Med Oncol, 1999 Apr, 16(1), 38 - 45 Linker-based GnRH-PE chimeric proteins inhibit cancer growth in nude mice; Ben-Yehudah A et al.; Since the number of cancer-related deaths has not decreased in recent years, major efforts are being made to find new drugs for cancer treatment . In this report we introduce the gonadotropin releasing hormone-Pseudomonas exotoxin (GnRH-PE) based chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 . These proteins are composed of a GnRH moiety attached to modified forms of Pseudomonas exotoxin via a polylinker (gly4ser)2 . The chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 have the ability to target and kill adenocarcinoma cell lines in vitro, whereas non-adenocarcinoma cell lines are not affected . We demonstrate that L-GnRH-PE66 and L-GnRH-PE40 efficiently inhibit cancer growth . Nude mice were injected subcutaneously with the SW-48 adenocarcinoma cell line to produce xenograft tumours . When the tumours were established and visible, the animals were injected with chimeric proteins for 10 days . At the end of this period, a reduction of up to 3-fold in tumor size was obtained in the treated mice, as compared with the control group, which received equivalent amounts of GnRH; the difference was even greater 13 days after termination of treatment . Thus, the chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 are promising candidates for treatment of a variety of adenocarcinomas and their use in humans should be considered. Biochemistry (Mosc), 1999 May, 64(5), 483 - 7 Some properties of dissimilatory nitrate reductases lacking molybdenum and molybdenum cofactor Antipov AN, Lyalikova NN, Khiznjak TV, L'vov NP. Novel periplasmic and membrane-bound nitrate reductases lacking molybdenum and molybdenum cofactor were isolated from the vanadate-reducing bacterium Pseudomonas isachenkovii, and their properties were studied . Both enzymes have some unusual features, i . e., the individual subunits (130-kD subunit of the membrane-bound enzyme and monomeric 55-kD subunit of the periplasmic enzyme) possess their own nitrate reductase activity . In addition, both enzymes are highly thermostable, their temperature optimum being at 70-80 degrees C, which is unexpectedly high for enzymes from mesophilic bacteria . Similarly to conventional molybdenum-containing nitrate reductases, these isolated enzymes are very sensitive to low concentrations of cyanide and azide . During anaerobic cell growth on medium with nitrate and vanadate, nitrate consumption is followed by a period of vanadate dissimilation, and this period is associated with some structural reorganizations of the nitrate reductases. Eur J Pediatr, 1999 Jun, 158(6), 455 - 9 Efficacy and safety of acarbose in patients with cystic fibrosis and impaired glucose tolerance; Kentrup H et al.; Impaired glucose tolerance (IGT) is an increasingly frequent complication of cystic fibrosis (CF) . In CF patients, a fast postprandial rise in plasma glucose is typically followed by a delayed but prolonged insulin response . Patients may develop symptoms of both hyper- and hypoglycaemia . The alpha-glucosidase inhibitor, acarbose, delays the hydrolysis and subsequent absorption of ingested carbohydrates . The aim of this study was to investigate the efficacy of acarbose in CF patients with IGT . During a 2-week inpatient period for treatment of Pseudomonas infection, 12 CF patients with IGT were studied in a double-blinded, randomized crossover trial . Each patient received acarbose (50 mg t.i.d.) for 5 days and placebo for 5 days (days 3-8 and days 10-14, respectively) . Glucose, insulin and C-peptide responses to a standardized nutritional load were measured at baseline and at the end of each study period (Days 2, 8 and 14) . Treatment with acarbose was associated with significant reductions in the mean value, mean peak values and the area under the curve of plasma glucose, insulin and C-peptide, compared to respective baseline values and placebo . Gastro-intestinal disturbances were recorded in 67% of patients during therapy with acarbose . CONCLUSION: Acarbose has a positive therapeutic effect on glucose tolerance in cystic fibrosis patients, as shown by attenuation of postprandial plasma glucose increase and a significant decrease in insulin secretion response . However, acarbose treatment was associated with adverse gastro-intestinal effects that may prevent patients from accepting long-term therapy. Plant J, 1999 May, 18(3), 321 - 7 Characterization of an Arabidopsis thaliana receptor-like protein kinase gene activated by oxidative stress and pathogen attack; Czernic P et al.; An Arabidopsis thaliana cDNA clone that encodes a putative receptor-like protein kinase gene (At-RLK3) was characterized . The deduced 667-amino acid protein consists of an amino-terminal signal sequence, an extracellular domain, a single transmembrane domain, and a cytoplasmic domain with characteristics of serine/threonine protein kinase . Because of the original features of its extracellular domain, the At-RLK3 protein is a member of a new class of receptor-like protein kinases . The At-RLK3 gene is present as a single copy within the Arabidopsis genome and its transcripts are detected in root, stem, leaf and flower . In cultured cells, the At-RLK3 gene is activated upon oxidative stress and salicylic acid treatment . In plants, the gene appears to be differentially regulated during various plant-pathogen interactions: upon inoculation with strains of Pseudomonas syringae pv . tomato harboring or not, different avr genes, At-RLK3 transcripts accumulate transiently at similar levels during both compatible and incompatible interactions . This gene is, however, preferentially expressed during the incompatible interaction induced by the soil-borne vascular bacteria, Ralstonia solanacearum . The involvement of At-RLK3 in signal transduction pathways during pathogen attack is discussed. Proc Natl Acad Sci U S A, 1999 Jun 22, 96(13), 7167 - 71 Substrate-induced closure of the flap domain in the ternary complex structures provides insights into the mechanism of catalysis by 3-hydroxy-3-methylglutaryl-CoA reductase; Tabernero L et al.; 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is the rate-limiting enzyme and the first committed step in the biosynthesis of cholesterol in mammals . We have determined the crystal structures of two nonproductive ternary complexes of HMG-CoA reductase, HMG-CoA/NAD+ and mevalonate/NADH, at 2.8 A resolution . In the structure of the Pseudomonas mevalonii apoenzyme, the last 50 residues of the C terminus (the flap domain), including the catalytic residue His381, were not visible . The structures of the ternary complexes reported here reveal a substrate-induced closing of the flap domain that completes the active site and aligns the catalytic histidine proximal to the thioester of HMG-CoA . The structures also present evidence that Lys267 is critically involved in catalysis and provide insights into the catalytic mechanism. Br J Cancer, 1999 Jun, 80(8), 1214 - 22 An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA') is a potent immunotoxin against a Hodgkin-derived cell line; Klimka A et al.; The human CD30 receptor is highly overexpressed on the surface of Hodgkin Reed-Sternberg cells and has been shown to be an excellent target for selective immunotherapy using monoclonal antibody-based agents such as immunotoxins . To construct a new recombinant immunotoxin for possible clinical use in patients with Hodgkin's lymphoma, we have chosen the murine anti-CD30 hybridoma Ki-4 to generate a high-affinity Ki-4 single-chain variable fragment (scFv) . Hybridoma V-genes were polymerase chain reaction-amplified, assembled, cloned and expressed as a mini-library for display on filamentous phage . Functional Ki-4 scFv were obtained by selection of binding phage on the Hodgkin lymphoma-derived, CD30-expressing cell line L540Cy . The selected recombinant Ki-4 scFv was shown to specifically bind to an overlapping epitope on the CD30 antigen with binding kinetics similar to those of the original antibody . The Ki-4 scFv was subsequently fused to a deletion mutant of Pseudomonas exotoxin A (ETA') . The resulting immunotoxin Ki-4(scFv)-ETA' specifically binds to CD30+ L540Cy cells and inhibits the protein synthesis by 50% at a concentration (IC50) of 43 pM . This recombinant immunotoxin is a promising candidate for further clinical evaluation in patients with Hodgkin's lymphoma or other CD30+ malignancies. Microbiology, 1999 May, 145 ( Pt 5), 1173 - 80 Butane metabolism by butane-grown 'Pseudomonas butanovora'; Arp DJ; The pathway of butane metabolism by butane-grown 'Pseudomonas butanovora' was determined to be butane --> 1-butanol --> butyraldehyde butyrate . Butane was initially oxidized at the terminal carbon to produce 1-butanol . Up to 90% of the butane consumed was accounted for as 1-butanol when cells were incubated in the presence of 5 mM 1-propanol (to block subsequent metabolism of 1-butanol) . No production of the subterminal oxidation product, 2-butanol, was detected, even in the presence of 5 mM 2-pentanol (an effective inhibitor of 2-butanol consumption) . Ethane, propane and pentane, but not methane, were also oxidized . Butane-grown cells consumed 1-butanol and other terminal alcohols . Secondary alcohols, including 2-butanol, were oxidized to the corresponding ketones . Butyraldehyde was further oxidized to butyrate as demonstrated by blocking butyrate metabolism with 1 mM sodium valerate . Butyrate also accumulated from butane when cells were incubated with 1 mM sodium valerate . The pathway intermediates (butane, 1-butanol, butyraldehyde and butyrate) and 2-butanol stimulated O2 consumption by butane-grown cells . 1-Butanol, butyraldehyde and butyrate supported growth of 'P . butanovora', as did 2-butanol and lactate. Mar Biotechnol (NY), 1999 Jan, 1(1), 102 - 106 Revelation of Antiviral Activities by Artificial Sulfation of a Glycosaminoglycan from a Marine Pseudomonas; Ahmad AS et al.; : Sulfated derivatives of a glycosaminoglycan containing l-glutamic acid produced by a marine Pseudomonas species, No . 42 strain, were prepared by the method of dicyclohexyl-carbodiimide-mediated reaction . Both low and high degrees of sulfation of the polysaccharides (products A1 and A2, respectively) were investigated for their antiviral activities against influenza virus type A (FluV-A) and B (FluV-B) in MDCK cells . Both preparations showed antiviral activity against FluV-A at the 50% antiviral effective concentration of 17.3 and 5.2 microg/ml, respectively, whereas they had no antiviral activity against FluV-B . No cytotoxicity of either product was noted against MDCK cells at the 50% cytotoxic concentration of 100 microg/ml. Mar Biotechnol (NY), 1999 Jan, 1(1), 68 - 73 Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives; Matsuda M et al.; : A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide . Among the antiviral activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration is 1.4 microg/ml) . Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration is 11.0 microg/ml; for another, 2.9 microg/ml) . Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1 . No cytotoxicity of these products was noted against host cells at the 50% cytotoxic concentration of 100 microg/ml, except that naturally occurring sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8-21 microg/ml. EMBO J, 1999 Jun 15, 18(12), 3232 - 40 Constitutively active Pto induces a Prf-dependent hypersensitive response in the absence of avrPto; Rathjen JP et al.; Resistance in tomato to Pseudomonas syringae pv tomato (avrPto) is conferred by the gene Pto in a gene-for-gene relationship . A hypersensitive disease resistance response (HR) is elicited when Pto and avrPto are expressed experimentally within the same plant cell . The kinase capability of Pto was required for AvrPto-dependent HR induction . Systematic mutagenesis of the activation segment of Pto kinase confirmed the homologous P+1 loop as an AvrPto-binding determinant . Specific amino acid substitutions in this region led to constitutive induction of HR upon expression in the plant cell in the absence of AvrPto . Constitutively active Pto mutants required kinase capability for activity, and were unable to interact with proteins previously shown to bind to wild-type Pto . The constitutive gain-of-function phenotype was dependent on a functional Prf gene, demonstrating activation of the cognate disease resistance pathway and precluding a role for Prf upstream of Pto. Toxicol Pathol, 1999 Jan-Feb, 27(1), 87 - 94 BR96 sFv-PE40 immunotoxin: nonclinical safety assessment; Haggerty HG et al.; BR96 sFv-PE40, a recombinant DNA-derived fusion protein composed of the heavy- and light-chain variable region domains of the monoclonal antibody BR96 and the translocation and catalytic domains of Pseudomonas exotoxin A, is being developed for the treatment of solid tumors expressing cell surface Lewis(y)-related antigens . Single- and repeat-dose intravenous toxicity studies in rats and dogs and a comparative ex vivo tissue-binding study with rat, dog, and human tissues were conducted to assess the toxicity of BR96 sFv-PE40 and to estimate a safe starting dose in humans . Additional studies were performed to investigate the prevention of pulmonary vascular-leak syndrome, the dose-limiting toxicity of BR96 sFv-PE40 in rats, and the immunogenicity of BR96 sFv-PE40 . In single-dose studies in rats, the vascular leak appeared to be primarily confined to the lungs; however, with a repeat-dose regimen (every other day for 5 doses) other organs including the brain and heart were involved at lethal doses (12-15 mg/m2 cumulative) . Single doses of 1.8 mg/m2 and a cumulative 3.8 mg/m2 dose (0.75 mg/m2, every other day for 5 doses) were generally well tolerated in rats . These doses are significantly greater than doses required to cure rodents bearing human tumor xenografts . In dogs, the major target organ following single or repeated doses (every 3 days for 5 doses) was the pancreas . Morphologic changes in the exocrine pancreas ranged from atrophy with single-cell necrosis to diffuse acinar necrosis . After a 1-mo dose-free observation period, no residual pancreatic toxicity was observed in dogs given single doses up to 6.0 mg/m2 or 5 doses of 2.4 mg/m2 (12 mg/m2 cumulative) . No significant pancreatic toxicity was observed at doses <0.6 mg/m2 in high Lewis(y)-expressing dogs . Assessment of trypsinlike immunoreactivity was useful in monitoring changes in pancreatic function . The immunogenicity of BR96 sFv-PE40 could be inhibited by combined treatment with an immunosuppressant in dogs, thus maintaining exposure to BR96 sFv-PE40. Toxicol Pathol, 1999 Jan-Feb, 27(1), 53 - 7 Development of a recombinant interleukin-4-Pseudomonas exotoxin for therapy of glioblastoma; Puri RK; About 12,000 Americans are diagnosed with malignant astrocytoma each year . Despite surgery, radiotherapy, and chemotherapy, the prognosis of these patients remains poor . Targeted toxins based on the identification of novel antigens or receptors provide a promising new approach to treating cancer . We have identified one such cell surface protein in the form of interleukin (IL)-4 receptors (IL-4R) on human malignant astrocytoma . Normal brain tissues from frontal cortex and temporal lobe cortex do not express IL-4R . To target IL-4R, we generated a chimeric fusion protein composed of IL-4 and Pseudomonas exotoxin (IL4-PE) . This toxin is highly cytotoxic to IL-4R-bearing human brain cancer cells . Preclinical toxicologic experiments were performed in mice, rats, and guinea pigs to determine an maximum tolerated dose . Intrathecal administration in cynomolgus monkeys produced high cerebrospinal fluid levels without any central nervous system or other abnormalities . When IL4-PE was injected into the right frontal cortex of rats, localized necrosis was observed at 1,000 but not < or =100 microg/ml doses . Intravenous administration of this biologic to monkeys produced reversible grade 3 or grade 4 elevations of hepatic enzymes in a dose-dependent manner . These results indicate that localized administration can produce nontoxic levels of IL4-PE that may have significant activity against astrocytoma . In vivo experiments with nude mice have demonstrated that IL4-PE has significant antitumor activity against human glioblastoma tumor model . Intratumor administration of IL4-PE has been initiated for the treatment of malignant astrocytoma in a phase I clinical trial. Curr Opin Biotechnol, 1999 Jun, 10(3), 279 - 85 Perspectives of medium chain length poly(hydroxyalkanoates), a versatile set of bacterial bioplastics Witholt B, Kessler B. Medium chain length (mcl) poly(hydroxyalkanoic acids) (PHAs) are polyesters accumulated by fluorescent Pseudomonads and other bacteria . Work on the genetics of mcl-PHA formation has led to polymer synthesis in recombinant bacteria and plants . Several high and medium cost applications are now emerging . With optimized bacterial mcl-PHA synthesis on inexpensive agro-substrates and the development of plant-based mcl-PHAs in the next decade, the production economics of these bioplastics will ultimately permit their sustainable production for bulk applications. Plant Physiol, 1999 Jun, 120(2), 529 - 38 Enhanced expression and activation of the alternative oxidase during infection of Arabidopsis with Pseudomonas syringae pv tomato; Simons BH et al.; Cyanide-resistant ("alternative") respiration was studied in Arabidopsis during incompatible and compatible infection with Pseudomonas syringae pv tomato DC3000 . Total leaf respiration increased as the leaves became necrotic, as did the cyanide-resistant component that was sensitive to salicylhydroxamic acid . Infiltration of leaves with an avirulent strain rapidly induced alternative oxidase (AOX) mRNA, whereas the increase was delayed in the compatible combination . The increase in mRNA correlated with the increase in AOX protein . Increased expression was confined to the infected leaves, in contrast to the pathogenesis-related protein-1, which was induced systemically . Virtually all of the AOX protein was in the reduced (high-activity) form . Using transgenic NahG and mutant npr1-1 and etr1-1 plants, we established that the rapid induction of the AOX was associated with necrosis and that ethylene, but not salicylic acid, was required for its induction . Increased pyruvate levels in the infected leaves suggested that increased substrate levels were respired through the alternative pathway; however, in the control leaves and the infected leaves, respiration was not inhibited by salicylhydroxamic acid alone . Increased respiration appeared to be associated primarily with symptom expression rather than resistance reactions. Mol Microbiol, 1999 Jun, 32(5), 927 - 41 Characterization of the Pseudomonas syringae pv . tomato AvrRpt2 protein: demonstration of secretion and processing during bacterial pathogenesis; Mudgett MB et al.; Pseudomonas syringae pv . tomato strain DC3000 (Pst DC3000) expressing avrRpt2 is specifically recognized by plant cells expressing RPS2 activity, resulting in localized cell death and plant resistance . Furthermore, transient expression of this bacterial avrRpt2 gene in plant cells results in RPS2-dependent cell death . This indicates that the AvrRpt2 protein is recognized inside RPS2 plant cells and is sufficient for the activation of disease resistance-mediated cell death in planta . We explored the possibility that Pst DC3000 delivers AvrRpt2 protein to plant cells via the hrp (type III) secretion pathway . We now provide direct evidence that mature AvrRpt2 protein is secreted from Pst DC3000 and that secretion is hrp dependent . We also show that AvrRpt2 is N-terminally processed when Arabidopsis thaliana plants are infected with Pst DC3000 expressing avrRpt2 . Similar N-terminal processing of AvrRpt2 occurred when avrRpt2 was stably expressed in A . thaliana . No cleavage of AvrRpt2 was detected in bacteria expressing avrRpt2 in culture or in the plant extracellular fluids . The N-terminus of AvrRpt2 was not required for RPS2 recognition in planta . However, this region of AvrRpt2 was essential for Pst DC3000-mediated elicitation of RPS2-dependent cell death in A . thaliana leaves. Antisense Nucleic Acid Drug Dev, 1999 Apr, 9(2), 183 - 90 Stable antisense RNA expression neutralizes the activity of low-density lipoprotein receptor-related protein and promotes urokinase accumulation in the medium of an astrocytic tumor cell line; Hussaini IM et al.; Low-density lipoprotein receptor-related protein (LRP) binds and internalizes multiple ligands that are structurally and functionally diverse . However, the effects of LRP on cellular phenotype remain unclear . To study LRP in human astrocytic tumor cells, we designed LRP antisense RNA expression constructs in which the antisense cDNA fragment was expressed under the control of the cytomegalovirus (CMV) promoter . U-1242 MG astrocytic tumor cells were transfected with the antisense constructs and cloned from single cells to yield multiple cell lines with decreased LRP expression . Further studies were performed with two cell lines in which LRP antigen was completely eliminated (L(alpha)42) or substantially decreased (Lalpha47), as determined by Western blot analysis . Untransfected U-1242 MG cells and cells that were stably transfected with empty vector (pBK-CMV) bound activated alpha2-macroglobulin (alpha2M) in a specific and saturable manner . The Bmax was about 5000 receptors/cell . Lalpha42 cells did not bind alpha2M, and binding was decreased by >60% in Lalpha47 cells . Lalpha42 and Lalpha47 cells also demonstrated reduced susceptibility to the cytotoxin, Pseudomonas exotoxin A, and accumulated greatly increased levels of urokinase-type plasminogen activator (uPA) in conditioned medium . The accumulation of uPA demonstrates a major role for LRP in the catabolism of this protein in astrocytic tumor cells . The LRP-deficient cell lines, developed using antisense technology, represent a new model system for studying LRP function in astrocytes. CLAO J, 1999 Apr, 25(2), 123 - 4 Bilateral Pseudomonas corneal ulcer in a disposable contact lens wearer; Lipener C et al.; PURPOSE: To describe a case of bilateral corneal ulcers caused by Pseudomonas in a disposable soft contact lens wearer . This case study discusses the role of patient examination, contact lens care instruction, and adequate patient supervision in reducing the risk of serious potential complications . METHODS AND RESULTS: A 17 year old student who had been using disposable soft contact lenses on an extended wear basis for 6 months presented complaining of pain in the left eye . When he was examined, a corneal ulcer with surrounding infiltrate was observed in the superior middle periphery of the left eye . Samples were collected for culture, and treatment with fortified cefalotin and gentamicin was started . After 8 hours the patient returned, now complaining of pain in the right eye . Examination of the right eye revealed a diffused keratitis with a mucopurulent discharge . A culture was taken, and the same treatment was instituted . The laboratory tests revealed Pseudomonas in both eyes . The bilateral corneal ulcers responded to therapy after 1 week of treatment . CONCLUSIONS: We discuss the factors involved in the occurrence of infectious keratitis in contact lens wearers, and stress that even disposable contact lens wear can be associated with serious complications . This case also highlights extended wear as one of the main risk factors for complications in disposable soft contact lens wear. Mol Cells, 1999 Apr 30, 9(2), 225 - 9 Cloning and sequencing of the fcbB gene encoding 4-chlorobenzoate-coenzyme A dehalogenase from Pseudomonas sp . DJ-12; Chae JC et al.; Pseudomonas sp . DJ-12 degrades 4-chlorobenzoate through hydrolytic dechlorination to produce 4-hydroxybenzoate and a chloride ion . The fcbB gene encoding the 4-chlorobenzoate-coenzyme A (4CBA-CoA) dehalogenase which catalyzes the nucleophilic substitution reaction to convert 4CBA-CoA to 4-hydroxybenzoate-coenzyme A (4HBA-CoA) in the consecutive steps of dechlorination was cloned from the chromosome of the organism . A nucleotide sequence analysis of the gene showed an open reading frame consisting of 810 nucleotides, which can encode for a polypeptide of molecular mass 30 kDa, containing 269 amino acid residues . A promoter-like sequence (-35 and -10 region) and a putative ribosome-binding sequence were identified . A deduced amino acid sequence of the 4CBA-CoA dehalogenase showed 86%, 50%, and 50% identity with those of corresponding enzymes in the Pseudomonas sp . CBS3, Arthrobacter sp . SU, and Arthrobacter sp . TM1, respectively. Cornea, 1999 May, 18(3), 366 - 9 Cyanoacrylate tissue adhesive augmented tenoplasty: a new surgical procedure for bilateral severe chemical eye burns; Sharma A et al.; PURPOSE: To report on cyanoacrylate tissue adhesive augmented tenoplasty, a new surgical procedure for bilateral severe chemical eye injuries . METHODS: A 26-year-old man presented with bilateral severe (grade IV) chemical burns involving the eye, periorbital tissues, face, and neck . Despite adequate medical therapy, corneal, limbal, and scleral ulceration progressed in both eyes . Secondary Pseudomonas keratitis necessitated therapeutic penetrating keratoplasty in the right eye . Tenoplasty and glued-on rigid gas permeable contact lens were unsuccessful to arrest progression of corneolimboscleral ulceration in the left eye . We applied n-butyl cyanoacrylate tissue adhesive directly on the ulcerating corneal, limbal, and scleral surface to augment tenoplasty . RESULTS: The left ocular surface healed with resultant massive fibrous tissue proliferation and symblepharon on the nasal side . Ocular surface rehabilitation resulted in a vascularized leukomatous corneal opacity with upper temporal clear cornea . The patient achieved visual acuity of 6/36 in the left eye . CONCLUSION: We suggest that cyanoacrylate tissue adhesive-augmented tenoplasty can be undertaken to preserve ocular integrity and retain visual potential in a severe chemical eye injury. J Surg Res, 1999 Jun 1, 84(1), 13 - 8 Glutamine-enriched total parenteral nutrition preserves respiratory immunity and improves survival to a Pseudomonas Pneumonia; DeWitt RC et al.; BACKGROUND: Addition of 2% glutamine (GLN), a specific lymphocyte fuel, prevents deleterious effects of TPN on gut-associated lymphoid tissue and IgA while preserving IgA-mediated upper respiratory immunity to influenza virus . We examined whether a 2% GLN-enhanced TPN solution preserves respiratory immunity to a lethal and clinically relevant pneumonia challenge . MATERIALS AND METHODS: Male ICR mice were randomized to chow (n = 20), TPN (n = 20), or an isonitrogenous, isocaloric TPN-2% GLN solution (n = 17) . All groups were immunized 10 days before surgery with Pseudomonas polysaccharide-containing liposomes (LIP) to confer immunity except for a nonimmune chow-fed LIP control group (n = 21) which received LIP without Pseudomonas . Mice received 5 days of diet and then were given an LD90 dose of 1.2 x 10(8) intratracheal Pseudomonas bacteria, and mortality was recorded . RESULTS: Immunization reduced mortality compared with LIP alone . TPN impaired immunity and reduced survival while GLN maintained immunization effectiveness . CONCLUSIONS: Pseudomonas immunization reduces mortality to Pseudomonas pneumonia, but this immunity is lost with TPN . Addition of 2% GLN to TPN preserves immunity in the respiratory tract and reduces mortality to a lethal bacterial challenge compared with standard TPN . Biochem Biophys Res Commun, 1999 May 19, 258(3), 732 - 6 Immunolocalization of betaine aldehyde dehydrogenase in porcine kidney; Figueroa-Soto CG et al.; Polyclonal anti-BADH serum was raised in rabbits against native BADH purified from porcine kidney . The antiserum cross-reacted strongly with BADH purified from kidney, Amaranthus palmierii, and Pseudomona aeuroginosa (1:1000), and weakly with Amaranthus hypochondriacus L (1:100) . Antibodies bound to purified native kidney BADH in immunoblots showed a major band of an apparent molecular mass of 340 kDa and a subunit with an apparent molecular mass of 52 kDa . Data on activity assays showed higher activity in cortex sections (81.3 nmol/min/mg protein) than in medulla sections (21.3 nmol/min/mg protein) . Immunolocalization of BADH in kidney tissue sections showed that BADH is found in cortex and medulla . In inner medulla, the enzyme was mainly localized in cells surrounding the tubules . Western blot analysis on extracts from the cortex and medulla sections showed higher concentration of BADH protein in cortex than in medulla . These results were in accordance with immunolocalization and activity analysis . Thromb Res, 1999 May 1, 94(3), 153 - 64 Evolutionary conservation of circulating soluble low density lipoprotein receptor-related protein-like ("LRP-like") molecules; Grimsley PG et al.; Low density lipoprotein receptor family members characteristically bind 39-kDa receptor associated protein (RAP) . Soluble forms of these receptors have been described in humans including the 515/85-kDa dimeric receptor, low density lipoprotein receptor-related protein (LRP/alpha2MR), which is involved in multiple processes including lipoprotein and protease metabolism . Here we demonstrate evolutionary conservation in the generation of these soluble RAP-binding proteins of high molecular weight, by identifying their presence in mammalian, avian, and reptilian sera as well as in the circulating haemolymph of a mollusc . Sera extracted on immobilized RAP, produced bands at approximately 500 kDa in radiolabeled ligand blots by using the LRP/alpha2MR-specific ligand, Pseudomonas exotoxin A (PEA) . These findings suggest that circulating RAP-binding proteins with high molecular weight in vertebrates share features of LRP/alpha2MR (LRP-like molecules) . RAP-binding molecules in the mammalian serum extracts were further characterized as LRP/alpha2MR homologues in Western blots by using antibodies against the 515-kDa alpha-chain of LRP/alpha2MR . Western blots of mammalian serum extracts using two monoclonal antibodies recognizing the 85-kDa transmembrane beta-chain suggested that a portion of the beta-chain's ectodomain remains associated with the alpha-chain, but the beta-chain's intracellular carboxy terminus is absent . These results are consistent with evolutionary conservation in the generation, composition, and ligand-binding ability of soluble LRP-like receptors and suggest that their presence is a necessary aspect of the receptor's function. J Bacteriol, 1999 May, 181(10), 3256 - 61 Isolation and characterization of the cis-trans-unsaturated fatty acid isomerase of Pseudomonas oleovorans GPo12; Pedrotta V et al.; Pseudomonas oleovorans contains an isomerase which catalyzes the cis-trans conversion of the abundant unsaturated membrane fatty acids 9-cis-hexadecenoic acid (palmitoleic acid) and 11-cis-octadecenoic acid (vaccenic acid) . We purified the isomerase from the periplasmic fraction of Pseudomonas oleovorans . The molecular mass of the enzyme was estimated to be 80 kDa under denaturing conditions and 70 kDa under native conditions, suggesting a monomeric structure of the active enzyme . N-terminal sequencing showed that the isomerase derives from a precursor with a signal sequence which is cleaved from the primary translation product in accord with the periplasmic localization of the enzyme . The purified isomerase acted only on free unsaturated fatty acids and not on esterified fatty acids . In contrast to the in vivo cis-trans conversion of lipids, this in vitro isomerization of free fatty acids did not require the addition of organic solvents . Pure phospholipids, even in the presence of organic solvents, could not serve as substrate for the isomerase . However, when crude membranes from Pseudomonas or Escherichia coli cells were used as phospholipid sources, a cis-trans isomerization was detectable which occurred only in the presence of organic solvents . These results indicate that isolated membranes from Pseudomonas or E . coli cells must contain factors which, activated by the addition of organic solvents, enable and control the cis-trans conversion of unsaturated acyl chains of membrane phospholipids by the periplasmic isomerase. J Bacteriol, 1999 May, 181(10), 3105 - 13 Diverse oxygenations catalyzed by carbazole 1,9a-dioxygenase from Pseudomonas sp . Strain CA10; Nojiri H et al.; Carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas sp . strain CA10 is a multicomponent enzyme that catalyzes the angular dioxygenation of carbazole, dibenzofuran, and dibenzo-p-dioxin . It was revealed by gas chromatography-mass spectrometry and 1H and 13C nuclear magnetic resonance analyses that xanthene and phenoxathiin were converted to 2,2',3-trihydroxydiphenylmethane and 2,2',3-trihydroxydiphenyl sulfide, respectively . Thus, for xanthene and phenoxathiin, angular dioxygenation by CARDO occurred at the angular position adjacent to the oxygen atom to yield hetero ring-cleaved compounds . In addition to the angular dioxygenation, CARDO catalyzed the cis dihydroxylation of polycyclic aromatic hydrocarbons and biphenyl . Naphthalene and biphenyl were converted by CARDO to cis-1, 2-dihydroxy-1,2-dihydronaphthalene and cis-2,3-dihydroxy-2, 3-dihydrobiphenyl, respectively . On the other hand, CARDO also catalyzed the monooxygenation of sulfur heteroatoms in dibenzothiophene and of the benzylic methylenic group in fluorene to yield dibenzothiophene-5-oxide and 9-hydroxyfluorene, respectively . These results indicate that CARDO has a broad substrate range and can catalyze diverse oxygenation: angular dioxygenation, cis dihydroxylation, and monooxygenation . The diverse oxygenation catalyzed by CARDO for several aromatic compounds might reflect the differences in the binding of the substrates to the reaction center of CARDO. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 469 - 78 DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp . nov . and Pseudomonas cannabina sp . nov . (ex Sutic and Dowson 1959); Gardan L et al.; A total of 48 pathovars of Pseudomonas syringae and eight related species were studied by DNA-DNA hybridization (S1 nuclease method) and ribotyping . The existence of nine discrete genomospecies was indicated . Genomospecies 1 corresponded to P . syringae sensu stricto and included P . syringae pathovars syringae, aptata, lapsa, papulans, pisi, atrofaciens, aceris, panici, dysoxyli and japonica . Genomospecies 2 included P . syringae pathovars phaseolicola, ulmi, mori, lachrymans, sesami, tabaci, morsprunorum, glycinea, ciccaronei, eriobotryae, mellea, aesculi, hibisci, myricae, photiniae and dendropanacis and nomenspecies Pseudomonas savastanoi, Pseudomonas ficuserectae, Pseudomonas meliae and Pseudomonas amygdali, which are thus synonymous . P . amygdali is the earliest valid name for this genomospecies . Genomospecies 3 included P . syringae pathovars tomato, persicae, antirrhini, maculicola, viburni, berberidis, apii, delphinii, passiflorae, philadelphi, ribicola and primulae . We recommend strain CFBP 2212 of P . syringae pv . tomato to serve as the type strain . Genomospecies 4 included 'Pseudomonas coronafaciens' and P . syringae pathovars porri, garcae, striafaciens, atropurpurea, oryzae and zizaniae and corresponds to 'P . coronafaciens' . Genomospecies 5 included P . syringae pv . tremae and corresponds to Pseudomonas tremae sp . nov . Genomospecies 6 included Pseudomonas viridiflava and the presently misidentified pathotype strains of P . syringae pv . ribicola and P . syringae pv . primulae and thus corresponds to P . viridiflava . Genomospecies 7 included P . syringae pv . tagetis and P . syringae pv . helianthi . We recommend strain CFBP 1694 of P . syringae pv . tagetis to serve as a reference strain . Genomospecies 8 included P . syringae pv . these and Pseudomonas avellanae and thus corresponds to P . avellanae . Genomospecies 9 included P . syringae pv . cannabina and corresponds to Pseudomonas cannabina sp . nov . Ribotyping (SmaI and HincII endonucleases) could separate seven of the nine genomospecies . The unnamed genomospecies 3 and 7 will be named when phenotypic data are available for identification . Two species are described, P . tremae sp . nov . and P . cannabina sp . nov . Other species will be named when phenotypic data are available for identification. Biochemistry (Mosc), 1999 Apr, 64(4), 481 - 2 PsiI, a novel restriction endonuclease recognizing the DNA sequence 5'-TTA downward arrow TAA-3'; Abdurashitov MA et al.; PsiI, a novel restriction endonuclease produced by the bacterial strain Pseudomonas sp . SE-G49, has been isolated and characterized . The enzyme cleaves DNA in the middle of its palindromic recognition sequence 5'-TTA downward arrow TAA-3' . Thus, PsiI belongs to a rare group of type II restriction endonucleases whose recognition sites consist of AT base pairs only. Biochemistry, 1999 May 4, 38(18), 5799 - 812 Solution structure of component B from methane monooxygenase derived through heteronuclear NMR and molecular modeling; Chang SL et al.; Methane monooxygenase (MMO) is a nonheme iron-containing enzyme which consists of three protein components: a hydroxylase (MMOH), an NADH-linked reductase (MMOR), and a small "B" component (MMOB) which plays a regulatory role . Here, 1H, 13C, 15N heteronuclear 2D and 3D NMR spectroscopy has been used to derive the solution structure of the 138 amino acid MMOB protein in the monomer state . Pulse field gradient NMR self-diffusion measurements indicate predominant formation of dimers at 1 mM MMOB and monomers at or below 0.2 mM . MMOB is active as a monomer . Aggregate exchange broadening and limited solubility dictated that multidimensional heteronuclear NMR experiments had to be performed at a protein concentration of 0.2 mM . Using 1340 experimental constraints (1182 NOEs, 98 dihedrals, and 60 hydrogen bonding) within the well-folded part of the protein (residues 36-126), MMOB structural modeling produced a well-defined, compact alpha/beta fold which consists of three alpha-helices and six antiparallel beta-strands arranged in two domains: a betaalphabetabeta and a betaalphaalphabetabeta . Excluding the ill-defined N- and C-terminal segments (residues 1-35 and 127-138), RMS deviations are 1.1 A for backbone atoms and 1.6 A for all non-hydrogen atoms . Compared to the lower resolution NMR structure for the homologous protein P2 from the Pseudomonas sp . CF600 phenol hydroxylase system (RMSD = 2.48 A for backbone atoms) (Qian, H., Edlund, U., Powlowski, J., Shingler, V., and Sethson, I . (1997) Biochemistry, 36, 495-504), that of MMOB reveals a considerably more compact protein . In particular, MMOB lacks the large "doughnut" shaped cavity reported for the P2 protein . This difference may result from the limited number of long-range NOEs that were available for use in the modeling of the P2 structure . This NMR-derived structure of MMOB, therefore, presents the first high-resolution structure of a small protein effector of a nonheme oxygenase system. J Mass Spectrom, 1999 Apr, 34(4), 281 - 90 A fast screening method for the identification of siderophores from fluorescent Pseudomonas spp . by liquid chromatography/electrospray mass spectrometry; Kilz S et al.; A screening method was developed for the fast identification of known pyoverdin-type siderophores produced by fluorescent Pseudomonas spp . It is based on reversed-phase high-performance liquid chromatography interfaced with electrospray ionization mass spectrometry of the Sep-Pak RP-C18 culture supernatant extracts . The siderophores of five bacterial strains were characterized by their molecular masses obtained from their doubly protonated molecular ions {M + 2H}2+ and their UV/visible spectra recorded with a diode-array detector . Additional structural information was gained by skimmer collision-induced dissociation experiments . For all strains new minor siderophores were found . A table of fully or partially identified pyoverdins and related siderophores is provided which will be the basis for screening studies. FEMS Microbiol Lett, 1999 Apr 15, 173(2), 379 - 88 Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae; Jagtap P et al.; The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue {Ray, M.K . et al . (1994) FEMS Microbiol . Lett . 122, pp . 49-54} . To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth . The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases . The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr) . The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases . The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor . The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P . syringae. Anticancer Res, 1999 Jan-Feb, 19(1A), 125 - 31 Pancreatic cancer cells express interleukin-13 and -4 receptors, and their growth is inhibited by Pseudomonas exotoxin coupled to interleukin-13 and -4; Kornmann M et al.; BACKGROUND: Interleukin (IL)-13 and -4 are multifunctional cytokines that bind to specific cell-surface receptors . The aim of this study was to determine whether pancreatic cancer cells express either receptor, and to assess the growth suppressive effects of chimeric proteins composed of a Pseudomonas exotoxin (PE) A mutant (PE38QQR) fused to IL-13 (IL-13-PE38QQR) or IL-4 (IL-4-PE38QQR) in these cells . MATERIALS AND METHODS: Northern and Western blot analysis were used to analyze the expression of IL-4/-13 receptors and the common gamma chain (gamma c) in pancreatic cancer cell lines . MTT growth assays were carried out to assess the effects of IL-4/-13 and IL-4/-13-PE38QQR on cell growth . RESULTS: All 6 pancreatic cancer cell lines examined expressed IL-13R alpha 1 and IL-4R alpha, one cell line expressed IL-13R alpha 2, and 5 pancreatic cancer cell lines expressed gamma c . IL-13 (5 nM) significantly enhanced the growth of 3 cell lines, whereas IL-4 (5 nM) enhanced the growth of 1 cell line . In contrast, IL-13-PE38QQR and IL-4-PE38QQR inhibited the growth of all 6 tested cell lines . There were large variations in the individual sensitivity of the cells, with LD50 values ranging from 100 ng/ml to 5 micrograms/ml for IL-13-PE38QQR and from 20 ng/ml to 10 micrograms/ml for IL-4-PE38QQR . IL-13 and -4 antagonized these inhibitory activities in some, but not all, of the cell lines . CONCLUSIONS: IL-13 and -4 may act as mitogens toward pancreatic cancer cells by activating IL-4- and IL-13-receptors and IL-13- and IL-4-coupled toxins may ultimately have a role in the treatment of pancreatic cancer. Mol Plant Microbe Interact, 1999 May, 12(5), 401 - 9 Conductive properties and gating of channels formed by syringopeptin 25A, a bioactive lipodepsipeptide from Pseudomonas syringae pv . syringae, in planar lipid membranes; Dalla Serra M et al.; Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes . Pore conductance is around 40 pS in 0.1 M NaCl . Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added . These pores open and close with a lifetime of several seconds . At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present . The voltage dependence of the rates of opening and closing of the stable pores is exponential . This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges . When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations . The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages . The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution . From this, an estimate of a minimal pore radius of 0.4 nm was derived. Mol Plant Microbe Interact, 1999 May, 12(5), 391 - 400 The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv . syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins; Dalla Serra M et al.; Pseudomonas syringae pv . syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25) . Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane . We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles) . All peptides induced red blood cell hemolysis . The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size . Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm . All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A) . They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film . These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein) . The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol . From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers . On average, syringopeptin oligomers were smaller than SRE and ST oligomers. Genetics, 1999 May, 152(1), 401 - 12 Identification of three putative signal transduction genes involved in R gene-specified disease resistance in Arabidopsis; Warren RF et al.; The RPS5 disease resistance gene of Arabidopsis mediates recognition of Pseudomonas syringae strains that possess the avirulence gene avrPphB . By screening for loss of RPS5-specified resistance, we identified five pbs (avrPphB susceptible) mutants that represent three different genes . Mutations in PBS1 completely blocked RPS5-mediated resistance, but had little to no effect on resistance specified by other disease resistance genes, suggesting that PBS1 facilitates recognition of the avrPphB protein . The pbs2 mutation dramatically reduced resistance mediated by the RPS5 and RPM1 resistance genes, but had no detectable effect on resistance mediated by RPS4 and had an intermediate effect on RPS2-mediated resistance . The pbs2 mutation also had varying effects on resistance mediated by seven different RPP (recognition of Peronospora parasitica) genes . These data indicate that the PBS2 protein functions in a pathway that is important only to a subset of disease-resistance genes . The pbs3 mutation partially suppressed all four P . syringae-resistance genes (RPS5, RPM1, RPS2, and RPS4), and it had weak-to-intermediate effects on the RPP genes . In addition, the pbs3 mutant allowed higher bacterial growth in response to a virulent strain of P . syringae, indicating that the PBS3 gene product functions in a pathway involved in restricting the spread of both virulent and avirulent pathogens . The pbs mutations are recessive and have been mapped to chromosomes I (pbs2) and V (pbs1 and pbs3). Appl Environ Microbiol, 1999 May, 65(5), 1876 - 82 Activation and inactivation of Pseudomonas stutzeri methylbenzene catabolism pathways mediated by a transposable element; Bolognese F et al.; The arrangement of the genes involved in o-xylene, m-xylene, and p-xylene catabolism was investigated in three Pseudomonas stutzeri strains: the wild-type strain OX1, which is able to grow on o-xylene but not on the meta and para isomers; the mutant M1, which grows on m-xylene and p-xylene but is unable to utilize the ortho isomer; and the revertant R1, which can utilize all the three isomers of xylene . A 3-kb insertion sequence (IS) termed ISPs1, which inactivates the m-xylene and p-xylene catabolic pathway in P . stutzeri OX1 and the o-xylene catabolic genes in P . stutzeri M1, was detected . No IS was detected in the corresponding catabolic regions of the P . stutzeri R1 genome . ISPs1 is present in several copies in the genomes of the three strains . It is flanked by 24-bp imperfect inverted repeats, causes the direct duplication of 8 bp in the target DNA, and seems to be related to the ISL3 family. Hum Gene Ther, 1999 Apr 10, 10(6), 923 - 34 Apoptosis induced by Pseudomonas exotoxin: a sensitive and rapid marker for gene delivery in vivo; Hafkemeyer P et al.; The rapid progress in gene therapy has expanded our ability to alter genetic structure, necessitating the development of methods for detecting the activity of new vectors . The central concept of a reporter gene is simple: it is a defined nucleotide sequence, which when introduced into a biological system, yields a readily measurable phenotype on expression . This provides a convenient parameter that is correlated to the molecular events associated with genetic expression . In this study we demonstrate that Pseudomonas exotoxin A (PE) can serve as a sensitive reporter gene to detect gene expression in single cells of mouse lung on cationic liposome delivery of PE-encoding DNA in vivo . Furthermore, we show that PE expression can be detected as early as 2 hr after systemic gene delivery in lungs of recipient mice . We compared PE with the widely used beta-galactosidase gene for this purpose . PE induces apoptosis that can be detected by TdT end labeling of DNA fragments (TUNEL assay) Since few expressed PE molecules are necessary to trigger the apoptosis cascade, the minimal amount of PE-encoding plasmid DNA needed for detection of apoptotic cells after systemic delivery was 0.1 microg per animal compared with at least 1 microg for the beta-galactosidase-encoding plasmid DNA . The maximum number of apoptotic cells detected in lungs was about 15-20 times higher than the maximum number of beta-galactosidase-positive cells . Specificity of apoptosis due to PE expression on delivery of the PE-encoding plasmid was shown by prevention of the apoptotic cascade by the caspase inhibitor Z-VAD-fmk. Arch Biochem Biophys, 1999 May 1, 365(1), 17 - 24 Oxaloacetate decarboxylase from Pseudomonas stutzeri: purification and characterization; Labrou NE et al.; Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxylation of oxaloacetate to pyruvic acid and carbon dioxide, was purified 245-fold to homogeneity from Pseudomonas stutzeri . The three-step purification procedure comprised anion-exchange chromatography, metal-chelate affinity chromatography, and biomimetic-dye affinity chromatography . Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, respectively, 63 and 64 kDa, suggesting a monomeric protein . OXAD required for maximum activity divalent metal cations such as Mn2+ and Mg2+ but not monovalent cations . The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5'-diphosphate, acetic acid, phosphoenolpyruvate, malic acid, and oxalic acid . Initial velocity, product inhibition, and dead-end inhibition studies suggested a rapid-equilibrium ordered kinetic mechanism with Mn2+ being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate . Inhibition data as well as pH-dependence profiles and kinetic parameters are reported and discussed in terms of the mechanism operating for oxaloacetate decarboxylation . FEMS Microbiol Lett, 1999 Apr 1, 173(1), 155 - 62 Diversity of Pseudomonas strains isolated with King's B and Gould's S1 agar determined by repetitive extragenic palindromic-polymerase chain reaction, 16S rDNA sequencing and Fourier transform infrared spectroscopy characterisation; Johnsen K et al.; King's B and Gould's S1 agar were compared with regard to the isolation of Pseudomonas from four environmental samples . In all samples, King's B gave the highest number of colony-forming units, and in some environments, there were more fluorescent colony-forming units on King's B as well . However, almost all types grew on Gould's S1, which enabled us to isolate a greater variety of groups than with King's B, fluorescent as well as non-fluorescent members of Pseudomonas . The Pseudomonas isolates were comparatively typed by repetitive extragenic palindromic-polymerase chain reaction and Fourier transform infrared spectroscopy, not previously used for environmental Pseudomonas . The two typing methods were similar in resolution, thus Fourier transform infrared spectroscopy proved fast and reproducible and is a good method for discrimination at subspecies level . Representative strains were identified by partial 16S rDNA sequencing . Thus, we suggest Gould's S1 agar be used for isolation of Pseudomonas because the results are reproducible, specific and give the most diverse recovery and the least work. Biochemistry, 1999 Apr 27, 38(17), 5666 - 75 A model for the solution structure of oxidized terpredoxin, a Fe2S2 ferredoxin from Pseudomonas; Mo H et al.; Terpredoxin (Tdx) is a 105-residue bacterial ferredoxin consisting of a single polypeptide chain and a single Fe2S2 prosthetic group . Tdx was first identified in a strain of Pseudomonas sp . capable of using alpha-terpineol as sole carbon source . The Tdx gene, previously cloned from the plasmid-encoded terp operon, that carries genes encoding for proteins involved in terpineol catabolism, has been subcloned and expressed as the holoprotein in E . coli . Physical characterization of the expressed Tdx has been performed, and a model for the solution structure of oxidized Tdx (Tdxo) has been determined . High-resolution homo- and heteronuclear NMR data have been used for structure determination in diamagnetic regions of the protein . The structure of the metal binding site (which cannot be determined directly by NMR methods due to paramagnetic broadening of resonances) was modeled using restraints obtained from a crystal structure of the homologous ferredoxin adrenodoxin (Adx) and loose restraints determined from paramagnetic broadening patterns in NMR spectra . Essentially complete 1H and 15N NMR resonance assignments have been made for the diamagnetic region of Tdxo (ca . 80% of the protein) . A large five-stranded beta-sheet and a smaller two-stranded beta-sheet were identified, along with three alpha-helices . A high degree of structural homology was observed between Tdx and two other ferredoxins with sequence and functional homology to Tdx for which structures have been determined, Adx and putidaredoxin (Pdx), a homologous Pseudomonas protein . 1H/2H exchange rates for Tdx backbone NH groups were measured for both oxidation states and are rationalized in the context of the Tdx structure . In particular, an argument is made for the importance of the residue following the third ligand of the metal cluster (Arg49 in Tdx, His49 in Pdx, His56 in Adx) in modulating protein dynamics as a function of oxidation state . Some differences between Tdx and Pdx are detected by UV-visible spectroscopy, and structural differences at the C-terminal region were also observed . Tdx exhibits only 2% of the activity of Pdx in turnover assays performed using the reconstituted camphor hydroxylase system of which Pdx is the natural component. J Bacteriol, 1999 May, 181(9), 2675 - 82 A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila BKME-9; Martin VJ et al.; Pseudomonas abietaniphila BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase . The ditA1, ditA2, and ditA3 genes, which encode the alpha and beta subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced . The ferredoxin gene is 9 . 2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditC . A Tn5 insertion in the alpha subunit gene, ditA1, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA . Disruption of the ferredoxin gene, ditA3, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941 . Sequence analysis of the putative ferredoxin indicated that it is likely to be a {4Fe-4S}- or {3Fe-4S}-type ferredoxin and not a {2Fe-2S}-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases . Expression in Escherichia coli of ditA1A2A3, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme . The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11, 12-dihydroxy-8,13-abietadien acid, which was identified by 1H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry . The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the alpha subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases. Postgrad Med J, 1998 Oct, 74(876), 604 - 6 Localised upper airway obstruction in a patient with acquired immunodeficiency syndrome; D'Arsigny CL et al.; We describe a case of rapidly progressive upper airway obstruction due to tracheal Pseudomonas abscesses in a patient with acquired immunodeficiency syndrome . The case highlights the aggressive nature of Pseudomonas infections and the difficulty of eradicating this organism in patients infected with the human immunodeficiency virus. Oncogene, 1999 Mar 4, 18(9), 1711 - 21 Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors; Schmidt M et al.; Cytotoxic strategies which are directed to tumor-associated antigens might be most beneficial for cancer patients with minimal tumor load such as in an adjuvant setting after initial therapy . We have recently described a highly potent single chain antibody-toxin, scFv(14E1)-ETA, which consists of the variable domains of the antibody 14E1 genetically fused to a truncated form of Pseudomonas exotoxin A . ScFv(14E1)-ETA specifically recognizes the human epidermal growth factor receptor (EGFR) and the oncogenically activated receptor variant EGFRvIII, which have been implicated in the development of various human malignancies . Here we have investigated the antimetastatic activity of bacterially expressed scFv(14E1)-ETA and its disulfide-stabilized derivative ds-scFv(14E1)-ETA in a novel model for disseminated disease which is based on murine renal carcinoma cells subsequently transfected with the E . coli beta-galactosidase gene, and human full-length or variant EGFR cDNAs . Intravenous injection of these Renca-lacZ/EGFR and Renca-lacZ/EGFRvIII cells in syngenic Balb/c mice led to the formation of pulmonary metastases which were readily detectable upon excision of the lungs and X-gal staining . Systemic treatment of mice with scFv(14E1)-ETA resulted in the complete suppression of Renca-lacZ/EGFRvIII metastasis formation and drastically reduced the number of pulmonary Renca-lacZ/EGFR tumor nodules . The ds-scFv(14E1)-ETA derivative where the antibody variable regions are connected by an artificial disulfide bond displayed improved thermal stability at physiological temperature but due to reduced cytotoxic activity was less potent than the original scFv(14E1)-ETA in metastasis suppression. Microbiology, 1999 Jan, 145 ( Pt 1), 135 - 41 A dispensable region of the chromosome which is associated with an avirulence gene in Pseudomonas syringae pv . pisi; Arnold DL et al.; Pseudomonas syringae pv . pisi comprises a number of races which fall into two phylogenetically distinct groups (designated I and II) . Races are based on cultivar specificity in the host plant, pea (Pisum sativum), and are specified by the presence of avirulence genes . The avirulence gene avrPpiA1 is present on the chromosome of all strains examined in race 2, which belongs to phylogenetic group II . A race 4B strain, from phylogenetic group I, lacks this avirulence gene and a comparative study was made of the chromosome in strains representing these two races . A race 2 cosmid clone (pAV270) carrying avrPpiA1 was used as a basis for collinearity analysis of races 2 and 4B . A region of the chromosome amounting to 8.5 kb and including avrPpiA1 was absent from race 4B compared with race 2 . A fragment spanning the junction of the discontinuity in race 4B was isolated, cloned and used to delimit the extent of the additional DNA present in race 2 . In both races the borders of the discontinuity contained DNA sequences which showed a high degree of conservation . A 7 bp slightly imperfect direct repeat (CCAGC(T)/(A)T) flanked the additional DNA in race 2, with a single copy in race 4B . The region flanking the additional DNA was present in all races of P . syringae pv . pisi . These results confirm the phylogenetic groupings in P . syringae pv . pisi. Clin Nutr, 1998 Oct, 17(5), 211 - 5 Nutrition in adults with cystic fibrosis; Bell SC et al.; Improved survival has been associated with better nutritional status in patients with cystic fibrosis (CF) . In this study we examined the relationship between nutritional state and other measures of clinical severity in adult patients with CF, attending a regional centre . Eighty-one patients (median age 21 years) were studied . Patients with CF were significantly under weight, compared to healthy individuals but were of similar height . Measurements of lung function, FEV1 and FVC were significantly related to body mass index . Lung function was poorer in patients with chronic pseudomonal infection but body weight and body mass index were not significantly different compared to those without such infection . In 53 patients who were alive 4 years later, FEV1 had declined by -10.5 (2.1)% (P < 0.001) but there was no significant change in body weight 1.5 (6.5) kgs . In 23 patients who died or had lung transplantation the change from 1994 to the date of death or transplantation the FEV1 was reduced by -7.9 (11.2)% (P = 0.004) and body weight -2.8 (4.4) kgs (P < 0.01) . In 12 patients who had supplemental enteral feeding, the median increase in body weight was 7|kgs over a period of 12 months . This study confirms that young adult patients with CF are significantly under weight and declining health is associated with significant weight loss . In patients with severe malnutrition significant improvement can be achieved by enteral feeding. Mol Microbiol, 1999 Mar, 31(5), 1385 - 93 A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly; Taupiac MP et al.; Pseudomonas exotoxin A (PE) is a cytotoxin composed of three structural domains . Domain I is responsible for cell binding, domain II for membrane translocation enabling access to the cytosol, and domain III for the catalytic inactivation of protein synthesis, which results in cell death . To investigate the role of the six alpha-helices (A-F) that form the translocation domain, we deleted them successively one at a time . All mutants showed native cell-binding and catalytic activities, indicating that deletions specifically affected translocation activity . This step of the intoxication procedure was examined directly using a cell-free translocation assay, and indirectly by monitoring cytotoxicity . Translocation activity and log(cytotoxicity) were highly correlated, directly indicating that translocation is rate limiting for PE intoxication . Deletion of B, C and D helices resulted in non-toxic and non-translocating molecules, whereas mutants lacking the A or E helix displayed significant cytotoxicity albeit 500-fold lower than native PE . We concluded that B, C and D helices, which make up the core of domain II, are essential, whereas the more peripheral A and E helices are comparatively dispensable . The last helix (F) is inhibitory for translocation because its deletion produced a mutant displaying a translocation activity 60% higher than PE, along with a three- to sixfold increase in cytotoxicity in all tested cell lines . This toxin is the most in vitro active PE mutant obtained until now . Finally, partial duplication of domain II did not give rise to a more actively translocated PE, but rather to a threefold less active molecule. Med Mycol, 1999 Feb, 37(1), 57 - 60 Paecilomyces lilacinus fungemia in an adult bone marrow transplant recipient; Chan-Tack KM et al.; Paecilomyces lilacinus is a rare fungal pathogen in humans . We report a case of fungemia caused by P . lilacinus in a non-neutropenic adult, 120 days after bone marrow transplant . The patient's primary risk factor was the presence of an indwelling vascular catheter . Her initial clinical course was characterized by fever, chills, and rigors . Blood cultures from the central line and peripheral veins were positive, as was a peripheral specimen drawn after removal of the catheter . Two initial peripheral specimens were positive for P . lilacinus only by blind subculture and/or sustained incubation . She developed peripheral pulmonary nodules following the fungemia, thus raising the possibility of disseminated disease, but definitive diagnosis was confounded by Pseudomonas bacteremia . The nodules cleared and she recovered following removal of the central line and treatment with amphotericin B and 5-fluorocytosine, despite in vitro resistance to these antifungal drugs . This case underscores the increasing importance of P . lilacinus as a human pathogen capable of producing disease in immunocompetent, as well as in immunocompromised hosts . Also of note is that blood culture systems may require extended incubation or subcultures in order to detect fungi. J Bacteriol, 1999 Apr, 181(8), 2315 - 22 NahW, a novel, inducible salicylate hydroxylase involved in mineralization of naphthalene by Pseudomonas stutzeri AN10; Bosch R et al.; Two genes, nahG and nahW, encoding two independent salicylate 1-hydroxylases have been identified in the naphthalene-degrading strain Pseudomonas stutzeri AN10 . While nahG resides in the same transcriptional unit as the meta-cleavage pathway genes, forming the naphthalene degradation lower pathway, nahW is situated outside but in close proximity to this transcriptional unit . The nahG and nahW genes of P . stutzeri AN10 are induced and expressed upon incubation with salicylate, and the enzymes that are encoded, NahG and NahW, are involved in naphthalene and salicylate metabolism . Both genes, nahG and nahW, have been cloned in Escherichia coli JM109 . The overexpression of these genes yields peptides with apparent molecular masses of 46 kDa (NahG) and 43 kDa (NahW), respectively . Both enzymes exhibit broad substrate specificities and metabolize salicylate, methylsalicylates, and chlorosalicylates . However, the relative rates by which the substituted analogs are transformed differ considerably. Bone Marrow Transplant, 1999 Jan, 23(2), 137 - 44 Prevention of graft-versus-host disease (GVHD) by elimination of recipient-reactive donor T cells with recombinant toxins that target the interleukin 2 (IL-2) receptor; Harris DT et al.; Graft-versus-host disease (GVHD), due to the presence of recipient-reactive T cells, limits the usefulness of bone marrow transplantation (BMT) and is a major contributor to patient mortality . To prevent GVHD, murine and human T cells were activated by antigen or mitogens and treated with a genetically engineered form of Pseudomonas exotoxin A (PE) directed against the IL-2 receptor . Treatment with the chimeric toxin eliminated alloreactive cytotoxic T lymphocytes (CTL) as determined by cytotoxicity and mixed lymphocyte culture assays . Precursor frequencies of alloreactive cytotoxic T cells and proliferative T cells were reduced up to 100-fold as shown by limiting dilution assays . Flow cytometric analyses revealed that treatment with the chimeric toxin completely eliminated CD25+ cells from the cultures . Toxin treatment had no significant effect on hematopoietic stem and progenitor cells as determined in vitro by colony-forming assays and in vivo by long-term hematopoietic recovery after 950 rad irradiation . Toxin treatment decreased GVHD in transplanted mice to less than 10% (as compared to 88% in untreated controls) . Thus, it is possible to prevent life-threatening GVHD after BMT by using a CD25 receptor-directed toxin to eliminate host-reactive T cells from bone marrow grafts. Vaccine, 1999 Mar 17, 17(11-12), 1425 - 33 Mucosal administration of a chimera composed of Pseudomonas exotoxin and the gp120 V3 loop sequence of HIV-1 induces both salivary and serum antibody responses; Mrsny RJ et al.; We have used a mouse immunization model to evaluate the potential for a chimera protein composed of a nontoxic form of Pseudomonas exotoxin (ntPE) to incite and sustain a mucosal immune response against an integrated antigen . The chimera, termed ntPE-V3MN26, contained 26 amino acids of the gp120 V3 loop region sequence of the MN strain of HIV-1 integrated in place of the Ib region of ntPE . Following either vaginal, rectal, oral or subcutaneous administration and boosting, anti-gp120-specific IgA and IgG levels in serum and saliva samples were assessed by ELISA . All dosing regimens stimulated significant and comparable salivary IgA and serum IgG responses at 1, 2 and 3 months after the initial inoculation . Following a boost at 16 months with ntPE-V3MN26, a strong memory response to the antigen was observed . Isotyping of serum antibodies at this time suggested that both a Thl and a Th2 response had been induced . Responses to ntPE-V3MN26 following subcutaneous injection in the presence or absence of Freund's adjuvant demonstrated that Freund's adjuvant resulted in a three-fold greater enhancement of immune response compared to administration of chimera alone . These results demonstrate that mucosal presentation of a chimera composed of a nontoxic form of Pseudomonas exotoxin can result in a strong mucosal and systemic antigen-specific immune response to an integrated antigen . The profound memory responses induced by this chimera may be particularly useful for practical vaccine applications. J Microbiol Methods, 1999 Mar, 35(2), 163 - 6 An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions; Martin VJ et al.; We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions . This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment . From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon . This method avoids the problem of amplifying or cloning long sequences flanking Tn5 . To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA) . The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified. Appl Environ Microbiol, 1999 Apr, 65(4), 1435 - 43 Location and survival of leaf-associated bacteria in relation to pathogenicity and potential for growth within the leaf Wilson M, Hirano SS, Lindow SE. The growth and survival of pathogenic and nonpathogenic Pseudomonas syringae strains and of the nonpathogenic species Pantoea agglomerans, Stenotrophomonas maltophilia, and Methylobacterium organophilum were compared in the phyllosphere of bean . In general, the plant pathogens survived better than the nonpathogens on leaves under environmental stress . The sizes of the total leaf-associated populations of the pathogenic P . syringae strains were greater than the sizes of the total leaf-associated populations of the nonpathogens under dry conditions but not under moist conditions . In these studies the surface sterilants hydrogen peroxide and UV irradiation were used to differentiate cells that were fully exposed on the surface from nonexposed cells that were in "protected sites" that were inaccessible to these agents . In general, the population sizes in protected sites increased with time after inoculation of plants . The proportion of bacteria on leaves that were in protected sites was generally greater for pathogens than for nonpathogens and was greater under dry conditions than under moist conditions . When organisms were vacuum infiltrated into leaves, the sizes of the nonexposed "internal" populations were greater for pathogenic P . syringae strains than for nonpathogenic P . syringae strains . The sizes of the populations of the nonpathogenic species failed to increase or even decreased . The sizes of nonexposed populations following spray inoculation were correlated with the sizes of nonexposed, internal populations which developed after vacuum infiltration and incubation . While the sizes of the populations of the pathogenic P . syringae strains increased on leaves under dry conditions, the sizes of the populations of the nonpathogenic strains of P . syringae, P . agglomerans, and S . maltophilia decreased when the organisms were applied to plants . The sizes of the populations on dry leaves were also correlated with the sizes of the nonexposed populations that developed following vacuum infiltration . Although pathogenicity was not required for growth in the phyllosphere under high-relative-humidity conditions, pathogenicity apparently was involved in the ability to access and/or multiply in certain protected sites in the phyllosphere and in growth on dry leaves. Appl Environ Microbiol, 1999 Apr, 65(4), 1786 - 8 Quantitative selective PCR of 16S ribosomal DNA correlates well with selective agar plating in describing population dynamics of indigenous Pseudomonas spp . in soil hot spots; Johnsen K et al.; We used a quantitative PCR method targeting 16S ribosomal DNA using competitive PCR for specific detection of indigenous Pseudomonas DNA in soil hot spots . The amount of Pseudomonas DNA corresponded to the number of culturable Pseudomonas bacteria on Gould's S1 agar . This represents the first use of PCR for quantification of indigenous bacteria in more than one sample of soil. Appl Environ Microbiol, 1999 Apr, 65(4), 1703 - 9 Relationship between succinate transport and production of extracellular poly(3-hydroxybutyrate) depolymerase in Pseudomonas lemoignei; Terpe K et al.; The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei . Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6 . Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis . The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6 . 8 . The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6 . The uptake of {14C}succinate was strongly inhibited by several monocarboxylates . Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2) . We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms . The inability to take up succinate2- accounts for the carbon starvation of P . lemoignei observed during growth on succinate at pH values above 7 . As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products. Development, 1999 May, 126(9), 1997 - 2005 Heparin-binding EGF-like growth factor interacts with mouse blastocysts independently of ErbB1: a possible role for heparan sulfate proteoglycans and ErbB4 in blastocyst implantation; Paria BC et al.; Blastocyst implantation requires molecular and cellular interactions between the uterine luminal epithelium and blastocyst trophectoderm . We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is induced in the mouse luminal epithelium solely at the site of blastocyst apposition at 16:00 hours on day 4 of pregnancy prior to the attachment reaction (22:00-23:00 hours), and that HB-EGF promotes blastocyst growth, zona-hatching and trophoblast outgrowth . To delineate which EGF receptors participate in blastocyst activation, the toxicity of chimeric toxins composed of HB-EGF or TGF-(&agr;) coupled to Pseudomonas exotoxin (PE) were used as measures of receptor expression . TGF-(&agr;) or HB-EGF binds to EGF-receptor (ErbB1), while HB-EGF, in addition, binds to ErbB4 . The results indicate that ErbB1 is inefficient in mediating TGF-(&agr;)-PE or HB-EGF-PE toxicity as follows: (i) TGF-(&agr;)-PE was relatively inferior in killing blastocysts, 100-fold less than HB-EGF-PE, (ii) analysis of blastocysts isolated from cross-bred egfr+/- mice demonstrated that HB-EGF-PE, but not TGF-(&agr;)-PE, killed egfr-/- blastocysts, and (iii) blastocysts that survived TGF-(&agr;)-PE were nevertheless killed by HB-EGF-PE . HB-EGF-PE toxicity was partially mediated by cell surface heparan sulfate proteoglycans (HSPG), since a peptide corresponding to the heparin-binding domain of HB-EGF as well as heparitinase treatment protected the blastocysts from the toxic effects of HB-EGF-PE by about 40% . ErbB4 is a candidate for being an HB-EGF-responsive receptor since RT-PCR analysis demonstrated that day 4 mouse blastocysts express two different erbB4 isoforms and immunostaining with anti-ErbB4 antibodies confirmed that ErbB4 protein is expressed at the apical surface of the trophectoderm cells . It is concluded that (i) HB-EGF interacts with the blastocyst cell surface via high-affinity receptors other than ErbB1, (ii) the HB-EGF interaction with high-affinity blastocysts receptors is regulated by heparan sulfate, and (iii) ErbB4 is a candidate for being a high-affinity receptor for HB-EGF on the surface of implantation-competent blastocysts. Biotechnol Bioeng, 1999 Mar 5, 62(5), 554 - 61 Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents; Secundo F et al.; The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane . The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined . Both rates depended on the lipase form, solvent employed, and aw value . Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent . At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer . Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer . The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media . Biotechnol Bioeng, 1998 Jul 20, 59(2), 171 - 7 Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase Drijfhout FP, Fraaije MW, Jongejan H, van Berkel WJ, Franssen MC. Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol, and 2-methoxy-4-propylphenol into 1-(4'-hydroxyphenyl)ethanol, 1-(4'-hydroxyphenyl)propanol, and 1-(4'-hydroxy-3'-methoxyphenyl)propanol, respectively, with an ee of 94% for the R enantiomer . The stereochemical outcome of the reactions was established by comparing the chiral GC retention times of the products to those of chiral alcohols obtained by the action of the lipases from Candida antarctica and Pseudomonas cepacia . Isotope labeling experiments revealed that the oxygen atom incorporated into the alcoholic products is derived from water . During the VAO-mediated conversion of 4-ethylphenol/4-propylphenol, 4-vinylphenol/4-propenylphenol are formed as side products . With 2-methoxy-4-propylphenol as a substrate, this competing side reaction is nearly abolished, resulting in less than 1% of the vinylic product, isoeugenol . The VAO-mediated conversion of 4-alkylphenols also results in small amounts of phenolic ketones indicative for a consecutive oxidation step . Biotechnol Bioeng, 1998 Jan 20, 57(2), 228 - 37 Synthesis of alkane hydroxylase of Pseudomonas oleovorans increases the iron requirement of alk+ bacterial strains; Staijen IE et al.; The alk genes enable Pseudomonas oleovorans to utilize alkanes as sole carbon and energy source . Expression of the alk genes in P . oleovorans and in two Escherichia coli recombinants induced iron limitation in minimal medium cultures . Further investigation showed that the expression of the alkB gene, encoding the integral cytoplasmic membrane protein AlkB, was responsible for the increase of the iron requirement of E . coli W3110 (pGEc47) . AlkB is the non-heme iron monooxygenase component of the alkane hydroxylase system, and can be synthesized to levels up to 10% (w/w) of total cell protein in E . coli W3110 (pGEc47) . Its synthesis is, however, strictly dependent on the presence of sufficient iron in the medium . Our results show that a glucose-grown E . coli alk+ strain can reach alkane hydroxylase activities of about 25 U/g cdw, and are consistent with the recent finding that catalytically active AlkB contains two, rather than one iron atom per polypeptide chain . Biotechnol Bioeng, 1998 Jan 5, 57(1), 62 - 70 Plasmid stability of recombinant Pseudomonas sp . B13 FR1 pFRC20P in continuous culture; Hempel C et al.; Plasmid stability of recombinant Pseudomonas sp . B13 FR1 pFRC20P, a strain capable of mineralizing 3- and 4-chlorobenzoate and 4-methylbenzoate, was investigated in continuous culture . The hybrid cosmid pFRC20P enables the strain to mineralize 4-methylbenzoate . Rapid plasmid loss was observed under nonselective conditions using 3-chlorobenzoate as the substrate . Plasmid stability decreased with increasing dilution rate . Despite the growth advantage of the generated plasmid free cells a total depletion of plasmid bearing cells was not observed . After approximately 50 generations the fraction of plasmid bearing cells reached a constant level of 10%, which was stably maintained during the next 25 generations . Cells from this stage were used to inoculate a new culture that resulted in a stable level of 50% plasmid bearing cells . By a temporary substrate change to selective conditions (4-methylbenzoate), this level could be further increased to 70% . Literature models on plasmid stability could not be applied to describe the experimental data . Therefore, a new but unstructured model was developed to describe the experimental results . The model is based on the existence of three subpopulations: a plasmid free one, an original plasmid bearing one with a growth disadvantage compared to plasmid free cells, and a second plasmid bearing subpopulation with increased stability that is generated from the original one and has a growth rate comparable to the plasmid free cells . Proc Natl Acad Sci U S A, 1999 Mar 30, 96(7), 3957 - 62 Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome; Baluna R et al.; The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS) . VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure . IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo . Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered . In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs . Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo . In contrast, the same peptides with a deleted or mutated sequence do not . Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa . In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules . Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins. Mol Microbiol, 1999 Feb, 31(4), 1217 - 28 The alarmone (p)ppGpp mediates physiological-responsive control at the sigma 54-dependent Po promoter; Sze CC et al.; Transcription from the Pseudomonas-derived sigma 54-dependent Po promoter of the dmp operon is mediated by the aromatic-responsive regulator DmpR . However, physiological control is superimposed on this regulatory system causing silencing of the DmpR-mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached . Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo . Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non-permissive conditions . Conversely (p)ppGpp-deficient Escherichia coli strains were found to be severely defective in DmpR-mediated transcription, demonstrating the requirement for this metabolic signal . A subset of mutations in the beta, beta' and sigma 70 subunits of RNA polymerase, which confer prototrophy on ppGpp0 E . coli, was also found to restore specific DmpR-mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the sigma 54-RNA polymerase . These data provide a direct mechanistic link between the physiological status of the cell and expression from sigma 54 promoters. J Bacteriol, 1999 Apr, 181(7), 2298 - 301 Cellular locations of Pseudomonas syringae pv . syringae HrcC and HrcJ proteins, required for harpin secretion via the type III pathway; Deng WL et al.; The complete hrp-hrc-hrmA cluster of Pseudomonas syringae pv . syringae 61 encodes 28 polypeptides . A saprophytic bacterium carrying this cluster is capable of secreting HrpZ-a harpin encoded by hrpZ-in an hrp-dependent manner, which suggests that this cluster contains sufficient components to assemble functional type III secretion machinery . Sequence data show that HrcJ and HrcC are putative outer membrane proteins, and nonpolar mutagenesis demonstrates they are all required for HrpZ secretion . In this study, we investigated the cellular localization of the HrcC and HrcJ proteins by Triton solubilization, sucrose-gradient isopycnic centrifugation, and immunogold labeling of the bacterial cell surface . Our results indicate that HrcC is indeed an outer membrane protein and that HrcJ is located between both membranes . Their membrane localization suggests that they might be involved in the formation of a supramolecular structure for protein secretion. Pneumologie, 1999 Jan, 53(1), 31 - 6 {Ambulatory vs . inpatient intravenous antibiotic therapy in mucoviscidosis patients--a controlled study}; Klettke U et al.; METHODS: 14 patients with cystic fibrosis and chronic pulmonary pseudomonas infection received four courses of two-week intravenous antibiotic therapy at home and during hospitalisation over an 18-month period . Following a controlled, intra-individual cross-over design, two courses of home therapy were followed by two courses of hospital treatment or vice versa . Parameters for inflammation, lung function, and body mass index were obtained at the beginning and end of each intravenous antibiotic therapy . Health-related quality of life, i.e . physical, emotional, social and functional components as well as happiness and medical care, was assessed at the end of each course . RESULTS: There was a trend towards better reduction of infection (p = 0.20 for leukocyte reduction) and improvement of lung function (p = 0.20 for FEV1 improvement) with hospital intravenous antibiotic therapy, although the differences did not attain statistical significance . Quality of life during therapy was significantly higher with home therapy regarding social (p < 0.01), functional and emotional subscales and happiness (all p < 0.05) . The necessity for professional help and support from family and/or partner was emphasised . Individual answers showed that home therapy has the advantage of self-determination and continuity of daily life . Parents and partners felt impaired by day and night intravenous therapy at home . CONCLUSION: From our data we conclude that home intravenous antibiotic therapy is a useful option for a selected subgroup of patients with cystic fibrosis, but professional support and family aid is important to gain an effect similar to hospital treatment. Graefes Arch Clin Exp Ophthalmol, 1999 Mar, 237(3), 177 - 80 The combination of topical ceftazidime and aminoglycosides in the treatment of refractory pseudomonal keratitis; Robinson A et al.; BACKGROUND: Pseudomonal keratitis is a serious and potentially blinding infection . METHODS: We treated 12 patients with culture-positive fulminant pseudomonal keratitis with a topical combination of ceftazidime ophthalmic solution (50 mg/ml) and aminoglycosides (14 mg/ml) . None of these patients had responded to the standard initial therapy with topical fortified gentamicin or tobramycin (14 mg/ml) combined with cefazolin (50 mg/ml) . RESULTS: Substitution of cefazolin by ceftazidime achieved a remarkable clinical improvement during the first 24-48 h of administration in all cases . The average time of healing after initiation of the combination of ceftazidime and fortified aminoglycosides was 21+/-15 days . No serious side effects accompanied ceftazidime administration . In vitro susceptibility testing showed resistance to gentamicin or tobramycin in 33% of cases (4/12) and sensitivity to ceftazidime in all cases . CONCLUSIONS: The combination of ceftazidime, in a 5% solution, and fortified aminoglycosides (1.4%) may be a useful, safe and effective topical therapy for the treatment of pseudomonal keratitis resistant to aminoglycosides. Biochem Biophys Res Commun, 1999 Mar 24, 256(3), 469 - 73 The phnIJ genes encoding acetaldehyde dehydrogenase (acylating) and 4-hydroxy-2-oxovalerate aldolase in Pseudomonas sp . DJ77 and their evolutionary implications; Hwang S et al.; The two final steps of meta-cleavage pathway for catechol degradation involve conversion of 4-hydroxy-2-oxovalerate, via acetaldehyde, to acetyl coenzyme A . We report here the complete nucleotide sequences and overexpression of the phnIJ genes for an acetaldehyde dehydrogenase (acylating) (ADA) and a 4-hydroxy-2-oxovalerate aldolase (HOA) from the meta-pathway operon of the phenanthrene-degrading bacterium, Pseudomonas sp . strain DJ77 . Additional partial sequence analysis of adjacent DNA shows the gene order within the operon to be phnHIJ, identical to the order found for the isofunctional genes in the other meta-pathway operons . The deduced amino acid sequences of the PhnI (312 amino acids) and PhnJ (343 amino acids) have identities of 51-71% with the corresponding genes of dmp, xyl, nah, bph_LB400, bph_KKS102, tod, cum, cmt, and MTCY03C7 operons . The phylogenetic analyses reveal the evolutionary relationships of HOA and ADA . Cryobiology, 1999 Feb, 38(1), 60 - 7 Ice-nucleating bacteria from the guts of two sub-antarctic beetles, hydromedion sparsutum and perimylops antarcticus (Perimylopidae) Worland MR, Block W. The site of ice nucleation in the freeze-tolerant, sub-Antarctic beetle Hydromedion sparsutum has been investigated . Ice+ bacteria, active at above -2.0 degrees C, were isolated from the guts of beetles and identified as a fluorescent Pseudomonas species . Other possible sites of nucleation, including the hemolymph, were examined but had a lower activity . Ice+ bacteria were isolated from mixed populations, isolated from the guts of adult beetles, and grown on nutrient agar plates and in nutrient broth . Nucleation activity of the broth culture peaked after only 2 days although the number of live cells continued to increase until day 6 . These cultures were used to determine the maximum nucleation activity of a bacterial suspension in sterile distilled water (-3.4 degrees C) and the dilution factor required to cause a 50% reduction in activity (10(4)) . The original bacterial suspension had an absorbance of 0.5 measured at 660 nm and contained 6 x 10(11) bacteria per milliliter . From this it is estimated that only 1 in 10(6) bacteria possessed the highest levels of ice-nucleating activity . Other insect species, including Perimylops antarcticus, which are found in habitats similar to that of H . sparsutum, were examined for the presence of ice+ bacteria . All contained ice-nucleating bacteria in their guts but with a lower level of activity than in H . sparsutum . Int J Cancer, 1999 Mar 31, 81(1), 148 - 55 Complete regression of human B-cell lymphoma xenografts in mice treated with recombinant anti-CD22 immunotoxin RFB4(dsFv)-PE38 at doses tolerated by cynomolgus monkeys; Kreitman RJ et al.; RFB4(dsFv)-PE38 is a recombinant immunotoxin in which the variable light domain (V(L)) is disulfide bonded via cysteine residues to the variable heavy domain (V(H)), which in turn is fused to PE38, a mutant form of Pseudomonas exotoxin A . RFB4 binds to CD22, which is a differentiation antigen expressed on the majority of B-cell leukemias and lymphomas . To examine the potential efficacy of RFB4(dsFv)-PE38 when administered at a dose schedule appropriate for phase I testing, mice bearing CA46 human CD22+ Burkitt's lymphoma xenografts were treated on alternate days i.v . for 3 doses (QOD x 3) . Complete regressions were observed in 80% and 100% of mice treated with 200 and 275 microg/kg QOD x 3, respectively . The higher dose was 27% of the LD50 and 34% of the LD10 in mice . Because RFB4(dsFv)-PE38 is stable at 37 degrees C, it could also be given by continuous infusion using pumps placed in the peritoneal cavity; complete regressions also resulted from this mode of administration . To study toxicology, a pilot toxicology study of RFB4(dsFv)-PE38 was undertaken in cynomolgus monkeys, which like humans but unlike mice have CD22, which binds RFB4 . Doses of 100 and 500 microg/kg i.v . QOD x 3 were well tolerated, indicating that a dose that cured tumors in mice was tolerated by primates . Based on these preclinical results, RFB4(dsFv)-PE38 is being developed for the treatment of patients with CD22-positive leukemias and lymphomas. Biol Pharm Bull, 1999 Feb, 22(2), 227 - 8 Nucleotide sequence of the gene cluster containing the mphB gene for macrolide 2'-phosphotransferase II; Katayama J et al.; Escherichia coli BM2506 produced macrolide 2'-phosphotransferase II {MPH (2') II} . A gene for MPH (2') II, designated mphB, is located on plasmids pTZ3721 and pTZ3723 in E . coli BM2506 . In the present study, we determined the nucleotide sequence of the 6.5-kb EcoRI-PstI fragment containing mphB on pTZ3721 . The DNA region of 6.5-kb EcoRI-PstI fragment contained five open reading frames (ORFs) . ORF4 corresponded to mphB . Respective products deduced from ORF1, ORF2, ORF3, and ORF5 were similar to the penicillin-binding protein 4 of Streptomyces lactamduras, the repressor protein AcrR of the acrAB operon, the enzyme RdmC involved in the biosynthesis of the antibiotic aklavinone, and IS801 transposase-like protein from Pseudomonas pseudoalcakigenes, respectively . Among these genes, ORF2, ORF3, and mphB formed a gene cluster with ORF2 in the lead sequence . Our results suggest that mphB may originate from an operon related to antibiotic biosynthesis. Microbiology, 1999 Feb, 145 ( Pt 2), 325 - 34 Replication regions from plant-pathogenic Pseudomonas syringae plasmids are similar to ColE2-related replicons; Gibbon MJ et al.; Many strains of the phytopathogen Pseudomonas syringae contain mutually compatible plasmids that share extensive regions of sequence homology and essential replication determinants . The replication regions of two compatible large plasmids involved in virulence or pathogenicity, pPT23A from P . syringae pv . tomato strain PT23 and pAV505 from P . syringae pv . phaseolicola strain HRI1302A, were isolated . DNA sequencing of the origins of replication revealed homologous ORFs, designated ORF-Pto and ORF-Pph, respectively . Both ORFs are 1311 bp long and encode peptides of 437 amino acids with predicted molecular masses of 48259 (Pto) and 48334 (Pph) Da . Expression of the two ORFs in Escherichia coli produced peptides of 50 kDa (Pto) and 56 kDa (Pph) . The predicted peptides showed an overall identity of 897 %, being highly conserved from residues 1 to 373, but showing considerable variation in their C-terminal regions (50% identity over the last 64 aa) . The two ORFs had significant similarity with the putative replication protein from plasmid pTiK12 of Thiobacillus intermedius and other CoIE2-related plasmids . However, both peptides were 100 residues longer than any of the known CoIE2-related rep sequences . Subcloning of fragments from the replication region of pPT23A revealed the presence of at least three incompatibility determinants, designated IncA, IncB and IncC . Partial sequencing of the region downstream of ORF-Pto revealed homology to the ru/AB genes, involved in UV resistance, from plasmid pPSR1 . It is proposed that the replication origin of pPT23A serves as the prototype of a family of related plasmids. J Bacteriol, 1999 Mar, 181(6), 1831 - 7 Aspartate 205 in the catalytic domain of naphthalene dioxygenase is essential for activity; Parales RE et al.; The naphthalene dioxygenase enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp . strain NCIB 9816-4 . The crystal structure of naphthalene dioxygenase (B . Kauppi, K . Lee, E . Carredano, R . E . Parales, D . T . Gibson, H . Eklund, and S . Ramaswamy, Structure 6:571-586, 1998) indicates that aspartate 205 may provide the most direct route of electron transfer between the Rieske {2Fe-2S} center of one alpha subunit and mononuclear iron in the adjacent alpha subunit . In this study, we constructed four site-directed mutations that changed aspartate 205 to alanine, glutamate, asparagine, or glutamine to test whether this residue is essential for naphthalene dioxygenase activity . The mutant proteins were very inefficient in oxidizing naphthalene to cis-naphthalene dihydrodiol, and oxygen uptake in the presence of naphthalene was below detectable levels . The purified mutant protein with glutamine in place of aspartate 205 had identical spectral properties to wild-type naphthalene dioxygenase and was reduced by NADH in the presence of catalytic amounts of ferredoxinNAP and reductaseNAP . Benzene, an effective uncoupler of oxygen consumption in purified naphthalene dioxygenase, did not elicit oxygen uptake by the mutant protein . These results indicate that electron transfer from NADH to the Rieske center in the mutant oxygenase is intact, a finding consistent with the proposal that aspartate 205 is a necessary residue in the major pathway of electron transfer to mononuclear iron at the active site. Plant Cell, 1999 Mar, 11(3), 431 - 44 The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants; Solomon M et al.; Programmed cell death (PCD) is a process by which cells in many organisms die . The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms . Cysteine proteases have emerged as key enzymes in the regulation of animal PCD . Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases . The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells . Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress . Similar expression of serine protease inhibitors was ineffective . A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases . Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors . We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants. Plant Physiol, 1999 Mar, 119(3), 935 - 50 Isolation of ethylene-insensitive soybean mutants that are altered in pathogen susceptibility and gene-for-gene disease resistance Hoffman T, Schmidt JS, Zheng X, Bent AF. Plants commonly respond to pathogen infection by increasing ethylene production, but it is not clear if this ethylene does more to promote disease susceptibility or disease resistance . Ethylene production and/or responsiveness can be altered by genetic manipulation . The present study used mutagenesis to identify soybean (Glycine max L . Merr.) lines with reduced sensitivity to ethylene . Two new genetic loci were identified, Etr1 and Etr2 . Mutants were compared with isogenic wild-type parents for their response to different soybean pathogens . Plant lines with reduced ethylene sensitivity developed similar or less-severe disease symptoms in response to virulent Pseudomonas syringae pv glycinea and Phytophthora sojae, but some of the mutants developed similar or more-severe symptoms in response to Septoria glycines and Rhizoctonia solani . Gene-for-gene resistance against P . syringae expressing avrRpt2 remained effective, but Rps1-k-mediated resistance against P . sojae races 4 and 7 was disrupted in the strong ethylene-insensitive etr1-1 mutant . Rps1-k-mediated resistance against P . sojae race 1 remained effective, suggesting that the Rps1-k locus may encode more than one gene for disease resistance . Overall, our results suggest that reduced ethylene sensitivity can be beneficial against some pathogens but deleterious to resistance against other pathogens. FEBS Lett, 1999 Feb 19, 445(1), 183 - 8 Bacterial formate dehydrogenase . Increasing the enzyme thermal stability by hydrophobization of alpha-helices; Rojkova AM et al.; NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied . To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein alpha-helices . Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101 . Residues Ser-131 and Ser-160 were replaced by Ala, Val and Leu . Residues Ser-168, Ser-184 and Ser-228 were changed into Ala . Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability . Mutant S168A was 1.7 times less stable than the wild-type FDH . Double mutants S(131,160)A and S(184,228)A and the four-point mutant S(131,160,184,228)A were also prepared and studied . All FDH mutants with a positive stabilization effect had the same kinetic parameters as wild-type enzyme . Depending on the position of the replaced residue, the single point mutation Ser/Ala increased the FDH stability by 5-24% . Combination of mutations shows near additive effect of each mutation to the total FDH stabilization . Four-point mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild-type enzyme. Plant J, 1999 Jan, 17(1), 41 - 50 Plants expressing the Pto disease resistance gene confer resistance to recombinant PVX containing the avirulence gene AvrPto; Tobias CM et al.; Elicitation of hypersensitive cell death and induction of plant disease resistance by Pseudomonas syringae pv . tomato (Pst) is dependent on activity of the Pst Hrp secretion system and the gene-for-gene interaction between the tomato resistance gene Pto and the bacterial avirulence gene avrPto . AvrPto was expressed transiently in resistant or susceptible plant lines via a potato virus X (PVX) vector . We found that while PVX is normally virulent on tomato, a PVX derivative expressing avrPto was only capable of infecting plants lacking a functional Pto resistance pathway . Mutations in either the Pto or Prf genes allowed systemic spread of the recombinant virus . These results indicate that recognition of AvrPto by Pto in resistant plant lines triggers a plant defense response that can confer resistance to a viral as well as a bacterial pathogen. Mol Genet Metab, 1999 Feb, 66(2), 122 - 7 Sequence comparisons reveal two classes of 3-hydroxy-3-methylglutaryl coenzyme A reductase; Bochar DA et al.; Both in eukaryotes and in archaebacteria the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (E.C . 1.1 . 1.34) is known to catalyze an early reaction unique to isoprenoid biosynthesis . In humans, the HMG-CoA reductase reaction is rate-limiting for the biosynthesis of cholesterol and therefore constitutes a prime target of drugs that reduce serum cholesterol levels . Recent advances in genome sequencing that permitted comparison of 50 HMG-CoA reductase sequences has revealed two previously unsuspected classes of this enzyme . Based on sequence and phylogenetic considerations, we propose the catalytic domain of the human enzyme and the enzyme from Pseudomonas mevalonii as the canonical sequences for Class I and Class II HMG-CoA reductases, respectively . These sequence comparisons have revealed, in addition, that certain true bacteria, including several human pathogens, probably synthesize isoprenoids by reactions analogous to those of eukaryotes and that there therefore exist two distinct pathways for isoprenoid biogenesis in true bacteria . J Urol, 1999 Mar, 161(3), 984 - 9 Recombinant oncotoxin AR209 (anti-P185erbB-2) diminishes human prostate carcinoma xenografts; Skrepnik N et al.; PURPOSE: Prostate cancer is the most common malignancy of males in the United States . Although the overall survival rate for early stage prostate cancer is good, if cancer recurs following curative therapies there is no adequate salvage therapy . Systemic chemotherapy has never been associated with any meaningful improvement in overall survival or overall objective benefit . There is a need to develop novel therapies for prostate cancer . MATERIALS AND METHODS: Two prostatic cancer cell lines, DU-145 and PC-3, were grown as subcutaneous xenografts in athymic nude mice . The recombinant oncotoxin AR209, formerly OLX-209 {e23(Fv)PE38KDEL}), has the specificity of an anti-p185erbB-2 antibody contained within a single-chain antibody domain (e23Fv) coupled to a portion of the Pseudomonas exotoxin A (PE38KDEL) . Using Western blot analysis, the cell lines were shown to express p185erbB-2 . The mice received either 3 i.v . injections, one every 2 days, of the recombinant oncotoxin AR209 or PBS, or were implanted with osmotic pumps that delivered a constant s.c . amount of AR209 or PBS . RESULTS: The oncotoxin was effective in reducing the size of s.c . prostatic xenografts in athymic nude mice . The data demonstrated that small tumors (<200