determined . Using transposon mutagenesis, the coding region has been identified, and the amino acid (aa) sequence deduced . Comparison of the pyr-4 aa sequence with URA3, the equivalent gene of Saccharomyces cerevisiae, showed extensive blocks of homology, with non-homologous sequences between these blocks being generally much longer in Neurospora than in yeast . Computer-predicted protein secondary structure of pyr-4 and URA3 was conserved within equivalent blocks . Upstream sequences of pyr-4 were compared with other sequenced Neurospora genes and possible promoter sequences identified.

Gene, 1986, 43(1-2), 41 - 50
High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector; Hammarskjold ML et al.; To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA . The vector replicates in both Escherichia coli and eukaryotic cells . Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells . Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection . Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV . A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells . These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells . In vivo labelling of transfected cells with {32P}orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation . The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos) . Expression of the EBNA1 protein was obtained in up to 20% of the cells.

Gene, 1986, 43(1-2), 103 - 10
Initiation of phage phi 29 DNA replication by mutants with deletions at the carboxyl end of the terminal protein; Zaballos A et al.; Series of deletions at the C end of p3, the phage phi 29 DNA terminal protein (TP), have been constructed and characterized . Measurements of the activity of those deletion mutants in the formation of the p3-dAMP initiation complex in vitro indicate the need of an intact C-end for the normal TP primer function in DNA replication . It appears that the region at the C-end between aa 240 and 262 of p3, or part of it, might be essential for the normal TP function.

Gene, 1986, 43(1-2), 1 - 11
Cloning and template activity of the origins of replication of phage phi 29 DNA; Gutierrez J et al.; A 73-bp fragment from the left end of phi 29 DNA and a 269-bp fragment from the right end have been cloned in plasmids pPLc28 and pKK223-3, respectively, after removal of the terminal protein p3 by treatment with piperidine . In addition, the 73- and 269-bp fragments were cloned together in plasmid pKK223-3 in such a way that the two termini of phi 29 DNA were joined . Treatment of the latter recombinant plasmid with AhaIII releases several fragments, two of which contain the phi 29 DNA terminal sequences at the DNA end . These two fragments initiated replication specifically at the ends of the DNA giving rise to the formation of the p3-dAMP complex . The activity was about 15% of that obtained with phi 29 DNA-protein p3 . All remaining recombinant plasmids were essentially inactive when tested as templates either in circular form or after cutting in such a way that placed the origin of phi 29 DNA replication close but not at the DNA end.

Cytobios, 1986, 47(188), 45 - 52
Probable occurrence of brain basic protein factor I, an activator of phosphoprotein phosphatases; Kuo WN et al.; Basic protein factor I (BPFI was purified to homogeneity from bovine brain by boiling and trichloroacetic acid-precipitation of tissue homogenate, followed by DEAE-cellulose, Sephadex G-150, Affi-Gel-phenothiazine, and Bio-Gel P-6DG chromatographic procedures . The preparation appeared as a single protein band in the SDS-polyacrylamide gel electrophoresis with a minimal Mr of 13,200 . The factor was a basic protein as indicated by an estimated isoelectric point of pH 8.3 and a high content of amino acids including arginine, histidine, lysine and others . In the absence of Mn2+, the factor stimulated the phosphoprotein phosphatases (PPase) from rabbit brains . Unlike histones or protamine, the factor was a poor substrate for megamodulin-dependent protein kinase . In addition, the factor did not interact significantly with E . coli megamodulin.

C R Acad Sci III, 1986, 303(3), 49 - 54
{A case of overlapping reading frames in Escherichia coli}; Aufrere R et al.; The two promoters and the first 65 nucleotides of a gene related to the rrnB operon are localized in the coding sequence of the btuB gene.

Biomed Biochim Acta, 1986, 45(6), 727 - 36
Probing of mRNA binding sites involved in interactions with rat liver ribosomes using poly(U) spin labeled at the ribose moiety; Damerau W et al.; The interaction of rat liver ribosomes with poly(U), spin labeled (SL) at the 2'OH groups of ribose residues by N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)-imidazole, has been studied by electron spin resonance (ESR) spectroscopy . The ESR spectra demonstrate that SL-poly(U) with a modification of 1 spin label per 20 ribose residues binds to 80S ribosomes as well as to 40S subunits in a 1:1 stoichiometry at 12 mM MgCl2 . The same result is found with highly modified poly(U) bearing 1 SL per 4 ribose residues . Addition of excessive amounts of unmodified poly(U) displaces bound SL-poly(U) from the ribosome which points to a competition for the same binding site at the ribosome . The biological activity of SL-poly(U) was tested with regard to trigger 80S ribosomes for binding of Phe-tRNAPhe and for poly(Phe) synthesis . SL-poly(U) bearing 1 SL group per 20 ribose residues directs the binding of only 50% of the amount of Phe-tRNAPhe bound to ribosomes in the presence of unmodified poly(U) . When SL-poly(U) bearing 1 SL group per 4 ribose residues is used, this value drops to 25% . Poly(Phe) synthesis is even more impaired: In the presence of poly(U) bearing 1 SL-group per 20 ribose residues only about 30% of the amount of poly(Phe) coded by unmodified poly(U) are synthesized and in the presence of SL-poly(U) bearing 1 SL group per 4 ribose residues poly(Phe) synthesis is completely abolished . The results suggest that modification of the ribose moiety has only a relatively small influence on the binding of mRNA to ribosomes but causes substantial impairment of the mRNA function, whereas, as shown earlier (Ebert et al., Acta Biol . Med . Germ . 41, 431, 1982), modification of the base moiety of poly(U) (in a proportion of 1 SL per 30 bases) does not influence coding efficiency for poly(Phe) synthesis.

Tierarztl Prax, 1986, 14(2), 211 - 6
{Pathology and control of virus-induced enteritis in calves}; Kirsch R; Considerable economic loss can arise from virus-caused enteritis in calves, in the form of so-called infectious factor diseases, which often develop more seriously when bacterial organisms, such as E . coli become involved . Rota-, corona- and parvoviruses are of particular interest . These pathogens have a marked predilection for intestinal epithelium . Rotavirus destroys the epithelial cells of the upper parts of the villi . Coronavirus penetrates to the base of the small intestinal villi and the superficial and crypt colonic epithelium is frequently affected . Infection of the small intestinal crypt epithelium is characteristic of parvovirus; loss of epithelium at the villus tip is also observed . Damage of the mucosa results in a reduction in digestive and absorbing capacity . It is not possible to treat these virus strains specifically . Great importance is therefore attached to the vaccination of dams as immune prophylaxis . Consumption of sufficient colostrum and milk from vaccinated dams affords the calves good protection . The mechanism is based on the presence of milk antibodies in the calf's intestine which neutralise orally ingested pathogens.

Gene, 1986, 42(3), 323 - 30
Polypeptides specified by the mercuric resistance (mer) operon of plasmid R100; Bhriain NN et al.; Overlapping deletion mutations were constructed in chimaeric plasmids carrying the mer operon of plasmid R100 . Polypeptides specified by the mutant plasmids in Escherichia coli minicells correlated with the mer genes as follows: merT, 17- and 16-kDa polypeptides; merP, 9.8- and 9.5-kDa polypeptides; merC, a 14-kDa polypeptide; merA, 65- and 62-kDa polypeptides . The products of the merR and merD genes were not identified . The revised nomenclature of the mer genes is explained.

Gene, 1986, 42(3), 253 - 63
Positive-selection vectors utilizing lethality of the EcoRI endonuclease; Kuhn I et al.; The construction and use of a series of positive-selection vectors are described . These plasmids encode EcoRI endonuclease, the synthesis of which is under the control of the lacUV5 promoter . The pKG2 plasmid encodes a wild-type EcoRI endonuclease . In the absence of EcoRI methylase, the endonuclease is lethal . Cloning into any of the unique restriction sites within the endonuclease-coding gene allows survival of the transformed EcoRI-methylase-less host . The pKGW and pKGS plasmids encode an altered EcoRI endonuclease which, when repressed in a lacIQ host, allows survival in the absence of the methylase . Induction with IPTG, however, results in cell death as a result of high-level EcoRI synthesis . Cloning into any of the unique restriction sites within the EcoRI gene of pKGW or pKGS allows survival of derepressed transformed cells . These vectors strongly select for cloning events which inactivate the endonuclease gene.

Gene, 1986, 42(3), 241 - 51
Expression of the E2 open reading frame of papilloma viruses BPV1 and HPV6b in Escherichia coli; Mallon RG et al.; A new expression vector, pRA10, has been constructed for the expression of the open reading frames (ORF) of bovine (B) and human (H) papilloma viruses (PV) . This vector is a derivative of pAJ pi and contains 15 restriction sites proximal to the lambda PL promoter, offering considerable versatility for insertion of different ORFs . This vector was used specifically to express the E2 ORF gene products from BPV1 and HPV6b at high level in Escherichia coli . The genuine nature of these proteins was demonstrated by restriction map analysis of expression vector plasmids to insure proper orientation, nucleotide sequence analysis to demonstrate in-frame insertion, E2 ORF protein production by expression-vector plasmids, and not by appropriate controls and, immunoprecipitation of E2 proteins by antibody specific for a common N-terminal sequence derived from the expression vector.

Gene, 1986, 42(2), 215 - 9
Spontaneous insertions into cosmid vector: a warning; Fernandez C et al.; During the course of characterization of a human genomic library we found that some of the selected pNNL cosmid clones carried only very short inserts . In addition, several clones that did contain full-length inserts were found to be unstable and generated repeated deletions of various portions of their inserts . Moreover, all the clones examined displayed rearrangements in the vector portions . The rearranged clones that were characterized by restriction mapping were found to have deletions starting between ClaI and HindIII in the region of the cos segment of the pNNL vector used in the construction of the library . We determined the nucleotide sequence of the 1300-bp EcoRI-PvuII segment located in the cos region, and by the computer search we found that it contains Escherichia coli insertion elements IS1.

Gene, 1986, 42(2), 201 - 8
The nucleotide sequence of the malT gene encoding the positive regulator of the Escherichia coli maltose regulon; Cole ST et al.; The molecular organization of the malT region of the Escherichia coli K-12 chromosome has been elucidated by nucleotide sequence studies . A single open reading frame of 901 codons comprises the malT gene which is separated by a repetitive extragenic palindromic unit from an unidentified gene, orfX, divergently oriented with respect to malT . The predicted Mr of the MalT protein is 102988, making it the largest transcriptional regulatory protein yet described in E . coli . By deleting in vitro the 3'-end of the gene or constructing malT-lacZ gene fusions, it was found that the integrity of the C-terminus of MalT is indispensable for the activity of the protein . Furthermore, it was found that truncated MalT proteins lacking up to 300 amino acids at the C-terminus blocked the activity of the wild type protein . No sequence homology can be found either with the other activators known in E . coli or with the other proteins of the maltose regulon.

Gene, 1986, 42(2), 169 - 73
A rapid, efficient method for isolating DNA from yeast; Holm C et al.; A method is described for the purification of chromosomal and plasmid DNA from the yeast Saccharomyces cerevisiae . This method is rapid, gives 75% of theoretical yield, and produces DNA that can be cut with restriction endonucleases . Yeast cells are treated with zymolyase, and the resulting spheroplasts are lysed in the presence of the chaotropic agent guanidine hydrochloride . After a brief ethanol precipitation, protein is removed by treatment with proteinase K followed by phenol-chloroform extraction . After ethanol precipitation, the DNA is sufficiently pure for restriction analysis or for the transformation of Escherichia coli.

Biochimie, 1986 Jan, 68(1), 157 - 66
Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity; Waugh R et al.; Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities . This mutant carries a mutation located near minute 58 in the genome . Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system . A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12 . Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated . These mutants were divided into three groups . Class I mutants were restored to the wild-type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1 . Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2 . Class III mutants were unaffected by both pRW1 and growth with NiCl2 . The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity . Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al . (1985) J . Bacteriol., 162, 353-360) . None of the three classes of mutants possess mutations in hydrogenase structural genes.

Biochimie, 1986 Jan, 68(1), 147 - 55
Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata; Colbeau A et al.; A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1 . More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length . Mutants deficient in uptake hydrogenase (Hup-) were obtained from R . capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis . The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis . Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay . The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4 . The cloned R . capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease . Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10 . Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R . capsulata.

Avian Dis, 1986 Jan-Mar, 30(1), 81 - 6
Pathology of avian adenovirus serotypes in the presence of Escherichia coli in infectious-bursal-disease-virus-infected specific-pathogen-free chickens; Dhillon AS; Ten strains of adenovirus representing 10 serotypes were administered intratracheally to 3-week-old specific-pathogen-free chickens, which also received 2.5 X 10(5) colony-forming units of a pathogenic Escherichia coli intranasally . Those birds had been inoculated by eye drop at 1 day of age with a virulent infectious bursal disease virus (IBDV) . Controls consisted of groups of chickens inoculated with: (a) IBDV and E . coli, (b) IBDV only, (c) E . coli only, and (d) no virus or E . coli . Gross pulmonary alterations at 5 days postinoculation (PI) included congestion and consolidation of one or both lungs of a chick inoculated with adenovirus serotype Ind-C and another inoculated with A-2 . Histopathologic alterations in the lungs were those of multifocal interstitial and occasionally diffuse pneumonia . All 10 adenovirus serotypes elicited multifocal or diffuse pneumonia and bronchiolitis in one or more of the five birds per group necropsied at 5 days PI . T-8 and A-2 serotypes induced marked to serve diffuse pneumonia within 5 days; Ind-C, Stein, Tipton, 75-1A, B-3, and X-11 incited a mild diffuse pneumonia . In all groups, the pneumonic lesions were more severe 5 days PI than 12 days PI . Tracheitis was incited by Ind-C, Stein, T-8, and A-2 at 5 days PI; the lesions were minimal to marked in severity . None of the four control groups exhibited gross or histopathologic alterations except the two IBDV-infected groups, which exhibited bursal change.

Gene, 1986, 42(1), 11 - 9
Intramolecular transposition of IS102; Bernardi F et al.; It has been postulated that deletions mediated by transposable elements are intramolecular transposition events . An implication of this hypothesis is that the deleted fragment may be recovered if it is capable of autonomous replication . We report here the characterization of the products of intramolecular transposition of the element IS102 in bireplicons . We show that when two origins (ori's) (of pSC101 and R6-5) generate the same copy numbers, two dissociated replicons are recovered as well as the inversions . On the contrary, when two ori's (of pSC101 and pBR322) have different copy numbers, intramolecular transposition results essentially in inversions . However, the very low frequency (5 X 10(-8)) at which intramolecular transpositions in the bireplicons occurs, as compared to the single replicon (10(-4)), suggests that a complete transposition reaction may not be necessary to generate deletions.

Drugs Exp Clin Res, 1986, 12(4), 355 - 67
A study on structure-activity relationships of nucleoside analogues; Beranek J; A selected series of nucleoside analogues were tested for inhibitory activity vs DNA and/or RNA synthesis at L1210 cells . The structure-activity relationship was studied among derivatives of 5-fluorouracil and arabinosylcytosine (araC), compounds clinically used in cancer chemotherapy . 5-Fluorouridine was found to be a selective, most potent inhibitor of RNA synthesis . Arabinosylcytosine was the most potent selective inhibitor of DNA synthesis . Only cyclocytidine stands very close to the carcinostatic antibiotics in its strong simultaneous inhibition of both DNA and RNA synthesis . A biologically interesting new group of 5'-deoxy derivatives of arabinosylcytosine was investigated . The inhibitory activity vs DNA and/or RNA synthesis is compared with the activity against growth of E . coli and against herpes simplex virus type I (HSV) . The system for inhibition of NA synthesis is proposed as a preliminary first step: an in vitro screening method promising for the discovery of new types of potential carcinostatic agents . Both transport and biotransformation processes of nucleoside analogues were studied in the isolated everted rat jejunum with a continuous perfusion technique . The 5'-substituted derivatives of araC and 5'-chloro-5-fluorouridine exhibited higher transport rates and higher metabolic stability during intestinal penetration than the parent nucleosides . There was found to be no correlation between lipophilicity and transport rate, but there is a correlation between lipophilicity and metabolic alterations among the nucleoside derivatives studied.

Ciba Found Symp, 1986, 120, 136 - 56
Immunization against bovine papillomavirus infection; Pilacinski WP et al.; The two large open reading frames denoted L1 and L2 in the non-transforming region of the bovine papillomavirus type 1 (BPV-1) genome have been molecularly cloned to expression in Escherichia coli . Antisera against the E . coli-derived L1 and L2 protein reacted with BPV-1 in both enzyme-linked immunosorbent assays and immunoprecipitation reactions . Neutralization of BPV-induced transformation of mouse C127 cells was demonstrated most consistently with antisera against the L1 protein . E . coli-derived L1 protein protected calves against BPV-1 challenge after vaccination.

J Gen Microbiol, 1986 Jan, 132 ( Pt 1), 61 - 70
Characterization of the ColE9-J plasmid and analysis of its genetic organization; Chak KF et al.; We have determined the restriction and functional map of the ColE9-J plasmid . By sub-cloning and transposon mutagenesis we have shown that the ColE9imm gene and the ColE5imm gene present on the ColE9-J plasmid are located on separate EcoRI fragments . Using an expression vector we have demonstrated the presence of two lys genes on the ColE9-J plasmid, both of which are dependent upon the colicin E9 structural gene promoter . Promoter mapping studies imply that the colicin E9 structural gene and the ColE5imm gene are transcribed in the same direction, but that the ColE9imm gene is transcribed in the opposite orientation.

Gene, 1986, 41(2-3), 349 - 53
Nucleotide sequence of the R26 chloramphenicol resistance determinant and identification of its gene product; Dorman CJ et al.; The cml gene of plasmid R26 is carried on a 1.9-kb HindIII fragment and specifies low-level, inducible resistance to chloramphenicol (Cm) . In this paper we report the identification of its product as an approx . 31 kDa protein in minicell experiments, and the determination of the nucleotide sequence of cml, which indicates that the gene product is a relatively hydrophobic protein of Mr 33,800 . The protein has no detectable homology to other characterised chloramphenicol-resistance (CmR) proteins, nor any to the membrane-associated tetracycline-resistance (TcR) proteins . The presumptive ribosome-binding site (RBS) of cml mRNA is within a region showing potential for secondary structure.

Gene, 1986, 41(2-3), 337 - 42
Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9; Spratt BG et al.; Analogues of the cloning vectors pUC8, pUC9, pEMBL8 +/- and pEMBL9 +/- that have kanamycin resistance (KmR) instead of ampicillin resistance (ApR) as the selectable marker have been developed . HindIII and SmaI sites within the KmR gene have been removed so that all of the cloning sites in the multi-linker region of these plasmids may be used except the AccI site.

Gene, 1986, 41(2-3), 249 - 60
Genes from the cyanobacterium Agmenellum quadruplicatum isolated by complementation: characterization and production of merodiploids; Porter RD et al.; The isolation of several biosynthetic genes from a cyanobacterium, Agmenellum quadruplicatum, by complementation of auxotrophic mutations in Escherichia coli, and their partial characterization, is described . Although our search for such genes has not been exhaustive, it appears that complementation of E . coli mutations may be of limited utility for the identification and/or isolation of cyanobacterial genes . Despite some overlap in the complementation abilities of these isolated cyanobacterial DNA fragments, the genes that we have studied in some detail are not located in operons . We have used mutagenized versions of these cyanobacterial DNA fragments to produce mutant phenotypes in the cyanobacterium, but clean auxotrophs were not obtained . Complementation of these mutant phenotypes can be obtained when the appropriate wild-type DNA fragment is introduced into the cyanobacterium on a shuttle vector . Recombination between two copies of a cyanobacterial gene occurs at high frequency in the cyanobacterium.

Gene, 1986, 41(2-3), 173 - 81
Relaxed specificity of the EcoRV restriction endonuclease; Halford SE et al.; The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC . In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence . But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide . Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG . However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli.

Acta Physiol Hung, 1986, 67(1), 65 - 9
Can neonatal treatment with a related hormone adapt the receptor for itself?
Csaba G, Nagy SU.
Treatment of rats with endotoxin immediately after birth caused destruction of the cell membrane, resulting in depression of the thyroxin level and of the response to thyrotropin in adulthood . The thyroid gland of the rats treated neonatally with endotoxin failed to differentiate TSH from gonadotropin . Neonatal treatment (imprinting) with thyrotropin or gonadotropin after preexposure to the endotoxin improved the adult response to the exogenous hormone presented for imprinting after endotoxin . It appears that during the reconstruction stage following upon membrane perturbation in the critical period of receptor maturation, the adequate hormone or a related molecule can equally adapt the receptor for itself, but neither can fully compensate the perinatal membrane injury, nor the consequent diminution of receptor activity.

Scand J Infect Dis, 1986, 18(2), 101 - 3
Nitroblue tetrazolium (NBT) reduction by neutrophilic granulocytes in patients with HTLV-III infection; Sonnerborg A et al.; The NBT reduction of granulocytes was determined in 11 patients with HTLV-III infection . The reaction was measured in granulocytes both in resting state and after stimulation with Escherichia coli, as well as with and without addition of plasma . In 5 patients with AIDS, the NBT reduction was significantly decreased in all these types of experiments, when compared with 12 healthy controls . In 6 patients with the lymphadenopathy syndrome (LAS), the NBT reduction of resting granulocytes was significantly higher than that of the controls . The findings could in general not be explained by superinfections . The elevated NBT test during the long term LAS stage reflects a release of free oxygen radicals, against which treatment might be directed.

Mol Gen Genet, 1986 Jan, 202(1), 96 - 101
Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli; Brandsch R et al.; The 160 kb plasmid pAO1 from Arthrobacter oxidans (Brandsch and Decker 1984) was subcloned in Escherichia coli with the aid of the plasmid vectors pUR222 and pBR322 . Screening of the recombinant clones for enzyme activity revealed that the flavoenzyme 6-hydroxy-D-nicotine oxidase (6-HDNO), one of the enzymes of the nicotine-degradative pathway in A . oxidans, is encoded on pAO1 . Immunoprecipitation of 35S-methionine-labelled E . coli cells with 6-HDNO-specific antiserum and expression of recombinant plasmid DNA in E . coli "maxicells" revealed that 6-HDNO is made as a 52,000 dalton protein, approximately 4,500 daltons larger than 6-HDNO from A . oxidans . The 6-HDNO activity was constitutively expressed in E . coli cells, possibly from an A . oxidans promoter, as shown by subcloning of the 6-HDNO gene in pBR322, using the expression vector pKK223-3 and the promoter probe vector pCB192.

Mol Gen Genet, 1986 Jan, 202(1), 75 - 82
Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast; Heinisch J; Yeast phosphofructokinase is an octamer composed of two different kinds of subunit . The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant . As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used . Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2) . Restriction maps of the respective insertions are provided . The genomic organization was confirmed by Southern analysis . Northern analysis showed hybridization to mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits . Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity . Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type . Overproduction of the protein subunits in this transformant was confirmed by SDS-polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue . This was not accompanied by an increased ethanol production . The sequences encoding the two subunits were shown to share homology.

Mol Gen Genet, 1986 Jan, 202(1), 35 - 41
Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12; Mogi T et al.; Two new mutants of E . coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport . They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate (pCMB) . The lesion was mapped at the putP gene, which is located at min 23 on the revised E . coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order . The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product) . Hybrid plasmids carrying the put region (Motojima et al . 1979; Wood et al . 1979) were used to construct the physical map of the put region . The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.

Mol Gen Genet, 1986 Jan, 202(1), 24 - 9
Effect of base substitutions in the colicin E1 gene on colicin E1 export and bacteriocin activity; Yamada M et al.; Base substitutions have been introduced into the segment of the colicin E1 gene corresponding to the polypeptide region between the 404th and the 502nd residues which was considered to participate in colicin E1 export and bacteriocin activity . The methods used were in vitro localized mutagenesis with sodium bisulphite and in vivo mutagenesis using either nitrosoguanidine or ethyl methane sulphonate . Cells carrying mutagenized plasmids were screened by their inability to form a clear zone on a lawn of colicin E1 sensitive cells . Mutation sites were determined from the nucleotide sequence analysis and the altered amino acid residues were reduced . The mutant proteins were analysed for their ability to be exported to the periplasmic space and for their bacteriocin activity . Out of eight mutants obtained, three had a single amino acid replacement . Mutant proteins that had Ser and Glu in place of Pro-462 and Gly-502, respectively, showed a decrease in both the export and the bacteriocin activity . A mutant protein having Arg in place of Gly-439 showed a decrease only in the bacteriocin activity . These results suggest that the target region of colicin E1 contributes to the export as well as the bacteriocin activity but the two functions are supported in part by different amino acid residues of the protein.

Mol Gen Genet, 1986 Jan, 202(1), 132 - 5
The repeated sequences (incB) preceding the protein E gene of plasmid mini-F are essential for replication; Disque-Kochem C et al.; At the XhoI site (45.08F) of plasmid mini-F a deletion of 649 bp was generated employing exonuclease Bal31 . By this deletion nucleotide sequences functioning as origin II and the four 19 bp direct repeats constituting the incB region in front of the E protein gene were removed from the plasmid . Analysis of proteins radioactively labelled in Escherichia coli mini-cells indicated that all mini-F encoded proteins are expressed . However, the plasmid carrying the deletion was not capable of replicating from the primary origin (origin I, 42.6F) . Recently a smaller deletion at the XhoI site (45.08F) of about 300 bp, removing only the region functioning as origin II and replicating from origin I, was described by Tanimoto and Iino (1984, 1985) . The data presented suggest that the incB repeats are essential for the initiation of replication from origin I, and possibly also from origin II, and seem not to be engaged in the autoregulation of E protein expression.

EMBO J, 1986 Jan, 5(1), 191 - 6
The structure of cruciforms in supercoiled DNA: probing the single-stranded character of nucleotide bases with bisulphite; Gough GW et al.; The single-stranded character of cytosine bases in three cruciform structures has been assessed by an examination of reactivity towards sodium bisulphite . Unpaired cytosine residues undergo deamination at C4 to give deoxyuracil, and propagation in an ung Escherichia coli host results in C-G----T-A transition mutations, detectable by restriction cleavage or sequence analysis . Very high frequencies of such mutations have been found at cruciform loops, confirming their unpaired character, with almost zero background mutation frequencies elsewhere . A low level of modification was observed at the four-way junction of a cruciform . The results indicate that the optimal cruciform loop size is four bases, with loose 'breathing' at the first base pair at the top of the cruciform stem at 37 degrees C, and little or no opening of base pairs at the four-way junction.

EMBO J, 1986 Jan, 5(1), 181 - 9
Endonuclease VII resolves Y-junctions in branched DNA in vitro; Jensch F et al.; Endonuclease VII (gp 49 of phage T4) resolves four-way junctions in branched DNAs . We have extended our investigations of the specificity of endo VII and tested its activity with three-way junctions (Y-structures) constructed in vitro . Both 'closed' and 'open' Y-structures were made, absolutely identical in sequence but differing from each other by a single nick in one of the three arms . Pure Y-structures were obtained on a preparative scale by annealing plus and minus strands from two M13mp strains . One strain has an inverted repeat of 2 X 31 nucleotides cloned into the single EcoRI site while in the other strain this repeat is absent . The structures were used in reactions with endo VII, which recognizes the branch point of both structures and introduces a characteristic number of nicks, 3' to the junction in each arm of the structure . Strong and weak sites could be distinguished and the cleavage pattern differed significantly between the two structures . The observed resolution of Y-junctions by endo VII in vitro is compatible with a model for the resolution of recombinant Y-branches in DNA.

EMBO J, 1986 Jan, 5(1), 167 - 73
The upstream operator of the Escherichia coli galactose operon is sufficient for repression of transcription initiated at the cyclic AMP-stimulated promoter; Kuhnke G et al.; Two operators are known to bind Escherichia coli galactose repressor with roughly equal affinity . A study of the control these two operators exert on the two overlapping gal promoters is reported . The experiments rest on a set of mutations specifically constructed to inactivate individual control units of the gal operon and on quantitation of gal promoter activities . Messenger RNAs initiated at one or other of the promoters in a cell-free transcription-translation system were determined by a primer extension assay with synthetic deoxyoligonucleotide primers . The main conclusions are: (i) the classical galactose operator O1, located upstream with respect to the two overlapping promoters is sufficient for negative control of the cAMP activated promoter P1; (ii) complete repression of the second promoter P2, on the other hand, needs the presence of both intact operators O1 and O2 . Thus, the two overlapping gal promoters (with only 5 bp separating their respective transcriptional start sites) are both subject to negative control by the galactose repressor . This regulation, however, is exerted by two different mechanisms.

Anal Biochem, 1986 Jan, 152(1), 66 - 73
Half-site editing: an in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site; Livak KJ et al.; Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions . Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single-stranded template DNA followed by polymerization with DNA polymerase I (Klenow) . Half-site editing differs from other techniques in two main ways . First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer . Second, a blunt-end ligation step is included . This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment . Half-site editing has been used to place ClaI restriction sites at the 3' end of the yeast pyruvate kinase promoter and at two positions at the 5' end of the yeast acetolactate synthase coding sequence.

Plasmid, 1986 Jan, 15(1), 8 - 18
Genetic analysis of promoters on the insertion sequence IS21 of plasmid R68.45; Schurter W et al.; Tandem duplication of a 2.1-kb DNA sequence on R68 leads to the active insertion element IS21 on the enhanced chromosome mobilizing plasmid R68.45 . The HindIII/SalI fragment which carries the single copy or the tandem duplication of IS21 was cloned from R68 and R68.45, respectively, into the multicopy plasmid pED815 . Promoters on the two HindIII/SalI fragments were subsequently identified by cloning Sau3A fragments into the BglII site of the promoter cloning vector pGA46 . Three promoters were identified on the HindIII/SalI fragment derived from R68 or R68.45, two of them mapped on Sau3A fragments of 214 bp and 82 bp, respectively, on IS21 . The promoter on the 82 bp Sau3A fragment which maps at the SmaI site close to the left end of IS21 reads inward . The Sau3A fragment of 214 bp contains the left end of IS21 and transcription from its promoter proceeds outward . In R68.45, readthrough from this preexisting promoter located near the junction of the tandem copies of IS21 proceeds from the right-hand copy into the left, opposing the reading direction of the promoter mapped at the SmaI site of IS21 . The expression of genes on one copy of IS21 by readthrough from a promoter on the other one is a possible explanation for the transpositional activity of the tandem configuration of IS21 . The similarity of IS21 to other insertion sequences and especially to "mobile promoters" is discussed.

Mol Biol (Mosk), 1986 Jan-Feb, 20(1), 278 - 87
{Cloning of the DNA fragment of a transgenic mouse containing an integrated recombinant plasmid}; Tarantul VZ et al.; The "plasmid rescue" method has been used to isolate the recombinant plasmid pMA3 fragment and flanking sequences from the transgenic mouse genome containing the fragment in integrated form . The "rescued" plasmid, pMAR1, lacks all virus sequences and retains only those regions of pBR322 that are responsible for the plasmid replication and Escherichia coli ampicillin resistance . The plasmid pMA3 deletion has occurred at its integration into the mouse genome after microinjection into the zygote . The integrated fragment of the plasmid is adjacent to the genome repeated sequence that is highly conservative in evolution.

Mol Biol (Mosk), 1986 Jan-Feb, 20(1), 185 - 91
{The role of ATP and membrane potential in the penetration of phage T17 DNA into the cell during infection}; Zimkus AZ et al.; The role of ATP and membrane potential in phage T7 DNA injection into E . coli during infection has been studied . Entrance of phage T7 genes of class II and III was shown to be prevented by arsenate, indicating the requirement for phosphorylated macroergs in the phage DNA injection . The injection process was also inhibited by exposition of the cells to the uncoupler of oxidative phosphorylation . Dependence of the injection efficiency on the membrane-potential value has been shown to be sigmoidal, which suggests a regulatory role of the membrane potential in phage T7 DNA injection from the virion into the host cell.

Mol Biol (Mosk), 1986 Jan-Feb, 20(1), 154 - 62
{Amplification of the adenylate cyclase gene in Escherichia coli cells}; Gol'tsmaier TA et al.; Amplification of the cya gene of E . coli on the plasmid pBR325 leads to an increase of adenylate cyclase activity proportional to the gene dosage . In strains harboring hybrid plasmids with cya gene the intracellular level of cAMP and the rate of nucleotide secretion are also elevated . The adenylate cyclase activity in cells with truncated cya gene cloned on pBR322 remains sensitive to glucose inhibition . Amplification of the cya gene leads to considerable resistance of beta-galactosidase synthesis to transient repression by alpha-methylglucoside, but does not influence the permanent repression caused by glucose.

Genetika, 1986 Jan, 22(1), 172 - 4
{New method for the synthesis and cloning of cDNA}; Borovkov AIu; A simple method for cloning cDNA has been suggested . The plasmid pUC18 was digested with Pst1 . A plasmid primer for cDNA synthesis was prepared by dT tailing with terminal transferase . After synthesis of cDNA, dG tails were added and then 3' ends blocked with rG . The plasmid was digested with Kpn1 and dC tails were added, after which annealing took place and RNA:DNA hybrids were used for Escherichia coli transformation . The efficiency of approx . 10(4) transformants per microgram of starting mRNA has been obtained.

Bioorg Khim, 1986 Jan, 12(1), 116 - 23
{Isolation and properties of recombinant DNA from a plasmid and filamentous phage}; Titeeva GR et al.; In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR) . Both parental DNAs contain a fragment of lac-operon (ca . 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment . E . coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs . A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio . Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation . Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiot Med Biotekhnol, 1986 Jan, 31(1), 28 - 31
{Characteristics of the derepressed mutant of the F-like plasmid pAP18-1 coding for tetracycline resistance in Escherichia coli}; Shchipkov VP et al.; A transfer function derepressed mutant of the F-like plasmid pAP18-1 (Tc, ColV) was induced with the help of N-methyl-N'-nitro-N-nitrozoguanidine . The mutant plasmid pAP18-1drd belongs to the FVII incompatibility group of the F-like plasmids . The plasmid pAP18-1drd is characterized by the loss of the capacity for inhibiting the tra-genes functions of the Flac plasmid and is sensitive to the Tra-function inhibitors of the reference plasmids of the FinV and FinW groups.

J Bacteriol, 1986 Jan, 165(1), 66 - 71
SOS-independent coupling between DNA replication and cell division in Escherichia coli; Jaffe A et al.; Inhibition of DNA synthesis in Escherichia coli mutants in which the SOS-dependent division inhibitors SfiA and SfiC were unable to operate led to a partial arrest of cell division . This SOS-independent mechanism coupling DNA replication and cell division was characterized with respect to residual division, particle number, and DNA content . Whether DNA replication was blocked in the initiation or the elongation step, numerous normal-sized anucleate cells were produced (not minicells or filaments) . Their production was used to evaluate the efficiency of this coupling mechanism, which seems to involve the cell division protein FtsZ (SulB), also known to be the target of the division inhibitors SfiA and SfiC . In the absence of DNA synthesis, the efficiency of coupling was modulated by the cyclic-AMP-cyclic-AMP receptor protein complex, which was required for anucleate cell production.

J Bacteriol, 1986 Jan, 165(1), 41 - 6
Analysis of cloned DNA from Leptospira biflexa serovar patoc which complements a deletion of the Escherichia coli trpE gene; Yelton DB et al.; To analyze the cloned region of the chromosome of the spirochete Leptospira biflexa serovar patoc which complemented a defect in the trpE gene of Escherichia coli, we performed a series of experiments involving subcloning, transposon mutagenesis, and maxicells . By subcloning into pBR322 we were able to isolate the Leptospira genes on a 9.7-kilobase pair plasmid (pYC6) . Transposon mutagenesis with Tn5 identified a 2.8-kilobase pair region of this plasmid as being necessary to complement a trpE deletion mutation in E . coli . Transformation of plasmid pYC6 into E . coli cells deleted for trpE and the proximal end of trpD showed that the Leptospira DNA complemented both defects . A maxicell analysis of various transposon-induced mutations of the plasmid revealed that three proteins (53.5, 23.6, and 22 kilodaltons) were encoded by the 2.8-kilobase pair region of the Leptospira genome . Two different promoters controlled the production of these three proteins.

J Bacteriol, 1986 Jan, 165(1), 331 - 3
Purification and properties of shikimate kinase II from Escherichia coli K-12; DeFeyter RC et al.; Shikimate kinase II was purified to near homogeneity from an Escherichia coli strain which overproduced the enzyme . The apparent Km of this isoenzyme for shikimate was 200 microM, and for ATP it was 160 microM . The Km for shikimate is approximately 100-fold lower than the Km of shikimate kinase I, suggesting that shikimate kinase II is the isoenzyme normally functioning in aromatic biosynthesis . Shikimate kinase II is dependent on metal ions for activity.

J Bacteriol, 1986 Jan, 165(1), 321 - 3
RNase H is not involved in the induction of stable DNA replication in Escherichia coli; Bialy H et al.; rnh mutations of Escherichia coli inactivating RNase H activity allow the initiation of rounds of DNA replication in the absence of protein synthesis (stable DNA replication) . However, levels of RNase H did not change during or after the induction of stable DNA replication in rnh+ strains by incubation with nalidixic acid or UV irradiation.

J Bacteriol, 1986 Jan, 165(1), 233 - 9
Nucleotide sequence of the transcription unit containing the aroL and aroM genes from Escherichia coli K-12; DeFeyter RC et al.; The nucleotide sequence of 2,021 base pairs (bp) of DNA containing the Escherichia coli aroLM operon was determined, and the coding regions of both aroL and aroM were identified . The 501-bp intercistronic region between aroL and aroM contains an open reading frame which might encode a 63-residue protein . Northern blots with RNA from strains carrying multicopy aroL+ plasmids detected one longer (2,000-base) and two shorter (950- and 1,100-base) transcripts which contained aroL . It was concluded that the longest transcript, which was not abundant, spanned the entire operon and that the shorter transcripts resulted from either termination or posttranscriptional processing in the intercistronic region . The DNA upstream of aroL contains a number of imperfect palindromes which are closely homologous to known sites of regulation by the TyrR protein in other operons.

J Bacteriol, 1986 Jan, 165(1), 226 - 32
Genetic and molecular analysis of aroL, the gene for shikimate kinase II in Escherichia coli K-12; DeFeyter RC et al.; The gene aroL in Escherichia coli K-12, specifying shikimate kinase II, was contransduced with proC at a frequency of 99% . The gene order is lac proC aroL . A 2.7-kilobase BamHI fragment containing aroL+ was cloned into pBR322 . This plasmid conferred highly elevated levels of shikimate kinase synthesis which were subject to repression control by tyrR . The aroL gene was localized within a 730-base-pair region by both subcloning and insertional mutagenesis with Tn1000 . A second gene, designated aroM and encoding a protein of molecular weight 26,000, is cotranscribed with aroL . Transcription proceeds in the order aroL aroM in a clockwise direction on the chromosome . The function of aroM remains unknown.

Histol Histopathol, 1986 Jan, 1(1), 75 - 87
Oxygen toxicity in the infant rhesus monkey lung . Light microscopic and ultrastructural studies; Ainsworth DM et al.; Eight monkeys were anesthetized with ketamine hydrochloride and positive pressure ventilated with greater than 95% oxygen (tests) or room air (controls) for 24 hours . Two test monkeys and one control were treated with E . coli endotoxin (500 micrograms/kg) IV at the start of the study and after 12 hours . Histopathological changes in the lung parenchyma were evaluated using light and electron microscopy . Interstitial edema was detected as early as 24 hours after the onset of hyperoxia but there was no significant increase in the alveolar-capillary distance (blood-air barrier) . Morphologic signs of oxygen toxicity further included swelling and disruption of vascular endothelium, and swelling of alveolar Type II pneumocytes . There was no difference in the number of macrophages per high power field between the four groups but significant differences were observed in the number of neuroepithelial bodies (NEBs) per cm2 and mast cells per high power field at the light microscopic level . Treatment with endotoxin did not protect against oxygen toxicity and was associated with an exacerbation of the morphological alterations in the lung parenchyma and swelling of alveolar Type I pneumocytes.

Gene, 1986, 50(1-3), 87 - 96
Boundaries of the nutL antiterminator of coliphage lambda and effects of mutations in the spacer region between boxA and boxB; Hasan N et al.; To study the relationship of sequence to function for the phage lambda nutL transcriptional antiterminator, we cloned the 354-bp HincII lambda DNA fragment (coordinates 35261-35615; Daniels et al., in Lambda II, 1983, pp . 485-486 and 618-619) between the plac promoter and Rho-dependent or Rho-independent terminators (t) in the plac-t-galK plasmid derived from vector pKO3, and assayed the galK expression in Escherichia coli hosts in the presence or absence of the N gene product . The 354-bp fragment displayed the complete antitermination activity, as did several shorter fragments obtained by restriction cutting, exonucleolytic deletions and ligations . The minimal length of cloned and fully active nutL comprises 43 bp and consists (in the 5'-to-3' order) of a 10-bp sequence upstream from boxA, the 8-bp boxA (5'-CGCTCTTA-3'), the 7-bp A-B spacer between boxA and boxB; the 17-bp boxB (nutL core; 5'-AGCCCTGAAGAAGGGCA-3') and 1-bp downstream from the nutL core . Deletion of the 10-bp sequence upstream from boxA reduces anti-termination by 40% . Deletion of both that sequence and boxA reduces antitermination by 90% at both 30 degrees C and 42 degrees C . Most of the deletions entering boxB abolish antitermination . Also, some of the small internal deletions within the 7-bp A-B spacer region have a strong negative effect on the nutL function, when transcription is from the plac promoter.

Gene, 1986, 49(3), 311 - 21
Dual-origin plasmids containing an amplifiable ColE1 ori; temperature-controlled expression of cloned genes; Wright EM et al.; Dual-origin plasmids comprising an inducible ColE1-derived origin of replication controlled by the lambda pR promoter, the cI857 temperature-sensitive repressor gene and the pSC101 origin of replication and its associated par sequence, were constructed . Such plasmids carrying cloned genes were stably maintained at four copies per chromosome, and were readily amplifiable by thermal induction . Cloned gene expression increased with copy number, and accumulation values of greater than 20% total cellular protein were detected . These vectors should prove useful for the production of foreign protein on a large scale, since they provide for stable plasmid maintenance during the growth phase, and high-level gene expression without plasmid loss during the production phase.

Gene, 1986, 49(2), 253 - 62
Host/vector interactions which affect the viability of recombinant phage lambda clones; Wertman KF et al.; A class of recombinant phage lambda clones are recovered from human genomic libraries on Escherichia coli recB21 recC22 sbcB15 cells, which fail to form plaques on wild-type cells . We report experiments which address the mechanism of this inhibition . The introduction of the recombination-stimulating sequence chi into one such clone allows growth of this phage on Rec+ cells . In addition, the insertion of lambda gam+ gene into a rec+-inhibited clone results in the ability of the phage to form plaques on wild-type cells . Since lambda Gam protein is an inhibitor of host RecBC enzyme, we tested a collection of such phage for growth on a variety of hosts altered in RecBC function . Host permissiveness correlated with the inactivation of the RecBC nucleolytic activities and not with the recombinational activities . These observations suggest that the inserted DNA sequences of these phage limit the production of packageable chromosomes . This conclusion is easily reconciled with our current knowledge of the interaction of the host recombination systems with lambda replication and encapsidation . Based on these experiments we have constructed strains, both recombination-proficient and recombination-deficient, which serve as improved hosts for the recovery of genomic sequences which are otherwise inhibitory to the growth of phage lambda.

Nucleic Acids Symp Ser, 1986, (17), 223 - 6
An NMR study on the structure of OR3 in the lambda cro-OR3 complex; Lee SJ et al.; 1H-NMR spectra of lambda-OR3 17mer in the complex with lambda-cro has been analysed and the change in its conformation on the complexation has been deduced . The changed conformation is retained until the duplex melts above 55 degrees C . Increase in heat stability of the cro dimer was observed . The result was compared with those of other DNA fragments phi 80-OR2 19 mer and the CRP binding site 22mer mixed with lambda-cro.

Gene, 1986, 48(1), 133 - 44
Structure of Physarum actin gene locus ardA: a nonpalindromic sequence causes inviability of phage lambda and recA-independent deletions; Nader WF et al.; Previously we reported that approx . 80% of the genome from the plasmodial slime mold Physarum polycephalum, including all the actin genes, can be cloned only in recBC- sbcB- Escherichia coli hosts {Nader et al., Proc . Natl . Acad . Sci . USA 82 (1985) 2698-2702} . We have now sequenced the actin gene locus ardA . The nucleotide sequence of its coding region is flanked by the typical putative regulatory sequences for transcription initiation and polyadenylation . The coding region is interrupted by five introns, all located at novel positions with regard to those of previously analysed actin genes . Within the ardA gene we have located a 360-bp fragment which comprises exon V and parts of its flanking introns . This region suppresses plaque formation of recombinant lambda phages and causes recA-independent deletions in phages and plasmids . In contrast to our previous hypothesis, this sequence is not a DNA palindrome, but consists of five (dA) X (dT)- and (dG) X (dC)-homopolymers . Both termination of replication and partial unwinding of duplex DNA under torsional stress were detected within the unstable 360-bp region in vitro.

Gene, 1986, 45(3), 253 - 63
Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene; Penttila M et al.; The filamentous fungus Trichoderma reesei produces several endoglucanases (EG) and cellobiohydrolases (CBH) which are involved in cellulose hydrolysis in a complex synergistic manner . We have cloned and sequenced the gene and the full-length cDNA coding for the major endoglucanase EG-I, and compared this to the cbh1 gene sequence to clarify the relationship between the EG and CBH classes of cellulases . The deduced 437-amino acids (aa) long EG-I protein with a 22-aa long signal peptide is 45% identical in aa sequence with CBH-I . The best conserved region is found at the C terminus and shows about 70% homology . The data suggest that the two enzymes have arisen from a common ancestor by gene duplication . Despite this, the intron positions have not been conserved in these genes which both contain two short introns . The deduced EG-I sequence contains six putative N-glycosylation sites, and a putative O-glycosylated region is found near the C terminus, closely resembling a similar region at the C terminus of CBH-I . Comparison of the aa sequences suggests that the evolutionary divergence of EG-I from CBH-I has involved four separate 10-20 aa "deletions" from the ancestral protein.

Gene, 1986, 44(2-3), 307 - 14
The nucleotide sequence, localization and transcriptional properties of a tRNALeuCUG gene from Drosophila melanogaster; Glew L et al.; The nucleotide sequence of a tRNALeuCUG gene from Drosophila melanogaster has been determined and compared with available tRNALeuCUG sequences from other eukaryotes, as well as with the tRNALeuUUG gene of D . melanogaster . The genomic location, determined by in situ hybridization, was found to be at site 66B on chromosome 3L . This localization probably places it within one of the known, but uncharacterized, clusters of tRNA genes in this organism . In addition, the transcriptional behaviour of this tRNALeuCUG gene in various in vitro systems is described and it seems that, although the gene is transcribed in all test systems, the very A + T-rich 5'-flanking sequence of this particular gene may be somewhat inhibitory to transcription in vitro.

Gene, 1986, 44(1), 147 - 50
A remarkable amino acid sequence homology between a phage T4 tail fibre protein and ORF314 of phage lambda located in the tail operon; Michel CJ et al.; We have found that the amino acid (aa) sequence of the tip of phage T4 tail fibre (gene 37) shows more than 50% homology with the aa sequence predicted from an open reading frame (ORF314) in the phage lambda genome . ORF314 is near the 3' end of the late morphogenetic operon, beyond gene J coding for the lambda tail fibre . The homologous sequences are for the most part composed of repeated aa, the most remarkable of which is a Gly-X-His-Y-His motif where X and Y are small, uncharged aa, found six times in the T4 protein and seven times in the lambda ORF314 sequence.

Gene, 1986, 42(1), 79 - 88
A cloned tryptophan-synthesis gene from the ascomycete Cochliobolus heterostrophus functions in Escherichia coli, yeast and Aspergillus nidulans; Turgeon BG et al.; A gene (TRP1) in the tryptophan biosynthetic pathway of the fungal plant pathogen Cochliobolus heterostrophus was isolated by complementation of an Escherichia coli trpF mutant which lacked phosphoribosylanthranilate isomerase (PRAI) activity . The cloned gene also complemented an E . coli trpC mutant lacking indoleglycerolphosphate synthase (IGPS) activity, a yeast trp1 mutant missing PRAI activity and an Aspergillus nidulans trpC mutant . It functioned in E . coli and A . nidulans without apparent rearrangement but in yeast only after the 5' end of the gene was deleted . The gene was subcloned on a 4.65-kb DNA fragment and the PRAI domain was localized to a 2.9-kb region . It showed homology to the A . nidulans trpC and Neurospora crassa trp-1 genes . There was one predominant transcript of C . heterostrophus TRP1, the same size (2.6-kb) as one of the two functional transcripts produced by A . nidulans trpC . The constitutive activity of the C . heterostrophus TRP1 gene was high whereas that of the A . nidulans trpC gene was low.

J Gen Microbiol, 1986 Jan, 132 ( Pt 1), 53 - 9
An accurate method for estimating sizes of small and large plasmids and DNA fragments by gel electrophoresis; Rochelle PA et al.; Several regression methods were tested for estimating the sizes of a wide range of plasmids (1.37-312 MDa) and restriction fragments (2.2-14.2 MDa) by agarose gel electrophoresis . The most accurate and least variable method was the multiple regression of log10 molecular size against log10 relative mobility and the reciprocal square root of the relative mobility . This method gave a good fit to all the data with low percentage errors of the molecular size estimates (less than or equal to 3.0 +/- 1.5%) . It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E . coli IR713 as standards.

Zh Mikrobiol Epidemiol Immunobiol, 1986 Jan, (1), 29 - 31
{Effect of factors in the cultivation of lysogenic Escherichia coli K12 (lambda) on the spontaneous level of phage}; Anosova MG et al.; The content of free phage in the culture of E . coli K12 (lambda) under different conditions of their cultivation, not affecting the M-concentration of these bacteria, may differ by several times . These differences are well reproduced in consecutive experiments and little affected by the fluctuations of the absolute values of the phage titer.

Circ Shock, 1986, 18(3), 193 - 203
Protective action of calcium entry blockers in endotoxin shock; Lee HC et al.; Calcium entry blockers (CEBs) have been reported to protect against cellular necrosis caused by experimental ischemia and the beneficial effect has been related to prevention of ischemia-induced calcium overload by the CEBs . Since circulatory shock can be expected to produce generalized tissue hypoxia, the possibility that CEBs might be beneficial in shock produced by endotoxin was investigated . In control male Wistar rats, a 10-mg/kg dose of E . coli endotoxin (Difco 0127:B8) produced a fall in blood pressure and a transient increase followed by a progressive slowing of the heart rate . Mortality 48 hours after endotoxin (10 mg/kg) was 84% . The calcium entry blockers (CEBs) verapamil, nitrendipine, and nilvadipine {corrected}, administered i.v . 15 min before endotoxin, produced a dose-dependent reduction in mortality . The CEBs were less effective when given as post-treatment (15 to 30 min after endotoxin) . Measurements of total tissue and mitochondrial calcium levels in control rats revealed that endotoxin did not produce an increase in the calcium content of heart, lung, pancreas, small intestine, kidney, and aorta . Since increased cellular calcium levels did not occur in response to endotoxin, the protection induced by CEBs in endotoxin shock does not appear to be related to prevention of calcium overload; however, the possibility that the CEBs may beneficially prevent an excessive accumulation of calcium in discrete cells or intracellular compartments (which may not be detected by our methods) cannot be excluded.

Int J Biochem, 1986, 18(3), 277 - 8
RNA molecular weight determination by agarose gel electrophoresis using formaldehyde as denaturant: comparison of RNA and DNA molecular weight markers; Wicks RJ; Reliable molecular weight measurements of RNA molecules as large as 4.0 X 10(6) dalton can be made on agarose gels containing 2.2 M formaldehyde as denaturant (Lehrach et al., 1977) . Both eucaryotic and procaryotic ribosomal RNAs have generally been used as molecular weight markers . However, Maniatis et al . (1982) have suggested the use of restriction fragments of DNA as convenient molecular weight markers for RNA samples run in formaldehyde/agarose gels . This communication compares RNA and DNA molecular weight markers run under identical conditions.

Mutat Res, 1986 Jan, 173(1), 19 - 24
Antimutagenic effects of 5-fluorouracil and 5-fluorodeoxyuridine on UV-induced mutagenesis in Escherichia coli; Ohta T et al.; Inhibitors of UV induction of the SOS function were screened . A log phase culture of E . coli PQ37 (sulA::lacZ, rfa, uvrA, Phoc) was irradiated with UV and then immediately subjected to culture for 2 h in a liquid LB medium containing each test compound . Expression of the SOS gene (sulA) was assayed by monitoring the levels of beta-galactosidase . In order to examine the inhibitory effects of test compounds on protein synthesis, the levels of the constitutive alkaline phosphatase were assayed in parallel . The total number of compounds tested was 233, including 44 food and feed additives, 23 naturally occurring compounds and derivatives, 21 antibiotics, 61 pesticides, 33 inorganics and 51 other chemicals . As a result, 5-fluorouracil and 5-fluorodeoxyuridine were found to inhibit considerably the UV induction of the SOS gene without any inhibition of protein synthesis . Mutagenesis induced by UV irradiation was depressed by the addition of either compound at non-toxic concentrations.

Gene, 1986, 49(3), 351 - 60
Organization of genes expressing the blood-group-M-specific hemagglutinin of Escherichia coli: identification and nucleotide sequence of the M-agglutinin subunit gene; Rhen M et al.; The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment . This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa . The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger . The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum . Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long . Although the organization of the M-agglutinin gene cluster resembles those of other E . coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E . coli.

Nucleic Acids Symp Ser, 1986, (17), 127 - 30
A most primitive primodial gene as a building block of the genes for the adenylate kinase, F1-ATPase subunits (epsilon and gamma), aminoacyl-tRNA synthetase, and core enzyme subunits; Ohnishi K; Internal homology units of F1-ATPase epsilon and gamma subunits were searched by computer-aided methods . The epsilon in E . coli (EC) and maize chloroplast (Ch1) was found to consist of three homologous domains, named domains I, II and III (amino acids 1-47, 48-95 and 96-139 for EC) . The gamma in E . coli was demonstrated to have at least six homologous domains, tentatively named here domains I-III and V-VII (I = aa 1-23, II = 26-69, III = 71-112, V = 150-192, VI = 196-242, VII = 285-329), with leaving a region IV (113-149) unclassified . Adenylate kinases (AK's) in pig and E . coli were found to have three internal homology units, named I, I' and II (I = aa 1-47, I' = 48-79, II = 80-124 for pig) . Statistical evaluations and dot matrix analyses at both base and amino acid sequence levels have confirmed that all of these repeating units, being about 46 amino acids long, are homologous with one another . Of these, epsilon III, II, gamma VII and AK II domains were most conservative and some of them showed homology to core enzyme alpha and an internal repeating unit of tryptophanyl-tRNA synthetase (Trp-RS) . Thus these homology unit-encoding gene segments must be relics of a primodial gene.

Anim Genet, 1986, 17(4), 305 - 21
Polymorphism and inheritance of swine small intestinal receptors mediating adhesion of three serological variants of Escherichia coli-producing K88 pilus antigen; Rapacz J et al.; Brush borders, enterocytes, or both preparations obtained from the small intestine of 345 pedigreed pigs, carrying components of seven breeds, were tested by adhesion assay in vitro with 6-32 enteropathogenic Escherichia coli strains, each expressing one of the three K88 pilus antigens, K88ab, K88ac and K88ad . With few exceptions, all pigs were classified as belonging to one of four adhesion phenotypes: I I--corresponding to K88ab(-),ac(-),ad(-); II--K88ab(-),ac(-),ad(+); III--K88ab(+),ac(+),ad(-); and IV--K88ab(+),ac(+),ad(+) . The non-adhering phenotype I was found to be the most frequent among the pigs tested, with the exception of one commercial herd, and this phenotype seems to be inherited as a recessive trait . The remaining three phenotypes are adhering, or are susceptible to adherence by one K88 variant, K88ad (phenotype II), by two variants, K88ab, ac (phenotype III), or by all three K88 variants, K88ab,ac,ad (phenotype IV) . Phenotype II was found to be at low frequency, whereas III and IV occurred with similar frequencies . While the prevailing phenomenon was the bacterial adhesion to all, or none, of the brush borders, some pigs exhibited both adhering and non-adhering brush borders, a mixed adherence phenotype . Preliminary segregation data, obtained from the F1 generation, seem to indicate that phenotypes III and IV correspond to two haplotypes with genes at two or three closely linked loci respectively . An alternative hypothesis is that the phenotypes III and IV are expressions of alleles at a single locus, each allele specifying a receptor able to bind two or three different serological types of K88 E . coli.

Dev Biol Stand, 1986, 64, 53 - 61
An enzyme immunoassay for measuring Escherichia coli pilus antigens; Brown AL et al.; Quantitative enzyme-linked antibody centrifuge (ELAC) assays have been developed for measuring Escherichia coli adhesion pilus antigens K99, K88, 987P and F41 in E . coli bacterins used for protecting newborn animals against neonatal enteric colibacillosis . The test consists of reacting an alkaline phosphatase conjugate of specific pilus antigen antibody directly with dilutions of the bacterin on test and a standard reference bacterin . Following incubation the unbound conjugate is removed by two centrifugation steps and the bacteria with bound conjugate are reacted with substrate . The optical density is measured at 405 nm . Unknown samples are compared to a standard bacterin known to develop sufficient immunity in sows to provide protective levels of passive maternal immunity to their piglets . A relative potency (RP) value is calculated and used for standardizing bacterial concentrates and for final potency testing . The advantages to be realized are: shortening of the test period, specificity, and reduction in test animal usage.

Gene, 1986, 44(1), 17 - 28
The sequence of the chloroplast atpB gene and its flanking regions in Chlamydomonas reinhardtii; Woessner JP et al.; The chloroplast (cp)-encoded CF1 ATPase beta-subunit gene (atpB) of Chlamydomonas reinhardtii and its flanking regions have been sequenced . The derived amino acid (aa) sequence is highly homologous to that of the beta-subunit gene in Escherichia coli, bovine heart mitochondria, and higher plant cp . In contrast to all other cp genomes, the CF1 epsilon subunit gene (atpE) does not lie at the 3' end of the atpB gene but maps to a position 92 kb away in the other single-copy region . Northern blots confirm that the beta subunit is not encoded as part of a dicistronic message as it is in higher plants . The region just upstream from the atpB gene in C . reinhardtii contains two small open reading frames (ORFs) and not the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase as is found in cp genomes of higher plants . No transcripts for either ORF were detected, but the codon usage in these ORFs as well as in the atpB gene follows the unique pattern of codon usage previously seen in other cp genes in C . reinhardtii.

Gene, 1986, 43(1-2), 79 - 84
Expression of a cloned 987P adhesion-antigen fimbrial determinant in Escherichia coli K-12 strain HB101; Morrissey PM et al.; The properties of three independent enterotoxigenic Escherichia coli isolates known to express 987P adhesion fimbriae in a manner subject to phase variation were examined . Phase variation could not be correlated with any major changes in the plasmid DNA content of these strains or with readily detectable changes in any other tested phenotypic markers . The 987P genetic determinant from one of these strains, E . coli 987, was cloned into the non-fimbriated E . coli K-12 strains HB101, and expressed, using the cosmid vector system . 987P fimbriae produced by cells harbouring these recombinant plasmids (987P+ phenotype) could not be distinguished from 987P fimbriae produced by strain 987 . Expression of 987P fimbriae from some recombinant plasmids was unstable but none of the recombinants exhibited the phase variation phenotype displayed by the parental strain . One recombinant plasmid, pPM200, contained an insert of strain 987 DNA of ca . 33 kb . The HB101{pPM200} displayed a rather stable 987P+ phenotype, but this was not true for several hosts, since pPM200 acquired approx . 20-kb deletions following transformations of E . coli K-12 strains other than HB101 . The deletions mapped to the same region of pPM200 irrespective of the host strain transformed . Cells harbouring the deleted plasmids did not express 987P fimbriae (987P- phenotype).

Microbiol Immunol, 1986, 30(6), 495 - 508
A probable new adhesive factor (F42) produced by enterotoxigenic Escherichia coli isolated from pigs; Yano T et al.; Three enterotoxigenic Escherichia coli (ETEC) strains (coded 62, 104, and 567/7) isolated from piglets with neonatal diarrhea produced only a thermostable enterotoxin . Although these strains showed mannose-resistant microhemagglutination (MRMH), the responsible factor was serologically different from the known hemagglutinating colonization factors from porcine strains (K88, K99, and F41) . Bacterial cells from these strains adhered to HeLa cells and pig brush borders . Electron microscope studies revealed the presence of fimbria-like structures on bacterial cells grown at 37 C but not on cells grown at 18 C . The antiserum prepared from partially purified fimbrial antigen (provisionally called F42) inhibited chicken erythrocyte MRMH caused by these strains as well as adherence of strain 567/7 to HeLa cells and to pig brush borders . These data taken together suggest the existence of a new hemagglutinating adhesin that is different from those so far described for porcine ETEC.

Gene, 1986, 43(3), 265 - 72
Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli; Garcia P et al.; Autolysins are enzymes that have several important biological functions and also seem to be responsible for the irreversible effects induced by the beta-lactam antibiotics . The pneumococcal autolysin gene (lyt) has been subcloned from the plasmid pGL30 {Garcia et al., Mol . Gen . Genet . 201 (1985) 225-230} and we have found that the E form of the autolysin is synthesized in Escherichia coli using its own promoter . The high amount of autolysin obtained in the heterologous system when the lyt gene is present in different orientations in the recombinant plasmids studied supports the idea that the autolysin promoter could be a strong one . The nucleotide sequence of the HindIII fragment of pGL80 (1213 bp) containing the autolysin structural gene has been determined . A unique open reading frame (ORF) has been found, a consensus ribosome-binding site and -10 and -35 promoter-like sequences as well as A + T-rich regions farther upstream were also identified . The lyt ORF encodes a protein of 318 amino acid residues having a calculated Mr of 36,532, which agrees with previous size estimates based on electrophoretic migration {Holtje and Tomasz, J . Biol . Chem . 251 (1976) 4199-4207; Briese and Hakenbeck, Eur . J . Biochem . 146 (1985) 417-427} . Our results also demonstrate that the lyt-4 marker represents the first example of a mutation in a structural gene of a bacterial autolysin . The polarity profile of the pneumococcal autolysin supports previous suggestions about the localization of this enzyme in the normal cell.

J Hosp Infect, 1986 Jan, 7(1), 78 - 81
A simple device for disinfecting endoscopes; Wagenvoort JH et al.; A method for disinfecting fibreoptic endoscopes with povidone-iodine and a simple cleaning device, consisting of a curved glass pipe and a peristaltic pump is described . If properly employed the system produces satisfactory results.

J Biochem (Tokyo), 1986 Jan, 99(1), 135 - 41
Human F1-ATPase: molecular cloning of cDNA for the beta subunit; Ohta S et al.; F1-ATPase is the major enzyme for ATP synthesis, and its beta subunit is the catalytic site . To date, no full-length cDNA for the eukaryotic F1 gene has been reported . Human F1 was studied because of its importance in medicine and cell biology . Here we report molecular cloning of a full-length cDNA for the human F1 beta subunit and purification of the human F1 beta subunit . The HeLa cell cDNA library constructed in an expression vector gamma gt11 was screened with antiserum against the yeast F1 beta subunit . One of the positive phage DNAs containing the human F1 beta gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method . The open reading frame started from a putative signal presequence, which was rich in both serine and arginine . There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1 beta . The precursor of F1 beta was expressed in E . coli harboring a plasmid which had been constructed with T5 promotor and the F1 beta cDNA . Both the precursor and mature form of F1 beta were detected in HeLa cells in a pulse-chase experiment . The amino acid sequence of 480 residues (51,568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1 beta (97.5%) and E . coli F1 beta (71.7%), but the codon usage in the human gene was very different from those of reported genes coding for F1 beta of other species.

Plasmid, 1986 Jan, 15(1), 19 - 34
Distribution of basic replicons having homology with RepFIA, RepFIB, and RepFIC among IncF group plasmids; Bergquist PL et al.; Plasmids encoding F-like pili have been divided into groups on the basis of their incompatibility behavior . Three basic replicons have been recognized previously in the IncFI plasmid group and we have now examined their distribution in representative plasmids from 22 of the currently recognized incompatibility groups . The occurrence of these basic replicons was found to be rare outside of the IncF group, and significant hybridization was shown only for RepFIA to IncH1 and I group plasmids . Homology to the RepFIC basic replicon was found in all but one of the IncF group plasmids examined but RepFIA and RepFIB have a more restricted distribution . It appears likely that some plasmids carry vestiges of replicons which still express incompatibility but are incapable of replication . We suggest that evolutionary divergence among the plasmids of the IncF group has resulted from various genetic rearrangements among these basic replicons.

Arch Biochem Biophys, 1986 Jan, 244(1), 361 - 72
Chemical crosslinking of alpha subunits in the F1 adenosine triphosphatase of Escherichia coli; Bragg PD et al.; The arrangement of the subunits in the F1 adenosine triphosphatase of Escherichia coli has been investigated using bifunctional chemical crosslinking agents to covalently link adjacent subunits in the enzyme molecule . The synthesis of the new cleavable crosslinking agent 2,2'-dithiobis(succinimidyl propionate) is described . The crosslinked products resulting from the reaction of the enzyme with 2,2'- and 3,3'-dithiobis(succinimidyl propionate), 3,3'-dithiobis(sulfosuccinimidyl propionate), disuccinimidyl tartrate, dimethyl adipimidate, 1-ethyl-3{3-(dimethylamino)propyl}carbodiimide, and 1,2:3,4-diepoxybutane were analyzed by "three-dimensional" polyacrylamide gel electrophoresis in which they were resolved first in a two-dimensional system . Following cleavage of the crosslinking bridge in the separated products, the constituent subunits were identified by a further one-dimensional gel electrophoresis step . This procedure greatly improved the precision with which crosslinked subunits could be identified . It largely overcame problems due to abnormal migration of crosslinked species on gel electrophoresis and to the formation of multiple species of the same crosslinked subunit dimers . The following crosslinked subunit dimers were identified: alpha alpha, alpha beta, beta gamma, alpha delta, beta epsilon, and gamma epsilon . The trimer alpha alpha delta was recognized . The formation of alpha alpha over alpha beta dimers was favored when more polar crosslinking agents were used . The constraints placed by the finding of adjacent alpha subunits upon current models for the arrangement of the subunits in the F1 ATPase are discussed.

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