J Bacteriol, 1996 Apr, 178(7), 2141 - 4 Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis: the cryV-encoded protein is expressed early in stationary phase; Kostichka K et al.; A CryV-type protein (CGCryV) has been isolated from supernatant fluids of Bacillus thuringiensis AB88 cultures . Previous reports have suggested the cryptic nature of the cryV-type genes on the basis of the absence of CryV-type proteins in parasporal crystals . The CryV-type protein reported here is expressed early in stationary phase, and evidence indicates that it is an exported protein . Analysis of the deduced protein sequence from this gene reveals the presence of an N-terminal domain that likely acts as a signal peptide . The CGCryV protein is the first reported case of a delta-endotoxin being a secreted protein, which may influence the biological relevance of these proteins. J Bacteriol, 1996 Apr, 178(7), 2108 - 17 Dynamics in oxygen-induced changes in S-layer protein synthesis from Bacillus stearothermophilus PV72 and the S-layer-deficient variant T5 in continuous culture and studies of the cell wall composition; Sara M et al.; Stable synthesis of the hexagonally ordered (p6) S-layer protein from the wild-type strain of Bacillus stearothermophilus PV72 could be achieved in continuous culture on complex medium only under oxygen-limited conditions when glucose was used as the sole carbon source . Depending on the adaptation of the wild-type strain to low oxygen supply, the dynamics in oxygen-induced changes in S-layer protein synthesis was different when the rate of aeration was increased to a level that allowed dissimilation of amino acids . If oxygen supply was increased at the beginning of continuous culture, synthesis of the p6 S-layer protein from the wild-type strain (encoded by the sbsA gene) was immediately stopped and replaced by that of a new type of S-layer protein (encoded by the sbsB gene) which assembled into an oblique (p2) lattice . In cells adapted to a prolonged low oxygen supply, first, low-level p2 S-layer protein synthesis and second, synchronous synthesis of comparable amounts of both types of S-layer proteins could be induced by stepwise increasing the rate of aeration . The time course of changes in S-layer protein synthesis was followed up by immunogold labelling of whole cells . Synthesis of the p2 S-layer protein could also be induced in the p6-deficient variant T5 . Hybridization data obtained by applying the radiolabelled N-terminal and C-terminal sbsA fragments and the N-terminal sbsB fragment to the genomic DNA of all the three organisms indicated that changes in S-layer protein synthesis were accompanied by chromosomal rearrangement . Chemical analysis of peptidoglycan-containing sacculi and extraction and recrystallization experiments revealed that at least for the wild-type strain, a cell wall polymer consisting of N-acetylglucosamine and glucose is responsible for binding of the p6 S-layer protein to the rigid cell wall layer. Clin Exp Immunol, 1996 Apr, 104(1), 44 - 7 Mice that carry the resistance allele of the Bcg gene (Bcgr) develop a superior capacity to stabilize bacille Calmette-Guerin (BCG) infection in their lungs and spleen over a protracted period in the absence of specific immunity; Medina E et al.; In mice, natural resistance to infection with BCG is under the influence of an autosomal gene designated Bcg . It is shown here in agreement with others that mice that possess the dominant resistant allele of the gene (Bcgr) are more capable than mice that possess the susceptible recessive allele (Bcgs) at restricting the growth of BCG in their lungs, as well as in their spleens, during the first 20 days of infection . It is shown, in addition, that in the absence of specific immunity the resistance difference between Bcgr and Bcgs mice became much more pronounced as infection progressed beyond day 20 . Whereas T cell-depleted Bcgr mice developed a capacity after day 20 to cause infection in their lungs and spleens to stabilize and plateau for at least 40 days, T cell-depleted Bcgs mice were unable to prevent infection from progressing in these organs . On the other hand, both types of T cell-depleted mice were capable of causing infection to plateau in their livers and kidneys . Moreover, this T cell-independent mechanism of resistance was essentially abolished in all organs in which it was expressed by treating the mice with hydrocortisone . In the lungs of immunocompetent Bcgs mice, failure to stabilize infection was associated with heavily infected macrophages and failure to contain BCG at original sites of infection. Clin Exp Immunol, 1996 Apr, 104(1), 37 - 43 Influence of the mouse Bcg, Tbc-1 and xid genes on resistance and immune responses to tuberculosis infection and efficacy of bacille Calmette-Guérin (BCG) vaccination; Nikonenko BV et al.; We have studied the role of three mouse distinct non-H-2 genes (Bcg, Tbc-1, xid) in several phenomena of antituberculosis immunity and resistance . On the basis of median survival time (MST) of mice following infection with virulent Mycobacterium tuberculosis H37Rv, Bcg gene did not control resistance to the lethal dose of H37Rv infection in non-vaccinated and Myco . bovis (BCG)-vaccinated mice . However, Bcgr allele, in comparison with Bcgs allele, determined more effective suppression of an early multiplication in spleens of H37Rv mycobacteria after a low dose (5x10(4) colony-forming units (CFU)) injection . CBA/N mice, which are not protected efficiently against tuberculous challenge by BCG vaccination, were characterized by a decreased in vitro proliferation of immune lymph node cells, both spontaneous and stimulated with mycobacterial antigens . The decreased proliferation was due to immunosuppression caused by interactions between responding T cells and CBA/N antigen-presenting cells (APC) . We have confirmed that the defective response to BCG-vaccination in CBA/N mice is linked with the X-chromosome and thus is presumably determined by the xid gene itself . I/St mice (Tbc-1s), supersusceptible to H37Rv infection, were not able to restrict the growth of H37Rv mycobacteria in spleens, even following infection with a low dose (5x10(4)), but restricted the growth of Myco.bovis BCG more effectively than Bcgs mice. Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 575 - 80 Inconsistencies in determining Bacillus thuringiensis toxin binding sites relationship by comparing competition assays with ligand blotting; Lee MK et al.; Receptor binding properties of Cry1Aa, Cry1Ab, and Cry1Ac Bacillus thuringiensis toxins to Lymantria dispar brush border membrane vesicles (BBMV) were investigated by competition assays and BBMV ligand blotting . Homologous competition binding assays demonstrated that all Cry IA toxins bound to L . dispar BBMV with high binding affinities . Heterologous competition assays suggested that all three toxins share the same binding sites . However, our ligand blotting experiments were not consistent with the heterologous competition assays . We identified a 120 kDa peptide as a Cry1Ac binding protein and a 210 kDa peptide as a Cry1Aa and Cry1Ab binding protein . These results indicated that determining the relationships between toxin binding sites by either competition assays or ligand blotting alone may not be conclusive. J Mol Biol, 1996 Mar 22, 257(1), 129 - 52 Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp . kyushuensis and implications for membrane pore formation; Li J et al.; The delta-endotoxin CytB, found in parasporal inclusions of Bacillus thuringiensis subspecies kyushuensis, is a membrane pore-forming protein which is lethal to the larvae of Dipteran insects and broadly cytolytic in vitro . The crystal structure of CytB in the protoxin form has been determined by isomorphous replacement using heavy-atom derivatives of both the wild-type protein and an engineered cysteine mutant . The atomic model comprising residues 19 to 245 and 28 bound water molecules has been refined at 2.6 angstrom resolution to a crystallographic R-factor of 19.7% and a free R-factor of 26.1% . CytB has a single domain of alpha/beta architecture but a novel connectivity comprising two outer layers of alpha-helix hairpins wrapped around a mixed beta-sheet . In the protoxin form, CytB is a dimer linked by the intertwined N-terminal strands in a continuous, 12-stranded beta-sheet . Proteolytic processing cleaves the intertwined beta-strands to release the active CytB as a monomer, as well as removing the C-terminal tail to uncover the three-layered core . The homologous toxin CytA should show the same fold . Mutations in CytA that inhibit expression map to the dimer contacts and to the tip of helix pair A-B in contact with the sheet, apparently preventing correct folding . Mutations that inhibit toxicity map to the edge of the beta-sheet adjoining the helix pair C-D and to the sheet face, while mutations on the helix surfaces have no effect . Therefore segments forming the sheet, rather than the amphiphilic but short helices, are responsible for membrane binding and pore formation . A conformational change is postulated by which the helix pair C-D peels away from the sheet to lie on the membrane surface, while the sheet region rearranges to form an oligomeric trans-membrane pore. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2343 - 7 Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface; Jucovic M et al.; Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens . Inhibition involves the formation of a very tight one-to-one complex of the two proteins . With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail . In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase . Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar . This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent . When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative . A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding . The mutations selected as suppressors could not have been predicted on the basis of the known protein structures . The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W . The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges. Structure, 1996 Mar 15, 4(3), 277 - 86 Protein-protein interactions in the pyruvate dehydrogenase multienzyme complex: dihydrolipoamide dehydrogenase complexed with the binding domain of dihydrolipoamide acetyltransferase; Mande SS et al.; BACKGROUND . The ubiquitous pyruvate dehydrogenase multienzyme complex is built around an octahedral or icosahedral core of dihydrolipoamide acetyltransferase (E2) chains, to which multiple copies of pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) bind tightly but non-covalently . E2 is a flexible multidomain protein that mediates interactions with E1 and E3 through a remarkably small binding domain (E2BD) . RESULTS . In the Bacillus stearothermophilus complex, the E2 core is an icosahedral assembly of 60 E2 chains . The crystal structure of the E3 dimer (101 kDa) complexed with E2BD (4 kDa) has been solved to 2.6 A resolution . Interactions between E3 and E2BD are dominated by an electrostatic zipper formed by Arg135 and Arg139 in the N-terminal helix of E2BD and Asp344 and Glu431 of one of the monomers of E3 . E2BD interacts with both E3 monomers, but the binding site is located close to the twofold axis . Thus, in agreement with earlier biochemical results, it is impossible for two molecules of E2BD to bind simultaneously to one E3 dimer . CONCLUSIONS . Combining this new structure for the E3-E2BD complex with previously determined structures of the E2 catalytic domain and the E2 lipoyl domain creates a model of the E2 core showing how the lipoyl domain can move between the active sites of E2 and E3 in the multienzyme complex. Biochemistry, 1996 Mar 12, 35(10), 3162 - 9 Subtilisin BPN' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity; Brode PF 3rd et al.; Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties . Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface . While most variants adsorb at a level very similar to that of native BPN', several variants were identified which adsorb either more or less . For surface-bound substrates we report a linear dependence between the concentration of adsorbed protease enzyme and substrate hydrolysis, similar to the linear dependence between enzyme solution concentration and hydrolysis of soluble substrates . On the basis of this knowledge we hypothesized that variants designed to adsorb at a higher level on a surface-bound peptide substrate would hydrolyze that surface-bound substrate faster . Contrary to our original expectations, the variants that adsorb more on the covalently bound peptide surface hydrolyze this substrate slower . In addition, variants of BPN' which adsorb at a lower level than native BPN' hydrolyze the surface-bound substrate faster . Enzyme adsorption and the subsequent peptide hydrolysis are altered by substituting amino acids that modify the surface charge or hydrophobicity of the native enzyme . This effect is most dramatic when the changes were made at surface-exposed sites around the binding pocket/active site of the enzyme . One mechanism that is consistent with the data is based on the relationship between the level of adsorption and the enzyme's affinity for the surface . In this mechanism weakly adsorbed enzymes are postulated to move more rapidly from site to site on the surface, thereby increasing substrate hydrolysis. J Mol Biol, 1996 Mar 8, 256(4), 667 - 75 Late events in translation initiation . Adjustment of fMet-tRNA in the ribosomal P-site; La Teana A et al.; The requirements for the adjustment of fMet-tRNA in the ribosomal P-site have been analyzed by studying the formation of fMet-puromycin in a Bacillus stearothermophilus system . The binding of fMet-tRNA to the 30 S ribosomal subunit is not drastically affected by the omission of GTP, mRNA, mRNA and GTP, or by replacing GTP with GTP analogues . The adjustment of fMet-tRNA in the P site has stricter requirements and fMet-puromycin formation occurred at its maximum rate and extent when fMet-tRNA was bound to 30 S subunits programmed with the AUG triplet or with an mRNA in the presence of GTP . Neither GTP nor the mRNA, however, were found to be essential . Omission of GTP caused only a slight reduction in the rate of fMet-puromycin formation without a significant change of the activation energy, while omission of the template resulted in a requirement for a higher activation energy . In the absence of both GTP and template, however, essentially no fMet-puromycin was formed, indicating that these components cooperate in the adjustment of the initiator tRNA in the P-site . The contribution of various structural elements of the mRNA in determining this adjustment was investigated . It was found that the codon-anticodon interaction and the filling of the ribosomal mRNA channel with a polyribonucleotide are necessary (but not sufficient singly) for the correct orientation of the initiator tRNA in the absence of GTP . The nature of the initiation triplet and the occurrence and/or the strength of the Shine-Dalgarno interaction were also found to contribute to the orientation of the bound fMet-tRNA. Riv Eur Sci Med Farmacol, 1996 Mar-Apr, 18(2), 67 - 71 {The use of BCG for the prevention of recurrence of superficial bladder tumors: 10 year's experience}; Sampalmieri G; The aim of this study was evaluate the usefulness of intravescical administration of Bacillus of Calmette Guerin (BCG) in patients with superficial bladder cancer . The BCG, an immunostimulating agent, has been introduced in routine clinical use; according to the international literature, it is reported to offer better result than the above-mentioned drugs . A retrospective analysis was made of 210 patients who had undergone a complete TUR for superficial bladder tumor . The irritative symptoms and systemic effects are acceptables considering the benefit of the BCG . The results are of great interest and encourage us to continue this study to confirm therapeutic strength. Appl Biochem Biotechnol, 1996 Mar, 56(3), 311 - 24 A novel synthesis method for cyclodextrins from maltose in water-organic solvent systems; Morita T et al.; A novel enzymatic synthesis method of cyclodextrin (CD) from low-mol-wt maltose using cyclomaltodextrin glucanotransferase (CGTase) from Bacillus macerans has been developed in various water-organic solvent systems . A beta-CD was synthesized in a two-phase system consisting of water and cyclohexane . However, no CDs could be synthesized in an aqueous buffer solution . A maximal yield of beta-CD has been obtained at a cyclohexane content volume of 44% . This synthesis has been obtained only at low temperatures, i.e., 7 degrees C, and did not take place at 50 degrees C . In addition, various organic solvents have been used for the enzymatic synthesis of CD from maltose . Consequently, beta-CD could be synthesized in various water-organic solvent systems, e.g., cyclohexane, benzene, xylene, and chloroform, but no enzymatic reaction occurred using aliphatic n-hydrocarbon solvents such as hexane, dodecane, and hexadecane . Furthermore, alpha- and beta-CD could be synthesized in water mixture solutions using organic solvents having an alcoholic group (e.g., ethanol, propanol, butanol, and pentanol) in a wide range of the reaction temperatures, typically 7-50 degrees C . In this temperature range, alpha- and beta-CD were also formed and the maximal yield from maltose to beta-CD of approx 13% was reached in 60 h. Appl Environ Microbiol, 1996 Mar, 62(3), 1107 - 11 Characterization of a restriction-modification system of the thermotolerant methylotroph Bacillus methanolicus; Cue D et al.; We report the isolation of a restriction endonuclease, BmeTI, an isoschizomer of BclI, that recognizes the DNA sequence 5' TGATCA 3' . We also report that BmeTI sites are modified to TGm6ATCA . These findings provide the basis for devising strategies to prevent BmeTI restriction of any DNA introduced into Bacillus methanolicus. J Appl Toxicol, 1996 Mar-Apr, 16(2), 139 - 45 Hypersensitivity reactions and specific antibodies in workers exposed to industrial enzymes at a biotechnology plant; Biagini RE et al.; Thirty-six employees who produced industrial enzymes from selected strains of bacteria and fungi were evaluated by epicutaneous threshold testing and enzyme-linked immunosorbent assays (ELISA) for specific IgE and IgG antibodies . The workers complained of 'asthma- and flu-like' symptoms, which generally lessened away from work . The enzymes evaluated were: alpha-amylase (1,4-alpha-d-glucan glucanohydrolase) from Bacillus licheniformis (alpha ABl), B . subtilis formation 1 (alpha A1Bs) and B . subtilis formation 2 (alpha A2Bs); purified alpha-amylase from B . licheniformis (C alpha ABl) and A . oryzae (C alpha AAo); alkaline protease from B . licheniformis (APBl) and purified alkaline protease (CAPBl); amyloglucosidase (1,4-alpha-d-glucan glucohydrolase) from A . niger (AGAn) and purified amyloglucosidase (CAGAn) . Statistically significant increases (P > 0.05) in the proportion of workers having positive skin tests to CAPBl, AGAn and CAGAn were found . Significantly elevated (P > 0.05) mean specific IgE results were observed for C alpha AAo CAGAn and AGAn, and elevated (P > 0.05) mean specific IgGs were observed for C alpha AAo, CAGAn, AGAn, alpha A1Bs, alpha AB1 and alpha A2Bs . These results indicate that occupational exposure to some industrial enzymes can cause immediate-onset cutaneous hypersensitivity reactions, pulmonary function deficits and significantly elevated specific antibody levels . Our results are equivocal as to whether work-related respiratory and cutaneous hypersensitivity reactions are antibody mediated, as there was no statistically significant association between these reactions and specific IgE or IgG levels. Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 63 - 71 Purification of alkaline proteases from a Bacillus strain and their possible interrelationship; Kobayashi T et al.; Alkalophilic Bacillus sp . KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE) . The major protease, designated M protease, was recently purified to homogeneity and its properties were characterized . In the present study, two minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism . H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55 degrees C . N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE . Its maximum activity was observed at pH 11.0 and at 60 degrees C . The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease . The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values and at 5 degrees C . M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 11 . N protease appeared to be the autolytic product of the M and H proteases. J Clin Microbiol, 1996 Mar, 34(3), 680 - 5 U.S . hospital mycobacteriology laboratories: status and comparison with state public health department laboratories; Tokars JI et al.; In response to the resurgence of tuberculosis, the Centers for Disease Control and Prevention recommended the use of certain mycobacteriology laboratory methods to improve the accuracy of diagnosis and/or minimize times to complete specimen processing . A study to determine the extent to which these recommended methods were being used in hospital laboratories was needed . In 1992, a survey was mailed to infection control and laboratory personnel at 1,076 hospitals with > or = 100 beds to determine the mycobacterial laboratory services being performed, the methods being used, the number of specimens being processed, and the times to completion during 1991 . In 1995, a 20% sample of hospital laboratories that responded to the initial questionnaire was resurveyed . Responses to the 1992 survey were received from personnel at 756 (70%) hospitals representing 750 laboratories . Among laboratories performing the services, the use of recommended methods was as follows: fluorochrome stain for acid-fast bacillus microscopy (47%); radiometric methods for primary culture (29%); rapid (radiometric methods, use of nucleic acid probes, high-performance liquid chromatography, or gas-liquid chromatography) methods for identification of Mycobacterium tuberculosis (59%); and radiometric methods for drug susceptibility testing (55%) . Reported times to complete specimen processing were shortest for laboratories that used recommended methods and longest for hospitals that referred specimens to outside laboratories . Only 46% of surveyed laboratories performed at least the minimal number of mycobacterial cultures (20/week) deemed necessary to maintain competence . Among 145 laboratories that performed the services and were resurveyed in 1995, use of recommended techniques increased from 44 to 73% for acid-fast bacillus microscopy, from 27 to 37% for primary culture, from 59 to 88% for M . tuberculosis identification, and from 55 to 75% for drug susceptibility testing . These changes were associated with reductions in reported specimen turnaround times . Use of the methods recommended by the Centers for Disease Control and Prevention increased at the resurveyed hospital mycobacteriology laboratories between 1991 and 1995 . However, continued efforts are needed to increase the use of recommended methods at moderate- and high-volume laboratories, encourage referral of specimens from low-volume laboratories, and transmit results rapidly from all laboratories. AIDS, 1996 Mar, 10(3), 299 - 309 An epidemiological study of tuberculosis and HIV infection in Tanzania, 1991-1993; Chum HJ et al.; OBJECTIVE: In Tanzania during the past 6 years reported tuberculosis (TB) cases have nearly doubled, with proportionately much greater increases in smear-negative and extrapulmonary cases compared with smear-positive cases . At the same time, HIV infection has become widespread throughout the country . This survey was undertaken in order to study the association of TB and HIV and to determine the impact of HIV on present and future TB cases in Tanzania . METHODS: The survey design provided for HIV testing of a representative country-wide sample of approximately one-sixth of all new and relapse cases registered between January 1991 and December 1993, with linkage to demographic, clinical and bacteriological data for these cases . HIV surveillance data were used for comparison purposes . RESULTS: A total of 6928 TB cases from all of the country's 20 mainland regions were tested . The overall HIV seroprevalence was 32% . Both crude and adjusted odds ratios (OR) for HIV infection were higher in women, those aged 25-44 years, urban residents, cases of smear-negative and extrapulmonary disease, and persons with a bacille Calmette-Guerin (BCG) vaccination scar . The age-and sex-adjusted relative risk for HIV infection in TB patients compared to blood donors in the same regions was 7.1 (95% confidence interval, 6.6-7.5), and was significantly higher among those aged 25-34 years . Of 3360 patients with bacteriological culture results 46% were culture-positive for Mycobacterium tuberculosis . Drug susceptibility tests were performed on 1164 isolates with the overall rate of drug resistance of 6.2% . Rates of initial resistance were low in both HIV-positive (4%) and HIV-negative (5.8%) patients . Rates of acquired resistance were higher (19% overall) and did not vary significantly by HIV serostatus . Initial combined resistance to both isoniazid and rifampicin was uncommon (0.4%) as was monoresistance to rifampicin (0.3%) . CONCLUSIONS: The higher OR for women and young adults reflect the higher rates of HIV infection in those populations . The finding that smear-positive relapse cases were no more likely to have HIV infection than new smear-positive cases suggests that the treatment regimen for new cases is effective in HIV-associated TB . The low rates of both initial and acquired drug resistance in HIV-positive patients is further evidence of adequacy of treatment . The higher relative risk for HIV infection among patients aged 25-34 years suggests increased HIV-related TB transmission . Finally, it is estimated that approximately two-thirds of the increase in the rate of smear-positive tuberculosis in the country can be directly attributed to HIV infectionPIP: The number of tuberculosis (TB) cases reported in Tanzania during the past six years has nearly doubled . Concurrently, HIV infection has become widespread throughout the country . This survey was conducted to study the association between TB and HIV, and to determine the impact of HIV upon present and future TB cases in the country . The survey design provided for HIV testing of a representative country-wide sample of approximately 17% of all new and relapse TB cases registered between January 1991 and December 1993 . 6928 TB cases were tested from all of the country's mainland regions to find an overall HIV seroprevalence of 32% . Both crude and adjusted odds ratios for HIV infection were higher in women, those aged 25-44 years, urban residents, cases of smear-negative and extrapulmonary disease, and persons with a BCG vaccination scar . The age- and sex-adjusted relative risk for HIV infection in TB patients compared to blood donors in the same regions was 7.1, and was significantly higher among those aged 25-34 years . 46% of the 3360 patients with bacteriological culture results were culture-positive for Mycobacterium tuberculosis . Drug susceptibility tests were performed on 1164 isolates with the overall drug resistance rate of 6.2% . Rates of initial resistance were 4% among HIV-positive patients and 5.8% among HIV-negative patients . There was a 19% overall rate of acquired resistance which did not vary significantly by HIV serostatus . Initial combined resistance to both isoniazid and rifampicin was 0.4%; there was a 0.3% monoresistance to rifampicin . The authors estimate that approximately two-thirds of the increase in the rate of smear-positive TB in Tanzania can be directly attributed to HIV infection . FEMS Microbiol Lett, 1996 Mar 1, 136(3), 325 - 8 Variable numbers of rRNA gene operons in Bacillus cereus strains; Johansen T et al.; Ribosomal RNA operon organisation was analysed in two Bacillus cereus strains of different chromosome size, ATCC 10987 (5.4 Mb) and F0837/76 (2.4 Mb) . We estimated that there were twelve and nine copies of the rRNA operons in these two strains, respectively . In B . cereus ATCC 10987 six rRNA operons were less than 10 kb apart, while in B . cereus F0837/76 four rRNA operons were similarly clustered . The origin of replication was located in the vicinity of a rRNA operon in both strains. J Appl Bacteriol, 1996 Mar, 80(3), 259 - 65 Biological indicators for low temperature steam and formaldehyde sterilization: investigation of the effect of change in temperature and formaldehyde concentration on spores of Bacillus stearothermophilus NCIMB 8224; Wright AM et al.; Five strains of Bacillus stearothermophilus have been studied to identify a spore strain to be used as a biological indicator organism for low temperature steam and formaldehyde sterilization . Three strains gave poor reproducibility of batch size and growth index and were discarded . The other two strains gave good reproducibility with a high growth index and gave rise to linear survivor curves when exposed to 5% aqueous formaldehyde . However, only NCIMB 8224 sporulates on a simpler medium and as it was the most resistant to formaldehyde, it was further studied . Tests were carried out in a modified miniclave and factors studied included temperature of the steam and formaldehyde concentration . All studies confirmed the suitability of this strain as a biological indicator organism. J Biochem (Tokyo), 1996 Mar, 119(3), 435 - 40 Cytochrome P450foxy, a catalytically self-sufficient fatty acid hydroxylase of the fungus Fusarium oxysporum; Nakayama N et al.; We have purified membrane-bound fatty acid (omega-1-omega-3) hydroxylase of the fungus Fusarium oxysporum MT-811 and found that the activity depends on a single polypeptide with an apparent M(r) value of 118,000 . The purified hydroxylase exhibited spectral characteristics of cytochrome P450 (P450), and could catalyze the hydroxylation without the aid of any other proteinaceous components, such as NADPH-P450 reductase . These properties of the fungal hydroxylase are the same as those of bacterial P450BM3 of Bacillus megaterium, a catalytically self-sufficient fused protein of P450 and its reductase . Other properties of the two enzymes, such as molecular weight, high catalytic turnover, and the regiospecificity of the hydroxylating position, were also almost identical . Further, the fungal hydroxylase reacted with the antibody to P450BM3 . It was thus shown that the fungal fatty acid hydroxylase reacted with the antibody to P450BM3 . It was thus shown that the fungal fatty acid hydroxylase structurally and functionally bears a close resemblance to P450BM3, although it is membrane-bound, unlike the bacterial counterpart . On the other hand, a unique phenomenon was found with the fungal hydroxylase: its NADPH-cytochrome c- or NADPH-menadione reductase activity was enhanced enormously upon binding of its substrate (fatty acid) . This appears to be the first instance in which the reactivity of P450 reductase against an artificial electron acceptor was enhanced by the binding of the substrate (to be hydroxylated) to P450 . These results raise interesting questions about the molecular evolution of P450 . Here we term the fungal hydroxylase cytochrome P450foxy. Proteins, 1996 Mar, 24(3), 370 - 8 Experimental and theoretical study of electrostatic effects on the isoelectric pH and the pKa of the catalytic residue His-102 of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase); Bastyns K et al.; Barnase, the guanine specific ribonuclease of Bacillus amyloliquefaciens, was subjected to mutations in order to alter the electrostatic properties of the enzyme . Ser-85 was mutated into Glu with the goal to introduce an extra charge in the neighborhood of His-102 . A double mutation (Ser-85-Glu and Asp-86-Asn) was introduced with the same purpose but without altering the global charge of the enzyme . A similar set of mutations was made using Asp at position 85 . For all mutants the pI was determined using the technique of isoelectric focusing and calculated on the basis of the Tanford-Kirkwood theory . When Glu was used to replace Ser-85, the correlation between the experimental and the calculated values was perfect . However, in the Ser-85-Asp mutant, the experimental pI drop is bigger than the calculated one, and in the double mutant (Ser-85-Asp and Asp-86-Asn) the compensation is not achieved . The effect of the mutations on the pKa of His-102 can be determined from the pH dependence of the kcat/KM for the hydrolysis of dinucleotides, e.g., GpC . The effect can also be calculated using the the method of Honig . In this case the agreement is very good for the Glu-mutants and the single Asp-mutant, but less for the double Asp-mutant . The global stability of the Asp-mutants is, however, the same as the wild type, as shown by stability studies using urea denaturation . Molecular dynamics calculations, however, show that in the double Asp-mutant His-102 (H+) swings out of its pocket to make a hydrogen bridge with Gin-104 which should cause an additional pKa rise . The effect of the Glu-mutations was also tested on all the kinetic parameters for GpC and the cyclic intermediate G > p at pH 6.5, for RNA at pH 8.0, and for poly(A) at pH 6.2 . The effect of the mutations is rather limited for the dinucleotide and the cyclic intermediate, but a strong increase of the KM is observed in the case of the single mutant (extra negative charge) with polymeric substrates . These results indicate that the extra negative charge has a strong destabilizing effect on the binding of the polymeric substrates in the ground state and the transition state complex . A comparison with the structure of bound tetranucleotides (Buckle, A.M . and Fersht, A.R., Biochemistry 33:1644-1653, 1994) shows that the extra negative charge points towards the P2 site. Immunology, 1996 Mar, 87(3), 339 - 42 Stimulation of human peripheral blood mononuclear cells with live Mycobacterium bovis BCG activates cytolytic CD8+ T cells in vitro; Turner J et al.; Experimental data have shown that Mycobacterium tuberculosis can survive within the host cell and in doing so may release secreted antigen into the endogenous antigen-processing pathway . If mycobacterial antigen can gain access to MHC class I molecules then CD8+ T cells may play a role in host defence against M . tuberculosis infection . To identify whether there is a role for the CD8+ T cell in mycobacterial infection we have stimulated peripheral blood mononuclear cells (PBMC) from bacillus Calmette-Guerin (BCG) vaccinated individuals with live M . bovis BCG . The activation state of the T cells was established by staining for the interleukin-2 (IL-2) receptor (CD25), HLA-DR or the transferrin receptor (CD71) . Using FACScan analysis we have shown that, in vitro, live M . bovis BCG activates significantly more CD8+ T cells in comparison to the soluble antigen purified protein derivative (PPD) . In addition, live M . bovis BCG activates more CD8+ T cells than a non-viable preparation of the same M . bovis BCG following irradiation . The function of the activated CD8+ T cells was addressed using positively selected cells in a cytotoxic T-cell assay . CD8+ T cells isolated from a 7-day M . bovis BCG-stimulated PBMC culture were shown to be cytolytic against target cells infected with live M . bovis BCG, dead M . bovis BCG, and to a lesser extent, PPD . These results suggest that CD8+ T cells may be activated by stimulation with live mycobacteria, and that this subset can play a cytolytic role in the immune response to mycobacterial infections. Zhonghua Yi Xue Za Zhi, 1996 Mar, 76(3), 203 - 6 {Role of nitric oxide in immunological liver injury in mice}; Wang G et al.; OBJECTIVE: To study the role of nitric oxide (NO) in mouse immunological liver injury . METHODS: Injection of either bacille calmette gurein (BCG) or lipopolysaccharide (LPS) alone in mice was used to induce moderate increase of plasma NO level and liver damage were seen after the RESULTS: Administration of LPS following BCG injection resulted in remarkable elevation of plasma NO level and severe liver damage . The elevation of NO level and liver damage induced by BCG or BCG + LPS were not affected by administration of L-arginine, and substrate of NO synthase . Inhibition of NO synthase by NG-monomethyl-arginine (NMMA) decreased the elevated plasma NO without effect on the elevated plasma GPT and GOT levels induced by BCG injection . The BCG + LPS induced elevation of plasma GPT and GOT levels were more pronounced after NO production was inhibited by NMMA treatment . The action of NMMA mentioned above was partially reversed by simultaneous administration of L-arginine . CONCLUSION: NO plays a protective action against liver injury induced by BCG + LPS in mice. Genet Anal, 1996 Mar, 12(5-6), 185 - 95 Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity; Aliotta JM et al.; A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity . The dominant species of the Bst DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels . The Bst polA gene that codes for Bst polymerase I was cloned and sequenced . Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs found in E . coli DNA polymerase I were missing in Bst DNA polymerase I . This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme . Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polymerase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain . These exonuclease assays demonstrated that Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonuclease activity but lacked any detectable 3'-->5' proofreading exonuclease activity . The lack of 3'-->5' exonuclease function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermostability at the expense of proofreading activity. Mem Inst Oswaldo Cruz, 1996 Mar-Apr, 91(2), 231 - 7 Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp . medellin; Orduz S et al.; Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt) subsp . medellin (Btmed) was performed and compared to solubilized crystal proteins of isolates 1884 of B . thuringiensis subsp . israelensis (Bti) and isolate PG-14 of B . thuringiensis subsp . morrisoni (Btm) . In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI) was lower than at alkaline pH . The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3 Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested . Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3 . An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200) . Three main protein peaks were observed on the chromatogram . The first peak had two main proteins that migrate between 90 to 100 kDa . These proteins are apparently not common to other Bt strains isolated to date . The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively . Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae . Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity . These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae . When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein . These data suggest that Btmed could be an alternative bacterium for mosquito control programs in case mosquito larval resistance emerges to Bti toxic proteins. Prikl Biokhim Mikrobiol, 1996 Mar-Apr, 32(2), 247 - 50 {Inhibition of phagolysis in a Bacillus thuringiensis culture by chitosan}; Kochkina ZM et al.; The ability of chitosan (poly-D-glucosamine) and two chitosan salts to prevent the phagolysis of Bacillus thuringiensis subsp . galleriae strain 1-97 was studied . Chitosan and its salts inhibited the productive infection caused by two nonrelated bacteriophages 1-97A and 1-97B and suppressed the culture lysis upon spontaneous prophage induction . The efficiency of inhibition depended on the chitosan concentration, medium composition, and bacteriophage type. Mikrobiologiia, 1996 Mar-Apr, 65(2), 235 - 40 {Scanning tunneling and electron microscopy of parasporal crystals in Bacillus thuringiensis}; Solontsov IL et al.; Comparison of the forms of parasporal inclusions in Bacillus thuringiensis subsp . kurstaki and B . thuringiensis subsp . tenebrionis under a scanning electron microscope showed that, in addition to bipyramidal and cuboid crystals, cells of the subspecies kurstaki are able to form crystals taking the shape of flat parallelopipeds typical of the subspecies tenebrionis . The inclusions in the subspecies tenebrionis vary in size and electron density . Examination of the structure of crystals in B . thuringiensis subsp . tenebrionis under a scanning tunneling microscope showed that delta-endotoxins in the crystals are assembled in globules 5.0 nm in diameter and 1.0 nm in height . The globules are connected by its bases and form chains . The subunits of tetragonal crystals in the subspecies tenebrionis are vertically oriented with respect to the chain axis, as was revealed by other methods for subunits of bipyramidal crystals of the subspecies kurstaki. J Am Mosq Control Assoc, 1996 Mar, 12(1), 33 - 8 Effect of tadpole shrimp, Triops longicaudatus, (Notostraca: Triopsidae), on the efficacy of the microbial control agent Bacillus thuringiensis var . israelensis in experimental microcosms; Fry-O'Brien LL et al.; Laboratory bioassays using Culex quinquefasciatus larvae evaluated the effect of tadpole shrimp on the persistence of Bacillus thuringiensis var . israelensis (B.t.i.) in water collected from the surface of field microcosms . Time elapsed since B.t.i . treatment, as well as presence or absence of soil and tadpole shrimp, affected B.t.i . persistence at the water surface in 15- and 30-cm total depths . The presence of tadpole shrimp slowed the natural decline in B.t.i . effectiveness over time, but this effect was depressed when soil was present . Tadpole shrimp foraged throughout the water column and stirred up the substrate, keeping more particles in suspension at the surface than in microcosms with no shrimp, in microcosms with water depths of 15 and 30 cm. Int J Urol, 1996 Mar, 3(2), 98 - 100; discussion 101 Local toxicity patterns associated with intravesical bacillus Calmette-Guérin: a Southwest Oncology Group Study; Berry DL et al.; BACKGROUND: While the efficacy of bacillus Calmette-Guerin (BCG) immunotherapy has been demonstrated, the relative benefit, given a seemingly high incidence and severity of toxicities, remains an issue . Adequate understanding and management of toxicities can maximize the safety of the treatment and enable the administration of required doses of BCG intravesical therapy . METHODS: All week-to-week symptoms recorded for the 143 immunotherapy-naive participants assigned to the BCG arm of SWOG-8216, BCG vs . Doxorubicin in Superficial Bladder Cancer were analyzed in order to document the pattern of toxicities in the first six week induction course of intravesical BCG treatments . The statistical analysis consisted of fitting logistic regression models to these data for the probability of irritative bladder symptoms (IBS) . RESULTS: In the optimal model, the probability of IBS depends only on whether there was IBS associated with the previous treatment, and not on which treatment . The estimated probability of having IBS when there were no IBS associated with the previous instillation is 0.136, whereas the estimated probability of having IBS when there was IBS associated with the previous instillation is 0.689 . CONCLUSIONS: Irritative bladder symptoms are unlikely in the week after the first intravesical BCG treatment . Once a patient experiences IBS, he or she is more likely to have IBS with the next and subsequent treatments . Clinicians can use the findings of this analysis when informing their patients about the treatment course and when making decisions about continuing treatments. Anticancer Res, 1996 Mar-Apr, 16(2), 979 - 80 Treatment of superficial bladder cancer with intravesical perfusion of rIL-2: a follow-up study; Ferlazzo G et al.; We have recently reported the results of a phase I study on the intravesical perfusion of recombinant interleukin-2 in patients with superficial bladder cancer . The treatment feasible with mild toxic effects, especially compared with the treatment using TUR and instillations of Bacillus Calmette-Guerin . The follow-up of these phase I study patients was continued for another twelve months . During this period, cytoscopy and cytological examination of cells washed from bladder were performed every four months . The results showed that three out of 9 patients relapsed, over a period ranging from 6 to 20 months after treatment . All these data clearly confirm that the intravesical perfusion of rIL-2 is feasible, safe and should be an effective and nontoxic treatment of patients with superficial bladder cancer. An Med Interna, 1996 Mar, 13(3), 111 - 4 {Epidemiological study of tuberculosis in the health area of Santiago de Compostella in 1992, 1993 and 1994}; Salgueiro M et al.; An exhaustive search for the clinical records of patients diagnosed with tuberculous disease was done in the hospitals of the area under study, which involves 392,000 population . During the years 1992, 1993 and 1994 . There were included: 1) patients who had positive bacilloscopy and/or positive Lowenstein's culture in any specimen: 2) patients younger than 35-years-old who had pleural effusion, significant Mantoux and adenosine deaminase (ADA) over 47 U/I in the pleural effusion . In total 814 patients remained in the study with an average age of 38.39(19.39 DE) in 1992, 39.02 (20.04 DE) in 1993, and 34.1 years-old (19.2 DE) in 1994, with extreme ages of 2 months and 87 years-old . The incidence/100,000 H was: in 1992: 67.86, in 1993: 66.58 and in 1994: 73.2 . The contagious forms incidence/100,000 H was: 1.5 in 1992 and 1993; and 1.79 in 1994 . The hospital mortality incidence/100,000 H was 2.04 in 1992, 2.30 in 1993 and 2.6 in 1994 . We conclude that tuberculosis is endemic in our area with moderately high and stationary incidence. J Exp Med, 1996 Mar 1, 183(3), 1045 - 51 Evidence inconsistent with a role for the Bcg gene (Nramp1) in resistance of mice to infection with virulent Mycobacterium tuberculosis; Medina E et al.; The superior resistance of some strains of mice over others to infection with certain intracellular pathogens, including the vaccine strain of Mycobacterium bovis, bacillus Calmette Guerin (BCG), is determined by a gene associated with a small segment of chromosome 1 designated by Ity/Lsh/Bcg locus, referred to here as the Bcg locus . DBA/2 mice containing the dominant resistant allele of the Bcg gene (Bcgr), major histocompatibility complex-compatible BALB/c mice containing the recessive susceptible allele (Bcgs), and congenic C.D2-N20 Bcgr, which are genetically the same as BALB/c mice except for possessing a small piece of DBA/2 chromosome 1 containing the Bcg locus, were used to determine whether the Bcg gene determines resistance to infection with the virulent H37Rv strain of Mycobacterium tuberculosis (Mtb) . According to the survival times of Bcgr and Bcgs mice infected via either the intravenous or respiratory route, Bcgr mice proved much less, rather than more, resistant to Mtb infection than Bcgs mice . Shorter survival times of Bcgr mice were associated with an inferior capacity to control Mtb growth in their lungs and to retard the development of Mtb-induced pathology in this organ . Resistance to Mtb infection was a dominant trait in the F1 progeny of Bcgr and Bcgs mice . The results show that resistance to Mtb is not determined by the resistance allele of the Bcg gene nor by the recently isolated candidate Bcg gene Nramp1, located in the Bcg locus. Int J Lepr Other Mycobact Dis, 1996 Mar, 64(1), 26 - 36 Immunotherapy of far-advanced lepromatous leprosy patients with low-dose convit vaccine along with multidrug therapy (Calcutta trial); Majumder V et al.; This report describes a promising mode of treatment of lepromin-unresponsive, far-advanced, lepromatous (LL) leprosy patients with antileprosy vaccines as an adjunct to multidrug therapy (MDT) . The Trial Groups included 50 highly bacilliferous, lepromin-negative, untreated LL patients . They were given MDT for 2 years . Of them, 30 patients were administered a mixed antileprosy vaccine containing killed Mycobacterium leprae of human origin plus M . bovis BCG . The remaining 20 patients were given M . bovis BCG . Depending on the severity of lepromin unresponsiveness, they were given one to six inoculations at 3-month intervals . Another 20 similar LL patients were taken in the Control Group . They were given only MDT for 2 years . From the start of the study, all patients belonging to the Trial and Control Groups were followed every 3 months for clinical, bacteriological and immunological outcomes . Within 2 years all 50 patients of the Trial Groups and 19 of the 20 patients of the Control Group became clinically inactive and bacteriologically negative . However, the clinical cure and the falls of the bacterial and morphological indexes were much faster in those patients receiving the mixed vaccine therapy than in those patients who were given BCG plus MDT or only MDT . The immunological improvements in the patients of the Trial and Control Groups were assessed by: a) lepromin testing at the beginning of the study and at 3-month intervals and also by b) the in vitro leukocyte migration inhibition (LMI) test at both the beginning and end of the study . As the patients were given more and more vaccinations, the incidence of lepromin conversion increased, more so in the patients receiving the mixed vaccine . Thus, 63%, 15% and 5% of the patients became lepromin positive in those patients receiving the mixed vaccine, BCG, and MDT only, respectively . Lamentably, the vaccine-induced lepromin positivity was temporary and faded away within several months . At the beginning of the study, the LMI test against specific M . leprae antigen was negative in all patients of both the Trial and Control Groups . After the end of the chemo-immunotherapy schedule, the LMI test became positive in 50% and 20% of LL patients receiving the mixed vaccine and BCG, respectively . None of the Control Group could show LMI positivity after completion of the MDT schedule . These results show that treatment of LL patients with the mixed vaccine and MDT could quickly reverse the clinical course of the disease, remove immunologic anergy in some patients, and induce a rapid decrease in the bacterial load in them. J Bacteriol, 1996 Mar, 178(6), 1742 - 9 Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a delta cyo delta cyd strain complemented by genes from Bacillus firmus OF4; Sturr MG et al.; Escherichia coli GK100, with deletions in the operons encoding its two terminal oxidases, cytochrome bo and ctyochrome bd, was complemented for growth on succinate by a recombinant plasmid (pMS100) containing a 3.4-kb region of DNA from alkaliphilic Bacillus firmus OF4 . The complementing DNA was predicted to encode five proteins, but neither sequence analysis nor complementation experiments with subclones provided insight into the basis for the complementation . Cytochrome difference spectra of everted membrane vesicles from the transformed strain had characteristics of a cytochrome bd spectrum but with features different from those observed for alkaliphile membranes . To determine the bacterial source and identity of the structural genes for the cytochrome bd in the transformed mutant, the complex was extracted and partially purified . On sodium dodecyl sulfate-polyacrylamide gels, two polypeptides were resolved from the preparation, 43 (subunit I) and 27 (subunit II) kDa . An internal peptide from subunit I was sequenced, and it yielded the same primary sequence as is found in positions 496 to 510 of E . coli appC . Consistent with the microsequencing results pMS100 failed to complement a triple mutant of E . coli carrying a deletion in appB as well as in the cyo and cyd loci . The deduced sequence of AppBC had been predicted to be very similar to the sequence of CydAB (J . Dassa et al., Mol . Gen . Genet . 229:341-352, 1991) but this is the first demonstration that the former is indeed a cytochrome bd terminal oxidase . The enzyme catalyzed oxygen uptake coupled to quinol or N,N,N',N'-tetramethyl-p-phenylenediamine oxidation, and the activity was sensitive to cyanide . No cross-reactivity to subunit-specific polyclonal antibodies directed against the two individual subunits of cyd-encoded cytochrome bd was detected . Since this is the second cytochrome bd discovered in E . coli, it is proposed that the two complexes be designated cytochrome bd-I (cydAB-encoded enzyme) and cytochrome bd-II (appBC-encoded enzyme) . In addition, cbdAB is suggested as a more appropriate gene designation for cytochrome bd than either appBC or cyxAB. J Bacteriol, 1996 Mar, 178(6), 1600 - 6 Cyclization reaction catalyzed by branching enzyme; Takata H et al.; The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on amylose was analyzed . The enzyme reduced the molecular size of amylose without increasing the reducing power . This result could not be explained by the normal branching reaction model . When the product was treated with glucoamylase (an exo++-type amylase), a resistant component remained . The glucoamylase-resistant component was easily digested by an endo-type alpha-amylase or by isoamylase plus glucoamylase . These results suggested that the glucoamylase-resistant component was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages . In other words, it was suggested that branching enzyme catalyzed cyclization of the alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic linkage, thereby forming two smaller molecules . Mass spectrometry also supported the cyclic nature of the product. Nat Med, 1996 Mar, 2(3), 334 - 7 Auxotrophic vaccines for tuberculosis; Guleria I et al.; Tuberculosis is responsible for the deaths of more people each year than any other single infectious disease, with greater than 7 million new cases and 2 million deaths annually . It remains the largest attributable cause of death in HIV-infected individuals, responsible for 32% of deaths of HIV-infected individuals in Africa . The only currently available vaccine for tuberculosis, bacille Calmette-Guerin (BCG) is the most widely used vaccine in the world, being administered to approximately 100 million children each year . Although untoward effects were not seen in several studies of HIV-seropositive children, the safety of live attenuated BCG vaccine in HIV-positive adults remains unknown and a matter of some concern . To obviate potential adverse affects of BCG vaccines in immunodeficient individuals, we have studied five auxotrophic strains of BCG produced by insertional mutagenesis for safety in administration to mice with severe combined immunodeficiency disease (SCID), and for protection in a susceptible strain of mice . The results indicate that viable BCG could no longer be detected in mice receiving the auxotrophs after 16-32 weeks, and that infected SCID mice survived for at least 230 days . In contrast, all SCID mice succumbed within eight weeks to conventional BCG vaccine . When susceptible BALB/c mice were immunized with auxotrophs and subsequently challenged with virulent Mycobacterium tuberculosis, several of the auxotrophs produced comparable protection against intravenous and intratracheal challenge with M . tuberculosis relative to conventional BCG . These results suggest that auxotrophic strains of BCG represent a potentially safe and useful vaccine against tuberculosis for populations at risk for HIV. J Trauma, 1996 Mar, 40(3 Suppl), S135 - 9 Multiple organ injuries and failures caused by shock and reperfusion after gunshot wounds; Fu X et al.; Experiments were performed to observe the changes of multiple system organ failure (MSOF) and gut barrier function caused by shock and reperfusion after gunshot wounds . Eighteen dogs were divided randomly into two groups . In the experimental group, the dogs were subjected to 60 minutes of shock (40mm Hg), followed by reinfusion of shed blood after hindlimb gunshot wounds . In the control group, the dogs experienced pure gunshot wounds without shock and reperfusion . The results showed that dogs in the experimental group developed multiple system organ injuries or failures compared with the control group . The levels of malondialdehyde (MDA) values in plasma were significantly elevated in the experimental group when compared with preinjury and the control group . Gut flora disorder, bacillus intestinalis overgrowth, and bacterial translocation occurred in the experimental group . The pathological results support the gut barrier function injury . The results indicated that pure gunshot wounds do not easily injure gut barrier function and produce MSOF . Gunshot wounds with shock and reperfusion are capable of causing gut flora disorder, bacillus intestinalis overgrowth, and lead to bacterial translocation, furthermore causing MSOF . Although fluid resuscitation is a potential treatment modality, pathogenically, it can lead to MSOF. Ophthalmology, 1996 Mar, 103(3), 390 - 7 Useful visual outcomes after treatment of Bacillus cereus endophthalmitis; Foster RE et al.; PURPOSE: Bacillus cereus endophthalmitis occurring after penetrating ocular trauma has been almost always associated with a poor visual outcome . The purpose of our study was to review and report patients who had useful visual acuity outcomes . METHODS: The study group consisted of five patients from a single medical center with penetrating ocular trauma and endophthalmitis caused by B . cereus . The study population was derived from a review of the microbiology records, clinical records, and operative reports of patients with culture-proven, post-traumatic endophthalmitis over a 15-year period . Patients were only included if the final visual acuity outcomes were 20/200 or better . RESULTS: All five patients had penetrating ocular injuries, and four patients had a retained intraocular foreign body . Endophthalmitis was diagnosed preoperatively in three patients and intraoperatively in two patients . All patients underwent pars plana vitrectomy and injection of intravitreal and periocular antibiotics . Postoperatively, a rhegmatogenous retinal detachment developed in three patients between 4 weeks and 12 months after the injury (average, 19 weeks); all retinal detachments were reattached with additional vitreoretinal surgery . Final postoperative visual acuities were 20/200 (two patients), 20/30 (one patient), and 20/25 (two patients) . The postoperative follow-up time interval ranged from 12 months to 30 months (average, 19.2 months) . CONCLUSION: The current series adds further support to the observation that certain eyes with post-traumatic B . cereus endophthalmitis may be associated with preservation of anatomic integrity and restoration of useful visual acuity. Curr Genet, 1996 Mar, 29(4), 404 - 9 Secretion of an enzymatically active Trichoderma harzianum endochitinase by Saccharomyces cerevisiae; Draborg H et al.; A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENCI) from a Trichoderma harzianum cDNA library by expression in yeast . The 1473-bp chi1 cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide . The deduced ENCI amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 from Bacillus circulans . The T . harzianum endochitinase I was secreted into the culture medium by the yeast Saccharomyces cerevisiae in a functionally active form . The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 degrees C. Mol Gen Genet, 1996 Feb 25, 250(3), 259 - 66 Contributions of XylR CcpA and cre to diauxic growth of Bacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose; Schmiedel D et al.; Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose . We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion . xylA is 13-fold repressed during growth on glucose . Induction occurs at the onset of the lag phase after glucose is consumed . Inactivation of xylR yields a two-fold increase in expression of xylA on glucose . Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold . When xylR and cre are inactivated together a residual two-fold repression of xylA is found . Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40 min . In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars . In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously. Proc Natl Acad Sci U S A, 1996 Feb 20, 93(4), 1434 - 8 The human lysozyme promoter directs reporter gene expression to activated myelomonocytic cells in transgenic mice; Clarke S et al.; The 5' region of the human lysozyme gene from -3500 to +25 was fused to a chloramphenicol acetyltransferase (CAT) reporter gene and three transgenic founder mice were obtained . All three transgenic lines showed the same pattern of CAT enzyme expression in adult mouse tissues that was consistent with the targeting of elicited, activated macrophages in tissues and developing and elicited granulocytes . In normal mice high CAT enzyme activity was found in the spleen, lung, and thymus, tissues rich in phagocytically active cells, but not in many other tissues, such as the gut and muscle, which contain resident macrophages . Cultured resident peritoneal macrophages and cells elicited 18 hr (granulocytes) and 4 days (macrophages) after injection of sterile thioglycollate broth expressed CAT activity . Bacillus Calmette-Guerin infection of transgenic mice resulted in CAT enzyme expression in the liver, which contained macrophage-rich granulomas, whereas the liver of uninfected mice did not have any detectable CAT enzyme activity . Although the Paneth cells of the small intestine in both human and mouse produce lysozyme, the CAT gene, under the control of the human lysozyme promoter, was not expressed in the mouse small intestine . These results indicate that the human lysozyme promoter region may be used to direct expression of genes to activated mouse myeloid cells. J Biol Chem, 1996 Feb 16, 271(7), 3445 - 52 Mechanistic studies on thiaminase I . Overexpression and identification of the active site nucleophile; Costello CA et al.; Thiaminase I (EC 2.5.1.2) catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles . Here we report the sequencing of a thiaminase I clone from Bacillus thiaminolyticus, the overexpression of the cloned gene in Escherichia coli, and the purification and characterization of the enzyme . Recombinant thiaminase I functions as a monomer with a Km for thiamin of 3.7 +/- 0.6 microM and a kcat of 34 s-1 . Electrospray ionization Fourier-transform mass spectrometry identified a single sequencing error and demonstrated heterogeneity, finding molecular weights of 42,127, 42,198, and 42,255 due to added Ala and Gly-Ala at the amino terminus . Similar analysis of the 4-amino-2-methyl-6-chloropyrimidine inactivated enzyme indicated that the active site nucleophile involved in catalysis of the substitution reaction is located between Pro79 and Thr177 . Subsequent cysteine-specific labeling and site-directed mutagenesis identified Cys113 as the active site nucleophile. Eur J Biochem, 1996 Feb 15, 236(1), 301 - 8 Mechanistic studies on the 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis; Ploux O et al.; The reaction mechanism of 8-amino-7-oxopelargonate (8-amino-7-oxononoate) synthase from Bacillus sphaericus, an enzyme dependent on pyridoxal 5'-phosphate (pyridoxal-P), which catalyzes the condensation of L-alanine with pimeloyl-CoA, the second step of biotin biosynthesis, has been studied . To facilitate mechanistic studies, an improved over-expression system in Escherichia coli, and a new continuous spectrophotometric assay for 8-amino-7-oxopelargonate synthase were designed . In order to discriminate between the two plausible basic mechanisms that can be put forth for this enzyme, that is: (a) formation of the pyridoxal-P-stabilized carbanion by abstraction of the C2-H proton of the alanine-pyridoxal-P aldimine, followed by acylation and decarboxylation, and (b) formation of the carbanion by decarboxylation followed by acylation, the fate of the C2-H proton of alanine during the course of the reaction has been examined using 1H NMR . Spectra of the 8-amino-7-oxopelargonate formed using either L-{2-2H}alanine in H2O or L-alanine in D2O, showed that the C2-H proton of alanine is lost during the reaction and that the C8-H proton of 8-amino-7-oxopelargonate is derived from the solvent, a result that is only consistent with mechanism (a) . Furthermore 8-amino-7-oxopelargonate synthase catalyzes, in the absence of pimeloyl-CoA, the stereospecific exchange, with retention of configuration, of the C2-H proton of L-alanine with the solvent protons . Similarly, 8-amino-7-oxopelargonate synthase catalyzes the exchange of the C8-H proton of 8-amino-7-oxopelargonate . In addition to these exchange reactions, 8-amino-7-oxopelargonate synthase catalyzes an abortive transamination yielding an inactive pyridoxamine 5'-phosphate (pyridoxamine-P) form of 8-amino-7-oxopelargonate synthase and pyruvate . Kinetic analysis gave a rate constant of kexch . = 1.8 min-1 for the exchange reaction which is 10 times lower than the catalytic constant and a rate constant of ktrans . = 0.11 h-1 for the transamination . Finally deuterium kinetic isotope effects (KIE) were measured at position 2 of L-alanine (DV = 1.3) and in D2O (D2OV = 4.0) . The magnitudes of the KIE are consistent with a partially rate-limiting abstraction of the C2-H proton of alanine and a partially rate-limiting reprotonation step . Taken together, all these results show that 8-amino-7-oxopelargonate synthase utilizes mechanism (a) . 8-Amino-7-oxopelargonate synthase and 5-aminolevulinate synthase, which has also been shown to use mechanism (a), belong to a class of pyridoxal-P-dependent enzymes that catalyze the formation of alpha-oxoamines . Based on the fact that all these alpha-oxoamine synthases share strong sequence similarities, we postulate that they also share the same reaction mechanism. Presse Med, 1996 Feb 10, 25(5), 199 - 201 {Visceral localizations of cat-scratch disease in an immunocompetent patient}; Bouchard O et al.; Locoregional expression of cat scratch disease is well known, but despite advances in microbiology over the last 10 years leading to the description of two new bacteria (Afipia felis, Bartonella henselae) the infective agent responsible for cat scratch syndrome remains unknown . Until the 80s, only one systemic disease was attributed to infection with a germ in the Bartonella genus: trench fever . With the onset of the AIDS epidemic, new clinical syndromes caused by Bartonella bacteria have been described: bacillary angiomatosis, hepatic peliosis, cases of recurrent septicemia, cases of endocarditis, etc . More recently, atypical forms of cat scratch disease including systemic diseases have been reported in immunocompetent subjects . Although quite rare (1% of the cases), such types of expression can raise questions as to diagnosis both in terms of clinical signs and in terms of bacteriological findings . Clinical and experimental data do not provide a clear direction for treatment but would suggest that prolonged use of aminoglycosides is useful. Schweiz Med Wochenschr, 1996 Feb 10, 126(6), 207 - 13 {Detection of Bartonella (Rochalimaea) henselae/B . quintana by polymerase chain reaction (PCR)}; Goldenberger D et al.; Bartonella (Rochalimaea) henselae and/or B . quintana are the causative agents of a variety of infections such as trench fever, bacillary angiomatosis, septicemia, peliosis hepatis and endocarditis . Recently, B . henselae has been identified as a major cause of cat scratch disease . Diagnosis of such infections is based on clinical information, histopathology, culture and serology . However, none of these methods alone is sufficiently sensitive or specific . We have used the PCR to search for DNA specific for B . henselae/B . quintana in 33 clinical samples and in 6 controls . In comparison with clinical data and histopathology, PCR was extremely specific (100%) and reasonably sensitive (61%) . Possible explanations for the limited sensitivity of PCR are discussed . We conclude that PCR provides a useful adjunct for the diagnosis of infections caused by B . henselae and B . quintana. Biochim Biophys Acta, 1996 Feb 7, 1305(1-2), 44 - 8 Cloning of a cluster of chitinase genes from Aeromonas sp . No . 10S-24; Shiro M et al.; A gene encoding chitinases from Aeromonas sp . No . 10S-24 was cloned into Escherichia coli DH5 alpha using pUC19, and its nucleotides were sequenced . The chitinase gene was clustered in ORFs (open reading frame) 1 to 4, in a 8-kb fragment of DNA . ORF-1 consisted of 1608 bp encoding 535 amino acid residues, and ORF-2 consisted of 1425 bp encoding 474 amino acid residues . ORF-3 was 1617 bp long and encodes a protein consisting of 538 amino acids . ORF-4 encodes 287 amino acids of the N-terminal region . The amino acid sequences of ORF-1 and ORF-3 share sequence homology with chitinase D from Bacillus circulans, and chitinase A and B from Streptomyces lividans . The amino acid sequence of ORF-2 shared sequence homology with chitinase II from Aeromonas sp . No . 10S-24, and chitinase from Saccharopolyspora erythraea . A region of the sequence starting from Ala-28 of the amino acid sequence of ORF-3 coincided with the N-terminal amino acid sequence of chitinase III from Aeromonas sp . No . 10S-24. Biochemistry, 1996 Feb 6, 35(5), 1581 - 8 Cooperative interactions of RNA and thiostrepton antibiotic with two domains of ribosomal protein L11; Xing Y et al.; Ribosomal protein L11 interacts with a 58-nucleotide domain of large subunit ribosomal RNA; both the protein and its RNA target have been highly conserved . The antibiotic thiostrepton recognizes the same RNA domain, and binds to the ribosome cooperatively with L11 . Experiments presented here show that RNA recognition and thiostrepton cooperativity can be attributed to C- and N-terminal domains of L11, respectively . Under trypsin digestion conditions that degrade Bacillus stearothermophilus L11 to small fragments, the target RNA protects the C-terminal 77 residues from digestion, and thiostrepton and RNA in combination protect the entire protein . A 76-residue C-terminal fragment of L11 was overexpressed and shown to fold into a stable structure binding ribosomal RNA with essentially the same properties as full-length L11 . An L11.thiostrepton.RNA complex was 100-200-fold more stable than expected on the basis of L11-RNA and thiostrepton-RNA binding affinities; similar measurements with the C-terminal fragment detected no cooperativity with thiostrepton . L11 function is thus more complex than simple interaction with ribosomal RNA; we suggest that thiostrepton mimics some ribosomal component or factor that normally interacts with the L11 N-terminal domain. Biochem Biophys Res Commun, 1996 Feb 6, 219(1), 6 - 13 High-yield synthesis of N-acetyllactosamine by regioselective transglycosylation; Vetere A et al.; The synthesis of N-acetyllactosamine (D-Galp beta 1-4D-GlcpNAc) with very low contamination of its isomer N-acetyllactosamine (D-Galp beta 1-6D-GlcpNAc) was obtained by use of regioselective transglycosylation activity of beta-galactosidase from Bacillus circulans using lactose as the donor of D-Galp and D-GlcpNAc as the acceptor . The reaction was conducted at 15 degrees C and at pH 5.0 . The incubation time was considerably reduced and the yield improved 100% with respect to the best results so far described in the literature. J Biol Chem, 1996 Feb 2, 271(5), 2390 - 6 Role of domain II, loop 2 residues of Bacillus thuringiensis CryIAb delta-endotoxin in reversible and irreversible binding to Manduca sexta and Heliothis virescens; Rajamohan F et al.; Site-directed mutagenesis was used to examine the role of domain II, loop 2 residues, 368RRPFNIGI375, of Bacillus thuringiensis insecticidal protein CryIAb . Alanine substitution of residues 368RRP370, called B4, abolished potency toward Manduca sexta and Heliothis virescens, and the loss of toxicity was correlated directly to substantially reduced binding affinity to brush-border membrane vesicles (BBMV) prepared from the target insect midguts . These results indicated that these positive charges might be essential to orient the toxin to midgut receptor molecule(s) . The role of residue Phe371 of CryIAb toxin to M . sexta was investigated by substituting a series of residues at this position . Irreversible binding and toxicity were affected significantly by hydrophilic, aliphatic, and smaller side-chain residues such as Cys, Val, Leu, and Ser but not by Tyr or Trp . A hydrophobic aromatic side-chain residue at position 371 was therefore essential for irreversible binding of CryIAb toxin in M . sexta . The role of residues 370PFNIGI375 of CryIAb toxin on H . virescens was also examined . Mutants D2 (deletion of residues 370-375), G374A (alanine substitution of Gly374), and I375A had reduced toxicity to H . virescens . In contrast to our findings with M . sexta, the reduction in toxicity of these mutants was correlated directly with loss of initial binding to H . virescens BBMV, indicating that these residues perform functionally distinct roles in binding and toxicity to different insects . In ligand blots, CryIAb recognized a major 210-kDa peptide in M . sexta BBMV and a 170-kDa peptide in H . virescens BBMV. Science, 1996 Feb 2, 271(5249), 642 - 5 Catalysis of amide proton exchange by the molecular chaperones GroEL and SecB; Zahn R et al.; Hydrogen-deuterium exchange of 39 amide protons of Bacillus amyloliquefaciens ribonuclease (barnase) was analyzed by two-dimensional nuclear magnetic resonance in the presence of micromolar concentrations of the molecular chaperones GroEL and SecB . Both chaperones bound to native barnase under physiological conditions and catalyzed exchange of deeply buried amide protons with solvent . Such exchange required complete unfolding of barnase, which occurred in the complex with the chaperones . Subsequent collapse of unfolded barnase to the exchange-protected folding intermediate was markedly slowed in the presence of GroEL or SecB . Thus, both chaperones have the potential to correct misfolding in proteins by annealing. J Mol Biol, 1996 Feb 2, 255(4), 628 - 40 Functional significance of loops in the receptor binding domain of Bacillus thuringiensis CryIIIA delta-endotoxin; Wu SJ et al.; Analysis of the three surface loops in domain II of Bacillus thuringiensis CryIIIA delta-endotoxin has been carried out to assess their role in receptor binding and toxicity . Site-directed mutagenesis was used to convert loop residues to alanine and the mutant proteins were analyzed for structural stability, toxicity to beetle larvae (Tenebrio molitor), binding to receptors on T . molitor brush border membrane vesicles (Tm-BBMV) and insertion into BBMV, as measured by irreversible membrane receptor binding . This study demonstrates the functional significance of loops for binding and insertion . Alanine replacements in loop I resulted in disruption of receptor binding or structural instability . The double mutation, Y350A, Y351A, could be suppressed by replacing a nearby R345 with alanine, and the resultant mutant protein also showed reduced receptor binding . Substitution of N353 and D354 in loop I with alanine residues caused the loss of binding ability and toxicity . A loop II double mutant, P412A, S413A, had no effect on binding or toxicity . A block mutation of loop III residues to alanine had the effect of reducing receptor binding while concomitantly increasing toxicity by 2.4-fold . We compared this up-mutant to wild-type toxin in each step of physiological processing of protoxin: solubility, proteolytic activation, and insertion into the Tm-BBMV . The loop III block mutant showed increased membrane insertion, but was similar to wild-type toxin in other parameters . These results reveal that loop I and loop III in domain II of CryIIIA delta-endotoxin are involved in receptor binding . In addition, the direct correlation between toxicity and irreversible binding of the loop III block mutant (despite the indirect relationship to reversible binding) suggests that loop III may play a role in membrane insertion. Arch Bronconeumol, 1996 Feb, 32(2), 100 - 2 {Miliary dissemination with pulmonary involvement secondary to intravesical immunotherapy with Calmette-Guérin bacillus}; Paredes Arranz C et al.; We describe the case of a 67-years-old man who underwent transurethral resection and immunotherapy with Calmette-Guerin bacillus solution (CGB) for superficial transitional carcinoma of the bladder . After a series of intravesical irrigations with CGB, the patient developed fever, asthenia and persistent anorexia and was hospitalized . After testing he was diagnosed of miliary tuberculosis due to CGB and died in spite of tuberculostatic therapy . Hematogenous dissemination, a rare but serious complication of vesical irrigation with CGB, is thought to be more common than previously suspected . It should be suspected in all patients receiving CGB when a compatible clinical picture presents . An understanding of this complication and early establishment of specific treatment is the only wat to improve prognosis. Immunol Cell Biol, 1996 Feb, 74(1), 32 - 7 T cell responses to Mycobacterium bovis in red deer, a large animal model for tuberculosis; Cross ML et al.; Red deer (Cervus elaphus) represent an appropriate large animal model to study the immunology of tuberculosis, being naturally susceptible to Mycobacterium bovis infection . Cell-mediated immune responses were investigated in deer displaying protective- or disease-type reactions, following immunization with M . bovis bacille Calmette-Guerin (BCG) or infection with virulent M . bovis, respectively . T cell responses were measured as antigen-dependent cell proliferation and production of T cell growth factor (TCGF) following in vitro stimulation with M . bovis antigens (live or heat-killed BCG, or PPD) . T cells from immunized deer proliferated less in response to soluble denatured culture antigen (purified protein derivative, PPD) than to particulate BCG, although there were no differences in the magnitude of these responses between the two groups of animals . Cells derived from immunized deer produced less TCGF than cells from infected deer when stimulated with PPD in vitro, although responses to BCG antigens were similar between the two groups . The majority of TCGF activity was neutralized by anti-IL-2 antibodies, regardless of the animal group or source of antigen used for in vitro stimulation . After 7 days in vitro culture with antigen, blast cells staining positively for alpha beta (CD4, CD8) and gamma delta T cell receptors were recorded . The majority of blasts were CD4+, although in immunized deer fewer CD4+ blasts were produced following in vitro stimulation with PPD than with BCG antigens . These results, together with previous reports from our laboratory, represent the only detailed examinations of T cell responses to M . bovis in this naturally-susceptible ruminant species. Microbiology, 1996 Feb, 142 ( Pt 2), 315 - 20 Regulatory sequences of two flagellin genes in Bacillus thuringiensis subsp . alesti; Ankarloo J et al.; Two highly homologous flagellin genes, flaA and flaB, are expressed in Bacillus thuringiensis subsp . alesti . Both genes were found to be transcribed during vegetative growth . After the onset of sporulation, transcripts could not be detected . Both flaA and flaB were found to be transcribed from sigma70-like promoters . In addition, the 3'-terminal half of flaA was cloned and sequenced, completing the sequence of flaA. Acta Virol, 1996 Feb, 40(1), 5 - 8 Detection of sugarcane bacilliform virus in sugarcane germplasm; Viswanathan R et al.; Sugarcane bacilliform virus (SCBV), a badnavirus was found in sugarcane genotypes of Saccharum officinarum L., S . barberi Jesw., S . sinense Roxb., S . robustum Brand and Jesw., and Saccharum hybrids . In most of the suspected genotypes the virus was found associated with clear foliar symptoms . However, certain symptom-free clones carried the virus too . The virus was detected by immuno-electron microscopy (IEM) and enzyme-linked immunosorbent assay (ELISA) in suspected clones . The virions measured about 108-118 x 20-21 nm in size . The virus was serologically closely related to another badnavirus, banana streak virus (BSV) . Virus titer was low in most of the genotypes . However, a close correlation between symptoms expression and virus titer existed in some genotypes. Biotechnol Appl Biochem, 1996 Feb, 23 ( Pt 1), 13 - 8 Effect of heterogeneity of hydrophobic moieties on surface activity of lichenysin A, a lipopeptide biosurfactant from Bacillus licheniformis BAS50; Yakimov MM et al.; Lichenysin A, a surface-active lipopeptide produced by Bacillus licheniformis, strain BAS50, contains longchain beta-hydroxy fatty acids . Regulation of the synthesis of fatty acids and beta-hydroxy fatty acids was studied by modifying the culture medium . Addition of branched-chain alpha-amino acids to the medium caused similar changes to both cellular fatty acid and to beta-hydroxy fatty acid composition in the lipophilic part of lichenysin A . Production of lichenysin A was enhanced about two- and four-fold by addition of L-glutamic acid and L-asparagine respectively . It is suggested that these amino acids may be involved in the control of lipopeptide formation . Elucidation of the structure-function relationship of surface-active lipopeptides by analysis of the activities of structurally characterized compounds is discussed . Fractions of lichenysin A with branched beta-hydroxy acids in the lipid tail demonstrated lower surface-tension activity than the fractions of lichenysin A having straight beta-hydroxy acids . The presence of a lichenysin A fraction with beta-hydroxymyristic {(C14)n} acid residues appears to have an important influence on the surface activity of a mixture of lichenysins A. Neuropathol Appl Neurobiol, 1996 Feb, 22(1), 44 - 53 A comparison of leucocyte responses to heat-killed bacillus Calmette-Guérin in different CNS compartments; Matyszak MK et al.; We have previously shown that heat-killed bacillus Calmette-Guerin (BCG) injected into the CNS parenchyma does not produce a typical delayed-type hypersensitivity (DTH) response {23} . In this paper we have compared the initial leucocyte response in the CNS parenchyma, ventricles and skin to gain insight into the mechanisms by which the DTH response in the CNS might be controlled . We have found that 10(5) organisms of heat-killed BCG injected into either the CNS parenchyma or the lateral ventricles produced a rapid neutrophil response at the site of the injection, which was comparable with that in the skin . The neutrophil response resolved within the first week . Unlike the neutrophil response, the mononuclear phagocyte response in the CNS parenchyma was much smaller than that seen in the ventricles and the skin and it resolved within 4 weeks . Furthermore, the myelomonocytic response in the CNS parenchyma failed to clear the BCG . The acute inflammatory response in the choroid plexus/ventricles and skin developed with a similar time-course into a typical DTH response . After the first week, lesions at these two sites were composed predominantly of T-cells and macrophages . DTH lesions were still detected at both sites after 6 weeks . The failure of the immune system to recognize foreign antigens sequestrated in the CNS parenchyma may have significant implications especially in studies of inflammatory responses in the CNS of unknown origin. Biochem Mol Biol Int, 1996 Feb, 38(2), 223 - 30 Induction and carbon catabolite repression in the biosynthesis of beta-amylase by Bacillus megaterium B6; Ray RR et al.; Biosynthesis of extracellular beta-amylase in Bacillus megaterium B6 was induced by starch, although maltodextrin was found to be the actual inducer . Amongst the carbon sources tested, glucose was found to be the most potent repressor and when added exogenously to a starch-induced culture it brought about an immediate fall in enzyme synthesis through carbon catabolite repression . The repression was not overcome after the fall of glucose concentration in the culture medium below a critical level . This "catabolite repression" exerted by glucose was partially relieved by exogenous cyclic guanosine monophosphate (CGMP) and its dibutyryl derivative . On the other hand, guanosine monophosphate (GMP) was found to restore the original extent of enzyme synthesis in glucose repressed cells. Antimicrob Agents Chemother, 1996 Feb, 40(2), 506 - 8 The erythromycin resistance gene from the Bacteroides conjugal transposon Tcr Emr 7853 is nearly identical to ermG from Bacillus sphaericus; Cooper AJ et al.; Tcr Emr 7853, from Bacteroides thetaiotaomicron 7853, is a large chromosomal conjugative transposon which encodes resistance to both tetracycline (Tcr) and erythromycin (Emr) . The erythromycin resistance gene of Tcr Emr 7853 did not cross-hybridize with ermF, the Emr gene found on previously studied Bacteroides regular and conjugative transposons . We have cloned and sequenced the erythromycin resistance gene from Tcr Emr 7853 . The DNA sequence of this gene was 99.6% identical to that of ermG from Bacillus sphaericus. Antimicrob Agents Chemother, 1996 Feb, 40(2), 400 - 7 Luciferase in vivo expression technology: use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice; Hickey MJ et al.; The development of new drugs and vaccines directed against Mycobacterium tuberculosis is severely impeded by the slow growth of this organism and the need to work under stringent biosafety conditions . These difficulties pose considerable obstacles when animal studies with M . tuberculosis are performed . We investigated whether a novel approach termed luciferase in vivo expression, using an enhanced luciferase-expressing mycobacterial strain, could be used to evaluate antimycobacterial activity in mice . Vectors that expressed firefly luciferase (lux gene) at high levels in the bacillus Calmette-Gu-erin (BCG) strain of Mycobacterium bovis were constructed for use in vivo . One recombinant BCG reporter strain (rBCG-lux) was selected for high-level expression of the lux gene product and for its ability to replicate in mice . Methodology to monitor in vivo growth of the rBCG-lux reporter strain in mice by direct assay of luciferase luminescence in organ homogenates was developed . The utility of this approach for assessing the in vivo efficacies of antimycobacterial compounds was evaluated . The activities of standard antimycobacterial drugs were directly apparent in mice infected with the rBCG-lux reporter strain by statistically significant reductions in spleen luminescence . In addition, antimycobacterial immunity was also evident in BCG-immunized mice, in which suppression of rBCG-lux growth in comparison with that in naive mice was clearly observed . The use of luciferase in vivo expression for the in vivo evaluation of antimycobacterial activity compared favorably with standard CFU determinations in terms of time, labor, expense, and statistical significance but permitted the evaluation of antimycobacterial drugs and immunity in mice in 7 days or less . Thus, the use of this technology can greatly accelerate the process of evaluation of antibiotics and immunogens in animal models for the slowly growing pathogenic mycobacteria. Mol Microbiol, 1996 Feb, 19(3), 495 - 503 Heterologous expression and self-assembly of the S-layer protein SbsA of Bacillus stearothermophilus in Escherichia coli; Kuen B et al.; The cell surface of Bacillus stearothermophilus PV72 is covered by a regular surface layer (S-layer) composed of single species of protein, SbsA, with a molecular weight of 130,000 . Recently, the sequence of the corresponding gene (sbsA) has been determined . The SbsA coding region including the signal sequence was cloned as a polymerase chain reaction (PCR) product into a low-copy-number vector under the transcriptional control of the lambda pL promoter . Expression of sbsA was shown to be thermally inducible from the resulting vector pBK4 in a strain of Escherichia coli expressing the lambda cl857 from the chromosome . As shown by ultrathin sectioning of whole cells and immunogold labelling using SbsA- specific antibodies, expression of sbsA in E . coli led to accumulation of sheet-like self-assembling products of the protein in the cytoplasm . No SbsA protein was detected either in the periplasm or in the supernatant fractions . Long-term expression of sbsA from pBK4, including in the late stationary phase, did not lead to degradation of SbsA. Anat Rec, 1996 Feb, 244(2), 235 - 45 Omental milky spots in the local immune response in the peritoneal cavity of rats; Van Vugt E et al.; BACKGROUND: Milky spots have been described as reactive structures, their classification varying from inflamed or haematopoietic tissue to lymphoid organs . In this study we investigated the reactivity of the milky spots in the omentum of rats upon induction of a chronic immune response in the peritoneal cavity . METHODS: At different time points after intraperitoneal administration of Bacillus Calmette-Guerin (BCG), a peritoneal lavage was made, and the omentum and the draining parathymic lymph nodes were taken out . The cellular composition of these tissues was examined on the light microscopic level, using a panel of monoclonal antibodies, and also by electron microscopy . RESULTS: During the first 4 months after administering BCG, the number and size of the milky spots increased enormously . Separate macrophage, T, and B cell areas were formed, but interdigitating cells and follicular dendritic cells were not observed . The number of cells in the peritoneal cavity also increased, and the cellular composition showed a strong similarity with that of the milky spots . Especially during the onset of the experiment, most bacteria were observed in the macrophages in the milky spots rather than in the draining lymph nodes . A cellular immune response was observed in the parathymic lymph nodes but not in the milky spots . CONCLUSIONS: Milky spots, either unstimulated or stimulated, should be classified as perivascular infiltrates . They play a role in the initial clearance of bacteria from the peritoneal cavity . Although the large increase in cell number is predominantly caused by immigration of cells, the results do support the role of milky spots as a site for local proliferation and maturation of especially macrophages and also B cells . The obtained data, however, do not support the earlier made assumption that milky spots function as a secondary lymphoid organ in the peritoneal cavity. J Clin Microbiol, 1996 Feb, 34(2), 460 - 2 Primary invasive cutaneous Microsporum canis infections in immunocompromised patients; King D et al.; Two cases of primary invasive cutaneous infections caused by the zoophilic dermatophytic species Microsporum canis are presented . The first case occurred in a liver transplant recipient who was receiving immunosuppressive therapy . Multiple erythematous papules were seen on both legs, and a biopsy revealed invasive fungal hyphae . The second case was diagnosed in a human immunodeficiency virus-positive individual with a CD4 lymphocyte count of 81 mm3 . Raised red nodules were seen on her scalp and face . Histopathology was consistent with bacillary angiomatosis, and in addition, invasive septate hyphae were observed . The two strains recovered from the biopsy specimens from both individuals had colony morphologies consistent with that of M . canis, but it was difficult to induce production of macroconidia . These cases serve to increase the awareness of this unusual infection, reinforce the need for cultures, and raise some interesting questions about the potential virulence of this dermatophyte species. J Clin Microbiol, 1996 Feb, 34(2), 355 - 7 Cost and time savings following introduction of rejection criteria for clinical specimens; Morris AJ et al.; We have evaluated the yield of several tests and have instituted specimen rejection criteria to reduce costs and save time . For a 12-month period, we recorded the reduction of these tests and calculated the resultant cost and time savings . Seven changes were analyzed: not performing fungal or mycobacterial (acid-fast bacillus) cultures on cerebrospinal fluid (CSF) specimens from patients without known immunosuppression when chemistry and cell count are normal; not performing routine stool culture or ovum and parasite examination on specimens from patients in the hospital for > 3 days; not culturing endotracheal suction aspirates when no organisms or > 10 squamous epithelial cells are present; discontinuing broth cultures on all specimens except for tissue, continuous ambulatory peritoneal dialysis fluid, and CSF from patients with shunts; and eliminating bacterial antigen tests . For each test, the number not performed (n), reagent savings, and technologist time saved, respectively, were as follows: CSF fungal culture, 267, $999, and 67 h; CSF acid-fast bacillus culture, 275, $1,662, and 124 h; stool cultures, 320, $2,991, and 98 h; ovum and parasite examinations, 216, $525, and 108 h; endotracheal suction aspirate cultures, 1,505, $4,447, and 306 h; broth cultures, 5,218, $4,931, and 80 h; and bacterial antigen tests, 2,598, $2,293, and 299 h . Overall, 5,181 tests were rejected and 5,218 broth cultures were omitted . Achievable savings were $28,000 in reagent costs and 1,082 h of technologist time . In conclusion, rejecting specimens of proven low yield saves reagent costs and, more importantly, saves technologist time . This time can be spent on specimens having greater diagnostic utility. Eur Respir J, 1996 Feb, 9(2), 288 - 92 Evaluation of a western blot serum test for the diagnosis of Mycobacterium tuberculosis infection; Rovatti E et al.; This study was designed to evaluate the possibility of monitoring Mycobacterium tuberculosis infection using a serological assay . A discriminant score comprising antigen fractions of 38, 28, 24 and 19 kDa, identified in western blots using the Mycobacterium bovis bacille Calmette-Guerin (BCG) A60 antigen complex was established in a sample of 57 purified protein derivative (PPD)-negative and 47 PPD-positive individuals . It was then tested in a group of 140 subjects undergoing BCG vaccination as a model of tuberculosis complex infection and in a group of human immunodeficiency virus (HIV)-infected individuals as a model of cell-mediated immunodeficiency-related risk of tuberculosis . The discriminant score identified 57 out of 57 (100%) PPD-positives and none (0%) of the 47 PPD-negatives . In the BCG vaccinated subjects, 1.4% tested positive before vaccination and 90% after vaccination . In the HIV-positive subjects, 90% of the PPD-positive and 5% of the PPD-negative subjects had a positive score . This study suggests that the western blot discriminant score is an accurate test to survey M . tuberculosis infection in serum samples. Wei Sheng Wu Xue Bao, 1996 Feb, 36(1), 69 - 72 {Construction of shuttle vector containing delta-endotoxin gene of Bacillus thuringiensis}; Sun L et al.; The Bacillus thuringiensis toxin gene CryIA(c) was inserted into the shuttle vector pBE-2 to construct pAMY for expressing the B.t . gene in both Gram-negative and -positive bacterial systems . pAMY was introduced into wild type Bacillus cereus, B.brevis and B.subtilis by electroporation . Transformants containing delta-endotoxin gene produced proteins reacted with B.t . crystal protein antibody . Upon biological toxicity tests, the transformants gave a mortality of 100% against Ostrinia furnacilis, 58.8% against Heliothis armigera and 100% against Heliothis assulta . The ability of promoting plant growth of the original strains is retained. Tuber Lung Dis, 1996 Feb, 77(1), 52 - 8 Tuberculin reactivity in Bacillus Calmette-Guérin vaccinated subjects; Miret-Cuadras P et al.; SETTING: The Centre for Prevention and Control of Tuberculosis in Barcelona, Spain, where the staff appointed to Training Centers are examined . AIMS: To check for tuberculin sensitivity due to Bacillus Calmette-Guerin (BCG) vaccine and ascertain its duration . METHOD: We compared the results of a tuberculin test (TT) on vaccinated and non-vaccinated subjects . The induration diameter and the time elapsed between BCG vaccination and the TT were determined . RESULTS: Of the 2424 vaccinated subjects, 1489 (61.4%) reacted to TT (> or = 5 mm) and of the 3135 non-vaccinated, 905 (28.9%) reacted, a significant difference . Of 1978 subjects vaccinated between 6 and 14 years of age, 63.3% were TT reactors, compared to 23.9% of the 1948 non-vaccinated . Induration diameters > or = 15 mm amounted to 11% for vaccinated subjects and 8% for those not vaccinated, a significant difference . The time from vaccination to TT was 13-25 years . Of the 446 subjects vaccinated at birth, 237 were reactors (53.1%); of the 887 non-vaccinated subjects of the same age, 154 (17.4%) reacted . Reactors > or = 15 mm amounted to 40 (9%) for vaccinated subjects and 46 for non-vaccinated (5.2%), a significant difference . The time elapsed between vaccination and TT was 20-25 years . For 124 vaccinated subjects with a previous negative TT, a second test was positive for 87 (70.2%), and for 257 non-vaccinated it was positive for 64 (24.9%) . The difference is due to a booster effect . CONCLUSIONS: BCG vaccination at birth and for school age children causes a reactivity to tuberculin which persists for 20 to 25 years . An induration diameter of > or = 15 mm does not exclude a vaccinal origin . For vaccinated subjects with a previous negative TT, it is necessary to exclude the booster effect. Semin Urol Oncol, 1996 Feb, 14(1 Suppl 1), 36 - 40 Nursing implications in the management of superficial bladder cancer; Games J; Intravesical therapy is preferred over systemic treatment for superficial bladder cancer . Four agents commonly used are thiotepa, doxorubicin, mitomycin, and bacille Calmette-Guerin . A thorough understanding of the unique methods involved in the instillation of these drugs and the special patient-management procedures associated with their use is critical for nursing personnel . Occupational Safety and Health Administration guidelines exist to ensure safe handling of intravesical agents . Nurses are urged to take precautions to limit their exposure to these potentially hazardous drugs . For example, bacille Calmette-Guerin, a live virus, represents a biohazard and extreme caution is required in all aspects of handling, particularly in its disposal . Because of the potential complications associated with intravesical therapy, a significant responsibility of the nurses who administer it is patient education . Patients need to know what to expect during the treatment process as well as how to recognize the signs and symptoms of complications . In addition to gaining a thorough knowledge of the techniques involved in the intravesical treatment of superficial bladder cancer, nurses can also serve their patients better by using sensitive to the unique psychosocial problems they face. Semin Urol Oncol, 1996 Feb, 14(1 Suppl 1), 17 - 22 Intravesical chemotherapy versus immunotherapy for superficial bladder cancer; Huben RP; The decision to treat superficial bladder cancer with intravesical therapy should be predicated primarily on disease stage and grade as well as the patient's clinical history . Once the decision to proceed with intravesical therapy has been made, the clinician must select the appropriate agent . Several agents are available and the choice of which agent to use should be based on careful consideration of the potential benefit of a given drug versus its inherent risk of complications . The first drug to be administered intravesically, thiotepa is an alkylating agent used as first-line treatment for low-grade lesions; it has limited use against higher-grade tumors or carcinoma in situ . In addition, the low molecular weight of thiotepa results in significant systemic absorption, which often results in myelosuppression . Mitomycin, also an alkylating agent, has shown significant activity as both first-line therapy and in patients with recurrent disease . Unlike thiotepa, mitomycin has a relatively high molecular weight, and the incidence of significant bladder absorption and systemic side effects is low . Doxorubicin, which also possesses a high molecular weight, is used intravesically against superficial bladder cancer more frequently in Europe and Japan than in the United States . Immunotherapy with bacille Calmette-Guerin is the treatment of choice for carcinoma in situ and high-grade T1 lesions . It is associated with the highest incidence of both minor and major adverse reactions, however, and its use should be tempered by its potential toxicity. Semin Urol Oncol, 1996 Feb, 14(1 Suppl 1), 1 - 9 Diagnosis and treatment of superficial bladder cancer: an update; Crawford ED; It is expected that approximately 50,500 new cases of bladder cancer will be diagnosed in 1995 . In addition, approximately 10,000 people are expected to die of this disease during the year . An 11% increase in incidence observed from 1957 to 1987 was accompanied by a 19.4% decrease in the death rate in American males . This rate change may be attributable to stage migration to more localized disease as well as increased survival owing to improved treatment . Transurethral resection is standard first-line treatment for transitional cell carcinoma of the bladder . Intravesical therapy with cytotoxic agents or immunomodulators has become an important element in the management of patients with superficial bladder cancer at risk for disease recurrence and progression . Study results confirm the efficacy of both mitomycin and bacille Calmette-Guerin when administered intravesically . Comparative studies of these two agents are warranted to further clarify their respective roles in the prophylaxis of recurrences of superficial bladder cancer . Studies should enroll large numbers of patients, particularly those with high-grade lesions at significant risk of recurrence and progression . Current research involves evaluation methods of optimizing the efficacy of intravesical chemotherapy . High drug concentrations, hyperthermia, and combination modalities are a few new avenues being studied . In addition, new drugs like bropirimine, interferons, mycobacteria-associated antigens, and keyhole-limpet hemocyanin are being explored. Int J Food Microbiol, 1996 Feb, 29(1), 1 - 10 Influence of pH on heat resistance of Bacillus licheniformis in buffer and homogenised foods; Palop A et al.; The influence of pH of heating menstruum (McIlvaine buffer) on the heat resistance of Bacillus licheniformis was investigated and compared with the heat resistance in homogenised tomato and asparagus at pH 7 and 4 in a wide range of temperatures . Heat resistance was in all mestrua smaller at acid pH . At 99 degrees C and pH 4, heat resistance was 1/20 lower than at pH 7 . However, the magnitude of this effect decreased as heat treatment temperatures were increased almost disappearing at 120 degrees C . z values increased from 6.85 at pH 7, to 10.75 at pH 4 . At 99 degrees C the effect of pH on heat resistance was constant along the range of pH's tested . The increase of one pH unit increased D99 by 180% . At pH 7 and 4, heat resistance was the same in buffer as in tomato and asparagus homogenates at all temperatures tested . The diminishing influence of the acidification of some foods on the heat resistance of B . licheniformis sterilisation temperatures should be taken into account when a raise in temperature is considered to shorten the duration of heat processes. Biokhimiia, 1996 Feb, 61(2), 357 - 68 {Bacillus cereus chitinases: isolation and characteristics}; Trachuk LA et al.; Three chitinases (M(r) = 68, 52 and 38 kDa) have been isolated from the cultural filtrate of Bacillus cereus strain VKPM B-6838 by stepwise hydrophobic chromatography on butyl-Toyopearl and gel filtration on Superdex 75 (FPLC) . The chitinases are stable in the pH range 4-10 and have the same pH optimum of activity . The 68 and 38 kDa enzymes display the highest activity at 60 degrees C . while the 52 kDa chitinase-at 50 degrees C . In contrast with the 68- and 52 kDa enzymes, the 38 kDa chitinase hydrolyzes not only colloidal but also "crystalline" chitin and chitosan . None of the chitinases hydrolyzes chitobiose . The N-terminal sequences (10 amino acids) of the 52 and 38 kDa chitinases do not reveal structural similarity between themselves or to other known bacterial chitinases . The 68 kDa chitinase is not immunologically related to the 52 and 38 kDa enzymes . The results obtained suggest that the 68, 52 and 38 kDa chitinase are the unique proteins. J Neurooncol, 1996 Feb, 27(2), 179 - 89 Autologous tumor cell vaccination combined with adoptive cellular immunotherapy in patients with grade III/IV astrocytoma; Holladay FP et al.; Brain tumors are highly resistant to treatment . Their diffuse infiltrative nature and the relative inaccessibility of the brain to blood and lymph are barriers to surgical and cytotoxic treatments alike . Preclinical animal studies demonstrated that intravenously administered tumor antigen-specific T lymphocytes will reject tumors growing in the brain . Specifically activated effector T lymphocytes may be generated by in vivo immunization followed by restimulation of antigen-primed T cells with autologous tumor cells in vitro . In order to apply these findings to humans, feasibility studies of combined active immunization and specific adoptive cellular immunotherapy were performed on fifteen patients with recurrent astrocytoma . The objective was to determine whether; 1) T cells could be grown from peripheral blood of patients immunized with autologous tumor cells, and 2) whether stimulated cells could be safely readministered to patients . Patients were immunized with a combination of their own irradiated tumor cells and Bacillus of Calmette and Guerin . Two weeks later a mononuclear cell-rich fraction of blood was obtained by leukapheresis . Mononuclear cells were cultured with irradiated autologous tumor cells and interleukin-2 . Selective expansion of CD4+ and CD8+ T lymphocytes occurred . Intravenous transfer of stimulated cells to the fifteen patients on twenty-four separate occasions with or without systemic administration of interleukin-2 was tolerated with limited toxicity . The studies established the feasibility of conducting controlled studies of the anti-tumor effects of tumor antigen-specific cellular immunotherapy. Med Hypotheses, 1996 Feb, 46(2), 163 - 71 Mycobacterial infections: are the observed enigmas and paradoxes explained by immunosuppression and immunodeficiency? Maes HH, Causse JE, Maes RF. The enigmas and paradoxes observed in tuberculous patients, in Bacille Calmette-Guerin-vaccinated people and in Bacille Calmette-Guerin-treated cancer patients have been examined, in an attempt to explain them through the mechanisms of immunodeficiency and immunosuppression . A dual effect is postulated: an immunosuppression induced by the infecting mycobacteria that adds to a pre-existing or emerging state of immunodeficiency of the infected individual . The immunological cellular and humoral anergies observed at the beginning of a tuberculous therapy are usually lifted after the first two weeks of treatment . This restoration of immune responsiveness may be attributed to the destruction or to the growth inhibition of immunosuppressive mycobacteria . The observation that drugs cytocidal in vitro do not always sterilize the patients under treatment whereas bacteriostatic drugs do, may find an explanation in the dual immunosuppression induced by cytocidal drugs and mycobacteria . The fact that Bacille Calmette-Guerin applied as an immunotherapy to residual cancer has either a favorable or an unfavorable action may