Cell Transplant, 1994 Nov-Dec, 3(6), 515 - 27 A device to measure the oxygen uptake rate of attached cells: importance in bioartificial organ design; Foy BD et al.; Quantification of the dependence of cellular oxygen uptake rate (OUR) on oxygen partial pressure is useful for the design and testing of bioartificial devices which utilize cells . Thus far, this information has only been obtained from suspended cells and from cells attached to microcarriers . In this work, a device was developed to obtain the dependence of OUR on oxygen partial pressure for anchorage-dependent cells cultured in standard culture dishes . The device is placed and sealed on the top of the culture dish, and holds a Clark polarographic mini-electrode flush with the bottom surface of the device . It also houses a motor to spin a magnetic stir bar within the cell chamber to insure that the medium is well-mixed . Several characteristics of the device--such as oxygen leakage into the device chamber, electrode-lag time, and linearity of the electrode at low oxygen partial pressures--were quantified and their potential effect on the values of Vm (maximal OUR) and K0.5 (oxygen partial pressure at which OUR is half-maximal) were evaluated . Comparison of Vm and K0.5 values obtained with this device with previously published values for suspended rat hepatocytes, Bacillus cereus, and E . coli indicated that the technique provides values accurate within 30% as long as the cell under study has a K0.5 greater than approximately 1.0 mmHg . For hepatocytes cultured on 0.05 mm thickness collagen gel for 1 day (n = 4) and 3 days (n = 6), Vm was found to be 0.38 +/- 0.12 and 0.25 +/- 0.09 nmol O2/S/10(6) cells, respectively, and K0.5 was found to be 5.6 +/- 0.5 and 3.3 +/- 0.6 mmHg, respectively . This technique should aid in predicting bioreactor conditions such as flow rate, cell density, distance of cell from flow, and gas phase oxygen partial pressure which can lead to oxygen limitations . In addition, further studies of the effect of factors such as extracellular matrix composition, metabolic substrate, and drugs on the dependence of OUR on oxygen partial pressure for many anchorage-dependent cell types can be pursued with this technique. Anal Biochem, 1994 Nov 1, 222(2), 461 - 4 A turbidometric assay for phospholipase C and sphingomyelinase; Torley L et al.; We describe a simple turbidometric assay for phosphatidylcholine-specific phospholipase C (PC-PLC) (EC 3.1.4.3), phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10), and sphingomyelinase (SMase) (EC 3.1.4.12), suitable for high-volume screening using unmodified substrates . Under the conditions described, 1 to 10 U/ml of PC-PLC (Bacillus cereus) induces a rapid and continuous increase in turbidity (0.4 to 0.6 AU at 410 nm) of phosphatidylcholine vesicles (1-10 mM) that highly correlates with hydrolysis . Turbidity increases with the formation of small homogenous particles, which is enzyme and substrate dependent . Analogously, PI-PLC (1-10 U/ml) causes a continuous increase in the turbidity of PI vesicles . SMase also causes a continuous increase in PC vesicle turbidity, but unlike like the glycerol phospholipases, SMase causes a discontinuous increase in vesicles of its proper substrate sphingomyelin (SM) . After 8-15% hydrolysis, SM vesicles are converted to large heterogeneous particles permitting detection of SMase activity by visual inspection . Thus, turbidity is a useful property to monitor SMases and C-type phospholipases that cleave vesicle-forming phospholipids . Furthermore, the assay is designed for the microtiter plate format, enabling the continuous and simultaneous monitoring of up to 96 wells. J Dairy Res, 1994 Nov, 61(4), 529 - 35 Identification of Bacillus strains isolated from milk and cream with classical and nucleic acid hybridization methods; Tatzel R et al.; A total of 529 bacterial strains have been isolated from milk and cream sampled at different sites in a dairy production plant under conditions selective for aerobic sporeforming bacteria . Identification with classical methods based on morphological, physiological and biochemical criteria showed Bacillus licheniformis to be the most frequently occurring Bacillus sp . The investigation also revealed 62 unidentified strains . Classical identification methods were time consuming (3-7 d), lacked specificity and--because of their dependence on phenotypic gene expression--sometimes produced ambiguous results . Consequently, a colony hybridization method developed for the identification of B . licheniformis strains and using nonradioactive labelled 23S rRNA targeted oligonucleotide probes was also used . Identification of B . licheniformis with both classical and hybridization methods revealed diverging identification results for 70 strains. Urologe A, 1994 Nov, 33(6), 540 - 6 {Cytokine therapy of superficial bladder carcinoma . Mechanisms of action and results of therapy}; Otto T et al.; The object of immunotherapy is the elimination of tumor cells mediated by modulation of the immune system . This can be achieved by different mechanisms, i.e . cell-mediated and humorally mediated immune reactions . Immunotherapy can be classified as passive, adoptive, active and non-conventional . Most clinical experience has been gathered with unspecific active immunotherapy . Superficial bladder carcinomas can be treated by intravesical application of Bacillus Calmette-Guerin (BCG) or of different cytokines, i.e., interferons or interleukin-2 . Since there has not so far been any standard immunotherapy for superficial bladder carcinoma, the efficacy of therapy with cytokines should be evaluated in clinical studies (phase II/III) only. Biol Mass Spectrom, 1994 Nov, 23(11), 675 - 81 Rapid identification of specific mutations in the sequence of an enzyme variant produced by protein engineering using high-performance liquid chromatographic/fast atom bombardment mass spectrometric techniques; van Dongen WD et al.; Unknown, specific mutations in the sequence of an enzyme variant (a Bacillus subtilisin protease) produced by protein engineering were identified using High-performance Liquid Chromatographic/Fast Atom Bombardment Mass Spectrometric (HPLC/FAB MS) techniques . The variant and the highly homologous wild-type enzyme were treated with CNBr followed by tryptic digestion . The resulting peptides were analysed using HPLC/frit FAB MS . The peptides with molecular masses beyond the range of the HPLC/MS system under the chosen scanning conditions were collected using HPLC and subsequently analysed 'off-line' using static FAB MS . This procedure allowed the complete amino acid sequence determination of the variant protease using the known amino acid sequence of the wild-type enzyme as reference. Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 358 - 63 Direct high-level secretion into the culture medium of tuna growth hormone in biologically active form by Bacillus brevis; Sagiya Y et al.; The characteristic features of the Bacillus brevis system developed by us are very high productivity of heterologous proteins and very low extracellular proteinase activity . However, the production level of eucaryotic proteins with this system was generally one or two orders of magnitude lower than that of bacterial proteins . Therefore, we have explored methods for increasing the production efficiency as to animal proteins . Signal peptide modification was found to be very effective for high-level secretion of tuna growth hormone (tGH) . Modification of the signal peptide with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a ten-fold increase in tGH production . Further elevation of the tGH yield to 240 mg/l was achieved by using a low proteinase mutant and a stable plasmid, and by culturing B . brevis under optimal conditions with the addition of some chemicals . Thus, biologically active tGH can be efficiently produced directly in the medium with this B . brevis system. Lett Appl Microbiol, 1994 Nov, 19(5), 353 - 6 Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin; Buchanan RL et al.; Two commercial serological kits (Oxoid BCET-RPLA and Tecra VIA) and a Chinese hamster ovary (CHO) cell cytotonicity assay for the detection of Bacillus cereus diarrhoeal enterotoxin were compared . Eleven B . cereus strains and one enterotoxigenic B . thuringiensis strain were evaluated . Both kits and the CHO cell assay yielded positive toxin responses for cell-free culture filtrates from eight out of 11 diarrhoeal enterotoxigenic strains . An emetic enterotoxin producing strain was negative with all three assays . Two B . cereus strains were negative using the BCET-RPLA kit, but positive with the Tecra VIA kit and CHO cell assay . The BCET-RPLA indicated significant levels of enterotoxin after samples were boiled, whereas the CHO cell and Tecra assays were negative . Overall, the cell culture assay was the most sensitive . However, the Tecra VIA kit provided similar results and was better suited for the rapid detection of B . cereus diarrhoeal enterotoxin. Curr Microbiol, 1994 Nov, 29(5), 301 - 7 An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis; Porteous LA et al.; A rapid direct-extraction method was used to obtain DNA from environmental soil samples . Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells . The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI . The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum . Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered . Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments . Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples. Mikrobiologiia, 1994 Nov-Dec, 63(6), 986 - 92 {Biosynthesis of extracellular ribonuclease by Bacillus pumilus}; Znamenskaia LV et al.; Conditions for the biosynthesis of alkaline extracellular ribonuclease by Bacillus pumilus were studied and their comparison with that of the biosynthesis of ribonuclease by Bacillus intermedius was made . It was estimated that ribonuclease of Bacillus pumilus is synthesized post-exponentially, its synthesis is inhibited by inorganic phosphate and is stimulated by actinomycin D, as the synthesis of ribonuclease by Bacillus intermedius . At the same time essential differences in dynamic of enzyme formation, extent of phosphate inhibition and spores forming conditions of the comparing cultures were found. Protein Eng, 1994 Nov, 7(11), 1379 - 86 Thermostabilization of the Bacillus circulans xylanase by the introduction of disulfide bonds; Wakarchuk WW et al.; The thermostability of the 20 396 Da Bacillus circulans xylanase was increased by the introduction of both intra- and intermolecular disulfide bridges by site-directed mutagenesis . Based on the 3-D structure of the enzyme, sites were chosen where favourable geometry for a bridge existed; in one case, to obtain favourable geometry additional mutations around the cysteine sites were designed by computer modelling . The disulfide bonds introduced into the xylanase were mostly buried and, in the absence of protein denaturants, relatively insensitive to reduction by dithiothreitol . The mutant proteins were examined for residual enzymatic activity after various thermal treatments, and were assayed for enzymatic activity at elevated temperatures to assess their productivity . We have examined one of these mutants by X-ray crystallography . All of the disulfide bond designs tested increased the thermostability of the B . circulans xylanase, but not all enhanced the activity of the enzyme at elevated temperatures. J Biochem (Tokyo), 1994 Nov, 116(5), 955 - 9 Purification and properties of L-ornithine delta-aminotransferase from gramicidin S-producing Bacillus brevis; Takechi M et al.; In gramicidin S-producing Bacillus brevis, the addition of L-ornithine to the minimal medium with L-glutamate as the sole carbon and nitrogen source caused an 8-fold induction of L-ornithine delta-aminotransferase {EC 2.6.1.13} . The enzyme was purified to homogeneity . The native enzyme had a molecular weight of about 88,000 after gel filtration and consisted of two subunits with an identical in molecular weight of about 45,000 . The enzyme was specific for L-ornithine (Km = 1.05 mM) as an amino donor and for 2-oxoglutarate (Km = 6.25 mM) as an amino acceptor, and catalyzed the conversion of L-ornithine and 2-oxoglutarate, respectively, to glutamic-gamma-semialdehyde, which is spontaneously cyclized to delta 1-pyrroline-5-carboxylate and L-glutamate . The enzyme exhibits an absorption maximum at 425 nm at neutral pH, and 1 mol of pyridoxal phosphate is bound per subunit . The enzyme activity was irreversibly inhibited by gabaculine, and L-ornithine protected the enzyme from the inhibition . The N-terminal amino acid sequence revealed a noteworthy similarity between human and yeast L-ornithine delta-aminotransferases in residues 17-28 of the B . brevis enzyme. J Mol Biol, 1994 Oct 28, 243(3), 530 - 2 Crystallization and preliminary X-ray diffraction studies of the lepidopteran-specific insecticidal crystal protein CrylA(a); Borisova S et al.; A trypsin-activated CrylA(a) protein from Bacillus thuringiensis has been purified and crystallized . Crystals belong to orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 53.3, b = 111.3 and c = 154.7 A . The crystals diffract to at least 2.2 angstrum resolution and are suitable for X-ray structural analysis . They contain a single molecule in the asymmetric unit. J Biol Chem, 1994 Oct 28, 269(43), 26836 - 41 Common pathways of cytochrome P450 gene regulation by peroxisome proliferators and barbiturates in Bacillus megaterium ATCC14581; English N et al.; Bacillus megaterium contains a barbiturate-inducible cytochrome P450BM-3, which catalyzes the hydroxylation of fatty acids . We report the intriguing finding that peroxisome proliferators, a major class of epigenetic carcinogen, are also extremely potent inducers of this enzyme being up to 50-fold more potent than one of the most effective barbiturates, secobarbital . Similar to barbiturates, the mechanism of induction appears to involve the direct binding of the peroxisome proliferator to the transcriptional repressor (Bm3R1), resulting in its dissociation from its DNA operator . These observations provide evidence that peroxisome proliferators can interact with a transcription factor to modulate gene expression . The data also demonstrate that the effects of these compounds are highly conserved through evolution and that there are important common denominators in the regulation of gene expression by peroxisome proliferators and the barbiturates . Evidence is presented to indicate that this may involve effects on unsaturated fatty acid homeostasis. Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10417 - 21 Native-like in vivo folding of a circularly permuted jellyroll protein shown by crystal structure analysis; Hahn M et al.; A jellyroll beta-sandwich protein, the Bacillus beta-glucanase H(A16-M), is used to probe the role of N-terminal peptide regions in protein folding in vivo . A gene encoding H(A16-M) is rearranged to place residues 1-58 of the protein behind a signal peptide and residues 59-214 . The rearranged gene is expressed in Escherichia coli . The resultant circularly permuted protein, cpA16M-59, is secreted into the periplasm, correctly processed, and folded into a stable and active enzyme . Crystal structure analysis at 2.0-A resolution, R = 15.3%, shows cpA16M-59 to have a three-dimensional structure nearly identical with that of the parent beta-glucanase . An analogous experiment based on the wild-type Bacillus macerans beta-glucanase, giving rise to the circularly permuted variant cpMAC-57, yields the same results . Folding of these proteins, therefore, is not a vectorial process depending on the conformation adopted by their native N-terminal oligopeptides after ribosomal synthesis and translocation through the cytoplasmic membrane. Biochemistry, 1994 Oct 25, 33(42), 12644 - 8 Structural and functional roles of the cysteine residues of Bacillus stearothermophilus farnesyl diphosphate synthase; Koyama T et al.; p-(Chloromercuri)benzoic acid inhibited the catalytic activity of Bacillus stearothermophilus farnesyl diphosphate synthase (FPP synthase), which contains only two cysteine residues at positions 73 and 289 . In order to explore the role of the cysteine residues, either or both of them were replaced with phenylalanine or serine . Five mutant enzymes, C73F, C73S, C289F, C289S, and C73S-C289S, were overproduced in Escherichia coli and purified to homogeneity . All of them were active as farnesyl diphosphate synthase, showing specific activities comparable to that of the wild-type enzyme . These results indicate that neither of the cysteines is essential for catalytic function . The C73F mutant, however, was very sensitive to heat treatment, while C73S was as highly stable as the wild type . The Km value of C289F for isopentenyl diphosphate is 10 times that of the wild type . The wild-type enzyme was converted into an oxidized form which was separable from the native enzyme on ion-exchange chromatography, and this conversion was accelerated by cupric ions . Similar conversion has previously been reported by several researchers, who found the occurrence of two forms of pig liver FPP synthase and who attributed this phenomenon to the oxidoreduction of sulfhydryl and disulfide groups . However, even the C73S-C289S mutant, which has no cysteine residues, was also converted into an oxidized form as in the case of the wild-type enzyme . These results provide evidence that residues other than cysteine are involved in the conversion of this enzyme into the oxidized form. FEBS Lett, 1994 Oct 24, 353(3), 259 - 63 A comparison and analysis of the toxicity and receptor binding properties of Bacillus thuringiensis CryIC delta-endotoxin on Spodoptera littoralis and Bombyx mori; Sanchis V et al.; The binding of L-{35S}methionine in vivo labelled CryIC toxin to its receptor in brush border membrane vesicle (BBMV's) prepared from Spodoptera littoralis and Bombyx mori was studied . Both insect species were highly susceptible to the CryIC toxin in bioassays, B . mori being 7-fold more sensitive to CryIC than S . littoralis (LC50's of 10 ng/cm2 and 70 ng/cm2, respectively) . Competition and direct binding experiments revealed saturable high-affinity binding sites on BBMV's from both insects which had similar binding characteristics for the CryIC toxin (Kd = 10 nM, Bmax = 8 to 9 pmol/mg BBMV's and IC50 = 37 nM for both inspect species) . Thus a specific receptor for the CryIC toxin is present in both insect species and the 7-fold greater potency of CryIC towards B . mori is not due to qualitative or quantitative differences in binding affinity or receptor site concentration . Dissociation experiments also indicated that the binding of {35S}CryIC to B . mori BBMV's is partially reversible. Med Clin (Barc), 1994 Oct 22, 103(13), 490 - 3 {How many cases of tuberculosis are not reported?}; Garcia Rodriguez JF et al.; BACKGROUND: The aim of this study was to evaluate the degree of registry of tuberculosis and the factors associated to the same . METHODS: A retrospective study of the cases of respiratory tuberculosis diagnosed in the hospital A . Marcide-Novoa Santos (El Ferrol . La Coruna . Spain) from 1990 to 1992 was carried out . Identification was obtained from the registries of microbiology and pathology and the clinical history files . Registered cases were obtained from the nominal notifications to the Epidemiology Department of the local health service department . Sex, age, place of residence, previous history of tuberculosis, HIV, diagnostic method, localization of the tuberculosis, registration and reporting physician were evaluated for each patient . RESULTS: Three hundred ninety-three cases were identified of which 78 (19.8%) had been registered . Age and pulmonary localization were the variable influencing the degree of registration . Reporting was greater in the age group from 0 to 10 years (p < 0.05) . Pulmonary tuberculosis was the most reported type although only 22.4% of the cases were declared . Bacilloscopy was positive in sputum in 190 patients and declared in 46 (24.2%) . The degree of registration increased significantly over three years (p < 0.000001) . Sex, previous history of tuberculosis, infection by the HIV and diagnostic method did not influence the degree of registration . CONCLUSIONS: Seventy-five percent of the cases with positive bacilloscopy in sputum were not declared . The degree of declaration has improved over time, however, remains deficient being 2.7 fold lower than the total number of cases diagnosed in 1992. Ugeskr Laeger, 1994 Oct 17, 156(42), 6175 - 80 {Cat-scratch disease and bacillary angiomatosis . An old and a new infectious disease with common etiology?}; Engbaek K et al.; A review of cat-scratch disease (CSD) and bacillary angiomatosis (BA) is presented on the basis of published articles . Two newly identified bacteria--Rochalimaea henselae and Afipia felis--have been isolated from patients with CSD . Preliminary investigations seem to indicate that A . felis is an uncommon cause of the disease . CSD may appear as a local suppurative lymphadenopathy or a systemic infection . BA is caused by Rochalimaea species and may appear as cutaneous, mucous or visceral angiomas or bacteremia . It may be a special manifestation of CSD in immunocompromised patients . A description is given of the various pathological pictures and differential diagnosis, and an evaluation is made of the different diagnostic methods, namely visualisation of bacteria in the lesions with Warthin-Starry's silver impregnation, isolation of bacteria, demonstration of bacteria with gene technique and detection of antibodies . The treatment of the disease is discussed. Biochem J, 1994 Oct 15, 303 ( Pt 2), 555 - 8 Effects of site-specific mutagenesis of tyrosine 105 in a class A beta-lactamase; Escobar WA et al.; Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site . Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis . This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties . In particular, in comparison to the wild-type, kcat . for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates . For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme . Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation . Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity . We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding. Biochem J, 1994 Oct 15, 303 ( Pt 2), 423 - 8 The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function . Evidence against the 'covalent switching' hypothesis of P-450 electron transfer; Munro AW et al.; The 'Covalent Switching' hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems {Baldwin, Morris and Richards (1991) Proc . R . Soc . London B 245, 43-51} . We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium . Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes . However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity . C.d . and e.p.r . spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation . These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem prosthetic group. Structure, 1994 Oct 15, 2(10), 945 - 51 Stability and function: two constraints in the evolution of barstar and other proteins; Schreiber G et al.; BACKGROUND: Barstar is the intracellular inhibitor of barnase, an extracellular RNAse of Bacillus amyloliquefaciens . The dissociation constant of the barnase-barstar complex is 10(-14) M with an association rate constant between barnase and barstar of 3.7 x 10(8) s-1 M-1 . The rapid association arises in part from the clustering of four acidic residues (Asp35, Asp39, Glu76 and Glu80) on the barnase-binding surface of barstar . The negatively charged barnase-binding surface of barstar effectively 'steers' the inhibitor towards the positively charged active site of barnase . RESULTS: Mutating any one of the four acidic side chains of barstar to an alanine results in an approximately two-fold decrease in the association rate constant, while the dissociation rate constant increases from five orders of magnitude for Asp39-->Ala, to no significant change for Glu80-->Ala . The stability of barstar is increased by all four mutations, the increase ranging from 0.3 kcal mol-1 for Asp35-->Ala or Asp39-->Ala, to 2.1 kcal mol-1 for Glu80-->Ala . CONCLUSIONS: The evolutionary pressure on barstar for rapid binding of barnase is so strong that glutamate is preferred over alanine at position 80, even though it does not directly interact with barnase in the complex and significantly destabilizes the inhibitor structure . This, and other examples from the literature, suggest that proteins evolve primarily to optimize their function in vivo, with relatively little evolutionary pressure to increase stability above a certain threshold, thus allowing greater latitude in the evolution of enzyme activity. J Immunol, 1994 Oct 15, 153(8), 3684 - 90 Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages; Jun CD et al.; The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined . Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity . When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner . This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting . The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma . Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages . Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma . In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester . When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone . On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma. Biochem Biophys Res Commun, 1994 Oct 14, 204(1), 428 - 35 Two o-type oxidases in Methylobacillus flagellatum KT; Muntyan MS et al.; Two oxidases of the o-type in membranes of the methanol-grown obligate methylotroph Methylobacillus flagellatum KT were distinguished . For this purpose the kinetic analysis of the laser flash-induced optical absorbance changes of CO-oxidase complexes under reducing conditions was used . The ratio of these oxidases in membranes greatly depended on the phases of bacterial growth . One of the oxidases appeared to belong to the Escherichia coli o-type oxidase family being more sensitive to KCN (Ki = 1 microM) . It showed monophasic CO recombination kinetics with tau 25-30 ms and was expressed in the early exponential phase of growth . The other oxidase seemed to be similar to the Bacillus sp . FTU o-type oxidase being less sensitive to KCN (Ki = 6 microM), having three-phasic CO reassociation kinetics with tau 35-70 microseconds, 0.25-0.5 ms and 2-4 ms and dominating in the stationary growth phase . Pyridine haemochrome spectra showed haems A and D to be absent from the bacterial membranes. J Med Chem, 1994 Oct 14, 37(21), 3668 - 70 Synthesis of N-acetylglucosamine-modified ara-C and its effect on ovarian cancer cells; Fujimoto H et al.; 1-beta-D-Arabinofuranosylcytosine (ara-C) was modified by reaction of tetra-N-acetylchitotetraose ((GlcNAc)4) using the transglycosylation activity of thermostable chitinase (EC 3.2.1.14) from Bacillus licheniformis X-7u . The structure of the modified ara-C was determined to be either beta 1-3'- or beta 1-5'-linked GlcNAc-ara-C or (GlcNAc)2-ara-C . The total yield of these glycosylated ara-Cs was about 10% . GlcNAc-ara-C and (GlcNAc)2-ara-C depressed the growth of G-401 cancer cells, while 5-O-beta-D-galactopyranosyl-beta-D-arabinofuranosylcytosine (Gal-ara-C) had no effect on G-401 cells. Biochemistry, 1994 Oct 11, 33(40), 12056 - 62 Evidence for conformational dynamics and molecular aggregation in cytochrome P450 102 (BM-3); Black SD et al.; The native molecular weight of affinity-purified cytochrome P450 102 from barbiturate-induced Bacillus megaterium has been studied by sedimentation methods and HPLC size-exclusion chromatography . Sedimentation velocity experiments yielded an s020,w = 9.244 S for the holocytochrome, but the diffusion coefficient was unexpectedly large and varied widely with centrifugal field, ionic strength, and protein concentration . Addition of 50 mM DL-dithiothreitol (DTT) caused a small decrease in the value of s020,w, but D20 still did not behave as expected . The sedimentation coefficients were consistent with a molecular weight of about 200,000, and the diffusion coefficients indicated molecular aggregation . Sedimentation equilibrium analyses suggested that the native enzyme was a mixture of monomer, dimer, trimer, and tetramer . However, after incubation of P450 102 with DTT, sedimentation equilibrium demonstrated that the enzyme was dimeric (molecular weight 236,000) . HPLC size-exclusion chromatography of the cytochrome showed the presence of four peaks, which corresponded to 1.45-mer, 2.06-mer, 3.02-mer, and a higher molecular weight fraction; aggregated forms accounted for about 52% of the P450 102 . Incubation of the enzyme with DTT caused a shift toward the 1.45-mer, but dimer, trimer, and the high molecular weight peak still persisted; the shift was not attributable to disulfide bond reduction . The 1.45-mer was determined to be a monomeric species of significantly asymmetric geometry . Together, the results indicated that cytochrome P450 exists with monomer, dimer, trimer, etc . in equilibrium, contrary to the expectation that this soluble P450 would be monomeric.(ABSTRACT TRUNCATED AT 250 WORDS) Virology, 1994 Oct, 204(1), 91 - 100 Rice tungro bacilliform virus: transcription and translation in protoplasts; Chen G et al.; Protoplasts from cell suspension cultures of Oryza sativa (monocot) and Orychophragmus violaceus (dicot) support transcription from the rice tungro bacilliform virus (RTBV) promoter and translation of the resulting mRNA despite the presence of a long leader sequence with strong secondary structure and 12 short open reading frames . Transcriptional elements located both upstream and downstream of the transcription initiation site are defined by deletion analysis and the functional TATA motif is determined . Expression of an open reading frame downstream of the entire leader is more efficient than that downstream of truncated derivatives . For optimal expression sequences in the 5' and 3' parts of the leader are required. J Urol, 1994 Oct, 152(4), 1275 - 80 Bacillus Calmette-Guérin interacts with the carboxyl-terminal heparin binding domain of fibronectin: implications for BCG-mediated antitumor activity; Cheng DL et al.; Intravesical bacillus Calmette-Guerin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder . The mechanisms by which BCG achieves this effect remain unclear . Reports have attributed an important role to fibronectin both in the initial attachment of BCG to bladder surfaces and in the limitation of tumor cell motility . In the present study, using limited protease cathepsin B degradation followed by Western blot analyses with antibodies to various domains of the fibronectin molecule, we showed that BCG appears to bind to fibronectin near the carboxyl terminal and adjacent to the heparin binding domain . Furthermore a 51-chromium release assay with human bladder cancer cell line T24 as target cells and lymphokine activated killer (LAK) cells as effector cells showed that fibronectin was needed for tumor cytotoxicity by the LAK cells . By using antibodies and peptides to various domains of the fibronectin molecule, the heparin binding domain, but not the cell binding domain, carboxyl terminal region, or the amino terminal region of the fibronectin molecule, was identified as essential to tumor cell lysis by the LAK cells . Flow cytometric analysis showed that both peripheral blood lymphocytes and the LAK cells express fibronectin receptors VLA-3, VLA-4 and VLA-5 on their surfaces . However, the numbers of receptors are not significantly different in the two cell populations . We conclude that, by binding near the carboxyl terminal region and adjacent to the heparin-binding domain of the fibronectin molecule, BCG may protect this region of the molecule from tumor proteases, and may thus allow the antitumor activity of the host immune cells to take place. Planta Med, 1994 Oct, 60(5), 414 - 6 Suppression of chemically and immunologically induced hepatic injuries by gentiopicroside in mice; Kondo Y et al.; Gentiopicroside (GPS), a main bitter secoiridoid constituent of roots of Gentiana macrophylla Pall., was tested for therapeutic effects on the two hepatic injury models, the CCl4-induced and lipopolysaccharide (LPS)/bacillus Calmette-Guerin (BCG)-induced hepatitides . An increase in serum level of hepatic aminotransferases (GOT: EC 2.6.1.1 . and GPT: EC 2.6.1.2.) induced by a p.o . treatment of CCl4 was suppressed by pretreatment with GPS at 30-60 mg/kg/day for 5 consecutive days . An increase of these enzymes triggered by an i.v . treatment with LPS in mice primed with bacillus Calmette-Guerin (BCG) was also inhibited by GPS pretreatment at the same dose of GPS . In the BCG/LPS model, tumor necrosis factor (TNF), a major inflammatory mediator, was increased in serum with a peak at 90-120 min, followed by an increase of serum transaminase activities . GPS treatment significantly suppressed the increase of TNF in serum at the therapeutic doses, suggesting that GPS protected against hepatitis by inhibiting the production of TNF. Hinyokika Kiyo, 1994 Oct, 40(10), 873 - 7 {Long-term results and complications of intravesical instillation of bacillus Calmette-Guerin for prophylaxis of bladder cancer recurrence}; Irie A et al.; Since 1982, we treated 22 patients with superficial bladder cancer via intravesical bacillus Calmette-Guerin (Tokyo 172 strain) for prophylaxis against tumor recurrence . To determine the long-term efficacy and complications of BCG therapy, retrospective analysis was performed . The BCG therapy was initiated one to two weeks after complete transurethral resection of visible tumors . One hundred twenty mg or 80 mg of BCG suspended in 40 ml of 0.9% NaCl solution was instilled into the bladder once a week for 6 times, then monthly for 10 times . Of 22 patients, 10 tumor recurrences were recognized in 4 patients (mean followup 74 months) . Recurrent free rate at 5 years was 78.5% . Common side effects were low grade fever and bladder irritability . Although these side effects were self-limiting, 15 patients (68%) refused instillation of BCG before completion of our protocol because of the bladder irritation . No relationship was observed between total dose of BCG instilled and tumor recurrence . Severe side effects such as permanent structural or functional alterations of the bladder or mycobacterial infection of other organs were not observed . Intravesical administration of Tokyo 172 strain of BCG seemed to be safe and useful for long-term prophylaxis of superficial bladder cancer recurrence. Plant Physiol, 1994 Oct, 106(2), 643 - 50 Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers; Menting JG et al.; NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-{(3-cholamidopropyl)dimethylammonio}-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography . Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P . hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies . Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts . Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase . The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively . We believe that the purified enzyme is a P . hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4). FEMS Microbiol Lett, 1994 Oct 1, 122(3), 203 - 10 Membrane-bound Bacillus cytochromes c and their phylogenetic position among bacterial class I cytochromes c; Sone N et al.; Gram-positive bacteria lack a periplasmic compartment and contain only membrane-bound cytochromes c . There are at least two types . One is found in subunit II of cytochrome oxidase, and the other is small cytochrome c which is also membrane-bound because of an unprocessed signal sequence or post-translational acylation at the N-terminal end of the protein . These Bacillus cytochromes c are compared with known class I cytochromes c, and a phylogenetic tree has been constructed by the neighbour-joining method. Plant Mol Biol, 1994 Oct, 26(1), 51 - 9 Insect resistance of transgenic plants that express modified Bacillus thuringiensis cryIA(b) and cryIC genes: a resistance management strategy; van der Salm T et al.; Tobacco and tomato plants were generated exhibiting insect resistance due to the introduction of modified cryIA(b) and cryIC genes of Bacillus thuringiensis . Limited modifications at selected regions of the coding sequences of both genes are sufficient to obtain resistance against Spodoptera exigua, Heliothis virescens and Manduca sexta . The criteria used to modify both genes demonstrate that the removal of sequence motifs potentially resulting in premature polyadenylation and transcript instability causes increased insect resistance . The expression of a cryIC-cryIA(b) fusion resulting in protection against S . exigua, H . virescens and M . sexta demonstrates the potential of expressing translational fusions, not only to broaden the insect resistance of transgenic plants, but also to simultaneously employ different gene classes in resistance management strategies. Plant Mol Biol, 1994 Oct, 26(1), 285 - 90 Biosynthesis of lysine in plants: the putative role of meso-diaminopimelate dehydrogenase; Chatterjee SP et al.; Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway . Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all . However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean . Moreover, it was not found in Chlamydomonas, corn and tobacco as well . In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus . The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants. Epidemiol Infect, 1994 Oct, 113(2), 297 - 306 Contamination of hospital linen by Bacillus cereus; Barrie D et al.; An investigation into two cases of post-operative Bacillus cereus meningitis revealed that hospital linen laundered by a batch continuous washing machine was heavily contaminated by B . cereus spores . The washing machine, detergents, other chemical additives and the water supply were eliminated as the source of contamination . It was found that the linen introduced into the washing machine had a high B . cereus spore content and that this was still present after the wash process . The spores were not killed by either the heat disinfection stage of the wash or the addition of chemical disinfectants and were not removed by the dilution in the process . The multiplication of B . cereus was thought to have occurred on used, damp linen stored in plastic bags, particularly when ambient temperatures were high . An increase in the water flow through the washing machine was the only measure associated with a decrease in B . cereus on laundered linen. Cancer Res, 1994 Oct 1, 54(19), 5178 - 85 Establishment, molecular rescue, and expression of 123AV16-1, a tumor-reactive human monoclonal antibody; Hall BL et al.; The human monoclonal antibody (mAb) 123AV16-1 was generated by Epstein-Barr virus transformation of peripheral blood lymphocytes from a colorectal patient undergoing active specific immunotherapy with an autologous tumor cell-Bacille Calmette-Guerin vaccine . Direct immunohistochemical staining of tumor and normal pairs of tissues indicated that this human IgA1, lambda 2 mAb preferentially reacted with colon tumor epithelium . To generate a recombinant derivative of this Epstein-Barr virus-transformed cell line, we isolated the expressed complete heavy and light chain genes by a novel strategy and cloned them into modified pSV2-neo and pSV2-gpt expression vectors . The recombinant 123AV16-1 human mAb was expressed in both a murine myeloma and a human-murine heteromyeloma and was secreted as both monomers and dimers . The recombinant 123AV16-1 mAb expressed by both cell lines reacted with human colon tumor xenografts in a manner similar to the mAb derived from the Epstein-Barr virus-transformed cell line, indicating that the antibody specificity was not appreciably altered during the molecular rescue, cloning, or expression. J Biochem (Tokyo), 1994 Oct, 116(4), 931 - 6 Construction and characterization of chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase; Kataoka K et al.; Phenylalanine dehydrogenase of Thermoactinomyces intermedius acts preferentially on L-phenylalanine and L-tyrosine, whereas leucine dehydrogenase of Bacillus stearothermophilus acts almost exclusively on L-leucine and some other branched-chain L-amino acids . The two enzymes share a sequence similarity (47%) . Aiming at elucidation of the mechanism of substrate recognition by the two amino acid dehydrogenases, we have genetically constructed a chimeric enzyme consisting of an N-terminal domain of phenylalanine dehydrogenase containing the substrate-binding region and a C-terminal domain of leucine dehydrogenase containing the NAD(+)-binding region . The chimeric enzyme purified to homogeneity acted on phenylalanine with a specific activity of 6% of that of the parental phenylalanine dehydrogenase and showed a broad substrate specificity in the oxidative deamination, like phenylalanine dehydrogenase . However, it acted much more effectively than phenylalanine dehydrogenase on isoleucine and valine . Its Km values for L-phenylalanine and L-leucine were similar to those of phenylalanine dehydrogenase . The substrate specificity of the chimeric enzyme in the reductive amination was an admixture of those of the two parent enzymes . These results suggest that the two domains of phenylalanine dehydrogenase and leucine dehydrogenase probably can fold independently . Accordingly, their chimera forms a new active enzyme which consists of their N- and C-terminal domains containing the substrate- and coenzyme-binding regions, respectively . However, the two domains of chimeric enzyme interact and communicate with each other to form a new active site and consistently show the new substrate specificity. Protein Eng, 1994 Oct, 7(10), 1255 - 9 C-terminal truncations of a thermostable Bacillus stearothermophilus alpha-amylase; Vihinen M et al.; A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases . The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues . The longer the truncation, the lower the specific activity of the enzyme . Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme . The Km values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding . The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile . The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling . The liquefying amylases seem to require approximately 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity. Pharm Res, 1994 Oct, 11(10), 1485 - 91 Gamma sterilization of a semi-solid poly(ortho ester) designed for controlled drug delivery--validation and radiation effects; Merkli A et al.; Radiation sterilization is becoming increasingly popular for the sterilization of many pharmaceutical products . Although this technique is not limited to the sterilization of polymers, it is probably the most suitable method for such materials . This method however suffers several drawbacks . The sterilization of a product must lead to a safety level of 10(-6), i.e . one chance in a million to find a contaminated sample . In many cases, this assurance of sterility can be achieved by using a uniform treatment dose of 2.5 Mrad, recommended by the pharmacopeia . We investigated the possibility of using doses of radiation inferior to 2.5 Mrad to sterilize a semi-solid poly(ortho ester) (POE) developed for use as carrier in controlled drug delivery . After determination of the initial bioburden, the polymer was intentionally contaminated with the bioindicator Bacillus pumilus E 601 . Following exposure to gamma irradiation, the D10 value of the radio resistant bioindicator was determined . Using the initial contamination value, the reduction factor D10 and the safety level, it is possible to calculate an optimal sterilizing dose for POE . All polymers are affected by ionizing radiation and the amount of radiation which produces a significant change in properties may vary from one polymer to the other . A molecular weight and dynamic viscosity decrease resulting from backbone cleavage was observed for this POE at a dose lower than 2.0 Mrad . Evaluation of the structure using 1H-NMR, 13C-NMR and IR analysis shows that for doses higher than 2.0 Mrad, another degredation process takes place.(ABSTRACT TRUNCATED AT 250 WORDS) In Vitro Cell Dev Biol Anim, 1994 Oct, 30A(10), 690 - 5 Cytolytic differences among lepidopteran cell lines exposed to toxins of Bacillus thuringiensis subsp . kurstaki (HD-263) and aizawai (HD-112): effect of aminosugars and N-glycosylation; McCarthy WJ; Comparison of lytic-dose response behavior of seven lepidopteran cell lines to the activated delta-endotoxin polypeptides of Bacillus thuringiensis subspecies kurstaki (HD-263) and aizawai (HD-112) indicated distinct differences among the lines . The lines derived from Spodoptera species S . exigua (URC-SE-1A) and S . littoralis (UIV-SL-575) were more susceptible to lysis by aizawai toxin (Bta) than kurstaki toxin (Btk) as were cells from the Lymantria dispar line (IPLB-LD652Y) . However, the concentrations of Bta required for lysis of 50% of URC-SE-1A and IPLB-LD652Y cells (LC50) were 0.2 to 0.8 micrograms/ml compared to 5 to 9 micrograms/ml for UIV-SL-575 cells . In comparison, Btk LC50 concentrations for the three lines were similar (14 to 19 micrograms/ml) . Cells from S . frugiperda (IPLB-SF-21AE) and Trichoplusia ni (TN368) were similar in their response to Bta (LC50 = 2.5 to 3.7 micrograms/ml) and Btk (LC50 = 1.0 to 2.8 micrograms/ml) whereas the lines derived from Heliothis spp . were the least susceptible to both toxins . The LC50 concentrations for Bta with the H . zea line (IPLB-HA-1075) and H . virescens line (BCIRL-HV-AM1) were > 50 micrograms/ml and for Btk were > 50 micrograms/ml and 42 to 50 micrograms/ml, respectively, yet for both lines Btk was the more cytolytic . Cytolysis of TN368 cells could be inhibited to varying extents by preincubation of the toxins with the aminosugars of galactose, mannose, and glucose and their N-acetyl derivatives . The unsubstituted hexoses were not inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS) Tuber Lung Dis, 1994 Oct, 75(5), 334 - 40 Empiric antituberculosis treatment: benefits for earlier diagnosis and treatment of tuberculosis; Anglaret X et al.; SETTING: Tuberculosis may be diagnosed too late, especially in HIV-infected patients, with consequences on bacillus transmission and survival . Empiric antibuberculosis treatment (EATT) may be started before diagnosis of tuberculosis is confirmed . As rifampicin is a broad spectrum antibiotic, EATT including rifampicin may be effective in infections other than tuberculosis, leading to misdiagnosis . OBJECTIVE: To define the efficiency criteria of EATT with or without rifampicin . DESIGN: Between 1988 and 1991, 20 febrile patients with suspected tuberculosis (including 15 who were HIV-positive) were started on EATT in the absence of bacteriological or histological proof of tuberculosis . 10 patients (50%) received a 4-drug non-specific EATT including rifampicin, isoniazid, pyrazinamide and ethambutol, and 10 (50%), received a 3-drug specific EATT without rifampicin . RESULTS: In 10 patients (50%), the diagnosis of tuberculosis was confirmed by positive cultures within a mean of 32 days (15-57 days) after the beginning of EATT (group TB 1) . Of the 10 patients whose cultures remained negative, 4 (20%) became afebrile and showed improvement under EATT (group TB 2), and 6 (30%) remained febrile and did not improve (group No TB) . Patients from groups TB 1 and TB 2 became afebrile within a mean of 11 days (1-54 days) . This delay was not different between patients receiving specific or non-specific EATT . In patients receiving specific EATT, rifampicin was added to the initial 3-drug treatment after resolution of fever . CONCLUSION: EATT appears to be a useful method for rapid presumptive diagnosis and treatment of tuberculosis. Immunology, 1994 Oct, 83(2), 227 - 31 Mycobacteria precipitate an SLE-like syndrome in diabetes-prone NOD mice; Baxter AG et al.; Non-obese diabetic (NOD) mice spontaneously develop organ-specific autoimmunity and are widely used as a model for diabetes . Aged NOD mice also exhibit some features of non-organ-specific autoimmune rheumatic disease such as anti-nuclear antibodies and late-onset haemolytic anaemia . Here, we report that a single dose of 2.6 x 10(7) heat-killed bacillus Calmette-Guerin (BCG) i.v . in 8-week-old NOD mice prevented diabetes but precipitated a syndrome similar to systemic lupus erythematosus (SLE) . Treated mice developed haemolytic anaemia, anti-DNA and anti-Sm anti-nuclear autoantibodies and an increased severity of sialadenitis . Perivascular lymphocytic infiltration in the kidneys and glomerular immune complex deposition were also found . The action of BCG appeared to be mediated by an adjuvant-like activity as treated mice showed a substantial increase in reticuloendothelial cell function and enhanced antigen presentation capacity. Mol Microbiol, 1994 Oct, 14(2), 381 - 9 Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var . tenebrionis; Adams LF et al.; NB176 is a Bacillus thuringiensis mutant derived by gamma-irradiation of NB125 Bacillus thuringiensis var . tenebrionis (Krieg) . It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein . Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment . Each cryIIIA gene-bearing HindIII fragment was cloned from NB176 . The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers . Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn1000 . These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by gamma-irradiation . Integration of additional copies of the cryIIIA gene into the native 90 MDa plasmid of the wild-type B . thuringiensis var . tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity. Mol Microbiol, 1994 Oct, 14(1), 41 - 50 Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins; Domenighini M et al.; Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity . This cavity consists of 18 contiguous amino acids that form an alpha-helix bent over a beta-strand . The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence . Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site . These are: a glutamic acid that is conserved in all toxins, and a nucleophilic residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus . The latter group of toxins presents an additional histidine that appears important for catalysis . This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins. Mol Microbiol, 1994 Oct, 14(1), 131 - 9 IS231A insertion specificity: consensus sequence and DNA bending at the target site; Hallet B et al.; In its natural host, Bacillus thuringiensis, the insertion sequence IS231A is preferentially inserted into the terminal inverted repeats of the transposon Tn4430 . Using a novel transposition assay, we demonstrate that the Tn4430 ends behave as insertion hot spots for IS231A in Escherichia coli . Sequence analysis reveals that IS231A insertion sites match the 5'-GGG(N)5CCC-3' consensus . However, this consensus is not the only determinant of IS231A insertion specificity . Although both Tn4430 ends have identical sequences, one is strongly preferred to the other and the orientation of insertion into this end is not random . We demonstrate that this preference is determined by the flanking regions of the site . These regions display a conserved periodic organization of their sequence which, by conferring anisotropic flexibility, would induce the DNA to bend in a roughly 'S'-shaped structure centered on the target consensus . DNA conformation analysis by polyacrylamide gel electrophoresis indeed shows that the preferred target site of IS231A is flanked by DNA segments curved in opposite directions . We present a model in which DNA bendability and curvature would contribute to the positioning of IS231A transposase on the target DNA. Skeletal Radiol, 1994 Oct, 23(7), 569 - 71 Case report 865 . Bacillary angiomatosis; Standiford KN et al.; A case of bacillary angiomatosis was presented, characterized by subcutaneous lesions, systemic symptoms, and impressive periostitis which initially masked a small cortical lytic lesion . Review of the reported cases suggests that a diagnosis of bacillary angiomatosis should be strongly considered when periostitis is identified in an AIDS patient with skin lesions . Additionally, deep surgical biopsy of the skin lesion to include the subcutaneous tissue should be performed to confirm the diagnosis if the initial punch biopsies are unrevealing. Pathologe, 1994 Oct, 15(5), 259 - 70 {Vascular tumors of the skin and soft tissue . Overview of newly characterized entities and variants}; Mentzel T et al.; This review summarizes vascular tumours of skin and soft tissues that have been characterised in recent years . Although most of them are very rare, knowledge about their reproducible clinicopathological features is important to avoid diagnostic pitfalls . These lesions include: bacillary angiomatosis, a vasoproliferative, pseudoneoplastic infection of immunocompromised patients caused by Rochalimaea henselae; tufted angioma, a variant of lobular capillary haemangioma characterised by a "cannon-ball" distribution of multiple lobules composed of packed capillaries and pericytes; microvenular haemangioma, a cutaneous haemangioma composed of thin-walled and irregularly branching blood vessels which dissect dermal collagen; sinusoidal haemangioma, a distinctive variant of cavernous haemangioma which may be located in the subcutaneous breast tissue and then may be confused with well-differentiated angiosarcoma; "hobnail haemangioma" (targetoid haemosiderotic haemangioma), a benign vascular tumour with a distinctive clinical targetoid appearance and a hobnail cytomorphology of the prominent endothelial tumour cells; retiform haemangioendothelioma, a very recently characterised low-grade angiosarcoma occurring most commonly in the extremities of adolescents which is characterised by arborising blood vessels arranged in a retiform pattern and lined by hobnail-like prominent endothelial cells; Kaposi-like infantile haemangioendothelioma, a borderline malignant tumour of infants mimicking Kaposi's sarcoma histologically; epithelioid angiosarcoma, a highly aggressive tumour in the spectrum of epithelioid vascular lesions which stains positively for endothelial and epithelial immunohistological markers; benign lymph-angioendothelioma (progressive lymphangioma), a benign, slowly growing macule or plaque which has to be distinguished from well-differentiated angiosarcoma and Kaposi's sarcoma; and lymphangiomatosis of the limbs, a poorly recognised angiomatosis occurring in young patients and limited mainly to the limbs with a favourable prognosis. Clin Infect Dis, 1994 Oct, 19(4), 751 - 5 Lytic vertebral lesions: an unusual manifestation of AIDS-associated Kaposi's sarcoma; Isenbarger DW et al.; The differential diagnosis of neovascular skin lesions in patients with AIDS includes Kaposi's sarcoma and bacillary angiomatosis . It has been suggested that the radiographic presence of lytic bone lesions in association with these skin lesions supports a diagnosis of bacillary angiomatosis . We present a case of disseminated Kaposi's sarcoma in which evidence of lytic vertebral disease was seen on computed tomography; the histopathologic characteristics of the osseous lesions are described . Findings of magnetic resonance imaging implied more diffuse marrow involvement . Human immunodeficiency virus-associated osseous manifestations of rochalimaea infection and Kaposi's sarcoma are reviewed. Appl Microbiol Biotechnol, 1994 Oct, 42(1), 78 - 84 Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus brevis; Louw ME et al.; The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced . The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively . Sequence comparisons at the amino acid level to the B . subtilis degS-degU genes showed 74% and 84% similarity, respectively . On a multicopy vector the B . brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B . subtilis rec- strain as well as in a B . subtilis sacU(HY) strain. Immunobiology, 1994 Oct, 191(4-5), 564 - 8 Cytokine regulation of disease progression in leprosy and tuberculosis; Kaplan G; Studies in our laboratory have focussed on the role of cytokines in the regulation of the cellular immune response and disease progression in two important mycobacterial infection of man, namely leprosy and tuberculosis . Our studies in leprosy have involved the use of key regulatory cytokines such as IFN-gamma in the modulation of the cellular response of infected patients . We have investigated the effect of intradermal administration of low dose IFN-gamma on the lesions of anergic lepromatous patients and have reported an accelerated bacillary clearance from the skin . This was associated with the local accumulation of mononuclear cells and killing of infected macrophages . However, IFN-gamma administration also resulted in the induction of erythema nodosum leprosum, a toxic syndrome associated with excess TNF-alpha production . Both the toxic symptoms and the high levels of TNF-alpha production could be inhibited by thalidomide treatment, a drug we have shown reduces the half life of TNF-alpha mRNA . In preliminary clinical trials in tuberculosis patients we have attempted to use thalidomide to reduce TNF-alpha production and toxicities . These results are discussed. Immunobiology, 1994 Oct, 191(4-5), 413 - 23 Transient inducible events in different tissues: in situ studies in the context of the development and expression of the immune responses to intracellular pathogens; Belkaid Y et al.; Intracellular pathogens whether facultative like Mycobacterium sp., e.g . Bacillus Calmette Guerin, Listeria monocytogenes or strictly intracellular like Leishmania sp . initiate either asymptomatic infectious processes or disease depending both on factors of the host (genetic as well as environmental ones) and the infectious/pathogenic agents . In this contribution, we first summarized informations which justify to develop in situ analysis to decipher the sequential events that result in different modes/classes of immune responses . How the mode of the immune response is determined remains a main question to address . Although it has recently become clear, in vitro, that immunocompetent cells and their cytokines are critical to set on a stable mode of immune response, acting on naive T cells, this area deserves more in vivo studies . Indeed, peripheral T cells, at different stages of differentiation, may exist in vivo (a) naive/virgin, (b) experienced, (c) effector T cells, depending on the level of stimulation of the immune system by either endogenous or exogenous (e.g . gut flora) signals . The three chosen examples illustrate our contributions in this field focusing on three different non-lymphoid tissues which may become infected: bone marrow (Bacille de Calmette Guerin), liver (Listeria monocytogenes), skin (Leishmania major) . These three illustrations also allow to attract attention on the interest of using mice of genetically different strains the immune response of which is set up under different modes. Int J Biol Macromol, 1994 Oct, 16(5), 265 - 75 Gamma-poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies; Birrer GA et al.; Cryogenically frozen vegetative cells of Bacillus licheniformis 9945a derived from young mucoid colonies were used to inoculate gamma-poly(glutamate) (gamma-PGA) production media containing L-glutamate, citrate and glycerol as carbon sources . A gel permeation chromatography (GPC) method was developed to determine gamma-PGA volumetric yield and molecular weight directly using culture filtrates . For GPC volumetric yield measurements, a calibration curve was generated using purified gamma-PGA to relate the gamma-PGA GPC peak area and polymer weight . Purified gamma-PGA was characterized by elemental analysis, 1H- and 13C-NMR spectroscopy . Cultures of B . licheniformis using all three carbon sources showed the following characteristics: cell growth mainly during the first 24 h; largest gamma-PGA volumetric productivity (approximately 0.12 gl-1 h-1) between 48 and 96 h; 11 g l-1 gamma-PGA volumetric yield by 96 h; reduction (utilization) of glycerol, glutamate and citrate during a 96 h cultivation time from 80 to 45 g l-1, 18 to 10 g l-1 and 12 to approximately 1 g l-1, respectively; a decrease in pH from 7.4 to approximately 5.5 by 42 h cultivation; acetic acid secretion into the medium at a maximum level of approximately 4.5 g l-1 and detection of the metabolite 2,3-butanediol (as acetoin) as a fermentation by-product at approximately 42 h and through a 96 h cultivation period . The presence of 2,3-butanediol indicated that the level of oxygen in the medium no longer supported a fully aerobic mode of metabolism . When the medium formulation was altered by removal of either citrate, L-glutamate or glycerol in shake flask experiments where pH was not controlled, 2.3, 9.0 and 4.0 g l-1, respectively, of gamma-PGA were formed . Variation of the medium ionic strength by the addition of up to 4% (w/v) NaCl led to the formation of gamma-PGA of relatively higher molecular weight but lower volumetric yield . Studies carried out on 5-day-old B . licheniformis cultures suggested that gamma-PGA depolymerase is intracellularly located or cell-bound . Culture filtrates showed no significant gamma-PGA depolymerase activity. Appl Environ Microbiol, 1994 Oct, 60(10), 3711 - 7 Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide; Von Tersch MA et al.; Bacillus thuringiensis toxin CryIIIB2 exhibits activity against two agriculturally important pests, the Colorado potato beetle, Leptinotarsa decemlineata, and the Southern corn rootworm, Diabrotica undecimpunctata . CryIIIB2 shows significant structural similarity to Colorado potato beetle-active toxin CryIIIA, whose crystal structure has been determined elsewhere {J . Li, J . Carrol, and D . J . Ellar, Nature (London) 353:815-821, 1991} . A clone limited to the putative 7-alpha-helical bundle domain I peptide of CryIIIB2 was constructed by PCR . The truncated protein was expressed at high levels in Escherichia coli . Domain I peptide was isolated and compared with native CryIIIB2 toxin in promoting ion efflux from synthetic phospholipid vesicles and formation of ion channels in black lipid membranes . The results showed that CryIIIB2 domain I peptide is sufficient for ion channel formation and promotes ion efflux . Both native CryIIIB2 toxin and domain I peptide were inefficient channel-forming proteins that produced noisy ion channels of various conductance states . In ion efflux assays, native toxin promoted greater ion efflux from synthetic vesicles than did the truncated peptide. Biochem Biophys Res Commun, 1994 Sep 30, 203(3), 1745 - 9 Flavin supported fatty acid oxidation by the heme domain of Bacillus megaterium cytochrome P450BM-3; Gonvindaraj S et al.; Cytochrome P450BM-3 is a fatty acid hydroxylase that consists of a heme domain covalently attached to a diflavin (FMN+FAD) cytochrome P450 reductase domain . The heme and flavin domains can be separately expressed and purified from E . coli recombinant expression systems . Normally P450s require a protein redox partner as a source of electrons . We now have found that the P450BM-3 heme domain can be reduced by NADPH+FMN and that reduced FMN can support the P450 catalyzed hydroxylation of a fatty acid substrate, myristic acid . HPLC profiles show that the "artificial" FMN supported hydroxylation gives the same products as does holo-P450BM-3. Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9544 - 8 Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts; Dent P et al.; The serine-threonine protein kinase Raf-1 is an important signal transducer in mitogenesis, phosphorylating and activating mitogen-activated protein (MAP) kinase kinase . Raf-1 activation in vivo is dependent on Ras, but the mechanism of Raf activation is unknown . The ability of preparations of plasma membranes to activate exogenous (His)6-Raf-1 was studied . Plasma membranes of v-Ras-transformed NIH 3T3 cells, but not parental cells, enhanced MAP kinase kinase kinase (MAPKKK) activity dependent on addition of (His)6-Raf-1 and ATP/Mg . Treatment of membranes with concentrations of Bacillus cereus phosphatidylcholine-specific phospholipase C that activated Raf-1 in vivo failed to enhance MAPKKK activity in vitro . Activation of (His)6-Raf-1 in vitro by membranes was dependent on binding to Ras . Membranes from v-Src-transformed cells also activated (His)6-Raf-1 and synergized with v-Ras membranes . Serum-treatment of NIH 3T3 cells stimulated the ability of membranes to activate (His)6-Raf-1 . Activated (His)6-Raf-1 could be recovered on Ni(2+)-agarose, and this methodology was used to demonstrate that activation by membranes was ATP dependent . These findings demonstrate Ras- and ATP-dependent step(s) for Raf-1 activation by plasma membranes in vitro. Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9407 - 11 Nitric oxide is an important mediator for tumoricidal activity in vivo; Farias-Eisner R et al.; When cultured in vitro, peritoneal macrophages, obtained from mice previously inoculated with bacillus Calmette-Guerin, release nitric oxide, which is cytostatic and/or cytolytic for tumor cells . However, it is not known whether nitric oxide has antitumor effects in vivo . Here we demonstrate that nitric oxide is an important mediator of host resistance to syngeneic and xenogeneic ovarian tumor grafts in C3HeB/FeJ mice . A murine ovarian teratocarcinoma cell line, utilized to study the mechanism of bacillus Calmette-Guerin-induced host resistance to a syngeneic ovarian tumor, proliferated when transplanted intraperitoneally . Marked tumoricidal activity was observed, however, when these murine ovarian teratocarcinoma cells were transplanted 8 days after intraperitoneal bacillus Calmette-Guerin inoculation . In studies related to xenogeneic ovarian tumor grafts, tumoricidal activity was observed after intraperitoneal transplantation of a human epithelial ovarian cancer cell line, NIH:OVCAR-3 . This cell line proliferates only in athymic nude (immunologically incompetent) mice . In both sets of experiments, tumoricidal activity was reduced by inhibition of nitric oxide synthesis . These results demonstrate the tumoricidal action of nitric oxide in vivo. J Mol Biol, 1994 Sep 23, 242(3), 193 - 202 Cavity mutants of Savinase . Crystal structures and differential scanning calorimetry experiments give hints of the function of the buried water molecules in subtilisins; Pedersen JT et al.; The subtilisin molecule possesses several internal water molecules, which may be characterised as an integral part of the protein structure . We have introduced specific mutations (T71I, T71S, T71V, T71A and T71G) at position 71 in the subtilisin variant Savinase from Bacillus lentus . This position is involved in a hydrogen bonded network with several internal water molecules, forming a water channel . The water channel and most of the other internal water molecules are positioned in the interface between two half-domains of the subtilisin molecule . The data presented here indicate that the internal water molecules are structural, and may be the result of trapping during the folding process. FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 43 - 8 Characterization of the type strain of Bacillus thuringiensis subsp . cameroun serotype H32; Juarez-Perez VM et al.; The strain 273 B, the type strain of a H serotype of Bacillus thuringiensis not yet characterized: B . thuringiensis subsp . cameroun, serotype H32, was isolated from soil samples collected in Cameroon . This strain produces cuboidal parasporal bodies composed of two major proteins of 53 kDa and 35 kDa . N-terminal sequences of the major proteins share no homology with published sequences . Only the 35 kDa protein is susceptible to digestion by trypsin . A complex array of 9 plasmids was revealed. Gene, 1994 Sep 15, 147(1), 1 - 11 Biosynthesis of butirosin in Bacillus circulans NRRL B3312: identification by sequence analysis and insertional mutagenesis of the butB gene involved in antibiotic production; Aubert-Pivert E et al.; As an approach to an analysis of the biosynthesis of the aminoglycoside antibiotic butirosin (But), we investigated the chromosomal regions flanking the ButR gene (aphA4/butA) of Bacillus circulans NRRL-B3312, and have identified, by nucleotide sequence analysis, a large open reading frame (ORF; ButB) upstream from the ButR gene . Hybridization was detected between butB and chromosomal DNA from other Bacillaceae that produce But-like compounds (but not from non-producers) . Interruption of this sequence by insertion of an erythromycin-resistance-encoding gene (erm) at either of two distinct sites eliminated the production (biosynthesis or export) of But, thus indicating a role for butB in antibiotic production . Gene butB is transcribed in the same direction as butA and encodes a protein of 1616 amino acid (aa) residues with a 30-aa N-terminal signal peptide . Comparison of the sequence for the translation product (ButB) with the aa compositions and sequences of known bacterial surface proteins, such as S-layer proteins, suggests that this protein is cell-wall associated . It is proposed that ButB plays a role in the export of But from the producing organism. Biochem J, 1994 Sep 1, 302 ( Pt 2), 611 - 6 Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC delta-endotoxin affects insecticidal specificity; Smith GP et al.; Site-directed mutagenesis was used to determine the role of two surface-exposed loops (Gly-317-Phe-320 and Gln-374-Pro-377) in the insecticidal specificity of the Bacillus thuringiensis CryIC delta-endotoxin . Mutant toxins were generated by PCR using degenerate oligonucleotide primers, and expressed in Escherichia coli . More than 50 mutant toxins were screened for toxicity to the lepidopteran Spodoptera frugiperda Sf9 cell line using an in vitro lawn assay . A panel of these mutant toxins, which included toxic and non-toxic variants from both loops, was further screened for activity towards Aedes aegypti larvae . The activity of these mutants to Sf9 cells was quantified more precisely using a cell lysis assay . Three categories of mutants were identified: (1) those non-toxic to either Sf9 cells or Aedes aegypti larvae; (2) those fully toxic to both genera; and (3) those which were only toxic to Sf9 cells . For the first loop, the differential specificity was not restricted to any single residue . In the second loop, two mutant toxins with a Pro-377-->Ala substitution displayed this phenotype . The time dependence of toxicity towards Sf9 cells was examined using the same panel of mutants . All toxic mutants displayed an identical time course to the wild-type toxin, with the exception of the two Pro-377-->Ala mutants of the second loop . These toxins displayed a lower time dependence, no cell death occurring within the first hour of incubation . These results show that the two loops are important determinants of both the activity and specificity of the CryIC delta-endotoxin. J Bacteriol, 1994 Sep, 176(18), 5654 - 64 Trehalose-6-phosphate hydrolase of Escherichia coli; Rimmele M et al.; The disaccharide trehalose acts as an osmoprotectant as well as a carbon source in Escherichia coli . At high osmolarity of the growth medium, the cells synthesize large amounts of trehalose internally as an osmoprotectant . However, they can also degrade trehalose as the sole source of carbon under both high- and low-osmolarity growth conditions . The modes of trehalose utilization are different under the two conditions and have to be well regulated (W . Boos, U . Ehmann, H . Forkl, W . Klein, M . Rimmele, and P . Postma, J . Bacteriol . 172:3450-3461, 1990) . At low osmolarity, trehalose is transported via a trehalose-specific enzyme II of the phosphotransferase system, encoded by treB . The trehalose-6-phosphate formed internally is hydrolyzed to glucose and glucose 6-phosphate by the key enzyme of the system, trehalose-6-phosphate hydrolase, encoded by treC . We have cloned treC, contained in an operon with treB as the promoter-proximal gene . We have overproduced and purified the treC gene product and identified it as a protein consisting of a single polypeptide with an apparent molecular weight of 62,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme hydrolyzes trehalose-6-phosphate with a Km of 6 mM and a Vmax of at least 5.5 mumol of trehalose-6-phosphate hydrolyzed per min per mg of protein . The enzyme also very effectively hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, but it does not recognize trehalose, sucrose, maltose, isomaltose, or maltodextrins . treC was sequenced and found to encode a polypeptide with a calculated molecular weight of 63,781 . The amino acid sequence deduced from the DNA sequence shows homology (50% identity) with those of oligo-1,6-glucosidases (sucrase-isomaltases) of Bacillus spp . but not with those of other disaccharide phosphate hydrolases . This report corrects our previous view on the function of the treC gene product as an amylotrehalase, which was based on the analysis of the metabolic products of trehalose metabolism in whole cells. Ann Emerg Med, 1994 Sep, 24(3), 418 - 21 Exposure of emergency department personnel to tuberculosis: PPD testing during an epidemic in the community; Sokolove PE et al.; STUDY OBJECTIVE: The present epidemic of tuberculosis has increased the risk of transmission of tuberculosis to health care workers in general and emergency department staff in particular, who often treat patients with tuberculosis before their diagnosis . The purpose of this study was to determine the risk of tuberculosis exposure among the nursing and physician staff of an urban ED . DESIGN: Observational study of self-reported purified protein derivative (PPD) skin test results and tuberculosis exposure . SETTING: Urban, public ED . PARTICIPANTS: Attending physicians, resident physicians, and registered nurses . INTERVENTIONS: None . RESULTS: Questionnaires were sent to all attending physicians, resident physicians, and registered nurses in the ED at Harbor-UCLA Medical Center requesting information on the subject's present and prior PPD status, known exposure to tuberculosis, and duration of time and average number of hours worked in the ED . Ninety-six of 129 questionnaires (74%) were returned . Five of the respondents had been immunized with Bacillus of Calmette and Guerin vaccine (BCG) and 10 of the respondents were PPD positive before beginning work in the ED . Of the other 81 respondents, 31% (25 of 81) had become PPD positive while working in the ED . The majority of these conversions (15 of 25) occurred in the first 6 months of 1993 . A Kaplan-Meier survival analysis revealed nearly a 40% risk of PPD conversion after 60 months of full-time work in the ED . CONCLUSION: As measured by self-reported PPD status, a high rate of exposure to tuberculosis has been observed among the ED staff at Harbor-UCLA Medical Center . The highest rate of PPD conversion has been noted most recently, suggesting that there has been a significant increase in staff exposure to tuberculosis during 1992 and the beginning of 1993 . Systematic monitoring of PPD conversion rates among ED staff is necessary to determine the adequacy of ED respiratory isolation procedures during the current tuberculosis epidemic. J Bacteriol, 1994 Sep, 176(17), 5571 - 3 The zymogen of the protease that degrades small, acid-soluble proteins of spores of Bacillus species can rapidly autoprocess to the active enzyme in vitro; Illades-Aguiar B et al.; The zymogen of the protease (GPR) that initiates protein degradation during spore germination in Bacillus species is not activated in vitro under normal physiological conditions . However, there is rapid, acid-pH-dependent, zero-order, proteolytic activation of the purified zymogen in high concentrations of dimethyl sulfoxide . These findings provide further evidence that GPR activates itself during sporulation. J Bacteriol, 1994 Sep, 176(17), 5554 - 9 Identification of amino acid residues of Bacillus thuringiensis delta-endotoxin CryIAa associated with membrane binding and toxicity to Bombyx mori; Lu H et al.; Alanine substitution (A3) or deletion (D3) of residues 365 to 371 of Bacillus thuringiensis CryIAa insect toxin removed nearly all toxicity for Bombyx mori (> 1,000-fold less active than the wild type) . The loss of larvicidal activity in the mutants was not caused by increased sensitivity to larval gut enzymes but could be attributed to significantly reduced binding to B . mori brush border membrane vesicles . Some or all of the affected amino acid residues may interact directly or indirectly with the B . mori membrane receptor(s) . Such receptor binding appears to be directly correlated with insect toxicity. J Appl Bacteriol, 1994 Sep, 77(3), 256 - 63 Studies on the Bacillus flora of milk and milk products; Crielly EM et al.; Bacillus licheniformis and B . cereus were the most commonly isolated species of Bacillus found in milk at all stages of processing . Bacillus licheniformis was ubiquitous in the farm environment and counts in raw milks heat-treated in the laboratory were higher during the winter months, whilst B . cereus was associated with cattle feed throughout the year, and tended to be more common in raw milks during the summer months . Although B . licheniformis was usually isolated in larger numbers than B . cereus, this pattern changed after raw and pasteurized milks and reconstituted milk powders were pre-incubated at ambient temperatures, and B . cereus came to dominate the Bacillus population, reaching levels associated with enterotoxin production . Investigation of the growth kinetics of strains of both species showed that B . cereus grew faster than B . licheniformis at ambient temperatures . It is suggested that post-pasteurization contamination, which is commonly blamed for spoilage of milk and milk products by B . cereus, is not necessarily the most important source of this organism. Acta Urol Belg, 1994 Sep, 62(3), 63 - 8 Evolution of cellular and humoral response against Tuberculin and antigen 85 complex during intravesical treatment with BCG of superficial bladder cancer; Zlotta A et al.; Prophylactic treatment with Bacillus Calmette-Guerin (BCG) is an established and effective therapy of bladder cancer . The antitumor effect of BCG seems to be largely related to cellular immunological mechanisms, although its precise mode of action is unknown . Antitumor response of BCG seems to be initiated by the attachment of BCG to bladder wall via Fibronectin (FN) . The cellular immune response against Tuberculin PPD and the major secreted BCG antigen (Fibronectin-binding AG 85 complex) has been tested in a control group of 20 untreated bladder tumor patients and before and after 6 weekly intravesical BCG instillations in a group of 20 superficial bladder tumor patients . A major increase in the lymphoproliferative response against PPD and AG 85 was observed in respectively 66% and 57% of the treated patients . In contrast, no detectable antibody response (IgA, IgM, IgG) was observed against AG 85 complex after BCG treatment . On the other hand, antibodies against Tuberculin increased in 13 of 20 patients . This study seems to demonstrate a specific cellular immune activation against AG 85 Fibronectin-binding complex during BCG treatment of superficial bladder tumors . Humoral response against the AG 85 is not activated after BCG treatment . Further studies are needed to elucidate the role of AG 85 in the cellular intravesical penetration of BCG . Presence or absence of cellular response against this antigen could be of clinical value. Trends Genet, 1994 Sep, 10(9), 328 - 33 Cell polarity in yeast; Chant J; Cell polarity is fundamental to the development and functioning of all organisms, from bacteria to humans . Examples of processes that involve cell polarity include the growth of axons, the interaction between T cells and their targets, the formation of buds by yeast, and sporulation in Bacillus spp . Recent work on budding yeast has provided valuable insights into the molecular machinery responsible for establishing and orienting cell polarity . Comparisons of the DNA sequences of genes involved in such pathways have raised the possibility that these mechanisms are conserved in all eukaryotic cells. Can J Microbiol, 1994 Sep, 40(9), 782 - 6 Transformation of Bartonella bacilliformis by electroporation; Grasseschi HA et al.; Bartonella bacilliformis is a member of the order Rickettsiales, family Bartonellaceae . The bacterium is an intracellular parasite of human erythrocytes . To date, members of the family Bartonellaceae have not been transformed by standard chemical methods . We report the first successful transformation of a member of the Bartonellaceae family, B . bacilliformis, by the method of electroporation . The optimal conditions for electroporation of B . bacilliformis include a field strength of 12.5 kV/cm and a time constant of 5 ms using 0.2-cm cuvettes . With these parameters and the cosmid pEST (RK2 replicon), a transformation efficiency of 7.8 x 10(5) was obtained . Transformants were readily cultured on medium containing kanamycin sulfate at concentrations ranging from 15 to 600 micrograms/mL . Bacterial survival was approximately 31% under optimal electroporation conditions, and the maximal number of transformants was obtained with 80 ng of pEST DNA . Bartonella bacilliformis was verified as the transformed organism by a comparison of transformant protein profiles with those of wild-type B . bacilliformis using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and detection of the exogenous plasmid in DNA from the transformed bacteria by DNA hybridization . Transformations using the plasmids pMK20, pML31, and pUCK18 (containing the replicons ColE1, F, and pMB1, respectively) were not successful. Appl Environ Microbiol, 1994 Sep, 60(9), 3096 - 104 Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus; Takata H et al.; Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood . The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli . The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method . About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C . The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5 . The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis . The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family . When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared . The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase . On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed. J Antibiot (Tokyo), 1994 Sep, 47(9), 959 - 68 Bacithrocins A, B and C, novel thrombin inhibitors; Kamiyama T et al.; Novel thrombin inhibitors, bacithrocins A, B and C, have been isolated from the culture broth of Bacillus laterosporus Laubach NR 2988 . The structures of these inhibitors have been determined to be N-acyl-L-phenylalanyl-DL-arginal by the 2D-NMR experiments on their oxidation products and by amino acid analysis . Bacithrocin A inhibits thrombin, factor Xa and trypsin with IC50s of 48, 13 and 0.65 microM, respectively, which are similar to those of bacithrocins B and C . Bacithrocins prolong the clotting time induced by thrombin and factor Xa. Res Microbiol, 1994 Sep, 145(7), 503 - 18 Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria; Hueck CJ et al.; One form of catabolite repression (CR) in the Gram-positive genus, Bacillus, is mediated by a cis-acting element (CRE) . We use here a consensus sequence to identify such elements in sequenced genes of Gram-positive bacteria . These are analysed with respect to position and type of gene in which they occur . CRE sequences near the promoter region are mainly identified in genes encoding carbon catabolic enzymes, which are thus likely to be subject to CR by a global mechanism . Functional aspects of CREs are evaluated. Mol Microbiol, 1994 Sep, 13(6), 965 - 72 Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis; Wu D et al.; The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp . israelensis and the PG-14 isolate of B . thuringiensis subsp . morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which it is composed (CytA, CryIVA, B, and D) . This high toxicity is postulated to be due to synergistic interactions among parasporal proteins . However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae . In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CryIVD toxins were cloned and expressed in acrystalliferous B . thuringiensis subsp . israelensis cells using the shuttle vector pHT3101 . CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bioassays against first instars of Aedes aegypti . The LC50 for the CytA inclusion was 60 ng ml-1, whereas the LC50 for the CryIVD was 85 ng ml-1 . In comparison, the LC50s for different combinations of CytA and CryIVD inclusions ranged from 12-15 ng ml-1, 4-5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins . These results suggest that the high toxicity of the wild-type parasporal bodies of B . thuringiensis subspp . israelensis and morrisoni is due to synergism among three or four of their major proteins. Pept Res, 1994 Sep-Oct, 7(5), 238 - 41 A general approach for identifying and cloning peptide synthetase genes; Turgay K et al.; A wide variety of bioactive peptides are synthesized nonribosomally by large multienzyme complexes employing the thiotemplate mechanism . Based on the known and highly conserved structures of several genes encoding multifunctional peptide synthetases, we developed a universal polymerase chain reaction (PCR) approach for amplifying, cloning and identifying parts of putative peptide synthetase genes . We showed, by cloning fragments of peptide synthetase genes from the phaseolotoxin-producing strain Pseudomonas syringe pv . phaseolica and from Bacillus licheniformis, which produces the branched peptide antibiotic bacitracin, that this approach is applicable . It gives a new and potentially general access to the biosynthetic genes of many nonribosomally synthesized peptides. J Gastroenterol Hepatol, 1994 Sep-Oct, 9(5), 433 - 6 Spontaneous bacterial peritonitis and culture negative neutrocytic ascites in patients with non-alcoholic liver cirrhosis; al Amri SM et al.; Medical records of 18 patients with spontaneous bacterial peritonitis (SBP) and 19 patients with culture negative neutrocytic ascites (CNNA) were reviewed . The diagnosis of SBP was based on a positive ascitic fluid culture, a polymorphonuclear cell count (PMN) greater than 250 cells/mm3 and the absence of an intra-abdominal source of infection . The diagnosis of CNNA was based on a PMN count greater than 250 cells/mm3, a negative ascitic fluid culture, the absence of an intra-abdominal source of infection and no antibiotic treatment in the preceding 30 days . All patients in both groups had liver cirrhosis, which was mainly (62.2%) due to HBV infection . A single strain, mostly 'a Gram-negative' bacillus, was recovered from the ascitic fluid culture in the vast majority of patients (83%) with SBP . There were no significant differences between the clinical data of both groups . However, the CNNA group had a significantly better Pugh score (P value = 0.01) with a mean score of 9.42 +/- 2.24, compared to the SBP group (10.94 +/- 2.88) . The only significant difference in the laboratory data was that the total bilirubin was higher in the SBP group (P < 0.01) . Hospital mortality was significantly higher in the SBP patients compared to those with CNNA, 50 and 16%, respectively (P < 0.03) . Recurrent ascitic fluid infection occurred in one of five patients who initially presented . In contrast no recurrence was documented in 12 patients with CNNA . Spontaneous bacterial peritonitis is a serious complication of liver cirrhosis with significantly higher mortality than CNNA . A single organism, usually enteric, is the most common causative agent. Biokhimiia, 1994 Sep, 59(9), 1393 - 400 {Secreted serine proteinase from the spore-forming bacteria Bacillus intermedius 3-19}; Balaban NP et al.; Extracellular serine proteinase has been isolated from the cultural medium of Bacillus intermedius 3-19 using CM-cellulose chromatography and affinity chromatography on bacitracin-Sepharose . The specificity of the proteinase with a wide range of natural and synthetic substrates has been investigated . The greatest activity was observed with tripeptides containing C-terminal Leu or Phe . The enzyme was completely inhibited by diisopropylfluorophosphate and partly inhibited by thiol-specific reagents . It is concluded that B . intermedius proteinase is a thiol-dependent serine proteinase pertaining to the subtilisin group . The amino acid composition of the enzyme has been determined . The enzyme contains one to three 1/2 cysteine residues, one of which is supposedly a Cys residue . The N-terminal amino acid sequences of the protein is AQTVPYGIPQIKAPA-. Med Hypotheses, 1994 Sep, 43(3), 135 - 7 Bartonella bacilliformis or a similar organism and cardiovascular disease; Sood FH et al.; Bartonella bacilliformis invades the endothelial lining of the cardiovascular system . Damage to the red blood cells and white blood cells, the effects of the toxins, invasion of the brain and electrical charges induced by the organism so interfering with normal electrical stimulation of the heart may explain many of the features of cardiovascular disease (1-5). Clin Infect Dis, 1994 Sep, 19(3), 528 - 40 The International Tuberculosis Campaign: a pioneering venture in mass vaccination and research; Comstock GW; If an American pediatrician's conversation with Dr . Johannes Holm, a Danish pathisiologist and future director of the International Tuberculosis Campaign, had not been interrupted, the campaign would probably not have become a monumental precedent for world health activities . The International Tuberculosis Campaign was conducted under the auspices of the United Nations International Children's Emergency Fund and three Scandinavian voluntary organizations . In a program that started in the war-torn areas of Europe, nearly 30 million persons underwent tuberculin testing, and almost 14 million were given BCG (bacille Calmette-Guerin) vaccine . In addition, a postgraduate school for physicians was initiated, new laboratories were established and old ones were improved, hundreds of young doctors and nurses were introduced to international public health, and, perhaps most important, research and service were successfully integrated . The success of the campaign led to its becoming the first major disease control and research activity of the World Health Organization. Int J Food Microbiol, 1994 Sep, 23(1), 99 - 109 Characteristics of Bacillus cereus related to safe food production; Dufrenne J et al.; Thirty Bacillus cereus strains, isolated from different sources, were characterized in relation to safe food production . The minimal growth temperatures of the strains varied from < or = 5 degrees C to 11 degrees C . Generation times at 7 degrees C of strains capable of growing at temperatures < or = 5 degrees C were approximately 8.2 h . The D90 degrees C-values of spores of strains with a minimal growth temperature of 11 degrees C determined in phosphate buffer at pH 7.0 ranged from 4.8 to > 200 min . Strains with the capacity to grow at temperatures < or = 9 degrees C, had a D90 degrees C value ranging from 4.6 to 14 min . Addition of either nisin (250 micrograms/ml) or diacetyl (1500 micrograms/ml) to the heating menstruem at the single concentrations investigated seemed not influence the thermal destruction of spores . Germination of spores of almost all strains occurred in all three media tested (Brain Heart Infusion, rice extract and milk) even at temperatures below the minimal growth temperature . All B . cereus strains tested yielded positive results with a commercial test kit for diarrhoeal enterotoxin . The results indicate that strains with the capacity to grow at temperatures < or = 7 degrees C are not essentially different from those with minimal growth temperatures of > 10 degrees C. Int J Food Microbiol, 1994 Sep, 23(1), 111 - 6 Stability of spores of Bacillus cereus stored on silicagel; Dufrenne J et al.; Spores of four different strains of Bacillus cereus were stored on silicagel at 22 degrees C and in physiological saline solution at -20 degrees C for a period of 260 days . At different intervals the spores were tested for survival, heat sensitivity and capacity to germinate . There was no clear change in any of the parameters tested, so storage on silicagel can be a good alternative for storage as a frozen suspension . Spores stored in this way can easily be exchanged for reference material and used for microbiological challenge testing. Int J Food Microbiol, 1994 Sep, 23(1), 1 - 15 Bacillus cereus in infant foods and dried milk products; Becker H et al.; Dried milk products and infant food are known to be frequently contaminated with Bacillus cereus . Sources of the organism and its behaviour in the product and in the equipment during processing are discussed . With regard to the incidence of B . cereus in infant food, 261 samples distributed in 17 countries were collected and examined for its B . cereus content . Fifty-four percent of the samples were contaminated with B . cereus reaching levels from 0.3 to 600/g . Counts higher than 10/g were found in only 27 samples (10%) . Most of the positive samples (44%) contained 0.3 to 10 B . cereus/g . Four samples (1.5%) were contaminated with more than 100 organisms/g reaching a maximum level of 600 B . cereus/g . When classified into different types of products about 50% of the infant formulae based on milk, the follow-on formulae and the weaning foods were contaminated with B . cereus as well as 69% of those based on soy protein and 92% of the special dietetic foods . Compared to our earlier studies from 1982/83, the percentage of contaminated samples from Germany increased from 31% to 70% in the case of infant formulae, from 28% to 55% in the case of follow-on formulae, and from 40% to 100% in the case of special dietetic food . The percentage of weaning food contaminated with B . cereus remained nearly unchanged . It should be stressed, however, that the numbers of B . cereus were almost the same in both studies with the highest count in 1982/83 being 460 and in 1992 600/g . Samples naturally contaminated with counts of about 100 B . cereus/g were reconstituted and incubated at a room temperature of 27 degrees C . Levels of 10(5) B . cereus/g were reached after 7-9 h . Toxigenicity of B . cereus in dried milk products and infant food as well as food poisoning outbreaks attributed to B . cereus are discussed. J Am Mosq Control Assoc, 1994 Sep, 10(3), 403 - 6 Emergency control of Aedes aegypti in the Dominican Republic using the Scorpion 20 ULV forced-air generator; Tidwell MA et al.; In an effort to develop a more effective measure for use in emergency control of the dengue vector Aedes aegypti . applications of a combination of a larvicide (Bacillus thuringiensis israelensis {B.t.i.}) and an adulticide (permethrin) were made using a truck-mounted forced-air generator (Scorpion 20) and evaluated in the Dominican Republic . This method has the potential to simultaneously control adults and larvae . In bioassay cages placed in household water containers at the time of application, larval mortalities were 95.1 and 100% for 2 application rates of permethrin mixed with B.t.i . Adult mortalities were not as impressive, probably because of resistance to permethrin . Higher adult mortality in caged specimens (78.5%) and a substantial reduction in the natural population (68.4%) of Ae . aegypti were obtained following a 2.1-g AI/ha application of deltamethrin alone. J Am Mosq Control Assoc, 1994 Sep, 10(3), 363 - 73 Integrated management of waste tire mosquitoes utilizing Mesocyclops longisetus (Copepoda: Cyclopidae), Bacillus thuringiensis var . israelensis, Bacillus sphaericus, and methoprene; Tietze NS et al.; This study evaluated the compatibility and efficacy of using a predatory copepod, Mesocyclops longisetus in concert with 3 "biorational" compounds for mosquito control in waste tires . The toxicity of Bacillus thuringiensis var . israelensis (B.t.i), Bacillus sphaericus, and methoprene to Mesocyclops longisetus was assessed in the laboratory using concentrations 10 times the maximum labeled or suggested rate and based on a water depth of 7.6 cm . Microbials were tested using mature copepods exposed for durations of 24, 48, and 72 h . Methoprene bioassays consisted of individually exposing newly hatched copepods (i.e., nauplius larvae) and monitoring their development to maturity . The toxicity tests indicated B.t.i., B . sphaericus, and methoprene were not deleterious to copepods at concentrations exceeding those expected in the field . Copepods exposed to methoprene matured normally, and when mated, 50% developed egg sacs . A 5-month field test, integrating the copepod and B.t.i., B . sphaericus, and methoprene provided better mosquito reduction together than either copepods or control agents alone . When copepods were combined with B.t.i . or methoprene, overall reduction of 3rd- and 4th-instar larvae during the 5-month interval was equal to or greater than 90% . Bacillus thuringiensis var . israelensis alone temporarily produced a high degree of larval reduction (up to 100%), however reapplications were necessary to maintain that level of control . Of all the treatments, B . sphaericus alone produced the lowest degree of mosquito suppression due to lack of toxicity to Aedes albopictus, the predominant species during the study . It is recommended that mosquito control managers consider integrating M . longisetus and B.t.i . or methoprene against mosquitoes in waste tires. Toxicon, 1994 Sep, 32(9), 1125 - 36 Biochemical and morphological changes in rat muscle cultures caused by 28,000 mol . wt toxin of Bacillus thuringiensis israelensis; Cahan R et al.; The 28,000 mol . wt protein of Bacillus thuringiensis israelensis showed a high degree of toxicity to rat muscle in culture . Application of 1 microgram/ml to the culture medium completely inhibited cell fusion . Reversibility of this effect was demonstrated by replacement of the culture medium with fresh medium, and the consequence was that cell fusion was resumed . When differentiated myotubes were treated with 1 microgram/ml of the toxin, the spontaneous contractile activity was abolished within 20 min . Cytotoxic effects were observed 1 hr after treatment was initiated, as manifested by creatine kinase (CK) release to the medium . Two hours after toxin was applied to the muscle culture, the myotubes were deteriorated whereas the mononucleated cells were not affected . Six or 7-day-old cultures which were treated by 1 microgram/ml of 28,00 mol . wt toxin revealed a change in the levels of Na+ and K+ within the fibres as analysed by X-ray microanalysis (XRMA) . Preincubation of the toxin for 20 min with phospholipids before application to the cells reduced the cytotoxic effect . Phosphatidylinositol and phosphatidylserine were the most efficient inhibitors, whereas phosphatidylcholine, sphingomyelin and phosphatidylethanolamine were less effective in protecting cultures from the cytotoxic effects of the 28,000 mol . wt protein. Biosci Biotechnol Biochem, 1994 Sep, 58(9), 1738 - 41 Enzymatic conversion of dethiobiotin to biotin in cell-free extracts of a Bacillus sphaericus bioB transformant; Ohshiro T et al.; The activity of biotin synthase, responsible for biotin synthesis from dethiobiotin, was demonstrated in a completely defined reaction mixture with cell-free extracts of a Bacillus sphaericus bioB transformant . Among the sulfur compounds tested, only S-adenosyl-L-methionine was active, while L-methionine and L-cysteine had no significant effect . Protein concentrations higher than 15 mg/ml in the reaction mixture were needed to detect biotin synthase activity . When dialyzed cell-free extracts were used for the reaction, NADH, NADPH, or FAD among the well-known cofactors tested enhanced the activity, and Fe2+, Mn2+, and Ca2+ among the metal ions tested also had some effects. Biotechnology (N Y), 1994 Sep, 12(9), 915 - 8 Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management; Bosch D et al.; To obtain Bacillus thuringiensis crystal proteins with new properties and to identify the regions involved in insecticidal activity, we generated hybrid genes composed of cryIC and cryIE by in vivo recombination . Analysis of the hybrid proteins showed that domain III of CryIC is involved in the toxicity towards Spodoptera exigua and Mamestra brassicae . Transfer of this domain to CryIE, which is not active against these insects, resulted in a new protein with a broader activity . This hybrid protein binds to different receptors than CryIC, suggesting its use as an alternative for CryIC in resistance management programs. Biochem Cell Biol, 1994 Sep-Oct, 72(9-10), 397 - 402 Effect of the antirheumatic agent Tenidap on CD3, CD4, and CD8 expression and interleukin-1 and leukotriene B4 secretion in human peripheral blood mononuclear cells; Conti P et al.; Thymocytes that express the complete CD3-T-cell receptor (TCR) complex are CD4- and CD8- . The CD4+ T-cell population can be subdivided into at least two quite distinct subsets, TH1 and TH2 cells, based upon cytokine expression . Interleukin-1 (IL-1) appears to be required for optimal proliferation of T cells in response to antigen and it seems that in the absence of IL-1, TH2 clones proliferate less in response to antigen . Tenidap is an antirheumatic agent that has an inhibitory effect on IL-1 production . In these studies, we show that isolated human peripheral blood mononuclear cells (PBMCs) treated in vitro with Tenidap (15 micrograms/mL) for 48-h incubations significantly (p < 0.05) enhanced the present of CD4+ expression compared with untreated cells (control), as determined by cytofluorimetric analysis . Lipopolysaccharide and Bacillus Calmette-Guerin were used as positive controls . When the cells were tested for CD3 or CD8 receptor expression, no differences were found between the untreated PBMCs and the treated (15 micrograms/mL Tenidap) cells . No change was found when cells were incubated for 72 h . Moreover, our data show a strong dose-dependent inhibitory effect of Tenidap (15 micrograms/mL) on IL-1 alpha, IL-1 beta, and leukotriene B4 secretion in PBMCs treated overnight . The increased CD4+ expression by Tenidap in PBMCs may suggest an important role for this new antirheumatic agent in immunity and may hold future therapeutic promise for diseases involving IL-1 and leukotriene B4 as mediators. Eur J Immunol, 1994 Sep, 24(9), 2237 - 42 Response of interleukin-6-deficient mice to tumor necrosis factor-induced metabolic changes and lethality; Libert C et al.; Whether interleukin (IL)-6 contributes to tumor necrosis factor (TNF)-induced lethal shock or whether,