Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 200 - 2 Purification, crystallization and preliminary X-ray crystallographic study of alpha-amylase from Bacillus stearothermophilus; Suvd D et al.; A recombinant alpha-amylase from Bacillus stearothermophilus was found to be produced as several isoforms arising from different N--terminal processing . Some of those isoforms were purified to homogeneity and crystallized at 293 K using the hanging-drop vapour-diffusion method under the following conditions: 35 mM sodium acetate (pH 4.6), 6.25%(v/v) 2-propanol, in the presence of 1.23%(w/v) acarbose (a pseudo-oligosaccharide inhibitor) in the drop . The crystals diffracted beyond 2.0 A resolution using synchrotron radiation at the Photon Factory, Tsukuba . They belong to the monoclinic space group P2(1), with unit-cell parameters a = 53.7 (2), b = 92.9 (4), c = 53.2 (2) A, beta = 109.4 (1) degrees. Acta Crystallogr D Biol Crystallogr, 2000 Feb, 56 ( Pt 2), 181 - 4 Crystallization and preliminary X-ray analysis of an intracellular xylanase from Bacillus stearothermophilus T-6; Teplitsky A et al.; Xylanases (1,4-beta-D-xylan xylanhydrolases; E.C . 3.2.1.8) hydrolyze the 1,4-beta-D-xylopyranosyl linkage of xylans . The structural characterization of xylanase active sites is of great interest, since it can lead to a better understanding of their catalytic mechanism and contribute significant knowledge to the rational design of specific oligosaccharide-binding sites via protein engineering . An intracellular xylanase gene (xynA2) from Bacillus stearothermophilus T-6 has recently been cloned and sequenced . The xynA2 gene encodes for an intracellular enzyme (IXT6) of 331 amino acids, with a calculated molecular weight of 38 639 Da and a pI of 5.72 . Based on sequence homology, the enzyme belongs to family 10 of the glycosyl hydrolases . The xynA2 gene product (IXT6) was overproduced in Escherichia coli and purified to homogeneity . Crystallographic studies of IXT6 were initiated in order to study the specificity and mechanism of catalysis of this unique xylanase, as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis . The M1 crystal form was found to be the most suitable for detailed crystal structure analysis . These crystals belong to a C--centered monoclinic crystal system (space group C2) with unit-cell parameters a = 170.6, b = 82.5, c = 80.0 A, beta = 91.43 degrees . They are mechanically strong, are fairly stable in the X-ray beam and diffract X--rays to better than 2.5 A resolution . A full 2.9 A resolution diffraction data set (97.9% completeness, R(merge) = 8.4%) has recently been collected from one crystal at room temperature using X-ray synchrotron radiation (lambda = 1.125 A) and a MAR300 imaging-plate area detector . A comparable 2.5 A data set was measured at 90 K using a rotating-anode X-ray source and an R-AXIS IIc imaging-plate area detector (97.2% completeness, R(merge) = 6.9%) . Molecular-replacement studies and multiple anomalous dispersion (MAD) experiments are currently in progress in order to determine the detailed three-dimensional structure of IXT6. J Virol, 2000 Mar, 74(5), 2073 - 83 The rice tungro bacilliform virus gene II product interacts with the coat protein domain of the viral gene III polyprotein; Herzog E et al.; Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein . The function of most of the viral proteins is still unknown . To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins . P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro . Domains involved in the P2-CP association have been identified and mapped on both proteins . To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants . The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3 . This study suggests that P2 could participate in RTBV capsid assembly. Folia Microbiol (Praha), 1999, 44(3), 271 - 5 Cell viability and protein turnover in nongrowing Bacillus megaterium at sporulation suppressing temperature; Kucerova H et al.; In Bacillus megaterium, a temperature that suppresses sporulation (43 degrees C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40-41 degrees C) . Here we show that, when cells grown at 35 degrees C and transferred to a sporulation medium, were subjected to shifts between 35 degrees C and the sporulation suppressing temperature (SST, 43 degrees C), their development and proteolytic activities were deeply affected . During the reversible sporulation phase that took place at 35 degrees C for 2-3 h (T2-T3), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation . During the following irreversible sporulation phase refractile heat-resistant spores appeared at T4-T5 . Protein turnover rate increased again after T2 and up to T8 60-70% prelabelled proteins were degraded . The SST suppressed sporulation at its beginning; at T3 no asymmetric septa were observed and the amount of heat-resistant spores at T8 was by 4-5 orders lower than at 35 degrees C . However, the cells remained viable and were able to sporulate when transferred to a lower temperature . Protein degradation was increased up to T3 but then its velocity sharply dropped and the amount of degraded protein at T8 corresponded to slightly more than one-half of that found at 35 degrees C . The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased . When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T2.5), the sporulation process was impaired . A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects . Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the amount of degraded protein was slightly lower than at 35 degrees C . A later (T4) shift to SST had no effect on the sporulation process. Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2232 - 5 Arginine-55 in the beta-arm is essential for the activity of DNA-binding protein HU from Bacillus stearothermophilus; Saitoh F et al.; DNA-binding protein HU (BstHU) from Bacillus stearothermophilus is a homodimeric protein which binds to DNA in a sequence-nonspecific manner . In order to identify the Arg residues essential for DNA binding, four Arg residues (Arg-53, Arg-55, Arg-58, and Arg-61) within the beta-arm structure were replaced either by Gln, Lys, or Glu residues, and the resulting mutants were characterized with respect to their DNA-binding activity by a filter-binding analysis and surface plasmon resonance analysis . The results indicate that three Arg residues (Arg-55, Arg-58, and Arg-61) play a crucial role in DNA binding as positively charged recognition groups in the order of Arg-55 > Arg-58 > Arg-61 and that these are required to decrease the dissociation rate constant for BstHU-DNA interaction . In contrast, the Arg-53 residue was found to make no contribution to the binding activity of BstHU. Biosci Biotechnol Biochem, 1999 Dec, 63(12), 2053 - 8 Catabolite repression of the xylanase gene (xynA) expression in Bacillus stearothermophilus no . 236 and B . subtilis; Cho SG et al.; Catabolite repression of the Bacillus stearothermophilus No . 236 xynA gene, encoding an extracellular xylanase, was investigated in this work . Expression of the xynA gene in the B . stearothermophilus strain was found to be subject to glucose catabolite repression, and the level of repression was about 50-fold when the relative amounts of xynA transcript synthesized on different carbon sources were analyzed . The experiments with the B . subtilis MW15 strains carrying plasmids containing the xynA::aprA fusion gene showed that the cloned xynA gene did not require any specific carbon source for its induction . Nevertheless, the expression of the cloned gene was repressed by the presence of glucose . From the nucleotide sequence of the cloned xynA gene, we found two potential catabolite responsive elements (cre) within its reading frame region (cre-1: nucleotides +160 to +173 and cre-2: +173 to +186) . Furthermore, by using various deletion derivatives of the xynA::aprA fusion plasmid (pMGW23), we suggested that only the cre-2 element might play a role in the glucose catabolite repression . Repression level of the xynA gene expression in the recombinant B . subtilis strain was estimated to be about 3-fold by analysis of the amounts of xynA transcript. Cytobios, 1998, 96(383), 133 - 9 Larvicidal activity of Bacillus thuringiensis isolated from Jordanian habitats against Drosophila melanogaster larvae; al-Momani F et al.; Bacillus thuringiensis was isolated from 23 of 37 samples obtained from different Jordanian habitats . Of the 37 samples, 187 different spore-forming colonies were selected and thirty (16%) were identified as B . thuringiensis based on their pathogenicity and production of parasporal inclusions . The lethal dose (LD50) of B . thuringiensis indicated a variation in their pathogenicity to Drosophila melanogaster larvae . Sensitivity of the first, the second and the third instar larvae of D . melanogaster showed slight variation in between . The third instar was the most sensitive stage to the pathogen spores. Eur J Pediatr, 2000 Mar, 159(3), 149 - 52 Isolated cutaneous response to granulocyte-monocyte colony stimulating factor in fatal idiopathic disseminated Bacillus-Calmette-Guerin infection; Sanal O et al.; Severe disseminated Bacillus-Calmette-Guerin (BCG) infection is very rare and has been regarded as idiopathic when no immunodeficiency is present . This entity seems to be due to several new types of inherited abnormalities in the pathways important in defence against Mycobacteria . Although improvement with interferon-gamma (IFN-gamma) has been reported in some patients, to our knowledge there are no reports on the effect of other cytokines in the treatment of these patients . We report here the clinical response to IFN-gamma and granulocyte-monocyte colony stimulating factor (GM-CSF) treatment in a patient with idiopathic disseminated BCG infection who failed to respond to multiple antimycobacterial agents . The patient showed partial and transitory response to IFN-gamma, however, GM-CSF treatment led to rapid improvement of skin lesions within 2 weeks without any effect on the progression of the disease in the other organ systems . CONCLUSION: The response of idiopathic disseminated Bacillus-Calmette-Guerin infection to granulocyte-monocyte colony stimulating factor treatment was limited to cutaneous lesions . Granulocyte-monocyte colony stimulating factor may have acted to promote wound healing or the levels of this factor achieved in other affected organs may have been inadequate. AIDS Res Hum Retroviruses, 2000 Jan 20, 16(2), 91 - 8 Recombinant bacillus Calmette-Guérin as a potential vector for preventive HIV type 1 vaccines; Falk LA et al.; In August 1997, the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) convened an expert working group to discuss current strategies for the development of HIV type 1 vaccines . Based on the recent findings of investigators from Japan's National Institute of Infectious Diseases (NIID) in Tokyo using recombinant bacillus Calmette-Guerin (rBCG) as a potential vectored vaccine for HIV, a recommendation was made that further work in this area is a priority . As a result, the working group reconvened in September 1998 to discuss the progress to date with this vaccine approach, as well as areas of related research to assess the feasibility of a BCG-vectored HIV vaccine . This report summarizes the discussions addressing the available scientific data on the potential use of rBCG as a vector for preventive HIV vaccines, the work necessary to move such candidate vaccines into Phase 1 clinical trials, and recommendations targeted at facilitating the long-term development of rBCG-vectored HIV vaccines. Braz J Med Biol Res, 2000 Feb, 33(2), 147 - 55 Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice; Vazquez-Padron RI et al.; The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice . The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route . The antibody fraction in serum and intestinal fluids consisted mainly of IgG1 . In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT) . Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected . The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins. J Mol Biol, 2000 Feb 11, 296(1), 87 - 102 Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors; Legendre D et al.; Many attempts have been made to endow enzymes with new catalytic activities . One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme . Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it . Selection of phage displayed enzymes for new catalytic activities remains a challenge . The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries . Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically . They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate . Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2 . The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS . The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester . Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4 . Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated . J Mol Biol, 2000 Jan 28, 295(4), 1023 - 37 Protein-protein interaction revealed by NMR T(2) relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus; Howard MJ et al.; T(2) relaxation experiments in combination with chemical shift and site-directed mutagenesis data were used to identify sites involved in weak but specific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus . The pyruvate decarboxylase component, a heterotetramer E1(alpha(2)beta(2)), is responsible for the first committed and irreversible catalytic step . The accompanying reductive acetylation of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between E1 and the lipoyl domain of the E2 polypeptide chain . The interactions between the free lipoyl domain (9 kDa) and free E1alpha (41 kDa), E1beta (35 kDa) and intact E1alpha(2)beta(2) (152 kDa) components, all the products of genes or sub-genes over-expressed in Escherichia coli, were investigated using heteronuclear 2D NMR spectroscopy . The experiments were conducted with uniformly (15)N-labeled lipoyl domain and unlabeled E1 components . Major contact points on the lipoyl domain were identified from changes in the backbone (15)N spin-spin relaxation time in the presence and absence of E1(alpha(2)beta(2)) or its individual E1alpha or E1beta components . Although the E1alpha subunit houses the sequence motif associated with the essential cofactor, thiamin diphosphate, recognition of the lipoyl domain was distributed over sites in both E1alpha and E1beta . A single point mutation (N40A) on the lipoyl domain significantly reduces its ability to be reductively acetylated by the cognate E1 . None the less, the N40A mutant domain appears to interact with E1 similarly to the wild-type domain . This suggests that the lipoyl group of the N40A lipoyl domain is not being presented to E1 in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain structure, especially in the lipoyl-lysine beta-turn region, as indicated by chemical shift data . Interaction with E1 and subsequent reductive acetylation are not necessarily coupled . CLAO J, 2000 Jan, 26(1), 26 - 9 Microbiological study of disposable soft contact lenses after photorefractive keratectomy; Dantas PE et al.; PURPOSE: To evaluate the bacterial contamination of bandage disposable soft contact lenses used in patients following photorefractive keratectomy (PRK) and to correlate our findings with clinical data . METHODS: Forty-six patients (81 eyes) underwent PRK . Immediately after each procedure, disposable soft contact lenses were positioned with sterile forceps . After 3 days, the lenses were removed in a sterile manner, placed in sterile Eppendorf pipettes containing 8 mL of enriched brain heart infusion broth, and analyzed for microbial contamination . RESULTS: Seven positive cultures were found: six gram positive cocci (7.4%) and one gram negative bacillus (1.2%) . There was no clinical correlation with these findings . CONCLUSION: Isolated microorganisms were similar to those described in the literature as agents of bacterial keratitis and are components of the normal ocular flora . Klebsiela pneumoniae--considered an occasional or transient flora--was the exception . All isolated microorganisms but K . pneumoniae were sensitive to most of the antibiotics tested . Our findings suggest that the risk of infectious keratitis after PRK related to soft contact lens wear for 3 days seems to be low, which may be because lenses were not manipulated by the patient during the wearing period, and the postoperative antibiotic regimen was strictly followed by patients . However, care should be taken to instruct patients in proper lens care practices to reduce the risk of bacterial keratitis in contact lens wear following PRK. Int J Tuberc Lung Dis, 2000 Jan, 4(1), 4 - 11 Tuberculosis in the urban poor settlements in the Philippines; Tupasi TE et al.; SETTING: Urban poor settlements in the Philippines . OBJECTIVE: To determine the magnitude of the tuberculosis problem in urban poor settlements in comparison with urban areas studied in the Nationwide Tuberculosis Prevalence Survey . STUDY DESIGN AND METHOD: A multistage cluster survey of BCG scar, tuberculin test, chest radiography and sputum examination for bacillary disease, in urban poor areas . RESULTS: The prevalences of culture-positive and smear-positive tuberculosis were 17.5 +/- 2.3 (95% CI 13.3-22.4) and 7.9 +/- 2.3 per thousand (95% CI 2.611.5), respectively . Extrapolated to the total population, the rates in the urban poor settlements were 12.4 +/- 1.7 (95% CI 9.6-16.2) and 5.6 +/- 1.6 per thousand population (95% CI 1.3-8.3), respectively . The prevalence of active pulmonary tuberculosis in subjects aged 10 years or more was 66 +/- 5.6/1000 (95% CI 55-77) . The BCG vaccination rate was 72% . The overall prevalence of tuberculosis infection was 66%, and 39% in those aged 5-9 years, corresponding to an annual risk of infection (ARI) of 6.5% . CONCLUSION: The problem of tuberculosis was substantial in the urban poor settlements, and was appreciably worse than that in the general urban population. Appl Environ Microbiol, 2000 Feb, 66(2), 825 - 7 Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B . lentus chromosome by an improved technique; Jorgensen PL et al.; A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined . The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique. Appl Environ Microbiol, 2000 Feb, 66(2), 638 - 42 A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis; Kajino T et al.; We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner . Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold) . Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms . We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. J Occup Environ Med, 2000 Jan, 42(1), 64 - 8 Evaluation of a western blot test as a potential screening tool for occupational exposure to Mycobacterium tuberculosis in health care workers; Franchi A et al.; Health care workers (HCWs) have a higher than average risk for contracting Mycobacterium tuberculosis (MTB) infection and tuberculosis (TB) . No markers of MTB-exposure are available, and TB risk assessment is performed by tuberculin screening, identifying individuals with acquired MTB infection . This study evaluated a western blot (WB) anti-M . bovis A60 complex antibody as a MTB-exposure marker . WB reactivity was evaluated on 127 exposed and 28 non-exposed HCWs from four divisions of the Policlinico Hospital of Modena, and 140 non-exposed bacille Calmette-Guerin-vaccinated controls . Excess of occupational TB risk according to the Occupational Safety and Health Administration (OSHA) was calculated in each division . WB-positivity (%) was: (1) significantly higher in exposed HCWs compared with non-exposed (72% vs 25%, P < 0.00001), (2) highly related (r = 0.99) to OSHA risk excess in all divisions, (3) higher than non-exposed in HCWs with short (< 5 years) MTB-exposure (purified protein derivative {PPD}, P > 0.18; WB, P < 0.04) . PPD-positivity (%) was higher than controls only in HCWs with longer (> 5 years) MTB-exposure . The study suggests that the WB antibody might represent a more sensitive biological marker of MTB contact among exposed HCWs, related to the level of TB risk and detectable earlier than the PPD skin test, thus providing new tools for TB risk assessment in health care facilities. Eur J Biochem, 2000 Feb, 267(3), 658 - 65 The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms; van der Veen BA et al.; Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate) . Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms . Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism . Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism . Michaelis-Menten kinetics, however, have been observed using shorter amylose chains . Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism . In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly . Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible . Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate . In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme . These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes. Br J Dermatol, 2000 Jan, 142(1), 72 - 6 Development of a polymerase chain reaction dot-blotting system for detecting cutaneous tuberculosis; Arora SK et al.; For a definitive diagnosis of cutaneous tuberculosis the demonstration of mycobacteria is essential, but this is generally not possible in skin lesions . Routinely available techniques have poor sensitivity and are time consuming, therefore, delaying the institution of timely therapy . The high sensitivity and speed of polymerase chain reaction (PCR) for the detection of infectious agents has prompted investigators to use this technique for the detection of Mycobacterium tuberculosis in body fluids such as cerebrospinal fluid or pleural fluid . In the present study, PCR was used to examine punch biopsy specimens from the affected skin of 10 patients with clinical diagnoses of tuberculosis verrucosa cutis, lupus vulgaris, scrofuloderma, papulonecrotic tuberculide and erythema induratum . A control group of 20 patients included individuals having skin manifestations with definite clinical diagnoses other than cutaneous tuberculosis, such as leprosy, fungal mycetoma, chronic bullous disease of childhood and pemphigus vulgaris . The PCR amplified products were dot hybridized with a probe which was random prime labelled with 32P . The results were compared with routine microbiological and histological findings . Among the test group, six of 10 (60%) were positive for M . tuberculosis by PCR, although their histopathology showed non-specific chronic inflammation with no definite diagnosis . Microbiological investigations, including acid-fast bacillus smear and culture, were positive in a single case of scrofuloderma . All patients in the control group were negative by PCR for M . tuberculosis . The data indicate that the combination of dot hybridization with PCR markedly increased the sensitivity and specificity of PCR . This may be a useful tool in the diagnosis of tuberculosis when conventional methods fail. Biochemistry, 2000 Feb 1, 39(4), 800 - 9 Novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp . strain OxB-1: purification, characterization, and molecular cloning of the gene; Kato Y et al.; A novel dehydratase that catalyzes the stoichiometric dehydration of Z-phenylacetaldoxime to phenylacetonitrile has been purified 483-fold to homogeneity from a cell-free extract of Bacillus sp . strain OxB-1 isolated from soil . It has a M(r) of about 40 000 and is composed of a single polypeptide chain with a loosely bound protoheme IX . The enzyme is inactive unless FMN is added to the assay, but low activity is also observed when sulfite replaces FMN . The activity in the presence of FMN is enhanced 5-fold under anaerobic conditions compared to the activity measured in air . The enzyme has maximum activity at pH 7.0 and 30 degrees C, and it is stable at up to 45 degrees C at around neutral pH . The aerobically measured activity in the presence of FMN is also enhanced by Fe(2+), Sn(2+), SO(3)(2)(-), and NaN(3) . Metal-chelating reagents, carbonyl reagents, electron donors, and ferri- and ferrocyanides strongly inhibit the enzyme with K(i) values in the micromolar range . The enzyme is active with arylalkylaldoximes and to a lesser extent with alkylaldoximes . The enzyme prefers the Z-form of phenylacetaldoxime over its E-isomer . On the basis of its substrate specificity, the enzyme has been tentatively named phenylacetaldoxime dehydratase . The gene coding for the enzyme was cloned into plasmid pUC18, and a 1053 base-pair open reading frame that codes for 351 amino acid residues was identified as the oxd gene . A nitrilase, which participates in aldoxime metabolism in the organism, was found to be coded by the region just upstream from the oxd gene . In addition an open reading frame (orf2), whose gene product is similar to bacterial regulatory (DNA-binding) proteins, was found just upstream from the coding region of the nitrilase . These findings provide genetic evidence for a novel gene cluster that is responsible for aldoxime metabolism in this microorganism. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 63 - 6 Identification and purification of the 69-kDa intracellular protease involved in the proteolytic processing of the crystal delta-endotoxin of Bacillus thuringiensis subsp . tenebrionis; Reddy ST et al.; The dynamics of appearance of intracellular proteases in relation to the synthesis of crystal delta-endotoxin was studied to identify the native intracellular protease(s) involved in the proteolytic processing of the 73-kDa protoxin of Bacillus thuringiensis subsp . tenebrionis . In vitro proteolytic activation of the 73-kDa protoxin indicated the possible role of 69-kDa protease in the proteolytic processing of 73-kDa protoxin . The purified 69-kDa protease was able to cause the proteolytic activation of the 73-kDa protoxin to 68-kDa toxin and this conversion was inhibited by ethylenediamine tetraacetic acid and 1,10-phenanthroline. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 1 - 7 Cellulase-free xylanases from Bacillus and other microorganisms; Subramaniyan S et al.; Xylanases are used mainly in the pulp and paper industries for the pretreatment of Kraft pulp prior to bleaching to minimize use of chlorine, the conventional bleaching agent . This application has great potential as an environmentally safe method . Hydrolysis by xylanases of relocated and reprecipitated xylan on the surface of cellulose fibres formed during Kraft cooking facilitates the removal of lignin by increasing permeability to oxidising agents . Most of the xylanases reported in the literature contained significant cellulolytic activity, which make them less suitable for pulp and paper industries . The need for large quantities of xylanases which would be stable at higher temperatures and pH values and free of cellulase activity has necessitated a search for novel enzymes . We have isolated and characterised several xylanase-producing cultures, one of which (an alkalophilic Bacillus SSP-34) produced more than 100 IU ml(-1) of xylanase activity . The SSP-34 xylanases have optimum activity at 50 degrees C in a pH range 6-8, with only small amounts of cellulolytic activity (CMCase (0.4 IU ml(-1), pH 7), FPase (0.2 IU ml(-1), pH 7) and no activity at pH 9). J Nat Prod, 2000 Jan, 63(1), 104 - 8 New phloroglucinol derivatives from Hypericum papuanum; Winkelmann K et al.; Bioactivity-guided fractionation of the petroleum ether extract of the aerial parts of Hypericum papuanum led to the isolation of five new tricyclic phloroglucinol derivatives . On the basis of extensive 1D and 2D NMR experiments as well as MS studies, their structures were elucidated as the C-3 epimers of 8-hydroxy-4,4, 7-trimethyl-9-(2-methylpropionyl)-3-(1-methylvinyl)-5beta -H-tricyclo{ 5.3.1.0(1,5)}undec-8-ene-10,11-dione (1,2); the C-3 epimers of 8-hydroxy-4,4, 7-trimethyl-9-(2-methylbutyryl)-3-(1-methylvinyl)-5beta-H-++ +tricyclo{5 . 3.1.0(1,5)}undec-8-ene-10,11-dione (3, 4), and 8-hydroxy-4,4, 7-trimethyl-9-(2-methylpropionyl)-5beta-H-tricyclo{5.3 .1.0(1, 5)}undec-8-ene-10,11-dione (5), and their corresponding tautomers (1a, 2a, 3a, 4a, 5a) . Compounds 1/1a-5/5a were named ialibinones A-E, respectively . Compounds 1/1a-4/4a showed antibacterial activity against Bacillus cereus, Staphylococcus epidermidis, and Micrococcus luteus. J Bacteriol, 2000 Feb, 182(4), 949 - 55 Cloning, expression, and purification of a thermostable nonhomodimeric restriction enzyme, BslI; Hsieh PC et al.; BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position) . The BslI restriction-modification system from Bacillus species was cloned and expressed in Escherichia coli . The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), the bslIRalpha gene, and the bslIRbeta gene . The alpha and beta subunits of BslI can be expressed independently in E . coli in the absence of BslI methylase (M.BslI) protection . BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together . Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (alphabeta), heterotetramers (alpha(2)beta(2)), and possibly oligomers in solution . Two beta subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (alpha(2)beta(2)) . In DNA mobility shift assays, neither subunit alone can bind DNA . DNA mobility shift activity was detected after mixing the two subunits together . Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is alpha(2)beta(2) . M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the beta group of aminomethyltransferase . Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in BslI sites in the first or second cytosine are resistant to BslI digestion . C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion . Two putative zinc fingers are found in the alpha subunit of BslI endonuclease. Plant Foods Hum Nutr, 1999, 54(1), 13 - 20 Influence of temperature on the fermentation of bambara groundnut (Vigna subterranea) to produce a dawadawa-type product; Amadi EN et al.; Bambara groundnut (Vigna subterranea) was fermented to produce a dawadawa-type product using a starter culture of Bacillus licheniformis isolated from naturally fermenting bambara groundnut beans . Fermentation was carried out at 30 and 37 degrees C for four days and at 45 degrees C for two days . The pH of the substrate decreased after 24 hours and then rose at 30 and 37 degrees C but remained constant at 45 degrees C after the initial drop . Total titratable acidity of the fermenting beans mimicked the pH values . Proximate analyses for moisture, protein and fat of the cotyledons showed an increase in all three constituent at each of the three fermentation temperatures . At the end of fermentation, total available carbohydrate was 55%, 59% and 62% of the original value at 30, 37 and 45 degrees C, respectively . Fermentation of bambara groundnut at 45 degrees C for two days is recommended as the ideal fermentation temperature and time. J Biol Chem, 2000 Jan 28, 275(4), 2447 - 54 The C-terminal subdomain (IF2 C-2) contains the entire fMet-tRNA binding site of initiation factor IF2; Spurio R et al.; Previous protein unfolding studies had suggested that IF2 C, the 24 . 5-kDa fMet-tRNA binding domain of Bacillus stearothermophilus translation initiation factor IF2, may consist of two subdomains . In the present work, the four Phe residues of IF2 C (positions 531, 599, 657, and 721) were replaced with Trp, yielding four variant proteins having intrinsic fluorescence markers in different positions of the molecule . Comparison of the circular dichroism and Trp fluorescence changes induced by increasing concentrations of guanidine hydrochloride demonstrated that IF2 C indeed consists of two subdomains: the more stable N-terminal (IF2 C-1) subdomain containing Trp-599, and the less stable C-terminal (IF2 C-2) subdomain containing Trp-721 . Isolated subdomain IF2 C-2, which consists of just 110 amino acids (from Glu-632 to Ala-741), was found to bind fMet-tRNA with the same specificity and affinity as native IF2 or IF2 C-domain . Trimming IF2 C-2 from both N and C termini demonstrated that the minimal fragment still capable of fMet-binding consists of 90 amino acids . IF2 C-2 was further characterized by circular dichroism; by urea-, guanidine hydrochloride-, and temperature-induced unfolding; and by differential scanning calorimetry . The results indicate that IF2 C-2 is a globular molecule containing predominantly beta structures (25% antiparallel and 8% parallel beta strands) and turns (19%) whose structural properties are not grossly affected by the presence or absence of the N-terminal subdomain IF2 C-1. Indian J Med Res, 1999 Oct, 110, 128 - 32 Field evaluation of Spicbiomoss, a biolarvicidal formulation of Bacillus sphaericus against immatures of Culex quinquefasciatus & Anopheles species; Mariappan T et al.; Spicbiomoss, an aqueous suspension formulation of Bacillus sphaericus was evaluated for its efficacy against immatures of Culex quinquefasciatus at the application rate of 120 l/ha in cement tanks, cesspits and drains in Pondicherry and in drains in Kochi, Kerala . The formulation was also tested against anophelines (Anopheles fluviatilis and An . culicifacies) breeding in stream pools in Malkangiri district, Orissa . In cement tanks and cesspits more than 80 per cent reduction in immature density was observed for a period of 6-13 days (mean 9.8 +/- 1.25 days) and 3-8 days (mean 5.2 +/- 0.7 days) respectively . The same level of reduction was found to last for 1-4 days (mean 2.2 +/- 0.52 days) in drains in Pondicherry and 2-9 days (mean 4.8 +/- 1.17 days) in Kochi . In bunded stream pools 40-79 per cent reduction in immature density of Anopheles was obtained for an average period of 1-7 (mean 3.83 +/- 0.98) days . There was no improvement in the efficacy of the formulation against anophelines even at the higher application rate (240 l/ha) . Thus, Spicbiomoss can be used against Cx . quinquefasciatus in an integrated vector management programme. J Gen Virol, 2000 Feb, 81(Pt 2), 307 - 16 Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions; van Hulten MC et al.; White spot syndrome is a worldwide disease of penaeid shrimp . The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb . Analysis of a 12.3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase . The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats . The first domain contained six direct repeats of 54 bp and is part of a coding region . The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location . This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome . Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor . The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family. Acta Trop, 2000 Jan 5, 74(1), 43 - 9 Development of Wuchereria bancrofti in Culex quinquefasciatus that survived the exposure of sub-lethal dose of Bacillus sphaericus as larvae; Gunasekaran K et al.; Development of Wuchereria bancrofti in Culex quinquefasciatus emerged from the larvae that survived the exposure of sub-lethal dose of Bacillus sphaericus was examined in the laboratory . Third instar larvae of Cx . quinquefasciatus were treated with B . sphaericus at a sub-lethal dose of 11.35 microg/250 ml . The female mosquitoes that emerged from the survived larvae were fed on microfilaraemic human blood and parasite development was monitored in the fed mosquitoes . Both treated and untreated mosquitoes could ingest microfilaria (mF) equally as there was no significant difference in mF density between them . But, density of developmental stages of the parasite in treated group was significantly lower . Since, there was no mortality of mosquitoes, the lower density of the developmental stages could be attributed to the loss of parasites in the treated mosquitoes . Consequently, the proportion of mosquitoes with infective larvae (L3) and number of L3 were also significantly lower in treated females . Delay in parasite development was also noticed in treated mosquitoes . The present study indicates that B . sphaericus, when applied at sub-lethal dose kills larvae, and in addition, inhibits development of the filarial parasite and consequently reduces L3 yield in adult mosquitoes that emerged from the survived larvae. Adv Dermatol, 1999, 14, 285 - 306 Cutaneous malignant epithelioid neoplasms; Sidbury R et al.; In summary, cutaneous malignancies with an epithelioid appearance form a diverse group of neoplasms that may be difficult to diagnose by utilizing routine microscopy alone . Cutaneous malignancies, including malignant melanoma and metastatic carcinoma, certain benign neoplasms such as mixed tumor of the skin and angiolymphoid hyperplasia with eosinophils (epithelioid hemangioma), and infectious conditions such as bacillary (epithelioid) angiomatosis can be considered in this differential . However, through recognition of the characteristic histologic, immunocytochemical, and ultrastructural findings outlined above, definitive diagnosis of these challenging neoplasms is usually possible. J Am Acad Dermatol, 2000 Feb, 42(2 Pt 1), 299 - 301 Bacillary angiomatosis by Bartonella quintana in an HIV-infected patient; Santos R et al.; Bacillary angiomatosis and bacillary peliosis are opportunistic infections caused by Bartonella henselae and Bartonella quintana, which occur in patients with late-stage infection . We report a case of bacillary angiomatosis in an HIV-infected patient with skin, bone, and probably liver involvement, The identification of the agent (B quintana ) was done by polymerase chain reaction in the skin specimen . The patient had complete regression of all lesions after a 6-month regimen of oral erythromycin. Biochem J, 2000 Feb 1, 345 Pt 3, 487 - 94 L-Pipecolic acid oxidase, a human enzyme essential for the degradation of L-pipecolic acid, is most similar to the monomeric sarcosine oxidases; Dodt G et al.; L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs) . Because its role, if any, in these disorders is unknown, we cloned the associated human gene and expressed its protein product . The cDNA was cloned with the use of a reverse genetics approach based on the amino acid sequence obtained from purified L-pipecolic acid oxidase from monkey . The complete cDNA, obtained by conventional library screening and 5' rapid amplification of cDNA ends, encompassed an open reading frame of 1170 bases, translating to a 390-residue protein . The translated protein terminated with the sequence AHL, a peroxisomal targeting signal 1 . Indirect immunofluorescence studies showed that the protein product was expressed in human fibroblasts in a punctate pattern that co-localized with the peroxisomal enzyme catalase . A BLAST search with the amino acid sequence showed 31% identity and 53% similarity with Bacillus sp . NS-129 monomeric sarcosine oxidase, as well as similarity to all sarcosine oxidases and dehydrogenases . No similarity was found to the peroxisomal D-amino acid oxidases . The recombinant enzyme oxidized both L-pipecolic acid and sarcosine . However, PBD patients who lack the enzyme activity accumulate only L-pipecolic acid, suggesting that in humans in vivo, this enzyme is involved mainly in the degradation of L-pipecolic acid. Biochem Pharmacol, 2000 Feb 15, 59(4), 317 - 20 Granulysin: a novel antimicrobial peptide of cytolytic T lymphocytes and natural killer cells; Krensky AM; Granulysin is a novel antimicrobial protein produced by human cytolytic T lymphocytes and natural killer cells . It is active against a broad range of microbes, including Gram-positive and Gram-negative bacteria, fungi, and parasites . The fact that it kills Mycobacterium tuberculosis is particularly important, since the current vaccine (Bacille Calmette-Guerin, BCG) is of limited efficacy and antibiotic resistance is increasing . Although functionally related to other antibacterial peptides, defensins and magainins, granulysin is structurally distinct . Like porcine NK lysin and amoebapores made by Entamoeba histolytica, granulysin is related to saposins, small lipid-associated proteins present in the central nervous system . The identification of this novel molecule indicates a broader and perhaps more significant role for T lymphocytes in both innate and acquired antimicrobial defenses. Bioseparation, 1999, 7(6), 327 - 31 Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus; Nandakumar R et al.; A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase . Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations . Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye . The dissociation constants of the enzyme-dye complexes were 7.2 +/- 0.2 microM and 11.2 +/- 0.2 microM at 5 degrees C and 20 degrees C respectively. Virology, 2000 Jan 20, 266(2), 227 - 36 Identification of two major virion protein genes of white spot syndrome virus of shrimp; van Hulten MC et al.; White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp . Two structural proteins of WSSV were identified . WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 x 120 nm, and a tail-like extension at one end . The double-stranded viral DNA has an approximate size 290 kb . WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively . VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope . N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome . To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis . A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28 . Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites . These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae . Infect Immun, 2000 Feb, 68(2), 990 - 3 MTSA-10, the product of the Rv3874 gene of Mycobacterium tuberculosis, elicits tuberculosis-specific, delayed-type hypersensitivity in guinea pigs; Colangeli R et al.; In a search for new skin test reagents specific for tuberculosis, we found that the antigen encoded by gene Rv3874 of Mycobacterium tuberculosis elicited delayed-type hypersensitivity in M . tuberculosis-infected guinea pigs but not in control animals immunized with Mycobacterium bovis bacillus Calmette-Guerin (BCG) or Mycobacterium avium . The antigen, which was named MTSA-10 (for M . tuberculosis-specific antigen 10), is a prime candidate for a component of a new tuberculin that will allow discrimination by a skin test of latent M . tuberculosis infection from vaccination with BCG or from sensitization with environmental, nontuberculous mycobacteria. Public Health, 1999 Nov, 113(6), 311 - 3 Differential protective effect of bacillus Calmette-Guerin vaccine against multibacillary and paucibacillary leprosy in Nagpur, India; Kulkarni HR et al.; For this paper we conducted a secondary data analysis to test the hypothesis that a linear trend exists in the protective effect of bacillus Calmette-Guerin (BCG) vaccine against types of leprosy . We used data from two previous case-control studies to perform an unmatched test for linear trend . We observed that both the studies revealed a significant linear trend (P<0.00001) . One study that estimated an insignificant protective effect of BCG against paucibacillary leprosy showed a significant departure from linearity . We conclude that, the protective effect of BCG vaccination is differential across severity of leprosy as it brings about a shift in the immune response to a higher level of cell mediated immunity . We recommend that future studies dealing with the protective effect of BCG against leprosy should also conduct an analysis for trend. Curr Pharm Des, 2000 Feb, 6(3), 345 - 59 Intravesical therapy of superficial bladder cancer; Melekos MD et al.; Transurethral resection (TUR) of the superficial transitional cell carcinoma (TCC) of the bladder is known to be insufficient in controlling the disease because of the unacceptable rates of recurrence, progression and ultimate cystectomy . Adjuvant intravesical chemo-and/or immunotherapy is administered in an effort to enhance the efficacy of surgery alone . The initial tumor stage and grade, the multifocality of this cancer and the history of previous recurrences remain the determinant factors in survival . It is important to decide exactly which patients are at risk, and, therefore, do need treatment . Knowledge of the natural history of the disease will facilitate this decision making, although the natural history of TCC is largely unpredictable owing to tumor heterogeneity . Several cytotoxic and immune modifying agents have been used intravesically in different treatment schedules . However, despite their effectiveness, no consensus exists about the optimal antineoplastic regimen . The selection of the latter is a subject of continuous investigation . Intravesical treatment with cytotoxic drugs has been demonstrated to achieve an acceptable reduction in short- and intermediate-term recurrence rates, but has no proven ability in preventing disease progression to muscle-invasive cancer or prolonging survival . On the other hand, bacillus Calmette-Guerin (BCG) currently appears to be the most effective agent for intravesical use, especially in patients with high grade and stage neoplasms but the optimum strain, dosage and duration schedule have not been determined . Clinical trials have shown that BCG provides long-term protection from tumor recurrence, while there is evidence that it may favorably alter the progression rate of the disease with prolongation of survival . Toxicity of intravesical chemo- and immunotherapy still remains a major problem and attempts at reducing the dosage, and, thus, toxicity without affecting efficacy are underway . This review endeavors to present updated information on intravesical chemotherapy in treating superficial bladder cancer, the expanding role of intravesical immunotherapy, the recent work comparing various immunotherapeutic regimens with chemotherapeutic intravesical therapies, and the progress made towards achieving optimal treatment regimens. Carbohydr Res, 1999 Dec 12, 322(3-4), 209 - 18 Towards regioselective synthesis of oligosaccharides by use of alpha-glucosidases with different substrate specificity; Mala S et al.; alpha-Glucosidase from two microbial sources, Bacillus stearothermophilus and Brewer's yeast, has been used to catalyze transglycosylation reactions and a comparative study was carried out to determine the regioselectivity of this reaction . Bacterial alpha-glucosidase exhibited higher transfer activity with maltose and was able to synthesize tri- and tetrasaccharides in high yield (27%) . In the case of yeast enzyme, only trisaccharides were synthesized in lower yield . Structure analysis of transglycosylation products by means of GC-MS and NMR spectroscopy revealed a correlation between the hydrolytic substrate specificity and the regioselectivity of transglycosylation reaction . Higher substrate specificity of bacterial enzyme, however, influenced its transglucosylation activity toward other saccharide acceptors. Rev Mal Respir, 1999 Dec, 16(6), 1161 - 3 {Phantom thoracic opacity}; Bakhatar A et al.; The etiology of the respiratory distress syndrome is dominated by pulmonary edema and the septic shock . We report a rare etiology of a respiratory distress secondary to a rupture of a well treated tuberculous latero-tracheal adenopathy . A 24-year-old woman was treated a year ago for a peripheral and mediastinal lymph node tuberculosis confirmed by the biopsy of a left supra clavicular adenopathy, by two months of isoniazid-rifampicin-pyrazinamide-ethambutol and seven months of isoniazid-rifampicin . The patient completed 9 month treatment with a good clinical and radiology course . Two months after stopping the antibacillary treatment, the patient was admitted to an intensive care unit with a respiratory distress syndrome requiring both intubation and artificial ventilation . The bronchial aspiration brought back plain pus . The telethorax from admission was normal and the retrospective history suggested the diagnostic of a ganglio-bronchial fistula which was confirmed by bronchial fibroscopy demonstrating right latero-tracheal fistula . The course was good with recovery of consciousness on the seventh day . Direct bacilloscopies and culture were negative . The digestive fibroscopy was normal . Finally, fistulization of a tuberculous adenopathy must be considered among the etiologies of respiratory distress even in a patient appropriately treated for mediastinal lymph node tuberculosis. Biosci Biotechnol Biochem, 1999 Nov, 63(11), 1902 - 9 Transcriptional regulation of the Bacillus ohbensis cyclodextrin glucanotransferase gene in B . subtilis; Nishida T et al.; The expression of the cyclodextrin glucanotransferase (CGTase) gene (cgt) of Bacillus ohbensis, when introduced into an alpha-amylase-defective strain of B . subtilis on a multicopy plasmid, pHY300PLK, was induced in the presence of starch and was subject to catabolite repression by glucose as well as in the original strain, B . ohbensis . We constructed a cgt'::'lacZ translational fusion to study the expression in B . subtilis, and this construct was confirmed to be subject to both starch induction and catabolite repression . In order to define the region involved in the regulation of the cgt gene, a series of cgt'::'lacZ gene with various lengths of deletion in the promoter region was constructed on pHY300PLK . DNA regions responsible for starch induction and catabolite repression by glucose could be separated in the deletion experiment . Primer extension analysis showed that the catabolite repression was controlled at the initiation of transcription, while the starch induction is likely to be controlled by a transcriptional termination-antitermination mechanism. Insect Mol Biol, 1999 Nov, 8(4), 557 - 64 Satellite DNA variation in parental and derived unisexual hybrids of Bacillus stick insects (Phasmatodea); Scali V et al.; The Bag320 sequence family of satellite DNA (satDNA) has been found in some stick insect taxa: the bisexual Bacillus grandii, the related parthenogen B . atticus and their hybrids with B . rossius . However, under the same experimental conditions, the Bag320 sequences were not found in B . rossius . Bag320 sequences of the clonal hybrid B . whitei (= B . rossius/grandii grandii) intermingled with those of B . grandii in all plotted dendrograms . On the whole, satDNA features (restriction pattern, sequence variation, fluorescent in situ hybridization (FISH)), allozymes and karyology support a relatively recent origin of B . whitei . Our investigations on unisexual hybrids of Bacillus also suggested that their origin and clonal reproduction allow the occurrence of different sequence subsets of limited variability in isolated populations stemming from the hybridization focus. Receptors Channels, 1999, 6(6), 477 - 91 Bacillus stearothermophilus lctB gene gives rise to functional K+ channels in Escherichia coli and in Xenopus oocytes; Wolters M et al.; We have cloned a small K+ channel subunit (LctB) of the gram-positive bacterium Bacillus stearothermophilus (B . stearo.) . The B . stearo . LctB protein is only 134 amino acids long . The sequence contains a typical K+ channel P-domain with a K+ channel GYGD signature sequence and two hydrophobic, possibly membrane-spanning segments M1 and M2 . Unexpectedly, LctB K+ channels exhibited properties which differed markedly from the ones reported for KcsA channels of the gram-positive bacterium Streptomyces lividans . LctB channels, when expressed in E . coli, were targeted to the outer membrane and not like KcsA channels to the inner membrane . After reconstitution in black lipid membrane, LctB channels mediated K+ currents at neutral pH . They were apparently not gated by pH like KcsA channels . Also, LctB cRNA produced functional LctB channels in the Xenopus oocyte expression system in marked contrast to KcsA . The results demonstrated that heterologous expression produced functional LctB channels both in E . coli and in Xenopus oocytes . It is proposed that bacterial LctB subunits can be properly handled by the Xenopus oocyte leading to the occurrence of functional LctB K+ channels in the oocyte plasma membrane. Int J Food Microbiol, 1999 Dec 15, 53(2-3), 159 - 67 Heterogeneity observed in the components of hemolysin BL, an enterotoxin produced by Bacillus cereus; Schoeni JL et al.; Hemolysin BL (HBL), a diarrheal enterotoxin originally isolated from Bacillus cereus strain F837/76, is composed of three antigenically distinct proteins designated B, L1, and L2 . All three components are required for biological activity . Here, we report antigenic and physical variations in HBL components produced by other B . cereus isolates . Reactions of partial identity were observed in double gel immunodiffusion assays using antibodies to highly purified B, L1, and L2 components of F837/76 and culture supernatants of strains F837/76 and S1C . Western blot analysis showed that F837/76 produced one 38-kDa B protein, one 38-kDa L1, and one 43-kDa L1 protein . In strain S1C, two B (38 and 42 kDa), two L1 (38 and 41 kDa), and one L1 (43 kDa) proteins were detected . Further Western blot analysis of 127 B . cereus isolates showed that 90 produced one or more of the three HBL components . Approximately half of these 90 isolates (43/90; 48%) produced protein profiles which differed from that of F837/76 . A total of four B, two L1, and three L2 component profiles with proteins of different sizes were observed . Individual strains produced various combinations of single or multiple bands of each component . In addition, some strains produced only one or two of the three HBL components . The public health significance of these strains is unknown, as all three components are required for biological activity . The data presented here demonstrates a high degree of heterogeneity in HBL and provide the basis for further studies to characterize the variations in HBL and to determine the role of the variant components in pathogenicity. J Bacteriol, 2000 Feb, 182(3), 734 - 41 Regulation by overlapping promoters of the rate of synthesis and deposition into crystalline inclusions of Bacillus thuringiensis delta-endotoxins; Sedlak M et al.; During sporulation, Bacillus thuringiensis produces intracellular, crystalline inclusions comprised of a mixture of protoxins active on insect larvae . A major class of these protoxin genes, designated cry1, is transcribed from two overlapping promoters (BtI and BtII) utilizing RNA polymerase containing sporulation sigma factors sigma(E) and sigma(K), respectively . Fusions of these promoters to lacZ were constructed in order to analyze transcription patterns . Mutations within the -10 region of the BtII promoter (within the spacer region of the BtI promoter) which departed from the consensus -10 sequence for either sigma(E) or sigma(K) resulted in inactivation of transcription from BtII and a fivefold stimulation of transcription from BtI . In contrast, transcription from both promoters was inhibited with a change to the sigma(E) consensus . One of the "promoter-up" mutations was fused to the cry1Ac1 gene, and enhanced transcription was confirmed by Northern blotting . There was an increase in the accumulation of Cry1Ac antigen at early but not later times in sporulation in the mutant . This shift was due to the rapid turnover of much of the excessively accumulated protoxin at the early times as measured by pulse-chase labeling . As a result of the turnover and the inactivation of the BtII promoter, the mutant produced smaller inclusions which contained two- to threefold-less protoxin than inclusions from the wild type . Promoter overlap is a mechanism for modulating protoxin synthesis, thus ensuring the efficient packaging of these protoxins into inclusions. Clin Exp Immunol, 2000 Feb, 119(2), 270 - 9 Identification of distinct lymphocyte subsets responding to subcellular fractions of Mycobacterium bovis bacille calmette-Guérin (BCG); Batoni G et al.; In order to investigate the ability of Mycobacterium bovis BCG vaccination to induce immune responses toward different classes of mycobacterial antigens and the cell populations involved in such responses, proliferation of distinct human lymphocyte subsets from BCG-vaccinated donors in response to different subcellular fractions of BCG was analysed and compared with that of not sensitized subjects . Proliferation of different cell subsets was evaluated by flow cytometric determination of bromodeoxyuridine incorporated into DNA of dividing cells and simultaneous identification of cell surface markers . Although a certain degree of variability was observed among different donors, after 6 days of in vitro stimulation BCG-vaccinated subjects displayed, as a mean, a stronger blastogenic response to all the classes of antigens compared with non-sensitized ones . PPD, culture filtrates and membrane antigens induced a predominant proliferation of CD4+ T cells . In contrast, preparations enriched in cytosolic antigens elicited strong proliferation of gammadelta+ T cells which, as a mean, represented 55% of the proliferating cells . Although to a lesser extent, proliferation of gammadelta+ T cells was also elicited by preparations enriched in membrane and cell wall antigens . In response to the latter preparation proliferation of CD4+ T cells and CD16+/CD3- (natural killer (NK)) cells was observed, as well . In particular, cell wall antigens were found to induce significantly higher levels of proliferation of NK cells compared with all the other classes of antigens. Bioorg Med Chem, 1999 Nov, 7(11), 2303 - 11 Altering the specificity of subtilisin Bacillus lentus through the introduction of positive charge at single amino acid sites; Davis BG et al.; The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments . As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have recently adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL) . We now describe the use of this strategy to introduce multiple positive charges . A series of mono-, di- and triammonium methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site . Kinetic parameters for these chemically modified mutants (CMM) enzymes were determined at pH 8.6 . The presence of up to three positive charges in the S1, S1' and S2 subsites of SBL resulted in up to 77-fold lowered activity, possibly due to interference with the histidinium ion formed in the transition state of the hydrolytic reactions catalyzed. Bioorg Med Chem, 1999 Nov, 7(11), 2293 - 301 The controlled introduction of multiple negative charge at single amino acid sites in subtilisin Bacillus lentus; Davis BG et al.; The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments . As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL) . A series of mono-, di- and triacidic acid methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site . Kinetic parameters for these chemically modified mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the unnatural amino acid side-chains introduced . The presence of up to three negative charges in the S1, S1' and S2 subsites of SBL resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion stabilization of the transition state of the hydrolytic reactions catalyzed . Each unit increase in negative charge resulted in a raising of K(M) and a reduction of k(cat) . However, no upper limit was observed for increases in K(M), whereas decreases in k(cat) reached a limiting value . Comparison with sterically similar but uncharged CMMs revealed that electrostatic effects of negative charges at positions 62, 156 and 217 are detrimental, but are beneficial at position 166 . These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-pNA, to SBL is reduced, but without drastic attenuation of catalytic efficiency, and show that SBL tolerates high levels of charge at single sites. Biochim Biophys Acta, 2000 Jan 15, 1463(1), 142 - 50 S-layer-coated liposomes as a versatile system for entrapping and binding target molecules; Mader C et al.; In the present study, unilamellar liposomes coated with the crystalline bacterial cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were used as matrix for defined binding of functional molecules via the avidin- or streptavidin-biotin bridge . The liposomes were composed of dipalmitoyl phosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4 and they had an average size of 180 nm . For introducing specific functions into the S-layer lattice without affecting substances encapsulated within the liposomes, crosslinking and activation reagents had to be identified which did not penetrate the liposomal membrane . Among different reagents, a hydrophilic dialdehyde generated by periodate cleavage of raffinose and a sulfo-succinimide activated dicarboxylic acid were found to be impermeable for the liposomal membrane . Both reagents completely crosslinked the S-layer lattice without interfering with its regular structure . Biotinylation of S-layer-coated liposomes was achieved by coupling p-diazobenzoyl biocytin which preferably reacts with the phenolic residue of tyrosine or with the imidazole ring of histidine . By applying this method, two biotin residues accessible for subsequent avidin binding were introduced per S-layer subunit . As visualized by labeling with biotinylated ferritin, an ordered monomolecular layer of streptavidin was formed on the surface of the S-layer-coated liposomes . As a second model system, biotinylated anti-human IgG was attached via the streptavidin bridge to the biotinylated S-layer-coated liposomes . The biological activity of the bound anti-human IgG was confirmed by ELISA. J Invertebr Pathol, 2000 Jan, 75(1), 69 - 75 Natural coprevalence of Strongwellsea castrans, Cystosporogenes deliaradicae, and Bacillus thuringiensis in the host, Delia radicum; Eilenberg J et al.; Adult cabbage root flies (Delia radicum) from three Danish localities were diagnosed microscopically for the natural prevalence of Strongwellsea castrans, Cystosporogenes deliaradicae, and Bacillus thuringiensis . C . deliaradicae was significantly coprevalent with S . castrans . B . thuringiensis sporangia were diagnosed in the hemolymph in two D . radicum which were also infected with S . castrans and proved to belong to serovar aizawai and serovar balearica . The biological characterization of S . castrans proved that at 17.5 degrees C flies developed an abdominal hole 7.9 days (mean) after infection and that 5.7 days (mean) passed from the emergence of the hole to the death of the infected host . No mortality effect among D . radicum subjected to B . thuringiensis serovar aizawai, balearica, and kurstaki isolates was detected . RAPD with DNA proved that six B . thuringiensis serovar balearica isolates (all from the same fly) were indistinguisable . This indicates that proliferation of B . thuringiensis in the abdomen of an S . castrans-infected D . radicum may be due to just one genotype . The profiles of one isolated aizawai strain did not correspond to the profiles of other serovar aizawai strains used for comparison . The biological significance of the interaction between the involved pathogens is discussed . Exp Parasitol, 2000 Jan, 94(1), 33 - 41 BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice; Streit JA et al.; Cellular immune responses are required for protective immunity against Leishmania chagasi . Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens . Previous studies demonstrated that the L . chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L . chagasi infection . To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1) . Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L . chagasi infection than LCR1 protein alone . BCG-LCR1 administered intraperitoneally did not protect . Splenocytes from mice immunized s.c . with either BCG-LCR1 or BCG-pMV261 and then infected with L . chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice . We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine . Biochemistry, 2000 Jan 18, 39(2), 463 - 9 Intersubunit location of the active site of farnesyl diphosphate synthase: reconstruction of active enzymes by hybrid-type heteromeric dimers of site-directed mutants; Koyama T et al.; Farnesyl diphosphate synthase is a homodimer of subunits having typically two aspartate-rich motifs with two sets of substrate binding sites for an allylic diphosphate and isopentenyl diphosphate per molecule of a homodimeric enzyme . To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interaction, we constructed several expression plasmids that overproduce hybrid-type heterodimers of Bacillus stearothermophilus FPP synthases constituting different types of mutated monomers, which exhibit little catalytic activity as homodimers, by combining two tandem fps genes for the manipulated monomer subunit with a highly efficient promoter trc within an overexpression pTrc99A plasmid . A heterodimer of a combination of subunits of the wild type and of R98E, a mutant subunit which exhibits little enzymatic activity as a dimer form (R98E)(2), exhibited 78% of the activity of the wild-type homodimer enzyme, (WT)(2) . Moreover, when a hybrid-type heterodimeric dimer of FPP synthase mutant subunits (R98E/F220A) was prepared, the FPP synthase activity was 18- and 390-fold of that of each of the almost inactive mutants as a dimeric enzymes, (R98E)(2) and (F220A)(2) {Koyama, T., et al . (1995) Biochem . Biophys . Res . Commun . 212, 681-686}, respectively . These results suggest that the subunits of the FPP synthase interact with each other to form a shared active site in the homodimer structure rather than an independent active site in each subunit. J Dairy Res, 2000 Nov, 67(4), 597 - 608 Effect of partial hydrolysis with an immobilized proteinase on thermal gelation properties of beta-lactoglobulin B; Otte J et al.; We have investigated the influence of partial hydrolysis with an immobilized proteinase from Bacillus licheniformis on the thermal gelation of isolated beta-lactoglobulin B . Gelation behaviour was determined by dynamic rheological measurements (small deformation) and the gels were characterized with respect to microstructure and water-holding properties . A fine-stranded gel with a complex modulus of approximately 2000 Pa was formed from beta-lactoglobulin (50 g/l in 75 mM-Tris-HCl, pH 7.5) . Limited hydrolysis prior to thermal gelation resulted in coarser gels with thicker protein strands and larger pores . Gel structure correlated with its permeability, proton mobility and water-holding capacity . Total stiffness gel increased with low degrees of hydrolysis, but decreased after prolonged hydrolysis . Maximal gel stiffness was 1.5-fold that gels made from of unhydrolysed beta-lactoglobulin . This was much lower than the stiffening effect obtained after partial hydrolysis of whey protein isolate, showing that the gel strengthening effect of partial hydrolysis was depedent on the protein composition and/or the hydrolysis and gelatin conditions . A mechanism to explain the observed effects of hydrolysis on gelation and gel properties is presented. Mol Gen Genet, 1999 Dec, 262(4-5), 738 - 48 Cloning and expression of two autolysin genes, cwIU and cwIV, which are tandemly arranged on the chromosome of Bacillus polymyxa var . colistinus; Ishikawa S et al.; The cwlV gene, which encodes Bacillus polymyxa var . colistinus autolysin was cloned and sequenced . cwlV comprises a 1497-bp ORF and encodes a polypeptide of 499 amino acid (aa) residues (Mr of 53,707 Da) . The N-terminal sequence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermined aa); this processed form corresponds to the C-terminal portion (183 aa, Mr of 20,050 Da) of the cwlV ORF . Sequencing of the flanking region revealed that another putative autolysin gene, cwlU, is located upstream of cwlV . cwlU encodes a polypeptide of 524 aa and its deduced sequence is 34.9% identical to the full-length sequence of CwlV . Downstream of cwlV, the genes for a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-homologous OrfX, a glutathione peroxidase homolog (Gpx), and the N-terminal region of OrfZ containing a ATP/GTP-binding site motif were found . Northern blotting and primer-extension analyses revealed that cwlU is transcribed as a single cistron, but cwlV is transcribed with orfW . The unprocessed forms of CwlV and CwlU (VdeltaS and UdeltaS, respectively) and their predicted mature forms (Vcat and Ucat, respectively) were expressed in, and purified from, Escherichia coli . Enzyme analysis indicated that VdeltaS and Vcat exhibit low and high cell wall hydrolase activities toward B . polymyxa cell wall, respectively, but UdeltaS and Ucat exhibit almost no and low cell wall hydrolase activities, respectively. Biochemistry, 2000 Jan 11, 39(1), 171 - 82 Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus; Bird LE et al.; For the first time, we demonstrate directly a stable complex between a bacterial DnaG (primase) and DnaB (helicase) . Utilizing fragments of both proteins, we are able to dissect interactions within this complex and provide direct evidence that it is the C-terminal domain of primase that interacts with DnaB . Furthermore, this C-terminal domain is sufficient to induce maximal stimulation of the helicase and ATPase activities of DnaB . However, the region of DnaB that interacts with the C-terminal domain of primase appears to comprise a surface on DnaB that includes regions from both of the previously identified N- and C-terminal domains . Using a combination of biochemical and physical techniques, we show that the helicase-primase complex comprises one DnaB hexamer and either two or three molecules of DnaG . Our results show that in Bacillus stearothermophilus the helicase-primase interaction at the replication fork may not be transient, as was shown to be the case in Escherichia coli . Instead, primase appears to interact with the helicase forming a tighter complex with enhanced ATPase and helicase activities. J Clin Oncol, 2000 Jan, 18(1), 148 - 57 Adjuvant active specific immunotherapy for stage II and III colon cancer with an autologous tumor cell vaccine: Eastern Cooperative Oncology Group Study E5283; Harris JE et al.; PURPOSE: A randomized phase III clinical trial of adjuvant active specific immunotherapy (ASI) with an autologous tumor cell-bacillus Calmette-Guerin (BCG) vaccine was conducted to determine whether surgical resection plus ASI was more beneficial than resection alone in stage II and III colon cancer patients . PATIENTS AND METHODS: Patients (n = 412) with colon cancer (297 with stage II disease, 115 with stage III disease) were randomly allocated to an observation arm or to a treatment arm in which they received three weekly intradermal vaccine injections of 10(7) irradiated autologous tumor cells beginning approximately 4 weeks after surgery . The first two weekly injections also contained 10(7) BCG organisms . Patients were observed for determination of time to recurrence and disease-free and overall survival . RESULTS: This was a negative study in that after a 7.6-year median follow-up period, there were no statistically significant differences in clinical outcomes between the treatment arms . However, there were disease-free survival (P =.078) and overall survival (P =.12) trends in favor of ASI when treatment compliance was evaluated, ie, patients who received the intended treatment had a delayed cutaneous hypersensitivity (DCH) response to the third vaccination (induration >/=5 mm) . Also, the magnitude of the DCH response correlated with improved prognosis . The 5-year survival proportion was 84.6% for those with indurations greater than 10 mm, compared with 45.0% for those with indurations less than 5 mm . CONCLUSIONS: When all randomized patients were evaluated, no significant clinical benefit was seen with ASI in surgically resected colon cancer patients with stage II or III colon cancer . However, there was an indication that treatment compliance with effective immunization results in disease-free and overall survival benefits. Biotechnol Bioeng, 2000 Feb 20, 67(4), 398 - 407 Secretory production of human leptin in Escherichia coli; Jeong KJ et al.; Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis . In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp . endoxylanase signal peptide . The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene . The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG . Using T7 promoter in E . coli BL21(DE3), most of protein produced was in a premature form . Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37 degrees C . However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E . coli . At 30 degrees C, ca . 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37 degrees C . When the periplasmic oxidoreductase of E . coli, DsbA, was co-expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37 degrees C without the decrease of the amount of leptin produced . FEMS Microbiol Lett, 2000 Jan 15, 182(2), 297 - 301 The gene encoding mycobacterial DNA-binding protein I (MDPI) transformed rapidly growing bacteria to slowly growing bacteria; Matsumoto S et al.; Pathogenic species of Mycobacterium are slowly growing intracellular bacteria . Slow growth is important for the parasitism of these organisms and chronicity of the disease, but its precise mechanism has not been elucidated . Recently, we found that a novel DNA-binding protein (MDPI) was expressed (7-10% in total protein) in mycobacteria, such as Mycobacterium bovis bacillus Calmette-Guerin, Mycobacterium tuberculosis, and Mycobacterium leprae . In this study, we observed that MDPI interfered with replication, transcription, and translation in the analysis in in vitro E . coli cell-free macromolecular biosynthesizing systems . Furthermore, MDPI inhibited the rapid growth of both Escherichia coli and Mycobacterium smegmatis, and NH(2)-terminal second amino acid, asparagine, was observed to be important in terms of this function . These data suggest an important role of MDPI for suppression of growth rates of mycobacteria. FEBS Lett, 1999 Dec 3, 462(3), 373 - 6 Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase-N receptor is not directly related to toxicity; Jenkins JL et al.; Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds a 115-kDa aminopeptidase-N purified from Manduca sexta midgut . Cry1Ac domain III mutations were constructed around a putative sugar-binding pocket and binding to purified aminopeptidase-N and brush border membrane vesicles (BBMV) was compared to toxicity . Q509A, R511A, Y513A, and 509-511 (QNR-AAA) eliminated aminopeptidase-N binding and reduced binding to BBMV . However, toxicity decreased no more than two-fold, indicating activity is not directly correlated with aminopeptidase-N binding . Analysis of toxin binding to aminopeptidase-N in M . sexta is therefore insufficient for predicting toxicity . Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo. Bull Acad Natl Med, 1999, 183(7), 41 - 50; discussion 50-1 {What can be expected from sequencing of the Mycobacterium tuberculosis genome?}; Cole ST; Mycobacterium tuberculosis, the scourge of humanity, is one of the most successful and scientifically challenging pathogens of all time . To catalyze the conception of new prophylactic and therapeutic interventions against tuberculosis, and to enhance our understanding of the biology of the tubercle bacillus, the complete genome sequence of the most widely used strain, H37Rv, has been determined . Bioinformatic analysis led to the identification of approximately 4,000 genes in the 4.41 Mb genome sequence and provided fresh insight into the biochemistry, physiology, genetics and immunology of this much-feared bacterium . Genomic information is centralised in TubercuList . Bull Acad Natl Med, 1999, 183(7), 25 - 37; discussion 37-40 {Which strategies for eradication of tuberculosis?}; Grosset J; Eradication of tuberculosis is biologically feasible because infectious tuberculosis is relatively easy to identify, is treatable and curable, with cure rates approaching 100% when modern short-course therapy is used . Early diagnosis and effective treatment reduce transmission . Infected persons at increased risk of developing infectious tuberculosis can be identified through tuberculin screening of high-risk populations and tuberculosis is, at least partly, preventable by the administration of preventive therapy (chemoprophylaxis) and BCG vaccination . Humans are the primary reservoir of the tubercle bacillus . In industrialized countries, tuberculin testing of dairy cattle and slaughter of infected animals and pasteurization of milk have virtually eliminated the problem . In industrialized countries, tuberculosis has retreated into focal pockets that can be targeted for intensified control efforts . Elimination of tuberculosis in these countries depends in part on global elimination because of imported cases . In developing countries, the immediate task is to control tuberculosis, and DOTS is the key detection and treatment strategy . Joint efforts should address the issue of the global implementation of DOTS strategy and of the research needs for effective vaccine, new drugs and new delivery services. Arch Biochem Biophys, 2000 Jan 15, 373(2), 394 - 400 Characterization of endo-alpha-N-acetylgalactosaminidase from Bacillus sp . and syntheses of neo-oligosaccharides using its transglycosylation activity; Ashida H et al.; Endo-alpha-N-acetylgalactosaminidase was purified to homogeneity from the culture fluid of Bacillus sp . isolated from soil and characterized . The molecular mass of the enzyme was estimated as 110 kDa . The enzyme was stable at pH 4.0-10.0, up to 55 degrees C, and was most active at pH 5.0 . The substrate specificity of the enzyme was strict for the disaccharide, galactosyl beta1, 3 N-acetyl-d-galactosamine, bound to aglycone in alpha configuration . On the other hand, the specificity of the enzyme for the aglycone structure was fairly relaxed . The enzyme could transfer the disaccharide from para-nitrophenyl substrate to various acceptors, such as monosaccharides, disaccharides, and sugar alcohols . Using this transglycosylation activity of the endoglycosidase, it may be possible to synthesize neo-oligosaccharides . Arch Biochem Biophys, 2000 Jan 1, 373(1), 110 - 5 Kinetics and inhibition of cyclomaltodextrinase from alkalophilic Bacillus sp . I-5; Kim MJ et al.; The cyclomaltodextrinase from alkalophilic Bacillus sp . I-5 (CDase I-5) was expressed in Escherichia coli and the purified enzyme was used for characterization of the enzyme action . The hydrolysis products were monitored by both HPLC and high-performance ion chromatography analysis that enable the kinetic analysis of the cyclomaltodextrin (CD)-degrading reaction . Analysis of the kinetics of cyclomaltodextrin hydrolysis by CDase I-5 indicated that ring-opening of the cyclomaltodextrin was the major limiting step and that CDase I-5 preferentially degraded the linear maltodextrin chain by removing the maltose unit . The substrate binding affinity of the enzyme was almost same for those of cyclomaltodextrins while the rate of ring-opening was the fastest for cyclomaltoheptaose . Acarbose and methyl 6-amino-6-deoxy-alpha-d-glucopyranoside were relatively strong competitive inhibitors with K(i) values of 1.24 x 10(-3) and 8.44 x 10(-1) mM, respectively . Both inhibitors are likely to inhibit the ring-opening step of the CD degradation reaction . Vaccine, 2000 Jan 31, 18(14), 1294 - 7 Inhibition of multiplication of Mycobacterium leprae in mouse foot pads by recombinant Bacillus Catmette-Guérin (BCG); Ohara N et al.; Immunization of mice with recombinant Mycobacterium bovis Bacillus Calmette-Guerin (rBCG) which over-produces a putative protective antigen candidate, the A component of antigen 85 complex (Ag85A), reduced the multiplication of Mycobacterium leprae in the foot pads of mice . The inhibition by this rBCG (rBCG/85A) was more evident than that with parental BCG . Repeated rBCG/85A immunization significantly could reduce M . leplae multiplication in mice . This is first report of rBCG to control mycobacterial infection in animal model . Therefore, rBCG technique may be useful for the development of a more effective mycobacteria vaccine. Clin Diagn Lab Immunol, 2000 Jan, 7(1), 1 - 5 Sonicated diagnostic immunoblot for bartonellosis; Mallqui V et al.; Two simple Bartonella bacilliformis immunoblot preparation methods were developed . Antigen was prepared by two different methods: sonication of whole organisms or glycine extraction . Both methods were then tested for sensitivity and specificity . Well-defined control sera were utilized in the development of these diagnostic immunoblots, and possible cross-reactions were thoroughly examined . Sera investigated for cross-reaction with these diagnostic antigens were drawn from patients with brucellosis, chlamydiosis, Q fever, and cat scratch disease, all of whom were from regions where bartonellosis is not endemic . While both immunoblots yielded reasonable sensitivity and high specificity, we recommend the use of the sonicated immunoblot, which has a higher sensitivity when used to detect acute disease and produces fewer cross-reactions . The sonicated immunoblot reported here is 94% sensitive to chronic bartonellosis and 70% sensitive to acute bartonellosis . In a healthy group, it is 100% specific . This immunoblot preparation requires a simple sonication protocol for the harvesting of B . bacilliformis antigens and is well suited for use in regions of endemicity. Appl Microbiol Biotechnol, 1999 Nov, 52(6), 845 - 52 Phage abortive infection of Bacillus licheniformis ATCC 9800; identification of the abiBL11 gene and localisation and sequencing of its promoter region; Tran LS et al.; The virulent bacteriophage BL11 infects almost all Bacillus licheniformis strains tested, including the industrial bacitracin-producing B . licheniformis 19 . B . licheniformis ATCC 9800, however, was virtually insensitive to phage BL11 infection, and all of the few surviving progeny phages proved to be mutants . The phage-resistance mechanism was neither inhibition of adsorption, nor restriction or exclusion provided by a resident prophage, but was, instead, of another type . Phage BL11 adsorbed well on to ATCC 9800 cells, its DNA was injected, but replication of phage DNA was inhibited and the infected cells died . Thus, the mechanism of phage resistance was identified as abortive infection (AbiBL11) . The so-called abiBL11 gene was identified on the chromosome of strain ATCC 9800 by Tn917PF1 transposon mutagenesis . Part of the abiBL11 gene from the phage-sensitive ATCC 9800::Tn917PFI was cloned . Gene-disruption analysis, based on Campbell-type integration, showed that a 0.3-kb EcoRI fragment contained the 5' end of abiBL11 . The promoter region of abiBL11 was identified using promoter- and terminator-probe plasmids . The deduced sequence (206 amino acids) of the N-terminal part of abiBL11 showed no significant homology to known abortive-infection genes, but did show homology to a Saccharomyces cerevisiae gene coding for a serine/threonine protein kinase (RCK1). J Paediatr Child Health, 1999 Dec, 35(6), 582 - 4 Clinical manifestations of Bacillus cereus meningitis in newborn infants; Tokieda K et al.; Bacillus cereus (B . cereus) meningitis sometimes occurs in patients with risk factors, which are associated with central nervous system (CNS) anomalies, surgical or anaesthetic access to CNS . We observed two cases of B . cereus meningitis in neonates without such risk factors . The clinical courses of both neonates were fulminant, and routine antibiotic therapy failed . Intracranial haemorrhage was evident at autopsy . According to the previous neonatal case reports and our experience, we found that six of seven neonates were premature babies admitted to the neonatal intensive care unit, five died within a week of onset of the disease, and six had intracranial haemorrhage . We speculate that B . cereus meningitis may occur in neonates, even without any of the risk factors previously described in adult case reports, and that the clinical manifestations of the meningitis might be characterized by the high incidence of intracranial haemorrhage and poor mortality. Syst Parasitol, 1999 Jun, 43(2), 123 - 31 Haycocknema perplexum n . g., n . sp . (Nematoda: Robertdollfusidae): an intramyofibre parasite in man; Spratt DM et al.; Haycocknema perplexum n . g., n . sp . (Nematoda: Robertdollfusidae) is described from a man in Tasmania, Australia . Adult male and female nematodes and larvae were recovered from myofibres following biopsy of the right vastus lateralis muscle and were associated with a polymyositis . H . perplexum is distinguished from all other genera of the Muspiceoidea by the presence of a large amorphous "cell" supporting a granule-filled, flask- or gourd-shaped reservoir in the rectal region of mature and gravid female nematodes, often containing one or more large, refractile, thick-rimmed "globules" on the external surface of the reservoir, by the small number of ova/eggs/larvae developing in each uterus, by the minute, weakly-sclerotised, almost tubular spicule, by the presence of a pair of ampulla-shaped glands posteriorly and by the presence of lateral bacillary bands comprised of a single row of pore cells spaced irregularly and extending posteriorly to the region of the vulva in immature females. Appl Environ Microbiol, 2000 Jan, 66(1), 428 - 30 Pulsed-electric field treatment enhances the bactericidal action of nisin against Bacillus cereus; Pol IE et al.; Vegetative cells of Bacillus cereus were subjected to low doses of nisin (0.06 microg/ml) and mild pulsed-electric field treatment (16.7 kV/cm, 50 pulses each of 2-micros duration) . Combining both treatments resulted in a reduction of 1.8 log units more than the sum of the reductions obtained with the single treatments, indicating synergy. Appl Environ Microbiol, 2000 Jan, 66(1), 304 - 9 Efficient production of artificially designed gelatins with a Bacillus brevis system; Kajino T et al.; Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system . The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy . Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli . Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells . Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography . The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin . These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method . This study revealed the possible industrial application of artificially designed repetitive proteins. Appl Environ Microbiol, 2000 Jan, 66(1), 118 - 24 Plasmid transfer between the Bacillus thuringiensis subspecies kurstaki and tenebrionis in laboratory culture and soil and in lepidopteran and coleopteran larvae; Thomas DJ et al.; Plasmid transfer between Bacillus thuringiensis subsp . kurstaki HD1 and B . thuringiensis subsp . tenebrionis donor strains and a streptomycin-resistant B . thuringiensis subsp . kurstaki recipient was studied under environmentally relevant laboratory conditions in vitro, in soil, and in insects . Plasmid transfer was detected in vitro at temperatures of 5 to 37 degrees C, at pH 5.9 to 9.0, and at water activities of 0.965 to 0.995, and the highest transfer ratios (up to 10(-1) transconjugant/donor) were detected within 4 h . In contrast, no plasmid transfer was detected in nonsterile soil, and rapid formation of spores by the introduced strains probably contributed most to the lack of plasmid transfer observed . When a B . thuringiensis subsp . kurstaki strain was used as the donor strain, plasmid transfer was detected in killed susceptible lepidopteran insect (Lacanobia oleracea) larvae but not in the nonsusceptible coleopteran insect Phaedon chocleriae . When a B . thuringiensis subsp . tenerbrionis strain was used as the donor strain, no plasmid transfer was detected in either of these insects even when they were killed . These results show that in larger susceptible lepidopteran insects there is a greater opportunity for growth of B . thuringiensis strains, and this finding, combined with decreased competition due to a low initial background bacterial population, can provide suitable conditions for efficient plasmid transfer in the environment. Ann Thorac Surg, 1999 Dec, 68(6), 2351 - 2 Bacillus cereus prosthetic valve endocarditis; Castedo E et al.; Bacillus cereus is a ubiquitous organism that often contaminates microbiological cultures but rarely causes serious infections . Reports of B . cereus endocarditis are infrequent . Infection in patients with valvular heart disease is associated with significant mortality and morbidity . We describe a case of B . cereus endocarditis involving a mechanical mitral prosthesis that resolved after replacement of the prosthetic valve . We also review the previous cases reported in the literature. J Gastroenterol, 1999, 34 Suppl 11, 28 - 31 Effect of culture conditions on morphological changes of Helicobacter pylori; Tominaga K et al.; The morphological conversion of Helicobacter pylori from the spiral form to the coccoid form may be the expression of a transitory adaptation to an unsuitable environment . The mechanism(s) of this conversion are not clear . In this study, we examined whether the morphological conversion of H . pylori is affected by various culture conditions, such as oxygen concentration, pH, temperature, or the presence of beta-cyclodextrin . H . pylori (NTCC11916) was cultured on Brucella agar, followed by culture in Brucella broth containing 1% agar under several conditions . Morphological conversion of individual H . pylori on the agar plate was investigated with time after incubation under phase contrast microscopy . When H . pylori was inoculated in Brucella broth containing beta-cyclodextrin, the spiral form of the organism was observed even after 6 days of incubation under standard culture conditions: 37 degrees C, pH 7, and microaerobic atmosphere (5% O2/10% CO2/85% N2) (control) . The morphological conversion of H . pylori was completed on day 3 in an aerobic atmosphere (20% O2 supply) and on day 2 in an undermicroaerobic atmosphere (<0.1% O2) . Its complete morphological conversion was observed at pH 8 on day 5 and at pH 4 on day 6 . All of the H . pylori (100%) incubated at 20 degrees or 42 degrees C had converted from the bacillary to the coccoid form on day 4 . Conditioned medium without beta-cyclodextrin caused complete conversion on day 5 . These results suggest that oxygen concentration, pH, temperature, and beta-cyclodextrin may be related to the H . pylori morphological conversion from the bacillary to the coccoid form. Vet Immunol Immunopathol, 1999 Dec 15, 72(1-2), 231 - 5 Selection for high immune response: an alternative approach to animal health maintenance? Wilkie B, Mallard B. To test the hypothesis that variation in ability to respond immunologically correlates with health, Yorkshire pigs were bred for high (HIR) and low (LIR) antibody (Ab) and cell-mediated immune response (CMI) . Selection was based on standardized measures of Ab (secondary response to hen egg white lysozyme, serum IgG concentration) and CMI (cutaneous delayed-type hypersenstivity to purified protein derivative of tuberculin after immunization with bacillus Calmette-Guerin and in vitro lymphocyte response to Con-A) . Differences in Ab and CMI by line were not restricted to the antigens used in the selection . Antibody response to vaccines was highest in HIR and non-responders were restricted to LIR pigs . The HIR pigs had the best rate of weight gain . After infection with Mycoplasma hyorhinis, HIR developed more severe arthritis and less polyserositis . Differences were associated with variation in cytokine message in joint-related cells . Following exposure to attenuated transmissible gastroenteritis virus, natural killer cells of the LIR pigs but not of HIR or control lines, were unresponsive . Genetic selection for Ab and CMI may provide health and productivity advantages and complement traditional health-maintenance methods. FEMS Microbiol Lett, 2000 Jan 1, 182(1), 119 - 24 Contact-dependent hemolytic activity distinct from deforming activity of Bartonella bacilliformis; Hendrix LR; Although Bartonella bacilliformis causes a severe anemia in humans, this study presents the first report of hemolytic activity by B . bacilliformis . The activity was not apparent in culture supernatants but was reliably detected when B . bacilliformis cells were centrifuged onto erythrocytes prior to incubation . Abrogation of hemolytic activity by proteinase K treatment suggested the hemolysin was a Bartonella protein . Even though hemolysis required relatively long incubation times, de novo protein synthesis was not required to produce the protein . A preparation containing factors released by B . bacilliformis, including deformin, a B . bacilliformis protein able to induce pits and invaginations in erythrocyte membranes, had some ability to lyse erythrocytes . However, pre-deformed erythrocytes did not lyse faster or to a greater extent than control erythrocytes after the addition of B . bacilliformis cells . Inhibition of deformation caused by B . bacilliformis cells with the erythrocyte ATPase inhibitor, vanadate, did not affect hemolytic activity . This study suggests hemolytic activity and deforming activity are attributable to different B . bacilliformis proteins. FEMS Microbiol Lett, 2000 Jan 1, 182(1), 29 - 34 Valine dehydrogenase from Streptomyces albus: gene cloning, heterologous expression and identification of active site by site-directed mutagenesis; Hyun CG et al.; A gene encoding valine dehydrogenase (Vdh) has been cloned from Streptomyces albus, a salinomycin producer, and expressed in Escherichia coli . The S . albus Vdh is composed of 364 amino acids that showed high homology with several other amino acid dehydrogenases as well as Vdhs from Streptomyces spp . and leucine and phenylalanine dehydrogenases (Ldh and Pdh) from Bacillus spp . A protein of 38 kDa, corresponding to the approximate mass of the predicted S . albus Vdh product (38.4 kDa) exhibiting specific Vdh activity, was observed when the S . albus vdh gene was overexpressed in E . coli under the controlled T7 promoter and was subsequently purified to homogeneity . Among branched- and straight-chain amino acids, L-valine and L-alpha-aminobutyrate were the preferred substrates for the enzyme . Lys-79 and Lys-91 of S . albus Vdh were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydrogenase sequences . To elucidate the functional roles of the lysyl residues, the Lys residues have individually been replaced with Ala by site-directed mutagenesis . Kinetic analyses of the Lys-79 and Lys-91-mutated enzymes revealed that they are involved in the substrate binding site and catalysis, respectively, analogous to the corresponding residues in the homologous Ldh and Pdh. J Burn Care Rehabil, 1999 Nov-Dec, 20(6), 482 - 6 Infestations and chronic infections in foreign pediatric patients with burns: is there a role for specific protocols? Barret JP, Dardano AN, Heggers JP, McCauley RL. Infestations by parasites such as Mycobacterium tuberculosis and other viral infections are common in third world countries . Consequently, the admission of a significant number of foreign patients to burn centers in the United States may pose new problems, not only for inpatients but also for health care workers . To document infestations in patients from third world countries and to determine the need for specific protocols, we studied 62 consecutive foreign patients admitted to our pediatric burn reconstruction service between July 1997 and December 1998 . All patients were evaluated with chest X-ray, hemogram with differential count, clinical and laboratory nutritional assessment, and skin test for tuberculosis, and stool samples were evaluated for ova and parasites . No pathologic findings were seen on chest radiographs . Only 1 patient had a positive skin test for tuberculosis, as a result of previous bacille Calmette-Guerin vaccine . Yet, 10 patients (16%) had positive stool cultures for ova and parasites that contained 29 isolates . The most frequently identified organism was Blastocystis hominis . All amoebas identified were nonpathogenic according to Centers for Disease Control criteria . Ascaris lumbricoides and 1 case of cysticercosis were found . None of the patients with parasites had clinical manifestations of parasitosis or chronic infections . However, parasite infestations had a positive correlation with eosinophilia, altered nutritional status, and altered mean corpuscular hemoglobin concentration, as defined by multiple linear regression . Although foreign patients admitted to burn centers from third world countries have a low rate of infestations, patients at risk can be identified by laboratory findings and studies of nutritional status . Simple hand washing prevents the spread of disease and protects health providers. Syst Parasitol, 1999 May, 43(1), 17 - 27 Aonchotheca musimon n . sp . (Nematoda : Capillariinae) from the mouflon Ovis musimon in the sub-antarctic Kerguelen archipelago, with comments on the relationships with A . bilobata (Bhalerao, 1933) Moravec, 1982 and other species of the genus; Pisanu B et al.; An intestinal capillariid nematode, Aonchotheca musimon n . sp., is described from Ovis musimon imported into the Kerguelen archipelago (Terres Australes et Antarctiques Francaises) . The comparison of this new material with other Aonchotheca spp . is based on the usual characters, i.e . spicule, caudal bursa, number of papillae, stichosome, bacillary bands, shape of the cirrus, and on the length of the ejaculatory duct which appears to be of some phylogenetic value . A . musimon, of which the spicule is 208-230 microm long, is close to A . bilobata, another parasite of bovids, which is redescribed here . It is distinct from this species because the posterior region of the female worm is cylindrical instead of conical, the lateral alae of the male worm are longer, quadrangular and vesicular instead of triangular and smooth, the caudal bursa has a folded dorso-lateral edge, there is a recurrent ventral fold of the cirrus, the slender distal part of the spicule is longer, the oesophagus is shorter in both sexes and the slightly larger eggs have a thicker shell . These two species from bovids and A . murissylvatici from murid rodents, of which the main characters are similar, represent a small group with a very elongate ejaculatory duct (1.9-2.5 mm) . This is in contrast to a larger group of species with a short ejaculatory duct (350-600 microm), which are parasites of Chiroptera (A . brosseti, A . chabaudi, A . landauae, A . gabonensis), Insectivora (A . erinacei), mustelid Carnivora (A . putorii, A . mustelorum) and glirid rodents (A . myoxinitelae, A . legerae) . A . bovis and A . dessetae, respectively parasites of bovids and lagomorphs, present an ejaculatory duct of intermediary length and do not belong to these groups . Several species are transferred to the genus Aonchotheca: A . kashmirensis (Raina & Kaul, 1982) n . comb., A . legerae (Justine, Ferte & Bain, 1987) n . comb., A . forresteri (Kinsella & Pence, 1987) n . comb., A . chabaudi (Justine, 1989) n . comb., A . landauae (Justine, 1989) n . comb., A . brosseti (Justine, 1989) n . comb., A . gabonensis (Justine, 1989) n . comb . and A . dessetae (Justine, 1990) n . comb. J Am Mosq Control Assoc, 1999 Dec, 15(4), 446 - 52 Field efficacy and nontarget effects of the mosquito larvicides temephos, methoprene, and Bacillus thuringiensis var . israelensis in Florida mangrove swamps; Lawler SP et al.; We compared the efficacy and nontarget effects of temephos, Bacillus thuringiensis var . israelensis (B.t.i.), and methoprene applied by helicopter to control mosquito larvae in mangrove swamps on Sanibel Island, FL, in May 1997 . Three sites per treatment and 3 untreated sites were used . Temephos (Abate) was applied at 37 ml/ha (43% active ingredient {AI}), B.t.i . granules (Vectobac G) were applied at 5.606 kg/ha (200 International Toxic Units/mg), and methoprene (Altosid ALL) was applied at 213 ml/ha (5% AI) . Efficacy was quantified by monitoring the survival of caged and uncaged larval Aedes taeniorhynchus . We quantified mortality of sentinel nontarget amphipods (Talitridae) at all sites, monitored the effect of temephos on flying arthropods using light traps, and collected dead insects in tarps suspended under mangroves in areas treated with either temephos or methoprene . Each pesticide showed good overall efficacy but occasional failures occurred . No detectable mortality of amphipods or flying insects attributable to pesticides was found . The inconsistent field efficacies of the pesticides indicate a need for reinspection of treated sites in this habitat. Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 15190 - 5 Mycobacterial infection of macrophages results in membrane-permeable phagosomes; Teitelbaum R et al.; Cell-mediated immunity is critical for host resistance to tuberculosis . T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacter