International journal of biochemistry and molecular biology最新文献

Thirty five years ago mesotrypsin was first isolated from the human pancreas. It was described as a minor trypsin isoform with the remarkable property of near total resistance to biological trypsin inhibitors. Another unusual feature of mesotrypsin was discovered later, when it was found that mesotrypsin has defective affinity toward many protein substrates of other trypsins. As the younger sibling of the two major trypsins secreted by the pancreas, cationic and the anionic trypsin, it has been speculated to represent an evolutionary waste with no apparent function. We know now that mesotrypsin is functionally very different from the other trypsins, with novel substrate specificity that hints at distinct physiological functions. Recently, evidence has begun to emerge implicating mesotrypsin in direct involvement in cancer progression. This review will explore the biochemical characteristics of mesotrypsin and structural insights into its specificity, function, and inhibition.

FXIII is a transglutaminase consisting of two catalytic (FXIIIA) and two non-catalytic subunits (FXIIIB) in plasma, where this enzyme is responsible for stabilizing fibrin clots. Although possible functions of intracellular FXIIIA have been proposed, these remain to be established. We show that a 40 kDa protein species of FXIIIA is present in the human neuroblastoma cell lines SH-SY5Y and LAN5. These data reveal the presence of a new uncharacterised variant of FXIIIA, possibly due to an alternative splicing, in nervous cells.

The transmembrane protein with epidermal growth factor (EGF) and two follistatin (FS) motifs 2 (TMEFF2) has a limited tissue distribution with strong expression only in brain and prostate. While TMEFF2 is overexpressed in prostate cancer indicating an oncogenic role, several studies indicate a tumor suppressor role for this protein. This dual mode of action is, at least in part, the result of metalloproteinase-dependent shedding that generates a soluble TMEFF2 ectodomain with a growth promoting function. While recent studies have shed some light on the biology of different forms of TMEFF2, little is known about the molecular mechanisms that influence its oncogenic/tumor suppressive function. In several non-prostate cell lines, it has been shown that a recombinant form of the TMEFF2 ectodomain can interact with platelet derived growth factor (PDGF)-AA to suppress PDGF receptor signaling and can promote ErbB4 and ERK1/2 phosphorylation. However, the role of the full length TMEFF2 in these pathways has not been examined. Using prostate cell lines, here we examine the role of TMEFF2 in ERK and Akt activation, two pathways implicated in prostate cancer progression and that have been shown to cross talk in several cancers. Our results show that different forms of TMEFF2 distinctly affect Akt and ERK activation and this may contribute to a different cellular response of either proliferation or tumor suppression.

Several plant-derived allergens have been identified which result in the formation of immunoglobulin E antibodies. Primarily, these allergens belong to the protein families including seed storage proteins, structural proteins and pathogenesis-related proteins. Several allergens are also reported from flower bulbs which cause contact dermatitis. Such symptoms are highly common with the bulb growers handling different species of Narcissus. Narcissus toxicity is also reported if the bulbs are consumed accidentally. The present study aimed to characterize the protein from the bulbs of Narcissus tazetta responsible for its allergenic response. A 13 kDa novel allergenic protein, Narcin was isolated from the bulbs of Narcissus tazetta. The protein was extracted using ammonium sulfate fractionation. The protein was further purified by anion exchange chromatography followed by gel filtration chromatography. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation. The allergenicity of the protein was measured by cytokine production using flow cytometry in peripheral blood mononuclear cells. Further estimation of total IgE was performed by ELISA method. This novel protein was found to induce pro-inflammatory cytokines and thus induce allergy by elevating total IgE level. The novel protein, Narcin isolated from Narcissus tazetta was found to exhibit allergenic properties.

Animal African trypanosomiasis (AAT) also known as Nagana is a devastating disease among domestic animals in large parts of Sub-Saharan Africa causing loses in milk and meat production as well as traction power. However, there is currently no commercial vaccine against AAT. The parasites have also developed resistance to some of the drugs in use. Moreover, the use of affordable computer-aided wet bench methods in the search for vaccine and/or new drug targets against this disease have not yet been fully explored in developing countries. This study, therefore, explored the use of PCR to screen a freshly prepared bloodstream form Trypanosoma brucei brucei (T. b. brucei) expression library for coding sequences followed by bioinformatics analyses specifying the functions and importance of these proteins to parasite survival. Eleven protein coding sequences were identified from twenty nine purified clones. The putative retro transposon hot spot protein 4 (RHSP 4) was the only protein with a fully annotated DNA sequence. All the others were hypothetical or had partial or unqualified annotations. RHSP 4 and pyruvate dehydrogenase E1 component, alpha sub-unit (PDE1α) are involved in aerobic respiration whereas succinyl-Co A-3-ketoacid-coenzyme A transferase mitochondrial precursor (SKTMP) is predicted to be involved in ketone body catabolism. Cystathionine beta-synthase (CBS) and alpha-1,3-mannosyltransferase (αMT) have been predicted in cysteine biosynthesis and vesicular transport respectively. The functions of the hypothetical proteins encountered have neither been experimentally determined nor predicted. We hypothesize that both CBS and PDE1α are good drug targets. Overall, about 300 plates are required to PCR screen the entire Trypanosoma brucei genome in approximately eight months. This method is therefore, applicable and affordable in the search for new drug targets under conditions of limited resources among developing countries.

TWO MAJOR PATHWAYS DEGRADE MOST CELLULAR PROTEINS IN EUKARYOTIC CELLS: the ubiquitin-proteasome system (UPS), which usually degrades the majority of proteins, and autophagy, primarily responsible for the degradation of most long-lived or aggregated proteins and cellular organelles. Disruption of these processes can contribute to pathology of a variety of diseases. Further, both pathways are critical for the maintenance of several aspects of cellular homeostasis, but, until recently, were thought to be largely distinct. Recent advances in this field, however, now strongly suggest that their activities are carefully orchestrated through several interfacing elements that are presented and discussed in this review.

RNA Aptamers refer to RNA oligonulceotides that are capable of binding to specific targets with high affinity and specificity. Through a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX), a number of RNA aptamers have been identified against various targets including organic compounds, nucleotides, proteins and even whole cells and organisms. RNA aptamers have proven to be of high therapeutic and diagnostic value with recent FDA approval of the first aptamer drug and additional ones in the clinical pipelines. It has also been found to be a particularly useful tool for cell-type specific delivery of other RNA therapeutics like siRNA. All these establish RNA aptamers as one of the pivotal tools of the emerging RNA nanotechnology field in the fight against human diseases including cancer, viral infections and other diseases. This article summarizes the current advancement in the identification of RNA aptamers and also provides some examples of their therapeutic and diagnostic applications.

Ceramidases cleave the N-acyl linkages of ceramide to generate sphingosine and its subsequent product sphingosine-1-phosphate (S1P). Ceramide and S1P are important bioactive lipids, and ceramidases are important in regulating the availability of these lipids. In this study, we report the purification and characterization of camel brain neutral ceramidase (CBCDase). The novel CBCDase was purified from camel brain using sequential chromatography of DEAE-Sepharose, Phenyl-Sepharose, Superdex, and Mono Q column. The Mono Q fractions containing ceramidase activity were used for enzyme characterization. The purified CBCDase showed a single band corresponding to a molecular weight of ~100 kDa, displaying classical Michaelis-Menten kinetics, with maximum enzymatic activity at pH 7.0. Deglycosylation of the enzyme yields an apparent molecular weight of ~80 kDa. The purified CBCDase was inhibited by Zn(2+) and Cu(2+), while Ca(2+) stimulates the activity. Phosphatidic acid, phosphatidylserine and phosphatidylcholine completely inhibited enzyme activity at low concentrations. Thiol-containing compounds inhibited the CBCDase activity. Among the nucleotides, ADP, UMP, and TMP inhibited the enzyme activity at low concentrations, whereas, ATP inhibited the activity at higher concentrations only. The CBCDase catalysed both ceramide hydrolysis and reverse CDase reactions. For the first time, we have purified to apparent homogeneity of a ~100 kDa nCDase from camel brain.

Objective: Interaction of genotype and environment results in a specific phenotype of the clinical course of asthma. TGFβ-1 gene belongs to the important group of genes involved in the regulation of proliferation, differentiation, adhesion, and migration of a variety of cell types. TGFβ-1 is inhibitory for B and T cells, as well as IgE production. In particular, it is engaged in inflammation of the bronchi and airway remodeling in asthma, which processes are critical in the pathogenesis of the disease. The aim of this study was to evaluate the correlation between the level of expression of TGFβ-1 and the severity of asthma.

Methods: The study included 39 participants (20 healthy subjects and 19 patients with asthma). Each sample was analysed by using real time PCR.

Results: There was statistical associations between the control group and the group of patients (p = 0,00007). It was demonstrated strong correlation between healthy and patients with severe asthma according GINA guidelines (p = 0,017). It was found the strong statistical correlation between healthy and patients with severe corticosteroid dependent asthma (p = 0,013). Correlations were observed between levels of asthma severity according to the ATS guidelines and controls. The influence of the level of TGFβ-1 mRNA expression and the severity of asthma (ATS) in the FEV1 (%) parameter value was found.

Conclusion: It was found that an important role is played by TGFβ-1 in the pathogenesis of asthma.

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is the principal receptor for the regulatory action of atrial and brain natriuretic peptides (ANP and BNP) and an important effector molecule in controlling of extracellular fluid volume and blood pressure homeostasis. We have utilized RNA interference to silence the expression of GC-A/NPRA gene (Npr1), providing a novel system to study the internalization and trafficking of NPRA in intact cells. MicroRNA (miRNA)-mediated small interfering RNA (siRNA) elicited functional gene-knockdown of NPRA in stably transfected human embryonic kidney 293 (HEK-293) cells expressing a high density of recombinant NPRA. We artificially expressed three RNA polymerase II-driven miRNAs that specifically targeted the Npr1 gene, but shared no significant sequence homology with any other known mouse genes. Reverse transcription-PCR (RT-PCR) and Northern blot analyses identified two highly efficient Npr1 miRNA sequences to knockdown the expression of NPRA. The Npr1 miRNA in chains or clusters decreased NPRA expression more than 90% as compared with control cells. ANP-dependent stimulation of intracellular accumulation of cGMP and guanylyl cyclase activity of NPRA were significantly reduced in Npr1 miRNA-expressing cells by 90-95% as compared with control cells. Treatment with Npr1 miRNA caused a drastic reduction in the receptor density subsequently a deceased internalization of radiolabeled (125)I-ANP-NPRA ligand-receptor complexes. Only 12%-15% of receptor population was localized in the intracellular compartments of microRNA silenced cells as compared to 70%-80% in control cells.