Appl Environ Microbiol, 1995 Apr, 61(4), 1649 - 52 Cocultivation of the amoeba Naegleria fowleri and the amoebicin- producing strain Bacillus licheniformis M-4; Lebbadi M et al.; Antagonism between Bacillus licheniformis M-4 and the pathogenic amoeba Naegleria fowleri HB-1 during cocultivation was influenced by the composition of the medium and the initial amoeba/bacterium ratio . While a ratio of 50 caused complete lysis of amoebae in soil extract with 0.3% glucose (SEG) before 72 h, this ratio had to be at least 12-fold lower in order to obtain similar results in Cline medium . Sporulation of B . licheniformis M-4 took place much earlier in SEG . Amoebicin production was stimulated by the presence of amoebae by either shortening the time of production (as in SEG) or increasing the amount of amoebicins released (as in Cline medium) . Electron microscopy showed that amoebae cocultivated in the Cline medium contained bacteria enclosed in digestive vacuoles, while amoebae from SEG cocultures did not. Appl Environ Microbiol, 1995 Apr, 61(4), 1469 - 74 Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1; Lin X et al.; Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers . By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease . The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined . A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA . Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers . PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions . The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B . licheniformis NCIMB 6816 . The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria . The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases . Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media. Am J Trop Med Hyg, 1995 Apr, 52(4), 354 - 9 A review of bartonellosis in Ecuador and Colombia; Alexander B; A review of the literature regarding bartonellosis or Carrion's disease in Colombia and Ecuador is presented, together with observations made by the author in areas of both countries from which the disease has been recorded . There is evidence from pre-Columbian artifacts that verruga peruana, the cutaneous form of the disease, was present in Ecuador at least 1,000 years prior to the arrival of Europeans . These artifacts were discovered in the coastal province of Manabi, a low-lying area very different from the high Andean valleys of Peru with which bartonellosis is normally associated . Most of the cases recorded in recent years from this coastal area . The disease does not appear to have occurred in Colombia before the 1930s and only one case has been reported during the past 40 years . The possibility of many more subclinical cases being present in both Ecuador and Colombia is discussed, together with the possibility that the acquired immunodeficiency syndrome epidemic will reveal a higher prevalence among the inhabitants of endemic areas than previously suspected . Although the suspected vector of Bartonella bacilliformis, the sand fly Lutzomyia verrucarum, has not been recorded from Ecuador or Colombia, related species are present in endemic areas and may be involved in transmission. Int J Syst Bacteriol, 1995 Apr, 45(2), 409 - 11 Antibiotic susceptibility as a taxonomic characteristic of the genus Bacillus; Reva ON et al.; A large number of Bacillus strains assigned to different species were tested to determine their susceptibilities to antibiotics . Some clear differences between species were observed . The antibiotic susceptibilities of strains isolated from natural sources seemed to be stable and to reflect adaptation of the strains to specific conditions in certain ecological niches . A method for data processing which can be used for rapid species identification is described. Int J Syst Bacteriol, 1995 Apr, 45(2), 212 - 7 Random amplified polymorphic DNA fingerprinting of mosquito-pathogenic and nonpathogenic strains of Bacillus sphaericus; Woodburn MA et al.; Random amplified polymorphic DNA fingerprinting was used to examine 31 mosquito-pathogenic and 14 nonpathogenic strains of Bacillus sphaericus . We verified that DNA bands that migrated the same distance in an agarose gel were homologous by using PCR-generated probes made from the random amplified polymorphic DNA bands . The band patterns obtained with eight primers were analyzed by using the Jaccard coefficient and unweighted pair group with arithmetic average clustering . Pathogenic strains belonging to DNA homology group IIA were similar to strains belonging to nonpathogenic homology groups at an average level of similarity of 6.3% . Individual serotypes were clearly identified among the pathogenic strains . This suggests that there is overall genetic homogeneity among strains within serotypes . It is also consistent with the uniform toxicity pattern found for each serotype (unlike the toxin diversity found in Bacillus thuringiensis serotypes) . These results, together with DNA homology data, support the proposal that a new species should be described for the pathogenic strains. J Econ Entomol, 1995 Apr, 88(2), 270 - 7 Increased efficacy of Bacillus thuringiensis subsp . kurstaki in combination with tannic acid; Gibson DM et al.; We identified tannic acid as an inexpensive additive that increased the efficacy of sublethal concentrations of Bacillus thuringiensis subsp . kurstaki (Berliner) . Tannic acid mimicked the active constituents contained in an aqueous, tannin-rich extract of Taxus baccata (L.) bark that retarded development of Heliothis virescens (F.) larvae at 10,000 ppm; most larvae remained in first and second stage when treated with 250-10,000 ppm of tannic acid . Instar development of Trichoplusia ni (Hubner) larvae was affected in a concentration-dependent manner by 2.5-500 ppm of tannic acid . In subsequent bioassays, tannic acid at 25-500 ppm in combination with B . thuringiensis (1.63 micrograms {AI}/ml diet) yielded mean mortalities of 57-75%, whereas treatments with B . thuringiensis alone produced 10% mortality . Mean mortalities in the 3.0, 4.5, and 6.75 micrograms (AI) B . thuringiensis per milliliter of diet treatments (5.5; 8.0, and 30%, respectively) were significantly higher in the presence of 250 and 2,500 ppm tannic acid; in these treatments we observed 78-94% mortality . Addition of tannic acid increased the activity of concentrations of 3-4.5 micrograms (AI) B . thuringiensis per milliliter of diet to approximately that of a concentration of 13 micrograms (AI) B . thuringiensis per milliliter of diet alone (85-95% mortality) . Although deaths caused by a formulation of B . thuringiensis + tannic acid occurred more slowly than with high rates of B . thuringiensis alone, such formulations would have the advantages of arresting development, minimizing foliar damage, and decreasing the concentration of B . thuringiensis used. J Bacteriol, 1995 Apr, 177(8), 1981 - 8 Analysis of a novel gene and beta-galactosidase isozyme from a psychrotrophic Arthrobacter isolate; Gutshall KR et al.; We have characterized a new psychrotrophic Arthrobacter isolate which produces beta-galactosidase isozymes . When DNA from this isolate was transformed into an Escherichia coli host, we obtained three different fragments, designated 12, 14, and 15, each encoding a different beta-galactosidase isozyme . The beta-galactosidase produced from fragment 12 was of special interest because the protein subunit was smaller (about 71 versus 116 kDa) than those typically encoded by the lacZ family . The isozyme encoded by fragment 12 was purified, and its activity and thermostability were examined . Although the enzyme is highly specific towards beta-D-galactoside substrates, its levels in the isolate do not increase in cells grown with lactose . Nucleotide sequence determination showed that the gene encoding isozyme 12 is not similar to the other members of the lacZ family but has regions similar to beta-galactosidase isozymes from Bacillus stearothermophilus and B . circulans . Addition of the isozyme 12 sequence to the database made it possible to examine these enzymes as possible members of a new, separate family . Our analysis of this new family showed some conserved amino acids corresponding to the lacZ acid-base catalytic region but no homology with the nucleophilic region . On the basis of these comparisons, we designated this a new lacG family. J Clin Psychiatry, 1995 Apr, 56(4), 161 - 6 Bacillary angiomatosis: a treatable cause of acute psychiatric symptoms in human immunodeficiency virus infection; Baker J et al.; BACKGROUND: Bacillary angiomatosis is a systemic infection that has been most commonly reported in the setting of immunosuppression, especially human immunodeficiency virus (HIV) disease . METHOD: We report two patients who had bacillary angiomatosis who presented with psychiatric symptoms . RESULTS: The first patient presented with marked exacerbation of previous depressive disease . The second patient presented with new psychotic symptoms . In both cases psychiatric symptoms did not resolve until antibiotic treatment was given . CONCLUSION: Our report expands the clinical spectrum of bacillary angiomatosis and identifies a new cause of treatable psychiatric disease in HIV-infected persons. Br J Cancer, 1995 Apr, 71(4), 801 - 7 Bacillus Calmette-Guerin (BCG) enhances monocyte- and lymphocyte-mediated bladder tumour cell killing; Pryor K et al.; A cytotoxicity assay was used to study the action of bacillus Calmette-Guerin (BCG) and cytokines on four human bladder cancer cell lines . Monocytes and lymphocytes from peripheral blood were incubated with or without BCG or cytokines for 24 h, after which {3H}thymidine-labelled target cells were added and the 72 h percentage specific release determined . BCG had a direct cytotoxic effect against tumour cells and significantly enhanced monocyte/macrophage and enhanced lymphocyte cytotoxicity against one cell line (UCRU-BL-17) . Supernatants (SNs) from BCG-activated monocytes/macrophages and lymphocytes increased the percentage specific release of {3H}thymidine from UCRU-BL-17 cells . Interferon alpha (IFN-alpha) and interleukin 2 (IL-2) were cytotoxic towards UCRU-BL-17 . No synergy occurred between BCG and cytokines at the concentrations tested . The results suggest that BCG is superior to IFN-alpha, interferon gamma (IFN-gamma) and IL-2 in enhancing cell-mediated cytotoxicity. Chest, 1995 Apr, 107(4), 1032 - 4 Sterilization of talc for pleurodesis . Available techniques, efficacy, and cost analysis; Kennedy L et al.; Although talc has been used as a pleurodesis agent since 1935, a sterilization protocol has not been established . We obtained USP asbestos-free talc from six different suppliers and sterilized each using dry heat, gamma irradiation, and ethylene oxide gas . Aerobic, anaerobic, and fungal cultures were obtained prior to sterilization, and 1, 30, and 90 days after sterilization . Bacillus species were cultured from all six unsterilized specimens and coagulase-negative Staphylococcus grew from two unsterilized specimens . No growth of organisms was found following any method of sterilization . The cost of sterilization per 5-g packet of talc was $4.74, $7.85, and $16.25 for heat, ethylene oxide, and gamma irradiation, respectively . In conclusion, untreated talc is not sterile . Sterilization by prolonged dry heat exposure, ethylene oxide gas, and gamma irradiation are all effective, with dry heat being the least expensive. Clin Exp Immunol, 1995 Apr, 100(1), 26 - 31 Induction of tumour necrosis factor-alpha (TNF-alpha) mRNA in bladders and spleens of mice after intravesical administration of bacillus Calmette-Guérin; Shin JS et al.; Intravesical bacillus Calmette-Guerin (BCG) therapy is highly effective in the therapy of carcinoma in situ of the bladder, but the mechanism of BCG immunotherapy is not clearly understood . We studied the production of TNF-alpha in spleens and bladders of mice after intravesical BCG or BCG/interferon-gamma (IFN-gamma) instillation . Significant change of TNF-alpha mRNA expression of spleens and bladders of C3H/He mice was observed after intravesical BCG instillation, although intravesical IFN-gamma therapy 3 days after BCG instillation to maintain the activated state of monocyte/macrophage lineage cells did not show a significant change of TNF-alpha mRNA, compared with that of BCG therapy alone . Maximal production of TNF-alpha mRNA in spleens of mice was seen after the first or second intravesical BCG instillation, and production of TNF-alpha mRNA in bladders was also increased after intravesical BCG instillation . The increment of TNF-alpha production by BCG stimulation in HL-60, a promyelocytic leukaemic cell line, and peripheral blood mononuclear cells in vitro may support the in vivo effect of BCG therapy on the bladder . These data show that local production of TNF-alpha as well as systemic production by intravesical BCG treatment may correlate with one of the mechanisms of BCG immunotherapy of superficial bladder cancer. Am J Infect Control, 1995 Apr, 23(2), 152 - 5 Implementing a tuberculosis control program; Williams J et al.; Between January 1989 and December 1990, 26 patients acquired multidrug-resistant tuberculosis at our institution . Their exposures occurred when they were admitted to a ward where a patient with acid fast bacillus smear-positive pulmonary tuberculosis was also admitted . In 20 cases, the infectious patients were not isolated until the sputum smears were positive . When the outbreak was recognized in the spring of 1990, the infection control department undertook a risk assessment and instituted measures that would become the tuberculosis control program . Since then, administrative and environmental controls have been implemented, education programs are ongoing, personal protective equipment is in use, and a more aggressive employee health testing program is underway . The steps we took and the barriers we had to overcome to implement our plan are included in this article. Arukoru Kenkyuto Yakubutsu Ison, 1995 Apr, 30(2), 69 - 79 {Effect of Bacillus natto-fermented product (BIOZYME) on blood alcohol, aldehyde concentrations after whisky drinking in human volunteers, and acute toxicity of acetaldehyde in mice}; Sumi H et al.; Effects of Bacillus natto-fermented product (BIOZYME) on blood alcohol and aldehyde concentrations after drinking whisky (corresponding to 30-65 ml ethanol) were studied in 21 healthy volunteers . When 100 ml of BIOZYME was orally administrated to the volunteers before drinking whisky, the time delay of both blood factors to attain maximum concentrations were observed . The maximum decrease in blood alcohol and aldehyde concentrations were about 23% and 45% (p < 0.005), respectively, at 1 hr after drinking whisky . The aldehyde lowering effect of BIOZYME was continued for at least 4 hr after whisky drinking . Concentration of the breath alcohol was also sharply decreased by BIOZYME administration . The breath alcohol concentration in the administered group (0.18 +/- 0.11 mg/l) was found to be lowered about 44% than that of the control group (0.32 +/- 0.11 mg/l) (p < 0.0005, n = 21), at 1 hr after drinking whisky . In acute toxicity experiments of aldehyde in mice (12 mmol AcH/mg), BIOZYME showed the survival effect as with alpha-D-Ala (134% increase of the living, at 40 min after i.p . administration) (p < 0.005, n = 22) . These findings reveal the Bacillus natto produced BIOZYME as a reasonable, safety and useful anti-hangover agent. Mol Microbiol, 1995 Apr, 16(2), 365 - 72 Saturation of penicillin-binding protein 1 by beta-lactam antibiotics in growing cells of Bacillus licheniformis; Lepage S et al.; With the help of a new highly sensitive method allowing the quantification of free penicillin-binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various beta-lactam antibiotics in growing cells of Bacillus licheniformis was studied . Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon . In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators . In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics . In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations. Int J Food Microbiol, 1995 Apr, 25(2), 131 - 9 Prevalence of Bacillus cereus in selected foods and detection of enterotoxin using TECRA-VIA and BCET-RPLA; Rusul G et al.; Enterotoxigenic Bacillus cereus was detected in cooked foods (17), rice noodles (3), wet wheat noodles (2), dry wheat noodles (10), spices (8), grains (4), legumes (11) and legume products (3) . One hundred ninety-four (42.3%), 70 (15.3%) and 23 (5.2%) of the 459 presumptive B . cereus colonies isolated from PEMBA agar were identified as B . cereus, Bacillus thuringiensis and B . mycoides, respectively . B . cereus isolates were examined for growth temperature, pH profile and enterotoxin production using both TECRA-VIA and BCET-RPLA kits . One hundred seventy-eight (91.8%) and 164 (84%) of the strains were enterotoxigenic as determined using TECRA-VIA and BCET-RPLA, respectively . Eighty-two (50%) of the enterotoxigenic strains were capable of growing at 5 degrees C, and 142 (86.6%) grew at 7 degrees C within 7 days of incubation . The enterotoxigenic strains did not grow at pH 4.0 but 69 (42.0%) of the strains were able to grow at pH 4.5 within 7 days at 37 degrees C . The isolates were resistant to ampicillin (98.8%), cloxallin (100%) and tetracycline (61.0%), and susceptible to chloroamphenicol (87%), erythromycin (77.4%), gentamycin (100%) and streptomycin (98.7%). Indian J Biochem Biophys, 1995 Apr, 32(2), 100 - 5 Activity and stability of Bacillus cereus penicillinase entrapped in aerosol OT reverse micelles; Chakravarty K et al.; The kinetics of enzyme catalyzed hydrolysis of penicillin G and stability of the enzyme alpha-penicillinase, entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically . Various physical parameters, such as, water pool size (related to Wo), pH and temperature, were optimized for maximum activity of penicillinase in water/aerosol OT/isooctane reverse micelles . The enzyme showed maximum activity of Wo - 14 and pH, 7.0 . At any temperature the enzyme was to be more active in reverse micelles than in aqueous solution . At optimum conditions of Wo, pH and temperature the enzyme was 100% more active in reverse micelles than its maximum activity in aqueous solution . In both the systems, the activity starts falling at and above 25 degrees C . CD Spectral studies showed that the enzyme in reverse micelles possesses more helical structure than it has in aqueous solution and at the optimum conditions in which it showed maximum activity, the alpha-helicity was also maximum . The enzyme was very stable in reverse micelles at and above room temperature compared to the same in aqueous solution. Biotechnol Appl Biochem, 1995 Apr, 21 ( Pt 2), 233 - 43 Cyclodextrin glycosyltransferase may be the only starch-degrading enzyme in Bacillus macerans; Nogrady N et al.; Cyclodextrin glycosyltransferase (CGTase) was released into the culture fluid by Bacillus macerans predominantly in the late stationary phase of growth and during autolysis in the presence of either glucose or starch as a carbon source . In both cases significant soluble intracellular enzyme activity could be observed in the early stationary phase, and a low non-soluble intracellular CGTase activity could be demonstrated also in the exponential growth phase in the presence of starch . At the end of the exponential phase the non-soluble specific intracellular enzyme activity was found to be constant with a value of 0.63 +/- 0.06 nkat/10(9) viable cells . Since amylase activity could not be detected in any intracellular or extracellular sample taken at any culture time, we conclude that cellbound CGTase is the only starch-digesting enzyme in growing B . macerans and, hence, may be fully responsible for the degradation of starch in the culture fluid. Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2805 - 9 Serological response patterns of melanoma patients immunized with a GM2 ganglioside conjugate vaccine; Kitamura K et al.; Gangliosides, such as GM2, GD2, GD3, and 9-O-acetyl GD3, are receiving attention as targets for antibody-based and vaccine-based therapies of melanoma . GM2 appears to be a particularly immunogenic ganglioside in humans, as indicated by the presence of naturally occurring IgM anti-GM2 antibodies in approximately 5% of humans and the fact that immunization with irradiated GM2-expressing melanoma cells or purified GM2 adherent to bacillus Calmette-Guerin elicits GM2 antibodies of low to moderate titers in a high proportion of vaccinated patients . To develop vaccines that consistently induce high titers of IgM as well as IgG anti-GM2 antibodies, vaccines containing GM2 conjugated to keyhole limpet hemocyanin as the carrier protein and QS-21 as the adjuvant have been constructed . The serological response of vaccinated patients was monitored by ELISA using purified GM2 ganglioside for IgM and IgG anti-GM2 antibodies and for GM2 cell surface-reactive antibodies by immune adherence assays and cytotoxic tests (IgM antibodies) and mixed hemadsorption assays (IgG antibodies) . The majority of vaccinated patients developed IgM and IgG antibodies detectable by ELISA . In most cases, the results of IgM ELISA correlated with assays for cell surface-reactive IgM antibodies . This was not true for IgG anti-GM2 antibodies, where strong discrepancies were seen between high titers in ELISA and little or no reactivity in mixed hemadsorption tests for cell surface-reactive antibodies . These IgG antibodies (and the less frequent IgM antibodies that show similar discrepancies) may be directed against GM2 determinants that are buried, hidden, or not present on GM2-expressing target cells . With regard to a major objective of ganglioside vaccines--i.e., generation of cytotoxic antibodies--the GM2-keyhole limpet hemocyanin/QS-21 vaccine is clearly superior to the previously tested GM2/bacillus Calmette-Guerin vaccine . However, variability in patient response and lack of persistence of high-titered IgM cytotoxic antibodies in many patients are problems that remain to be solved. Biochim Biophys Acta, 1995 Mar 22, 1234(2), 173 - 83 Purification of deformin, an extracellular protein synthesized by Bartonella bacilliformis which causes deformation of erythrocyte membranes; Xu YH et al.; A factor capable of deforming erythrocyte membranes, found in the culture supernatants of Bartonella bacilliformis, was purified 1840-fold using hydrophobic, ion exchange and gel exclusion chromatography . The final fractions contained a single detectable polypeptide species, referred to as deformin, having a molecular weight of 67000 by SDS-PAGE and a native molecular weight of 130,000 by gel exclusion chromatography or velocity sedimentation in a glycerol gradient . Erythrocytes treated with deformin acquire trenches, indentations, and invaginations which could be reversed by vanadate, dilauroylphosphatidylcholine (DLPC), or by raising the internal Ca2+ concentrations with the inophore A23187 . Internal vacuoles also form . Erythrocytes treated with trypsin or neuraminidase are much more sensitive to deformin than untreated erythrocytes; erythrocytes treated with phospholipase D are less sensitive to deformin . This protein may play a role in causing the severe anemia which can result as a consequence of infection by B . bacilliformis. Med Clin (Barc), 1995 Mar 18, 104(10), 365 - 8 {The epidemiology of tuberculosis in El Ferrol}; Garcia Rodriguez JF et al.; BACKGROUND: This study was undertaken to know the frequency of tuberculosis in El Ferrol and to contribute to the knowledge of the situation in Spain . METHODS: A retrospective study of all the cases of tuberculosis diagnosed in the Hospital A . Marcide-Novoa Santos (El Ferrol, Spain) from 1990 to 1993 was performed . RESULTS: Seven hundred twenty-four patients were diagnosed, with a mean annual prevalence of 83.3/100,000 inhabitants . Six hundred sixty-four cases (430 males {64.8%}) were evaluated . The mean age was 35.5 +/- 19 years with 58.9% under the age of 35 . 98.7% of the patients lived in the health care area and 73.2% were admitted, with 13.7% having previous history of tuberculosis . Sixty-one cases (11.1%; Cl: 8.25-13.7) had HIV infection . Diagnosis was microbiological in 505 cases (76%), anatomopathological in 60 (9%) and in 99 (14.9%) diagnosis was achieved by clinical and radiological criteria . Pulmonary localization (67.2%) was the most frequent form and was predominant in males, while lymph node and osteoarticular localizations were more frequent in women . The incidence of bacilliferous patients was 30.7/100,000 inhabitants . A delay of more than one month took place in the diagnosis of 66.4% of the bacilliferous patients . CONCLUSIONS: The incidence of tuberculosis in El Ferrol is very high with an important delay in the diagnosis of bacilliferous patients . The high percentage of patients admitted to hospital carries considerable costs in the treatment of the disease. J Biol Chem, 1995 Mar 17, 270(11), 6412 - 9 Mutations in domain I of Bacillus thuringiensis delta-endotoxin CryIAb reduce the irreversible binding of toxin to manduca sexta brush border membrane vesicles; Chen XJ et al.; Site-directed mutagenesis was used to generate CryIAb mutants at the selected N-terminal positions to study the function of domain I . Structurally stable mutant proteins were tested for toxicity, receptor binding kinetics, and pore function . Substitutions of tyrosine at position 153 with arginine (Y153R) or alanine (Y153A) did not affect toxicity appreciably, whereas replacing this tyrosine with aspartic acid (Y153D) resulted in a great loss of toxicity . Mutation of alanine at position 92 to glutamic acid (A92E) almost completely abolished toxicity . The initial receptor binding was unchanged as measured by competition binding assays among all mutant proteins . Reduced pore function, however, was observed for mutants A92E and Y153D as tested by voltage clamping . Further studies with specially designed association and dissociation binding assays showed that irreversible binding of these two mutant toxins to Manduca sexta brush border membrane vesicles was significantly reduced . The decrease in irreversible binding was correlated with the changes in toxicity and may reflect a severely disturbed membrane insertion process in these two mutant toxins, leading to reduced pore function and toxicity . The results support the model that domain I is involved in membrane integration and pore formation. J Immunol, 1995 Mar 15, 154(6), 2753 - 63 Involvement of IFN-gamma in Bacillus Calmette-Guérin-induced but not in tumor-induced sensitization to TNF-induced lethality; Cauwels A et al.; In healthy mice, murine (m) TNF is fairly lethal, whereas human (h) TNF (a selective murine TNF-R55 agonist) is rather harmless . However, we and others observed that mice suffering from a bacterial infection, such as Bacillus Calmette-Guerin (BCG), or bearing i.m . some types of tumor, develop a hypersensitivity to the IL-6-inducing and lethal properties of hTNF . This is a cardinal problem as it severely limits the potential use of hTNF-R55-specific agonists for systemic treatment of human cancer . Using mice carrying a targeted disruption in the gene encoding the IFN-gamma receptor (IFN-gamma Ro/o), we here report that endogenous IFN-gamma plays a crucial role in the development of TNF hypersensitivity during BCG infection . Indeed, both the lethality and the IL-6 induced by hTNF were drastically reduced in IFN-gamma Ro/o mice as compared with control mice . These results demonstrate that the enhancement of TNF effects is at least an equally important mechanism by which IFN-gamma contributes to BCG-induced hypersensitivity as the previously described augmentation of TNF production . Experiments in athymic nude mice, either depleted of NK cells or not, revealed that the latter cell population is an important source of the sensitizing IFN-gamma during BCG infection . In contrast, IFN-gamma Ro/o mice were as susceptible as control mice to the sensitizing effects of tumors . mTNF, which interacts with both mTNF-R55 and mTNF-R75 and causes lethality on its own, is as toxic in IFN-gamma Ro/o mice as in wt control mice; this means that TNF-induced IFN-gamma does not play a role in mTNF-induced lethality. Biochem J, 1995 Mar 15, 306 ( Pt 3), 727 - 33 Interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: stoichiometry and specificity in self-assembly; Lessard IA et al.; The interaction between the pyruvate decarboxylase (E1) component and a di-domain (lipoyl domain plus peripheral subunit-binding domain) from the dihydrolipoyl acetyltransferase (E2) component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex was investigated . Only 1 mol of di-domain (binding domain) was bound to 1 mol of heterotetrameric E1 (alpha 2 beta 2) and the binding was without effect on the kinetic activity of E1 . Similarly, the di-domain bound to separate E1 beta subunits at a maximal polypeptide chain ratio of 1:2, but no detectable interaction was found with the E1 alpha subunit . However, addition of the monomeric E1 alpha subunit to an E1 beta-di-domain complex generated a fully functional E1 (alpha 2 beta 2)-di-domain complex, indicating that the E1 beta subunit plays the critical part in binding the E1 component to the di-domain and suggesting that no chaperonin is needed in vitro to promote the assembly of the three separate proteins . Mixing the E1 and dihydrolipoyl dehydrogenase (E3) components in the presence of di-domain revealed that E1 and E3 cannot bind simultaneously to the same molecule of di-domain, a new feature of the assembly pathway and an important factor in determining the ultimate structure of the assembled enzyme complex. Biochemistry, 1995 Mar 14, 34(10), 3368 - 76 Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity; Penninga D et al.; Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans strain 251 . Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft {Strokopytov, B., et al . (1995) Biochemistry 34, (in press)}, suggested that Tyr195 plays an important role in cyclization of oligosaccharides . Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G) . Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 angstrum resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein . The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions . Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins . These mutants produced a considerable amount of linear maltooligosaccharides . The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products . Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1995 Mar 10, 270(10), 5490 - 4 Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis; Vadlamudi RK et al.; Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects . However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity . We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B . thuringiensis . The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B . thuringiensis subsp . berliner and leads to death of the hornworm . BT-R1 shares sequence similarity with the cadherin superfamily of proteins. FEBS Lett, 1995 Mar 6, 360(3), 217 - 22 Delta-endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers; Lorence A et al.; Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV) . The initial response and the sustained permeability change are cationic, not very K+ selective, and occur at in vivo lethal doses (nM) . The toxin response has a different ion selectivity and is more sensitive to Ba2+ than the intrinsic cation permeability of BBMV . Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS) . A 2-40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV . Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved . Thus, Bacillus thuringiensis delta-endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV-containing functional receptors. J Mol Biol, 1995 Mar 3, 246(4), 545 - 59 Crystal structure of calcium-depleted Bacillus licheniformis alpha-amylase at 2.2 A resolution; Machius M et al.; The three-dimensional structure of the calcium-free form of Bacillus licheniformis alpha-amylase (BLA) has been determined by multiple isomorphous replacement in a crystal of space group P4(3)2(1)2 (a = b = 119.6 A, c = 85.4 A) . The structure was refined using restrained crystallographic refinement to an R-factor of 0.177 for 28,147 independent reflections with intensities FObs > 0 at 2.2 A resolution, with root mean square deviations of 0.008 A and 1.4 degrees from ideal bond lengths and bond angles, respectively . The final model contains 469 residue, 237 water molecules, and one chloride ion . The segment between Trp182 and Asn192 could not be located in the electron density, nor could the N and C termini . Cleavage of the calcium-free form of BLA was observed after Glu189, due to a Glu-C endopeptidase present in trace amounts in the preparation . BLA did not crystallize without this cleavage under the conditions applied . BLA exhibits the characteristic overall topological fold observed for other alpha-amylases and related amylolytic enzymes: a central domain A containing an alpha/beta-barrel with a large protrusion between beta-strand 3 and alpha-helix 3 (domain B) and a C-terminal greek key motif (domain C) . Unlike in the other enzymes, domain B possesses a beta-sheet made up of six loosely connected, twisted beta-strands forming a kind of a barrel with a large hole in the interior . Topological comparisons to TAKA-amylase, pig pancreatic alpha-amylase and cyclodextrin glycosyltransferase reveal a very high structural equivalence for large portions of the proteins and an exceptionally pronounced structural similarity for calcium binding, chloride binding and the active site . None of the theories proposed to explain the enhanced thermostability of BLA showed a satisfactory correlation with the three-dimensional structure . Instead, sequence comparisons to the less thermostable bacterial alpha-amylase from Bacillus amyloliquefaciens (BAA) indicate that some ionic interactions present in BLA, but which cannot be formed in BAA, might be responsible for the enhanced thermostability of BLA. J Mol Biol, 1995 Mar 3, 246(4), 511 - 21 The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima at 2.5 A resolution; Korndorfer I et al.; The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophile Thermotoga maritima was determined by Patterson search methods using the known structure of the Bacillus stearothermophilus enzyme . The structure was refined at a resolution of 2.5 A to an R-factor of 16.63% for 26289 reflections between 8.0 A an 2.5 A with F > 2 sigma(F) . The crystallographic asymmetric unit contains two monomers related by approximate 2-fold symmetry and a tetramer is built up by crystallographic symmetry . The root-mean-square deviation of Ca positions of glyceraldehyde-3-phosphate dehydrogenase from T . maritima and B . stearothermophilus is 0.83 A in the NAD+ binding domains and smaller close to the cofactor . In contrast, the largest deviations in the catalytic domains are found at residues involved in coordination of sulphate ion SO4 339, which most likely marks the site of the attacking inorganic phosphate ion in catalysis . A large number of extra salt-bridges may be an important factor contributing to the high thermostability of this protein. MMWR Morb Mortal Wkly Rep, 1995 Mar 3, 44(8), 137 - 40 Exposure of passengers and flight crew to Mycobacterium tuberculosis on commercial aircraft, 1992-1995; Effect of inorganic salts et al.; Vector Control Research Centre, Indian Council of Medical Research, Pondicherry, IndiaVarious inorganic salts and commonly used soaps and detergents were tested in the laboratory for their effect on the dissolution and larvicidal residual activity of a slow-release alginate encapsulated granular formation of Bacillus sphaericus . Fluoride, chloride and sulphate salts and a detergent powder affected the residual activity of this formulation drastically by rupturing it but did not effect its larvicidal activity . Nitrates and phosphates of sodium and potassium also had the same effect but to a moderate level . The safest concentration of these water impurities for effective functioning of the alginate encapsulated B . sphaericus formulation have been determined. Am J Gastroenterol, 1995 Mar, 90(3), 485 - 8 Malignant histiocytosis in a patient presenting with hepatic dysfunction and peliosis hepatis; Fine KD et al.; In this article, we report the case of a 36-yr-old patient presenting with manifestations of portal hypertension, hepatic dysfunction, and fever who proved to have peliosis hepatis on liver biopsy . A thorough work-up revealed no obvious etiology . At autopsy, malignant histiocytosis of the liver and bone marrow was diagnosed . This case represents the first report of the association of peliosis hepatis with this rare histiocytic neoplasm and exemplifies the need for persistence in the search for malignancy, particularly hematological malignancy, in the patient with unexplained peliosis . The clinical similarity of peliosis hepatis associated with hematological malignancy and bacillary peliosis is also discussed. Pediatrics, 1995 Mar, 95(3), 414 - 8 Bacillus Calmette-Guérin complications in children born to HIV-1-infected women with a review of the literature; O'Brien KL et al.; OBJECTIVE . To compare the risk of complications following Bacillus Calmette-Guerin (BCG) vaccination among children by maternal and infant HIV-1 infection status as part of an investigation of an outbreak of BCG complications . METHODS . A nonconcurrent cohort study of BCG complications among 125 infants born to HIV-1 seropositive and 166 infants born to HIV-1 seronegative mothers was conducted in Cite Soleil, Haiti . Infants were examined at regular intervals until 15 months of age, and complications from BCG were documented . An investigation of BCG vaccination practices was conducted . RESULTS . Mild or moderate complications occurred among 16 of 166 (9.6%) infants born to HIV-1 seronegative mothers compared with 4 of 13 HIV-1-infected infants (30.8%, P = .04) and 10 of 75 (13.3%, P = .39) uninfected infants born to HIV-1-infected mothers . No serious complications were noted . The outbreak of complications was associated with administration of 2.0 to 2.5 times the recommended dose of BCG vaccine . CONCLUSIONS . This and five other cohort studies indicate that there may be a small increased risk of complications following BCG vaccination among HIV-1-infected children, but the reactions are usually mild and the risk does not outweigh the benefits of BCG vaccination in populations at high risk of tuberculosis during infancy and childhood. J Urol, 1995 Mar, 153(3 Pt 2), 929 - 33 A randomized study of intravesical mitomycin C, bacillus Calmette-Guerin Tice and bacillus Calmette-Guerin RIVM treatment in pTa-pT1 papillary carcinoma and carcinoma in situ of the bladder; Vegt PD et al.; Results of a randomized prospective study are reported in which mitomycin C, Tice bacillus Calmette-Guerin (BCG) and RIVM-BCG were compared in 437 patients with primary or recurrent pTa and pT1 bladder tumors, including carcinoma in situ . The followup (or time in study) varied from 2 to 81 months (mean 36 months) . After complete transurethral resection of all visible tumors the patients were treated with 30 mg . mitomycin C once a week for 4 consecutive weeks and thereafter every month for a total of 6 months, and 5 x 10(8) colony-forming units Tice BCG or RIVM-BCG once a week for 6 consecutive weeks . For papillary tumors mitomycin C and RIVM-BCG treatments were equally effective (p = 0.53), and mitomycin C was more effective than Tice BCG therapy (p = 0.01). Appl Environ Microbiol, 1995 Mar, 61(3), 959 - 65 Characterization and substrate specificity of an endo-beta-1,4-D-glucanase I (Avicelase I) from an extracellular multienzyme complex of Bacillus circulans; Kim CH; An endo-1,4-beta-D-glucanase I (Avicelase I; EC 3.2.1.4) was purified to homogeneity from an extracellular celluloxylanosome of Bacillus circulans F-2 . The purification in the presence of 6 M urea yielded homogeneous enzyme . The enzyme had a monomeric structure, its relative molecular mass being 75 kDa as determined by gel filtration and 82 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The pI was 5.4, and the N-terminal amino acid sequence was ASNIGGWVGGNESGFEFG . The optimal pH was 4.5, and the enzyme was stable at pH 4 to 10 . The enzyme has a temperature optimum of 50 degrees C, it was stable at 55 degrees C for 46 h, and it retains approximately 20% of its activity after 30 min at 80 degrees C . It showed high-level activity towards carboxymethyl cellulose (CMC) as well as p-nitrophenyl-beta-D-cellobioside, 4-methylumbelliferyl cellobioside, xylan, Avicel, filter paper, and some cello-oligosaccharides . Km values for birch xylan, CMC, and Avicel were 4.8, 7.2, and 87.0 mg/ml, respectively, while Vmax values were 256, 210, and 8.6 mumol x min-1 x mg-1, respectively . Cellotetraose was preferentially cleaved into cellobiose (G2) plus G2, and cellopentaose was cleaved into G2 plus cellotriose (G3), while cellohexaose was cleaved into cellotetraose plus G2 and to a lesser extent G3 plus G3 . G3 was not cleaved at all . G2 was the main product of Avicel hydrolysis . Xylotetraose (X4) and xylobiose (X2) were mainly produced by the enzyme hydrolysis of xylan . G2 inhibited the activity of carboxymethyl cellulase and Avicelase, whereas Mg2+ stimulated it . The enzyme was completely inactivated by Hg2+, and it was inhibited by a thiol-blocking reagent . Hydrolysis of CMC took place, with a rapid decrease in viscosity but a slow liberation of reducing sugars . On the basis of these results, it appeared that the cellulase should be regarded as endo-type cellulase, although it hydrolyzed Avicel. Appl Environ Microbiol, 1995 Mar, 61(3), 941 - 3 Isolation and characterization of a cytotoxic metabolite of Talaromyces bacillosporus; Ishii K et al.; A cytotoxic metabolite, talarotoxin, was isolated from a fungus, Talaromyces bacillosporus IFO 8397, cultured on rice . The structure of the toxin was elucidated and found to contain a pyrrolizidinedione connected with a trans delta 1-octalin through a conjugated triene. Appl Environ Microbiol, 1995 Mar, 61(3), 855 - 9 Stabilization of microbial cytochrome P-450 activity by creation of station-phase conditions in a continuously operated immobilized-cell reactor; Dror Y et al.; Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-steroid hydroxylation . This activity belongs to the widespread ferredoxin reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity . The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers . Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450-based detoxification . It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor . The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in "freezing" of a physiological steady-state analog under stationary phase conditions, allowing stable performance of continuous hydroxylation for several weeks . It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities. Sante, 1995 Mar-Apr, 5(2), 89 - 94 {The recycling of waste water and mosquitoes}; Karch S et al.; Recycling waste water in the Acheres complex (North-West Paris) is based on both sophisticated industrial techniques and simple agricultural methods . The sewage farms and settling pools provide suitable breeding sites for more than ten mosquito species . Aedes caspius is the major pest for the local population . Moreover Culex pipiens (anthropophilic form) breeds in the sewers of the neighbouring towns . Mosquito control is based early ground treatment of breeding sites . Temephos and fenotrothion are used against A . caspius . Spherimos (Bacillus sphaericus) is used to control C . pipiens . The two insecticides have no adverse effect on humans or the environment and Spherimos is harmless . Aedes pest have been virtually eliminated . Urban Culex control is generally good despite being performed by less well trained municipal employees . In the areas treated by the specialized team of the SIAAP, pest mosquitoes have disappeared . The techniques used in the Acheres complex do not require sophisticated equipment . Thus, if adapted to local ecological, epidemiological and financial conditions they could be transferred to developing countries. Biosci Biotechnol Biochem, 1995 Mar, 59(3), 529 - 31 The action of Bacillus circulans WL-12 chitinases on partially N-acetylated chitosan; Mitsutomi M et al.; Both chitinase A1 and D from Bacillus circulans WL-12 specifically hydrolyzed the N-acetyl-beta-D-glucosaminidic bonds in 50% N-acetylated chitosan molecules to produce hetero-oligosaccharides with GlcNAc at the reducing end residues, together with GlcNAc and (GlcNAc)2 . GlcN-GlcNAc and GlcN-GlcNAc-GlcNAc were produced as major hydrolysis products with chitinase A1 and D, respectively, but GlcN-GlcNAc was not detected in the digest of 50% N-acetylated chitosan with chitinase D. Biotechnol Prog, 1995 Mar-Apr, 11(2), 231 - 4 Penicillin-G enhanced production of thuringiensin by Bacillus thuringiensis sp . darmstadiensis; Tzeng YM et al.; The effect of penicillin-G on the production of the potential microbial insecticide thuringiensin by Bacillus thuringiensis sp . darmstadiensis was studied . Shake flask and 3-L jar fermentor studies showed that the addition of 360 units/mL penicillin-G at 9 h, when the fermentable sugar in the medium was about to be mostly consumed, improved thuringiensin production by more than 1-fold relative to the control . The dosage of 360 units/mL penicillin-G had only a modest effect on the growth of the microorganism . However, cell growth was inhibited at higher dosages of the antibiotic . Since penicillin-G could interfere with cell wall synthesis, which facilitated the release of thuringiensin, a high thuringiensin productivity of 2600 mg/L was attained in this study, which is about 2-10-fold higher than those values reported in the literature. Appl Microbiol Biotechnol, 1995 Mar, 42(6), 878 - 83 One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing alpha-amylase, glucoamylase and pullulanase; Janse BJ et al.; A recombinant strain of Saccharomyces cerevisiae was constructed that contained the genes encoding a bacterial alpha-amylase (AMY1), a yeast glucoamylase (STA2) and a bacterial pullulanase (pulA) . The Bacillus amyloliquefaciens alpha-amylase and S . cerevisiae var . diastaticus glucoamylase genes were expressed in S . cerevisiae using their native promoters and the encoded enzymes secreted under direction of their native leader sequences . In contrast, the Klebsiella pneumoniae pullulanase gene was placed under the control of the yeast alcohol dehydrogenase gene promoter (ADC1P) and secreted using the yeast mating pheromone alpha-factor secretion signal (MF alpha 1S) . Transcription termination of the pullulanase gene was effected by the yeast tryptophan synthase gene terminator (TRP5T), whereas termination of the glucoamylase and alpha-amylase genes was directed by their native terminators . Pullulanase (PUL1) produced by recombinant yeasts containing ADC1P MF alpha 1S pulA TRP5T (designated PUL1) was further characterized and compared to its bacterial counterpart (PulA) . The different genes were introduced into S . cerevisiae in different combinations and the various amylolytic Saccharomyces transformants compared to Schwanniomyces occidentalis . Introduction of PUL1 into a S . cerevisiae strain containing both STA2 and AMY1, resulted in 99% assimilation of starch. Appl Microbiol Biotechnol, 1995 Mar, 42(6), 871 - 7 Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement; Wittchen KD et al.; An efficient method for gene replacement in Bacillus megaterium was developed and used to inactivate the chromosomal neutral protease gene (nprM) from strain DSM319 . A temperature-dependant suicide vector was constructed to allow replacement of the normal chromosomal copy with an altered version of the nprM gene . One mutant B . megaterium MS941 was selected for further characterization . Measurement of extracellular protease activity from strain MS941 indicated the existence of an additional minor extracellular protease in B . megaterium . Inhibitor studies revealed that this minor protease, comprising only 1.4% of the wild-type total extracellular protease activities, is a serine-type enzyme. Bull Pan Am Health Organ, 1995 Mar, 29(1), 37 - 58 Epidemiology of AIDS and tuberculosis; Garcia Garcia ML et al.; This article reviews literature on the epidemiology, pathogenicity, and control of HIV and Mycobacterium tuberculosis coinfection . Regarding pathogenicity, immune system deterioration makes HIV-infected people more likely to develop active tuberculosis on primary or secondary exposure to the bacillus or to suffer reactivation of latent infections, and to experience considerably higher rates of extrapulmonary manifestations, relapses, and death . Regarding epidemiology, as of 1990 there were an estimated 3 million people coinfected with HIV and M . tuberculosis, with some 300,000 active tuberculosis cases and 120,000-150,000 tuberculosis deaths occurring annually among those coinfected . Over 500,000 coinfected people are thought to reside in the Americas, over 400,000 of them in Latin America . In general, the impact of coinfection is evident . Relatively high and increasing prevalences of HIV infection have been detected among tuberculosis patients around the world, and tuberculosis has become a frequent complication of AIDS cases . Moreover, there is no longer any doubt that coinfection obstructs tuberculosis prevention and control . Among other things, it affects BCG vaccination policies, suggests the need to administer preventive chemoprophylaxis to HIV-infected individuals at high risk of harboring or contracting tuberculosis infections, and complicates both detection and treatment of active tuberculosis cases . The recent proliferation of M . tuberculosis strains resistant to multiple drugs, most notably in the United States, compounds the problem . Tuberculosis prevention and control are still technically and economically feasible . However, more must be done to establish surveillance programs with laboratory support . More research is needed to determine what case prevention measures are best-suited to current circumstances and the HIV/AIDS presence . More effective preventive treatment regimens that are well tolerated, well complied with, and do not pose the risk of multiresistance need to be devised . More health workers need to be trained to suspect tuberculosis and to conduct timely and appropriate tests confirming this diagnosis . And finally, more must be done to standardize the types and durations of the various curative treatment regimens employed. Clin Infect Dis, 1995 Mar, 20(3), 629 - 33 Bacillus licheniformis bacteremia: five cases associated with indwelling central venous catheters; Blue SR et al.; Bacillus species are being more frequently recognized as pathogens in immunocompromised hosts or in patients with cancer and central venous catheters . Only nine cases of Bacillus licheniformis infection have been reported in the English-language literature since 1966 . In a retrospective study we describe six patients and 17 episodes of B . licheniformis bacteremia over a 5-year span . All six patients had either a Hickman or a Broviac catheter in place for more than 3 months . Five of the six patients had multiple clinically significant episodes of bacteremia due to B . licheniformis . The six patients ranged in age from 4 years to 62 years . Two patients had leukemia or lymphoma and three patients had solid tumors, but only one patient was neutropenic . No deaths were related to B . licheniformis bacteremia . B . licheniformis should be considered as a potential pathogen in immunocompromised patients, especially when bacteremia is associated with the presence of long-term central venous catheters . Mortality due to B . licheniformis bacteremia is low, but recurrent bacteremia due to this organism causes significant morbidity and usually necessitates removal of the catheter. AIDS, 1995 Mar, 9(3), 243 - 51 Safety and immunogenicity of a V3 loop synthetic peptide conjugated to purified protein derivative in HIV-seronegative volunteers; Rubinstein A et al.; OBJECTIVES: To develop a peptide-based model for a preventive vaccine for HIV-1 infection . DESIGN: Phase I trial in HIV-1-seronegative volunteers . PARTICIPANTS: Adult healthy subjects HIV-1-antibody-seronegative in an enzyme-linked immunosorbent assay, screened for tuberculin {purified protein derivative (PPD)} reactivity with 2 tuberculin units PPD-administered intradermally . INTERVENTIONS: Submicrogram doses of a PPD conjugate with a peptide of the primary neutralizing domain (PND) of HIV-1MN (PPD-MN-PND) were administered intradermally to tuberculin skin-test-positive and -negative volunteers . RESULTS: Antibodies to the MN-PND were measured after two immunizations in 10 out of 11 PPD skin-test-positive volunteers . After the fourth immunization high-affinity antibodies were detected, which persisted for over 1 year . High titers of MN-PND-specific immunoglobulin (Ig) G and IgA were detected in the serum and saliva of all volunteers tested . Serum antibodies were cross-reactive with PND peptide from some other HIV-1 strains but neutralized only the HIV-1MN prototype . Human leukocyte antigen (HLA)-B7-restricted MN-PND-specific cytotoxic T lymphocytes (CTL) were also detected . CONCLUSIONS: The PPD-MN-PND vaccine at submicrogram doses is safe and immunogenic in PPD skin-test-positive healthy adult volunteers . Long lasting humoral immune responses in the serum and saliva were possibly accompanied by HLA-B7-restricted CTL responses . This is a vaccine prototype that can be rapidly and inexpensively modified to include other peptide epitopes . It is especially suitable for use in a worldwide multibillion Bacillus Calmette-Guerin (BCG)-primed or tuberculosis-exposed population at risk for HIV-1 infection. J Clin Microbiol, 1995 Mar, 33(3), 636 - 40 Bacillus cereus phage typing as an epidemiological tool in outbreaks of food poisoning; Ahmed R et al.; Bacillus cereus is responsible for an increasing number of food poisoning cases . By using 12 bacteriophages isolated from sewage, a typing scheme for B . cereus isolates from outbreaks or sporadic cases of food poisoning was developed . The phages belonged to three morphotypes . Ten phages with contractile tails and icosahedral heads were members of the Myoviridae family, and two phages with noncontractile tails belonged to the Siphoviridae family . Phage 11 represented a new species . It had an isometric head and a very long contractile tail with long wavy tail fibers and was one of the largest viruses known . The vast majority of 166 B . cereus strains (161, or 97%) isolated from food poisoning cases were typeable . Of 146 strains isolated from 18 outbreaks, 142 (97%) could be divided into 17 phage types . A good correlation, on the order of 80 to 100%, between phage types of strains isolated from suspected foods and those of strains isolated from stools of symptomatic patients was observed . Most Bacillus thuringiensis strains were also typeable, providing further evidence of the close relatedness of B . cereus and B . thuringiensis . This phage typing scheme can be a valuable epidemiological tool in tracing the origins of food poisoning caused by B . cereus. J Antibiot (Tokyo), 1995 Mar, 48(3), 226 - 32 Inhibition of the binding of oxidized low density lipoprotein to the macrophages by iturin C-related compounds; Park JK et al.; Binding of modified lipoproteins including oxidized low density lipoprotein (oxidized LDL) to cell surface receptors is an initial step of conversion of monocyte-derived macrophages into lipid-laden foam cells, a key cellular component in the early lesions of atherosclerosis . We have searched for microbial metabolites that inhibit oxidized LDL-induced lipid accumulation in macrophages and isolated three compounds from a strain of Bacillus sp . as inhibitors of oxidized LDL binding . By chemical and spectroscopic analyses, these metabolites were shown to be related to the cyclic lipopeptide iturin C . Two of these compounds were novel metabolites having long chain beta-amino acid moieties of different length . These agents, at concentrations ranging from 5 to 20 microM, inhibited cell surface binding of oxidized 125I-LDL, resulting in reduced intracellular accumulation and degradation of the lipoprotein as well as in reduced cholesteryl ester formation from {14C}oleate in macrophages J774. J Invertebr Pathol, 1995 Mar, 65(2), 162 - 73 The insecticidal CryIB crystal protein of Bacillus thuringiensis ssp . thuringiensis has dual specificity to coleopteran and lepidopteran larvae; Bradley D et al.; The crystals found in sporulation extracts of Bacillus thuringiensis (Berliner) contain proteins that are highly toxic to insects . Different crystal proteins exhibit distinct specificities for restricted groups of insects . An uncharacterized strain of B . thuringiensis (BtS2), derived from China, was found to carry several crystal protein genes and to be toxic to a wide variety of insects, including some coleopterans . Surprisingly, the coleopteran toxicity was traced to a CryIB-class protein . The previously cloned CryIB protein from B . thuringiensis ssp . thuringiensis strain HD-290-I, which was believed to be lepidopteran-specific, was also found to be toxic to at least two species of coleopteran larvae under certain conditions . In contrast to CryIB toxicity toward lepidopterans, the coleopteran activity of CryIB is enhanced by solubilization and by truncation with trypsin prior to administration . The magnitude of this effect varies with the host species and is reversed for the one lepidopteran tested . These results suggest that, for at least some insects, the apparent host specificity of CryIB may depend both on differences in midgut environment and on differences in toxin-receptor interaction . The results of insect toxicity experiments with a series of deletion mutants allowed definition of a CryIB protein fragment of ca . 65 kDa as the smallest peptide that retains bioactivity against both lepidopteran and coleopteran larvae . Deletions smaller than this resulted in the production of a protein that was nontoxic to both lepidopteran and coleopteran larvae. Microbiology, 1995 Mar, 141 ( Pt 3), 649 - 54 Effects of signal peptide mutations on processing of Bacillus stearothermophilus alpha-amylase in Escherichia coli; Suominen I et al.; Bacillus stearothermophilus alpha-amylase has a signal peptide typical for proteins exported by Gram-positive bacteria . There is only one signal peptidase processing site when the protein is exported from the original host, but when it is exported by Escherichia coli, two alternative sites are utilized . Site-directed mutagenesis was used to study the processing in E . coli . Processing sites for 13 B . stearothermophilus alpha-amylases carrying mutations in their signal peptide were determined . Processing of the signal peptide was remarkably tolerant to mutations, because switching between the alternative sites was possible . The length and the sequence of the region between the hydrophobic core and the cleavage site was crucial for determining the choice of the processing site . Some mutations more distal to the cleavage site also affected the site preference. Microbiology, 1995 Mar, 141 ( Pt 3), 629 - 39 Bacillus thuringiensis protoxin: location of toxic border and requirement of non-toxic domain for high-level in vivo production of active toxin; Wabiko H et al.; Insecticidal crystal proteins, or protoxins, of Bacillus thuringiensis are composed of two domains, an amino-terminal half essential for toxicity, and a carboxy-terminal half with an as yet unassigned function . To define the boundary of the two domains, sequential termination codons were introduced from the 3'-end of the DNA sequence encoding the toxic domain of the 1155-residue cry1A(b) gene product . The mutated and the intact genes were placed under the control of the Escherichia coli inducible promoter PrecA, and toxicity of the cell extracts was determined using silkworm larvae . Under non-induced conditions, in which the gene products accumulated to a limited degree, mutations encoding 606 amino acid residues or more were toxic, whereas those encoding 605 residues or less were non-toxic . Comparison of the toxicities and the levels of the toxic proteins suggested that the mutant proteins had comparable activity to that of the intact protoxin . Furthermore, the non-toxic protein seemed to be unstable in the extracts . To investigate the roles of the non-toxic domain, the mutant proteins were overproduced in both E . coli and B . thuringiensis . The intact and the mutated genes carrying natural promoters were introduced into acrystalliferous B . thuringiensis . Upon induction of PrecA in E . coli, and upon sporulation in B . thuringiensis, there was a large accumulation of gene products which formed inclusion bodies . The inclusion bodies of the intact protoxin were active, whereas those of the mutant proteins were inactive . Inclusion bodies of the intact protein could be solubilized in alkali, whereas the mutant inclusion bodies were insoluble . Since solubilization under alkaline conditions in the insect midgut is considered to be the first step of toxic action, the non-toxic domain is required to direct the synthesis of inclusion bodies as an active soluble form. Eur J Immunol, 1995 Mar, 25(3), 838 - 46 Contribution of alpha/beta and gamma/delta T lymphocytes to immunity against Mycobacterium bovis bacillus Calmette Guérin: studies with T cell receptor-deficient mutant mice; Ladel CH et al.; Mutant mice with defined T cell deficiencies were infected with Mycobacterium bovis bacillus Calmette Guerin (BCG) and the relative contribution of alpha/beta T cells and gamma/delta T cells to the host immune response was assessed . Recombinase activating gene (RAG-1)-/- mutants as well as T cell receptor (TcR) beta-/-, but not TcR-delta-/-, mutants succumbed to M . bovis BCG infection and failed to develop granulomatous lesions . Antigen-induced IFN-gamma production by spleen cells in vitro was abrogated in RAG-1-/- mutants and markedly diminished in TcR-beta-/- and TcR-delta-/- mice . Reconstitution experiments suggest that both alpha/beta and gamma/delta T cells are essential for antigen-specific IFN-gamma secretion . Our data formally prove the crucial role of alpha/beta T cells and reveal accessory functions of gamma/delta T cells in optimum immunity against M . bovis BCG. Eur J Immunol, 1995 Mar, 25(3), 672 - 6 Interleukin-12 is required for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice; Wysocka M et al.; Several cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS) . Priming of mice with the avirulent Bacille Calmette Guerin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production . In response to low doses (1 microgram/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-gamma production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-gamma production . However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1-3 ng/ml at 3-6 h after LPS injection, whereas IFN-gamma production was observed only in BCG-primed mice . The priming effect of BCG on IFN-gamma production appears to be mostly due to its ability to increase TNF-alpha production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-gamma production, as shown by the ability of injection of TNF-alpha and LPS (1 microgram/mouse), but not LPS alone, to induce IFN-gamma production . However, in addition to TNF-alpha, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-gamma production, because co-injection of TNF-alpha and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-gamma production in vivo . Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-gamma production, also completely protect BCG-primed mice injected with up to 10 micrograms of LPS from shock-induced death . Thus, IL-12 is required for IFN-gamma production and lethality in an endotoxic shock model in mice. Br J Ophthalmol, 1995 Mar, 79(3), 250 - 6 Is leprosy blindness avoidable? The effect of disease type, duration, and treatment on eye damage from leprosy in Uganda; Waddell KM et al.; AIMS--The study was designed to measure the prevalence, range, and severity of eye involvement in leprosy patients; to relate this to disease type, duration, and treatment to identify risk factors; and to provide practical guidelines for programme managers and field staff on the prevention of blindness . METHODS--The visual outcome was assessed in a population based sample of patients in Kasese District, Uganda followed for up to two decades, and related to disease features and treatment . A total of 678 patients responded to an invitation out of 2715 registered since 1973 . RESULTS--Low vision was present in 4.4% of people and blindness in 1.3%, with 1.5% and 0.6% respectively being due to leprosy . Some 12.4% of patients had iritis, of whom 33% had visual loss in one or both eyes, 3.7% of patients had lagophthalmos, and 11.7% had lens opacity . For multi-bacillary (PB) cases, the adjusted odds ratios were: for iritis 4.6 (95% CI 2.6-8.2), for lagophthalmos 1.4 (0.6-3.2), and for lens opacity 1.7 (1.0-3.0) . Potentially sight threatening (PST) lesions were present in 16.8% of patients (95% CI 14.0-19.6) . CONCLUSION--Levels of eye involvement in this study are low compared with many surveys . Visual loss is uncommon and is more often caused by other diseases; in the present era of multidrug therapy (MDT) it is very unlikely to be caused by leprosy . It is more common with advancing age . PST lesions, especially iritis, may occur in both PB and MB cases, even if the diagnosis of leprosy is made early and MDT started immediately; they may occur also after completion of MDT . But eye complications need not proceed to loss of sight if treated promptly, and blindness can be avoided . Training of front line staff is therefore crucial. J Biochem (Tokyo), 1995 Mar, 117(3), 467 - 70 Homogeneity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus; Hiromasa Y et al.; The pyruvate dehydrogenase multienzyme complex was purified from Bacillus stearothermophilus by means of six gel-filtration column chromatographies; once on Cellulofine GCL-2000, twice on Sepharose CL-2B, and three times on Sephacryl S-500HR . The molecular size distribution of the complex was examined in detail by gel-filtration chromatography, analytical and sucrose-density ultracentrifugations, and dynamic light scattering . The complex was found to be homogeneous; a dimeric complex was undetectable even with a high concentration of protein (below 6.8 mg/ml). Mol Microbiol, 1995 Mar, 15(6), 1049 - 53 Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria; Deutscher J et al.; CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag . CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46 . HPr, a phospho-carrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase . Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA . The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate . These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism . Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression . The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity. Gastroenterol Hepatol, 1995 Mar, 18(3), 125 - 8 {Gastric tuberculosis: a report of 2 cases}; Fernandez de la Puebla Gimenez RA et al.; Two patients with gastric tuberculosis are described . The first presented toxic syndrome, multiple abdominal adenopathies, microcytic anemia and a subcardial ulcer with malignant characteristics at endoscopy . Diagnosis was based on the positivity of Ziehl-Neelsen staining and on the growth of Mycobacterium tuberculosis in the culture of the gastric mucosa . The second patient presented toxic syndrome, fever and a miliary pattern on thoracic radiography . Endoscopy demonstrated an ulcerated nodular lesion with granulomas with acid alcohol resistant bacillus being observed on biopsy . Mycobacterium tuberculosis was found in both the sputum and bronchoaspirate . The evolution of both cases was favorable with specific treatment. J Am Mosq Control Assoc, 1995 Mar, 11(1), 86 - 9 Control of Anopheles stephensi breeding in construction sites and abandoned overhead tanks with Bacillus thuringiensis var . israelensis; Kumar A et al.; Bacillus thuringiensis (H-14), strain 164 (Bactoculicide) when applied at 1 g/m2 surface area successfully controlled Anopheles stephensi breeding in construction sites, abandoned overhead tanks, and curing waters . Subsequent to application, no pupal production was observed in these habitats for 3, 18, and 21 days, respectively . Based on these findings, inclusion of Bactoculicide in the bioenvironmental vector control strategy is suggested and fortnightly spraying in construction sites at 1 g/m2 surface area is recommended for the containment of vector breeding . However, frequent retreatment of abandoned overhead tanks would be uneconomical and operationally impractical. J Am Mosq Control Assoc, 1995 Mar, 11(1), 72 - 6 Comparative toxicity of selected larvicides and insect growth regulators to a Florida laboratory population of Aedes albopictus; Ali A et al.; Five organophosphates (OPs) (chlorpyrifos, chlorpyrifos methyl, fenthion, malathion, and temephos), 3 pyrethroids (bifenthrin, cypermethrin, and permethrin), and 2 microbial pesticides (Bacillus thuringiensis serovar.israelensis {B.t.i.} and Bacillus sphaericus) were tested as larvicides against a Florida Aedes albopictus population colonized in the laboratory . In addition, 3 insect growth regulators (IGRS) (diflubenzuron, methoprene, and pyriproxyfen) were evaluated . All OPs, except for malathion, were highly effective as indicated by low LC90s ranging from 0.0069 ppm (chlorpyrifos) to 0.026 ppm (fenthion); the larvae were considered tolerant to malathion (LC90 = 1.043 ppm) . LC90 values of pyrethroids were: 0.0175 ppm (bifenthrin), 0.0079 ppm (cypermethrin), and 0.0031 ppm (permethrin) . Commercial products of B.t.i., Vectobac and Bactimos were considered economically effective against Ae . albopictus larvae but products of B . sphaericus were ineffective (LC90s > 28 ppm) . The IGRs showed exceptional activity . Pyriproxyfen (LC90 = 0.000376 ppm), was 2.23 and 21.5 times more toxic than diflubenzuron and methoprene, respectively . In general, toxicity ranking of chemicals and microbials tested was: IGRs > pyrethroids > OPs > microbials. J Am Mosq Control Assoc, 1995 Mar, 11(1), 107 - 10 Effect of low temperature on feeding rate of Aedes stimulans larvae and efficacy of Bacillus thuringiensis var . israelensis (H-14); Walker ED; Experiments were conducted to determine the effects of low temperature (0 and 4 degrees C), vs . a high temperature (22 degrees C), on the feeding rate of Aedes stimulans larvae, and their susceptibility to Bacillus thuringiensis var . israelensis (H-14) (B.t.i.) . Third-instar Ae . stimulans slowed but did not halt feeding at 0 and 4 degrees C compared to 22 degrees C . Susceptibility of larvae, as measured by LC50 values, to B.t.i . was highest at 22 degrees C (LC50 = 0.1 ppm), and lower at 4 degrees C (LC50 = 0.2 ppm) and 0 degree C (LC50 = 0.9 ppm) . The data from the feeding and susceptibility experiments suggest that decreased efficacy of B.t.i . at low temperatures may occur because the rate of larval feeding decreases . Low water temperature should be a consideration during operational applications of B.t.i . for control of larvae in cold-water habitats, such as the spring Aedes species. J Am Mosq Control Assoc, 1995 Mar, 11(1), 1 - 5 Development of a high level of resistance to Bacillus sphaericus in a field population of Culex quinquefasciatus from Kochi, India; Rao DR et al.; Field resistance to Bacillus sphaericus was observed in a population of Culex quinquefasciatus in Kochi, India, exposed to 35 rounds of spraying with a formulation of B . sphaericus 1593 M over a 2-year period . Larvae from the sprayed area gave LC50 and LC90 values that were 146 and 180 times greater than corresponding values for a susceptible strain from an unsprayed locality . When the resistant strain was colonized in the laboratory and subjected to moderate selection pressure at each generation, resistance rapidly increased and by the 18th generation it was 6,223 and 31,325 times greater at the LC50 and LC90 levels in comparison with the susceptible strain . There were no significant differences among 6 susceptible strains tested . Tests were repeated and validated using the standard primary powder SPH88, B . sphaericus 2362 . No cross resistance was observed against B . thuringiensis H-14. J Mol Cell Cardiol, 1995 Mar, 27(3), 893 - 900 Phospholipid degradation in hypoxic/reoxygenated cardiomyocytes in response to phospholipase C from Bacillus cereus; Forsdahl K et al.; In the present study, we investigated possible mechanisms behind exogenous phospholipase C-induced glycerol production in irreversibly damaged myocytes . Rat ventricular myocytes were preincubated for 60 min in substrate-free Krebs-Henseleit bicarbonate buffer equilibrated with 95% N2-5% CO2 (37 degrees C, pH = 7.4), resulting in exhaustion of cellular high energy phosphates and loss of rod-shaped morphology . At the end of the preincubation period, the incubation vials were divided into two groups; one receiving 10 mU/ml phospholipase C (PC-PLC), whereas the other received an equivalent volume of buffer (control incubations) . Incubation was then continued for another 60 min under 95% air-5% CO2 atmosphere . Samples for measurement of metabolite levels were taken immediately after cell isolation, at the end of the preincubation period and at the end of the normoxic incubation period . During the 60 min incubation period following reoxygenation, glycerol output was markedly higher from PC-PLC treated than from control myocytes . However, the elevated glycerol output from these cells was not accompanied by a simultaneous rise in glycerol-3-phosphate, nor was it inhibited by inclusion of pyruvate in the incubation buffer . On the other hand, glycerol output from PC-PLC treated myocytes was effectively inhibited by a diacylglycerol lipase inhibitor (U-57908, The Upjohn Company) . Analysis of cellular lipids revealed a 22% reduction of phospholipid in PC-PLC treated myocytes (P < 0.02), while the content of triacylglycerol, diacylglycerol and unesterified fatty acids increased by 76, 261 and 103%, respectively (P < 0.02) . No significant changes were observed for these parameters in control myocytes.(ABSTRACT TRUNCATED AT 250 WORDS) J Pharm Pharmacol, 1995 Mar, 47(3), 177 - 81 Interaction between fibronectin-bearing surfaces and Bacillus Calmette-Guérin (BCG) or gelatin microparticles; Lou Y et al.; Gelatin, prepared commercially by degradation of animal collagen, was studied to see whether it had an affinity for fibronectin, which has a known affinity for collagen, and whether gelatin-based drugs could be used to target fibronectin-excreting tumours . Bacillus Calmette-Guerin (BCG) vaccine, an attenuated strain of Mycobacterium bovis, is currently the most effective treatment for superficial transitional cell carcinoma of the bladder . The living cells of the BCG vaccine associate with the fibronectin-bearing surfaces of the tumour . Using a multi-well culture plate technique, gelatin microparticles were shown to be adsorbed onto murine S180 sarcoma cells and this reaction was substantially inhibited by the addition of human plasma fibronectin . The avidities of various BCG substrains and gelatin microparticles for glass-bound fibronectin were measured and the association constants determined . The gelatin microparticles associated with the fibronectin with equal avidity as the BCG cells . The results suggest that this model system may allow the investigation of gelatin-based drug delivery devices capable of targeting fibronectin-bearing surfaces associated with some tumours. Dig Dis, 1995 Mar-Apr, 13(2), 108 - 18 Whipple's disease; Fantry GT et al.; Whipple's disease is a chronic systemic infectious disease caused by Tropheryma whippelii that typically involves the small intestine and causes malabsorption . Extraintestinal manifestations such as arthritis and fever are common and often exist prior to the onset of gastrointestinal symptoms . Involvement of the central nervous system can occur and lead to permanent sequelae . Weight loss, hyperpigmentation, and lymphadenopathy are frequent findings . The definitive diagnosis is made by biopsy of the small intestine mucosa which reveals infiltration of the lamina propria of the small intestine with periodic acid-Schiff positive macrophages . Treatment with trimethoprim combined with sulfamethoxazole for 1 year usually results in clinical remission and an excellent prognosis . Recent advances using molecular techniques to identify the uncultured bacillus of Whipple's disease should lead to a better understanding of the pathophysiology and allow for the development of a sensitive noninvasive diagnostic test. J Clin Microbiol, 1995 Mar, 33(3), 742 - 4 A chemically defined liquid medium that supports primary isolation of Rochalimaea (Bartonella) henselae from blood and tissue specimens; Wong MT et al.; Rochalimaea (Bartonella) henselae is a fastidious, slowly growing, gram-negative bacillus that is an etiologic agent of bacillary angiomatosis, cat scratch disease, and related syndromes . Accumulation of direct microbiologic evidence of the relationship between the organism and the syndromes compatible with cat scratch disease has been hindered by the difficulties in the primary isolation of the organism from infected tissue specimens . A chemically defined liquid medium was developed to support the growth of Rochalimaea species to facilitate study of the organism . This medium was also used successfully to isolate R . henselae from clinical specimens from infected patients and a domestic cat . Recovery of R . henselae in this was more successful than when recovery was attempted on solid agar . This cell-free, extract-free, defined medium additionally supported the growth of Rochalimaea quintana and Afipia felis. J Bacteriol, 1995 Mar, 177(6), 1444 - 51 The S-layer from Bacillus stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase; Egelseer E et al.; The S-layer lattice from Bacillus stearothermophilus DSM 2358 completely covers the cell surface and exhibits oblique symmetry . During growth of B . stearothermophilus DSM 2358 on starch medium, three amylases with molecular weights of 58,000, 98,000, and 184,000 were secreted into the culture fluid, but only the high-molecular-weight enzyme was found to be cell associated . Studies of interactions between cell wall components and amylases revealed no affinity of the high-molecular-weight amylase to isolated peptidoglycan . On the other hand, this enzyme was always found to be associated with S-layer self-assembly products or S-layer fragments released during preparation of spheroplasts by treatment of whole cells with lysozyme . The molar ratio of S-layer subunits to the bound amylase was approximately 8:1, which corresponded to one enzyme molecule per four morphological subunits . Immunoblotting experiments with polyclonal antisera against the high-molecular-weight amylase revealed a strong immunological signal in response to the enzyme but no cross-reaction with the S-layer protein or the smaller amylases . Immunogold labeling of whole cells with anti-amylase antiserum showed that the high-molecular-weight amylase is located on the outer face of the S-layer lattice . Because extraction of the amylase was possible without disintegration of the S-layer lattice into its constituent subunits, it can be excluded that the enzyme is incorporated into the crystal lattice and participates in the self-assembly process . Affinity experiments strongly suggest the presence of a specific recognition mechanism between the amylase molecules and S-layer protein domains either exposed on the outermost surface or inside the pores . In summary, results obtained in this study confirmed that the S-layer protein from B . stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase. Biochemistry, 1995 Feb 28, 34(8), 2560 - 5 Role of glycine 212 in the allosteric behavior of phosphofructokinase from Bacillus stearothermophilus; Zhu X et al.; Crystallographic studies indicate that the loop between alpha-helix 8 and beta-strand H (the 8H loop) which borders the effector site of Bacillus stearothermophilus phosphofructokinase (BsPFK) is involved in the allosteric mechanism of the enzyme {Schirmer, T., and Evans, P.R . (1990) Nature 343, 140-145} . The residue at one end of this loop, glycine 212, has been proposed to be a pivot about which the loop hinges . Using site-directed mutagenesis, glycine 212 was replaced with valine (G212V) . Steady-state kinetic analysis and ligand binding studies on the altered and native PFKs showed that the G212V substitution resulted in discernible changes at the effector site . The mutated PFK required a 3-fold higher concentration of the allosteric inhibitor phosphoenolpyruvate than did the native enzyme to cause the same level of inhibition . The altered PFK had a 2-fold higher dissociation constant for the allosteric activator GDP than the wild-type enzyme . More importantly, whereas the native PFK was fully activated by 1 mM GDP from its PEP-inhibited T-state, the altered enzyme was only marginally activated . On the other hand, the G212V mutation resulted in no changes at the catalytic site of BsPFK . The catalytic rate constant kcat remained unchanged . The altered PFK had the same Km values for ATP and fructose-6-phosphate (Fru-6-P) as did the wild-type enzyme . Furthermore, starting from the same PEP-inhibited T-state, both enzymes gave identical sigmoidal responses to increasing Fru-6-P concentration, indicating that Fru-6-P can activate both to the R-state. J Biol Chem, 1995 Feb 24, 270(8), 3828 - 35 A chimeric bacterial phosphofructokinase exhibits cooperativity in the absence of heterotropic regulation; Byrnes WM et al.; The phosphofructokinases (PFKs) from the bacteria Escherichia coli and Bacillus stearothermophilus differ markedly in their regulation by ATP . Whereas E . coli PFK (EcPFK) is profoundly inhibited by ATP, B . stearothermophilus PFK (BsPFK) is only slightly inhibited . The structural basis for this difference could be closure of the active site via a conformational transition that occurs in the ATP-binding domain of EcPFK, but is absent in BsPFK . To investigate the role of this transition in ATP inhibition of EcPFK, we have constructed a chimeric enzyme that contains the "rigid" ATP-binding domain of BsPFK grafted onto the remainder of the EcPFK subunit . The chimeric PFK has the following characteristics: (i) tetrameric structure and kinetic parameters similar to those of the native enzymes, (ii) insensitivity to regulation by the effector phosphoenolpyruvate despite its ability to bind to the enzyme, and (iii) a sigmoidal (nH around 2) fructose 6-phosphate saturation curve . From the results, it is concluded that the active site regions of the two native enzymes are remarkably similar, but their effector sites and their mechanisms of heterotropic regulation are different . The chimeric subunit is locked in a structure resembling that of activated E . coli PFK, yet the enzyme can exist in two different conformational states . Mechanisms for its sigmoidal kinetics are discussed. Biochim Biophys Acta, 1995 Feb 22, 1247(1), 97 - 103 The role of histidine residues in the catalytic act of cyclomaltodextrin glucanotransferase from Bacillus circulans var . alkalophilus; Mattsson P et al.; Our previous study on cyclomaltodextrin glucanotransferase (CGTase) by chemical modification implied the importance of one or two histidine residues in the cyclization reaction of the enzyme . Based on a computer modelled three-dimensional structure of the CGTase, five histidine residues were chosen as targets for the site-directed mutagenesis . The histidine residues 98, 140, 233 and 327 were replaced by aspartate and His-177 by proline using polymerase chain reaction-mediated techniques . The CGTase variants H98D, H140D, H233D and H327D resulted in a profound decrease in the cyclizing and amylolytic activities, while mutation H177P had little influence on the activities but affected the thermal stability and the width of the pH optimum . It is suggested that His-98 functions as (or as a significant part of) the subsite 2 for the binding of the substrate in CGTase and therefore H98D destabilizes the intermediate for cyclization, but does not markedly affect the hydrolytic reactions . Mutants H140D and H233D produced only minor amounts of alpha-cyclodextrin, did not exhibit substrate inhibition with maltotriose and showed non-Michaelis-Menten kinetics . It is proposed that the variants H140D, H233D and H327D cause steric hindrances near the active center, while mutation H177D has similar consequences on the same site spatially. Biochemistry, 1995 Feb 21, 34(7), 2234 - 40 X-ray structure of cyclodextrin glycosyltransferase complexed with acarbose . Implications for the catalytic mechanism of glycosidases; Strokopytov B et al.; Crystals of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans strain 251 were soaked in buffer solutions containing the pseudotetrasaccharide acarbose, a strong amylase- and CGTase inhibitor . The X-ray structure of the complex was elucidated at 2.5-A resolution with a final crystallographic R value of 15.8% for all data between 8.0 and 2.5 A . Acarbose is bound near the catalytic residues Asp229, Glu257, and Asp328 . The carboxylic group of Glu257 is at hydrogen bonding distance from the glycosidic oxygen in the scissile bond between the B and C sugars (residue A is at the nonreducing end of the inhibitor) . Asp328 makes hydrogen bonds with the 4-amino-4,6-dideoxyglucose (residue B), and Asp229 is in a close van der Waals contact with the C1 atom of this sugar . From this we conclude that in CGTase Glu257 acts as the proton donor and Asp229 serves as the general base or nucleophile, while Asp328 is involved in substrate binding and may be important for elevating the pKa of Glu257 . On the basis of these results it appears that the absence of the C6-hydroxyl group in the B sugar is responsible for the inhibitory properties of acarbose on CGTase . This suggests that the C6-hydroxyl group of this sugar plays an essential role in the catalytic mechanism of CGTase.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1995 Feb 17, 270(7), 3081 - 8 Crystal structure and site-directed mutagenesis of Bacillus macerans endo-1,3-1,4-beta-glucanase; Hahn M et al.; In beta-glucans those beta-1,4 glycosidic bonds which are adjacent to beta-1,3 bonds are cleaved by endo-1,3-1,4-beta-glucanases (beta-glucanases) . Here, the relationship between structure and activity of the beta-glucanase of Bacillus macerans is studied by x-ray crystallography and site-directed mutagenesis of active site residues . Crystal structure analysis at 2.3-A resolution reveals a jelly-roll protein structure with a deep active site channel harboring the amino acid residues Trp101, Glu103, Asp105, and Glu107 as in the hybrid Bacillus beta-glucanase H(A16-M) (Keitel, T., Simon, O., Borriss, R., and Heinemann, U . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 5287-5291) . Different mutant proteins with substitutions in these residues are generated by site-directed mutagenesis, isolated, and characterized . Compared with the wild-type enzyme their activity is reduced to less than 1% . Several mutants with isosteric substitutions in Glu103 and Glu107 are completely inactive, suggesting a direct role of these residues in glycosyl bond hydrolysis . The kinetic properties of mutant beta-glucanases and the crystal structure of the wild-type enzyme are consistent with a mechanism where Glu103 and Glu107 are the catalytic amino acid residues responsible for cleavage of the beta-1,4 glycosidic bond within the substrate molecule. Eur J Biochem, 1995 Feb 15, 228(1), 206 - 10 Resistance in a laboratory population of Culex quinquefasciatus (Diptera: Culicidae) to Bacillus sphaericus binary toxin is due to a change in the receptor on midgut brush-border membranes; Nielsen-Leroux C et al.; Direct binding experiments with isolated brush border membrane fractions (BBMF) from larvae of a susceptible laboratory strain of Culex quinquefasciatus Say, indicated the presence of a single class of Bacillus sphaericus binary toxin receptors . The dissociation constant (Kd) was approximately 11 nM and the maximum binding capacity (Bmax) approximately 8 pmol/mg BBMF protein . Similar binding experiments with a field population of C . quinquefasciatus that had been selected in the laboratory to more than 100,000-fold resistance to B . sphaericus binary toxin failed to reveal the presence of any specific binding . Thus this resistant strain had lost the functional receptor for B . sphaericus toxin . The binding characteristics of BBMF from the F1 larval progeny (susceptible females x resistant males) were very close to those of the parental susceptible strain, consistent with the resistance being recessive. FEMS Microbiol Lett, 1995 Feb 15, 126(2), 133 - 7 Characterization of a DNA fragment carrying the raw starch-digesting alpha-amylase and salt-dependent alpha-amylase genes from Bacillus circulans F-2; Kim CH; A 5.4 kb HindIII DNA fragment carrying the gene encoding raw starch-digesting alpha-amylase (RSDA), has been previously cloned from Bacillus circulans F-2 and expressed in Escherichia coli {Kim et al . (1990) Biochim . Biophys . Acta 1048, 2233-2238} . Interestingly, when the cell extract of E . coli harboring a plasmid carrying this fragment was incubated with 1 M NaCl, it exhibited about 10 times higher enzyme activity than when assayed without NaCl . Differential zymograms showed two different amylase activities: one for RSDA and the other for a salt-dependent alpha-amylase (SDA) . Even though RSDA activity was detected without NaCl, SDA activity was detected only in high concentrations of NaCl . SDA activity was fully detected at above 1 M NaCl . Results from subcloning of the genes, fractionation analysis of cell extracts, and immunological assays clearly suggested that the two amylases are genetically distinct and that genes for both enzymes are closely linked on the 5.4 kb DNA fragment. Biochem Pharmacol, 1995 Feb 14, 49(4), 567 - 74 Activation and cytotoxicity of 2-alpha-aminoacyl prodrugs of methotrexate; Smal MA et al.; In an effort to improve the selectivity of the anticancer drug methotrexate (MTX), a series of potential prodrugs in which the 2-amino group was acylated with various alpha-amino acids (as well as L-pyroglutamic acid) was synthesized . Such derivatives are anticipated to be hydrolysed to MTX by appropriate aminopeptidases localized (over-expressed naturally or targeted as anti-tumor antibody conjugates) in the vicinity of the tumor . The L-leucyl, L-valyl, L-isoleucyl, D-alanyl and L-pyroglutamyl derivatives were assessed as to their suitability as prodrugs . Except for the L-pyroglutamyl compound, all derivatives decomposed slowly when incubated in phosphate buffer, pH 7.3; the formation of MTX was minimal . No major differences were observed when serum was included in the incubation medium, except for the L-leucyl compound, which was hydrolysed to MTX . The L-leucyl, L-valyl and L-isoleucyl derivatives were hydrolysed readily to MTX by aminopeptidase M (EC 3.4.11.2), while the L-pyroglutamyl and D-alanyl compounds were activated by pyroglutamate aminopeptidase (EC 3.4.19.3) (from Bacillus amyloliquefaciens) and D-aminopeptidase (from Ochrobactrum anthropi), respectively . When tested for inhibition of the target enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3), 2-L-valyl-MTX showed inhibition two orders of magnitude poorer than that given by MTX, in agreement with the expectation that acylation of the 2-amino group reduces binding to DHFR . After treatment of this derivative with aminopeptidase M, the extent of inhibition correlated with the amount of MTX formed . MTX derivatives alone or in combination with the complementary peptidase were tested for cytotoxicity on murine L1210 cells in culture . The above-listed derivatives were considerably less cytotoxic than MTX, except for the L-leucyl derivative which showed considerable cytotoxicity . When the appropriate exogenous peptidase was included, the cytotoxicity of the activated prodrugs approached that of MTX . These results indicate that 2-L-leucyl-MTX is unsuitable as a prodrug since it is activated prematurely by serum enzymes . Although the L-valyl and L-isoleucyl derivatives do not hydrolyse to MTX in serum and are readily activated, they are not ideal prodrugs since they decompose under physiological conditions; the properties of the decomposition product will have a bearing on the ultimate suitability of these compounds . 2-L-Pyroglutamyl-MTX is the best candidate prodrug, showing stability and ready activation by the appropriate aminopeptidase. J Biol Chem, 1995 Feb 10, 270(6), 2571 - 8 The assembly and organization of the alpha 5 and alpha 7 helices from the pore-forming domain of Bacillus thuringiensis delta-endotoxin . Relevance to a functional model; Gazit E et al.; The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA delta-endotoxin contains two helices, alpha 5 and alpha 7, that are highly conserved within all different Cry delta-endotoxins . To gain information on the mode of action of delta-endotoxins, we have used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability to self-assemble and to co-assemble within lipid membranes of alpha 5 and alpha 7 . Circular dichroism (CD) spectroscopy revealed that alpha 7 adopts a predominantly alpha-helical structure in methanol, similar to what has been found for alpha 5, and consistent with its structure in the intact molecule . The hydrophobic moment of alpha 7 is higher than that calculated for alpha 5; however, alpha 7 has a lesser ability to permeate phospholipids as compared to alpha 5 . Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated that alpha 7 binds to phospholipid vesicles with a partition coefficient in the order of 10(4) M-1 similar to alpha 5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity found with alpha 5 . Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in their membrane-bound state, alpha 5 self-associates but alpha 7 does not, and that alpha 5 coassembles with alpha 7 but not with an unrelated membrane bound alpha-helical peptide . Furthermore, resonance energy transfer experiments, using alpha 5 segments, specifically labeled in either the N- or C-terminal sides, suggest a parallel organization of alpha 5 monomers within the membranes . Taken together the results are consistent with an umbrella model suggested for the pore forming activity of delta-endotoxin (Li, J., Caroll, J., and Ellar, D . J . (1991) Nature 353, 815-821), where alpha 5 has transmembrane localization and may be part of the pore lining segment(s) while alpha 7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane. J Biol Chem, 1995 Feb 10, 270(6), 2517 - 24 Glycan requirements of glycosylphosphatidylinositol phospholipase C from Trypanosoma brucei . Glucosaminylinositol derivatives inhibit phosphatidylinositol phospholipase C; Morris JC et al.; Glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol phospholipase C (PI-PLC) from Bacillus sp . both cleave glycosylphosphatidylinositols (GPIs) . However, phosphatidylinositol, which is efficiently cleaved by PI-PLC, is a very poor substrate for GPI-PLC . We examined GPI-PLC substrate requirements using glycoinositol analogs of GPI components as potential inhibitors . Glucosaminyl (alpha 1-->6)-D-myo-inositol (GlcN(alpha 1-->6)Ins), GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate, GlcN(alpha 1-->6)-2-deoxy-Ins, and GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate inhibited GPI-PLC . GlcN(alpha 1-->6)Ins was as effective as Man-(alpha 1-->4)GlcN(alpha 1-->6)Ins; we surmise that GlcN(alpha 1-->6)Ins is the crucial glycan motif for GPI-PLC recognition . Inhibition by GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate suggests product inhibition since GPIs cleaved by GPI-PLC possess a GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate at the terminus of the residual glycan . The effectiveness of GlcN(alpha 1-->6)-2-deoxy-Ins indicates that the D-myo-inositol (Ins) 2-hydroxyl is not required for substrate recognition, although it is probably essential for catalysis . GlcN(alpha 1-->6)-2-deoxy-L-myo-inositol, unlike GlcN(alpha 1-->6)-2- deoxy-Ins, had no effect on GPI-PLC; hence, GPI-PLC can distinguish between the two enantiomers of Ins . Surprisingly, GlcN(alpha 1-->6)Ins 1,2-cyclic phosphate was not a potent inhibitor of Bacillus cereus PI-PLC, and GlcN(alpha 1-->6)Ins had no effect on the enzyme . However, both GlcN(alpha 1-->6)Ins 1-phosphate and GlcN(alpha 1-->6)Ins 1-dodecyl phosphonate were competitive inhibitors of PI-PLC . These observations suggest an important role for a phosphoryl group at the Ins 1-position in PI-PLC recognition of GPIs . Other studies indicate that abstraction of a proton from the Ins 2-hydroxyl is not an early event in PI-PLC cleavage of GPIs . Furthermore, both GlcN(alpha 1-->6)-2-deoxy-Ins 1-phosphate and GlcN(alpha 1-->6)-2-deoxy-L- myo-inositol inhibited PI-PLC without affecting GPI-PLC . Last, the aminoglycoside G418 stimulated PI-PLC, but had no effect on GPI-PLC . Thus, these enzymes represent mechanistic subclasses of GPI phospholipases C, distinguishable by their sensitivity to GlcN(alpha 1-->6)Ins derivatives and aminoglycosides . Possible allosteric regulation of PI-PLC by GlcN(alpha 1-->6)Ins analogs is discussed. Biochem Biophys Res Commun, 1995 Feb 6, 207(1), 55 - 61 Terminal oxidases of the bb- and caa3-types in Bacillus sp . FTU; Muntyan MS et al.; We previously identified two oxidases in the membranes of bacterium Bacillus sp . FTU . One of them slowly (caa3) and the other rapidly (bo) recombines with carbon monoxide (CO) after laser flash photolysis, in this respect resembling the Escherichia coli bo- and bd-type oxidases, respectively . In the present study we found three copper atoms in the slowly CO-recombining oxidase from Bacillus sp . FTU . In the other oxidase, the copper content is very low and clearly substoichiometric . Reversed-phase chromatography revealed the presence of haems A and C in the Bacillus sp . FTU copper-containing oxidase and haems B and C in the non-copper-containing one . We thus suggest that the Bacillus sp . FTU oxidase rapidly reacting with CO previously attributed to bo-type by analogy in redox spectrum with the E . coli enzyme be redefined as bb-type oxidase. Biochem Biophys Res Commun, 1995 Feb 6, 207(1), 20 - 4 Improvement of thermal stability of subtilisin J by changing the primary autolysis site; Bae KH et al.; The thermostability of subtilisin J, an extracellular serine protease secreted from Bacillus stearothermophilus, has been improved by changing the primary autolysis site of the Asp-49 mutant protein . Previously we have shown that the Asp-49 mutant protein has proteolytic activity, but so unstable that it was primarily autolyzed in Tyr-58-Gln-59 peptide bond during cultivation (Jang et al . Biochim . Biophys . Acta . 1162, 233-235 1993) . In the present study, to mitigate the autolytic degradation and increase the thermostability, we deleted the Tyr-58 residue using the Asp-49 mutant as a template . This mutant (Asp-49/delta Tyr-58 mutant) protein showed an improved resistance to heat treatment without changing the catalytic efficiency of the enzyme . These results show that change of primary autolysis site can stabilize the subtilisin. Mol Gen Genet, 1995 Feb 6, 246(3), 301 - 8 Factors regulating cryIVB expression in the cyanobacterium--Synechococcus PCC 7942; Soltes-Rak E et al.; The expression of the larvicidal Bacillus thuringiensis subsp . israelensis cryIVB gene in cyanobacteria has been suggested to be an effective means of controlling mosquito populations . Using a variety of cryIVB constructs, in this study we have examined the effect of Synechococcus PCC 7942 culture age on intracellular toxin levels and have attempted to determine the mechanisms by which cryIVB gene expression is regulated . The data suggest that specific degradation of the cryIVB mRNA limits toxin production; however, the addition of cyanobacterial 3' untranslated DNA sequences to the cryIVB gene did not improve mRNA stability or toxin levels . An analysis of the cryIVB sequence and comparison of codon usage patterns with highly expressed cyanobacterial genes suggest that inefficient translation and intragenic ribosomal binding sites impede protein synthesis and result in rapid turnover of the toxin mRNA. Hindustan Antibiot Bull, 1995 Feb-Nov, 37(1-4), 16 - 24 Production of free amino acids by some earthworm-borne microorganisms; Joy EK et al.; Production of free amino acids by some earthworm-borne microorganisms was investigated in three different synthetic media . Among the fungi tried Gliocladium roseum and Heterocephallum aurantiacum; among bacteria screened Bacillus macerans and B . mycoides; and among actinomycetes tested Streptomyces rimosus, S . violans, S . antibiticus, S . corchorusii and S . atroolivaceus produced significant amount of free amino acids . No correlations could be observed between vegetative growth and free amino acid production. Hindustan Antibiot Bull, 1995 Feb-Nov, 37(1-4), 1 - 8 A carbon-limited medium for growth and sporulation of Bacillus thuringiensis var . kurstaki; Liu WM et al.; A culture medium for batch production of d-endotoxin by Bacillus thuringiensis (B., t.) has been modified . Through batch and continuous cultivation studies, the original medium was diagnosed to be limited in organic nitrogen . Corn steep liquor was found to be an excellent source for the organic nitrogen and its addition resulted in a carbon limited medium and in a significant increase in the amount of spore-toxin complex formed in shake flasks . Results of bioassay, conducted on Trichoplusia ni, suggest enhancement of larvicidal efficacy under carbon-limited growth conditions. J Econ Entomol, 1995 Feb, 88(1), 97 - 105 Variation in tolerance to Bacillus thuringiensis among and within populations of the spruce budworm (Lepidoptera: Tortricidae) in Ontario; van Frankenhuyzen K et al.; Variation in tolerance to Bacillus thuringiensis Berliner subsp . kurstaki (strain HD-1-S-1980) among and within populations of the spruce budworm, Choristoneura fumiferana (Clemens), was assessed in the laboratory . Force-feeding assays using offspring of females collected as pupae from nine locations throughout Ontario and from a laboratory colony (DCF) demonstrated limited variation in tolerance among populations . Variation among populations was comparable with the variation observed among repeated assays with different batches of larvae from the DCF colony . Population LC50s were not significantly associated with age of the outbreak, host-plant species, incidence of the microsporidian Nosema fumiferanae (Thomson), or size of the female parent . Upper limits for genetic variation in tolerance were estimated by examining variation among full-sibling families within same populations . Mortality of individual families ranged from 6.5 to 70.9% within five field populations and from 2.7 to 93.3% within two laboratory colonies in response to a dose that caused a mean mortality of 40% . Familial factors accounted for 32.8% of the phenotypic variation in response across field populations, as compared with 3% for population factors . These data suggest that the phenotypic variation in tolerance to B . thuringiensis has a substantial genetic component and may provide a basis for evolution of resistance given sufficient selection pressure. J Econ Entomol, 1995 Feb, 88(1), 21 - 6 Inheritance of resistance to Bacillus thuringiensis subsp . tenebrionis CryIIIA delta-endotoxin in Colorado potato beetle (Coleoptera: Chrysomelidae); Rahardja U et al.; We investigated the genetic inheritance of Colorado potato beetle, Leptinotarsa decemlineata (Say), resistance to Bacillus thuringiensis CryIIIA delta-endotoxin . Standard reciprocal crosses and backcrosses between susceptible (S) and resistant (R) strains were used to determine the characteristics of resistance . Analysis of probit lines from the F1 reciprocal crosses indicated that B . thuringiensis delta-endotoxin resistance was inherited autosomaly without maternal effects . We estimated the degree of dominance to be 0.77 and 0.76 for the (R x S) and (S x R) F1 generations, respectively, indicating that B . thuringiensis CryIIIA delta-endotoxin resistance is conferred by incompletely dominant genes . Chi-square analysis of mortality responses of backcrossed offspring suggested that resistance might be caused by more than one locus . The stability of resistance was also studied by testing seventeen generations of resistant beetles after the selection pressure was removed . When the selection pressure was removed,