Lett Appl Microbiol, 2003, 36(5), 282 - 7 Physical properties of an extracellular polysaccharide produced by Bacillus sp . CP912; Yun UJ et al.; AIMS: In this study, some physical properties of Bacillus sp . exo-polysaccharide were investigated . METHODS AND RESULTS: An extracellular polysaccharide was purified by sequential precipitations after homogenization of the diluted culture supernatant of Bacillus sp . CP912 . Its physical properties were examined such as lipid emulsifying effect on several vegetable oils and flocculating activity against the activated carbon suspension . The melting point and endothermic calories of the polysaccharide were 128.7 degrees C and 50.864 kCal mol-1, respectively . Its pyrolysis temperature was 284.58 degrees C . The polysaccharide showed high lipid emulsifying activity on oil-water emulsion, against olive, peanut, sunflower and corn oils . It exhibited high flocculating activity as well against activated carbon . CONCLUSIONS: The present findings suggest that the extracellular polysaccharide produced by Bacillus sp . CP912 has a great industrial potential because of its high lipid emulsifying and flocculating activity . SIGNIFICANCE AND IMPACT OF THE STUDY: These data represent a novel Bacillus sp . extracellular polysaccharide possessing high emulsifying and flocculating effects. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Mar, 38(3), 483 - 92 Production of biosurfactant and its role in the biodegradation of oil hydrocarbons; Lu XX et al.; Based on indigenous bacteria from the oil-contaminated site of Dawu water source area in Zibo city of China, the production of biosurfactant and its role in the biodegradation of oil hydrocarbons have been investigated . Batch experiments were performed with paraffin as the sole substrate under temperature of 30 degrees C and pH of 7 . Two high-effective species of bacteria (Z1 and Z2) were isolated . During the growth of both species, biosurfactants were produced, as indicated by the decrease of surface tensions of the enrichment medium from 61 to 45 mN/m . The critical micelle concentration (CMC) of Z1 ferment liquid was 0.4 (volume content) . When reaching the CMC, Z1 ferment liquid demonstrated the effect of dissolution enhancement, that is, it could enhance the dissolved concentration of phenanthrene to above 1 mg/L, which was higher than the saturated solubility of phenanthrenene under standard condition . Elementary identification results revealed that both Z1 and Z2 belongs to bacillus . Z1 was 0.5 microm in diameter, 0.5-3 microm in length, and bearing flagellum, while Z2 was 0.2 microm in diameter, 0.5-1 microm in length and no flagellum. J Biomed Mater Res B Appl Biomater, 2003 May 15, 65(2), 239 - 44 Optimization of a RF-generated CF4/O2 gas plasma sterilization process; Lassen KS et al.; A sterilization process with the use of RF-generated (13.56 MHz) CF(4)/O(2) gas plasma was optimized in regards to power, flow rate, exposure time, and RF-system type . The dependency of the sporicidal effect on the spore inoculum positioning in the chamber of the RF systems was also investigated . Dried Bacillus stearothermophilus ATCC 7953 endospores were used as test organisms . The treatments were evaluated on the basis of survival curves and corresponding D values . The only parameter found to affect the sterilization process was the power of the RF system . Higher power resulted in higher kill . Finally, when the samples were placed more than 3-8 cm away from a centrally placed electrode in System 2, the sporicidal effect was reduced . The results are discussed and compared to results from the present literature . The RF excitation source is evaluated to be more appropriate for sterilization processes than the MW source . J Urol, 2003 May, 169(5), 1706 - 8 Defining bacillus Calmette-Guerin refractory superficial bladder tumors; Herr HW et al.; PURPOSE: We define bacillus Calmette-Guerin (BCG) refractory, high risk, superficial bladder cancer . MATERIALS AND METHODS: A total of 93 patients received a 6-week induction course of BCG . They were evaluated for response after 3 and 6 months . Half of the patients received monthly maintenance BCG for 2 years and half did not . In both groups the initial responses to BCG at 3 and 6 months were correlated with subsequent tumor recurrence and progression . RESULTS: Of the 93 cases 57% were negative for tumor at 3 months and 43% had residual tumor resected . At 6 months 80% of the patients were tumor-free and 20% had persistent or recurrent tumor . Maintenance BCG did not decrease tumor recurrence further than induction BCG . Subsequent tumor-free interval during 24 months of followup were best predicted by response to BCG after 6 months . CONCLUSIONS: A minimum treatment and followup time of 6 months is required to identify high risk, superficial bladder tumors as truly BCG refractory. J Urol, 2003 May, 169(5), 1702 - 5 The predictive value of purified protein derivative results on complications and prognosis in patients with bladder cancer treated with bacillus Calmette-Guerin; Bilen CY et al.; PURPOSE: We investigate the correlation of purified protein derivative (PPD) results before intravesical bacillus Calmette-Guerin (BCG) instillations with prognosis and complications of BCG . MATERIALS AND METHODS: A total of 57 men and 4 women with proven intermediate or high risk superficial bladder cancer received 6 courses of intravesical BCG instillations following complete resection of tumors . Skin reactivity to a PPD derivative of Mycobacterium tuberculosis was tested before starting and 1 week after BCG . The test was considered positive if the induration was 10 mm . or more in diameter after 48 or 72 hours . The patients were grouped according to PPD responses and symptoms . The statistical analyses were performed between PPD positive and negative groups, and also between symptomatic and asymptomatic patients . The groups were compared for relapse rates, time to first recurrence, complication rates and clinical outcome . RESULTS: Most of the patients with systemic side effects were in the PPD positive group but only fever had a statistically significant difference and was more frequent in the positive group (p <0.05) . The recurrence-free intervals after intravesical BCG therapy did not differ significantly between PPD positive and negative groups . However, the trend of longer recurrence-free survival was evident for symptomatic patients (p = 0.056) . The numbers of tumor recurrences were 10 (52%) in the PPD negative group and 19 (51%) in the PPD positive group, which was statistically insignificant . CONCLUSIONS: Patients with systemic reactions to BCG had the longest disease-free survival . It seems that patients with an augmented reaction to BCG probably have better antitumor activity . Furthermore, although larger groups of patients are mandatory for statistical analysis, this study shows that hypersensitivity reaction against tuberculin could alert physicians of severe complications. Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 24 - 9 Structure-function relationship in serine hydroxymethyltransferase; Appaji Rao N et al.; Serine hydroxymethyltransferase (SHMT), a pyridoxal-5'-phosphate (PLP)-dependent enzyme catalyzes the tetrahydrofolate (H(4)-folate)-dependent retro-aldol cleavage of serine to form 5,10-methylene H(4)-folate and glycine . The structure-function relationship of SHMT was studied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site . The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure . The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis . E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an 'open' to 'closed' conformation rather than proton abstraction from the hydroxyl group of serine . K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure . Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate . The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s) complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT. J Plant Physiol, 2003 Feb, 160(2), 105 - 13 Systemic induction of the biosynthesis of terpenic compounds in Digitalis lanata; Gutierrez Manero FJ et al.; A bacterial screening was carried out in the rhizosphere of two Digitalis species, D . thapsi and D . parviflora, both at the vegetative stage and at flowering . A total of 480 isolates were characterised at genus level, Bacillus being the dominant genera in all cases . Fifty percent of the Bacillus strains isolated from each species were analysed by PCR-RAPDs . At 85% similarity, 12 groups separated for D . thapsi and 18 for D . parviflora . One strain of each group was selected for biological assay on D . lanata, evaluating growth promotion and cardenolide content in leaves after inoculation performed in the root system . The plant parameters evaluated were leaf surface area, shoot and root dry weight and leaf number . Lanatoside C content was evaluated by HPLC . Only 17 strains caused significant increases in at least one of the parameters evaluated . The most striking result was that some strains promoted growth and increased cardenolide content at the same time . This effect was detected on leaves while inoculation was carried out on roots . Interestingly, these two parameters are not enhanced simultaneously under regular conditions in pot or in tissue cultures. Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 569 - 73 Epub 2003 Apr 03. In vitro reduction of hexavalent chromium by a cell-free extract of Bacillus sp . ES 29 stimulated by Cu2+; Camargo FA et al.; Chromium-resistant bacteria (CRB) isolated from soils can be used to reduce toxic Cr(VI) from contaminated environments . This study assessed in vitro reduction of hexavalent Cr using a cell-free extract (CFE) of CRB isolated from soil contaminated with dichromate . One isolate, ES 29, that substantially reduced Cr(VI) was identified as a Bacillus species by 16S rRNA gene-sequence homology . The isolate reduced Cr(VI) under aerobic conditions, using NADH as an electron donor and produced a soluble Cr(VI)-reducing enzyme stimulated by copper (Cu2+) . The CFE of the bacterial isolate reduced 50% of Cr(VI) in 6 h . The Cr(VI)-reduction activity of the CFE had a Km of 7.09 microM and a Vmax of 0.171 micromol min(-1) mg(-1) protein . Mercury inhibited the enzyme, but not competitively, with a Vmax of 0.143 micromol min(-1) mg(-1) protein, a Km of 7.07 microM and a Ki of 1.58 microM . This study characterizes the enzymatic reduction of Cr(VI) by Bacillus sp . ES 29 which can be used for the bioremediation of chromate. Plant Mol Biol, 2003 Mar, 51(5), 789 - 801 Microbial starch-binding domains as a tool for targeting proteins to granules during starch biosynthesis; Ji Q et al.; Modification of starch biosynthesis pathways holds an enormous potential for tailoring granules or polymers with new functionalities . In this study, we explored the possibility of engineering artificial granule-bound proteins, which can be incorporated in the granule during biosynthesis . The starch-binding domain (SBD)-encoding region of cyclodextrin glycosyltransferase from Bacillus circulans was fused to the sequence encoding the transit peptide (amyloplast entry) of potato granule-bound starch synthase I (GBSS I) . The synthetic gene was expressed in the tubers of two potato cultivars (cv . Kardal and cv . Karnico) and one amylose-free (amf) potato mutant . SBDs accumulated inside starch granules, not at the granule surface . Amylose-free granules contained 8 times more SBD (estimated at ca . 1.6% of dry weight) than the amylose-containing ones . No consistent differences in physicochemical properties between transgenic SBD starches and their corresponding controls were found, suggesting that SBD can be used as an anchor for effector proteins without having side-effects . To test this, a construct harbouring the GBSS I transit peptide, the luciferase reporter gene, a PT-linker, and the SBD (in frame), and a similar construct without the linker and the SBD, were introduced in cv . Kardal . The fusion protein accumulated in starch granules (with retainment of luciferase activity), whereas the luciferase alone did not . Our results demonstrate that SBD technology can be developed into a true platform technology, in which SBDs can be fused to a large choice of effector proteins to generate potato starches with new or improved functionalities. Recenti Prog Med, 2003 Apr, 94(4), 177 - 85 {Bartonellosis}; Mansueto P et al.; In the last years the number of Bartonella species significantly raised, often with an epidemiological profile of emergent disease . B . bacilliformis is the etiological agent of Carrion's disease . B . clarridgeiae has been associated, together with B . henselae, to Cat-Scratch Disease (CSD), whereas B . elizabethae to endocarditis and B . grahamii to neuroretinitis . B . henselae has been associated to CSD and, in patients with immunodeficiency, together with B . quintana, to bacillary angiomatosis, to peliosis and to endocarditis . B . quintana is the etiological agent of "trench fever", in its "classical" and "urban" form . B . vinsonii subsp . arupensis and subsp . berkhoffii have been associated to bacteremia and endocarditis . The diagnosis foresees pathological examination of the lesions, germ isolation and use of specific serological test and of polymerase chain reaction . Prognosis is, generally, favourable . Therapy foresees the use of numerous antibacterial agents, like: penicillins, cephalosporins, aminoglycosides, tetracyclines, macrolides, quinolones, trimethoprim-sulfomethoxazole and rifampicin. Arch Microbiol, 2003 May, 179(5), 363 - 7 Epub 2003 Apr 02. Activity of wild-type and hybrid Bacillus thuringiensis delta-endotoxins against Agrotis ipsilon; de Maagd RA et al.; Twelve Cry1 and two Cry9 delta-endotoxins from Bacillus thuringiensis were tested for their activity against black cutworm ( Agrotis ipsilon) . A . ipsilon was not susceptible to many toxins, but three toxins had significant activity . Cry9Ca was the most toxic, followed by Cry1Aa and Cry1Fb . Hybrids between these three active proteins were made by in vivo recombination and analyzed for activity against A . ipsilon . Analysis of hybrids between Cry1Aa and Cry1Fb indicated that domain I of Cry1Aa protein was involved in its higher activity. Appl Environ Microbiol, 2003 Apr, 69(4), 2245 - 52 Killing of bacillus spores by aqueous dissolved oxygen, ascorbic acid, and copper ions; Cross JB et al.; An approach to decontamination of biological endospores is discussed . Specifically, the performance of an aqueous modified Fenton reagent is examined . A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions . The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions . Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu(2+)) and then subsequent conversion to hydroxyl radicals . Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii . A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature . This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O(2) is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action. Appl Environ Microbiol, 2003 Apr, 69(4), 2065 - 72 Effects of inactivation and constitutive expression of the unfolded- protein response pathway on protein production in the yeast Saccharomyces cerevisiae; Valkonen M et al.; One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones . We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccharomyces cerevisiae . The effects of HAC1 deletion and overexpression on the production of a native protein, invertase, and two foreign proteins, Bacillus amyloliquefaciens alpha-amylase and Trichoderma reesei endoglucanase EGI, were studied . Disruption of HAC1 caused decreases in the secretion of both alpha-amylase (70 to 75% reduction) and EGI (40 to 50% reduction) compared to the secretion by the parental strain . Constitutive overexpression of HAC1 caused a 70% increase in alpha-amylase secretion but had no effect on EGI secretion . The invertase levels were twofold higher in the strain overexpressing HAC1 . Also, the effect of the active form of T . reesei hac1 was tested in S . cerevisiae . hac1 expression caused a 2.4-fold increase in the secretion of alpha-amylase in S . cerevisiae and also slight increases in invertase and total protein production . Overexpression of both S . cerevisiae HAC1 and T . reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation. Mol Ecol, 2003 Mar, 12(3), 765 - 75 Degradation of the Cry1Ab protein within transgenic Bacillus thuringiensis corn tissue in the field; Zwahlen C et al.; Large quantities of Bacillus thuringiensis (Bt) corn plant residue are left in the field after harvest, which may have implications for the soil ecosystem . Potential impacts on soil organisms will also depend on the persistence of the Bt toxin in plant residues . Therefore, it is important to know how long the toxin persists in plant residues . In two field studies in the temperate corn-growing region of Switzerland we investigated degradation of the Cry1Ab toxin in transgenic Bt corn leaves during autumn, winter and spring using an enzyme-linked immunosorbent assay (ELISA) . In the first field trial, representing a tillage system, no degradation of the Cry1Ab toxin was observed during the first month . During the second month, Cry1Ab toxin concentrations decreased to approximately 20% of their initial values . During winter, there was no further degradation . When temperatures again increased in spring, the toxin continued to degrade slowly, but could still be detected in June . In the second field trial, representing a no-tillage system, Cry1Ab toxin concentrations decreased without initial delay as for soil-incorporated Bt plants, to 38% of the initial concentration during the first 40 days . They then continued to decrease until the end of the trial after 200 days in June, when 0.3% of the initial amount of Cry1Ab toxin was detected . Our results suggest that extended pre- and post-commercial monitoring are necessary to assess the long-term impact of Bt toxin in transgenic plant residues on soil organisms. Mol Microbiol, 2003 Apr, 48(2), 417 - 27 Multiplication of a restriction-modification gene complex; Sadykov M et al.; Previous works have suggested that some gene complexes encoding a restriction (R) enzyme and a cognate modification (M) enzyme may behave as selfish mobile genetic elements . RM gene complexes, which destroy 'non-self' elements marked by the absence of proper methylation, are often associated with mobile genetic elements and are involved in various genome rearrangements . Here, we found amplification of a restriction-modification gene complex . BamHI gene complex inserted into the Bacillus chromosome showed resistance to replacement by a homologous stretch of DNA . Some cells became transformed with the donor without losing BamHI . In most of these transformants, multiple copies of BamHI and the donor allele were arranged as tandem repeats . When a clone carrying one copy of each allele was propagated, extensive amplification of BamHI and the donor unit was observed in a manner dependent on restriction enzyme gene . This suggests that restriction cutting of the genome participates in the amplification . Visualization by fluorescent in situ hybridization revealed that the amplification occurred in single cells in a burst-like fashion that is reminiscent of induction of provirus replication . The multiplication ability in a bacterium with natural capacity for DNA release, uptake and transformation will be discussed in relation to spreading of RM gene -complexes. Biotechnol Prog, 2003 Mar-Apr, 19(2), 652 - 4 Improved catalytic performance of Bacillus megaterium epoxide hydrolase in a medium containing Tween-80; Gong PF et al.; A new epoxide hydrolase with high enantioselectivity toward (R)-glycidyl phenyl ether (R-GPE) was partially purified from Bacillus megaterium strain ECU1001 . The maximum activity of the isolated enzyme was observed at 30 degrees C and pH 6.5 in a buffer system with 5% (v/v) of DMSO as a cosolvent . The enzyme was quite stable at pH 7.5 and retained full activity after incubation at 40 degrees C for 6 h . Interestingly, when the cosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative additive to help disperse the water-insoluble substrate, the apparent activity of the epoxide hydrolase significantly increased by about 1.8-fold, while the temperature optimum shifted from 30 to 40 degrees C and the half-life of the enzyme at 50 degrees C increased by 2.5 times . The enzymatic hydrolysis of rac-GPE was highly enantioselective, with an E-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously higher than that (41.2) observed in the DMSO-containing system. Biotechnol Prog, 2003 Mar-Apr, 19(2), 493 - 4 Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) of alpha-amylases using a modified alginate; Mondal K et al.; The crude extracts of alpha-amylases when mixed with alginate, tert-butyl alcohol, and ammonium sulfate resulted in an interfacial precipitate containing polymer-bound amylase . The precipitate was dissolved in 1 M maltose to recover alpha-amylase activity . The recovery of alpha-amylases were 74%, 77%, and 92% in the case of Bacillus amyloliquefaciens, wheat germ, and porcine pancreas, respectively . All purified preparations showed a single band on SDS-PAGE. Biol Chem, 2003 Feb, 384(2), 295 - 304 Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin; Poerio E et al.; The proteinase inhibitor WSCI, active in inhibiting bacterial subtilisin and a number of animal chymotrypsins, was purified from endosperm of exaploid wheat (Triticum aestivum, c.v . San Pastore) by ion exchange chromatography and its complete amino acid sequence was established by automated Edman degradation . WSCI consists of a single polypeptide chain of 72 amino acid residues, has a molecular mass of 8126.3 Da and a pl of 5.8 . The inhibition constants (Ki) for Bacillus licheniformis subtilisin and bovine pancreatic alpha-chymotrypsin are 3.92 x 10(-9) M and 7.24 x 10(-9) M, respectively . The inhibitor contains one methionine and of tryptophan residue and has a high content of essential amino acids (41 over a total of 72 residues), but no cysteines . The primary structure of WSCI shows high similarity with barley subtilisin-chymotrypsin isoinhibitors of the Cl-2 type and with maize subtilisinchymotrypsin inhibitor MPI . Significant degrees of similarity were also found between sequences of WSCI and of other members of the potato inhibitor I family of the serine proteinase inhibitors . The wheat inhibitor WSCI has a single reactive site (the peptide bond between methionyl-48 and glutamyl-49 residues) as identified by affinity chromatography and sequence analysis. Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 687 - 92 {Cloning and expression product of vip3A gene from Bacillus thuringiensis and analysis of inseceicidal activity}; Chen JW et al.; The vip3 A gene in a size of 2.3 kb amplified from wild-type Bacillus thuringiensis strain S184 by PCR was cloned into pGEM-T Easy vector and its sequence was analysized by DNASTAR . The plasmid pOTP was constructed by inserting vip3A-S184 gene into the expression vector pQE30 and then was transformed into E . coli M15 . E . coli M15 cells harbouring the plasmid pOTP were induced with 1 mmol/L IPTG to express 89 kD protein which was confirmed to be Vip3A-S184 by Western blot . Experiments showed that about 19% of Vip3A-S184 proteins were soluble, and others were insoluble proteins and formed inclusion bodies observed by transmission electron microscopy(TEM) . The target protein was purified under the native condition and the polyclonal antibody was prepared by immunizing rabbits . The polyclonal antibody was used to detect Vip3A proteins expressed in Bacillus thuringiensis . Bioassay showed that Vip3A-S184 showed a high toxicity against 3 tested insect larvae including Spodoptera exigua, Spodoptera litura and Helicoverpa armigera. J Am Mosq Control Assoc, 2003 Mar, 19(1), 63 - 6 Ice granules containing endotoxins of microbial agents for the control of mosquito larvae--a new application technique; Becker N; This study investigated a new method for delivering microbial mosquito control agents into aquatic sites as ice granules for mosquito control . Solutions containing powder formulations of Bacillus thuringiensis israelensis (Bti) or Bacillus sphaericus were transformed into ice pellets (named IcyPearls) using a special ice-making machine . This new technique was demonstrated to have the following advantages over Bti sand granules: 1) the Bti ice pellets melted on the water surface and released the microbial crystals there; 2) the control agent remained inside the ice pellets during the application and were not lost by friction in the spraying equipment; and 3) the ice formulation resulted in increased swath widths, significantly reducing the cost of application . In large field tests . IcyPearls have been applied at dosages of 5 and 10 kg/ha containing 400 g as well as 100, 200, and 400 g of VectoBac WDG (3,000 ITU/mg), respectively, against larvae of Aedes vexans . Mortality rates of 91-98% were achieved. J Am Mosq Control Assoc, 2003 Mar, 19(1), 39 - 46 Emergence of resistance and resistance management in field populations of tropical Culex quinquefasciatus to the microbial control agent Bacillus sphaericus; Mulla MS et al.; In recent years, highly potent mosquitocidal strains of the microbial agent Bacillus sphaericus (Bsph) have been isolated and developed for the control of mosquito larvae around the world . Laboratory selection experiments with the most active strains and their use in large-scale operational mosquito control programs resulted in the emergence of resistance in larvae of the Culex pipiens complex . This generated great concern among vector control agencies around the world, who feared reduced efficacy of this highly active larvicidal agent . To address this issue, the current studies were started to find practical strategies for controlling resistant mosquitoes and more importantly to develop resistance management strategies that would prevent or delay development of resistance . We initiated field studies in 3 low-income communities in Nonthaburi Province, Thailand . In 1 of the communities, larvae of Culex quinquefasciatus that were highly resistant (>125,000-fold) to Bsph strain 2362 were successfully controlled with applications of Bacillus thuringiensis var . israelensis (Bti) alone or in combination with Bsph . To prevent or delay resistance to Bsph, 2 other sites were selected, 1 treated with Bsph 2362 alone and the other treated with a mixture of Bsph 2362 and Bti . Mosquitoes treated with Bsph 2362 alone showed some resistance by the 9th treatment and almost complete failure of control occurred by the 17th treatment . After 9 treatments with the mixture over a 9-month period at another site, no noticeable change in susceptibility to Bsph was detected . During this period, the site treated with Bsph alone required 19 treatments, whereas the site treated with mixtures took only 9 treatments because of slower resurgence of larvae at the site treated with the mixture than at the site treated with Bsph alone . This is the 1st field evidence for delay or prevention of resistance to microbial agents in larval Cx . quinquefasciatus by using mixtures of Bti and Bsph . Further studies on the use of mixtures for the management of field resistance are warranted. Can J Microbiol, 2003 Jan, 49(1), 37 - 44 Antibacterial activity of Cry- and Cyt-proteins from Bacillus thuringiensis ssp . israelensis; Yudina TG et al.; Mosquitocidal endotoxins Cry4B, Cry11A, and CytA from Bacillus thuringiensis ssp . israelensis as well as the products of their limited proteolysis display antibacterial activity relative to Micrococcus luteus . The endotoxin Cry11A also induces the lysis of the micrococcus protoplasts . Potassium and sodium ions and N-acetylgalactosamine increased the antibacterial effect of Cry11A, whereas glucose and N-acetylglucosamine inhibited it . The endotoxin Cry11A displays the antibacterial effect on some other microorganisms. Cancer, 2003 Apr 15, 97(8 Suppl), 2099 - 108 Southwest Oncology Group studies in bladder cancer; Crawford ED et al.; Over 50,000 patients are diagnosed annually with bladder cancer, and approximately 10,000 eventually will die of their disease . Thus, the Southwest Oncology Group (SWOG) Genitourinary Cancer Committee is committed to the study of therapeutic interventions in patients with superficial, invasive, and metastatic bladder cancer . In the past 15 years, SWOG has completed six Phase III, randomized trials . Studies in patients with superficial disease have established the role of bacillus Calmette-Guerin in patient management; and a large, randomized trial has outlined the value of neoadjuvant chemotherapy and cystectomy in patients with advanced disease . SWOG plans to build on this model by evaluating patients with residual disease after chemotherapy for possible bladder preservation while evaluating more chemotherapy for patients with persistent disease . The Genitourinary Cancer Committee will continue to seek new, active agents for metastatic disease and will participate in and support large, Phase III, international trials that seek to improve current regimens . SWOG accomplishments in bladder cancer are highlighted, and future strategies are discussed . Cancer, 2003 Apr 15, 97(8 Suppl), 2090 - 8 Overview of bladder cancer trials in the Cancer and Leukemia Group B; Small EJ et al.; The Cancer and Leukemia Group B (CALGB) Genitourinary Committee has developed a broad range of clinical trials across most stages of bladder cancer . Recurrence rates of superficial bladder cancer after transurethral resection range from 50-70% . Although adjuvant bacillus Calmette-Guerin reduces the risk of disease recurrence or progression, only 30% of patients have long-term disease-free survival . Because the development of novel secondline agents is needed, the CALGB is evaluating the utility of intravesicle gemcitabine as well as an oral proapoptotic agent (CP-461) . In patients with locally advanced disease with an increased risk of disease recurrence after cystectomy, a randomized trial of conventional chemotherapy versus sequential dose-dense therapy is under development . The gemcitabine/cisplatin combination has become a commonly used regimen for the treatment of advanced transitional cell carcinoma (TCC) . The CALGB is undertaking a Phase II study that incorporates a fixed dose rate gemcitabine infusion in this regimen, together with a selective epidermal growth factor receptor tyrosine kinase inhibitor, Iressa (Astra Zeneca, Wilmington, DE) . In patients with renal insufficiency, a regimen of carboplatin, gemcitabine, and Iressa is planned . Novel agents, including arsenic trioxide and trastuzumab (Herceptin; Genentech, Inc., South San Francisco, CA), are being evaluated as secondline therapy in patients with advanced TCC who have disease progression after frontline therapy . Pediatr Res, 2003 Jul, 54(1), 105 - 12 Epub 2003 Apr 02. BCG promotes cord blood monocyte-derived dendritic cell maturation with nuclear Rel-B up-regulation and cytosolic I kappa B alpha and beta degradation; Liu E et al.; Mycobacterium bovis bacillus Calmette-Guerin (BCG) is given to millions of neonates in developing countries as a vaccine against Mycobacterium tuberculosis; however, little is known about the initiation of response in neonatal dendritic cells (DCs) to BCG . To address this issue, the interaction of BCG with human cord blood monocyte-derived DCs was studied . We showed that BCG could promote cord blood monocyte-derived DC maturation by up-regulation of CD80, CD83, CD86, CD40, and MHC class II molecules and down-regulation of mannose receptor . BCG was able to induce similar levels of tumor necrosis factor-alpha and IL-10 but no bioactive IL-12p70 production from cord blood DCs as from adult blood DCs . Functionally BCG-treated cord blood DCs had higher ability to induce mixed lymphocyte reaction than non-BCG-treated cord blood DCs . Both non-BCG-treated and BCG-treated cord blood DCs efficiently induced a high level of IL-10, medium level of interferon-gamma, but little IL-4 production by cord blood naive CD4+ T cells . Heat shock protein 65, a key component of BCG, had no effect on cord blood DC maturation in terms of CD86, MHC class II, and mannose receptor up-regulation . During the BCG-induced maturation process of cord blood DCs, nuclear transcription factor Rel-B was up-regulated and cytosolic Rel-B down-regulated with cytosolic IkappaB alpha and beta degradation . These results suggest that BCG can promote cord blood monocyte-derived DC maturation, and that the mechanism is through the up-regulation of nuclear Rel-B secondary to the degradation of cytosolic IkappaB alpha and beta. Acta Pol Pharm, 2002 Nov-Dec, 59(6), 433 - 5 Sterility and antibacterial activity of some antibiotics sterilized by irradiation; Muszynski Z et al.; Sterility and antibacterial activity of several antibiotics (including some penicillins and their salts, gramicidin and neomycin) subjected to sterilization by irradiation has been studied . The compounds in solid phase have been exposed to gamma irradiation in air atmosphere at room temperature, with a dose of 25 kGy, and afterwards they have been subjected to tests recommended by FP V (volume I, 1990) checking their sterility and activity . The results have shown that the majority of initial compounds have been to a slight degree contaminated by bacteria from the genera Bacillus and Micrococcus, the number of bacteria did not exceed 10(2) CFU, and fungi up to 10 CFU in 1 g of the compound . All compounds subjected to sterilization with a dose of 25 kGy were sterile and preserved the activity required by FP V . The decrease in activity observed for some compounds was always within the limits of FP specification . The results have proved that the penicillins analysed, gramicidin and neomycin can be sterilized by irradiation with a dose of 25 kGy, without any detrimental effect on their properties and antibacterial activity. J Biol Chem, 2003 Jun 6, 278(23), 20659 - 66 Epub 2003 Mar 31. Coordination geometries of metal ions in d- or l-captopril-inhibited metallo-beta-lactamases; Heinz U et al.; d- and l-captopril are competitive inhibitors of metallo-beta-lactamases . For the enzymes from Bacillus cereus (BcII) and Aeromonas hydrophila (CphA), we found that the mononuclear enzymes are the favored targets for inhibition . By combining results from extended x-ray absorption fine structure, perturbed angular correlation of gamma-rays spectroscopy, and a study of metal ion binding, we derived that for Cd(II)1-BcII, the thiolate sulfur of d-captopril binds to the metal ion located at the site defined by three histidine ligand residues . This is also the case for the inhibited Co(II)1 and Co(II)2 enzymes as observed by UV-visible spectroscopy . Although the single metal ion in Cd(II)1-BcII is distributed between both available binding sites in both the uninhibited and the inhibited enzyme, Cd(II)1-CphA shows only one defined ligand geometry with the thiolate sulfur coordinating to the metal ion in the site composed of 1 Cys, 1 His, and 1 Asp . CphA shows a strong preference for d-captopril, which is also reflected in a very rigid structure of the complex as determined by perturbed angular correlation spectroscopy . For BcII and CphA, which are representatives of the metallo-beta-lactamase subclasses B1 and B2, we find two different inhibitor binding modes. J Virol Methods, 2003 Apr, 109(1), 89 - 93 Detection of molecular variability in rice tungro bacilliform viruses from India using polymerase chain reaction-restriction fragment length polymorphism; Joshi R et al.; Rice tungro bacilliform virus (RTBV) with rice tungro spherical virus (RTSV) causes the destructive tungro disease of rice . In order to ascertain the molecular variability of RTBV in India, primers were designed to amplify a polymorphic DNA fragment of the virus . Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis on a number of field isolates indicated mixed infections and molecular heterogeneity in the viral genome. Microb Pathog, 2003 Apr, 34(4), 179 - 86 Differential expression of the invasion-associated locus B (ialB) gene of Bartonella bacilliformis in response to environmental cues; Coleman SA et al.; Bartonella bacilliformis is the causative agent of the biphasic human disease, Oroya fever . During the primary disease phase, up to 100% of the circulating erythrocytes can be parasitized and 80% lysed . During the secondary phase of this disease, bacterial invasion shifts to endothelial cells lining the vasculature . B . bacilliformis is transferred between human hosts by the sandfly, Lutzomyia verrucarum . To investigate the regulation of ialB by environmental cues signaling vector-to-host transmission; nuclease protection assays were performed to compare the amount of ialB mRNA in bacteria subjected to temperature shift, pH change, oxidative stress, or hemin limitation . The amount of ialB mRNA increased by 223-310% in acid-treated samples and decreased by 28-39% in base-treated samples as compared to bacteria kept at pH 7.2 . B . bacilliformis samples showed a 56-63% and 74-80% decrease in ialB mRNA when shifted to 37 degrees C from growth temperatures of 20 and 30 degrees C, respectively . Oxidative stress (1 mM H(2)O(2)) and hemin limitation had no significant effect on mRNA levels . Determination of IalB protein amounts using SDS-PAGE and immunoblotting showed the greatest amounts of IalB under acidic conditions or at 20 degrees C . The least amount of IalB was synthesized under basic conditions or at 37 degrees C . The viability of wild-type B . bacilliformis under the various experimental culture conditions was determined and found not to affect ialB mRNA amounts in these experiments . Finally, we compared the survival of wild-type and ialB mutant B . bacilliformis and found no difference in the viability of these two strains, demonstrating that IalB does not aid bacterial survival under these conditions . Curr Opin Plant Biol, 2003 Apr, 6(2), 191 - 8 Advances in plant biotechnology and its adoption in developing countries; Toenniessen GH et al.; Developing countries are already benefiting and should continue to benefit significantly from advances in plant biotechnology . Insect-protected cotton containing a natural insecticide protein from Bacillus thuringiensis (Bt cotton) is providing millions of farmers with increased yields, reduced insecticide costs and fewer health risks . Many other useful plant biotechnology products that can benefit poor farmers and consumers are in the research and development pipelines of institutions in developing countries, and should soon reach farmers' fields. Eur Urol, 2003 Apr, 43(4), 351 - 60; discussion 360-1 Do prognostic parameters of remission versus relapse after Bacillus Calmette-Guérin (BCG) immunotherapy exist? . analysis of a quarter century of literature; Saint F et al.; OBJECTIVE: To review prognostic factors identified in clinical trials for remission versus relapse after intravesical adjuvant Bacillus Calmette-Guerin (BCG) immunotherapy for superficial bladder cancer (Ta, T1, and carcinoma in situ) . MATERIALS AND METHODS: Information was retrieved by a MEDLINE search of the English literature . Indexing terms comprised bladder cancer, bladder neoplasm, BCG vaccine, superficial bladder cancer, immunotherapy, intravesical therapy, prognostic marker, and Bacillus Calmette-Guerin . Fifty clinical studies were assessed for the strength of their results on the therapeutic response to BCG instillation . Emphasis was placed on clinical trials that assessed tumor and/or host characteristics, immunological reactions, recurrence rates, progression rates and disease-specific survival after BCG . RESULTS: The predictive value of host factors is extremely controversial, but marked adverse reactions to BCG instillation appear to be associated with a better tumor response . Traditional pathological tumor characteristics, molecular markers (p53) and immunological status (PPD skin test) do not appear to have prognostic value in this setting . There is increasing evidence that immunologic markers are predictive of the BCG response, but most of them have not yet been assessed in large prospective studies . Histologic/cytologic response criteria are the critical determinant of post-BCG outcome . CONCLUSIONS: After a quarter century of clinical research, no independent prognostic factor for the bladder tumor response to BCG has yet been identified . Sophisticated individual therapeutic approaches (SITA) appear to be the most promising . Nomograms based on host, tumor and immunological characteristics may help with clinical decision-making and with rationalized BCG schedule design. Biochim Biophys Acta, 2003 Apr 7, 1621(1), 31 - 40 A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag; Ferrandon S et al.; Site-directed mutagenesis was carried out to investigate the roles of a number of highly conserved residues of the chitin-binding domain (ChBD) of Bacillus circulans chitinase A1 (ChiA1) in the binding of chitin . Analysis of single alanine replacement mutants showed that mutation of an exposed tryptophan residue (Trp(687)) impaired the binding to chitin, while mutation of other highly conserved residues, most carrying aromatic or hydrophobic side chains, did not significantly affect the binding activity . Interestingly, replacement of Trp(687) with phenylalanine significantly reduced chitin-binding activity at lower salt concentrations (0-1 M NaCl) but allowed strong binding to chitin at 2 M NaCl . Since Trp(687) is conserved among the ChBDs belonging to the bacterial ChiA1 subfamily, the data presented suggest a general mechanism in which this exposed tryptophan, which is located in the cleft formed between two beta-sheets as revealed by the solution structure {J . Biol . Chem . 275 (2000) 13654}, makes a major contribution to ligand binding presumably through hydrophobic interactions . Furthermore, modulation of the chitin-binding activity by the conserved amino acid replacement (W687F) and a shift in the ionic strength of buffer has led to the development of an elutable affinity tag for single column purification of recombinant proteins. Immunology, 2003 Apr, 108(4), 548 - 55 A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis; Skinner MA et al.; The Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis . There is a need for improved vaccines or vaccine strategies for control of these diseases . A recently developed prime-boost strategy was investigated for vaccination against M . bovis infection in mice . BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT-6 and antigen 85 A and boosted with attenuated M . bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge . Before challenge, the antigen-specific production of interferon-gamma (IFN-gamma) was evaluated by ELISPOT and antibody responses were measured . The prime-boost stimulated an increase in the numbers of IFN-gamma producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used . Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1 . BCG and WAg520 alone generally gave a 1-2 log10 reduction in bacterial load in lungs or spleen, compared to non-vaccinated or plasmid DNA only control groups . The prime-boost regimen was not more effective than BCG or WAg520 alone . These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis . However, priming with the DNA vaccine and boosting with an attenuated M . bovis vaccine enhanced IFN-gamma immune responses compared to vaccinating with an attenuated M . bovis vaccine alone, but did not increase protection against a virulent M . bovis infection. Przegl Epidemiol, 2002, 56(4), 577 - 86 {Eradication of leprosy and public health . Vaccination and multidrug therapy}; Janaszek W; Leprosy is a disease, which still affects large populations in the developing countries particularly in Africa, Asia and Latin America . For the last 15 years significant advances have been made towards leprosy elimination . The most effective strategy for leprosy control is an early identification of cases and an effective treatment with multidrug therapy (MDT) . The vaccination against leprosy plays only an additional role . There are two possible approaches to develop vaccine against leprosy . One is to produce a vaccine based on organisms related to M . leprae, such as: BCG, ICRC bacillus, Mycobacterium w, Mycobacterium vaccae, Mycobacterium habana . However, these organisms related to M . leprae are not very promising in experimental animal studies . In 1970s a new vaccine was prepared based on killed M . leprae . This vaccine, tested alone and together with BCG revealed little impact on increasing vaccine efficacy . The success in cloning and expressing the M . leprae genome in E . coli created the possibility of moving towards a second generation vaccine using peptide antigens . Up till now only MDT has essential impact on decline of global leprosy prevalence . Out of 122 endemic countries in 1985, 107 countries have reached elimination of leprosy at country level . At the end of 2000 leprosy was a public health problem only in 15 countries (prevalence rate > 1/10.000) . Currently leprosy remains a problem mainly in 6 major endemic countries . Among these, India alone accounts for 64% of prevalence and 78% of detection worldwide. Biopolymers, 2003 Apr, 68(4), 486 - 96 Elongational flow studies on conformational change in DNA induced by DNA-binding protein HU; Endoh T et al.; Interaction of DNA-binding protein HU from Bacillus stearothermophilis (HUBst) with coliphage T2 DNA was investigated by observing an elongational flow-induced birefringence, Deltan, of a T2-phage DNA aqueous solution at various HU concentrations . Localized flow birefringence was observed in the pure elongational flow region, and the strain rate dependence of Deltan had a critical strain rate epsilon;(c) for the appearance of flow birefringence at all of the HU concentrations examined, indicating that a coil-stretch transition occurred at epsilon;(c) in each DNA-HU system . For strain rates larger than epsilon;(c), Deltan increased rapidly and then gradually, approaching a plateau value . The value of epsilon;(c) increased with an increase in HU concentration . Analysis based on the relationship between epsilon; (c) and the Rouse-Zimm relaxation time revealed that the increase in epsilon;(c)with increase in HU can be explained by the decrease in the size of the DNA-HU complex . The plateau birefringence value, Deltan(p), decreased at small HU concentrations but did not change at larger HU concentrations . Considering that Deltan(p) is related to the orientational order parameter of segments, it was concluded that there were at least two stages in the process of compaction of DNA induced by HU . West Afr J Med, 2002 Oct-Dec, 21(4), 310 - 2 Neonatal intensive care unit: reservoirs of nosocomial pathogens; Newman MJ; Improvement in the care and treatment of neonates had contributed to their increased survival . Nosocomial infection remains an important problem in intensive care units . Hospital wards had been shown to act as reservoirs of pathogenic microorganisms associated with infection . To assess the prevalence of pathogenic organisms in the environment of the neonatal unit, 92 swabs were randomly collected from cots, incubators and various equipments in the unit and were cultured on Blood agar and MacConkey agar plates . Air contamination was detected by exposing the same types of agar plates for 3 hours in several areas of the unit . After 48 hours incubation, isolates were identified biochemically . There is marked congestion in the unit . Ninety one percent of swabs yielded growth, with coagulase negative Staphylococcus being the predominant organism (44%), followed by Bacillus species (20%), E . coli (12.5%), and Klebsiella (8.5%), Pseudomonas species (3.6%) and moulds (3.6%) . Sedimentation plates had colony counts of from 10 - 100 per plate and the majority of the cultures were polymicrobial cultures . The presence of various Gram-negative bacili including known neonatal pathogens (like E . Coli and Pseudomonas) especially on ward equipment and congestion in the ward has the potential to cause nosocomial infection. Life Sci Space Res, 1970, 8, 77 - 81 The effect of high vacuum on oxidative reactions in bacteria and the activity of certain enzymes; Imshenetsky AA et al.; Among the physical factors which might influence micro-organisms one of the most potentially interesting is high vacuum . The effect of high vacuum is less studied as compared with other physical factors . It is impossible to achieve, under laboratory conditions, a vacuum of the order 10(-16) mm Hg which is probably characteristic of space . Earlier, the effect of high vacuum was studied on different bacteria, yeasts, molds and algae . It appeared that spores and fungal conidia were not killed by high vacuum . Later, the effect of high vacuum on physiological processes in micro-organisms was studied . The ability to oxidize glucose or ethanol was studied with Sarcina flava and Bacillus simplex cells after they were subjected for 72 hr to vacuum (10(-8) to 10(-9) mm Hg) . The oxidation rate was followed polarographically . The oxidative ability of S . flava cells diminished {correction of dimished} after their subjection to vacuum, while B . simplex spores were unchanged in that respect . The following crystalline enzymes were subjected for 72 hr to the same vacuum: alpha-amylase, catalase, ribonuclease, trypsine and urease . Then the activity of the above enzymes was tested on corresponding substrates . Not a single enzyme was totally inactivated . About 50% of activity was lost with alpha-amylase; 25--35% of activity with catalase, ribonuclease and urease . Trypsine retained its total activity . Thus, high vacuum cannot be listed among factors rapidly inactivating enzymes of micro-organisms. J Clin Pathol, 2003 Apr, 56(4), 316 - 8 Anaerobiospirillum succiniciproducens bacteraemia; Pienaar C et al.; This report describes a case of bacteraemia caused by Anaerobiospirillum succiniciproducens . Anaerobiospirillum succiniciproducens is a rare cause of bacteraemia in humans, and when encountered usually occurs in immunocompromised patients . The organism is an anaerobic, spiral shaped, Gram negative bacillus with bipolar tufts of flagella . In this report, the morphology, with special reference to electron microscopic features, culture characteristics, and antimicrobial susceptibility are described. J Zhejiang Univ Sci, 2003 Mar-Apr, 4(2), 142 - 51 Influence of medium components on elastase production using crude sources by Bacillus sp . EL31410; He GQ et al.; A newly isolated strain EL31410, producing elastase (E.C3.4.4.7) with high elastolytic activity was identified as Bacillus sp . In the medium optimization, it was found that wheat bran and soybean flour hydrosate were the best crude carbon and nitrogen source for enzyme production, respectively . Addition of corn steep flour can affect the bacterium growth and elastase production . A fractional factorial design was applied to study the main factors that affect the enzyme production, and central composite experimental design and response surface methodology were adopted to derive a statistical model for the effect of wheat bran and soybean flour hydrosate on elastase production . The experimental results showed that wheat bran had positive effect but soybean flour hydrosate had negative effect, on enzyme production . An initial concentration of 3.4% (w/v) wheat bran and 9.4% (v/v) soybean flour hydrosate were found to be optimal for enzyme production in batch culture . The time course of elastase production in the optimized medium composition was also described. N Z Med J . 2003 Mar 14;116(1170):U354. Symptom complaints following aerial spraying with biological insecticide Foray 48B; Petrie K et al.; AIM: To investigate the effect of aerial Bacillus thuringiensis (Foray 48B) spraying on self-reported symptom complaints, health perceptions, and visits to healthcare providers . METHODS: Two hundred and ninety two residents within the Ministry of Agriculture and Forestry (MAF) West Auckland spray zone were recruited by a door-to-door survey of homes within the most intensively sprayed area ten weeks prior to the first aerial spraying . Participants completed a symptom checklist and a questionnaire measuring health perceptions . Three months after the start of spraying, 181 (62%) of the original participants responded to a similar postal questionnaire . Symptom reports, health perceptions and visits to healthcare providers were compared between the baseline and the follow-up questionnaire . Rates of symptom complaints in respondents with previously diagnosed asthma, hay fever, or other allergies were compared to those in respondents without these prior health conditions . RESULTS: Symptom complaints increased significantly following the aerial spraying, in particular: sleep problems, dizziness, difficulty concentrating, irritated throat, itchy nose, diarrhoea, stomach discomfort, and gas discomfort . Analyses showed a significant increase in symptoms in those participants with a previous history of hay fever . While overall self-ratings of health decreased following the spraying, most residents saw their health as unaffected by the spray programme, and there was no significant increase in visits to general practitioners or alternative healthcare providers . CONCLUSIONS: Aerial spraying with Foray 48B is associated with some adverse health consequences in terms of significant increases in upper airway, gastrointestinal, and neuropsychiatric symptoms, as well as a reduction in overall perception of health in the exposed population./ Int J Urol, 2003 Apr, 10(4), 183 - 9 Sufficient prophylactic efficacy with minor adverse effects by intravesical instillation of low-dose bacillus Calmette-Guérin for superficial bladder cancer recurrence; Irie A et al.; BACKGROUND: Intravesical instillation of bacillus Calmette-Guerin (BCG) is the most efficient strategy for prophylaxis of superficial bladder cancer recurrence . Adverse effects of BCG are major obstacles, but the reduction of BCG dose could minimize these effects . The efficacy and adverse effects of half-dose (40 mg) BCG, Tokyo 172 strain, were prospectively evaluated . METHODS: A total of 93 patients with superficial bladder cancer (pTa or pT1) were sequentially assigned to receive either 40 or 80 mg of BCG after transurethral resection . BCG was administered weekly for 6 weeks postoperatively . Eighty patients observed longer than 12 months after BCG therapy (41, 40 mg group; 39, 80 mg group) were analyzed . RESULTS: BCG therapy course was completed in 71 patients . Tumor recurrence was recognized in 11 of 40 patients in the 40 mg group and in 5 of 31 patients in the 80 mg group . There was no significant difference in tumor recurrence rate between the two groups (P = 0.547) . BCG therapy was withdrawn in 1 patient in the 40 mg group and in 8 patients in the 80 mg-group because of BCG-related adverse effects . The morbidity of BCG-related toxicity was significantly higher in the 80 mg group . CONCLUSION: Half-dose of BCG Tokyo 172 strain had a similar efficacy and its toxicity was significantly lower compared to the standard dose . Thus, half-dose of this strain might be suitable, at least for initial BCG therapy, for the prophylaxis of bladder cancer recurrence . Further study would be necessary to clarify the efficacy of low-dose instillation in high-risk patients. Mol Microbiol, 2003 Apr, 48(1), 77 - 84 Genes required for mycobacterial growth defined by high density mutagenesis; Sassetti CM et al.; Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide . Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents . Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth . These genes include those that can be assigned to essential pathways as well as many of unknown function . The genes important for the growth of M . tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria . In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories. Kekkaku, 2003 Jan, 78(1), 45 - 9 {HIV infection and tuberculosis}; Nagai H; The number of people infected with human immunodeficiency virus (HIV) is gradually increasing in Japan, and the morbidity rate from tuberculosis in the Japanese people is high . Accordingly, the number of cases with both infections is considered to increase in the future . Our hospital has already encountered 31 cases of HIV associated tuberculosis . HIV infects mainly CD4-positive cells . The extreme decrease in the cell count results in serious cellular immunological disorder . CD4-positive cell disorder induces disorders of B lymphocytes, cytotoxic T cells, natural killer cells, and macrophage functions . These destructive conditions show the state of immunodeficiency including macrophage that are most important for defense of acid-fast bacterial infection . Migration and activation of macrophages with cytokines derived from T cells are impaired to induce the following phenomena: hypoplasia of granuloma, failure of tubercule bacillus suppression, the spread to regional lymph nodes (hilar or mediastinal lymph nodes), and hematogenous dissemination . On this occasion, caseous necrosis and cavitation are unlikely to occur, and false-negative tuberculin reaction is often observed . The incidence of severe cases, which include miliary tuberculosis, tuberculous meningitis, etc., and extrapulmonary tuberculosis, are high among acquired immunodeficiency syndrome (AIDS)-associated tuberculosis cases . HIV-infected tuberculosis cases are generally regarded as endogenous exacerbation, but they include primary infection and reinfection as well . Even during the treatment for drug-sensitive strains particularly, some cases may have reinfection with multidrug-resistant bacteria, suggesting that caution should be taken against this point . Conversely, the association of tuberculosis is a factor for the poor prognosis of HIV infection, since it facilitates the development of HIV infection . If the bacteria belong to a drug-sensitive strain, the infection with them responds well to antituberculous drugs, the same as in tuberculosis cases without HIV infection, showing a favorable prognosis . However, the mortality rate of infection with multi-drug-resistant tuberculosis is extremely high . The combined use of a protease inhibitor, i.e., anti-HIV drug, with rifampicin is regarded as contraindication for the treatment because rifampicin strongly induces hepatic cytochrome P-450 and increases the metabolism of protease inhibitors and nonnucleoside reverse transcriptases to markedly decrease the blood concentrations . Accordingly, the treatment for tuberculosis should take priority over that for HIV infection in HIV-infected tuberculosis, and highly active antiretroviral therapy (HAART) may be administered after the treatment of tuberculosis . When HAART is necessary for the treatment during the tuberculosis treatment, rifampicin had better be exchanged to rifabutin because the effect of rifabutin to induce cytochrome P-450 is less potent than that of rifampicin . A report has recently shown that the exacerbation of pyrexia and chest X-ray findings was transiently observed approximately 2 weeks after potent anti-HIV therapy for HIV-infected tuberculosis, which included a protease inhibitor . The reason for the exacerbation has been believed to be that the impaired function of CD4-positive cells is improved by the administration of anti-HIV drugs to raise temporarily the reaction of the vital part to M . tuberculosis . A tuberculin skin test (TST) reaction size of > or = 5 mm of induration is considered positive (i.e., indicative of M . tuberculosis infection) in persons who are infected with HIV . Persons with a TST reaction size > or = 5 mm who have not previously received treatment for M . tuberculosis infection should receive tuberculosis preventive treatment . Prevention by BCG vaccination is regarded as contraindications for HIV-infected patients, because disseminated M . bovis infection may be associated with them . Many HIV-positive patients infected with tuberculosis show uneventful healing, when M . tuberculosis is the sensitive strain . However, since some patients show the rapid course of tuberculosis, clinical physicians keep the early detection of tuberculosis for HIV-infected patients and the association of HIV infection for tuberculosis patients in their mind, respectively. J Vet Med Sci, 2003 Feb, 65(2), 187 - 91 Effects of Bacillus thuringiensis Cry1Ab toxin on mammalian cells; Shimada N et al.; The Cry proteins produced by Bacillus thuringiensis are considered to be highly specific insecticidal proteins . Judged to be safe for humans and farm animals due to their insect-oriented selective toxicity, the proteins have been utilized as a biological pesticide and introduced into genetically modified plants . However, some critical fundamental characters of the Cry proteins remain unclear, and the direct effects of activated Cry proteins on mammalian cells have not yet been fully confirmed . Therefore, in this study we employed primary cultured bovine hapatocytes as a model system to determine if Cry1Ab, a Cry protein, affects mammalian cells . There were no significant changes in the secretion of albumin or the morphology of the Cry1Ab-treated cells . The LDH release showed a tendency to increase after the administration of Cry1Ab, but not significantly . Taking these results on bovine hepatocytes into consideration, Cry1Ab has little acute toxicity on mammalian cells. Infect Immun, 2003 Apr, 71(4), 1763 - 73 Differential effects of control and antigen-specific T cells on intracellular mycobacterial growth; Worku S et al.; We investigated the effects of peripheral blood mononuclear cells expanded with irrelevant control and mycobacterial antigens on the intracellular growth of Mycobacterium bovis bacillus Calmette-Guerin (BCG) in human macrophages . More than 90% of the cells present after 1 week of in vitro expansion were CD3(+) . T cells were expanded from purified protein derivative-negative controls, persons with latent tuberculosis, and BCG-vaccinated individuals . T cells expanded with nonmycobacterial antigens enhanced the intracellular growth of BCG in suboptimal cultures of macrophages . T cells expanded with live BCG or lysates of Mycobacterium tuberculosis directly inhibited intracellular BCG . Recent intradermal BCG vaccination significantly enhanced the inhibitory activity of T cells expanded with mycobacterial antigens (P < 0.02), consistent with the induction of memory-immune inhibitory T-cell responses . Selected mycobacterial antigens (Mtb41 > lipoarabinomannan > 38kd > Ag85B > Mtb39) expanded inhibitory T cells, demonstrating the involvement of antigen-specific T cells in intracellular BCG inhibition . We studied the T-cell subsets and molecular mechanisms involved in the memory-immune inhibition of intracellular BCG . Mycobacteria-specific gammadelta T cells were the most potent inhibitors of intracellular BCG growth . Direct contact between T cells and macrophages was necessary for the BCG growth-enhancing and inhibitory activities mediated by control and mycobacteria-specific T cells, respectively . Increases in tumor necrosis factor alpha, interleukin-6, transforming growth factor beta, and vascular endothelial growth factor mRNA expression were associated with the enhancement of intracellular BCG growth . Increases in gamma interferon, FAS, FAS ligand, perforin, granzyme, and granulysin mRNA expression were associated with intracellular BCG inhibition . These culture systems provide in vitro models for studying the opposing T-cell mechanisms involved in mycobacterial survival and protective host immunity. Infect Immun, 2003 Apr, 71(4), 1656 - 61 Virulence, immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis bacillus Calmette-Guérin strains expressing the antigen ESAT-6 from Mycobacterium tuberculosis; Bao L et al.; We constructed two recombinant Mycobacterium bovis BCG (rBCG) strains expressing ESAT-6 of Mycobacterium tuberculosis, named rBCG-1 and rBCG-2 . rBCG-1 contained the ESAT-6 gene linked to BCG hsp60 and expressed a fusion protein, while rBCG-2, with a secretory sequence, could secret ESAT-6 into the culture medium . There was no evidence for increased virulence of the two rBCG strains when we made a comparison between them and BCG with regard to organ bacterial loads, lung histology, and survival time . rBCG-1 induced significantly higher specific antibody titers and stronger cellular immune response than BCG, whereas rBCG-2 had immunogenicity similar to that of the parental BCG strain . Both rBCG-1 and rBCG-2 conferred marked protection against M . tuberculosis infection, yet in terms of protective efficacy, they showed no significant improvements upon conventional BCG vaccine. J Microbiol Methods, 2003 May, 53(2), 221 - 33 New biochip technology for label-free detection of pathogens and their toxins; Grow AE et al.; microSERS is a new biochip technology that uses surface-enhanced Raman scattering (SERS) microscopy for label-free transduction . The biochip itself comprises pixels of capture biomolecules immobilized on a SERS-active metal surface . Once the biochip has been exposed to the sample and the capture biomolecules have selectively bound their ligands, a Raman microscope is used to collect SERS fingerprints from the pixels on the chip . SERS, like other whole-organism fingerprinting techniques, is very specific . Our initial studies have shown that the Gram-positive Listeria and Gram-negative Legionella bacteria, Bacillus spores and Cryptosporidium oocysts can often be identified at the subspecies/strain level on the basis of SERS fingerprints collected from single organisms . Therefore, pathogens can be individually identified by microSERS, even when organisms that cross-react with the capture biomolecules are present in a sample . Moreover, the SERS fingerprint reflects the physiological state of a bacterial cell, e.g., when pathogenic Listeria and Legionella were cultured under conditions known to affect virulence, their SERS fingerprints changed significantly . Similarly, nonviable (e.g., heat- or UV-killed) microorganisms could be differentiated from their viable counterparts by SERS fingerprinting . Finally, microSERS is also capable of the sensitive and highly specific detection of toxins . Toxins that comprised as little as 0.02% by weight of the biomolecule-toxin complex produced strong, unique fingerprints when spectra collected from the complexes were subtracted from the spectra of the uncomplexed biomolecules . For example, aflatoxins B(1) and G(1) could be detected and individually identified when biochips bearing pixels of antibody or enzyme capture biomolecules were incubated in samples containing one or both aflatoxins, and the spectra were then collected for 20 s from an area of the biomolecule pixel approximately 1 microm in diameter . In the future, we plan to investigate the use of hyperspectral imaging Raman microscopy for collecting fingerprints from all the pixels on the biochip, individually yet simultaneously, to enable the rapid detection of diverse pathogens and their toxins in a sample, using a single biochip. Anal Biochem, 2003 Mar 15, 314(2), 227 - 34 Isothermal titration calorimetric procedure to determine protein-metal ion binding parameters in the presence of excess metal ion or chelator; Nielsen AD et al.; Determination of binding parameters for metal ion binding to proteins usually requires preceding steps to remove protein-bound metal ions . Removal of bound metal ions from protein is often associated with decreased stability and inactivation . We present two simple isothermal titration calorimetric procedures that eliminate separate metal ion removal steps and directly monitor the exchange of metal ions between buffer, protein, and chelator . The concept is to add either excess chelator or metal ion to the protein under investigation and subsequently titrate with metal ion or chelator, respectively . It is thereby possible in the same experimental trial to obtain both chelator-metal ion and protein-metal ion binding parameters due to the different thermodynamic "fingerprints" of chelator and protein . The binding models and regression routines necessary to analyze the corresponding binding isotherms have been constructed . Verifications of the models have been done by titrations of mixtures of calcium chelators (BAPTA, HEDTA, and EGTA) and calcium ions and they were both able to account satisfactorily for the observed binding isotherms . Therefore, it was possible to determine stoichiometric and thermodynamic binding parameters . In addition, the concept has been tested on a recombinant alpha-amylase from Bacillus halmapalus where it proved to be a consistent procedure to obtain calcium binding parameters. Int J Dermatol, 2003 Mar, 42(3), 197 - 200 Incidence of cutaneous tuberculosis in patients with organ tuberculosis; Kivanc-Altunay I et al.; BACKGROUND: Tuberculosis continues to be a health problem in some countries . The development of resistance to antituberculitic drugs and the increase in diseases and conditions associated with immunodeficiency such as AIDS and chemotherapy have caused tuberculosis to increase recently . As a result, the incidence of cutaneous tuberculosis has been increasing as well . AIM: To detect cutaneous tuberculosis in patients with organ tuberculosis and to establish some characteristics of the relation between organ and cutaneous TB . MATERIAL AND METHODS: A total of 370 patients (145 females and 225 males), aged 2-76 years (mean age 27.5), enrolled for this screening study . These patients were hospitalized patients who already had pulmonary or extrapulmonary tuberculosis diagnosed before admission . All patients underwent a general skin examination, and, if needed, cutaneous biopsies were taken from involved skin areas . RESULTS: Three hundred and forty-seven (93.78%) out of 370 patients had pulmonary tuberculosis only or in association with one of other organ tuberculoses . Twenty-three patients had extrapulmonary TB: nine were TB adenitis, six were TB peritonitis, three were bone tuberculosis, and five were TB meningitides . Of 370 patients, only 13 (3.51%) had cutaneous TB: seven scrofuloderma (SCD; 2.16%), four lupus vulgaris (LV; 1.35%), one LV and SCD, and one Bacille Calmette-Guerin (BCG) adenitis (0.027%) . Cutaneous tuberculosis was observed in seven out of 260 patients with parenchymal tuberculosis (2.96%) . Four out of nine patients with TB adenitis (44.4%), one out of 12 pulmopleuretic (8.3%), and one out of 67 pleuresic patients (1.40%) had cutaneous TB as well . Mean age of the 13 patients was 32.46 years: mean age of SCD and LV was 24.8 and 48 years, respectively . The one patient with BCG adenitis was 7 months old . Five (62.5%) out of eight patients with SCD, and only one (20%) out of five patients with LV were new cases . Four patients with SCD had a positive family history, while LV patients did not . CONCLUSIONS: Organ tuberculosis is rarely associated with cutaneous tuberculosis . Scrofuloderma and LV are the most frequent forms of skin TB associated with organ TB in this population . Tuberculosis adenitis is the organ TB that causes cutaneous TB most often among other organ tuberculoses . More than one form of cutaneous TB affected only one patient with pulmonary TB; therefore, it is very rare . Tuberculids were not observed in any of the patients. Biochemistry, 2003 Apr 1, 42(12), 3457 - 63 A high-resolution NMR study of long-lived water molecules in both oxidation states of a minimal cytochrome c; Bertini I et al.; The interaction of water with oxidized and reduced cytochrome c from the Gram-positive bacterium Bacillus pasteurii (a 71-amino acid long monoheme cytochrome) is investigated through CLEANEX experiments and (15)N-edited ePHOGSY and Tr-ROESY experiments . It appears that a water molecule gives rise to dipolar cross-relaxation with the amide protons of Gly74 and Ile75, with a residence time longer than 0.4 ns, to account for a negative NOE . Such water molecule is present in both the oxidized and reduced species and in the X-ray structure . It appears to have a structural role . Other possible roles are discussed by comparison with the water molecules present in other c-type cytochromes . The amide proton of Cys35 is found to exchange rapidly with the solvent in the oxidized but not in the reduced protein, at variance with H/D exchange experiments, which probe a different time scale . The present data confirm that electron-transfer proteins evolved to minimize reorganization energy upon change of the oxidation state, even though the consequent variation of charge of the metal ion may induce some changes in the structure and/or dynamics of the protein. Biotechnol Bioeng, 2003 Jun 5, 82(5), 517 - 24 Accelerated process development for protease production in continuous multi-stage cultures; Raninger A et al.; A fermentation process was developed and optimized for the production of a specific protease from Bacillus licheniformis PWD-1 . Media formulations were constructed and crucial environmental parameters were optimized to enhance growth and product formation . Process dynamics of substrate consumption, biomass-, product-, as well as by-product formation were determined under controlled conditions in a bioreactor . Using kinetic data from batch- and continuous-culture experiments, a fed-batch process was developed producing proteolytic activities 10 times those found during regular batch culture . In one stage continuous stirred tank culture protease formation was completely decoupled from sporulation . Shift experiments in one-stage continuous cultures led to the development of a two-stage continuous stirred tank fermentation process using optimized conditions for growth in the first stage and protease formation in the second stage . Accordingly, the basis for a continuous production of the enzyme on a pilot scale was accomplished . Ecotoxicol Environ Saf, 2003 Mar, 54(3), 290 - 5 Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae); Moreno-Garrido I et al.; A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed . This microalgae showed a good growth rate during the experimental period, even when low enriched media were used . Sediment spiked with heavy metals {cadmium (Cd), copper (Cu), and lead (Pb)} was employed to determine the EC(50) values, using microalgal growth inhibition as the endpoint . The obtained results were as follows: Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests . The EC(50) values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg(-1) for Cd, 26 mg kg(-1) for Cu, and 29 mg kg(-1) for Pb (in experimental sediment) . The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment) . The diatom C . closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media. Protein Expr Purif, 2003 Mar, 28(1), 190 - 5 Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate; Mondal K et al.; Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) . In this process, a crude preparation of the enzyme was mixed with alginate . On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase . Enzyme activity from this interfacial precipitate was recovered using 1M maltose . Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively . Both the purified preparations showed a single band on SDS-PAGE. Protein Expr Purif, 2003 Mar, 28(1), 63 - 8 Secretory expression in Escherichia coli and single-step purification of a heat-stable alkaline protease; Fu Z et al.; Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium . The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction . The recombinant F1 protease was efficiently excreted into the culture medium using E . coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs . Both vectors contain the E . coli lac promoter-operator system . In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium . This opens the way for the large-scale production of this protease in E . coli . The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity . The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0 . The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Protein Sci, 2003 Apr, 12(4), 861 - 70 Structure and dynamics of the DNA-binding protein HU of B . stearothermophilus investigated by Raman and ultraviolet-resonance Raman spectroscopy; Serban D et al.; The histone-like protein HU of Bacillus stearothermophilus (HUBst) is a 90-residue homodimer that binds nonspecifically to B DNA . Although the structure of the HUBst:DNA complex is not known, the proposed DNA-binding surface consists of extended arms that project from an alpha-helical platform . Here, we report Raman and ultraviolet-resonance Raman (UVRR) spectra diagnostic of subunit secondary structures and indicative of key side-chains lining the proposed DNA-binding surface . Raman conformation markers show that the DNA-binding arms of the dimer contain beta-stranded structure in excess (eight +/- two residues per subunit) of that reported previously . Important among side-chain markers are Met (701 cm(-1)), Ala (908 cm(-1)), Arg (1082 cm(-1)), and Pro (1457 cm(-1)) . The Ala marker undergoes a substantial shift (908 --> 893 cm(-1)) on deuteration of alanyl peptide sites, indicating a coupled side-chain/main-chain mode of diagnostic value in the identification of exchange-protected alanines . A large subset of alanines (67%) in the alpha-helical core exhibits robust resistance to exchange . A quantitative study of NH --> ND exchange exploiting newly identified amide II' markers of helical (1440 cm(-1)) and nonhelical (1472 cm(-1)) conformations of HUBst indicates unexpected flexibility at the dimer interface, which is manifested in rapid exchange of 80% of peptide sites . The results establish a basis for subsequent Raman and UVRR investigations of HUBst:DNA complexes and provide a framework for applications to other DNA-binding architectural proteins. Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3895 - 900 Epub 2003 Mar 20. Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations; Johnson SJ et al.; DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations . Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I . The 6-bp extension involves a 20-A translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal . In addition, we obtained the structure of a "closed" conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an "open" conformation . Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail . The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site . We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases . The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed. Cancer Res, 2003 Mar 15, 63(6), 1280 - 7 Development and preclinical evaluation of a Bacillus Calmette-Guérin-MUC1-based novel breast cancer vaccine; Chung MA et al.; Due to the high incidence of breast cancer and associated mortality rate,the development of an effective vaccine may be beneficial for the prevention or adjuvant treatment of this malignancy . We have constructed a novel breast cancer vaccine, Bacillus Calmette-Guerin (BCG)-hIL2MUC1, that consists of BCG and expresses a truncated form of MUC1 and human interleukin (IL)-2 . In vitro analysis of the BCG-hIL2MUC1 construct confirmed coexpression of MUC1 and human IL-2 . The ability of BCG-hIL2MUC1 to inhibit breast cancer growth was evaluated in hu-PBL-SCID mice (severe combined immunodeficient mice reconstituted with 50 x 10(6) human peripheral blood lymphocytes) that received three biweekly injections of BCG-hIL2MUC1 (0.5 colony-forming unit) . Control animals received PBS, MUC1 peptide (100 microg), or empty vector BCG-261 (0.5 colony-forming unit) vaccination . After immunization, hu-PBL-SCID mice (n = 8 in each group) were xenografted with 4 x 10(6) ZR75-1 human breast cancer cells . Whereas mice receiving the control vaccines developed a tumor, only 87% of BCG-hIL2MUC1-immunized animals developed a palpable tumor with a slower rate of tumor growth (P < 0.001) . Histological analysis of the primary tumors in BCG-hIL2MUC1-immunized animals revealed areas of reduced MUC1 expression . CD8-positive human lymphocytes were detected only in tumors grown in BCG-hIL2MUC1-immunized animals . These results imply a critical role of coexpressed IL-2 and MUC1 in eliciting tumor-specific immune response . To our knowledge, this is the first report of BCG engineered to express a tumor-associated antigen . Our results suggest that BCG-hIL2MUC1 immunization inhibited breast cancer growth in hu-PBL-SCID mice . Therefore, BCG-hIL2MUC1 may be a promising candidate as a breast cancer vaccine. J Immunol, 2003 Apr 1, 170(7), 3696 - 706 Conservation of nonpeptide antigen recognition by rhesus monkey V gamma 2V delta 2 T cells; Wang H et al.; We have previously found that monkey Vgamma2Vdelta2(+) T cells mount adaptive immune responses in response to Mycobacterium bovis bacillus Calmette-Guerin infections . We have now analyzed rhesus monkey gammadelta T cell responses to nonpeptide Ags and superantigens . Like human Vgamma2Vdelta2(+) T cells, rhesus monkey gammadelta T cells are stimulated when exposed to prenyl pyrophosphate, bisphosphonate, and alkylamine Ags . Responsiveness was limited to gammadelta T cells expressing Vgamma2Vdelta2 TCRs . Rhesus monkey Vgamma2Vdelta2(+) T cells also responded to the superantigen, staphyloccocal enterotoxin A . Sequencing of the rhesus monkey Vgamma2Vdelta2 TCR revealed a strong sequence homology to human Vgamma2Vdelta2 TCR that preserves important sequence motifs . Moreover, chimeric TCRs that pair human Vgamma2 with monkey Vdelta2 and monkey Vgamma2 with human Vdelta2 retain reactivity to nonpeptide Ags and B cell lymphomas . A molecular model of the rhesus monkey Vgamma2Vdelta2 TCR has a basic region in the complementarity-determining region 3 binding groove that is similar to that seen in the human Vgamma2Vdelta2 TCR and preserves the topology of the complementarity-determining region loops . Thus, recognition of nonpeptide prenyl pyrophosphate, bisphosphonate, and alkylamine Ags is conserved in primates suggesting that primates can provide an animal model for human gammadelta T cell Ag responses. Biochem Biophys Res Commun, 2003 Mar 28, 303(1), 31 - 6 Cloning of a novel one-repeat calcium channel-like gene; Arias JM et al.; We describe the cloning of a cDNA from a human testis library that encodes a novel protein with similarity to one repeat of voltage-gated Ca(2+) channels (Ca(v)) . Northern and dot blot analyses indicate that the novel Ca(v)-like gene is expressed predominantly in testis and at lower levels in many other tissues . Heterologous expression of the Ca(v)-like protein did not lead to the induction of any detectable ionic current and failed to modify intracellular Ca(2+) concentrations . Similar one-repeat Ca(v)-like proteins have been cloned from Bacillus, Mus, and Homo, and appear to encode ion channels involved in renal function, axis determination, and sperm motility. Carbohydr Res, 2003 Mar 28, 338(7), 687 - 92 Carbohydrate esterase family 4 enzymes: substrate specificity; Caufrier F et al.; The substrate specificity of selected enzymes classified under Carbohydrate Esterase family 4 (CE4) has been examined . Chitin deacetylase from Mucor rouxii and both a native and a truncated form of acetyl xylan esterase from Streptomyces lividans were found to be active on both xylan and several soluble chitinous substrates . Furthermore, the activities of all enzymes examined were significantly increased in the presence of Co(2+) when chitinous substrates were employed . However, the presence of this metal ion did not result in enhancing the activities of the enzymes when xylan was used as substrate . An acetyl xylan esterase from Bacillus pumilus, classified under Carbohydrate Esterase family 7, was found to be inactive towards all chitinous substrates tested . Finally, all enzymes examined were inactive towards cell wall peptidoglycan. FEMS Microbiol Lett, 2003 Mar 14, 220(1), 127 - 31 Isolation and characterisation of a novel bacteriocin produced by Bacillus thuringiensis strain B439; Ahern M et al.; Bacillus thuringiensis strain B439 produces a bacteriocin-like inhibitory substance in its growth medium . This antimicrobial peptide, referred to as thuricin 439, acts as a bacteriocidal peptide and exhibits an apparent narrow range of inhibitory activity, essentially only affecting growth of Bacillus cereus and B . thuringiensis strains . It remains active over a relatively wide pH and temperature range, showing no loss of activity following heat treatments up to 80 degrees C . Purification of thuricin 439 was achieved using several chromatographic steps, which resulted in the identification of two peptides with inhibitory activity . These two peptides were shown to possess identical N-terminal sequences, but different molecular masses. Bioconjug Chem, 2003 Mar-Apr, 14(2), 440 - 8 Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody; Pleschberger M et al.; Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species . Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports . For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen . For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-Lys3 . The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements . Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein . Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B . sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall . Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding . Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology. PDA J Pharm Sci Technol, 2003 Jan-Feb, 57(1), 3 - 11 Effect of carrier materials on the resistance of spores of Bacillus stearothermophilus to gaseous hydrogen peroxide; Sigwarth V et al.; The testing of the H2O2 decontamination process using spores of Bacillus stearothermophilus has gained widespread acceptance . Usually, commercially available Biological Indicators (BIs) with a specified resistance to H2O2 are challenged to qualify the process . The question arises whether the resistance of test spores is dependant on the type of carrier material and whether the resistance is representative for the system under test . The objective of the study is to quantify the effect of different carrier materials on the resistance of spores of Bacillus stearothermophilus to H2O2 . Materials from which isolators were built, as well as those used in disposables during daily work were investigated . These materials were inoculated with 106 spores of Bacillus stearothermophilus (ATCC 7953) . The spore resistance was tested to a well defined H2O2 decontamination cycle by determining the D-value using the "Fractional Negative" method . This paper reports on the effect of different carrier materials to the resistance of the test organism against H2O2 . Various materials have significantly increased resistance of the spores and should be avoided in isolator systems . If commercially available BIs are used for process qualification, the resistance of the BI used, the fluctuation in resistance caused by isolator materials, the required log reduction, and at least the bioload of isolator surfaces need to be known. Lett Appl Microbiol, 2003, 36(4), 251 - 7 Importance of Gram-positive naphthalene-degrading bacteria in oil-contaminated tropical marine sediments; Zhuang WQ et al.; AIMS: The aim of this study was to isolate, characterize and evaluate the importance of naphthalene-degrading bacterial strains from oil-contaminated tropical marine sediments . METHODS AND RESULTS: Three Gram-positive naphthalene-degrading bacteria were isolated from oil-contaminated tropical intertidal marine sediments by direct isolation or enrichment using naphthalene as the sole source of carbon and energy . Bacillus naphthovorans strain MN-003 can also grow on benzene, toluene, xylene and diesel fuel while Micrococcus sp . str . MN-006 can also grow on benzene . Staphylococcus sp . str . MN-005 can only degrade naphthalene and was not able to use the other aromatic hydrocarbons tested . Strain MN-003 possessed the highest maximal specific growth rate with naphthalene as sole carbon source . An enrichment culture fed with naphthalene as sole carbon source exhibited a significant increase in the relative abundances of the three isolates after 21 days of incubation . The three isolates constituted greater than 69% of the culturable naphthalene-degrading microbial community . Strain MN-003 outcompeted and dominated the other two isolates in competition studies involving batch cultures inoculated with equal cell densities of the three isolates and incubated with between 1 and 10 mg l-1 of naphthalene . CONCLUSIONS: Three Gram-positive naphthalene-degrading bacteria were successfully isolated from oil-contaminated tropical marine sediments . Gram-positive bacteria might play an important role in naphthalene degradation in the highly variable environment of oil-contaminated tropical intertidal marine sediments . Among the three isolates, strain MN-003 has the highest maximal specific growth rate when grown on naphthalene, and outgrew the other two isolates in competition experiments . SIGNIFICANCE AND IMPACT OF THE STUDY: This research will aid in the development of bioremediation schemes for oil-contaminated marine environments . Strain MN-003 could potentially be exploited in such schemes. Bull World Health Organ, 2003, 81(1), 61 - 70 Epub 2003 Mar 11. Immunization of children at risk of infection with human immunodeficiency virus; Moss WJ et al.; This paper reviews the English language literature on the safety, immunogenicity and effectiveness in children infected with the human immunodeficiency virus (HIV) of vaccines currently recommended by WHO for use in national immunization programmes . Immunization is generally safe and beneficial for children infected with HIV, although HIV-induced immune suppression reduces the benefit compared with that obtained in HIV-uninfected children . However, serious complications can occur following immunization of severely immunocompromised children with bacillus Calmette-Gu rin (BCG) vaccine . The risk of serious complications attributable to yellow fever vaccine in HIV-infected persons has not been determined . WHO guidelines for immunizing children with HIV infection and infants born to HIV-infected women differ only slightly from the general guidelines . BCG and yellow fever vaccines should be withheld from symptomatic HIV-infected children . Only one serious complication (fatal pneumonia) has been attributed to measles vaccine administered to a severely immunocompromised adult . Although two HIV-infected infants have developed vaccine-associated paralytic poliomyelitis, several million infected children have been vaccinated and the evidence does not suggest that there is an increased risk . The benefits of measles and poliovirus vaccines far outweigh the potential risks in HIV-infected children . The policy of administering routine vaccines to all children, regardless of possible HIV exposure, has been very effective in obtaining high immunization coverage and control of preventable diseases . Any changes in this policy would have to be carefully examined for a potential negative impact on disease control programmes in many countries. J Ethnopharmacol, 2003 Apr, 85(2-3), 283 - 7 Inhibitory effect of aqueous extract from the gall of Rhus chinensis on alpha-glucosidase activity and postprandial blood glucose; Shim YJ et al.; The present study examined the inhibitory effect of aqueous extract from the gall of Rhus chinensis (AEGRC) on alpha-glucosidase activity, an enzyme responsible for digestion of carbohydrate to monosaccharides in the process of intestinal absorption . AEGRC inhibited Bacillus alpha-glucosidase acitvity with an IC(50) of 0.9 micro g/ml . Its inhibition on alpha-glucosidase was determined to be noncompetitive and reversible when the enzyme-substrate mixture was simultaneously treated with AEGRC as an inhibitor . In addition, when it was orally administered to rats with sucrose (2g/kg), AEGRC (250-1000mg/kg) significantly suppressed the increase of blood glucose levels after sucrose loading in a dose dependent manner . These results suggest that AEGRC might exert anti-diabetic effect by suppressing carbohydrate absorption from intestine, and thereby reducing the postprandial increase of blood glucose. J Food Prot, 2003 Mar, 66(3), 441 - 5 Evaluation and interlaboratory validation of a selective agar for phosphatidylinositol-specific phospholipase C activity using a chromogenic substrate to detect Listeria monocytogenes from foods; Jinneman KC et al.; Phosphatidylinositol-specific phospholipase C (PI-PLC) activity is a potential virulence factor and is exhibited only by the Listeria species Listeria monocytogenes and Listeria ivanovii . A chromogenic substrate for the direct detection of PI-PLC activity is available in a new medium (BCM L . monocytogenes plating agar) . The use of a chromogenic substrate offers a mechanism with which to directly screen for L . monocytogenes and L . ivanovii other than the esculin used in Oxford (OXF) and Palcam (PAL) agars, which screen for all Listeria species . The specificity levels of BCM plating agar and of BCM confirmation and rhamnose agars were evaluated with 107 Listeria and 10 Bacillus species isolates . In addition, BCM L . monocytogenes plating agar was compared with standard Listeria selective agars (OXF and PAL agars) with regard to the recovery of L . monocytogenes from 2,000 food and environmental samples obtained from eight participating laboratories . A Listeria species was isolated from at least one of the agars in 209 analyses, and L . monocytogenes was isolated in 135 of these analyses . In 27 of the analyses in which L . monocytogenes was isolated, one or more of the selective differential agars used failed to isolate L . monocytogenes, and therefore the results of these analyses were discrepant . Relative to a reference method involving the use of all three agars (OXF, PAL, and BCM agars), the OXF-BCM, PAL-BCM, and OXF-PAL combinations had sensitivities of 99.3, 99.2, and 90.2%, respectively . In statistical analyses of the different combinations of agars, the OXF-BCM and BCM-PAL combinations were found to be superior to the OXF-PAL combination for the detection of L . monocytogenes. Int J Med Microbiol, 2003 Feb, 292(7-8), 441 - 51 Novel vaccination strategies based on recombinant Mycobacterium bovis BCG; Dietrich G et al.; In this manuscript, we will review the utilization of Mycobacterium bovis Bacille Calmette-Guerin (BCG) as a vaccine against tuberculosis (TB) and as a carrier system for heterologous antigens . BCG is one of the most widely used vaccines . Novel techniques in genome manipulation allow the construction of virulence-attenuated recombinant (r)-BCG strains that can be employed as homologous vaccines, or as heterologous antigen delivery systems, for priming pathogen-specific immunity against infectious diseases, including TB . Several approaches are available for heterologous antigen expression and compartmentalization in BCG and recent findings show the potential to modulate and direct the immune responses induced by r-BCG strains as desired . Recent achievements in complete genome analysis of various target pathogens, combined with a better understanding of protective pathogen-specific immune responses, form the basis for the rational design of a new generation of recombinant mycobacterial vaccines against a multitude of infectious diseases. Heredity, 2003 Feb, 90(2), 141 - 9 Host-plant-associated genetic differentiation in Northern French populations of the European corn borer; Martel C et al.; The phytophagous insects that damage crops are often polyphagous, feeding on several types of crop and on weeds . The refuges constituted by noncrop host plants may be useful in managing the evolution in pest species of resistance to the Bacillus thuringiensis toxins produced by transgenic crops . However, the benefits of these refuges may be limited because host-plant diversity may drive genetic divergence and possibly even host-plant-mediated sympatric speciation . The European corn borer, Ostrinia nubilalis Hubner (Lepidoptera: Crambidae), is the main pest of maize in Europe and North America, where it was introduced early in the 20th century . It has a wide host range but feeds principally on mugwort (Artemisia vulgaris L.) and maize (Zea mays L.) . O . nubilalis is found on mugwort only in the northern part of France, whereas it is found on maize throughout France . The extent of genetic variation at allozyme markers was investigated in populations collected from the two host plants over the entire geographical distribution of the European corn borer on mugwort in France . Allelic differentiation between pairs of populations and hierarchical analyses of pools of samples from each host plant indicate that the group of populations feeding on maize differed from the group of populations feeding on mugwort . Our results suggest (1) host-plant-related divergent selection at the genomic region surrounding the Mpi locus and (2) limited gene flow between the populations feeding on mugwort and those infesting maize fields . These data indicate that adults emerging from mugwort would not be useful for managing the evolution of resistance to the B . thuringiensis toxins in European corn borer populations. Microbiology, 2003 Mar, 149(Pt 3), 729 - 37 Correlation of the rate of protein synthesis and the third power of the RNA : protein ratio in Escherichia coli and Mycobacterium tuberculosis; Cox RA; In order to further understand the different physiological states of the tubercle bacillus, a frame of reference was sought by first correlating the macromolecular compositions of Escherichia coli with specific growth rates and also with the rates of protein synthesis . Data for DNA : protein : RNA were converted to the average amounts of DNA {m(DNA(av))}, protein {m(p(av))} and RNA {m(RNA(av))} per cell . The specific growth rate micro was found to be directly proportional to m(RNA(av))/m(p(av)) . The specific protein synthesis rate per average cell {omega(p(av))} was shown to be directly proportional to the third power of the ratio m(RNA(av))/m(p(av)) which reflects the ribosome concentration . The equations derived were shown apply to both E . coli ( micro =1.73 h(-1)) and Mycobacterium bovis BCG ( micro =0.029 h(-1)). FEBS Lett, 2003 Mar 13, 538(1-3), 29 - 34 A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae; Hara H et al.; Aminopeptidase N (APN) and cadherin-like protein (BtR175) from Bombyx mori larvae were examined for their roles in Cry1Aa- and Cry1Ac-induced lysis of B . mori midgut epithelial cells (MECs) . APNs and BtR175 were present in all areas of the midgut, were particularly abundant in the posterior region, and were found only on columnar cell microvilli and not on the lateral membrane that makes cell-cell contacts . This distribution was in accordance with the distribution of Cry1A-susceptible MECs in the midgut . The lytic activity of Cry1Aa and Cry1Ac on collagenase-dissociated MECs was linearly dependent on toxin concentration . Although pre-treatment of MECs with anti-BtR175 antibody was observed to partially inhibit the lytic activity exerted by 0.1-1 nM Cry1Aa toxin or 5 nM Cry1Ac toxin, no significant inhibition was observed when MECs were pre-treated with anti-APN antibody . These results suggest that BtR175 functions as a major receptor for Cry1A toxins in the midgut of B . mori larvae. Tunis Med, 2002 Oct, 80(10), 645 - 9 {Abdomino-pelvic actinomycosis: a case report}; Bedoui R et al.; The actinomycosis is a chronic suppurative granulomatosis disease . It is owed to a bacillus gram positive; actinomyces israelli . The cervical and thoracic localizations are most frequent . The digestive localization represents 20% of cases . It interest very rarely the pelvis and the genital tracts . We bring back the observation of a patient old of 30 years admitted for mass abdominal . To the exam, the patient had a sensibility of the left hypochondriac area and we found a mass of 6 cm of diameter . To the rectal touch, we found a mass in the bag of Douglas . The echography and the computed tomography revealed a collection under the spleen and a pelvic collection . A rectotomy is performed . The bacteriological study isolates actinomyces israelli . The collection under the spleen is drained under radiological control . Actinomyces israelli is also recovered in the pus brought back by the puncture . The patient is treated by Penicillin . The patient had a favourable evolution . No etiology is found at this patient . For this observation, the collection was accessible to a drainage permitting the diagnosis and the treatment of the actinomycosis while avoiding a mutilated surgery. World J Gastroenterol, 2003 Mar, 9(3), 516 - 20 Virulence of water-induced coccoid Helicobacter pylori and its experimental infection in mice; She FF et al.; AIM: To explore the virulence and the infectivity of coccoid Helicobacter pylori (H . pylori) transformed from spiral form by exposure to sterile tap water . METHODS: Three strains of H . pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid form by exposure to sterile tap water . Both spiral and coccoid forms of H . pylori were tested for the urease activity, and the adherence to Hep-2 cells . The presence of flagella was examined under electron microscopy . In the experimental animal infection, the spiral and coccoid forms of H . pylori originated from the same strain F49 were inoculated intragastrically into BALB/c mice respectively four times at a 3-day interval . Half of the mice from each group were sacrificed at Day 21 and Day 28 after the last inoculation . Histology and H . pylori colonization were detected by urease test of gastric mucosa, cultures of H . pylori, and electron microscopy and so on . RESULTS: The urease activity and the ability of adherence to Hep-2 cells were found to be lower in coccoid H . pylori than that in its spiral form . For example, the transformation in strain F(44) led to a significant decrease of the adherence rate and adherence index from 70.0+/-5.3 % to 30.2+/-3.5 % (P<0.01), and from 2.6+/-0.4 to 0.86+/-0.3 (P<0.01), respectively . The flagella of coccoid H . pylori were observed under electron microscope . In the experimental infection in mice, the positive rate of gastric mucosa urease test was 93.8 % (15/16) in the group infected by spiral H . pylori and 50 % (8/16) in the group infected by coccoid H . pylori, and the estimated coccoid H . pylori colony number was 1.75 vs 0.56 . The positive rates of H . pylori culture were 87.5 % (14/16) in spiral H . pylori group and 68.8 % (11/16) in coccoid H . pylori group . There was no significant difference in either urease test or bacterial culture rate between the groups examined at Day 21 and Day 28 after inoculation . Electron microscopic examination of the samples taken from both groups showed the adherence of H . pylori in spiral, bacillary and coccoid shapes to the epithelial cells of gastric wall . Histological examination showed the occurrence of gastric mucosal injury as indicated by various degrees of erosion, ulcer, and inflammatory cell infiltration . Mucosal injury was slighter in the mice infected by coccoid H . pylori . No positive result was obtained in the control group that received intragastrical administration of sterile tap water . CONCLUSION: Although the virulence of coccoid H . pylori induced by water decrease, coccoid H . pylori still remains a considerable urease activity and the adhering ability to epithelial cells . Furthermore, the flagella, an important component responsible for bacterial movement and infection, were still observed as a cellular structure of coccoid H . pylori under electron microscope . The coccoid H . pylori induced by water is capable of colonizing in gastric mucosa and causing gastrititis in mice. Biotechnol Appl Biochem, 2003 Apr, 37(Pt 2), 165 - 71 Regulatory effects of alanine-group amino acids on serine alkaline protease production by recombinant Bacillus licheniformis; Calik P et al.; Influences of the concentration and addition time of alanine-group amino acids, i.e . alanine, leucine and valine, on serine alkaline protease (SAP) synthesis were investigated by Bacillus licheniformis (DSM 1969) carrying pHV1431:: sub C in a defined medium to identify the amino acids creating intracellular reaction-rate limitation in SAP production . While the precursors of alanine-group amino acids, pyruvate and alanine, did not affect SAP production considerably within the range 0-15 mM, the addition of leucine decreased both SAP production and cell formation, because of the inhibition of valine synthesis . Although valine inhibits reactio