Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8960 - 4 Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine; Kawata M et al.; smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins . In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous {3H}mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L-{methyl-3H}methionine . In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1 . In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester . Extensive digestion of smg p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1 . Both peptides were modified by a thioether-linked geranylgeranyl group . One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH . Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu184, these results indicate that smg p21B is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group. J Biochem (Tokyo), 1990 Nov, 108(5), 816 - 21 Cloning and sequence analysis of cDNA for Trimeresurus flavoviridis phospholipase A2, and consequent revision of the amino acid sequence; Oda N et al.; A cDNA clone of Trimeresurus flavoviridis phospholipase A2 was isolated from the venom gland cDNA library using DNA amplified by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminal sequences of this enzyme . The amino acid sequence deduced from the cDNA nucleotide sequence determined by the dideoxy termination method was found to be inconsistent in the segment comprising the 69th to 81st positions from that reported previously {Tanaka, S . et al . (1986) J . Biochem . 99, 281-289} . Reinvestigation of the amino acid sequence of the peptide covering the sequence in question showed that the amino acid sequence predicted from the nucleotide sequence is correct . The sequence contains Asn-Asn-Gly (positions 69-71) and it was found that the Asn-Gly bond easily undergoes alpha-beta transpeptidation when digested with Achromobacter protease I at pH 9.0 but not seriously at pH 6.8 . It is likely that the transpeptidation reaction caused a failure in the previous sequence determination . The cDNA clone obtained was 597 base pairs long and contained an open reading frame of 414 base pairs coding for 138 amino acid residues . A typical signal peptide sequence (16 amino acid residues long), located at the N-terminal moiety of the deduced sequence, was immediately followed by a polypeptide which corresponds to the mature enzyme . Northern blot analysis showed a single transcript only in the poly(A)+ RNA fraction of the venom gland but not in those of many other organs tested. J Biol Chem, 1990 Oct 25, 265(30), 18345 - 50 Identification of lysine 134 in the steroid-binding site of the sex steroid-binding protein of human plasma; Namkung PC et al.; The sex steroid-binding protein of human plasma SBP (or sex hormone-binding globulin, SHBG) was specifically inhibited with the alkylating affinity label, 17 beta-{{( 2-14C}bromoacetyl)oxy}-5 alpha-androstan-3-one . The natural ligand, 5 alpha-dihydrotestosterone, was shown to protect against inactivation and labeling . The steroid-binding activity of the protein was abolished when approximately 1 mol of label was incorporated into 1 mol of dimeric SBP . In order to identify and locate the labeled amino acid in the steroid-binding site, the steroidal portion of the bound label was first removed and the protein was digested with Achromobacter protease and subdigested with trypsin . Seven radioactive peptides were isolated, sequenced, and found to contain the common sequence QVSGPLTSXR . Residue X was identified as lysine-134 from the SBP amino acid sequence (Walsh, K . A., Titani, K., Kumar, S., Hayes, R., and Petra, P . H . (1986) Biochemistry 25, 7584-7590) . The results indicate that only 1 of the 2 lysine-134 residues in the homodimer was labeled . This suggests that the steroid-binding site is constructed from an association of the two subunits in an AB to BA "sandwich" configuration with lysine-134 residue of one subunit on one surface near the D-ring and the lysine-134 of the other subunit at the opposite end of the steroid, or well away from the steroid-binding site . Although the nature of the data does not allow description of a specific role for lysine-134, its proximity to the 17 beta-OH of the steroid nucleus suggests participation in the binding process through direct or indirect hydrogen bonding. Experientia, 1990 Oct 15, 46(10), 1066 - 8 Inhibitory effect of halocyamine, an antimicrobial substance from ascidian hemocytes, on the growth of fish viruses and marine bacteria; Azumi K et al.; Halocyamine A, an antimicrobial substance isolated from hemocytes of the solitary ascidian Halocynthia roretzi, inhibited in vitro the growth of fish RNA viruses (infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus) . Pretreatment of RNA virus with halocyamine A reduced the infectivity of the virus toward host cells . The growth of marine bacteria, Achromobacter aquamarinus and Pseudomonas perfectomarinus, was also inhibited by halocyamine A but that of Alteromonas putrefaciens and Vibrio anguillarum was not . These results suggest that halocyamine may have a role in the defense mechanisms of H . roretzi against marine viruses and bacteria. J Biochem (Tokyo), 1990 Oct, 108(4), 604 - 8 Purification and complex formation analysis of a cysteine proteinase inhibitor (cystatin) from seeds of Wisteria floribunda; Hirashiki I et al.; Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin) . We purified and characterized one of these inhibitors, named WCPI-3 . The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively . The isoelectric point was 5.7 . WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM . Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis . A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK . The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases . The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds. J Mol Recognit, 1990 Oct-Dec, 3(5-6), 181 - 6 Enzymatic semisynthesis of human insulin: an update; Morihara K; Peptide bond formation can be enzymatically catalysed by the reverse reaction of proteases . Application is seen in the industrial production of human insulin . Human insulin derivative can be enzymatically prepared using either porcine insulin or the single chain B(1-29)-A(1-21) insulin precursor as the starting material . This is accomplished by either (1) digesting the starting material at Lys29 with Achromobacter lyticus protease I (Ach) and then coupling with Thr-X (X = blocking residue) (two-step reaction) or (2) subjecting Ala-B30 of porcine insulin or Gly-A1 of the single chain insulin precursor to transpeptidation with Thr-X (one-step reaction) . Trypsin and Ach can be used for either type of reaction and, in the immobilized form, for the two-step reaction . Since the single chain insulin precursor can be produced by gene technology (yeast), use of immobilized trypsin or Ach and the two-step reaction using the single chain insulin precursor as the starting material ensures the continuous production of human insulin making it a feasible method for industrial manufacture. J Bacteriol, 1990 Sep, 172(9), 4945 - 50 Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG; Jin SG et al.; Agrobacterium tumefaciens virulence genes are induced by plant signals through the VirA-VirG two-component regulatory system . The VirA protein is a membrane-spanning sensor molecule that possesses an autophosphorylating activity, and the VirG protein is a sequence-specific DNA-binding protein . In this report, we demonstrate that the VirG protein is phosphorylated by the VirA protein and that the phosphate is directly transferred from the phosphorylated VirA molecule (phosphohistidine) to the VirG protein . The chemical stability of the phospho-VirG bond suggested that the VirG protein was phosphorylated at the aspartate and/or glutamate residue . The phosphorylated VirG protein was reduced with tritiated sodium borohydride and subjected to proteolytic digestion with the Achromobacter protease I enzyme . The resulting peptide fragments were separated by C8 reversed-phase high-pressure liquid chromatography, and the tritium-labeled peptide was sequenced . Amino acid sequence data showed that the aspartate residue at position 52 was the only site phosphorylated . Changing this aspartate into asparagine resulted in a nonphosphorylatable and biologically nonfunctional gene product . As a control, a randomly chosen aspartate was changed into an asparagine (position 72), and no effect on its phosphorylation or biological activity was observed . Unlike its homologs, including CheA-CheY, EnvZ-OmpR, and NtrB-NtrC, the phospho-VirG molecule was very stable in vitro . The possible implications of these observations and the function of VirG phosphorylation in vir gene activation are discussed. Bioorg Khim, 1990 Aug, 16(8), 1040 - 4 {Site-specific endonucleases LplI and AagI}; Sokolov NN et al.; New site-specific endonucleases LplI and AagI have been isolated from the Lactobacillus plantarum and Achromobacter agile cells, respectively . The enzymes' purification stages included treatment of cell-free extracts with polyethylenimine, fractionation in two-phase system by Albertsson's method, chromatography on blue Sepharose and DEAE-cellulose . The results of cleavage of a 5'-32P-labelled oligodeoxynucleotide duplex by restriction endonucleases LplI and AagI indicate that these enzymes recognize and cut the sequence AT decreases CGAT, being therefore true isoschizomers of the ClaI restriction endonuclease from Caryophanon latum . The L . plantarum strain has 400 fold endonuclease productivity as compared with the ClaI producent and is perspective for preparative isolation of LplI. J Biochem (Tokyo), 1990 Jul, 108(1), 139 - 43 Amino acid sequence of a lectin from Japanese frog (Rana japonica) eggs; Kamiya Y et al.; The complete amino acid sequence and the location of disulfide bonds of a lectin from Japanese frog (Rana japonica) eggs, which specifically agglutinates transformed cells, are presented . The sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with Achromobacter protease I, or chymotrypsin, and by chemical cleavage with BNPS-skatole or cyanogen bromide . The lectin is a single-chain protein consisting of 111 residues, with a pyroglutamyl residue at the amino terminus . Four disulfide bonds link half-cystinyl residue 19 to 72, 34 to 82, 52 to 97, and 94 to 111 . The sequence and the location of the disulfide bonds are highly homologous to those of bull frog (Rana catesbeiana) egg S-lectin . They are also homologous to human angiogenin, a tumor angiogenesis factor, and a family of pancreatic ribonucleases. J Biochem (Tokyo), 1990 Jul, 108(1), 133 - 8 Complete amino acid sequence of rat kidney ornithine aminotransferase: identity with liver ornithine aminotransferase; Oyama R et al.; The complete amino acid sequence of rat kidney ornithine aminotransferase {EC 2.6.1.13} is presented . The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease . Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA {Mueckler, M.M . & Pitot, H.C . (1985) J . Biol . Chem . 260, 12993-12997} . The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation. Biochem Biophys Res Commun, 1990 Jun 29, 169(3), 1235 - 41 EPR spectra of ferric cytochromes c' from five strains of Achromobacter xylosoxidans at low temperature and their temperature dependence; Yoshimura T et al.; The EPR spectra at low temperature (6 K) and their temperature dependence (10-93 K) for five ferric cytochromes c' isolated from chemoheterotrophic bacteria, Achromobacter xylosoxidans NCIB 11015 (formerly Alcaligenes sp . NCIB 11015), GIFU 543, GIFU 1048, GIFU 1051, and GIFU 1764 are reported . The EPR spectral results indicate that the ground state of the heme iron(III) of cytochromes c' from these chemoheterotrophic bacteria can appear to be in an admixed spin state which consists of predominant S = 5/2 with a slight S = 3/2 character . The EPR spectra were compared with those for ferric cytochromes c' from photosynthetic bacteria and the other ferric hemoproteins. Blood, 1990 Jun 15, 75(12), 2349 - 56 Rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein Ib; Shimomura T et al.; Glycoprotein V (GPV) is a membrane-associated, 82 Kd platelet glycoprotein that is hydrolyzed during thrombin activation to yield 69 Kd fragment . We have developed a rapid and simple method for isolation of the protein from platelet extracts using a combination of gel permeation, anion-exchange, and lectin affinity chromatography . The partial amino acid sequence was determined by analysis of peptides generated by digestion of the S-carboxyamido-methylated protein with Achromobacter protease I or cyanogen bromide . The sequence shows a remarkable periodicity of leucine residues, which is homologous to the consensus sequence of a highly diversified protein super-family with a common repetitive module . Thrombin cleavage site was determined to be located at the C-terminal region of GPV by analysis of the products separated by sizing and reversed-phase high performance liquid chromatography . By lectin blot analysis, the existence of mucin-type carbohydrate chains was indicated, as well as the existence of asparagine-linked carbohydrate chains shown by the amino acid sequence analysis . From these data, we report a structural model of GPV that is analogous to glycoprotein Ib. J Biol Chem, 1990 Jun 5, 265(16), 9188 - 93 A distinct human testis and brain mu-class glutathione S-transferase . Molecular cloning and characterization of a form present even in individuals lacking hepatic type mu isoenzymes; Campbell E et al.; mu-Class glutathione S-transferases (GSTs) were identified in all 13 human testes and 28 brains examined; even subjects whose livers were devoid of mu-GSTs expressed extrahepatic GSTs of this class . Testes and brains from individuals with mu-class GSTs in their livers had additional forms that also reflected the liver phenotypes . An isoenzyme with an isoelectric point of 5.2, which was a major GST in testis and present as well in cerebral cortex but not detected in any livers, was identified and purified . Sequence analysis of peptides derived by cleavage of the testicular mu-class GST by Achromobacter protease I revealed distinct aspects of primary structure not found previously in any mammalian mu-class GSTs . These unique features included a blocked and extended amino terminus and 3 additional residues (Pro-Val-Cys) at the carboxyl terminus . This structure was confirmed by molecular cloning and sequencing of cDNAs derived from human testis and brain libraries . In the coding region the mRNA of the brain-testis mu-class GST was 75% homologous with that of the liver form, and its 3'-untranslated sequence was mostly divergent, indicating that it is the product of a separate gene . Distinct catalytic and structural properties of the testis-brain mu-class GSTs suggest that these GSTs may be uniquely involved in blood-barrier functions common to both organs. J Appl Bacteriol, 1990 May, 68(5), 495 - 504 Numerical analysis of electrophoretic protein patterns of 'Achromobacter' group B, E and F strains from human blood; Holmes B et al.; Thirty-two clinical strains representing 'Achromobacter' groups B, E and F were characterized by one-dimensional SDS-PAGE of cellular proteins . All the strains were isolated from blood samples from hospital patients in the United Kingdom . The protein patterns, which contained 40 to 45 discrete bands, were highly reproducible and were used as the basis for a numerical analysis which included all the protein bands . The 32 'Achromobacter' strains formed two clusters at the 77% S level . The first, phenon 1, included the 28 group B and the two group E strains and the second, phenon 2, contained the two strains of group F . The strains in each phenon were characterized by a clearly distinct pattern of protein bands . Phenon 1 could be further divided at the 87% S level into three subphenons which correlated with differences in the principal bands found between 40.0 and 42.5 kD . Strains of group E clustered with group B strains from which they could not be distinguished by protein patterns . We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides a useful method for the classification of this group of bacteria . Reference strains of each of the PAGE types identified are available from NCTC for inclusion in future studies. Electrophoresis, 1990 May, 11(5), 382 - 91 Numerical analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoretic protein patterns for the classification, identification and typing of medically important bacteria; Costas M; A numerical analysis of one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoretic protein patterns of three separate bacterial groups is described . Similarity between patterns was estimated using a correlation coefficient and is represented diagramatically by the unweighted pair-group with arithmetic averages method of clustering . Protein patterns were scanned using a laser densitometer interfaced directly to a microcomputer, where calculations were performed semi-automatically with specially written software . A method for the normalization of patterns from different gels is utilized . Whole cell protein patterns clearly distinguished strains representing various groups of Achromobacter . Similarly the two biovars of the Group EF-4 bacteria could be separated after analysis of partial "background patterns" as could the different species of Providencia . In addition, whole patterns could be used to type the various strains of P . rettgeri . These examples serve to illustrate the power, versatility and potential of the technique when applied to problems of classification, identification and typing of bacteria of medical interest . Its advantages and disadvantages, when compared to other available electrophoretic methodologies, are discussed, as is the future application of such technology to the hospital laboratory. Biol Chem Hoppe Seyler, 1990 May, 371(5), 441 - 6 Susceptibility of baboon aorta elastin to proteolysis; Desfontaines L et al.; Elastin was purified from baboon aorta using Achromobacter collagenase and its susceptibility to proteolysis by various enzymes was studied . Human leukocyte elastase (HLE) hydrolysed baboon aortic elastin 8 times faster than human cathepsin G . Bovine chymotrypsin had virtually no activity against this substrate . The kinetic constants V and {S50} of aortic elastin hydrolysis by HLE (0.15 microM) were 0.00286 mg x ml-1 x min-1 and 0.158 mg x ml-1, respectively . One mg of this elastin could be saturated with 5.6 micrograms of HLE . As with elastins isolated from other sources, the hydrolysis of baboon aortic elastin by HLE was highly sensitive to ionic strength, and a biphasic effect was obtained with increasing NaCl concentrations . A nearly 2-fold stimulation of elastolysis was observed at a 0.15M NaCl concentration . Further increase in ionic strength led to a continuous decrease of the rate of elastolysis which paralleled the decrease of adsorption of elastase to baboon aortic elastin . Cathepsin G, but not bovine alpha-chymotrypsin, was able to stimulate the rate of hydrolysis of baboon aortic elastin by HLE . A 1.7 fold stimulation was observed for a 1:1 molar ratio of the two proteinases and rose to 2.1 for a HLE/Cat . G ratio equal to 8. Diagn Microbiol Infect Dis, 1990 May-Jun, 13(3), 253 - 9 Antimicrobial activity of Ro 24-6778, a covalent bonding of desmethylfleroxacin and desacetylcefotaxime; Jones RN; A new "dual action" cephalosporin (Ro 24-6778), representing an ester-linked desacetylcefotaxime and desmethylfleroxacin was tested against 287 aerobic bacteria . Ro 24-6778 was found to be very active (minimal inhibitory concentrations {MIC90}, less than or equal to 0.5 micrograms/ml) against Enterobacteriaceae, Streptococcus spp., and Aeromonas hydrophila . Moderate Ro 24-6778 activity (MIC90, 1-8 micrograms/ml) was demonstrated against Bacillus spp., Staphylococcus spp . (including oxacillin-resistant strains), Flavobacterium spp., Enterococcus durans, and Acinetobacter anitratus . More Ro 24-6778-resistant strains (MIC90, 16- greater than 32 micrograms/ml) were usually found among the enterococci, Xanthomonas hydrophila, Pseudomonas spp., and Achromobacter xyloxidans isolates . These preliminary Ro 24-6778 MIC test results show a spectrum superior (93.4% of strains susceptible at less than or equal to 8 micrograms/ml) to the comparison drugs (cefotaxime or fleroxacin) and possible clinical utility for therapy of many fluoroquinolone- or cephalosporin-resistant strains. J Biochem (Tokyo), 1990 Feb, 107(2), 292 - 7 Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells; Minamitake Y et al.; The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure . Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis . After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence . The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86 . In addition, we concluded that Thr-3 and Asn-28 were glycosylated . The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate. J Biochem (Tokyo), 1990 Feb, 107(2), 236 - 41 Purification, amino acid sequence, and some properties of rabbit kidney lysozyme; Ito Y et al.; The lysozyme (rabbit kidney lysozyme) from the homogenate of rabbit kidney (Japanese white) was purified by repeated cation-exchange chromatography on Bio-Rex 70 . The amino acid sequence was determined by automated gas-phase Edman degradation of the peptides obtained from the digestion of reduced and S-carboxymethylated rabbit lysozyme with Achromobacter protease I (lysyl endopeptidase) . The sequence thus determined was KIYERCELARTLKKLGLDGYKGVSLANWMCLAKWESSYNTRATNYNPGDKSTDYGIFQ INSRYWCNDGKTPRAVNACHIPCSDLLKDDITQAVACAKRVVSDPQGIRAWVAWRNHCQ NQDLTPYIRGCGV, indicating 25 amino acid substitutions from human lysozyme . The lytic activity of rabbit lysozyme against Micrococcus lysodeikticus at pH 7, ionic strength of 0.1, and 30 degrees C was found to be 190 and 60% of those of hen and human lysozymes, respectively . The lytic activity-pH profile of rabbit lysozyme was slightly different from those of hen and human lysozymes . While hen and human lysozymes had wide optimum activities at around pH 5.5-8.5, the optimum activity of rabbit lysozyme was at around pH 5.5-7.0 . The high proline content (five residues per molecule compared with two prolines per molecule in hen or human lysozyme) is one of the interesting features of rabbit lysozyme . The transition temperatures for the unfolding of rabbit, human, and hen lysozymes in 3 M guanidine hydrochloride at pH 5.5 were 51.2, 45.5, and 45.4 degrees C, respectively, indicating that rabbit lysozyme is stabler than the other two lysozymes . The high proline content may be responsible for the increased stability of rabbit lysozyme. Biol Chem Hoppe Seyler, 1990 Feb, 371(2), 137 - 44 Collagenase activation of latent matrix-degrading proteinases from human plasma fibronectin; Imhoff JM et al.; Fibronectin contains two latent gelatinolytic enzymes, FN-gelatinase and FN-laminase that can be activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment . The results of this work show that Achromobacter collagenase cleaves fibronectin and generates an active FN-gelatinase . In contrast to the cathepsin D digest, the collagenase digest directly exhibits gelatinolytic activity without additional activation . The gelatinolytic activity of the total collagenase digest can be inhibited by phenylmethanesulfonyl fluoride, a serine proteinase inhibitor and by pepstatin A, an aspartic-acid proteinase inhibitor . FN-laminase activity, when assayed with its synthetic substrate GPAGPR and also with laminin was revealed after separation of the collagenase digest of fibronectin on heparin Ultrogel . FN-gelatinase and FN-laminase activities were found in heparin unretained and heparin strongly retained fractions . These results have demonstrated that in contrast to cathepsin D, Achromobacter collagenase activates two matrix-degrading proteinases from fibronectin, FN-Gelatinase und FN-Laminase. J Hosp Infect, 1990 Feb, 15(2), 149 - 55 Absence of role for plasmids in resistance to multiple disinfectants in three strains of bacteria; Nagai I et al.; Three chlorhexidine-resistant strains, two of Achromobacter xylosoxidans, one of Pseudomonas cepacia, were isolated from clinical sources . The three strains showed cross-resistance to benzethonium chloride, benzalkonium chloride and alkyldiaminoethylglycine . Resistance persisted after treatment of the strains with acridine orange (12.5 mg-25 mg l-1) . Conjugation to appropriate recipients of Escherichia coli was unsuccessful and plasmid DNA could not be detected . These results suggest that plasmids do not play a role in resistance to multiple disinfectants in the strains of bacteria studied. Biochem Biophys Res Commun, 1990 Jan 30, 166(2), 729 - 35 Isolation of a high specific activity pink, monomeric nitrous oxide reductase from Achromobacter cycloclastes; Hulse CL et al.; Nitrous oxide reductase from Achromobacter cycloclastes has been purified to homogeneity under aerobic conditions via DEAE-cellulose, phenyl-Sepharose, hydroxyapatite, and Sephacryl S-200 chromatography . It consists of a single polypeptide of MW 72 kDa, and contains 3.8 +/- 0.1 copper atoms per molecule . The enzyme is pink as isolated, yet exhibits a specific activity (86 U/mg) that is ca . 40 times greater than that observed for other N2O reductases under similar conditions . Double integration of the anomalous EPR spectrum at 77K showed the presence of 2.0 +/- 0.1 spins per molecule, implying the presence of EPR-silent copper atoms and/or spin-coupled mixed-valent centers. Toxicon, 1990, 28(1), 43 - 54 Purification and amino acid sequence of basic protein I, a lysine-49-phospholipase A2 with low activity, from the venom of Trimeresurus flavoviridis (Habu snake); Yoshizumi K et al.; A basic protein (pI 10.2), named basic protein I, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps . The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides produced by chemical (cyanogen bromide and formic acid) and enzymatic (chymotrypsin, Achromobacter protease I, and Staphylococcus aureus V8 protease) cleavages . The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid and viperid snakes . A most striking feature of this protein is that aspartic acid at the 49th position common in phospholipases A2 is replaced by lysine . When the protein acted on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, oleic acid was preferentially released, indicating that the protein has phospholipase A2 activity . Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was 1.5% that of T . flavoviridis phospholipase A2 isolated previously . The fact that both affinity to Ca2+ and reactivity to p-bromophenacyl bromide of basic protein I are approximately one order of magnitude lower than those of T . flavoviridis phospholipase A2 might explain the low activity of basic protein I. CLAO J, 1990 Jan-Mar, 16(1), 49 - 52 Microbial keratitis and corneal ulceration associated with therapeutic soft contact lenses; Kent HD et al.; We reviewed the records of 22 patients whose corneal ulcers were associated with therapeutic soft contact lens wear . The patients required hospitalization on the Cornea Service at Wills Eye Hospital between January 1, 1978 and September 1, 1988 . A majority of the ulcers were associated with pseudophakic or aphakic bullous keratopathy (9 of 22 cases; 41%); neurotrophic/exposure keratitis was the second most common diagnosis (7 of 22; 32%) . Most patients used topical antibiotics (15 of 22; 68%) and/or corticosteroids (13 of 22; 59%) . Cultures were positive in 15 of 22 cases (68%) . Gram-positive organisms were isolated in 60% the culture-positive cases (9 of 15) . Streptococcus was the most common organism isolated (6 of 15 culture positive-cases; 40%) . Gram-negative organisms were found in four of 15 culture-positive ulcers (27%) . There was only one Pseudomonas infection in the series . Uncommon organisms--including Candida, atypical mycobacteria, Achromobacter, Acinetobacter and Micrococcus--were isolated in five cases . Therapeutic soft contact lens wearers are at risk for developing corneal ulcers; most often these are caused by gram-positive bacteria, especially streptococci, and uncommon organisms. Agric Biol Chem, 1990 Jan, 54(1), 131 - 7 Limited proteolysis of silkworm antitrypsin by several proteinases; Sasaki T et al.; Silkworm antitrypsin (sw-AT) isolated from larval hemolymph was limitedly digested by Achromobacter lysylendopeptidase, alpha-chymotrypsin, subtilisin BPN', subtilisin Carlsberg, papain, or Pseudomonas elastase . Each proteinase could cleave specific site(s) around the reactive site identified for the reaction of sw-AT and bovine trypsin . Among these proteinases, only subtilisin BPN' was inhibited by sw-AT, although weakly . By the cleavable amino acid sequence in sw-AT, it was suggested that whether or not these proteinases were inhibited by sw-AT did not solely depend on their substrate specificities . The susceptibility to the attack of proteinase should indicate that this region is exposed on the molecular surface . The amino acid sequence in the COOH-terminal region slightly away from the reactive site in sw-AT had homology with that in the corresponding region of the serine proteinase inhibitor (serpin) group. J Biol Chem, 1989 Dec 5, 264(34), 20625 - 31 Cloning, nucleotide sequence, and expression of Achromobacter protease I gene; Ohara T et al.; Achromobacter protease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond . A gene coding for API was cloned from Achromobacter lyticus M497-1 . Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids . The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing . Escherichia coli carrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr 50,000 (API') into the periplasm . This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well . The N-terminal amino acid sequence of API' was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain . The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivo as a precursor protein bearing long extended peptide chains at both N and C termini. Cornea, 1989 Dec, 8(4), 267 - 9 Achromobacter xylosoxidans corneal ulcer in a therapeutic soft contact lens wearer; Fiscella R et al.; Achromobacter xylosoxidans is an opportunistic organism that is usually seen in immunocompromised or immunosuppressed patients . It is an aerobic gram-negative rod, often confused with other more commonly seen gram-negative bacteria such as Pseudomonas aeruginosa . The organism is usually sensitive to extended spectrum penicillins such as carbenicillin and usually resistant to aminoglycosides and first generation cephalosporins . We wish to describe a corneal ulcer from A . xylosoxidans that developed in a patient wearing a therapeutic soft contact lens . The patient did not have a preexisting microbial keratitis and was not receiving corticosteroid therapy. Protein Seq Data Anal, 1989 Dec, 2(6), 441 - 4 Determination of partial amino acid sequence of lipoate acetyltransferase of rat pyruvate dehydrogenase complex; Matuda S et al.; The partial amino acid sequence of rat lipoate acetyltransferase was determined using the intact protein and the peptides derived from a digest with Achromobacter protease I . The results showed the amino-terminal sequence of the mature enzyme to be (N) Ser-Leu-Pro-Pro-His-Gln-Lys-Val-Pro-Leu-Pro-Ser- Leu-Ser-Pro-Thr-Met-Gln-Ala-Gly-Thr-Ile-Ala-Arg-Trp-Glu-Lys . In addition, the sequences of two possible lipoyl-binding sites in the subunit, which are very similar to each other, were established. J Biol Buccale, 1989 Dec, 17(4), 269 - 74 Action of a bacterial Achromobacter collagenase on the soft carious dentine: an in vitro study with the scanning electron microscope; Goldberg M et al.; Samples of carious human teeth treated in vitro for 48-92 hours with a bacterial Achromobacter collagenase in solution in borate buffer at 33 degrees were examined with the scanning electron microscope . The enzyme was seen to destroy soft carious dentine but not sound layers of dentine beneath the lesion . This finding may have clinical implications. S Afr Med J, 1989 Nov 18, 76(10), 571 - 3 Fatal neonatal meningitis and ventriculitis caused by multiresistant Achromobacter xylosoxidans . A case report; Manjra AI et al.; Meningitis and ventriculitis in a 6-day-old neonate caused by a Gram-negative glucose-non-fermenting organism, Achromobacter xylosoxidans, was resistant to most antibiotics except ceftazidime and imipenem . The organism became resistant after 28 days treatment with ceftazidime . When the infant was 7 weeks old, imipenem became available but, in spite of 3 days of intravenous treatment, the organism was still recovered from ventricular cerebrospinal fluid and the child died . This would appear to be only the second report of neonatal meningitis caused by this organism. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1366 - 72 Spectroscopic evidence for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase; Suzuki S et al.; The reactions of nitrogen monoxide (NO) with the blue copper-containing nitrite reductases from Alcaligenes sp . NCIB 11015 and Achromobacter cycloclastes IAM 1013 were investigated spectroscopically . The electron paramagnetic resonance (EPR) signals of the blue coppers vanished in the presence of NO at 77 K, being fully restored by the removal of NO . The additions of NO to the enzyme solutions resulted in the substantial bleaching of the visible absorption bands at room temperature . The reactions were also completely reversible . These results suggest the formation of a cuprous nitrosyl complex (Cu+-NO+), which is likely the intermediate in the enzymatic nitrite reduction. J Biochem (Tokyo), 1989 Oct, 106(4), 548 - 51 The primary structure of porcine liver acylamino acid-releasing enzyme deduced from cDNA sequences; Mitta M et al.; Two overlapping cloned cDNAs encoding the entire amino acid sequences of the subunits of acylamino acid-releasing enzyme (AARE) {EC 3.4.19.1} have been isolated from porcine liver cDNA lambda gt10 cDNA libraries and sequenced . Sequence analyses of the cDNA and several Achromobacter protease I-digested peptides of the purified protein revealed that porcine liver AARE consists of four identical subunits, and each comprising a single chain of 732 amino acids with acetylmethionine at the N-terminus. J Bacteriol, 1989 Oct, 171(10), 5467 - 72 Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon; Furukawa K et al.; All the genes we examined that encoded biphenyl/polychlorinated biphenyl (PCB) degradation were chromosomal, unlike many other degradation-encoding genes, which are plasmid borne . The molecular relationship of genes coding for biphenyl/PCB catabolism in various biphenyl/PCB-degrading Pseudomonas, Achromobacter, Alcaligenes, Moraxella, and Arthrobacter strains was investigated . Among 15 strains tested, 5 Pseudomonas strains and one Alcaligenes strain possessed the bphABC gene cluster on the XhoI 7.2-kilobase fragment corresponding to that of Pseudomonas pseudoalcaligenes KF707 . More importantly, the restriction profiles of these XhoI 7.2-kilobase fragments containing bphABC genes were very similar, if not identical, despite the dissimilarity of the flanking chromosomal regions . Three other strains also possessed bphABC genes homologous with those of KF707, and five other strains showed weak or no significant genetic homology with bphABC of KF707 . The immunological cross-reactivity of 2,3-dihydroxybiphenyl dioxygenases from various strains corresponded well to the DNA homology . On the other hand, the bphC gene of another PCB-degrading strain, Pseudomonas paucimobilis Q1, lacked genetic as well as immunological homology with any of the other 15 biphenyl/PCB degraders tested . The existence of the nearly identical chromosomal genes among various strains may suggest that a segment containing the bphABC genes has a mechanism for transferring the gene from one strain to another. Nippon Juigaku Zasshi, 1989 Oct, 51(5), 1003 - 10 Biological effects of lipopolysaccharide from Achromobacter stenohalis on lymphocytes and macrophages; Isogai H et al.; The immunopotentiating activities of lipopolysaccharide from Achromobacter stenohalis (A-LPS) were examined . A-LPS was structurally atypical and gave no endotoxin shock in A-LPS-inoculated mice . Analysis in vitro showed that A-LPS was a potent activator of both macrophages and B-lymphocytes . After macrophage stimulation with A-LPS, interleukin-1 (IL-1) secretion, interferon (IFN) production and chemiluminescence (CL) response were induced . A-LPS was a potent mitogen for spleen lymphocytes . However, induction of interleukin-2 (IL-2) secretion in T lymphocytes was not observed . These activities of A-LPS were similar to or higher than that of enterobacterial LPS. J Biol Chem, 1989 Sep 25, 264(27), 16282 - 91 Cytoplasmic orientation and two-domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence-specific antibodies; Yoshimura A et al.; The predicted cytoplasmic orientation and two-domain structure of the multidrug efflux pump P-glycoprotein were demonstrated with sequence-specific antibodies . We synthesized peptides corresponding to amino acid residues, Glu393-Lys408 (anti-P) and Leu1206-Thr1226 (anti-C) in P-glycoprotein from human mdr1 cDNA and used these peptides to produce polyclonal antibodies . From the primary structure of P-glycoprotein, and anti-C antibody is expected to recognize another position, Leu561-Thr581, in the duplicate structure of P-glycoprotein, but anti-P recognizes only one site . These antibodies bind to multidrug-resistant cells (KB-C2) with permeabilized plasma membrane but do not bind to nonpermeabilized KB-C2 cells or parental KB cells, supporting the predicted cytoplasmic orientation of these sequences . With immunoblotting of the membrane fractions from KB-C2 cells, a major 140-kDa polypeptide of the P-glycoprotein was detected with both anti-P and anti-C . Two minor polypeptides with molecular mass of 95 and 55 kDa were also detected . When membrane vesicles were digested mildly with trypsin, the amount of these two polypeptides increased . Anti-P detected only the 95-kDa polypeptide, and anti-C detected both 95- and 55-kDa polypeptides . Achromobacter lyticus protease I (lysyl endopeptidase) and Staphylococcus aureus V8 protease also produced two polypeptides with similar molecular weights . Absorption into lectin-agarose beads and labeling with {3H}glucosamine indicated that the 95-kDa polypeptide was glycosylated but that the 55-kDa polypeptide was not . These two polypeptides as well as P-glycoprotein were photoaffinity-labeled with a calcium channel blocker, {3H}azidopine, but most of the label was found in the 55-kDa polypeptide . The yield of labeled fragments from membrane vesicles photolabeled after digestion with trypsin was similar to that from membrane vesicles digested with trypsin after photolabeling . These data indicate 1) that the 95-kDa polypeptide is the fragment corresponding to the amino-terminal half of P-glycoprotein containing sugar chains; 2) that the 55-kDa polypeptide is the carboxyl-terminal half which was mainly labeled with {3H}azidopine; and 3) that P-glycoprotein has a relatively rigid structure with a small number of protease-sensitive sites and its global structure is not destroyed by tryptic cleavage. J Clin Microbiol, 1989 Jul, 27(7), 1538 - 42 Application of gas-liquid chromatography to the routine identification of nonfermenting gram-negative bacteria in clinical specimens; Veys A et al.; A total of 430 strains of glucose-nonfermenting gram-negative bacteria representing 35 species were analyzed for their cellular fatty acid composition by gas-liquid chromatography (GLC) . On the basis of qualitative differences in their cellular fatty acid composition, these bacteria could be divided into 19 distinct chromatographic groups . Eight Pseudomonas species, Achromobacter xylosoxidans, group Vd, and Agrobacterium radiobacter were identified from their fatty acid compositions alone . The other glucose-nonfermenting gram-negative bacterial species studied here, classified within nine distinct GLC groups, were easily recognized by using the GLC fatty acid analysis supplemented with a limited number of conventional biochemical tests . The results support the hypothesis that bacterial fatty acid composition is rather specific and that qualitative GLC fatty acid analysis can be adapted in the clinical laboratory either to provide additional criteria for differentiation of closely related groups or to serve as a rapid and highly reproducible method for their routine identification. J Bacteriol, 1989 Jul, 171(7), 4038 - 44 Cloning of a carbofuran hydrolase gene from Achromobacter sp . strain WM111 and its expression in gram-negative bacteria; Tomasek PH et al.; A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp . strain WM111 . When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp . strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded . Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment . There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer . Western blotting (immunoblotting) of the protein products produced by low-level expression in P . putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase. J Biochem (Tokyo), 1989 Jun, 105(6), 986 - 91 Comparative studies on the primary structure of human cystatin as from epidermis, liver, spleen, and leukocytes; Takeda A et al.; We have studied the primary structure of human cystatin As from epidermis, liver, spleen, and leukocytes . These molecules were indistinguishable on PAGE in the presence and absence of SDS, by fast protein liquid chromatography (FPLC) chromatofocusing on a Mono P column, and in amino acid composition . The NH2- and COOH-terminal amino acid sequences of human cystatin As from epidermis, liver, and spleen were identical with those of human leukocyte cystatin A previously reported except for the lack of the NH2-terminal methionine residue in human epidermal cystatin A . The peptides obtained upon digestion of four human cystatin As with Achromobacter protease I (AP) showed identical peptide maps on HPLC except for different retention times of the NH2-terminal peptides . Furthermore, the amino acid compositions of corresponding separated peptide quartets were identical . We also determined the complete amino acid sequence of human epidermal cystatin A by sequencing peptides obtained from AP digestion and cyanogen bromide (CNBr) cleavage . It consisted of 97 amino acid residues, and was identical with those of human cystatin As from liver, spleen, and leukocytes except for the lack of the NH2-terminal methionine residue. Rev Cubana Med Trop, 1989 May-Aug, 41(2), 274 - 83 {Identification of non-fermenting gram-negative bacteria . "Havana Center" Pediatric Teaching Hospital . December 1986 through May 1987}; Azahares Romero LE et al.; A study is made of non-fermenting gram-negative bacterial strains isolated at the Microbiology Lab, "Centro Habana" Pediatric Teaching Hospital . The strains studied come from various types of samples with prevalence of exudates and smears . A definite biotyping of 100% of strains is made . 90.0% are classified within the genus Pseudomonas . In addition, organisms of the genera Acinetobacter, Alcaligenes, Achromobacter, and Moraxella are identified . The most frequent species turned out to be Pseudomonas aeruginosa, which accounted for 68.4% of the total. J Biochem (Tokyo), 1989 Apr, 105(4), 573 - 6 Identification of glutamic acid 186 affinity-labeled by 2,3-epoxypropyl alpha-D-glucopyranoside in soybean beta-amylase; Nitta Y et al.; Soybean beta-amylase was modified with 2,3-epoxypropyl alpha-D-{U-14C}glucopyranoside ({14C}alpha-EPG), a radioactive affinity-labeling reagent for beta-amylase, until it lost 95% of its enzyme activity . After S-carboxymethylation at pH 8.0 of SH groups, the modified enzyme was digested at pH 7.0 with Achromobacter protease I and the digest was fractionated by reverse-phase HPLC . A radioactive peptide was finally isolated and its amino acid sequence was determined to be 181Leu-Gly-Pro-Ala-Gly-Glu186 . Radioactivity derived from {14C}-alpha-EPG was found exclusively at Glu-186, the gamma-carboxyl group of which is esterified with the affinity label . It was concluded that the carboxylate of Glu-186 is a functional group at the catalytic site of soybean beta-amylase. J Biol Chem, 1989 Mar 5, 264(7), 3832 - 9 The primary structure and structural characteristics of Achromobacter lyticus protease I, a lysine-specific serine protease; Tsunasawa S et al.; The complete amino acid sequence of Achromobacter lyticus protease I (EC 3.4.21.50), which specifically hydrolyzes lysyl peptide bonds, has been established . This has been achieved by sequence analysis of the reduced and S-carboxymethylated protease and of peptides obtained by enzymatic digestion with Achromobacter protease I itself and Staphylococcus aureus V8 protease and by chemical cleavage with cyanogen bromide . The protease consists of 268 residues with three disulfide bonds, which have been assigned to Cys6-Cys216, Cys12-Cys80, and Cys36-Cys58 . Comparison of the amino acid sequence of Achromobacter protease and other serine proteases of bacterial and mammalian origins has revealed that Achromobacter protease I is a mammalian-type serine protease of which the catalytic triad comprises His57, Asp113, and Ser194 . It has also been shown that the protease has 9- and 26-residue extensions of the peptide chain at the N and C termini, respectively, and overall sequence homology is as low as 20% with bovine trypsin . The presence of a disulfide bridge between the N-terminal extension Cys6 and Cys216 close to the putative active site in the C-terminal region is thought to be responsible for the generation of maximal proteolytic function in the pH range 8.5-10.7 and enhanced stability to denaturation. Antibiot Khimioter, 1989 Jan, 34(1), 7 - 10 {Production of antibiotic substances by natural variants of the marine bacterium Vibrio Fischeri}; Baranova NA et al.; It was shown that under definite conditions there was competition between natural variants of sea bacteria belonging to V . fischeri . Natural variants of V . fischeri, strain 6 differed in their resistance to streptomycin and had different growth rates under conditions of limited aeration . Morphologically all the variants were identical . V . fischeri P-0, V . fischeri P-1 and V . fischeri P-2 were studied . The study revealed that V . fischeri P-0 produced a non-dialysing thermostable trypsin-sensitive substance inhibiting the growth of V . fischeri P-1 and V . fischeri P-2 . The maximum activity of the antibacterial substance was observed when V . fischeri P-0 was grown in a liquid medium with peptone and yeast extract without agitation at 26 degrees C . The inhibiting substance was also active against V . fischeri BKM B995 and V . fischeri P-7 isolated as a result of V . fischeri P-0 exposure to ethidium bromide . The substance had no effect on the following bacterial species: Aeromonas liquefaciens 301, Achromobacter liquefaciens, Pseudomonas putida 15, Pseudomonas fluorescence 7, Escherichia coli AH-32 and Staphylococcus aureus. Bioorg Khim, 1989 Jan, 15(1), 5 - 17 {Catalytic component of calmodulin-independent adenylate cyclase from bovine brain . Isolation and determination of partial amino acid sequence}; Lipkin VM et al.; The catalytic component of calmodulin-independent adenylate cyclase of cattle cerebral cortex was solubilized and purified to the homogeneous state . The conditions for preparative obtaining of the enzyme on the column with immobilized antibodies to adenylate cyclase were found . These antibodies were proved to interact with the calmodulin-independent rather than the calmodulin-dependent form of the enzyme . Molecular mass of the calmodulin-independent adenylate cyclase determined electrophoretically is 140 +/- 10 kDa . Amino acid composition of the enzyme and sequences of its fragments (in total 300 amino acid residues) obtained upon treatment with lysyl-specific proteinase from Achromobacter liticus were determined . Clone containing a cDNA 605 bp insertion coding for the 183 amino acid residue fragment of adenylate cyclase was isolated from the bovine brain cDNA library . Homology of this fragment to the known sequences of Escherichia coli and Bordetella pertussis adenylate cyclases was revealed. FEMS Microbiol Lett, 1989 Jan 1, 48(1), 61 - 4 Extracellular product of Nocardia amarae induces bacterial cell flocculation; Koizumi J et al.; The fact that Nocardia amarae YK1 produced a bacterial flocculation-inducing substance (designated as FIX) was discovered . FIX had a function of flocculating proliferous cells . FIX-induced flocculation was inhibited by making cells resting, but not completely by adding chloramphenicol . FIX worked widely on Gram-positive to -negative bacteria . In the presence of FIX, Achromobacter cycloclastus IAM1013, Acinetobacter calcoaceticus IAM1517, Bacillus subtilis IAM1069, Escherichia coli C600-1, E . coli IAM1239, Flavobacterium lutescens IAM1667, Klebsiella pneumoniae IAM1102, Micrococcus luteus IAM1313 and Pseudomonas putida IAM1002 formed flocs . B . cereus IAM1029, however, exhibited no flocculation. G Batteriol Virol Immunol, 1989 Jan-Dec, 82(1-12), 34 - 8 {Bacterial infections of the ear: case contribution on clinical and microbiological aspects}; Cavallo GP et al.; Ciprofloxacin was given in oral doses of 250 mg . each 8 hours to 21 patients with otitis . The efficacy of treatment was assessed by clinical and bacteriological parameters . The 61.9% of isolated bacteria are opportunistic . A case of chronic otitis by Achromobacter xylosoxidans is described. J Clin Microbiol, 1988 Nov, 26(11), 2425 - 6 Achromobacter xylosoxidans (Alcaligenes xylosoxidans subsp . xylosoxidans) meningitis associated with a gunshot wound; D'Amato RF et al.; The microbiological and clinical features of a case of Achromobacter xylosoxidans (Alcaligenes xylosoxidans subsp . xylosoxidans) meningitis associated with a gunshot wound are described . To our knowledge, this is the third confirmed case report of meningitis caused by this organism. Appl Environ Microbiol, 1988 Nov, 54(11), 2874 - 6 Microbial transformation of the pyrethroid insecticides: permethrin, deltamethrin, fastac, fenvalerate, and fluvalinate; Maloney SE et al.; Pure cultures of Bacillus cereus, Pseudomonas fluorescens, and Achromobacter sp . were shown to transform five pyrethroid insecticides in the presence of Tween 80 . One of the major products, 3-phenoxybenzoic acid, was further transformed to 4-hydroxy-3-phenoxybenzoic acid . Permethrin was the most rapidly transformed of the pyrethroids, with a half-life of less than 5 days. Microb Pathog, 1988 Nov, 5(5), 57 - 69 Identity of molecular structure of Shiga-like toxin I (VT1) from Escherichia coli O157:H7 with that of Shiga toxin; Takao T et al.; The primary structures of the A and B subunits of Shiga toxin and of Shiga-like toxin I (VT1), isolated from the culture supernatants of Shigella dysenteriae 1 and Escherichia coli O157:H7, respectively, were analyzed by Edman degradation of intact proteins and peptides in their digests with trypsin or Achromobacter protease I and also by fast atom bombardment mass spectrometry of the digests . The results indicated that the A and B subunits of Shiga toxin and Shiga-like toxin I have the same primary structures . The identity of their primary structures was confirmed by determining the nucleotide sequence of the gene encoding Shiga-like toxin I cloned from a Shiga-like toxin I converting phage . This nucleotide sequence was different from that reported by Jackson et al . (Microbial Pathogenesis 1987; 2: 147-153), by Calderwood et al . (Proc Natl Acad Sci USA 1987; 84: 4364-8) and by Grandis et al . (J Bacteriol 1987; 169: 4313-9) in one base at position 231, which was found to be adenine instead of thymine, which they reported . The amino acid residue at position 45 from the N-terminus of the A subunit of Shiga-like toxin I deduced from the nucleotide sequence determined in this study is threonine, which corresponds with that found by amino acid sequencing, whereas from previous reports by other investigators it is serine . Edman degradation of the intact A subunit of Shiga toxin indicated that the A subunit was nicked between Ala253 and Ser254 to form A1 and A2 fragments linked by a disulfide bond. J Biol Chem, 1988 Oct 15, 263(29), 14625 - 8 Resonance Raman spectra of the copper-sulfur chromophores in Achromobacter cycloclastes nitrite reductase; Dooley DM et al.; Resonance Raman spectroscopy at ambient temperature and 77 K has been used to probe the structures of the copper sites in Achromobacter cycloclastes nitrite reductase . This enzyme contains three copper ions per protein molecule and has two principal electronic absorption bands with lambda max values of 458 and 585 nm . Comparisons between the resonance Raman spectra of nitrite reductase and blue copper proteins establish that both the 458 and 585 nm bands are associated with Cu(II)-S(Cys) chromophores . A histidine ligand probably is also present . Different sets of vibrational frequencies are observed with 457.9 nm (ambient) or 476.1 nm (77 K) excitation as compared with 590 nm (ambient) or 593 nm (77 K) excitation . Excitation profiles indicate that the 458 and 585 nm absorption bands are associated with separate {Cu(II)-S(Cys)N(His)} sites or with inequivalent and uncoupled cysteine ligands in the same site . The former possibility is considered to be more likely. Eur J Biochem, 1988 Oct 1, 176(3), 683 - 97 Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae; Kawata Y et al.; The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined . This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and alpha-chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S-carboxymethylated or S-aminoethylated protein . Digestion with A . lyticus protease I was successfully used to degrade the N-terminal half of the S-aminoethylated protein at cysteine residues . RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29,155 . The sugar content is 7.9% (by mass) . Three glycosylation sites were determined at Asns 15, 76 and 239 . Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1 . These similar residues may be important for the catalytic activity of RNase T2. Anal Biochem, 1988 Oct, 174(1), 54 - 64 Generation of peptides suitable for sequence analysis by proteolytic cleavage in reversed-phase high-performance liquid chromatography solvents; Welinder KG; A methodological study of practical importance to protein sequencing has been carried out . Peptide mapping and sequence analysis of the cleavage products of reduced and carboxymethylated ribonuclease have been applied to the study of the activity and specificity of trypsin, chymotrypsin, elastase, lysyl endopeptidase (Achromobacter protease I), endoproteinase Arg-C (from mouse submaxillary gland), Staphylococcus aureus V8 protease, pepsin, and thermolysin in the presence of 20% methanol, ethanol, 2-propanol, and acetonitrile at 22 and 37 degrees C . The peptide bond specificities were retained, and the activities were generally unaffected or moderately reduced at 22 degrees C and pH 8 . At 37 degrees C the activity of chymotrypsin, endoproteinase Arg-C, V8 protease at pH 4, and pepsin was substantially reduced and decreased in the order methanol, ethanol, 2-propanol, and acetonitrile . The activity of thermolysin at 55 degrees C was reduced very little in the presence of 20% organic solvent and 50 mM Ca2+ . In low calcium and 20% 2-propanol at 22 degrees C the activity of thermolysin was restricted to the complete and specific cleavage of peptide bonds N-terminally of Phe, Ile, and Leu . The experiments suggest that secondary proteolytic digestions can be carried out directly in reversed-phase-HPLC fractions, and that organic cosolvents can be applied to control the degree of proteolysis . Moreover, the denaturing potential of these solvents might be useful in the degradation of proteins resistant to proteolysis, for example, in studies aimed at identification of disulfide bridges. J Clin Microbiol, 1988 Sep, 26(9), 1901 - 3 Evaluation of 9-chloro-9-{4-(diethylamino)phenyl}- 9,10-dihydro-10-phenylacridine hydrochloride (C-390) in broth and agar media for identification of Pseudomonas aeruginosa; Fader RC et al.; Brain heart infusion broth and Mueller-Hinton agar containing the compound 9-chloro-9-{4-(diethylamino)phenyl}- 9,10-dihydro-10-phenylacridine hydrochloride (C-390) were evaluated as selective media for identifying Pseudomonas aeruginosa . Of the 192 Pseudomonas spp . and 68 additional oxidase-positive or glucose-nonfermenting gram-negative organisms tested, only P . aeruginosa (120 of 121 isolates) and certain strains of Pseudomonas cepacia, Aeromonas hydrophila, and Achromobacter xylosoxidans were able to grow in brain heart infusion broth containing 15 micrograms of C-390 per ml . When production of green or yellow-green pigment after overnight growth on Mueller-Hinton agar containing 30 micrograms of C-390 per ml was used as the criterion for identification, only P . aeruginosa produced a positive test (96% sensitivity and 100% specificity) . The agar medium, therefore, can provide a single differential medium for the overnight identification of P . aeruginosa. Anal Biochem, 1988 Aug 1, 172(2), 420 - 6 A spectrophotometric assay for dissimilatory nitrite reductases; Hulse CL et al.; A spectrophotometric assay for dissimilatory nitrite reductases has been developed utilizing mammalian cytochrome c (equine heart) as reductant and spectrophotometric agent . The copper-containing nitrite reductase from Achromobacter cycloclastes has been shown to have apparent Km's for reduced cytochrome c and nitrite of 86 +/- 5 and 5.63 +/- 0.03 microM, respectively . The heme cd-containing enzyme from Pseudomonas stutzeri was shown to have apparent Km's for reduced cytochrome c and nitrite of 260 +/- 60 and 1.8 +/- 0.8 microM, respectively . This assay represents a simple, general method for consistently evaluating the activity of the copper- and heme cd-containing nitrite reductases that are capable of utilizing readily available mammalian cytochrome c as electron donor and should be useful for mechanistic studies of these enzymes. J Hosp Infect, 1988 Jul, 12(1), 1 - 6 Outbreak of infection with Achromobacter xylosoxidans from contaminated intravascular pressure transducers; Gahrn-Hansen B et al.; Achromobacter xylosoxidans contaminating transducers caused 15 cases of hospital infection . In the eight patients with bacteraemia the interval from inoculation to fever was an average of 6.6 days . All the infected patients recovered . Computerization of laboratory records allowed retrieval of previous isolates, and review of clinical records focused the problem on patients with cardiac and aortic diseases . The problem arose from the re-use of disposable equipment after disinfection with a benzalcone. Biochim Biophys Acta, 1988 Jun 29, 955(1), 43 - 9 New Achromobacter collagenase and its immunological relationship with a vertebrate collagenase; Nguyen TT et al.; Evidence is presented that Achromobacter iophagus produces two distinct collagenases . Achromobacter collagenases A and B were separated by high-performance liquid chromatography from partially purified enzyme . The main collagenase, A (EC 3.4.24.8), which has been already described, was eluted in the region of molecular mass 110-90 kDa . A minor collagenase B eluted in the region of 320 kDa, although in SDS-gel electrophoresis the apparent molecular masses of its main active forms were estimated as 55 and 110 kDa . The specificities of collagenases A and B are different . Collagenase A splits in its synthetic substrate Pz-Pro-Leu-Gly-Pro-DArg the bond Leu-Gly, collagenase B does not split this substrate . Both collagenases split bonds Gln-Gly and Leu-Gly in synthetic peptides DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-DArg-OH and DNP-Pro-Leu-Gly-Ile-Ala-Gly-DArg-NH2, respectively . Collagenase B is twice as active as A on the native collagen type I . Both enzymes are inhibited by EDTA . The antibodies raised against the human tooth collagenase specifically inhibited the collagenase B, but did not influence the activity of collagenase A . These results indicate, to our knowledge for the first time, an immunological relationship between a bacterial and a vertebrate collagenase. Eur J Clin Microbiol Infect Dis, 1988 Jun, 7(3), 428 - 31 Comparative in vitro activity of the new oral cephalosporin cefixime; Counts GW et al.; Cefixime was 8 to 10 times more active than cefaclor and augmentin against isolates of Escherichia coli, Klebsiella pneumoniae and Salmonella typhi, MIC90 values ranging from 0.06 to 0.25 micrograms/ml . However, none of these three drugs was particularly active against isolates of more resistant gram-negative bacilli such as Enterobacter, Serratia, Citrobacter, Acinetobacter, Providencia and Achromobacter spp . The lowest MIC values for gram-negative bacilli were seen with ciprofloxacin, except for isolates of Acinetobacter, where cotrimoxazole was the most active of the five drugs studied . Augmentin and ciprofloxacin exhibited the lowest MICs for isolates of streptococci and corynebacteria . Although cefixime may be among the most active oral beta-lactam drugs, it does not appear to be useful for treatment of infections caused by more resistant gram-negative bacilli. Biochem Biophys Res Commun, 1988 May 31, 153(1), 74 - 80 Near stoichiometric, irreversible inactivation of bacterial collagenases by o-chloranil (3,4,5,6-tetrachloro-1,2-benzoquinone); Makinen PL et al.; The hydrogen-abstracting quinone derivative 3,4,5,6-tetrachloro-1,2-benzoquinone (o-chloranil) caused a strong, near stoichiometric, irreversible inactivation of the collagenases from Bacillus cereus, Clostridium histolyticum and Achromobacter iophagus . p-Chloranil was a weaker inactivator . o-Chloranil reacted rapidly with a site that affected substrate binding . Amino acid analyses of native and totally inactivated enzymes, and the pH-profile of inactivation suggest that the dissociated form of a tyrosine residue was modified. J Biol Chem, 1988 May 15, 263(14), 6731 - 7 Characterization of the major protein-tyrosine-phosphatases of human placenta; Tonks NK et al.; In the preceding article (Tonks, N . K., Diltz, C . D., and Fischer, E . H . (1988) J . Biol . Chem . 263, 6722-6730), the purification of the major protein-tyrosine-phosphatases from human placenta, some to apparent homogeneity, was described . This report compares the characteristics of these enzymes and clearly identifies at least two distinct protein-tyrosine-phosphatase catalytic subunits . All were absolutely specific for phosphotyrosyl residues and showed no activity on any of the phosphoseryl/phosphothreonyl-containing proteins tested; they exhibited a high affinity for substrate with Km values in the submicromolar range . All were absolutely dependent on sulfhydryl compounds and appeared to contain at least one highly reactive cysteinyl residue essential for activity . Subtypes 1A and 1B could be distinguished by their response to polyanionic and polycationic compounds . The 1B enzymes were activated by EDTA, spermine, spermidine, and myelin basic protein to a greater extent than the 1A subtypes . Furthermore, they were inhibited by approximately 2 orders of magnitude lower concentrations of heparin (IC50 approximately 20 nM) and 1:1 or 4:1 poly (glutamate/tyrosine) (IC50 approximately 50 nM) than the 1A subtypes . Surprisingly, inhibition by the glutamate/tyrosine copolymers was strictly noncompetitive . Peptide mapping following digestion with Achromobacter protease I or Staphylococcus aureus V8 protease supported the view that, whereas protein-tyrosine-phosphatase subtypes 1A and 1B are different, their soluble and particulate counterparts are closely related structurally and are distinct from serine/threonine phosphatases 1 and 2A. FEBS Lett, 1988 Apr 25, 231(2), 347 - 51 Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence; Yokoyama C et al.; The complete amino acid sequence of prostaglandin endoperoxide synthase from sheep vesicular gland has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA . The results were confirmed by digestion of the enzyme with carboxypeptidase Y and by automated Edman degradation of the intact enzyme polypeptide and peptide fragments obtained by limited proteolysis of the enzyme with Achromobacter proteinase I . Mature sheep prostaglandin endoperoxide synthase is shown to be composed of 576 amino acids with an Mr of 66,175 . The precursor peptide is predicted to contain a 24-residue signal peptide . The serine residue susceptible to acetylation by aspirin is found to be located near the C-terminus of the enzyme polypeptide. J Biol Chem, 1988 Apr 25, 263(12), 5724 - 31 A phospholipase A2 in the supernatant fraction of rat spleen . Its similarity to rat pancreatic phospholipase A2; Tojo H et al.; Rat spleen supernatant contained two forms of calcium-dependent cellular phospholipase A2 which could be separated from each other by TEAE-cellulose chromatography . The phospholipase A2, named PLA2 S-1, present in the major flow-through fraction was purified to homogeneity . The structural and catalytic properties of splenic PLA2 S-1 were systematically compared with those of rat pancreatic phospholipase A2 . Structural evidence, including the sequence of the N-terminal 32 residues, peptide maps obtained on Achromobacter protease I digestion and cyanogen bromide cleavage, and the amino acid composition, showed the close similarity of the two enzymes . Their catalytic and immunochemical properties were also similar . These results demonstrated the existence of a pancreatic type phospholipase A2 in a non-pancreatic organ as a member of the cellular phospholipases A2 and suggest the potential functional involvement of pancreatic type phospholipase A2 in cellular phospholipid metabolism. J Biochem (Tokyo), 1988 Apr, 103(4), 596 - 605 Amino acid sequence of a coagulant enzyme, flavoxobin, from Trimeresurus flavoviridis venom; Shieh TC et al.; The amino acid sequence of a coagulant enzyme, named flavoxobin, isolated from the venom of Trimeresurus flavoviridis (the habu snake) was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides generated by chemical (cyanogen bromide and hydroxylamine) and enzymatic (clostripain, Staphylococcus aureus V8 protease, Achromobacter protease I, and elastase) cleavages . Hydrazinolysis was also employed to determine the C-terminal amino acid . The enzyme consisted of 236 amino acids and had a calculated molecular weight of 25,744 . Flavoxobin was found to be highly (69%) homologous in sequence to batroxobin, a coagulant enzyme from the venom of Bothrops atrox, and 27, 39, and 31% homologous to bovine thrombin, bovine trypsin, and human kallikrein, respectively . The sequence around the active site serine residue deduced from the homology relationship was Phe-Asp-Ser-Gly-Thr, which is different from the common sequence, Gly-Asp-Ser-Gly-Gly, for most serine proteases . Flavoxobin appears to be similar in secondary structure composition to batroxobin. J Clin Microbiol, 1988 Mar, 26(3), 598 - 9 Achromobacter xylosoxidans (Alcaligenes xylosoxidans subsp . xylosoxidans) bacteremia associated with a well-water source: case report and review of the literature; Spear JB et al.; A case of community-acquired Achromobacter xylosoxidans bacteremia in a patient with metastatic breast carcinoma is described . The patient's home drinking water was identified as the source of her bacteremia . The case represents the first in which a community-acquired infection due to this organism has been attributed to a documented water source. J Biol Chem, 1988 Feb 25, 263(6), 2651 - 7 Primary structure of chicken liver acetyl-CoA carboxylase deduced from cDNA sequence; Takai T et al.; The complete amino acid sequence of acetyl-CoA carboxylase from chicken liver has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA . The results were confirmed by Edman degradation of peptide fragments obtained by digestion of the enzyme polypeptide with Achromobacter proteinase I or staphylococcal serine proteinase . Chicken liver acetyl-CoA carboxylase is predicted to be composed of 2,324 amino acid residues, having a calculated molecular weight of 262,706 . The biotin carboxyl carrier protein domain is located in the middle region of the enzyme polypeptide . The amino-terminal portion of the acetyl-CoA carboxylase has been found to exhibit a homologous primary structure to that of carbamyl phosphate synthetase . Localization of possible functional domains including biotin carboxylase subsite in the acetyl-CoA carboxylase polypeptide is discussed. APMIS, 1988 Feb, 96(2), 185 - 7 Studies on ampicillin resistance in Achromobacter xylosoxidans . Brief report; Gahrn-Hansen B et al.; Population analyses of susceptibility to ampicillin in ampicillin-susceptible and ampicillin-resistant strains of Achromobacter xylosoxidans revealed the existence of ampicillin-resistant subpopulations in ampicillin-susceptible isolates . Bacteria resistant to a concentration four times the one that inhibited the majority population had a frequency of 10(-3) to 10(-4) . Strains isolated from aqueous environments are often found susceptible to ampicillin, while sporadic cases of infections with A . xylosoxidans are often caused by ampicillin-resistant strains . We suggest that the isolation from clinical specimens of ampicillin-susceptible strains, therefore, may be an indication of nosocomial infections due to recently contaminated aqueous solutions or medical equipment. Infect Control Hosp Epidemiol, 1988 Feb, 9(2), 84 - 7 Nosocomial Achromobacter xylosoxidans infections; Schoch PE et al.; A xylosoxidans is being recognized as an important nosocomial pathogen . As more patients are rendered immunosuppressed by chemotherapy, this organism's increasing role in hospital-acquired infections will be assured . Achromobacter is a water-borne organism, highly resistant to most antibiotics, and even to some disinfectant solutions, and easily establishes itself in the hospital aquatic environment . Achromobacter infections and outbreaks should be recognized and approached as serious problems requiring the institution of appropriate infection control measures . A xylosoxidans infections should be empirically treated with a combination of a third generation cephalosporin and TMP-SMX pending susceptibility testing. J Biol Chem, 1988 Jan 15, 263(2), 839 - 49 Complete amino acid sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Lively MO et al.; The complete amino acid sequence of 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was determined by direct analysis of the S-carboxamidomethyl protein . A complete set of nonoverlapping peptides was produced by cleavage with a combination of cyanogen bromide and specific proteolytic enzymes . The active enzyme is a dimer of two identical polypeptide chains composed of 470 amino acids each . The NH2-terminal amino acid residue of the polypeptide chain was shown to be N-acetylserine by fast atom bombardment mass spectrometry of the purified N-terminal tetradecapeptide isolated after cleavage of the intact S-carboxamidomethylated protein with lysyl endoproteinase (Achromobacter protease I) . Alignment of the set of unique peptides was accomplished by the analysis of selected overlapping peptides generated by proteolytic cleavage of the intact protein and the larger purified cyanogen bromide peptides with trypsin, Staphylococcus aureus V8 protease, and lysyl endoproteinase . Four nonoverlapping peptides were aligned by comparison with the amino acid sequence predicted from a partial cDNA clone encoding amino acid positions 166-470 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Colosia, A.D., Lively, M., El-Maghrabi, M . R., and Pilkis, S . J . (1987) Biochem . Biophys . Res . Commun . 143, 1092-1098) . The nucleotide sequence of the cDNA corroborated the peptide sequence determined by direct methods . A search of the Protein Identification Resource protein sequence database revealed that the overall amino acid sequence appears to be unique since no obviously homologous sequences were identified . However, a 100-residue segment of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (residues 250-349), including the active site histidine residue of the bisphosphatase domain, was found to be homologous to the active site regions of yeast phosphoglycerate mutase and human bisphosphoglycerate mutase. Connect Tissue Res, 1988, 17(2), 153 - 8 Bacterial collagenase and collagen identification; Berry L et al.; A method is described for the cleavage of collagenous molecules by bacterial collagenase in the presence of sodium dodecyl sulphate (SDS) and urea . Comparison of three commercially available preparations of bacterial collagenase showed that the most efficient cleavage under these conditions was by the enzyme isolated from Achromobacter iophagus (E.C . 3.4.24.8) . No non-specific proteinase activity was apparent in conditions where all collagen types showed some susceptibility to attack . This method represents a simple one-stage identification of collagenous molecules in complex mixtures of proteins and where limited amounts of a protein are available. J Clin Microbiol, 1987 Oct, 25(10), 1952 - 5 Diversity of plasmids in Achromobacter xylosoxidans isolates responsible for a seemingly common-source nosocomial outbreak; Arroyo JC et al.; Achromobacter xylosoxidans, an uncommon yet highly resistant opportunistic pathogen, was isolated from nine hospitalized patients during an 8-month period . It had been isolated from only seven patients with either nonfatal infection or colonization from 1981 to 1984 . From June 1985 to January 1986, A . xylosoxidans was isolated 18 times from seven different sites (sputum, 7 times; urine, 4 times; blood, 3 times; and lung, pleural fluid, wound tissue, and tracheal aspirate, 1 time each) . Four patients died, including the three with bacteremia . All but two patients had nosocomial infections and either were on the same ward or were cared for by the same staff members . Eleven A . xylosoxidans strains yielded eight distinct plasmids (8, 21, 23, 26, 38, 50, 51, and 64 megadaltons) . Whole-cell peptide patterns of 10 of these strains were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Isolates from the same patient contained the same plasmids and had identical peptide patterns but differed from other strains in both parameters . Plasmids were absent from the two community-acquired isolates . Although nosocomial strains showed similar antibiotic resistance patterns (only moxalactam and ticarcillin-clavulanic acid were uniformly active) and cross-contamination was strongly suggested epidemiologically, results of plasmid and peptide analyses did not support the possibility of a single-strain outbreak. Rev Infect Dis, 1987 Sep-Oct, 9(5), 1001 - 5 Achromobacter xylosoxidans bacteremia; Mandell WF et al.; Achromobacter xylosoxidans is a rare cause of bacteremia . A case of community-acquired pneumonia and bacteremia due to Achromobacter in a patient with concomitant pulmonary tuberculosis is reported herein . The majority of patients who have developed achromobacter bacteremia have had a predisposition to infection (although the predisposing conditions have been diverse) . Immunosuppression has been reported in only one of the seven patients with achromobacter bacteremia for whom detailed information is available . Achromobacter is usually resistant to ampicillin, cephalosporins, and aminoglycosides . Antipseudomonal penicillins and trimethoprim-sulfamethoxazole inhibit most isolates . Multiple-drug resistance is common, and optimal therapy is not known. Proc Natl Acad Sci U S A, 1987 Aug, 84(16), 5610 - 4 Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib; Titani K et al.; We report the amino acid sequence of a 299-residue segment from the alpha chain of the human platelet membrane glycoprotein Ib . This includes the complete sequence of the amino-terminal tryptic fragment of 290 residues comprising the von Willebrand factor-binding domain . Two primary sets of overlapping fragments were obtained by cleavage of the S-carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively . Additional fragments were obtained by treatment of native glycocalicin with trypsin, Staphylococcus aureus V8 protease, and Serratia marcescens protease . Analysis of all these fragments provided data that allowed determination of the continuous sequence corresponding to approximately half of the alpha-chain polypeptide . This region of glycoprotein Ib is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains . A hydrophilic region exists between residues 215 and 299, which contains a cluster of 10 negatively charged residues at 269-287 . This area is likely to attract positively charged molecules . The hydrophilic, highly glycosylated (at serine and threonine residues) region corresponding to the previously described "macroglycopeptide" and representing the carboxyl-terminal half of the alpha chain is likely to begin at residue 292 . The determined sequence of the alpha chain of glycoprotein Ib contains a region (residues 29-193) with seven repeats, which is indicative of gene duplication and is highly homologous to human leucine-rich alpha 2-glycoprotein . This protein sequence agrees completely with that deduced from the cDNA sequence reported by Lopez et al . {Lopez, J.A., Chung, D.W., Fujikawa, K., Hagen, F.S., Papayannopoulou, T . & Roth, G.J . (1987) Proc . Natl . Acad . Sci . USA 84, 5615-5619}. J Biochem (Tokyo), 1987 Jul, 102(1), 111 - 22 Bacterial synthesis of recombinant alpha-human atrial natriuretic polypeptide; Saito Y et al.; The high-level synthesis of alpha-human atrial natriuretic polypeptide hormone in Escherichia coli has been achieved based on the idea that the yield of a small, basic and unstable polypeptide, such as the natriuretic polypeptide, would be improved by fusion with an appropriate protective polypeptide to construct a neutral fused polypeptide . We prepared an expression vector, pCLaHtrp3t, coding a neutral polypeptide containing 130 amino acid residues in which the polypeptide hormone was fused to a newly designed protective polypeptide through lysine as an enzymatically cleavable residue . The fused polypeptide was synthesized at the high level of 32% of total cellular proteins and at 4.7 X 10(6) molecules per single cell . It was recovered as cellular insoluble fraction and purified to homogeneity . For the isolation of the peptide hormone from the resultant fused polypeptide, Achromobacter protease I, a lysine-specific endopeptidase was used, because it has sufficient activity even in 8 M urea . The recombinant natriuretic polypeptide was indistinguishable from native alpha-human atrial natriuretic polypeptide as regards amino acid sequence as well as biological activity. Am J Ophthalmol, 1987 Jun 15, 103(6), 745 - 8 Chronic bacterial endophthalmitis; Ficker L et al.; We studied a specific syndrome of uveitis secondary to intraocular bacterial pathogens of low virulence after extracapsular cataract extraction and intraocular lens implantation in three eyes . The onset of photophobia, visual impairment, conjunctival redness, and uveitis was delayed for four days to 12 weeks after surgery . Chronic inflammation persisted for five weeks to 16 months before a definitive diagnosis was made . Signs and symptoms were suppressed by administration of topical and systemic corticosteroids . Intraocular biopsy and antibiotic injection both established the cause as bacterial endophthalmitis and resulted in resolution of signs and symptoms . Staphylococcus epidermidis was cultured in two eyes and Achromobacter was cultured in one. Pathol Biol (Paris), 1987 Jun, 35(5 Pt 2), 839 - 42 {Treatment of post-traumatic and post-neurosurgical bacterial meningitis with ceftriaxone alone or in combination with fosfomycin}; May T et al.; From 1984 to 1986, 13 patients (10 adults, 3 children) with bacterial meningitis following neurosurgery or traumatism were given ceftriaxone alone 6 times at a dose of 40 mg/kg one IV injection per day, or in association 7 times with fosfomycin at a dose of 200 mg/kg/day, 3 IV perfusions every 4 h . The bacteriological diagnosis was confirmed in 9 cases (3 Staphylococcus aureus, 4 Streptococcus pneumoniae, 1 Klebsiella, 1 Peptococcus) . In vitro neither synergy nor antagonism were observed between the two antimicrobial agents . The acute infections episode resolved in all patients except on who died with a negative CSF culture . One superinfection meningitis with Achromobacter was seen . CSF concentrations of ceftriaxone were assayed and found to be comparable with those reported by most authors . Tolerance was excellent for all our patients. Biochem Biophys Res Commun, 1987 May 29, 145(1), 563 - 8 Kinetic studies of the copper nitrite reductase from Achromobacter cycloclastes and its interaction with a blue copper protein; Kashem MA et al.; Transient state, burst and steady state kinetics of reactions of the blue copper nitrite reductase (NIR) and blue copper protein from Achromobacter cycloclastes are investigated . The two copper-containing species are reacted with each other and where possible with dithionite, ascorbate and nitrite . Both copper proteins are fully reduced by dithionite with both S2O4(2-) and SO2- . species active . NIR is only partially reduced by ascorbate in an unusual biphasic reaction consistent with complete reduction of type-one copper followed by partial reduction of type-two copper . The rate of reduction of the type-one copper is accelerated using phenazine methosulfate as mediator . Nitrite can oxidize dithionite-reduced NIR but cannot reduce oxidized NIR . Rate constants were determined for all observed reactions. Biochemistry, 1987 Apr 21, 26(8), 2189 - 94 Amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) eggs; Titani K et al.; The complete amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) egg is presented . The 111-residue sequence was determined by the analysis of peptides generated by digestion of the S-carboxymethylated protein with Achromobacter protease I, chymotrypsin, or cyanogen bromide . The sequence is unique and not homologous to any known protein sequence . The protein may represent a new type of lectin. J Biol Response Mod, 1987 Apr, 6(2), 194 - 204 Resistance to biological and chemical challenge in rodents treated with xerosin II, a natural product of Achromobacter xerosis; Boylan ES et al.; The biological response-modifying activity of acid-precipitable material from Achromobacter xerosis was first described as suppression of viral pneumonia in mice . Later, this acid-precipitable material (xerosin) was found to have antiinflammatory activity and to induce tumor regression in chickens infected with Rous sarcoma virus . Here, we report further purification of xerosin resulting in a product (xerosin II) that retains high biological activity against viral and endotoxin-induced pneumonia in mice . In addition, we describe new activities of xerosin II in two rat tumor systems . Female CD rats received gastric intubations of 7,12-dimethylbenz(a)anthracene; 2 weeks later, half began 4 weeks of treatment with xerosin II, while others received saline only . Xerosin II treatment significantly delayed the appearance of the first palpable mammary tumors per rat . In female F344 rats implanted with the 13762 mammary tumor, 4 weeks of xerosin II treatment prolonged the survival of rats by an average of 5-11 days (12-24%) in two separate trials . Tumor growth and incidence of metastasis appeared unaffected by xerosin II treatment . Thus, this refined bacterial extract proved to be a potent biological response modifier in four different rodent systems. Blood, 1987 Feb, 69(2), 560 - 4 Purification and partial amino acid sequence of human platelet membrane glycoproteins IIb and IIIa; Hiraiwa A et al.; The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes by immunoaffinity chromatography using a monoclonal antibody specific for GP IIb-IIIa . GP IIb and IIIa were further separated in the presence of sodium dodecyl sulfate (SDS) by gel filtration high-performance liquid chromatography (HPLC) . Two cycles of this procedure yielded almost complete separation of homogeneous preparations of GP IIb and IIIa . Each protein was then digested with lysyl endopeptidase (Achromobacter protease I), which cleaves at the carboxyl side of lysine residues, and the resulting oligopeptides from GP IIb and IIIa were fractionated with HPLC using a C18 reverse-phase column . Comparison of the elution profiles showed no obvious homology between the two proteins . Amino acid sequences of selected oligopeptides from each glycoprotein were determined using a gas-phase protein sequencer . Sixty amino acid residues (26 residues for IIb and 34 residues for IIIa) were identified. Chemioterapia, 1987 Feb, 6(1), 32 - 7 Emerging microorganisms in cystic fibrosis; Fabbri A et al.; Pseudomonas aeruginosa is the most common bacterial isolate obtained from patients with cystic fibrosis of the lungs . Recently, however, new multiresistant organisms have emerged, whose identification may be difficult and whose pathogenic role proves hard to define . Of the 71 strains isolated from 24 patients with cystic fibrosis during acute flareups of pulmonary symptoms, 48 turned out to be Pseudomonas aeruginosa (67.6%); 11 were Pseudomonas non-aeruginosa (15.5%); and 12 were Achromobacter xylosoxidans (16.9%) . Each bacterial isolate was tested for sensitivity to nine antibiotics (ceftazidime, azlocillin, piperacillin, aztreonam, cefsulodin, cefoperazone, amikacin, tobramycin, and sisomycin) in terms of minimum inhibitory concentration and minimum bactericidal concentration values . In this series, Achromobacter xylosoxidans proved the species least responsive to treatment, and ceftazidime the most active antibiotic both against Achromobacter and against strains of the genus Pseudomonas . Twenty-three different associations of ceftazidime with aminoglycosides, tested for activity on the multiresistant strains, failed to show synergism of action. J Biochem (Tokyo), 1987 Jan, 101(1), 111 - 21 Amino acid sequence of thermostable direct hemolysin produced by Vibrio parahaemolyticus; Tsunasawa S et al.; The complete amino acid sequence of the subunit of thermostable direct hemolysin, a dimeric protein composed of identical subunits isolated from Vibrio parahaemolyticus, was determined by sequencing BrCN-peptides, their tryptic peptides, and overlaps obtained by Achromobacter protease I digestion . The subunit consists of 165 amino acid residues with the sole disulfide bond between Cys 151 and Cys 161 . It is deduced that the biologically active hemolysin is formed by noncovalent association of subunits which are not linked together by disulfide bonds . The primary structure of hemolysin elucidated in the present study is essentially the same as that deduced from the nucleotide sequence of a gene encoding the protein but differs in 9 amino acid residues, suggesting the possibility of the presence of multiple genes for the thermostable direct hemolysin in Vibrio parahaemolyticus. Biochem J, 1986 Dec 15, 240(3), 803 - 10 Influence of temperature on the enzymic semisynthesis of human insulin by coupling and transpeptidation methods; Morihara K et al.; The influence of temperature of enzymic semisynthesis of human insulin ester was determined by using coupling and transpeptidation methods with trypsin and Achromobacter lyticus proteinase I as catalysts . The optimal reaction conditions were studied at the selected temperatures of 25, 12 and 4 degrees C . The results showed that the synthesis rates by both methods with trypsin increased as the temperature increased, but the final product yield correspondingly decreased . Therefore the reaction with trypsin should be done below 12 degrees C, preferably at 4 degrees C . This agrees well with the stability of trypsin at these temperatures . When the catalyst was Achromobacter lyticus proteinase I, no such complex temperature effects were observed, and the findings indicated that the reactions should be conducted below 37 degrees C for enzyme stability. Infection, 1986 Nov-Dec, 14(6), 279 - 82 Infections due to Achromobacter xylosoxidans . Case report and review of the literature; Chandrasekar PH et al.; Achromobacter xylosoxidans is an uncommon nosocomial pathogen known to cause many serious infections . A 69-year-old woman with diabetes mellitus and chronic renal failure was admitted with pulmonary edema . The patient developed fever and pulmonary infiltrate with bilateral pleural effusions while she was on a respirator in the intensive care unit . Culture of sputum, pleural fluid and blood grew A . xylosoxidans . Bilateral chest tubes were inserted and the patient was treated for one month with piperacillin and trimethoprim-sulfamethoxazole . Gradual response, both clinically and radiologically, was noted after prolonged therapy . A review of the literature on infections due to A . xylosoxidans, the unique susceptibility pattern of the organism to various antibiotics and the use of combination therapy in Achromobacter infections are discussed. Eur J Biochem, 1986 Oct 15, 160(2), 413 - 8 New thiol inhibitor of Achromobacter iophagus collagenase . Specificity of the enzyme's S3' subsite; Yiotakis A et al.; New synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) which inhibit Achromobacter iophagus collagenase were produced in order to obtain more powerful bacterial collagenase inhibitors than currently available, and to investigate the specificity of the S3' subsite of the enzyme . Since similar binding constants were found for inhibitors carrying uncharged residues of various sizes in the P3' position (Yaa = Ala, Leu, Phe, Pro, Hyp) steric hindrance at the collagenase S3' appears relatively limited . The compound (HS-CH2-CH2-CO-Pro-Arg), which carries an arginine residue in the position P3' and had the highest inhibition constant of the series tested (Ki = 0.5 microM), proved to be the strongest inhibitor so far reported in the literature . The weakest in the present series was the compound (HS-CH2-CH2-CO-Pro-Asp) which carries an aspartic residue in position P3' and had a Ki = 70 microM . The present work revealed that the charged groups in the P3' position play a key role in the interaction of the inhibitors with the enzyme. Int J Pept Protein Res, 1986 Oct, 28(4), 411 - 9 Enzymatic semisynthesis of {LeuB30} insulin; Sakina K et al.; Experimental conditions for the preparation of {LeuB30} insulin by coupling of des-AlaB30 insulin with Leu-OBu(t) were determined using Achromobacter protease I and trypsin as catalysts . Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35-50%) and neutral pH of the reaction mixture . The coupling yield of Achromobacter protease I after 24 h at 37 degrees C was almost the same or a little higher than that at 25 degrees C . With trypsin, the coupling yield at 37 degrees C after 24 h was considerably lower than at 25 degrees C . This was partly ascribed to the difference in concentration of organic cosolvent at 37 degrees C and 25 degrees C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures . The maximum product yield was about 90% with both enzymes under optimal conditions . A preparative scale experiment was performed with Achromobacter protease I; the yield of {LeuB30} insulin was 51% using porcine insulin as the starting material . This semisynthetic insulin was identified by HPLC and amino acid analysis . No difference was observed in CD spectra between {LeuB30} insulin and human insulin. J Biochem (Tokyo), 1986 Jun, 99(6), 1749 - 52 Purification and some properties of cytochrome c' from a strain of Achromobacter xylosoxidans; Shidara S et al.; Cytochrome c' was crystallized from Achromobacter xylosoxidans GIFU 543 . The cytochrome was a basic protein and its molecular weight was 28,000 . The pyridine ferrohemochrome showed absorption peaks at 415, 521, and 551 nm . The absorption spectra of the oxidized and reduced forms at neutral pH were almost the same as those of other cytochromes c' reported already . The reduced cytochrome c' reacted with CO and NO, and the NO complex showed a characteristic absorption spectrum . The midpoint redox potential of the hemoprotein was measured to be + 110 mV at pH 7.2. J Biochem (Tokyo), 1986 May, 99(5), 1289 - 98 Amino acid sequence of copper,zinc-superoxide dismutase from spinach leaves; Kitagawa Y et al.; The complete amino acid sequence of Cu,Zn-superoxide dismutase (SOD) from spinach leaves has been determined on the basis of peptides obtained by cyanogen bromide (BrCN) cleavage and by enzymic hydrolyses with Achromobacter lyticus lysylendopeptidase, Staphylococcus aureus V8 protease, trypsin, and thermolysin . The spinach SOD consists of a total of 154 amino acid residues with alanine as the amino(N)-terminus and valine as the carboxy(C-)terminus . The present sequence, which has been established for the enzyme from a plant, is also highly homologous to those of the enzymes from other species . Especially, the residues essential for metal binding and enzyme activity have been extensively conserved among all of the Cu,Zn-SODs hitherto analyzed. Biochem Cell Biol, 1986 May, 64(5), 394 - 9 Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase; Sadana JC et al.; Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy . The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes . These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51 . Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide . When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative . The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme . The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT . The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine . Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the {15N}nitrosyl compound . A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate. J Biochem (Tokyo), 1986 Feb, 99(2), 407 - 22 Complete amino acid sequence of NADH-cytochrome b5 reductase purified from human erythrocytes; Yubisui T et al.; The complete amino acid sequence of soluble NADH-cytochrome b5 reductase purified from human erythrocytes was determined . The enzyme, which contained 8 methionine residues, was cleaved by cyanogen bromide . The resulting nine peptides were separated by gel filtration and purified further by high-performance liquid chromatography . The purified peptides were sequenced by automated Edman degradation . Three large CNBr peptides, residues 1-101, 109-151, and 169-231, were further fragmented with trypsin, Staphylococcus aureus V8 protease or a lysyl endopeptidase of Achromobacter lyticus . The peptides obtained from the tryptic digest of citraconylated FAD-depleted apoprotein completed the alignments of the other peptides . The enzyme was composed of 275 amino acid residues . The 4 functionally important cysteine residues were located in the COOH-terminal portion . The molecular weight of the protein was calculated to be 31,260 without FAD . A prediction of the secondary structure was made by the method of Chou and Fasman . The protein was hydrophilic as a whole (43% polarity), but some regions were rich in hydrophobic residues . From the sequence homology of this enzyme with the pyridine nucleotide-binding sites of other flavoproteins, three candidates for the FAD and NADH-binding domains were suggested. J Biochem (Tokyo), 1986 Jan, 99(1), 281 - 9 Amino acid sequence of Trimeresurus flavoviridis phospholipase A2; Tanaka S et al.; The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined . The enzyme subunit has a molecular weight of 13,764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds . The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively . Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides . The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone . T . flavoviridis phospholipase A2 was found to be highly (65-67%) homologous in sequence to the enzymes from T . okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas . The T . flavoviridis enzyme appears to be similar in secondary structure composition to the C . atrox enzyme. Ann Biol Clin (Paris), 1986, 44(2), 176 - 80 {Peptide inhibitors of a bacterial collagenase}; Dive V et al.; The properties of the cleavage site of collagen by collagenases remain discussed . The authors report their studies on the active site of the collagenase of a bacteria, achromobacter, based on two types of methods . The first type uses synthetic substrates, the second one enzymatic inhibitors . The results of both methods are reviewed . The inhibitors seem more sensitive than substrates to study the relation between structure and activity of the enzyme. J Clin Microbiol, 1985 Sep, 22(3), 470 - 1 Neonatal meningitis caused by Achromobacter xylosoxidans; Namnyak SS et al.; The clinical and bacteriological findings in a case of neonatal meningitis caused by Achromobacter xylosoxidans are presented . This appears to be only the second report of meningitis caused by this species. J Biochem (Tokyo), 1985 Sep, 98(3), 585 - 603 Subtilosin A, a new antibiotic peptide produced by Bacillus subtilis 168: isolation, structural analysis, and biogenesis; Babasaki K et al.; Subtilosin A, a new antibiotic produced by Bacillus subtilis 168, was extracted from culture medium with n-butanol and purified to homogeneity by a combination of gel filtration and thin-layer chromatography . The yield was 5.5 mg from a liter of culture . It had bacteriocidal activity against some gram-positive bacteria . Amino acid analysis and mass spectrometry showed that it was a peptide with a molecular weight of 3398.9, consisting of 32 usual amino acid and some non-amino acid residues . Its amino- and carboxyl-termini were blocked . By analysis of the fragments obtained by partial acid hydrolysis, as well as by chymotryptic and thermolysin digestions of reduced and S-carboxymethylated samples and Achromobacter protease I digestion of performic acid-oxidized samples, the amino acid sequence was determined to be as follows: X-Gly-Leu-Gly-Leu-Trp-Gly-Asn-Lys-Gly-Cys-Ala-Thr-Cys-Ser-(sequence; see text) Ile-Gly-Ala-Ala-Cys-Leu-Val-Asp-Gly-Pro-Ile-Pro-Asp-Glx-Ile-Ala-Gly-Ala . The analyses of cross-linking structures revealed that there were linkages between the amino- and carboxyl-termini and between the Cys-19 and the Glx-28 residues through an unknown residue with a residue weight of 163 . Consequently, subtilosin A was deduced to be a cyclic peptide antibiotic with a novel cross-linking structure . The production of subtilosin A begins at the end of vegetative growth and finishes before spore formation . Studies on the correlation between the production of subtilosin A and spore formation with decoyinine in the original strain and in asporogenous mutants of B . subtilis 168 suggested that there was no close correlation between the two phenomena . The production of subtilosin A was repressed by inhibitors of protein and RNA synthesis in contrast to that of many other antibiotic peptides, suggesting that it is synthesized by the mechanism of usual protein synthesis. J Biochem (Tokyo), 1985 Sep, 98(3), 799 - 805 Exposed tyrosine residues of lambda cro repressor protein evidenced by nitration and photo CIDNP experiments; Shirakawa M et al.; The tyrosine residues of lambda cro repressor were partially nitrated with tetranitromethane under mild conditions . After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis . Tyr 26 was most easily nitrated and Tyr 51 followed it . Tyr 10 was resistant to nitration . By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues . The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization). FEBS Lett, 1985 Jul 1, 186(1), 41 - 5 Complete amino acid sequence of bovine colostrum low-Mr cysteine proteinase inhibitor; Hirado M et al.; The complete amino acid sequence of bovine colostrum cysteine proteinase inhibitor was determined by sequencing native inhibitor and peptides obtained by cyanogen bromide degradation, Achromobacter lysylendopeptidase digestion and partial acid hydrolysis of reduced and S-carboxymethylated protein . Achromobacter peptidase digestion was successfully used to isolate two disulfide-containing peptides . The inhibitor consists of 112 amino acids with an Mr of 12787 . Two disulfide bonds were established between Cys 66 and Cys 77 and between Cys 90 and Cys 110 . A high degree of homology in the sequence was found between the colostrum inhibitor and human gamma-trace, human salivary acidic protein and chicken egg-white cystatin. Diagn Microbiol Infect Dis, 1985 Mar, 3(2), 149 - 58 Occurrence and antimicrobial susceptibility of gram-negative nonfermentative bacilli in cystic fibrosis patients; Klinger JD et al.; Isolation of nonfermentative gram-negative bacilli (other than Pseudomonas aeruginosa) from respiratory tract cultures of cystic fibrosis (CF) patients has increased in recent years . Species recovered include Pseudomonas cepacia, P . maltophilia, P . fluorescens/putida, P . alcaligenes, P . pseudoalcaligenes, P . stutzeri, Acinetobacter spp., Achromobacter xylosoxidans, Flavobacterium spp., and CDC groups IVe and Ve . Although colonization with most of these organisms is sporadic, P . cepacia (and to a lesser extent, P . maltophilia) is usually isolated consistently, and can be associated with significant clinical deterioration . Occurrence of P . cepacia in CF respiratory tract cultures obtained close to the time of death rose nearly ten-fold from 1979 to 1982 . Strains representing all nonfermentative gram-negative species encountered were assayed for susceptibility to 17 newer antimicrobial agents . Ceftazidime, n-formimidoyl thienamycin, and aztreonam were most active; cefsulodin, ceforanide, and ceftriaxone were not active against these isolates. J Hosp Infect, 1985 Mar, 6(1), 81 - 8 Prevention of bacterial contamination of water in dental units; Furuhashi M et al.; A study of the bacterial contamination of water in dental units was conducted in five dental practitioners' clinics and one university dental hospital in Tokyo . Various bacteria in numbers of 10(2)-10(5)/ml were detected in the water delivered from all 42 of the dental units examined, though very few bacteria were detected in the tap water upstream of the units . Most of the organisms isolated from the water were Gram-negative bacilli, predominantly Alcaligenes, Achromobacter and Pseudomonas spp . A new decontamination apparatus that could be incorporated into the pipe system of dental units was devised and its performance was evaluated in one unit . It effectively prevented bacterial contamination of the water. Mikrobiologiia, 1985 Jan-Feb, 54(1), 108 - 13 {Nature of factors stimulating cobalamin production by Achromobacter cobalamini}; Shteinberg BI et al.; The object of this work was to study the nature of factors contained in molasses and maize extract and stimulating cobalaminogenesis in Achromobacter cobalamini . The activity of substrate fractions was analyzed to show that the stimulating substance was precipitated on the cation exchanger and eluted from it with HCl . The factor was found to be an organic nitrogen base readily soluble in water and ethanol but insoluble in ether, chloroform and methanol . It was stable upon heating in concentrated HCl . Betaine in the composition of molasses and choline in the composition of maize extract had similar properties . Their addition to the growth medium produced the same effect as that of molasses and maize extract . It is concluded therefore that cobalaminogenesis is stimulated in A . cobalamini by betaine in molasses and by choline in maize extract. Appl Environ Microbiol, 1984 Dec, 48(6), 1214 - 20 In situ identification of bacterial species in marine microfouling films by using an immunofluorescence technique; Zambon JJ et al.; An