Synthesis and in vitro α-chymotrypsin inhibitory activity of 6-chlorobenzimidazole derivatives

A library of benzimidazole derivatives 1–20 were synthesized, and studied for their α-chymotrypsin (α-CT) inhibitory activity in vitro. Kinetics and molecular docking studies were performed to identify the type of inhibition. Compound 1 was found to be a good inhibitor of α-chymotrypsin enzyme (IC₅₀ = 14.8 ± 0.1 μM, K_i = 16.4 μM), when compared with standard chymostatin (IC₅₀ = 5.7 ± 0.13 μM). Compounds 2–8, 15, 17, and 18 showed significant inhibitory activities. All the inhibitors were found to be competitive inhibitors, except compound 17, which was a mixed type inhibitor. The substituents (R) in para and ortho positions of phenyl ring B, apparently played a key role in the inhibitory potential of the series. Compounds 1–20 were also studied for their cytotoxicity profile by using 3T3 mouse fibroblast cells and compounds 3, 5, 6, 8, 12–14, 16, 17, 19, and 20 were found to be cytotoxic. Molecular docking was performed on the most active members of the series in comparison to the standard compound, chymostatin, to identify the most likely binding modes. The compounds reported here can serve as templates for further studies for new inhibitors of α-chymotrypsin and other chymotrypsin-like serine proteases enzymes.

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Asymmetric acetalation of α,α-trehalose: Synthesis of α-d-galactopyranosyl α-d-glucopyranoside and 6-deoxy-6-fluoro-α-d-glucopyranosyl α-d-glucopyranoside

Selective acetalation of α,α-trehalose with ethyl or methyl isopropenyl ether and toluene-p-sulphonic acid in N,N-dimethylformamide gave the 4,6-isopropylidene acetal as the major product, isolated as its hexa-acetate 1 (38%). The gluco-galacto analogue 6 of α,α-trehalose was synthesized from 1 by the sequence: hydrolysis of the isopropylidene group with trifluoroacetic acid, mesylation of the resulting diol, benzoate displacement, and saponification of the product. Deacetylation of 1 followed by benzylation and hydrolysis of the acetal group furnished a hexa-O-benzyl derivative 9. Tosylation of the primary hydroxyl group in 9, treatment of the product with tetrabutylammonium fluoride in acetonitrile, and subsequent catalytic hydrogenolysis of the benzyl groups gave 6-deoxy-6-fluoro-α,α-trehalose (12). Compounds 6 and 12 and 6-deoxy-6-iodo-α,α-trehalose are substrates for cockchafer trehalase, but have very low V_max values.     

Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1(-/-) mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-β1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis.     

Expression of α2, α5 and α6 subunits of integrin in de-differentiated NIH3T3 cells by cell-free extract of embryonic stem cells

Generation of patient specific stem cells is among the ultimate goals in regenerative medicine. Such a cell needs to be functional when it transplants. Interaction between the matrix proteins and integrin adjust many cells' function such as adhesion, migration, cell cycle and self renewal in stem cells. In this study, NIH3T3 cells were dedifferentiated by mouse Embryonic Stem Cell (mESC) extract. The expression of pluripotency markers as well as a2, a5 and a6 integrin subunits were determined. NIH3T3 cells treated with mESC extract showed noticeable changes in cell morphology as early as day 2 post-treatment forming colonies similar to typical mESC morphology by day 8, after three passages. Alkaline phosphatase (ALP) assay and immunocytochemistry staining were performed for the induced reprogrammed cells. The results indicated that these colonies showed the ALP activity and they express Sox2 and Nanog. RT-PCR revealed that the colonies also express Oct3/4. NIH3T3 cells, ESC and reprogrammed cells expressed a2 integrin. a5 integrin expression was greatest in reprogrammed cells followed by the expression of this integrin in NIH3T3 which in turn was more than in ESC. a6A integrin was expressed in NIH3T3 cells while a6B integrin was expressed in ESC and in very low quantity was expressed in reprogrammed cells. These data provide evidence for both the generation of ES like cells from differentiated somatic cells and the expression profile of integrins after de-differentiation by mESC extract.     

Unexpected role of α-fetoprotein in spermatogenesis

Background

Heat shock severely affects sperm production (spermatogenesis) and results in a rapid loss of haploid germ cells, or in other words, sperm formation (spermiogenesis) is inhibited. However, the mechanisms behind the effects of heat shock on spermatogenesis are obscure.

Methodology/Principal Findings

To identify the inhibitory factor of spermiogenesis, experimental cryptorchid (EC) mice were used in this study. Here we show that α-fetoprotein (AFP) is specifically expressed in the testes of EC mice by proteome analysis. AFP was also specifically localized spermatocytes by immunohistochemical analysis and was secreted into the circulation system of EC mice by immunoblot analysis. Since spermatogenesis of an advanced mammal cannot be reproduced with in vitro, we performed the microinjection of AFP into the seminiferous tubules of normal mice to determine whether AFP inhibits spermiogenesis in vivo. AFP was directly responsible for the block in spermiogenesis of normal mice. To investigate whether AFP inhibits cell differentiation in other models, using EC mice we performed a partial hepatectomy (PH) that triggers a rapid regenerative response in the remnant liver tissue. We also found that liver regeneration is inhibited in EC mice with PH. The result suggests that AFP released into the blood of EC mice regulates liver regeneration by inhibiting the cell division of hepatocytes.

Conclusions/Significance

AFP is a well-known cancer-specific marker, but AFP has no known function in healthy human beings. Our findings indicate that AFP expressed under EC conditions plays a role as a regulatory factor in spermatogenesis and in hepatic generation.     

Distribution of acetylated α-tubulin in brain

Summary We studied the solubility properties of brain acetylated -tubulin, as well as the localization of this tubulin in brain tissue. Endogenous unpolymerized tubulin and cytoskeletal tubulin were fractionated after brain Triton-solubilization. Using the immunoblotting technique, we found that acetylated -tubulin was recovered in the cytoskeletal fraction, and that most (92%) of the acetylated microtubules of this fraction were depolymerized by cold/Ca²⁺ treatment. In another set of experiments, axonal and soma-dendritic preparations were found to have equivalent amounts of acetylated -tubulin. By immunogold electron microscopy, we established that acetylated microtubules are widely distributed in dendrites of the central nervous system.     

Mechanism of selection of side-chain rotamers in α-helices

In this study, a possible mechanism of selection of side-chain rotamers based on the rotamer distributions in known coiled-coil proteins is suggested. According to this mechanism, interhelical hydrophobic, polar, and packing interactions bring alpha-helices closer to each other and this effect squeezes side chains out of the helix-helix interface. As a result, in dimeric coiled coils and long alpha-alpha-hairpins where alpha-helices are packed in a face-to-face manner, most side chains occupying the a-positions have t-rotamers and those in the d-positions g(-)-rotamers. In tetramers, where alpha-helices are packed side-by-side, most side chains in the a-positions adopt g(-)-rotamers and those in the d-positions t-rotamers.     

Spermatotoxic effects of α-chlorohydrin in rats

This study was conducted to investigate the potential effects of α-chlorohydrin (ACH) on epididymal function and antioxidant system in male rats. The test chemical was administered to male rats by gavage at doses of 0, 3, 10, and 30 mg/kg/day for 7 days. Twenty-four male rats were randomly assigned to four experimental groups, with six rats in each group. Spermatotoxicity was assessed by measurement of reproductive organ weight, testicular sperm head count, epididymal sperm motility and morphology, histopathologic examination, and oxidative damage analysis in rats. At 30 mg/kg/day, an increase in the incidence of clinical signs, epididymis weight, and gross necropsy findings of the epididymis, a decrease in the sperm motility, and an increased incidence of histopathological changes of the epididymis were observed in a dose-dependent manner. At 10 mg/kg/day, an increased incidence of clinical signs and histopathological changes and decreased sperm motility were observed. In the oxidative damage analysis, an increase in the malondialdehyde concentration and a decrease in the glutathione content and glutathione peroxidase and catalase activities in the epididymal tissue were detected at ≥3 mg/kg/day. The results show that graded doses of ACH elicit depletion of the antioxidant defense system and that the spermatotoxicity of ACH may be due to the induction of oxidative stress.     

Molecular basis of α-thalassemia in Sicily

To evaluate the allelic frequency and genetic diversity of α-thalassemia defects in Sicily, both epidemiological and patient-oriented studies were carried out. For the epidemiological study, phenotypic data were collected on more than 1000 Sicilian individuals. Among them, 427 were explored at the molecular level for nine α-thalassemic variants known to be common in the Mediterranean region. Our data reveal an allele frequency of 4.1% for α⁺-thalassemia matching that of β-thalassemia in this region. The presence of α°-thalassemia (––^MEDI and ––^CAL) was observed only in the group of referred patients. Newly acquired nucleotide sequence data on the deletional breakpoint of ––^CAL allowed us to design a simple PCR-based procedure for exploring this allele. The data also provide additional information concerning the genetic mechanisms involved in such large deletions. Received: 8 August 1996 / Revised: 16 October 1996     

Structure assembly of Bcl-xL through α5-α5 and α6-α6 interhelix interactions in lipid membranes

Lipid bilayer membrane is the main site where Bcl-x_L executes its anti-apoptotic function. Here we used site-directed mutagenesis and cysteine-directed cross-linking to trap the structure of Bcl-x_L upon membrane insertion. Cys151 on α5-helix and Asn185 on α6-helix of two neighboring Bcl-x_L are found in close positions, respectively. The FRET based binding assay indicated that the BH3-peptide binding pocket in Bcl-x_L is disrupted after its membrane insertion. Co-immunoprecipitation experiments showed that the membrane-bound Bcl-x_L sequestered tBid by direct interaction at physiological pH. If Bcl-x_L behaves similarly at low pH as it does at physiological pH, the membrane-bound Bcl-x_L should bind to tBid through protein regions other than the BH3 domain of tBid and the hydrophobic pocket of Bcl-x_L. Previously, a crystallography study demonstrated that Bcl-x_L formed homodimers through domain swapping in water, where Cys151 and Asn185 of two monomeric subunits are far apart from each other and the BH3-peptide binding pocket is intact. Our results indicated that Bcl-x_L dimer trapped by cross-linking in lipids is distinct from the domain swapped dimer, suggesting that Bcl-x_L transits through a structural change from the water-soluble state to the membrane-bound state and there are multiple possibilities for structural reorganization of Bcl-x_L protein.     

Functional Elements in Molecular Chaperone α-Crystallin: Identification of Binding Sites in αB-Crystallin

α-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in α-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3′ dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine α-crystallin at 48°C. The complex was photolyzed and reduced with DTT and the subunits of α-crystallin, αA- and αB-, were separated. Fluorescence analysis showed that both αA- and αB-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of αB-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.     

Expression of α-amylase in cultured callus of french bean

α-Amylase (EC 3.2.1.1) expression was found in calli of French bean (Phaseolus vulgaris L. cv Goldstar). We examined enzyme activity in the calli to investigate influence of gibberellin and sugars on enzyme expression. After subculture of the calli, α-amylase activity decreased, and then increased at a stationary phase of callus growth. Exogenous application of gibberellin and an inhibitor of gibberellin synthesis, uniconazole, did not have any significant effects on the enzyme expression. Sugar starvation increased the activity, while addition of metabolizable sugars, such as sucrose, glucose and maltose, to the medium repressed expression. Addition of 6% mannitol, a non-metabolizable sugar, to the medium induced higher α-amylase expression as compared to addition of 3% mannitol. This result suggests that osmotic stress enhances α-amylase activity in the calli. Furthermore, high concentrations of agar in the medium increased α-amylase activity in the calli. It is probable that high concentrations of agar prevented incorporation of nutrient into the calli and induced the α-amylase activity in the calli.     

Stability of fungal α-amylase in sodium dodecylsulfate

Unfolding of a fungal -amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the -amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90°C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49°C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the -amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50°C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the -amylase is a slow process.     

A survey of sRNA families in α-proteobacteria

We have performed a computational comparative analysis of six small non-coding RNA (sRNA) families in α-proteobacteria. Members of these families were first identified in the intergenic regions of the nitrogen-fixing endosymbiont S. meliloti by a combined bioinformatics screen followed by experimental verification. Consensus secondary structures inferred from covariance models for each sRNA family evidenced in some cases conserved motifs putatively relevant to the function of trans-encoded base-pairing sRNAs i.e., Hfq-binding signatures and exposed anti Shine-Dalgarno sequences. Two particular family models, namely αr15 and αr35, shared own sub-structural modules with the Rfam model suhB (RF00519) and the uncharacterized sRNA family αr35b, respectively. A third sRNA family, termed αr45, has homology to the cis-acting regulatory element speF (RF00518). However, new experimental data further confirmed that the S. meliloti αr45 representative is an Hfq-binding sRNA processed from or expressed independently of speF, thus refining the Rfam speF model annotation. All the six families have members in phylogenetically related plant-interacting bacteria and animal pathogens of the order of the Rhizobiales, some occurring with high levels of paralogy in individual genomes. In silico and experimental evidences predict differential regulation of paralogous sRNAs in S. meliloti 1021. The distribution patterns of these sRNA families suggest major contributions of vertical inheritance and extensive ancestral duplication events to the evolution of sRNAs in plant-interacting bacteria.     

Ribosomal biosynthesis of α-amanitin in Galerina marginata

Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerinamarginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerinahybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G.marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary relationship between AMA1 in the genera Amanita and Galerina is unclear.     

The role of α-synuclein in the pathophysiology of alcoholism

Alcoholism has complex etiology and there is evidence for both genetic and environmental factors in its pathophysiology. Chronic, long-term alcohol abuse and alcohol dependence are associated with neuronal loss with the prefrontal cortex being particularly susceptible to neurotoxic damage. This brain region is involved in the development and persistence of alcohol addiction and neurotoxic damage is likely to exacerbate the reinforcing effects of alcohol and may hinder treatment. Understanding the mechanism of alcohol’s neurotoxic effects on the brain and the genetic risk factors associated with alcohol abuse are the focus of current research. Because of its well-established role in neurodegenerative and neuropsychological disorders, and its emerging role in the pathophysiology of addiction, here we review the genetic and epigenetic factors involved in regulating α-synuclein expression and its potential role in the pathophysiology of chronic alcohol abuse. Elucidation of the mechanisms of α-synuclein regulation may prove beneficial in understanding the role of this key synaptic protein in disease and its potential for therapeutic modulation in the treatment of substance use disorders as well as other neurodegenerative diseases.     

Role of α-fetoprotein in differentiation of regulatory T lymphocytes

The effect of native α-fetoprotein (AFP) on the expression of T-regulatory lymphocyte (Treg) markers by activated CD4⁺ lymphocytes with different proliferative status was studied. α-Fetoprotein did not affect the ratio of proliferating and non-proliferating activated CD4⁺ cells. In the study of Treg differentiation, it was found that AFP at concentrations of 50 and 100 μg/mL significantly inhibited the number of nonproliferating CD4⁺FOXP3⁺ and CD4⁺FOXP3⁺HELIOS⁺ lymphocytes without affecting the expression of Treg markers by proliferating CD4⁺ lymphocytes.     

Secretion of N-terminal domain of α-dystroglycan in cerebrospinal fluid

α-Dystroglycan (α-DG) plays crucial roles in maintaining the stability of cells. We demonstrated previously that the N-terminal domain of α-DG (α-DG-N) is secreted by cultured cells into the culture medium. In the present study, to clarify its function in vivo, we generated a monoclonal antibody against α-DG-N and investigated the secretion of α-DG-N in human cerebrospinal fluid (CSF). Interestingly, we found that a considerable amount of α-DG-N was present in CSF. α-DG-N in CSF was a sialylated glycoprotein with both N- and O-linked glycan. These observations suggest that secreted α-DG-N may be transported via CSF and have yet unidentified effects on the nervous system.