产几丁质酶褐色喜热裂孢菌的分离鉴定及产酶特性初步研究

采用选择性培养基从土壤中分离到1株产几丁质酶的微生物菌株YX,经形态和分子鉴定为褐色喜热裂孢菌(Thermobifida fusca)。进一步在摇瓶中比较了T.fusca YX在纤维二糖、几丁质、或羧甲基纤维素钠为碳源的培养基中的产酶特性,YX菌株在5 L发酵罐中以几丁质为碳源的培养基发酵到22 h左右时发酵液几丁质酶活即可达到1.7 U/m L。本文首次报道褐色喜热裂孢菌能够产生几丁质酶,具有潜在的应用价值。     

Binding of Thermobifida fusca CDCel5A, CDCel6B and CDCel48A to easily hydrolysable and recalcitrant cellulose fractions on BMCC

The binding of the catalytic domain of Thermobifida fusca endoglucanase (EC 3.2.1.4) CD_Cel5A and exoglucanases (EC 3.2.1.91) CD_Cel6B and CD_Cel48A to BMCC was studied. At 5 °C, the binding of these CDs to BMCC is rapid with maximum binding occurring just after CD loading. An observed desorption of the bound CDs with time was attributed to the loss of binding sites due to hydrolysis of the easily hydrolysable BMCC. This conclusion was supported by prehydrolysis experiments where the easily hydrolysable BMCC fraction was removed, and binding to the recalcitrant fraction was observed. More of the CDs were bound to the recalcitrant fraction. This was especially true for the exoglucanases, CD_Cel6B and CD_Cel48A, where 81–76% of the total CD binding was to the recalcitrant BMCC fraction. The binding to the easily hydrolysable fraction for the endo CD_Cel5A was 1.5–4 times higher than that of the exo CD_Cel6B and CD_Cel48A at the same enzyme concentration. Although CD_Cel5A saturated the easily hydrolysable fraction of BMCC first, the ratio of the bound to the recalcitrant substrate was constant for CD_Cel6B and CD_Cel48A as 81 and 76%, respectively. As the reaction temperature was increased to 50 °C, CD_Cel5A and CD_Cel48A exhibited a rapid increase in the rate and extent of binding, while CD_Cel6B exhibited a decrease in the extent of binding.     

Involvement of interleukin-1β mediated nuclear factor κB signalling pathways to down-regulate prostate-specific antigen and cell proliferation in LNCaP prostate cancer cells

Involvement of NF-κB (nuclear factor κB) mediated by IL-1β (interleukin-1β) on cell proliferation and PSA (prostate-specific antigen) production of LNCaP prostate cell lines and the possible cross-talk with Akt (also known as protein kinase B) signalling pathway has been investigated. NF-κB and Akt were analysed by Western blotting from LNCaP cells treated by IL-1β before proliferation and PSA production were measured. IL-1β inhibited proliferation and decreased PSA production. The Akt pathway was not sensitive, whereas NF-κB phosphorylation occurred as a result of treatment. PSA production and proliferation of LNCaP cells were down-regulated by NF-κB mediated by IL-1β promoting anti-apoptotic signalling and co-suppressor factors of PSA expression. IL-1β through NF-κB activation provides a rationale for therapeutic approaches in the anticancer treatment of prostate.     

Studies on intramolecular hydrogen bonding between the pyridine nitrogen and the amide hydrogen of the peptide: synthesis and conformational analysis of tripeptides containing novel amino acids with a pyridine ring.

For the first time tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1) and AA(3) = Gly or Aib, Xaa = 2Pmg and 2Pyg) were prepared containing alpha-methyl-alpha-(2-pyridyl)glycine (2Pmg) and alpha-(2-pyridyl)glycine (2Pyg) by solid-phase Ugi reaction. These results clearly indicate that for the preparation of tripeptides containing an amino acid with a pyridine ring, the solid-phase Ugi reaction is very useful.NMR analysis clarified that 2Pmg-containing tripeptides adopt a unique conformation with an intramolecular hydrogen bond between 2Pmg-NH and the pyridine nitrogen. However, in the case of Z-Gly-2Pyg-Gly-OMe, the intramolecular hydrogen bonding between 2Pyg-NH and the pyridine nitrogen was not observed, whereas Z-Aib-2Pyg-Aib-OMe adopts a unique conformation with an intramolecular hydrogen bond between 2Pyg-NH and a pyridine nitrogen. Conformational analysis of the tripeptides, Z-AA(1)-Xaa-AA(3)-OMe (AA(1), AA(3) = Gly or Aib, Xaa = alpha,alpha-di(2-pyridyl)glycine (2Dpy), alpha-phenyl-alpha-(2-pyridyl)glycine (2Ppg), 2Pmg and 2Pyg), clarified that when an alpha,alpha-disubstituted glycine with a 2-pyridyl group at an alpha-carbon atom is introduced into any peptide, an intramolecular hydrogen bond between a pyridine nitrogen and an amide proton is formed and conformational mobility of the peptide backbone is restricted.     

Purification and characterization of an exo-type β-N-acetylglucosaminidase from Pseudomonas fluorescens JK-0412

Aims: To purify and characterize an exo‐acting chitinolytic enzyme produced from a Gram‐negative bacterium Pseudomonas fluorescens JK‐0412. Methods and Results: A chitinolytic bacterial strain that showed confluent growth on a minimal medium containing powder chitin as the sole carbon source was isolated and identified based on a 16S ribosomal DNA sequence analysis and named Ps. fluorescens JK‐0412. From the culture filtrates of this strain, a chito‐oligosaccharides‐degrading enzyme was purified to apparent homogeneity with a molecular mass of 50 kDa on SDS–PAGE gels. The kinetics, optimum pH and temperature, and substrate specificity of the purified enzyme (named as NagA) were determined. Conclusions: An extracellular chitinolytic enzyme was purified from the Ps. fluorescens JK‐0412 and shown to be an exo‐type β‐N‐acetylglucosaminidase yielding GlcNAc as the final product from the natural chito‐oligosaccharides, (GlcNAc)_n, n = 2–5. Significance and Impact of the Study: As NagA is secreted extracellularly in the presence of colloidal chitin, Ps. fluorescens JK‐0412 can be recognized as a potent producer for industry‐level and cost‐effective production of chitinolytic enzyme. This enzyme appears to have potential applications as an efficient tool for the degradation of chitinous materials and industry‐level production of GlcNAc. To the best of our knowledge, this is the first report on an exo‐type chitinolytic enzyme of Pseudomonas species.     

Chirality transferring [3,3] sigmatropic rearrangement of (1-Acyloxy-2-alkenyl)trianlkylsilane synthesis of optically active vinylsilane-containing alpha-amino acid

A vinylsilane-containing alpha-amino acid and alpha,alpha-disubstituted alpha-amino acid 2 having two contiguous asymmetric carbon centers at their alpha and beta positions were synthesized in an optically active form by ester-enolate Claisen rearrangement of the alpha-acyloxysilane 1 as the key step, where the chirality of an alpha-acyloxy-TBDMS group was completely transferred to the rearranged product.     

A new active vitamin D analog,ED-71, causes increase in bone mass with preferential effects on bone in osteoporotic patients

As a candidate for active vitamin D analogs that have selective effects on bone, 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71) has been synthesized and is currently under clinical trials. In ovariectomized rat model for osteoporosis, ED-71 caused an increase bone mass at the lumbar vertebra to a greater extent than 1alpha-hydroxyvitamin D3 (alfacalcidol), while enhancing calcium absorption and decreasing serum parathyroid hormone levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers.An early phase II clinical trial was conducted with 109 primary osteoporotic patients. The results indicate that oral daily administration of ED-71 (0.25, 0.5, 0.75, and 1.0 microgram) for 6 months increased lumbar bone mineral density in a dose-dependent manner without causing hypercalcemia and hypercalciuria. ED-71 also exhibited a dose-dependent suppression of urinary deoxypyridinoline with no significant reduction in serum osteocalcin. These results demonstrate that ED-71 has preferential effects on bone with diminished effects on intestinal calcium absorption. ED-71 offers potentially a new modality of therapy for osteoporosis with selective effects on bone.     

BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response

Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 α (IRE1α). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.