Effect of glucose and citrate on α-amylase production in Bacillus licheniformis

Studies on the α-amylase synthesis were carried out with two strains of Bacillus licheniformis: a mutant strain 44MB82 and its selected variant 44MB82-G. The cells were cultivated in a nutrient medium containing glucose or citrate as a carbon source. The results obtained indicated that α-amylase was produced in both strains without inducers in the medium. The variant strain 44MB82-G, which synthesized α-amylase in the presence of 6% glucose, showed 58-fold increased α-amylase activity compared to strain 44MB82 grown in the presence of 2% glucose. When cells of both strains were cultivated in a medium with 1% citrate, α-amylase activity of the variant strain was 5 times higher than that of strain 44MB82. The effects of starch and ATP on α-amylase synthesis are also discussed.     

Purification and properties of α-amylase from Micrococcus varians

An extracellular α-amylase from Micrococcus varians was partially purified (63 fold) by ammonium sulphate precipitation followed by dialysis and separated by molecular exclusion into three components with molecular weights ranging from approximately 14,000 to 56,000. The amylase had a pH optimum of 7.0 and apparent Km of approximately 0.5 mg ml^-1 for starch. The enzyme activity was stimulated by Ca²⁺ and Mg²⁺ ions. Optimum temperature was 45 °C while there was a complete absence of activity at 70 °C in 20 min. Ethylene diaminetetra acetic acid (EDTA) and iodoacetic acid (IA) inhibited the activity of the enzyme.     

Formation of extracellular α-amylase by Bacillus subtilis in relation to guanosine polyphosphates

The kinetics of growth, extracellular α-amylase formation and pool sizes of guanosine polyphosphates (p)ppGpp and adenosine phosphates (ATP and AMP) were determined during discontinuous cultivation of Bacillus subtilis 44. The results indicate a positive involvement of (p)ppGpp in the regulation of the expression of the α-amylase gene.     

Bacillus subtilis α-amylase,lichenase, β-mannanase and xylanase: Screening for their induction by agro-industrial byproducts and precipitation by organic solvents

Current research focuses on the utilization of low value agro-industrial byproducts for targeting B. subtilis towards different exoenyzeme lines. This includes α-amylase, β-mannanase, xylanase, and lichenase. As an orientation step, growth and α-amylase activity were monitored in three different nutritional media. A medium which showed low levels of catabolite repression and spore development was selected as a basal fermentation medium. Different patterns of exoenzyme induction were obtained when beet pulp, corn cob, rice husk, wheat bran, and wheat straw were separately used to partially replace the nutrient contents of the selected medium. α-Amylase and lichenase were maximally expressed in the presence of corn cob or beet pulp. On the other hand, considerable levels of the four polysaccharide hydrolyzing enzymes were induced by wheat bran. β-mannanase and xylanase expression responded more significantly towards inducers than α-amylase and lichenase. The effects of five different organic solvents as precipitative agents on these exoenzymes were also studied.     

Production and control of extracellular α-amylase in Micrococcus varians

An extracellular α-amylase was induced in cultures of Micrococcus varians during growth in a liquid medium containing starch as sole carbon source. Synthesis of this enzyme was repressed by the addition of glucose or fructose to starch metabolizing cells and was induced in a glucose or fructose metabolizing culture by the addition of starch. Glycine was found to enhance growth and production of α-amylase while the least growth and enzyme activity was recorded in medium containing ammonium sulphate as nitrogen source.     

Appearance and distribution of laminin a chain isoforms and integrin α2, α3, α6, β1, and β4 subunits in the developing human small intestinal mucosa

Background: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. Methods: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins α2β1, α3β1,α6β1, and α6β4, four potential recptors, at the epithelial-mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. Results: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins α6β1 and α6β4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the α2β1 and α3β1 integrins was found time- and site-restricted. The α2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the α3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19–20-week-old crypts. Conclusions: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development. © 1995 Wiley-Liss, Inc.     

Polymeric chiral crown ethers, 7. Synthesis of polymers incorporating methyl glycosides of α-D-altrose, α-D-galactose, α-D-glucose and α-D-mannose residues via copolymerization of divinyl ethers

Polymers with chiral asymmetric crown ether units ( 5, 6, 7 and 8 ) were synthesized via cationic cyclopolymerization of methyl 2,3-bis{O-[2-(2-vinyloxyethoxy)ethyl]}-4,6-O-benzylidene α-D -altro-, α-D -galacto-, α-D -gluco- and α-D -manhopyranosides ( 1, 2, 3 and 4 ), respectively. The enantioselective transport of the methyl ester of phenylglycine (PhGlyOCH₃) and phenylalanine (PhAlaOCH₃) was examined through a bulk chloroform solution of chiral polymers from one aqueous solution to another. The transport rate of PhAlaOCH₃ was larger than that of PhGlyOCH₃ for every host polymer. For polymer 7 , the optical purity of PhAlaOCH₃ transported from one to the other phase was 12,6%, and the ratio of rate constants for the faster moving enantiomer A and the slower moving enantiomer B (k_A^*/k_B^*) was 1,48. The faster moving enantiomer was the L -isomer except for the systems polymer 7 ? PhAlaOCH₃ and polymer 8 ? PhAlaOCH₃. This enantioselectivity is caused by the diastereotopic faces of the crown ether units in the host polymers.     

Preparation and investigation of optically active polyesters from α-ethyl-α-phenyl-β-propiolactone, 1

α-Ethyl-α-phenyl-β-propiolactone ( 7 ) of an optical purity up to 95% was prepared; the resolution of the two optical antipodes was carried out with the optical active precursor ethyl 2-aminomethyl-2-phenylbutyrate ( 5 ), and the optical purity was determined by ¹H NMR analysis using europium salt shift reagents. The solution and thermal properties of the corresponding polyesters 8 , as obtained by anionic ring opening polymerization, were investigated. It was found that the inherent viscosity increased with the optical purity of the lactone. Distinct differences in the melting behaviour were found between the racemic and the optically active polymers; the ideal melting temperature T_M⁰ for the pure enantiomeric polymer was estimated to about 535 K.     

Murine T Cell Determination of Pregnancy Outcome: I. Effects of Strain, αβ T Cell Receptor, γδ T Cell Receptor,and γδ T Cell Subsets

PROBLEM: T cells bearing αβ T cell receptor (TcR) and γδ TcR are present at the fetomaternal interface, and the latter, which express surface activation markers, can react with fetal trophoblast cell antigens. What is the role of these cells? METHOD: Using stress-abortion-prone DBA/2-mated CBA/J and abortion-resistant C57/B16 mice, αβ, γδ, and CD8^+/- T cell subsets were measured in spleen and uterine decidua. The effect of immunization against abortion and administration of anti-TcR antibody in vivo was examined. Cytokine synthesis was measured by intracellular staining of Brefeldin A-treated cells. RESULTS: Abortion-prone matings showed an unexpected accumulation of γδ T cells beginning in the peri-implantation period and this was suppressed by immunization against abortion. The immunization deleted γδ T cells producing the abortogenic cytokines, TNF-α and γ-interferon, and increased production of the anti-abortive cytokines, IL-10 and transforming growth factor-β2 (TGF-β2). Immunization also boosted the number of αβ T cells which were present in the decidua as early as 2 days after implantation. In vivo injection of GL4 (anti-δ) depleted γδ T cells producing Th1 cytokines in the peri-implantation period, and prevented abortions, whereas H57 (anti-β) decreased the number of αβ T cells and led to 100% abortions. CD8⁺ T cells present in peri-implant decidua before onset of abortions were mostly αβ TcR⁺, although some were γδ⁺. Changes in γδ and αβ T cells in pregnancy were most dramatic in uterine tissue. CONCLUSION: Although decidual γδ T cells after formation of a distinct placenta and fetus produce anti-abortive TGF-β2-like molecules and IL-10, prior events can lead to abortion. High local production of TNF-α and γ-interferon develop during the peri-implantation phase because of an excessive increase in the Th1 cytokine⁺ subset of γδ cells; these cytokines may be contributed by other tissues in decidua, and the contribution of bioactive factors by γδ T cells may augment the cytokine pool. In contrast, αβ T cells (which may be inactivated by stress that causes abortions) may mediate the anti-abortive effect of alloimmunization. Alloimmunization involves a shift from a Th1 to a Th2 pattern in the γδ T cells in decidua.     

An immunohistochemical examination of the expression of E-cadherin, α- and β/γ-catenins,and α2- and β1-integrins in invasive breast cancer

This study examines the expression of the cell–cell adhesion molecules E-cadherin and its associated proteins, the catenins and the matrix–cell adhesion molecules β1- and α2-integrins, in primary invasive breast carcinoma. Expression was assessed immunohistochemically on frozen sections by semi-quantitative scoring of the intensity and proportion of immunoreactivity in 55 cases. Associations with each other and with other histological and prognostic features and survival were sought. There was a significant association between loss of E-cadherin expression and loss of α- and β/γ-catenin immunostaining. In 20 per cent of cases, membranous immunoreactivity with E-cadherin antibody was absent. Absent cytoplasmic expression of α- and β/γ-catenins was seen in 24 and 22 per cent of breast cancers, respectively. The intensity of reactivity with E-cadherin showed a significant association with histological grade (p = 0·002) and tumour type (p < 0·001). Lobular carcinomas frequently showed loss of expression of E-cadherin, as reported elsewhere; loss of catenin expression was also found in these tumours. α-Catenin intensity also showed a relationship with grade (p = 0·008) and with oestrogen receptor (ER) status (p = 0·006). β/γ-Catenin expression was not associated with other known prognostic factors. Forty-nine per cent and 42 per cent of cases showed no membrane immunostaining with β1- and α2-integrin, respectively, and co-ordinated loss of β1- and α2-integrin expression was found. Both β1- and α2-integrin expression were associated with histological grade (p = 0·003 and p = 0·031, respectively) and β1 immunoreactivity with tumour type (p = 0·010). None of the variables examined showed a statistically significant association with tumour size or lymph node stage, or with overall survival, although a trend was seen (p = 0·087) towards poorer survival of patients with tumours with absent or weak expression of β1-integrin. The expression of these markers is of biological interest, but appears to be of little additional use in predicting clinical behaviour. Copyright © 1999 John Wiley & Sons, Ltd.     

Antibacterial and Anticancer activity of ε ‐poly‐L‐lysine (ε ‐PL) produced by a marine Bacillus subtilis sp.

A marine Bacillus subtilis SDNS was isolated from sea water in Alexandria and identified using 16S rDNA sequence analysis. The bacterium produced a compound active against a number of gram negativeve bacteria. Moreover, the anticancer activity of this bacterium was tested against three different human cell lines (Hela S3, HepG2 and CaCo). The highest inhibition activity was recorded against Hela S3 cell line (77.2%), while almost no activity was recorded towards CaCo cell line. HPLC and TLC analyses supported evidence that Bacillus subtilis SDNS product is ?;‐poly‐L‐lysine. To achieve maximum production, Plackett‐Burman experimental design was applied. A 1.5 fold increase was observed when Bacillus subtilis SDNS was grown in optimized medium composed of g/l: (NH₄)₂SO₄, 15; K₂HPO₄, 0.3; KH₂PO₄, 2; MgSO₄ · 7 H₂O, 1; ZnSO₄ · 7 H₂O, 0; FeSO₄ · 7 H₂O, 0.03; glucose, 25; yeast extract, 1, pH 6.8. Under optimized culture condition, a product value of 76.3 mg/l could be obtained. According to available literature, this is the first announcement for the production of ?;‐poly‐L‐lysine (?;‐PL) by a member of genus Bacillus. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A TCR α chain transgene induces maturation of CD4− CD8− α β+ T cells from γ δ T cell precursors

The proportion of CD4⁻ CD8⁻ double-negative (DN) α β T cells is increased both in the thymus and in peripheral lymphoid organs of TCR α chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to α β and γ δ T cells. We show that the transgenic DN cells are phenotypically similar to γ δ T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCRα genes nor been negatively selected by the Mls^a antigen, suggesting that they originate from a differentiation stage before the onset of TCR α chain rearrangements and CD4/CD8 gene expression. Neither in-frame VδDδJδ nor VγJγ rearrangements are over-represented in this population. However, since peripheral γ δ T cells with functional TCRβ gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to γ δ lineage-committed precursors can be delivered via TCR α β heterodimers.     

Cationic polymerization of p-substituted α-methylstyrenes, 2. Crystalline polymers from p-methyl- and p-isopropyl-α-methylstyrene

Under identical conditions of cationic polymerization at low temperature 2-(p-tolyl)-1-propene (α,p-dimethylstyrene) gave polymers with much higher syndiotactic contents than unsubstituted α-methylstyrene. All samples of poly(α,p-dimethylstyrene) were crystalline, but poly(α-methylstyrene) did not crystallize even at high degrees of stereoregularity. Similarly polymers of p-isopropyl-α-methylstyrene were crystalline, and the crystalline properties of copolymers of the three monomers were investigated and compared to those of the homopolymers. The rôles of stereoregularity and crystallizability in forming crystalline products were considered.     

CD45RA expression on γδ and αβ T cells emigrating from the fetal and postnatal thymus

We have compared the expression of CD45RA on αβ and γδ T cells emigrating from the fetal and postnatal thymus. The fetal and postnatal thymus export both CD45RA⁺ and CD45RA^- T cells. The number of γδ⁺CD45RA⁺ T cells was remarkably constant regardless of stage of ontogeny or T cell maturity. Around 5--8% of γδ thymic emigrants, thymocytes and peripheral blood lymphocytes expressed CD45RA in both fetal and postnatal animals. In contrast to γδ T cells, up to one quarter of both fetal and postnatal αβ emigrants expressed CD45RA. Post-thymic maturation of CD45RA expression on αβ emigrants, which occurred both before and after birth, appeared to be antigen independent.     

Transendothelial chemotaxis of human αβ and γδ T lymphocytes to chemokines

Two subpopulations of human T lymphocytes expressing different antigen receptors, α / β and γ / δ, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of α / β and γ / δ T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β stimulated similar, dose-dependent chemotaxis of purified γ / δ T cells, whereas MCP-1, RANTES, and MIP-1α pro duced greater chemotaxis of purified α / β T cells than MIP-1β. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-γ inducible protein-10 (IP-10) did not promote chemotaxis of either α / β or γ / δ T cells. Three γ / δ T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mono nuclear cells confirmed the results with purified γ / δ T cells. Our data demonstrate that human peripheral blood α / β and γ / δ T cells can transmigrate to MCP-1, RANTES, MIP-1α, and MIP-1β, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.     

Polymerization of α,α-disubstituted β-propiolactones and lactams, 12. Properties and crystalline structure of poly(β-propiolactam)s

A series of α,α-disubstituted β-propiolactams (substituted nylon 3 polymers) was prepared from non-optically active lactams. Their thermal characteristics, densities, and x-ray fiber diffraction patterns were recorded. One of the α-substituents was always a methyl group while the other was either CH₃, C₂H₅, n-C₃H₇, or n-C₄H₉. The melting point for the dimethyl substituted polymer was 268°C; the others were respectively 76, 74, and 72°C. X-ray fiber diffraction data on all four samples yielded the same fiber repeat of ≈8,4 Å. A minimum energy based conformational analysis for the α,α-dimethyl derivative indicates that two conformations correspond to this repeat, each of which is a 2₁ helix.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.