Protein Eng, 1994 Apr, 7(4), 559 - 62 Analysis of the structure of Pseudomonas glumae lipase; Noble ME et al.; The lipase produced by Pseudomonas glumae is monomeric in the crystalline state and has a serine protease-like catalytic triad; Ser87-His285-Asp263 . The largest domain of the protein resembles closely a subset of the frequently observed alpha/beta-hydrolase fold and contains a well-defined calcium site . This paper describes structural analysis of this protein, focusing on (i) structural comparison with the lipase from Geotrichum candidum, (ii) the probable nature of the conformational change involved in substrate binding and (iii) structural variations amongst the family of Pseudomonas lipases . This analysis reveals similarities between P . glumae lipase and G . candidum lipase involving secondary structural elements of the hydrolase core and the loops carrying the catalytic serine and histidine residues . A possible functional equivalence has also been identified between parts of the two molecules thought to be involved in a conformational change . In addition, determination of the structure of P . glumae lipase has allowed rationalization of previously reported protein engineering experiments, which succeeded in improving the stability of the enzyme with respect to proteolysis. Appl Environ Microbiol, 1994 Apr, 60(4), 1121 - 8 Analysis of competition in soil among 2,4-dichlorophenoxyacetic acid-degrading bacteria; Ka JO et al.; Competition among indigenous and inoculated 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was studied in a native Kansas prairie soil following 2,4-D additions . The soil was inoculated with four different 2,4-D-degrading strains at densities of 10(3) cells per g of soil; the organisms used were Pseudomonas cepacia DBO1(pJP4) and three Michigan soil isolates, strain 745, Sphingomonas paucimobilis 1443, and Pseudomonas pickettii 712 . Following 2,4-D additions, total soil DNA was extracted and analyzed on Southern blots by using a tfdA gene probe which detected three of the strains and another probe that detected the fourth strain, S . paucimobilis 1443, which belongs to a different class of 2,4-D degraders . P . cepacia DBO1(pJP4), a constructed strain, outcompeted the other added strains and the indigenous 2,4-D-degrading populations . The S . paucimobilis population was the secondary dominant population, and strain 745 and P . pickettii were not detected . Relative fitness coefficients determined in axenic broth cultures predicted the outcome of competition in soil for some but not all strains . Lag time was shown to be a principal determinant of competitiveness among the strains, but the lag times were significantly reduced in mixed broth cultures, which changed the competitive outcome . Plasmids containing the genes for the 2,4-D pathway were important determinants of competitiveness since plasmid pKA4 in P . cepacia DBO1 resulted in the slower growth characteristic of its original host, P . pickettii, rather than the rapid growth observed when this strain harbors pJP4. Neurosurgery, 1994 Apr, 34(4), 649 - 55; discussion 655-6 In vivo efficacy of intrathecal transferrin-Pseudomonas exotoxin A immunotoxin against LOX melanoma; Hall WA et al.; Neoplastic meningitis due to the dissemination of systemic cancer or primary central nervous system tumors through the cerebrospinal fluid carries a very poor prognosis . Current treatments for this disease are ineffective, and new therapeutic modalities such as immunotoxins may be beneficial . We created an animal model of human carcinomatous meningitis with LOX melanoma-derived tissue-culture cells in athymic rats for testing the efficacy of intrathecal therapy with transferrin-Pseudomonas exotoxin A (Tfn-PE) immunotoxin . An injection of 5 x 10(5) LOX cells into the intrathecal space through an indwelling catheter resulted in the reproducible development of lower-extremity paraplegia at 9.24 +/- 1.77 days because of focal deposits of tumor growth adjacent to the thoracic and lumbar spinal cord . A dose of 2.5 or 5 micrograms of intrathecal Tfn-PE immunotoxin was neurotoxic and resulted in the deaths of 8 of 10 animals within 24 hours . Histological evidence of central nervous system damage was seen as hemorrhagic degeneration around the central canal or a pathological cleft at the level of the cervical spinal cord . Because no neurotoxicity was seen with 1 microgram of intrathecal Tfn-PE immunotoxin, this dose was administered in treatment experiments . Twenty-four hours after the intrathecal instillation of LOX cells, 10 animals received intrathecally either 1 microgram of Tfn-PE or phosphate-buffered saline with 0.1% human serum albumin (control group).(ABSTRACT TRUNCATED AT 250 WORDS) Appl Environ Microbiol, 1994 Apr, 60(4), 1093 - 1100 Restriction fragment length polymorphism evidence for genetic homology within a pathovar of Pseudomonas syringae; Scholz BK et al.; Pseudomonas syringae pv . phaseolicola NPS3121 hrp sequences were used as hybridization probes in a restriction fragment length polymorphism (RFLP) analysis of 24 P . syringae pv . tabaci strains as a means to evaluate the genetic and taxonomic relationship of pathovars of P . syringae . Southern blot analyses of genomic restriction digests, with hrpA-S sequences as hybridization probes, and restriction analyses of PCR-amplified DNA of regions within hrpD were conducted . The resulting RFLP patterns were uniform for 23 of the 24 isolates tested, with strain BR2R having a unique pattern . BR2R is a pathogen of bean which was classified as pathovar tabaci because of its ability to produce tabtoxin, but unlike the other 23 tabaci strains in this study, it does not incite disease symptoms on tobacco . When a DNA fragment containing hrpM sequences was used as a hybridization probe, the tabaci isolates could be divided into three groups on the basis of the RFLP patterns : BR2R, Pt11528R and Pt113R, and the remaining strains . For all of the above analyses, BR2R shared identical RFLP patterns with P . syringae pv . phaseolicola NPS3121, also a bean pathogen which does not cause disease on tobacco . However, BR2R AND NPS3121 could be differentiated from each other on the basis of the RFLP patterns from restriction analysis of PCR-amplified DNA of argF, while the remaining tabaci strains had a third pattern . These studies indicate that hrp genes and argF are conserved in strains of P . syringae pathogenic to tobacco, suggesting that P . syringae strains pathogenic to specific hosts may have a high level of genetic similarity . We believe that these analyses have shown that distinct identifiable genetic differences may be correlated with host range and suggest that such information may be useful for assigning pathovar designations. Biosci Biotechnol Biochem, 1994 Apr, 58(4), 752 - 5 Purification and characterization of a carboxylesterase from Pseudomonas sp . KWI-56; Sugihara A et al.; An intracellular carboxylesterase from Pseudomonas sp . was overproduced in E . coli, and purified to homogeneity by a combination of hydrogen bond chromatography, gel filtration, and hydrophobic interaction chromatography . Gel filtration and SDS-PAGE suggested that the purified enzyme consisted of two subunits of molecular mass of 28 kDa . Its isoelectric point was 5.9 . The enzyme was thermolabile, and showed its maximum activity at 22 degrees C (pH 7.5) . Methyl propionate was hydrolyzed at the highest rate among the fatty acid methyl esters tested . PMSF, DFP, PCMB, and HgCl2 inhibited the enzyme markedly, suggesting that serine and/or cysteine is in or near the active site. J Clin Microbiol, 1994 Apr, 32(4), 924 - 30 Linkage analysis of geographic and clinical clusters in Pseudomonas cepacia infections by multilocus enzyme electrophoresis and ribotyping; Johnson WM et al.; Multilocus enzyme electrophoresis and ribotyping were used to characterize 83 strains of Pseudomonas cepacia, mostly isolated from cystic fibrosis (CF) patients, although a number of isolates from non-CF nosocomial infections and reference environmental strains were represented . Twenty enzyme electrophoretic types (ETs) were determined; of these, one clone (ET12) was associated with six of nine ribotypes (RTs) said to be geographically representative of the United Kingdom and all of the Ontario (Canada) isolates from CF patients . This clone was not associated with nosocomial infections or environmental strains and was never found in CF isolates from British Columbia or Nova Scotia, Canada, or a center in the eastern United States . Individual isolate EcoRI RT signatures did not cluster geographically as did the ET signatures by clonal analysis . Frequently RTs occurred in more than a single ET . Known point source focal nosocomial outbreaks were typified by single ETs and stable RTs . Dendrographic analysis of the strains grouped those strains from CF patients, nosocomial outbreaks, and environmental sources into separate ET families, and diversity analysis indicated that, with the exception of ET17, CF isolates clustered in unique and closely related ETs different from those from nosocomial and environmental sources . This study has also shown the potential of multilocus enzyme electrophoresis to monitor the intercontinental spread of P . cepacia strains in CF patients, and this may have a significant impact on plans for CF patient summer camps and design of infection control practices . Whether the intercontinental ET12 clone, which predominates in the United Kingdom and the province of Ontario, linked by summer camp acquisition, has increased virulence for CF patients remains to be established. Transplantation, 1994 Mar 27, 57(6), 823 - 6 Retransplantation in hepatitis B--a multicenter experience; Crippin J et al.; Hepatitis B has become one of the most challenging diseases in liver transplantation . Infection of the allograft with subsequent graft failure is common and may prompt consideration of repeat liver transplantation . The aims of this study were to examine the experience in the United States with retransplantation in hepatitis B patients with recurrent disease as well as for other reasons . Questionnaires were mailed to adult liver transplant centers in the United States, requesting information on retransplantation in HBV patients . Responses were received from 71% of the centers . Thirty-eight patients were retransplanted, 20 for recurrent HBV and 18 for other reasons . The survival rate following retransplantation for HBV was poor . Nine patients (55%) died within 60 days . Eleven patients survived 60 days or longer, though eight died at a mean of 4.1 +/- 2 months, one required a third transplant for recurrent HBV infection at 4 months, and one died at 35 months . Only a single (5%) long-term survivor exists . Recurrent histologic disease occurred earlier in the second transplant at 2.8 +/- 2.9 months versus 6.1 +/- 5.2 months in the first allograft, though this difference did not reach statistical significance (P = .058) . Patients transplanted for other reasons (primary non-function {9}, hepatic artery thrombosis {6}, persistent rejection {2}, and a Pseudomonas graft infection {1}) had a better survival rate . Four patients survived less than 60 days . Of the 14 surviving longer than 60 days, 11 patients are alive at a mean of 21.2 +/- 14.8 months . Retransplantation for recurrent HBV appears to be contraindicated due to a high mortality . Retransplantation in HBV patients with graft failure due to other causes, however, should be considered, since over 60% of these patients appear to have good long-term survival . Additional studies examining risk factors for recurrent disease should be considered. J Biol Chem, 1994 Mar 11, 269(10), 7610 - 6 Pseudomonas exotoxin A mutants . Replacement of surface exposed residues in domain II with cysteine residues that can be modified with polyethylene glycol in a site-specific manner; Kuan CT et al.; Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recognition of receptors on eukaryotic target cells, domain II promotes translocation of PE into the cytosol, and domain III enzymatically ADP-ribosylates elongation factor 2 . Modification of proteins with polyethylene glycol (PEG) has been shown to prolong circulating plasma lifetime and may reduce or eliminate immunogenicity . However, in the case of toxins, PEG may interfere with or block toxin activity . To investigate the effect of polyethylene glycolation on specific residues located on the surface of PE domain II, we substituted cysteine, for each of the five most exposed surface amino acids (H276, E282, N306, R313, and E327) in domain II . These cysteines can serve as unique sites for PEG modification . The PE-Cys proteins retained most of their cytotoxicity even when the free sulfhydryl group was blocked by 5,5'-dithiobis(nitrobenzoic acid) or glutathione . When the PE-Cys proteins were conjugated with ovalbumin using a cleavable disulfide linkage, cytotoxicity was retained, but it was lost with a non-cleavable thioether linkage . In contrast, cytotoxicity was maintained when PE-Cys mutants were coupled to 5- or 20-kDa mPEG, using either a disulfide or a thioether linkage . Unexpectedly in some cases, the thioether conjugate was more active than the disulfide linkage . Pharmacokinetic studies on one of the polyethylene-glycolated molecules (R313C) showed that the mean residence time (t 1/2) was prolonged to 72 min, compared to 20 min for unpolyethylene glycolated PE-Cys(R313C) . These studies show it is possible to derivatize PE at specific residues in domain II, maintain significant cytotoxic activity, and alter pharmacokinetics . These studies also suggest that large mPEG molecules can be translocated to the cytosol while still attached to domain II of PE. Biochemistry, 1994 Mar 8, 33(9), 2682 - 7 Mechanism of the reaction catalyzed by delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: kinetic properties of a modified enzyme in which tyrosine 14 is replaced by 3-fluorotyrosine; Brooks B et al.; Tyrosine 14 of delta 5-3-ketosteroid isomerase plays an important role in the function of the enzyme, since its replacement by phenylalanine results in a decrease in kcat by a factor of 10(-4.7) . This result and the fact that this residue resides in the enzyme's substrate binding site and is in close proximity to C-2 of the bound steroid suggests that it functions as an electrophile in the catalytic mechanism by protonation of or hydrogen bonding to the C-3 carbonyl oxygen of the substrate . In order to obtain more information about the role of tyrosine 14, we have prepared a modified form of the enzyme in which tyrosine 14 has been substantially replaced in vivo by exogenously supplied 3-fluorotyrosine, a tyrosine derivative in which the pKa' of the phenol hydroxyl should be decreased by about 1.5 log units . Site specificity of this modification has been ensured by mutation of the codons for the nonessential tyrosines 55 and 88 to phenylalanine . We find that replacement of tyrosine 14 by 3-fluorotyrosine in the Y55,88F modified form of the isomerase results in a 4-fold decrease in kcat . We interpret this result in terms of a mechanism in which the transition state for enolization is dienolate-like, characterized by relatively little proton transfer from tyrosine 14 in the transition state, and the intermediate in the overall reaction is dienol-like . An alternative mechanism in which the intermediate is stabilized by a short, strong hydrogen bond can also be consistent with the data. Biochemistry, 1994 Mar 8, 33(9), 2672 - 81 Extent of proton transfer in the transition states of the reaction catalyzed by the delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: site-specific replacement of the active site base, aspartate 38, by the weaker base alanine-3-sulfinate; Holman CM et al.; Previous studies of the mechanism of the steroid isomerase of Comamonas (Pseudomonas) testosteroni have identified aspartate 38 as the proton porter which transfers the substrate's 4 beta proton to the 6 beta position of the product . Consequently, aspartate 38 functions as a base in the deprotonation of the substrate to form a dienol or dienolate intermediate, which then undergoes reprotonation from protonated aspartate 38 at C-6 beta to give the product . We have tried to characterize the transition states for the proton transfers by altering the pKa' of aspartate 38 and then determining the effect of the alteration on the kinetics of the enzyme . Alteration of the pKa' was accomplished by replacement of the carboxyl carbon of aspartate 38 by sulfur, a change which converts the carboxylate group to the much less basic sulfinate group . Employing Bronsted catalysis theory as applied to the individual steps of the isomerase mechanism, we find that in the enolization step of the reaction proton transfer to aspartate 38 is well advanced in the transition state . In the subsequent ketonization step, proton transfer from aspartate 38 has barely started when that transition state is reached . A series of mutant KSIs with alternative bases at position 38 have been constructed using a combination of site-directed mutagenesis and chemical modification: Asp-38 to Glu (D38E), His (D38H), and S-(carboxymethyl)cysteine (D38CMC) . While the D38H and D38E mutants both retain significant isomerase activity, D38CMC is essentially inert . From the results of kinetic experiments it is possible to get a qualitative idea of the sensitivity of the enzyme's catalytic ability to the location of the base responsible for proton transfer. Biochemistry, 1994 Mar 8, 33(9), 2620 - 7 Evidence for an equilibrium intermediate in the folding-unfolding pathway of a transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein; Gress JO et al.; TP40 is a chimeric protein containing transforming growth factor-alpha (TGF-alpha) at the N-terminus and a Cys-->Ala mutant (PE40 delta Cys) of a 40,000-dalton segment (PE40) of Pseudomonas exotoxin (PE) . The guanidine hydrochloride (Gdn-HCl)-induced unfolding of TP40 and PE40 delta Cys has been studied by tryptophan fluorescence, circular dichroism (CD), and high-performance size exclusion chromatography (HPSEC) . The equilibrium unfolding of both proteins involves at least one intermediate (I) . In the I state(s), which may be induced by 1.3-2.0 M Gdn-HCl, the tertiary structure is fully or partially collapsed as detected by tryptophan fluorescence and near-UV CD, but the protein largely retains the native secondary structure and a semicompact shape as judged by far-UV CD and HPSEC, respectively . Soluble aggregates of TP40 and PE40 delta Cys are observed in addition to monomers at these intermediate (but not at higher) Gdn-HCl concentrations, suggesting that self-association is possibly mediated by thermodynamically stable, partially unfolded I states . The kinetics of refolding of TP40 upon dilution of Gdn-HCl involve two or more phases . Re-formation of secondary structure occurs rapidly (t 1/2 < 10 s) as determined by CD and is followed by a biphasic refolding of the native tertiary structure as detected by changes in tryptophan fluorescence . The midpoint (Tm) of the thermal unfolding transition occurs at a lower temperature when measured by tryptophan fluorescence than when detected by DSC and CD . These data suggest that Gdn-HCl and temperature can induce conformation(s) of TP40 that are distinct from native (N) and unfolded (U) states.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1994 Mar 4, 236(4), 1169 - 85 Crystal structure and refinement of cytochrome P450terp at 2.3 A resolution; Hasemann CA et al.; Cytochrome P450terp is a class I (mitochondrial/bacterial) P450 that catalyzes the hydroxylation of alpha-terpineol as part of the catabolic assimilation of this compound by a pseudomonad species . Crystals grown from the purified protein have the symmetry of space group P6(1)22, and cell dimensions a = b = 69.4 A, c = 456.6 A, alpha = beta = 90 degrees, gamma = 120 degrees . Diffraction data were collected at the Cornell High Energy Synchrotron Source, and the structure of P450terp was solved by a combination of molecular replacement and multiple isomorphous replacement techniques . A model of P450terp was built and refined against native data, to an R-factor of 18.9% for data with I > or = sigma(I) between 6.0 A and 2.3 A resolution . This model contains 412 of the 428 P450terp amino acid residues; the loop between helices F and G is disordered in the crystal . While the overall fold of P450terp is very similar to that of P450cam, only three-quarters of the C alpha positions can be superimposed, to a root-mean-square deviation of only 1.87 A . The mode of substrate binding by P450terp can be predicted, and probable substrate contact residues identified . The heme environment and side-chain positions in the adjacent I-helix suggest possible modes of proton delivery in the catalytic cycle of the enzyme. Appl Environ Microbiol, 1994 Mar, 60(3), 966 - 72 Comparative biochemical and genetic analysis of naphthalene degradation among Pseudomonas stutzeri strains; Rossello-Mora RA et al.; Of a 49-strain collection of Pseudomonas stutzeri species, 11 isolates were able to degrade naphthalene and 1 isolate was able to use m- and p-toluate as sole carbon and energy sources . Of these 12 strains, 10 shared a highly homologous set of naphthalene catabolic genes, even though they belong to four different genomovars . These genes differed from those present in plasmid NAH7 . In only one of these degraders could a plasmid-encoded pathway be demonstrated, and a chromosome-encoded pathway is proposed for the remaining strains . meta cleavage of catechol was only observed in those strains able to metabolize alkyl derivatives of catechol. J Bacteriol, 1994 Mar, 176(6), 1689 - 94 Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp . strain KKS102; Kikuchi Y et al.; The nucleotide sequence of the downstream region of the bph operon from Pseudomonas sp . strain KKS102 was determined . Two open reading frames (ORF1 and ORF2) were found in this region, and the deduced amino acid sequence of ORF2 showed homology with the sequences of four ferredoxin reductases of dioxygenase systems . When this region was inserted just upstream of the bph operon, which does not contain a gene encoding ferredoxin reductase, biphenyl dioxygenase activity was detected . The 24- and 44-kDa polypeptides predicted from the two open reading frames were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Crude extract which contained the products of ORF2 and bphA1A2A3 showed cytochrome c reduction activity . These data clearly suggest that ORF2 encodes ferredoxin reductase . The deduced amino acid sequence of ORF1 does not show significant homology with the sequences of any other proteins in the SWISS-PROT data bank, and the function of ORF1 is unknown. J Bacteriol, 1994 Mar, 176(5), 1374 - 82 A mutation in the indole-3-acetic acid biosynthesis pathway of Pseudomonas syringae pv . syringae affects growth in Phaseolus vulgaris and syringomycin production; Mazzola M et al.; Homologs of the genes for indole-3-acetic acid (IAA) biosynthesis from Pseudomonas syringae pv . savastanoi were retrieved from a genomic library of P . syringae pv . syringae, and their nucleotide sequences were determined . Sequence relatedness between the P . syringae pv . syringae and P . syringae pv . savastanoi iaa operons is greater than 90% within the iaaM and iaaH loci but declines dramatically at a position approximately 200 bp 5' of the iaaM translation initiation codon . A third open reading frame was detected downstream of iaaH . Production of IAA was undetectable in mutant strain Y30-53.29, which was generated by transposition of Tn5 into the iaaM gene of P . syringae pv . syringae Y30 . The IAA-deficient (IAA-) mutant retained the ability to colonize the bean phylloplane and induced disease symptoms on bean which were similar to those produced by the parental strain . However, the population dynamics of the IAA- strain during the parasitic phase in leaves differed from those of both the parental strain and the mutant genetically restored for IAA biosynthesis . The mutant was capable of inducing disease symptoms when established in bean tissues at a lower initial cell density than either IAA-producing strain . Syringomycin biosynthesis by the IAA- strain was diminished in comparison with the parental strain or the mutant genetically restored for IAA production . The results indicate that bacterially derived IAA, or its biosynthesis, is involved in the regulation of in planta growth and in the expression of other factors that affect the host-pathogen interaction. Infect Immun, 1994 Mar, 62(3), 1109 - 17 Sequence determination and mutational analysis of the lly locus of Legionella pneumophila; Wintermeyer E et al.; The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene . The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined . The lly loci exhibited identical nucleotide sequences . They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa . N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced . The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp . 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms . An Lly-negative mutant of L . pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed . The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis . Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence . Intracellular survival of L . pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus. Mol Biochem Parasitol, 1994 Mar, 64(1), 111 - 20 Molecular cloning of ras and rap genes from Entamoeba histolytica; Shen PS et al.; To better understand growth regulation in the protozoan parasite Entamoeba histolytica, ameba genes homologous to the ras oncogene and rap (Krev-1) anti-oncogene were cloned . Two putative ameba ras genes (Ehras1 and Ehras2) were identified, which contain 205 and 203 amino acid (aa) open reading frames (ORFs), respectively . The Ehras1 ORF shows an 91% positional identity with that of Ehras2, a 55% identity with Dictyostelium discoideum (Dd) ras, and a 47% identity with human (Hs) ras . Two ameba rap genes (Ehrap1 and Ehrap2) were identified, both of which contain 184-aa ORFs . The Ehrap1 ORF shows a 93% positional identity with that of Ehrap2, a 60% identity with Dd rap, a 61% identity with Hs Krev-1, and a 45% identity with that of Ehras1 . Conserved aa in each ameba ras and rap ORF include GTP-binding sites, effector site, site of ADP-ribosylation by Pseudomonas exoenzyme S, and COOH-terminus CAAX . As all Xs = Leu or Phe, ameba ras and rap proteins may be gerenylgerenylated and not farnesylated . Both ras and rap genes are transcribed by trophozoites . A single 21-kDa ameba ras protein reacts with the rat Y13-259 anti-ras monoclonal antibody, which is located on the cytosolic side of the plasma membrane . These are the first ras and rap genes identified from a protozoan parasite. Leuk Lymphoma, 1994 Mar, 13(1-2), 1 - 10 Recombinant single-chain immunotoxins against T and B cell leukemias; Kreitman RJ et al.; Interleukin 2 (IL2) receptors (IL2R's) are found on malignant cells in many human leukemias and lymphomas and are expressed by activated T cells in many autoimmune disorders . Anti-Tac(Fv), a single-chain protein composed of the variable heavy and light domains of the anti-IL2R monoclonal antibody anti-Tac, can be genetically fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to form potent immunotoxins . We have shown that anti-Tac(Fv) binds to low affinity IL2R's on fresh chronic lymphocytic leukemia (CLL) and adult T-cell leukemia (ATL) cells and can target either toxin to kill those cells . Anti-Tac(Fv)-PE40, containing the truncated form of PE without its binding domain, was cytotoxic to malignant cells from 8 of 8 ATL patients tested, with IC50's ranging from 0.11 to 5.5 ng/ml . Anti-Tac(Fv)-PE40KDEL, a derivative of anti-Tac(Fv)-PE40 which contains the KDEL carboxyl terminus, was more cytotoxic toward cells from all ATL patients and also killed CLL cells from 8 of 16 patients . DT388-anti-Tac(Fv), containing amino acids 1-388 of DT fused to the amino terminus of anti-Tac(Fv), was less cytotoxic than anti-Tac(Fv)-PE40 on ATL cells from 4 of 5 patients, but was cytotoxic toward CLL cells from 12 of 16 patients . DT388-IL2, where IL2 is substituted for anti-Tac(Fv), is similar to DAB389IL2, an IL2-toxin currently in clinical trials . DT388-IL2 and DAB389IL2 differ by only a few amino acids and have equal cytotoxic activity . DT388-IL2 was cytotoxic toward ATL cells from all patients tested, but usually required much higher concentrations than anti-Tac(Fv)-PE40 and was poorly active against CLL cells . Thus, recombinant toxins containing anti-Tac(Fv) are cytotoxic toward freshly isolated CLL and ATL cells and will be studied further as potential therapy for IL2R-related disorders. J Clin Pharmacol, 1994 Mar, 34(3), 255 - 9 Absolute bioavailability and absorption characteristics of aerosolized tobramycin in adults with cystic fibrosis; Cooney GF et al.; Administration of antibiotics by the inhalational route has become part of standard protocols for treatment of and prophylaxis for Pseudomonal pneumonias in patients with cystic fibrosis . For tobramycin, however, limited data are available on the aerosol absorption patterns, and no absolute bioavailability data for tobramycin exist . The purpose of this study was to measure the absolute bioavailability and systemic absorption characteristics of tobramycin when administered in high doses by a nebulizer . Multiple serum concentrations of tobramycin were measured after administration of an intravenous dose (mean, 2.9 mg/kg every 6 hours) and after an inhalational dose (5.6 mg/kg over 1 hour) . Inhalational doses were superimposed over the "tail" of a steady-state intravenous dose to improve the sensitivity of the assay procedure (Abbott-TDX) . Absolute bioavailability (F) was determined from AUC ratios normalized for dose . Model-independent pharmacokinetic parameters (volume of distribution {Vss} and total clearance {CLt}) were determined for each subject . Absorption characteristics (absorption rate constant {Ka} and mean absorption time {MAT}) were assessed after calculation of the cumulative fraction of drug absorbed, amount of bioavailable drug, and percent remaining to be absorbed per unit time using the Loo-Riegelman method . Three men and three women completed the study, and all received concurrent doses of ceftazidime . Mean absolute bioavailability (+/- standard deviation) was 9.13% (+/- 3.82), and the rate of absorption into the systemic circulation was consistent with a zero-order model profile for all subjects . Mean absorption time values reflected a wide degree of subject variability and ranged from approximately 15 to 150 minutes.(ABSTRACT TRUNCATED AT 250 WORDS) Microbiology, 1994 Mar, 140 ( Pt 3), 509 - 16 Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification; Pelletier I et al.; The gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer Streptomyces aureofaciens ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in Streptomyces lividans TK64 . The cloned bromoperoxidase was over-produced up to 2800-fold by the S . lividans TK64 transformant . By taking advantage of the over-production of BPO-A1 and the heat stability of the enzyme, a new and simple purification procedure was developed . Subcloning into the vector pIJ487 and screening of recombinants by a newly developed histochemical assay located the bpoA1 gene on a 2.1 kb BamHI-HindIII fragment . The nucleotide sequence of the 2.1 kb fragment was determined; the bpoA1 gene was identified within the sequence on the basis of the biased codon usage of Streptomyces genes and the presence of a nucleotide sequence encoding the N-terminal amino acid sequence obtained from the purified BPO-A1 . Comparison of the deduced primary structure of BPO-A1 with those deduced for the non-haem chloroperoxidase CPO-P from Pseudomonas pyrrocinia and the bromoperoxidase BPO-A2 from S . aureofaciens ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively. Microbiology, 1994 Mar, 140 ( Pt 3), 499 - 508 The lower pathway operon for benzoate catabolism in biphenyl-utilizing Pseudomonas sp . strain IC and the nucleotide sequence of the bphE gene for catechol 2,3-dioxygenase; Carrington B et al.; Pseudomonas sp . strain IC is able to grow on biphenyl, 3-methylbiphenyl and 4-methylbiphenyl . These are converted to benzoate and the corresponding methylbenzoates . The lower pathway genes for the catabolism of the benzoates were cloned on a 22 kb HindIII fragment . Hybridization with gene-specific probes from the meta pathways of other catabolic plasmids showed that the gene order was identical to that of the operons carrying the same function from TOL plasmids . The nucleotide sequence of a 1241 bp region carrying the whole of the bphE gene (for a catechol 2,3-dioxygenase) and the 5' end of the downstream bphG gene (for 2-hydroxy semialdehyde dehydrogenase) was determined . Both genes showed a high degree of homology with genes encoding isofunctional proteins from other Pseudomonas strains . The upper pathway genes for the conversion of biphenyl to benzoate have also been cloned but no linkage with the lower pathway operon has been detected . Pseudomonas strain IC contains a large plasmid pWW110 (> 200 kb) and there are indications that this plasmid carries the bph genes. Southeast Asian J Trop Med Public Health, 1994 Mar, 25(1), 144 - 51 Effects of tunicamycin on the pH-activity pattern of acid phosphatase in Pseudomonas pseudomallei; Kondo E et al.; The liquid culture of Pseudomonas pseudomallei shows a complex feature in in the pH-activity pattern of acid phosphatase, not a single peak curve . There was an evident tendency that the higher activity shifted to the higher pH range with the growth of culture . The culture in the presence of tunicamycin (20 micrograms/ml) showed a decreased activity selectively in the higher pH range, while the activity in the lower pH was more heat-labile . The bacterial cells grown on agar plates containing tunicamycin were more heat-labile than the untreated control cells . The glucosidase-treatment reduced the enzymatic activity (of the phosphatase-active fractions from the living cells) with the shift of the optimum pH to lower pH . These observations together with some collateral findings suggest that the pH-activity pattern of acid phosphatase in P . pseudomallei is associated with the development of precursor enzyme proteins to mature glycoproteins. Plasmid, 1994 Mar, 31(2), 138 - 47 Characteristics of IS401, a new member of the IS3 family implicated in plasmid rearrangements in Pseudomonas cepacia; Byrne AM et al.; We have determined the nucleotide sequence of IS401, an insertion sequence implicated in rearrangements of a 170-kb cryptic plasmid from Pseudomonas cepacia . Our analysis focused on a 4066-bp plasmid fragment containing adjacent copies of IS401 and of IS408, an element reported previously to activate gene expression in P . cepacia . One objective was to determine if an apparent increase in the copy number of IS401 in strains carrying adjacent plasmid copies of these two elements might be due to readthrough transcription of an IS401 transposase gene from an outwardly directed promoter within IS408 . This possibility was ruled out by nucleotide sequence analysis of the 4066-bp plasmid fragment, which indicated that the major open reading frames of IS401 were oriented in the direction of IS408 . IS401 was 1316 bp in length and had 26-bp terminal inverted repeats flanked by 3-bp direct duplications of adjacent DNA . It was closely related to the IS3 family elements IS51 from P . savastanoi and IS3411 from Escherichia coli . Pertinent features of IS408 are also discussed. Chest, 1994 Mar, 105(3), 837 - 41 Single lung transplantation in patients with systemic disease; Levine SM et al.; OBJECTIVE: To report functional results and survival in patients undergoing single lung transplantation (SLT) for pulmonary involvement associated with systemic disease or prior malignancy, criteria traditionally considered contraindications to SLT . DESIGN: Case series . SETTING: The University of Texas Health Science Center at San Antonio . PATIENTS: Nine patients who have undergone SLT for end-stage lung disease: four patients with sarcoidosis; two patients with limited scleroderma; and three patients with prior malignancies (two with prior lymphoma and bleomycin-induced pulmonary fibrosis and one who received two bone marrow transplants for acute lymphocytic leukemia and subsequently developed chemotherapy-induced pulmonary fibrosis) . MEASUREMENTS: Pulmonary function testing, exercise oximetry, quantitative ventilation-perfusion lung scanning . Actuarial survival . RESULTS: All patients had marked improvement in pulmonary function, exercise oximetry, and quantitative ventilation perfusion to the SLT . One patient with scleroderma died 90 days postoperatively from Pseudomonas pneumonia with a sepsis syndrome . One patient with sarcoidosis died 150 days postoperatively from disseminated aspergillosis . At autopsy, there was no evidence of recurrent fibrosis or sarcoidosis in the transplanted lungs in either of these two patients . The seven surviving patients have returned to work or school and are conducting all activities of daily living without pulmonary disability . The 1- and 2-year actuarial survival rates in these nine patients is 68.6 percent as compared with the 1- and 2-year actuarial survival rates of 66.3 percent and 55.8 percent in the remainder of our SLT group as a whole (n = 49) . Despite pharmacologic immunosuppression, there is no evidence of recurrent malignancy in the 3 patients with prior malignancies . CONCLUSIONS: We conclude that carefully selected patients with end-stage lung involvement related to systemic disease or chemotherapy-induced fibrosis may benefit from SLT. Biochem Biophys Res Commun, 1994 Feb 28, 199(1), 41 - 5 Alcoholysis of epsilon-decalactone with polyethylene glycol-modified lipase in 1,1,1-trichloroethane; Furukawa M et al.; Lipase from Pseudomonas cepacia was modified with 2,4-bis{O-methoxypoly(ethylene glycol)}-6-chloro-s-triazine, activated PEG2, to form PEG-lipase . The PEG-lipase is soluble and active in organic solvents . It catalyzes alcoholysis of racemic epsilon-decalactone with ethanol in 1,1,1-trichloroethane to form (R)-hydroxydecanoic acid ethyl ester . No alcoholysis of (S)-decalactone takes place . These results were discussed in relation to carbon number of n-alcohol, optimum temperature and comparison with modified and non-modified lipases. J Mol Biol, 1994 Feb 25, 236(3), 759 - 85 High resolution structures of holo and apo formate dehydrogenase; Lamzin VS et al.; Three-dimensional crystal structures of holo (ternary complex enzyme-NAD-azide) and apo NAD-dependent dimeric formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp . 101 have been refined to R factors of 11.7% and 14.8% at 2.05 and 1.80 A resolution, respectively . The estimated root-mean-square error in atomic co-ordinates is 0.11 A for holo and 0.18 A for apo . X-ray data were collected from single crystals using an imaging plate scanner and synchrotron radiation . In both crystal forms there is a dimer in the asymmetric unit . Both structures show essentially 2-fold molecular symmetry . NAD binding causes movement of the catalytic domain and ordering of the C terminus, where a new helix appears . This completes formation of the enzyme active centre in holo FDH . NAD is bound in the cleft separating the domains and mainly interacts with residues from the co-enzyme binding domain . In apo FDH these residues are held in essentially the same conformation by water molecules occupying the NAD binding region . An azide molecule is located near the point of catalysis, the C4 atom of the nicotinamide moiety of NAD, and overlaps with the proposed formate binding site . There is an extensive channel running from the active site to the protein surface and this is supposed to be used by substrate to reach the active centre after NAD has already bound . The structure of the active site and a hypothetical catalytic mechanism are discussed . Sequence homology of FDH with other NAD-dependent formate dehydrogenases and some D-specific dehydrogenases is discussed on the basis of the FDH three-dimensional structure. J Biol Chem, 1994 Feb 18, 269(7), 5346 - 57 The substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase . Polar probes of the enzyme's myristoyl-CoA recognition site; Lu T et al.; Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is a monomeric enzyme that is essential for vegetative growth . Nmt1p catalyzes the co-translational transfer of myristate from CoA to the amino-terminal Gly of cellular proteins in an ordered Bi Bi reaction mechanism that initially involves binding of myristoyl-CoA to the apoenzyme . Forty one fatty acid analogs were synthesized to define features in the acyl chain of myristoyl-CoA which are important determinants of its recognition by Nmt1p's acyl-CoA binding site as well as to help us deduce the structure of the binding site itself . These analogs included dicarboxylic acids, omega-nitrocarboxylic acids, analogs equivalent in length to C13:0-C15:0 which contain electronegative halogens at their omega-termini, hydroxytetradecanoic acids with hydrogen replaced by OH from C3 to C13, and azidophenyl-containing fatty acids with the linear azide unit attached either meta or para to phenyl and with variations in the length of their methylene chains . These compounds were converted to their CoA derivatives using Pseudomonas acyl-CoA synthetase and then surveyed as substrates for purified Nmt1p in an in vitro assay system that included an octapeptide derived from residues 1-8 of the human immunodeficiency virus Pr55gag polyprotein precursor . The results suggest that the myristoyl-CoA binding site contains a conical-shaped "receptor" that interacts with the omega-terminus of the bound acyl chain of acyl-CoAs . The acuteness of this cone determines the enzyme's capacity to accommodate steric bulk at the omega-terminus as well as Nmt1p's sensitivity to the distance between the eclipsed C5-C6 bond of a bound acyl chain and its omega-terminus . The activity profile of the various analog-CoAs also indicates that the enzyme's myristoyl-CoA binding site can accommodate fatty acid analogs with marked increases in polarity at their omega-terminus (compared to C14:0) as long as their chain length is equivalent to that of myristate. Cancer Res, 1994 Feb 15, 54(4), 1059 - 64 Cytotoxicity of recombinant Fab and Fv immunotoxins on adult T-cell leukemia lymph node and blood cells in the presence of soluble interleukin-2 receptor; Saito T et al.; Single-chain immunotoxins anti-Tac(Fv)-PE40 and anti-Tac(Fv)-PE40KDEL, composed of variable domains of the anti-Tac monoclonal antibody and truncated forms of Pseudomonas exotoxin, have shown potent cytotoxic activity against malignant peripheral blood mononuclear cells (PBMCs) from adult T-cell leukemia (ATL) patients originating from the Caribbean . However, several clinically important issues have not previously been addressed . These include the potential of soluble interleukin 2 receptor in ATL patients to block immunotoxin effectiveness, the relative sensitivity of malignant lymph node cells (LNCs) versus PBMCs, the effect of an immunotoxin with a prolonged half-life, and finally whether ATL cells from patients in Japan have toxin sensitivity equal to those of the Caribbean patients . To resolve these questions, we studied 32 malignant PBMC and LNC samples from 30 ATL patients from Japan . PBMCs from 27 of 27 patients were very sensitive with 50% inhibition of protein synthesis achieved with 0.02-0.85 ng/ml (0.3-13 pM) of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fv)-PE40 . LNCs had sensitivity very similar to that of PBMCs in the five patients tested . The fully recombinant immunotoxin, anti-Tac(Fab)-PE40, which has 8-10 times the t1/2 alpha and beta compared to the Fv-immunotoxins, was also very cytotoxic toward cells from 27 of 27 patients tested with 50% inhibition of protein synthesis of 0.08-25 ng/ml . It was found that purified soluble interleukin 2 receptor added to the cytotoxicity assay decreased the cytotoxic activity of anti-Tac(Fv)-PE40KDEL or anti-Tac(Fab)-PE40, but that 1 x 10(4) units/ml or less had minimal competitive effects . It was found that ATL patients who have responded even incompletely to conventional chemotherapy have soluble interleukin 2 receptor levels lower than this at posttreatment . We conclude that recombinant immunotoxins containing anti-Tac(Fv) are effective against Japanese ATL PBMCs or LNCs and might be most effective if used in vivo after conventional chemotherapy . If it is found in humans that the effectiveness of single-chain recombinant toxins is limited by short half-life, anti-Tac(Fab)-PE40 should be considered as an alternative agent. Cancer Res, 1994 Feb 15, 54(4), 1008 - 15 Transforming growth factor-alpha-Pseudomonas exotoxin fusion protein (TGF-alpha-PE38) treatment of subcutaneous and intracranial human glioma and medulloblastoma xenografts in athymic mice; Phillips PC et al.; Epidermal growth factor receptor (EGFR) is amplified or overexpressed in many malignant gliomas and other primary brain tumors but is low or undetectable in normal brain . In the present study, this differential expression has been exploited for targeted brain tumor therapy using a TGF-alpha-Pseudomonas exotoxin recombinant toxin, TGF-alpha-PE38 . In vitro experiments demonstrate that the cytotoxicity of this fusion protein is primarily determined by tumor EGFR expression and that TGF-alpha-PE38 cytotoxicity is abolished by pretreatment with excess epidermal growth factor . Treatment with i.p . TGF-alpha-PE38 in nude mice bearing glioblastoma or medulloblastoma s.c . xenografts produced tumor regression and growth delay . For intracranial xenograft implants treated with i.p . TGF-alpha-PE38, significant increases in median survival were noted only for tumors with the highest EGFR expression . However, intracranial tumors treated with a single intratumoral injection of TGF-alpha-PE38 showed increased survival in all xenografts tested . These results indicate that TGF-alpha-PE38 is active against primary human brain tumors ranging from moderate to high EGFR expression . For intracranial tumors, however, the higher survival rates produced by intracranial injection of TGF-alpha-PE38 than by continuous i.p . administration suggest that increased drug clearance or impaired drug delivery reduces the efficacy of systemic TGF-alpha-PE38 . Direct delivery of TGF-alpha-PE38 into brain tumors by controlled-release biodegradable polymers or intratumoral implanted catheters, or intrathecal administration into the colony stimulating factor of patients with leptomeningeal metastasis, may represent clinically useful applications of recombinant toxin therapy in tumors with high EGFR expression. J Mol Biol, 1994 Feb 11, 236(1), 377 - 8 Crystallization of catechol-1,2 dioxygenase from Pseudomonas arvilla C-1; Earhart CA et al.; The metalloenzyme catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 consists of three isozymes formed by combinations of two non-identical subunits; alpha alpha, alpha beta and beta beta; with molecular masses of 59,000, 63,000 and 67,000 Da, respectively . The alpha alpha isozyme crystallizes in the orthorhombic space group C222(1) with unit cell dimensions a = 62.7 A, b = 71.5 A, c = 187.1 A . The rectangular plates diffract to 2.6 A resolution . This is the first dioxygenase to be crystallized that uses catechol as a substrate . Comparison of the structure of this enzyme with protocatechuate 3,4-dioxygenase will provide basic information about the mechanisms of subunit association, substrate selectivity, and the origins of metabolic diversity in enzymes. Am J Pediatr Hematol Oncol, 1994 Feb, 16(1), 22 - 6 Bone marrow transplantation for sickle cell disease . The United States experience; Johnson FL et al.; PURPOSE: As of June 1992, five patients with sickle cell disease had been treated by matched sibling bone marrow transplantation in the United States . PATIENTS AND METHODS: Three patients underwent transplantations for complications related to sickle cell disease, two with previous cerebrovascular accidents (CVAs) and one who had had multiple severe vasoocclusive crises . Two patients had other indications for allogeneic bone marrow transplantation: one had acute myeloid leukemia and the other had Morquio's disease . The patients' ages ranged from 3 to 10 years, and four were girls . Ages of the donors ranged from 4 to 13 years; four of the donors were boys and three carried the sickle cell trait . For four patients, the preparative regimen consisted of busulfan and cyclophosphamide given either alone or combined with antithymocyte globulin (ATG) . The patient with leukemia was prepared with cyclophosphamide and total body irradiation (TBI) . The regimens for prophylaxis of graft-versus-host disease (GVHD) included various combinations of cyclosporine A, methotrexate, and prednisone . RESULTS: The patient with Morquio's disease failed to engraft but underwent a successful retransplantation from the same donor . All patients eventually demonstrated donor engraftment and the donor's hemoglobin electrophoretic pattern posttransplant . Two patients had moderately severe GVHD of the skin and gastrointestinal tract, which resolved with prednisone therapy . One of these patients developed transient chronic GVHD involving the skin . Other acute complications included mild venoocclusive disease of the liver, central line infection with bacteremias, uterine hemorrhage in one patient, and pseudomonas sepsis in another . CONCLUSIONS: Both patients who underwent transplantation after CVAs have experienced subsequent neurological events . However, with a median follow-up of 16 months (range 8 months to 9.3 years), all patients are surviving in good to excellent clinical condition and appear to have benefitted from treatment by bone marrow transplantation. Am Fam Physician, 1994 Feb 1, 49(2), 427 - 31 Malignant external otitis: a case report and review; Evans P et al.; Malignant external otitis is an unusual but serious and potentially fatal condition that has only recently been described . It is an invasive pseudomonal infection of the external auditory canal and deep periauricular tissues that characteristically involves the bone and adjacent cartilaginous structures, and it may lead to osteomyelitis of the base of the skull . It typically occurs in elderly diabetic patients . Malignant external otitis can cause severe pain, necrosis of the external auditory canal and progressive palsies of the facial and cranial nerves . Treatment consists of debridement of external auditory canal granulation tissue and long-term therapy with an antipseudomonal cephalosporin or an antipseudomonal penicillin plus an aminoglycoside. Mol Microbiol, 1994 Feb, 11(3), 489 - 500 VsrA, a second two-component sensor regulating virulence genes of Pseudomonas solanacearum; Schell MA et al.; The wilt-inducing phytopathogen Pseudomonas solanacearum produces several extracellular virulence factors, both polysaccharides (EPS I) and proteins (EXPs), which are independently regulated by a LysR-type transcriptional regulator, PhcA, and a histidine kinase sensor, VsrB . Here we characterize a third locus, vsrA, which is also required for normal production of EPS I, some EXPs and wilt disease . Analysis of eps::lacZ reporters in vsrA mutants showed that, like vsrB and phcA, vsrA is required for maximal expression (transcription) of eps, which contains some of the genes necessary for production of EPS I . Unlike vsrB and phcA mutants, however, eps transcription (and EPS I production) by vsrA mutants varies from 3 to 17% of wild-type levels, depending on growth conditions . Inactivation of vsrA also causes a dramatic reduction in production of three species of EXPs (28 kDa, 48 kDa, and 66 kDa), and an apparent increase in production of a few other EXPs . Unlike most other EPS-deficient P . solanacearum strains, vsrA mutants caused almost no disease symptoms when 10(4) cells were stem-inoculated into tomato plants . This correlated with a greater than 10-fold reduction in their ability to grow in planta . vsrA was cloned from a P . solanacearum genomic library by complementation of the vsrA mutant and was further subcloned on a 2.3 kb DNA fragment . PhoA fusion analysis and subcellular localization of the vsrA gene product in Escherichia coli maxicells suggest that it is a 53 kDa membrane-associated protein . Analysis of the nucleotide sequence of vsrA revealed a 502 residue open reading frame with homology to the histidine kinase domain of sensors in the two-component regulator family . This discovery shows that EPS I production by P . solanacearum is simultaneously controlled by dual two-component sensors. Arch Biochem Biophys, 1994 Feb 1, 308(2), 400 - 6 New subunit in L-phenylalanine oxidase from Pseudomonas sp . P-501 and the primary structure; Mukouyama EB et al.; L-phenylalanine oxidase from Pseudomonas sp . P-501 was shown by isoelectric focusing and HPLC experiments to consist of two kinds of nonidentical subunits . The newly identified subunit is designated as alpha, and the larger subunit, which has been reported previously, as beta . The apparent molecular weight of alpha subunit was estimated to be 8200 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . From the molecular mass of each subunits and their contents in the enzyme, the enzyme was shown to be composed to two alpha and two beta subunits . The amino acid sequence analysis of alpha subunit showed that the subunit consists of 92 amino acid residues and contains considerably hydrophobic arrangements and the candidate for the common sequence characteristic of the AMP binding in the FAD binding domain. J Bacteriol, 1994 Feb, 176(4), 1025 - 36 Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv . syringae Pss61 hrp and hrmA genes; Xiao Y et al.; The Pseudomonas syringae hrp and hrmA genes controlling pathogenicity and elicitation of the hypersensitive response and the avr genes controlling host range have been shown previously to be regulated by carbon, nitrogen, pH, osmolarity, and hypothetical plant factors . In P . syringae pv . syringae Pss61, inactivation of hrp complementation groups II and XIII reduced expression of a plasmid-borne hrmA'-lacZ fusion . The hrp regions II and XIII were cloned on separate plasmids and shown to enhance the activity of the hrmA promoter in Escherichia coli MC4100 transformants at least 100-fold . The nucleotide sequence of region XIII revealed two open reading frames (hrpR and hrpS) whose deduced products share homology with P . syringae pv . phaseolicola NPS3121 HrpS and are both related to the NtrC family of two-component signal transduction systems . HrpR and HrpS differ from most members of the protein family by lacking an amino-terminal domain which modulates the regulatory activity . A single open reading frame, hrpL, whose product shares homology with AlgU, a putative alternate sigma factor of P . aeruginosa, as well as with the related alternate sigma factors was identified within region II . Key domains are partially conserved . Inactivation of hrpS in Pss61 repressed expression of a plasmid-borne hrpL'-lacZ fusion carried by pYXPL1R, and transformation of MC4100(pYXPL1R) with a plasmid carrying hrpRS increased hrpL promoter activity at least 200-fold . Neither hrpS nor hrpR, when cloned on separate plasmids, activated the hrpL promoter activity individually . The expression of hrpL when directed by a lac promoter was sufficient to express a set of plasmid-borne hrmA'-, hrpJ'-, and hrpZ'-lacZ fusions independently of other hrp genes . The results indicate that hrpRS and hrpL are part of a regulatory cascade in which HrpR and HrpS activate expression of hrpL and HrpL, a putative sigma factor, induces expression of HrpL-responsive genes. Res Microbiol, 1994 Feb, 145(2), 121 - 7 Cloning and expression of the mercury resistance genes of marine Pseudomonas sp . strain MR1 plasmid pMR1 in Escherichia coli; Rani DB et al.; The mercury resistance determinant of marine Pseudomonas sp . strain MR1 plasmid pMR1 was cloned into a narrow-host-range vector pUC18 . A direct selection of mercury resistant clones was successful and 12 clones were evolved; 9 from direct selection on mercury agar plates and 3 from ampicillin-resistant white colonies . All the predicted clones efficiently volatilized mercury . One of the hybrid plasmids pMRD5, containing a 15.5-kb insert, conferred inducible resistance to both HgCl2 and phenyl mercury acetate with over a 40-fold increase in mer resistance in Escherichia coli HB101 . No DNA homology existed between the mer operon of Pseudomonas sp . strain MR1 and the characterized determinants of Tn501 mer DNA. Bull Acad Natl Med, 1994 Feb, 178(2), 249 - 57; discussion 258-62 {Therapeutic hopes in excretory azoospermia and genetic risks in congenital aplasia of the vas deferens}; Cognat M et al.; Therapeutical solutions now can be proposed to some excretory azoospermia, using in vitro-fertilization with epididymal sperm . The encouraging results obtained with this new approach should be analyzed with the genetic risk, sometimes encountered in the specific form of azoospermia due to the congenital absence of the vas deferens . This abnormality is to day supposed to represent a moderate form of cystic fibrosis (CF), corresponding to a genital phenotype of this disease . This suggestion has been firstly induced by similar anatomical findings in the male individuals presenting a classical form of CF . The hypothesis has mainly been confirmed by the real progress in the genetic analysis of this disease . So an abnormally high percentage of known mutations of CF has been demonstrated in the patients with congenital absence of vas deferens . Other arguments such as positive sweat chloride tests, high percentage of sinusitis or presence of anti-pseudomonas antibodies, reinforce this hypothesis . It is the reason why a clinical and biological check-up, prior to any decision of therapy for infertility, in this specific indication should be done in order to propose a genetic counselling to the couple. Mol Cell Probes, 1994 Feb, 8(1), 1 - 9 Construction of a specific DNA probe for diagnosis of melioidosis and use as an epidemiological marker of Pseudomonas pseudomallei; Sermswan RW et al.; Pseudomonas pseudomallei is a causative agent of melioidosis . The disease manifestations range from fulminant sepsis to asymptomatic seroconversion . In septicemic cases, a mortality rate of 80-90% is reported . Rapid and specific diagnosis has become important to the clinical microbiology laboratory . We have developed a P . pseudomallei-specific DNA probe . The cloned fragment, herein designated pKKU-S23L, contained 1.5 kb of P . pseudomallei chromosomal DNA . A radioactively labelled pKKU-S23L insert could detect 1.5 ng of its genomic DNA or 40,000 P . pseudomallei cells . The probe was highly specific for P . pseudomallei DNA and did not cross-hybridize with DNAs prepared from other related bacteria . Using pKKU-S23L as a probe in total cellular DNA digestions and Southern blot hybridization, we were able to classify 60 P . pseudomallei clinical isolates obtained from individual melioidosis patients into eight categories . Therefore, this probe has a potential not only for use in development of specific detection of bacterial DNA in clinical specimens but also for application in epidemiological studies of P . pseudomallei. J Appl Bacteriol, 1994 Feb, 76(2), 142 - 8 Physiological aspects of disinfection resistance in Pseudomonas cepacia; Pyle BH et al.; A Pseudomonas cepacia population was isolated which had reduced susceptibility to iodine and maintained resistance when subcultured several times in phosphate buffer . This population was also resistant to iodine after growth in a minimal medium containing glycerol but not glucose . Addition of cAMP to glucose-grown cells caused increased resistance to iodine . Iodine-resistant cultures also demonstrated reduced susceptibility to chlorination but not to heat or metals (Cu/Ag) . The results indicate that halogen resistance can be expressed in varying degrees, dependent on the carbon source, and cAMP may promote this expression . Thus, a catabolite repression-like mechanism may cause resistant cultures grown in some media to become more sensitive to halogens. Am J Physiol, 1994 Feb, 266(2 Pt 1), C360 - 6 Heterologous expression of delta F508 CFTR results in decreased sialylation of membrane glycoconjugates; Dosanjh A et al.; The cystic fibrosis transmembrane conductance regulator (CFTR) is commonly mutated in cystic fibrosis to the delta F508 CFTR . CFTR has been shown to function as a adenosine 3',5'-cyclic monophosphate-dependent Cl- channel at the cell surface, and there is evidence to suggest that CFTR may also have a role in transmembrane Cl- conductance in intracellular membrane compartments . Studies using cells from cystic fibrosis patients or heterologous expression systems have demonstrated that defective Cl- conductance at the cell surface and defective acidification of the Golgi compartment are associated with the presence of mutant forms of CFTR . It is possible that mutation of CFTR could also result in altered Golgi function, consistent with reports of changes in the glycosylation of cell surface and secreted glycoproteins in cystic fibrosis . Glycosylation of cell surface glycoproteins, particularly levels of sialylation, may also be related to the increased binding of Pseudomonas to cystic fibrosis cells . The current study was undertaken to compare the sialylation of cell surface glycoconjugates in heterologous cells overexpressing normal CFTR or delta F508 CFTR . The presence of sialylated residues on cells was assessed by the surface binding of the specific lectins, wheat germ agglutinin and elderberry bark lectin . A fluorescent cholera toxin B subunit probe was used to measure surface binding to sialylated gangliosides in transfected cells . Our studies show that cells lacking CFTR and cells expressing normal CFTR have unaltered levels of sialylation . In contrast, cells expressing the delta F508 CFTR have significantly decreased amounts of sialylated glycoproteins and gangliosides on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS) Gene, 1994 Jan 28, 138(1-2), 27 - 33 Construction of hybrid biphenyl (bph) and toluene (tod) genes for functional analysis of aromatic ring dioxygenases; Hirose J et al.; Multicomponent enzyme complexes of biphenyl (BP) dioxygenase (Dox) encoded by the gene cluster, bphA1A2A3A4 in Pseudomonas pseudoalcaligenes strain KF707 {Taira et al., J . Biol . Chem . 267 (1992) 4844-4858} and toluene Dox encoded by the gene cluster, todC1C2BA in P . putida strain F1 {Zylstra et al., J . Biol . Chem . 264 (1989) 14940-14946}, show high homologies (approx . 60%) for the corresponding subunit component in spite of the fact that they have discrete substrate specificities . We constructed hybrid gene clusters by replacing the gene component(s) between the large and small subunits of terminal Dox in the bph and tod gene clusters, and analyzed the function of a novel hybrid aromatic ring Dox . Escherichia coli cells expressing the hybrid gene clusters, todC1::bphA2A3A4, todC1C2::bphA3A4 and bphA1::todC2::bphA3A4, gained the ability to convert benzene-toluene and their derivatives to the dihydrodiols, indicating that the hybrid terminal Dox composed of TodC1::BphA2 and BphA1::TodC2 forms a functionally active multicomponent Dox associated with ferredoxin (Fer) (BphA3) and Fer reductase (BphA4) . Moreover, hybrid Dox (composed of TodC1::BphA2A3A4 and TodC1C2::BphA3A4) showed a wide substrate specificity rather similar to that of the wild-type toluene Dox (TodC1C2BA) . On the other hand, the hybrid Dox (BphA1::TodC2::BphA3A4) showed oxidative activities for the same compounds, but the rate of oxidation was dependent upon the substrate . These results suggest that (i) the two subunits of terminal Dox are critically involved in the substrate specificity for BP, benzene and their derivatives, and (ii) the electron transport proteins, Fer and Fer reductase, are exchangeable with one another between the BP Dox and toluene Dox complexes. Gene, 1994 Jan 28, 138(1-2), 119 - 21 Cloning of cmpE, a plasmid-borne catechol 2,3-dioxygenase-encoding gene from the aromatic- and chloroaromatic-degrading Pseudomonas sp . HV3; Yrjala K et al.; Pseudomonas sp . strain HV3 degrades aromatics and chloroaromatics . It harbours a mega-plasmid, designated pSKY4, from which the gene cmpE, encoding a catechol 2,3-dioxygenase (C23O) catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde, was cloned and sequenced . The deduced amino acid (aa) sequence shows the highest homology, 52%, to the deduced aa sequences of xylE1 and dmpB . The deduced 307-aa sequence of cmpE contains the extradiol ring-cleavage signature in the same position as other 307-aa C23O-encoding genes. Proc Natl Acad Sci U S A, 1994 Jan 18, 91(2), 664 - 8 Tat-mediated delivery of heterologous proteins into cells; Fawell S et al.; The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture . To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to beta-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity . The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment . In mice, treatment with Tat-beta-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain . The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages . Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells. Blood, 1994 Jan 15, 83(2), 426 - 34 Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interleukin-2 receptor-expressing human carcinoma; Kreitman RJ et al.; Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin composed of the variable domains of the monoclonal antibody anti-Tac, which binds to the p55 subunit of the interleukin-2 receptor (IL-2R), and a truncated form of Pseudomonas exotoxin (PE), which does not bind to the PE receptor (Chaudhary et al, Nature 339:394, 1989) . Whereas its cytotoxic activity toward autoimmune and malignant target cells has been established, its efficacy in vivo remains unknown . To establish an animal model, we produced ATAC-4 cells by transfecting the gene encoding the low-affinity IL-2R (p55) into A431 epidermoid carcinoma cells . ATAC-4 cells contained low-affinity IL-2Rs (2 x 10(5)/cell) and formed tumors in nude mice . In tissue culture, protein synthesis in ATAC-4 cells was inhibited 50% (IC50) at 0.06 ng/mL (0.9 pmol/L) of anti-Tac(Fv)-PE40 . IC50s for the derivatives anti-Tac(Fv)-PE38, which is missing PE amino acids 365-380, and anti-Tac(Fv)-PE38KDEL, which contains the same deletion plus the KDEL carboxyl terminus, were 0.04 and 0.025 ng/mL, respectively . All the agents produced complete tumor regressions in ATAC-4 tumor-bearing mice and anti-Tac(Fv)-PE38KDEL had significant antitumor activity at 1% of the LD50 . The dose limiting toxicity of anti-Tac(Fv)-PE38KDEL was from hemorrhagic liver necrosis, which was observed at approximately 55% of the LD50. Carbohydr Res, 1994 Jan 3, 251, 251 - 67 New acyclic analogues of lipid A: synthesis of 4-phosphonoxybutyl and 3-phosphonoxypropyl glycosides of 2-amino-2-deoxy-D-glucose; Eustache J et al.; Several analogues of lipid A have been synthesized, in which the reducing monosaccharide moiety of the parent molecule has been replaced by an acyclic spacer . The new compounds show high endotoxic activity and are able to protect neutropenic mice against pseudomonas infection, two properties characteristic of LPS-like molecules. Life Sci, 1994, 54(7), 445 - 53 TGF alpha-PE40 inhibits non-small cell lung cancer growth; Draoui M et al.; The ability of a chimeric toxin containing transforming growth factor alpha (TGF alpha) and truncated Pseudomonas exotoxin A to inhibit NSCLC growth was investigated . TGF alpha-PE40 inhibited binding of 125I-EGF to NSCLC cell lines with an IC50 value of 0.5-3 micrograms/ml . Similarly, other forms of the fusion protein, TGF alpha-PE38 and TGF alpha-PE40Asp553, which have active TGF alpha binding domains, inhibited specific 125I-EGF binding to NSCLC cells with IC50 values of 0.1-2 and 0.05-05 microgram/ml respectively . TGF alpha-PE40 inhibited 35S-methionine uptake by NSCLC cells with an ED50 value of 1-30 ng/ml . TGF alpha-PE38, which has one of the two disulfide pairs of PE40, inhibited amino acid uptake with ED50 values of 3-50 ng/ml whereas TGF alpha-PE40Asp553, which lacks ADP ribosylation activity, had an ED50 > 100 ng/ml . TGF alpha-PE40 inhibited colony formation of NSCLC cells with an LD50 value of 0.008-0.1 ng/ml . Similarly, TGF alpha-PE38 inhibited NSCLC colony formation with LD50 values of 0.002-0.1 ng/ml whereas TGF alpha-PE40Asp553 had an LD50 > 10 ng/ml . Also, TGF alpha-PE40 and TGF alpha-PE38 inhibited NSCLC xenograft formation in nude mice whereas TGF alpha-PE40Asp553 was inactive . These data suggest that TGF alpha-PE40 and TGF alpha-PE38 may be useful agents to inactivate NSCLC cells. J Fam Pract, 1994 Jan, 38(1), 30 - 2 Swimming and grommets; Cohen HA et al.; BACKGROUND . Traditionally, children with tympanostomy ventilating tubes, or grommets, were advised that water should not enter their ears in order to prevent ear infections . This group of children has been considered somewhat handicapped regarding swimming . We conducted a prospective study to determine if there is a relation between suppurative otitis media and surface swimming in children with grommets . METHODS . Forty-two children with tympanostomy ventilating tubes were included in this study . Of the 42 children, 22 were swimmers and 20 were nonswimmers, who served as the control group . The age range was 3 to 12 years, and there was no difference in the age distribution between the groups . Surface swimming was allowed without earplugs or a bathing cap, although it was mandatory to use polymyxin B-neomycin-hydrocortisone eardrops at bedtime on the day of swimming . No diving was allowed . RESULTS . Three of 22 swimmers and 2 of 20 nonswimmers developed otorrhea . In 4 of the 5 children, the otorrhea was followed by an upper respiratory tract infection . In all cases, a bacterial culture revealed Pseudomonas . The ear drainage was easily controlled with local otic treatment in all the patients . CONCLUSIONS . Taking into consideration the possible risks of infection and bearing in mind the value and joy of swimming to children and parents, families should be reassured that surface swimming does not increase the risk of infection in children with tympanostomy tubes. Mol Gen Genet, 1994 Jan, 242(1), 9 - 16 Nucleotide sequence analysis and potential environmental distribution of a ferric pseudobactin receptor gene of Pseudomonas sp . strain M114; Morris J et al.; The nucleotide sequence of the Pseudomonas sp . strain M114 pbuA gene, encoding the outer membrane receptor for ferric pseudobactin M114, has been determined . The region sequenced spans 2788 bases of plasmid pCUP3, within which the receptor gene had previously been localised . A single open reading frame, potentially encoding 826 amino acids and including a leader peptide of 44 amino acids, is evident and is followed by an inverted repeat segment, which may act as a transcriptional terminator . A 20 bp region of DNA, having significant homology with the E . coli Fur-binding consensus sequence, is located upstream of the open reading frame . PbuA displays characteristics in common with other outer membrane proteins and displays strong homology with the TonB boxes of both E . coli and Pseudomonas receptors . More extensive homologies were found with the PupA receptor of P . putida WCS358 and the FhuE and BtuB receptors of E . coli . It is suggested that areas exhibiting the least homology between these receptors may represent ferric siderophore-specific recognition sites of the PbuA protein . The deduced amino acid sequence of pbuA was compared with that of pupX, encoding the outer membrane receptor for ferric pseudobactin B10, of Pseudomonas sp . strain B 10 . A direct alignment of the two proteins gave an identity score of 92.5% . The distribution of PbuA-like receptors among Pseudomonas isolates was investigated by DNA-DNA hybridisation analysis . The results suggest that a PbuA-like receptor may be widely distributed among Pseudomonas rhizosphere isolates. Bioconjug Chem, 1994 Jan-Feb, 5(1), 40 - 6 An immunotoxin with increased activity and homogeneity produced by reducing the number of lysine residues in recombinant Pseudomonas exotoxin; Debinski W et al.; Pseudomonas exotoxin A (PE) is a protein composed of 613 amino acids arranged into three major, and one minor, domains . Immunotoxins (ITs) containing PE38, a mutant form of PE which lacks the cell binding domain (Ia, amino acids 1-252) and 16 amino acids from domain Ib (amino acids 365-380), are extremely potent cytotoxic agents which can cause a complete regression of various human carcinomas grown in nude mice . However, these ITs are a mixture of several different chemical forms since the coupling between the antibody and the toxin may occur between either the light or heavy chain of the antibody and one of the four primary amino groups present on the truncated toxin . To modify the toxin with heterobifunctional crosslinking reagents only at specific sites, we replaced lysines 590 and 606 with glutamines and lysine 613 with arginine (PE38QQR) . We also added two different peptide sequences, each containing a lysine residue, at the N-terminus of PE38 . In one of these the sequence is ANLAEEAFK ("Lys" peptide), and in the other, the sequence is LQGTKLMAEE ("NLys" peptide) . The mutant toxins were coupled using a thioether linkage to monoclonal antibody B3 which recognizes an antigen present in large amounts on many human cancers . PE38QQR-containing recombinant toxins can only be linked to an antibody through the N-terminal methionine or the lysine within the peptide.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 140 - 7 Amino acid residues required for the activity of avrD alleles; Yucel I et al.; Certain Pseudomonas syringae pathovars harbor avrD alleles belonging to two different homology classes . The nonfunctional avrD allele of P . s . pv . glycinea is highly homologous to active class II avrD alleles but has five unique amino acid substitutions . Three of these five amino acid changes were shown to be absolutely required for restoration of avrD activity to the P . s . pv . glycinea allele by oligonucleotide site-directed mutagenesis . They were cysteine 19 to arginine, alanine 280 to valine, and leucine 304 to serine . In addition, changing leucine 301 to phenylalanine was required for high activity . However, alteration of the leucine at position 245 of the P . s . pv . glycinea allele to serine, present in the active alleles, did not affect avrD activity . Results from recombinant gene constructs between the nonfunctional P . s . pv . glycinea avrD gene and the functional allele from P . s . pv . phaseolicola identified six other amino acid residues that may form contextual motifs important for AvrD function . These were a four amino acid stretch comprised of glutamate 41, alanine 42, asparagine 43 and arginine 44 in addition to aspartate 243 and phenylalanine 301 . Some divergence is tolerated within the four amino acid motif, but phenylalanine 301 appears to be necessary for highly active class II avrD proteins. Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 131 - 9 Two different classes of avrD alleles occur in pathovars of Pseudomonas syringae; Yucel I et al.; Considerable variation was observed in the occurrence of avirulence gene D (avrD) in different isolates and pathovars of Pseudomonas syringae . Three functional alleles of avrD were cloned and characterized from P . s . pv . phaseolicola and P . s . pv . lachrymans . These avrD genes occurred on indigenous plasmids in both pathovars, like the allele originally cloned from P . s . pv . tomato . P . s . pv . lachrymans was unique in that it carried two different alleles on plasmids of different sizes . These alleles were cloned on 5.6- or 3.8-kb HindIII fragments that are conserved in several other P . syringae pathovars . Surprisingly, the two avrD alleles from P . s . pv . lachrymans were the most divergent of those compared, with only 85% amino acid identity . Allele 1 from P . s . pv . lachrymans was 95% identical to avrD from P . s . pv . tomato but less similar to the other three avrD genes . These two alleles were accordingly called homology class I . The avrD gene from P . s . pv . phaseolicola and allele 2 from P . s . pv . lachrymans were 97 and 98% identical, respectively, at the amino acid level with the nonfunctional P . s . pv . glycinea allele . These three alleles were therefore grouped into homology class II . Comparison of all the avrD alleles permitted the identification of four amino acid substitutions unique to the P . s . pv . glycinea allele at positions 19, 245, 280, and 304. Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 121 - 30 Characterization of a negative regulator of exopolysaccharide production by the plant-pathogenic bacterium Pseudomonas solanacearum; Kao CC et al.; Wild-type strains of the bacterial wilt pathogen Pseudomonas solanacearum exhibit reduced exopolysaccharide production and virulence when transformed with plasmids carrying the epsR locus . To understand the function of epsR, we used mutagenesis and DNA sequencing to identify the gene responsible for the shutoff of exopolysaccharide production . The epsR gene encodes a 236-amino-acid polypeptide that, based on polypeptide sequence homology, has significant similarity to other proteins of the luxR family of environmentally responsive, two-component regulatory systems . When a mutated copy of the epsR gene was marker-exchanged into the wild-type P . solanacearum chromosome, however, we observed no effect on growth in culture or on exopolysaccharide production . This suggests that the EpsR phenotype becomes apparent only via overproduction of the EpsR protein . By means of an antiserum directed against the EpsR protein, we detected the overproduction of EpsR in cell lysates of a strain of P . solanacearum harboring a multicopy plasmid with an active epsR gene but not in one harboring the same plasmid with a mutated epsR gene. J Wildl Dis, 1994 Jan, 30(1), 107 - 9 Protocalliphora braueri (Diptera: Calliphoridae) induced pathogenesis in a brood of marsh wren (Cistothorus palustris) young; Warren Y; Infestation with blow fly larvae (Protocalliphora (Trypocalliphora) braueri Hendel) was pathogenic to marsh wren (Cistothorus palustris) young . The mechanism of pathogenicity was Pseudomonas spp . infection of subdermal myiasis-induced lesions and subsequent sepsis . Neither internal organ involvement nor muscle destruction was seen on necropsy of fledglings . Multifocal hepatic necrosis was seen histologically and Pseudomonas sp . was isolated from myiasis sites, liver, and peritoneal cavities. Prikl Biokhim Mikrobiol, 1994 Jan-Feb, 30(1), 55 - 63 {Tn5-mutagenesis of the styrene-degrading strain Pseudomonas sp . Y2 . Analysis of transformation products and DNA-scopy of the mutants obtained}; Iakimov MM et al.; The bacterium Pseudomonas sp . Y2 using styrene as a sole source of carbon and energy was subjected to transposon Tn5(Kmr) mutagenesis . The mutants were divided into three classes by the ability to grow on styrene, 2-phenylethanol, and phenylacetate . It is shown that 2-phenylethanol is not an obligate metabolite of styrene transformation . This allows the conclusion that in Pseudomonas sp . Y2 styrene can be degraded via consistent oxidation of the side chain by two pathways . For the comparison of strains/mutants and determination of genetic markers, DNA-fingerprinting of total DNA was carried out using electrophoresis of restriction fragments and blot-hybridization with different 32P-DNA-probes. Postgrad Med J, 1994 Jan, 70(819), 47 - 8 Successful treatment of Pseudomonas paucimobilis haemodialysis catheter-related sepsis without catheter removal; Saltissi D et al.; Infection is the most common complication of long-term central venous access lines . We report a case of central venous haemodialysis catheter infection with Pseudomonas paucimobilis, successfully treated by prolonged antibiotic administration without catheter removal . The implications are discussed. Biometals, 1994 Jan, 7(1), 30 - 40 Silver resistance in Pseudomonas stutzeri; Slawson RM et al.; Silver resistance was studied in a silver-resistant Pseudomonas stutzeri AG259 strain and compared to a silver-sensitive P . stutzeri JM303 strain . Silver resistance was not due to silver complexation to intracellular polyphosphate or the presence of low molecular weight metal-binding protein(s) . Both the silver-resistant and silver-sensitive P . stutzeri strains produced H2S, with the silver-resistant AG259 strain producing lower amounts of H2S than the silver-sensitive JM303 strain . However, intracellular acid-labile sulfide levels were generally higher in the silver-resistant P . stutzeri AG259 strain . Silver resistance may be due to formation of silver-sulfide complexes in the silver-resistant P . stutzeri AG259 strain. Appl Environ Microbiol, 1994 Jan, 60(1), 235 - 42 Self-mobilization and organization of the genes encoding the toluene metabolic pathway of Pseudomonas mendocina KR1; Wright A et al.; The toluene metabolic pathway of Pseudomonas mendocina KR1 is chromosomally encoded, but the pathway could be transferred by conjugation from strain KR1 to the chromosome of P . aeruginosa or P . putida . Such transconjugants utilized toluene, p-cresol, and p-hydroxybenzaldehyde . However, transconjugants were unable to further transfer toluene genes to other recipients unless Pseudomonas sex factor R68.45 was present in trans . Although the genes encoding the upper pathway for toluene metabolism in P . mendocina KR1 are sufficiently linked to permit their coordinate mobilization, they were found to be encoded in three independently regulated units: one encoding toluene-4-monooxygenase, a second encoding p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase, and a third encoding p-hydroxybenzoate hydroxylase . The last two regulatory units were cloned from the chromosome of a P . aeruginosa transconjugant onto a plasmid designated pRO1999 . Analysis of pRO1999 showed that genes encoding p-cresol methylhydroxylase and p-hydroxybenzaldehyde dehydrogenase are organized as an operon; the gene encoding p-hydroxybenzaldehyde dehydrogenase is transcribed first, and this is followed by transcription of the gene encoding p-cresol methylhydroxylase . This operon is regulated by a positively acting regulator . The P . mendocina KR1 gene encoding p-hydroxybenzoate hydroxylase was linked to, but independently regulated from, the genes encoding toluene-4-monooxygenase, p-cresol methylhydroxylase, and p-hydroxybenzaldehyde dehydrogenase. J Clin Pediatr Dent, 1994 Spring, 18(3), 215 - 7 A dental complication involving Pseudomonas during chemotherapy for acute lymphoblastic leukemia; Cheatham BD et al.; This is a case report of an 11-year-old white female, diagnosed with acute lymphoblastic leukemia, who developed a dental complication involving endodontic failure and Pseudomonas infection during induction chemotherapy . This unusual set of circumstances resulted in alteration of the child's medical management . Although the oral infection, neutropenia and delayed healing response resulted in interruption of the course of chemotherapy, the child did eventually achieve remission . This case does however demonstrate the importance of dental consultation for oncology patients. J Cancer Res Clin Oncol, 1994, 120(9), 507 - 12 Cytotoxic effect of a fusion protein from transforming growth factor alpha and Pseudomonas exotoxin on rat and human bladder carcinoma cells in vitro; Kameyama S et al.; A protein formed by fusion of transforming growth factor alpha with Pseudomonas exotoxin (TGF alpha-PE40) has been shown to have the ability to kill or inhibit the growth of several carcinoma cell lines . This study was designed to evaluate the in vitro cytotoxic effects of TGF alpha-PE40 on rat and human bladder carcinoma cell lines with different biological potential, and normal rat urothelial cells . The rat cell lines used were D44c, LMC19, and MYU3L, which were established in our laboratory . Human cell lines used were RT4, T24, and 253J . As a normal control, we used the first-passage culture of normal rat bladder urothelium (RU-P1) . We examined the number and affinity of epidermal growth factor receptors (EGFR) in these cells, the ability of TGF alpha-PE40 to bind EGFR, and the cytotoxic effect of TGF alpha-PE40 and PE40 . Rat cell lines, D44c, LMC19, and MYU3L (EGFR = 4.9 x 10(3)-11.4 x 10(3)/cell) had ED50 values (the concentration of TGF alpha-PE40 needed to reduce the viable cell population by 50%) of 180 pM, 540 pM and 6000 pM respectively; for cI (the concentration required to achieve complete inhibition of growth under continuous serum stimulation) TGF alpha-PE40 concentrations of 10(4) pM, 10(4) pM and higher than 10(4) pM respectively were required . Human cell lines, RT4, T24, and 253J (EGFR = 32 x 10(3)-126 x 10(3)/cell) had ED50 values of 20 pM, 66 pM, and 330 pM respectively and T24 showed cI values of 10(3) pM . RU-P1 (EGFR = 92.6 x 10(3)/cell) had the highest ED50 value of 8000 pM . These data indicate that the susceptibility to TGF alpha-PE40 does not always depend on the number of EGFR, that cells having a relatively small number of EGFR respond well to TGF alpha-PE40, and that normal urothelial cells are more resistant to TGF alpha-PE40 than are cancer cells . The differential effect of TGF alpha-PE40 on normal and neoplastic cells provides a rational basis for its use in vivo to control tumor growth. J Basic Microbiol, 1994, 34(2), 77 - 85 Pseudomonas acidovorans: a bacterium capable of mineralizing 2-chloroaniline; Hinteregger C et al.; Prolonged adaptation of Ca-alginate immobilized cells of Pseudomonas acidovorans CA28 to a mixture of 3-chloroaniline (3-CA)1) and 2-CA and subsequently to 2-CA as sole substrate led to the isolation of another strain, termed CA50 with the additional capability of utilizing 2-CA as sole source of carbon, nitrogen, and energy . Batch-degradation of 190 mg/l of 2-CA at pH 6.1 by this newly isolated strain was achieved within 3 days, at higher concentrations up to 0.6 g/l increasing lag-phases and degradation periods were observed . Except chloride and ammonium no further metabolites were detectable in the medium . Mineralization of 2-CA proceeds via the modified ortho-cleavage pathway as demonstrated by the presence of catechol 1,2-dioxygenase (C120) activity, which is characterized by its substrate specificity and elution behaviour on DEAE-cellulose. Arch Virol, 1994, 137(1-2), 185 - 90 Bacteriophages from Bombyx mori; Ackermann HW et al.; Preparations of silkworm larvae contained two large phages with contractile tails (Myoviridae) . One phage was active on Pseudomonas paucimobilis . The other, not cultivated, was one of the largest viruses known. Rev Argent Microbiol, 1994 Jan-Mar, 26(1), 28 - 35 A decalin-consuming bacterial community; Vitale AA et al.; A bacterial community able to degrade cis- and trans-decalin (decahydronaphthalene) in the presence of n-decane could be isolated . It was composed by a couple of Pseudomonas strains (D1 and D2, respectively) . Neither the community nor the isolated strains were able to grow on decalin alone . D1 grew on decalin plus n-decane . Both could grow on n-decyl alcohol, acetate and adipate . From hydrocarbonated substrates it would be generated a metabolic chain which allows the growth of the community members, and then it resulted to be stable . Characteristics of both strains are described. Arch Microbiol, 1994, 162(1-2), 75 - 9 Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes; West TP; The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440 . A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine . Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement . When the wild-type strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed . Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5'-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil . It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines . In vitro regulation of aspartate transcarbamoylase activity in P . pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by Pi, PPi, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP. Plasmid, 1994 Jan, 31(1), 21 - 30 Characterization of high-frequency deletions in the iaa-containing plasmid, pIAA2, of Pseudomonas syringae pv . savastanoi; Soby S et al.; The phytopathogenic bacterium Pseudomonas syringae pv . savastanoi causes olive and oleander knot disease . The bacterium induces the formation of tumorous galls by the synthesis and secretion of the plant hormones trans-zeatin riboside and indole-3-acetic acid into host intercellular spaces . An Italian oleander isolate, PB213, has been observed to lose the ability to synthesize IAA at high frequency, thus becoming non-pathogenic . The IAA genes, located on the 72-kb iaa-containing plasmid, pIAA2 were lost mainly due to two classes of deletions: 18 or 22 kb in length . Both classes of deletions had a common endpoint upstream of the IAA genes . The other endpoints were in areas that flanked the insertion sequence element IS51 . The endpoints are in regions of repetitive DNA of at least 271 bp that have been designated a/b. Mol Plant Microbe Interact, 1994 Jan-Feb, 7(1), 78 - 90 Syringomycin production among strains of Pseudomonas syringae pv . syringae: conservation of the syrB and syrD genes and activation of phytotoxin production by plant signal molecules; Quigley NB et al.; The syrB and syrD genes of Pseudomonas syringae pv . syringae are predicted to encode proteins that function in the synthesis and export of syringomycin, respectively . Using portions of the syr genes as DNA probes, both genes were shown to be conserved as single copies within a 15-kb or smaller DNA region among a broad spectrum of P . s . pv . syringae strains that produce syringomycin or one of its amino acid analogs, syringotoxin and syringostatin . Strains representative of P . viridiflava and six pathovars of P . syringae failed to hybridize with the gene probes, demonstrating that syr sequences are highly specific to P . s . pv . syringae and related nonpathogenic strains . Maximum parsimony analysis of restriction fragment length polymorphism profiles was used to evaluate relatedness among strains within the syrB and syrD gene region . A tree, conveying the smallest number of evolutionary changes among strains, revealed considerable diversity within the syr gene region; subclusters of strains were identified that appear to share specific qualities relevant to the plant-pathogen interaction . Because both the syrB gene and syringomycin production can be induced in response to plant signal molecules, 42 strains containing homologous syr sequences were tested for signal-mediated induction of toxin production . Over 90% of the toxigenic strains produced larger quantities of toxin when the plant signal molecules, arbutin and D-fructose, were added to syringomycin-minimal medium; 13 of the strains produced > or = 10-fold higher toxin levels . Some strains, such as 5D428, produced toxin only in the presence of these signals.(ABSTRACT TRUNCATED AT 250 WORDS) Ann Oncol, 1994, 5 Suppl 1, 97 - 103 Immunotoxins: is there a clinical value? Gottstein C, Winkler U, Bohlen H, Diehl V, Engert A. Drug targeting is an attractive new approach to killing malignant cells, thereby leaving normal tissue unharmed . A decisive breakthrough was the advent of hybridoma technology, making monoclonal antibodies (MoAb) available in limitless supply . To construct reagents with selectivity for certain tumor cells, MoAbs or Fab' fragments were chemically linked to ribosome-damaging toxins derived from plants or bacteria like ricin, abrin, saporin, Pseudomonas exotoxin (PE), and diphtheria toxin (DT) to form immunotoxins, which combined the selectivity of the carrier moiety with the potency of the toxin moiety . The first generation of these immunotoxins showed impressive results in vitro but in most cases disappointing antitumour effects in animals or humans . By contrast, the second generation of immunotoxins, consisting of either A chain immunotoxins with a greatly improved stability in vivo or so-called 'blocked' ricin immunotoxins, have been demonstrated to be extremely effective in several animal models . Preliminary results of the current clinical trials suggest a possible clinical use of immunotoxins in leukemia and lymphoma patients . Genetically engineered fusion toxins have become available, which consist of a growth factor or a cytokine fused to a toxin moiety . In this paper, we will review the features of the three groups of immunotoxins which are most frequently used, i.e., ricin A chain and similar immunotoxins, blocked ricin immunotoxins, and recombinant toxins constructed with Pseudomonas exotoxin or diphtheria toxin. Chin J Biotechnol, 1994, 10(4), 233 - 9 Construction and expression of high efficiency expressing plasmid of transforming growth factor alpha-Pseudomonas aeruginosa exotoxin fusion protein TGF alpha-PE40; Xu Y et al.; The XbaI/EcoRI cleaved TGF alpha-PE40 gene from plasmid pXY382 was inserted into the same cloning site of the expression vector pCB604 resulting in the plasmid p2X-TP1 . E . coli BL21 (lambda DE3) cells were transformed with the p2X-TP1 and then induced with IPTG . The product expressed was accumulated mainly in the form of inclusion bodies . The expression level was closely related to the cell density, induction temperature and medium but not to the inductor dosage and the induction period within certain range . The expressed amount of the fusion protein TGF alpha-PE40 was about 50 mg/L. Planta, 1994, 195(2), 175 - 81 Extensin gene expression is induced by mechanical stimuli leading to local cell wall strengthening in Nicotiana plumbaginifolia; Tire C et al.; Nicotiana plumbaginifolia Viv . harbors a single extensin gene, although related hydroxyproline-rich sequences are present in the genome . Northern analysis showed that the gene is highly expressed in roots and to a lesser extent in stems . Expression in leaves is low but mRNA levels are increased upon infection with the incompatible bacterium Pseudomonas syringae . Extensin transcript levels in leaves were slightly enhanced after wounding and salicylic acid treatment . In-situ hybridization experiments showed high accumulation of extensin mRNA in cells which, at certain stages of development, require reinforcement of their cell walls . The cortical cells in stem nodes and roots, which are put under severe mechanical stress by adjacent developing tissues, tend to express the gene to high levels . Immunolocalization of the extensin protein in stems and roots demonstrated a close association of the protein with lignin deposition . Mature tissues contained more extensin than younger tissues . The extensin promoter was fused to the beta-glucuronidase gene. Acta Biol Hung, 1994, 45(1), 17 - 23 Investigation of chromium (VI) tolerant bacteria; Vincze G et al.; Pseudomonas strains were isolated from a heavy metal contaminated sludge . Some of the strains grew on chromium (VI) containing medium, and others were inhibited in their growth . Cells were able to remove chromium from the medium . This ability was independent of the rate of their growth . The concentration of chromium (VI) of the media decreased with 15-20 ppm in presence of 10(9) cells . The chromium (VI) was converted into a reduced form of chromium by bacteria . The reduced form of chromium was bound in the cell wall . The bound form of chromium could be recovered more or less with different chemicals . The best percentages of recovery were achieved with NaOH solution. Cell Biophys, 1994, 24-25, 9 - 14 Removal of endotoxin from antibody preparations for clinical use . Assessment of polymyxin-sepharose CNBr affinity chromatography; Lahiri VL et al.; Despite attempts to maintain asepsis, good manufacturing practices, and the use of terminal sterilization by millipore filtration, the nuclear practitioner is always worried about the possibility of endotoxin contamination . Methods, such as ion-exchange chromatography, have been tried for removing endotoxins during the preparation of radiolabeled antibodies, and so on . As suggested by Stevenson (1990), we evaluated the Issekutz technique (1) of endotoxin removal by affinity chromatography using a polymyxin cyanogen bromide (CNBr) Sepharose column . The endotoxin content of millipore filtrates of heat killed/sonicated suspensions of Pseudomonas pyocyaneus, E . coli were measured using a Sigma (St . Louis, MO) Endotoxin Assay Kit before and after filtration through such columns and compared with the results obtained using gel exclusion and ion-exchange columns of the same length and diameter . Reduction of endotoxin content to undetectable levels by the polymyxin column was observed . The use of such columns for terminal endotoxin removal analogous to terminal sterilization is advocated especially when developing a radiopharmaceutical such as radiolabeled antibodies for in house use. Cell Biophys, 1994, 24-25, 249 - 57 Targeting phosphodiesterases as a strategy for killing tumor cells; Deonarain MP et al.; Ribonucleases (RNases) are being employed as alternative cytotoxic proteins to the conventionally used ones such as ricin and Pseudomonas exotoxin . Mammalian RNases are attractive enzymes because of their comparable cytotoxicity when suitably directed and the likelihood of lower immunogenicity compared to plant and bacterial toxins . Bovine seminal RNase (BSRNase) is a member of the RNase superfamily, but differs in many interesting ways . Unlike the rest of the family it is dimeric, and possesses antitumor and immunosuppressive properties . These features make it a choice candidate for a single-chain antibody (scFv) based immunotoxin . This work describes preliminary data on the construction, expression in Escherichia coli and characterization of a tumor-specific scFv (directed against human placental alkaline phosphatase)-BSRNase chimeric molecule . It is shown that the created molecule has RNA degrading activity and antigen-binding activity when refolded from bacterial inclusion bodies. Antonie Van Leeuwenhoek, 1994, 66(4), 307 - 12 Pyrimidine ribonucleoside catabolic enzyme activities of Pseudomonas pickettii; West TP; Pyrimidine ribonucleoside catabolic enzyme activities of the opportunistic pathogen Pseudomonas pickettii were examined . Of the pyrimidine and related compounds tested, only dihydrouracil (nitrogen source) and ribose (carbon source) supported growth . Thin-layer chromatographic separation of the uridine and cytidine catabolities produced by P . pickettii extracts indicated that this pseudomonad contained nucleoside hydrolase activity . Its presence was confirmed by enzyme assay . Hydrolase activity was elevated in both glucose- and ribose-grown cells relative to succinate-grown cells . Nucleoside hydrolase activity was depressed when dihydrouracil served as a nitrogen source . Cytosine deaminase activity was present in extracts prepared from succinate-, glucose- or ribose-grown cells when (NH4)2SO4 served as the nitrogen source although cells grown on glucose or ribose exhibited a higher enzyme activity . Cytosine deaminase activity was not detected in extracts prepared from cells grown on dihydrouracil as a nitrogen source . Both dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were measurable in P . pickettii . The dehydrogenase activity was higher with NADH than with NADPH as its nicotinamide cofactor when uracil served as its substrate . Carbon source did not affect dehydrogenase or dihydropyrimidinase activity greatly but both activities were diminished in cells grown on the nitrogen source dihydrouracil. Microbiol Immunol, 1994, 38(1), 1 - 9 Characterization of hemolytic and antifungal substance, cepalycin, from Pseudomonas cepacia; Abe M et al.; Hemolytic and antifungal substances, cepalycin I and cepalycin II, have been isolated from Pseudomonas cepacia JN106 . A large amount of cepalycins were produced by growing the cells on 1% glycerin-nutrient agar medium covered with a cellophane membrane . The cell-washed supernatant was applied to an Amberlite XAD2 column, and cepalycins were eluted with 70% ethanol containing 1mM HCl . Cepalycins were separated by reverse phase HPLC in two fractions which were designated as cepalycin I and cepalycin II . The two cepalycins have indistinguishable UV absorption spectra but have different levels of hemolytic activity relative to the UV absorption . From the inhibition of hemolytic activity of cepalycin by sterols, both cepalycins were suggested to interact with cholesterol in erythrocyte membrane . Such an interaction may contribute to their hemolytic and antifungal activities. Bioconjug Chem, 1994 Jan-Feb, 5(1), 77 - 83 In vivo activities of acidic fibroblast growth factor-Pseudomonas exotoxin fusion proteins; Siegall CB et al.; Fibroblast growth factor receptors are highly expressed in a variety of cancer cells and activated vasculature . Using chimeric toxins targeted to cell-surface a FGF receptors, we have demonstrated specific cytotoxic activity to these cell types . These molecules, aFGF-PE40 and aFGF-PE40 KDEL, are fusion proteins containing acidic FGF and either a 40- or a 66-kDa binding defective form of Pseudomonas exotoxin, respectively . Both aFGF-toxin fusion proteins were able to inhibit protein synthesis in vitro in a variety of carcinoma cell lines . The half-life of aFGF-PE40 in serum was found to be 41 min when coadministered with heparin . Administration of aFGF-PE40 or aFGF-PE4E KDEL with heparin inhibits the growth of established KB and preestablished A431 epidermoid carcinoma xenografts in athymic mice . The antitumor activities of the two aFGF-toxin fusion proteins were equivalent against the KB tumor xenografts . While we were able to slow the growth of the KB tumor xenografts, we were unable to cause tumor regressions . Histochemical analysis of treated versus untreated tumor tissue revealed a difference in tumor size but not of vascularity . We conclude that aFGF-PE40 and aFGF-PE4E KDEL have in vivo antitumor activity that targets the tumor cell mass rather than vascular structures in mice xenografted with human epidermoid carcinoma. Br J Cancer, 1994 Jan, 69(1), 32 - 9 Immunotoxins recognising a new epitope on the neural cell adhesion molecule have potent cytotoxic effects against small cell lung cancer; Zangemeister-Wittke U et al.; The present study describes a comparison of two potent immunotoxins which utilise an identical targeting component, a monoclonal antibody (SEN7) specific for small cell lung cancer (SCLC), conjugated to two different effector components, blocked ricin (bR) and Pseudomonas exotoxin A (PE) . SEN7 recognises a novel epitope on the neural cell adhesion molecule (NCAM) which is highly associated with SCLC . The immunotoxins SEN7-PE and SEN7-bR were selectively and potently active against a number of SCLC cell lines, of both classic and variant morphologies, inhibiting the incorporation of {3H}leucine with IC50 values ranging between 22 pM and 85 pM and between 7 pM and 62 pM for SEN7-PE and SEN7-bR respectively . Intoxication by both immunotoxins proceeded rapidly following short 2 h lag phases; the initial rates of protein synthesis inhibition occurred with t50 values of 6.5 h for SEN7-PE and 5.5 h for SEN7-bR . Monensin drastically enhanced the cytotoxic activity of the weakly active SEN7-ricin A-chain by 2,100-fold and of SEN7-bR by 80-fold but had no effect on SEN7-PE . In limiting dilution assays, four and more than 4.5 logs of clonogenic SW2 tumour cells were selectively eliminated from the cultures during continuous exposure to the immunotoxins SEN7-PE and SEN7-bR respectively, while antigen-negative cells required up to 1,000-fold more drug for a similar cell kill . SW2 cells surviving SEN7-bR treatment in the cultures did not express NCAM and consequently were not selectively killed by SEN7 immunotoxins . SW2 cells surviving continuous exposure to SEN7-PE showed no alteration in NCAM expression but were more resistant to intoxication mediated by PE . These cells were still sensitive to SEN7-bR. Cancer Res, 1994 Jan 1, 54(1), 209 - 14 Comparison of two antibody-based methods for elimination of breast cancer cells from human bone marrow; Myklebust AT et al.; Three monoclonal antibodies reactive with antigens abundantly expressed on human carcinoma cells were used to develop and compare the efficacy of immunotoxins (ITs) and immunobeads for purging breast cancer cells from bone marrow . ITs constructed as conjugates of the monoclonal antibodies and Pseudomonas exotoxin A showed high specific cytotoxicity against three breast cancer cell lines, inhibiting protein synthesis by 50% at concentrations of 4 x 10(-13) M to 1 x 10(-10) M . Tested in a reproducible clonogenic assay, two of the ITs used at a concentration of 0.1 microgram/ml killed > 5 log units of MCF7 cells, the maximal sensitivity for assessing cytotoxic effects, and 1.5 log of T-47D tumor cells . At 1 microgram/ml, each of the three ITs eliminated > 5 log of both cell lines . The immunobead procedure removed 2.0-4.1 log of tumor cells with one purging cycle and up to 6.0 log with two cycles . The mixture of the three ITs or immunobeads was not clearly superior in efficacy, compared to the use of individual molecules, probably reflecting an overlap in expression of the respective antigens in these cell lines . For both methods, the purging efficacy was not reduced when the tumor cells were admixed with normal bone marrow cells at a ratio of 1:10 . The survival of colony-forming units, granulocyte/macrophage, was 49-86% with the immunobeads and 44-75% even at high concentrations (up to 2.5 micrograms/ml x 3) of the ITs . The results indicate that each of the two immunological methods can be safely used for effective elimination of tumor cells from the graft of breast cancer patients undergoing autologous bone marrow transplantation. Carbohydr Res, 1993 Dec 28, 250(2), 275 - 87 Studies of O-specific polysaccharide chains of Pseudomonas solanacearum lipopolysaccharides consisting of structurally different repeating units; Kocharova NA et al.; The structures of the O-antigenic polysaccharide chains of lipopolysaccharides of a number of Pseudomonas solanacearum strains were elucidated mainly with the help of methylation analysis and 13C NMR spectroscopy, including a computer-assisted 13C NMR-based analysis . Six structurally distinct but related polysaccharides were identified . They have a backbone which is built up of three L-rhamnopyranosyl residues and one 2-acetamido-2-deoxy-D-glucopyranosyl residue, and is unsubstituted or substituted with a residue of L-xylopyranose or L-rhamnopyranose as a monosaccharide side chain . The lipopolysaccharides of most of the strains contain polysaccharide chains consisting of at least two structurally different types of repeating units . Three of the polysaccharides are common to more than one strain. FEMS Microbiol Lett, 1993 Dec 15, 114(3), 339 - 42 Yeast genes involved in growth inhibition by Pseudomonas syringae pv . syringae syringomycin family lipodepsipeptides; Takemoto JY et al.; Saccharomyces cerevisiae genes encoding functions necessary for inhibition by the Pseudomonas syringae pv . syringae cyclic lipodepsipeptide, syringomycin-E, were identified by mutant analyses . Syringomycin-E-resistant mutants were isolated, shown to contain single recessive mutations, and divided into eight gene complementation groups . Representative strains from five groups were resistant to nystatin, and deficient in the plasma membrane lipid, ergosterol . All of the mutant strains were resistant to the related cyclic lipodepsipeptides, syringotoxin and syringostatin . The findings show that: 1) at least eight gene-encoded functions participate in the inhibitory response to syringomycin; 2) ergosterol is important for this response; 3) the three related lipodepsipeptides have similar modes of action. Biochem J, 1993 Dec 15, 296 ( Pt 3), 867 - 75 Interaction of a 39 kDa protein with the low-density-lipoprotein-receptor-related protein (LRP) on rat hepatoma cells; Iadonato SP et al.; We have recently described a PAI-1-independent pathway of tissue-type plasminogen