Discovery of a new inhibitor lead of adenovirus proteinase: steps toward selective, irreversible inhibitors of cysteine proteinases

Using the computer docking program EUDOC, in silico screening of a chemical database for inhibitors of human adenovirus cysteine proteinase (hAVCP) identified 2,4,5,7-tetranitro-9-fluorenone that selectively and irreversibly inhibits hAVCP in a two-step reaction: reversible binding (Ki = 3.09 microM) followed by irreversible inhibition (ki = 0.006 s(-1)). The reversible binding is due to molecular complementarity between the inhibitor and the active site of hAVCP, which confers the selectivity of the inhibitor. The irreversible inhibition is due to substitution of a nitro group of the inhibitor by the nearby Cys122 in the active site of hAVCP. These findings suggest a new approach to selective, irreversible inhibitors of cysteine proteinases involved in normal and abnormal physiological processes ranging from embryogenesis to apoptosis and pathogen invasions.     

Compositional changes of cottonseed hull substrate during P. ostreatus growth and the effects on the feeding value of the spent substrate

The enzymic synthesis of alkyl-beta-glucosides by water-immiscible alcohols was studied in stirred flasks as well as in a tubular enzymatic reactor. In the first case, direct alkylation of beta-D-glucose from hexanol using immobilized beta-glycosidase gave a higher conversion yield and final product concentration than that using the enzyme in its free state (yield 10 against 8% mol/mol, concentration 2 against 1.6 g/l). Direct glycosylation of beta-D-glucose from hexanol resulted in a higher reaction performance (yield 10%) than that from octanol (yield 5%). However, the two different incubation temperatures tested (37 degrees C and 50 degrees C), showed no significant differences concerning final product concentrations. The more interesting results were obtained by transglycosylation of methyl-1-beta-glucose from hexanol, with a conversion yield of 21% mol/mol (product amount 4 g/l). However, the transgalactosylation of lactose from hexanol, catalyzed by a fungal beta-galactosidase, showed only a feeble reactivity. The feasibility of enzymic alkylation was also tested in a tubular enzymatic reactor; hexyl-1-beta-glucoside was produced via direct glycosylation from hexanol catalyzed by free beta-glycosidase with a final concentration 1.3-2.3 g/l and a yield varying between 11% and 20% mol/mol.     

Prolonged membrane potential depolarization in cingulate pyramidal cells after digit amputation in adult rats

The anterior cingulate cortex (ACC) plays an important role in higher brain functions including learning, memory, and persistent pain. Long-term potentiation of excitatory synaptic transmission has been observed in the ACC after digit amputation, which might contribute to plastic changes associated with the phantom pain. Here we report a long-lasting membrane potential depolarization in ACC neurons of adult rats after digit amputation in vivo. Shortly after digit amputation of the hind paw, the membrane potential of intracellularly recorded ACC neurons quickly depolarized from ~-70 mV to ~-15 mV and then slowly repolarized. The duration of this amputation-induced depolarization was about 40 min. Intracellular staining revealed that these neurons were pyramidal neurons in the ACC. The depolarization is activity-dependent, since peripheral application of lidocaine significantly reduced it. Furthermore, the depolarization was significantly reduced by a NMDA receptor antagonist MK-801. Our results provide direct in vivo electrophysiological evidence that ACC pyramidal cells undergo rapid and prolonged depolarization after digit amputation, and the amputation-induced depolarization in ACC neurons might be associated with the synaptic mechanisms for phantom pain.     

Nucleotide sequences in the RNA of mammalian leukemia and sarcoma viruses.

The nucleotide sequences in viral RNA from purified murine sarcoma and hamster leukemia viruses (S+H+) from HTG-1 cells and Rauscher leukemia virus (RLV) from JLS-V 9 cells have been examined by polynucleotide agarose affinity chromatography. There is at least one copy of poly(A) sequences per genomic viral RNA molecule. After heat denaturation of genomic viral RNA (S+H+), there are two types of viral subunits for 34S and 28S species: one that contains poly(A) sequences and one that does not. There are no detectable poly(U) tracts in the viral RNA. However, poly(C) sequences and poly(G) tracts were detected in viral RNA, although less poly(G) than poly(C) tracts were observed. In addition, heat-denatured genomic viral RNA has a greater affinity for poly(G) agarose column than native genomic viral RNA.     

Capsule Design for Blue Light Therapy against Helicobacter pylori

A photo-medical capsule that emits blue light for Helicobacter pylori treatment was described in this paper. The system consists of modules for pH sensing and measuring, light-emitting diode driver circuit, radio communication and microcontroller, and power management. The system can differentiate locations by monitoring the pH values of the gastrointestinal tract, and turn on and off the blue light according to the preset range of pH values. Our experimental tests show that the capsule can operate in the effective light therapy mode for more than 32 minutes and the wireless communication module can reliably transmit the measured pH value to a receiver located outside the body.     

Bone‐marrow mesenchymal stem cell transplantation to treat diabetic nephropathy in tree shrews

Diabetic nephropathy (DN) is a common microvascular complication of diabetes. We used a new DN model in tree shrews to validate the use of bone‐marrow mesenchymal stem cell (BM‐MSC) transplantation to treat DN. The DN tree shrew model was established by a high‐sugar and high‐fat diet and four injections of streptozotocin. 4',6‐Diamidino‐2‐phenylindole labelled BM‐MSCs were injected into tree shrews. The DN tree shrew model was successfully established. Blood glucose was significantly increased ( p < 0.01) during the entire experiment. DN tree shrews showed dyslipidemia, insulin resistance and increased 24‐h proteinuria. At 21 days after BM‐MSC transplantation, glucose and levels of triglycerides, total cholesterol and 24‐h urine volume were lower than in tree shrews with DN alone ( p < 0.01) but were still higher than control values ( p < 0.01). Levels of creatinine and urea nitrogen as well as 24‐h proteinuria were lower for DN tree shrews with BM‐MSCs transplantation than DN alone ( p < 0.05). High‐sugar and high‐fat diet combined with STZ injection can induce a tree shrew model of DN. BM‐MSCs injection can home to damaged kidneys and pancreas, for reduced 24‐h proteinuria and improved insulin resistance. Copyright © 2014 John Wiley & Sons, Ltd.     

Citrus Vascular Proteomics Highlights the Role of Peroxidases and Serine Proteases during Huanglongbing Disease Progression

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Highlights

• •HLB is the most devastating citrus disease associated with the pathogen CLas.
• •Proteases and peroxidases accumulated in plant vascular tissue after CLas infection.
• •Dynamic changes in serine protease activity occur across genotypes and environments.

     

In vivo functional analysis of the structural domains of FLUORESCENT (FLU)

The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme protochlorophyllide oxidoreductase (POR). The interruption of Chl synthesis during darkness requires suppression of the synthesis of 5-aminolevulinic acid (ALA), the first precursor molecule specific for Chl synthesis. The inactivation of glutamyl-tRNA reductase (GluTR), the first enzyme in tetrapyrrole biosynthesis, accomplished the decreased ALA synthesis by the membrane-bound protein FLUORESCENT (FLU) and prevents overaccumulation of protochlorophyllide (Pchlide) in the dark. We set out to elucidate the molecular mechanism of FLU-mediated inhibition of ALA synthesis, and explored the role of each of the three structural domains of mature FLU, the transmembrane, coiled-coil and tetratricopeptide repeat (TPR) domains, in this process. Efforts to rescue the FLU knock-out mutant with truncated FLU peptides revealed that, on its own, the TPR domain is insufficient to inactivate GluTR, although tight binding of the TPR domain to GluTR was detected. A truncated FLU peptide consisting of transmembrane and TPR domains also failed to inactivate GluTR in the dark. Similarly, suppression of ALA synthesis could not be achieved by combining the coiled-coil and TPR domains. Interaction studies revealed that binding of GluTR and POR to FLU is essential for inhibiting ALA synthesis. These results imply that all three FLU domains are required for the repression of ALA synthesis, in order to avoid the overaccumulation of Pchlide in the dark. Only complete FLU ensures the formation of a membrane-bound ternary complex consisting at least of FLU, GluTR and POR to repress ALA synthesis.     

Ribosomal L22-like1 (RPL22L1) Promotes Ovarian Cancer Metastasis by Inducing Epithelial-to-Mesenchymal Transition

Double minute chromosomes (DMs) have important implications for cancer progression because oncogenes frequently amplified on them. We previously detected a functionally undefined gene amplified on DMs, Ribosomal L22-like1 (RPL22L1). The relationship between RPL22L1 and cancer progression is unknown. Here, RPL22L1 was characterized for its role in ovarian cancer (OC) metastasis and its underlying mechanism was examined. DNA copy number and mRNA expression of RPL22L1 in OC cells was analyzed using data obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database. An immunohistochemical analysis of clinical OC specimens was performed and the relationships between expression level and clinicopathological factors were evaluated. Additionally, in vivo and in vitro assays were performed to understand the role of RPL22L1 in OC. RPL22L1 expression was higher in OC specimens than in normal tissues, and its expression level was highly positively correlated with invasion and lymph node metastasis (P < 0.05). RPL22L1 over-expression significantly enhanced intraperitoneal xenograft tumor development in nude mice and promoted invasion and migration in vitro. Additionally, RPL22L1 knockdown remarkably inhibited UACC-1598 cells invasion and migration. Further, RPL22L1 over-expression up-regulated the mesenchymal markers vimentin, fibronectin, and α-SMA, reduced expression of the epithelial markers E-cadherin, α-catenin, and β-catenin. RPL22L1 inhibition reduced expression of vimentin and N-cadherin. These results suggest that RPL22L1 induces epithelial-to-mesenchymal transition (EMT). Our data showed that the DMs amplified gene RPL22L1 is critical in maintaining the aggressive phenotype of OC and in triggering cell metastasis by inducing EMT. It could be employed as a novel prognostic marker and/or effective therapeutic target for OC.