Arginine substitution by alanine at the P1 position increases the selectivity of CmPI-II,a non-classical Kazal inhibitor

CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine.     

The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny Lobster Panulirus argus

The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (K_i = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.     

Ultrasound evaluation of visceral fat and metabolic risk factors during early pregnancy

Objective: The objective was to study the relationships between ultrasound estimated visceral fat and metabolic risk factors during early pregnancy. Research Methods and Procedures: Thirty consecutive healthy pregnant women at 11 to 14 weeks of gestation were studied. Maximum subcutaneous fat thickness (SFT) and visceral fat thickness (VFT) were successfully measured by ultrasound. Fasting plasma glucose, insulin, triglycerides, total cholesterol, high‐density lipoprotein cholesterol (HDL‐C), and blood pressure were measured. Insulin resistance was calculated by using the homeostasis model assessment (HOMA). Results: VFT significantly correlated with diastolic blood pressure (r = 0.37, p = 0.04), glycemia (r = 0.37, p = 0.04), insulinemia (r = 0.59, p = 0.001) insulin sensitivity (HOMA; r = 0.59, p = 0.001), triglycerides (r = 0.58, p = 0.03), HDL‐C (r = ?0.39, p = 0.03), and total cholesterol/HDL‐C ratio (p = 0.002), whereas SFT was significantly correlated with only diastolic blood pressure (p = 0.03). VFT better significantly correlated with the metabolic risk factors than pre‐gestational BMI [r = 0.39, p = 0.03 for insulinemia, r = 0.42, p = 0.02 for insulin sensitivity (HOMA), and r = 0.49, p = 0.01 for triglycerides and not significant for the rest]. Discussion: Visceral fat thickness can be easily measured by ultrasound at early pregnancy and correlates better than BMI with metabolic risk factors.     

Actual and potential natural vegetation on the Canary Islands and its conservation status

The main vegetation units of the Canary Islands are briefly described and their current surface area established. The area of potential natural vegetation remnants is compared with the supposed original area, and expressed also as percent persistence, (area now/potential) × 100. We state that although the islands have between 40 and 50% of their surface under protection, several units of the most representative vegetation (e.g. like Euphorbia scrubs, thermo-sclerophyllous woodland and laurel forest) have retreated greatly, sometimes with little possibility of recovery. Willow, palm, tamarisk, and Plocama pendula communities, sandy beach vegetation, and small-area littoral ecosystems are also severely diminished in area. Comments are made about the conservation status of the most representative communities.     

Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris

Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris.     

Biological effects of recombinant human zona pellucida proteins on sperm function

The initial interaction between gametes takes place at the level of the sperm surface and the zona pellucida (ZP), the extracellular matrix of the egg in mammals. Successful fertilization requires the proper molecular recognition of the ZP by the sperm. Recently, human ZP was demonstrated to be composed of four proteins: ZP1, ZP2, ZP3, and ZP4. The goals of this study were to determine the effects of recombinant human ZP2, ZP3, and ZP4 on human sperm acrosomal exocytosis and sperm motility. Exposure of sperm to ZP proteins, alone or in combination, promoted acrosomal exocytosis in a time-dependent manner. This effect occurred in parallel with a considerable decrease in progressive motility, coincident with an increase in nonprogressive sperm motility. An analysis of kinetic parameters of ZP-treated sperm demonstrated that a characteristic motility pattern could be defined by values of curvilinear velocity > 63.9 mum/s and linearity 相似文献   

Relevant aspects of the Hcp100 molecular marker of Histoplasma capsulatum and its potential therapeutic use in histoplasmosis

BackgroundFungal pathogens have developed strategies, involving genes expression that favors their persistence and multiplication in the host. The absence of molecules encoded by these genes could interfere with the growth and death of these fungi. In the past, a coactivator protein coding gene (Hcp100) of the fungus Histoplasma capsulatum was reported, which is overexpressed after 1 h of contact between fungal yeast-cells and murine macrophages. The product of this gene, a protein of 100 kDa (Hcp100) of H. capsulatum, is probably a regulatory protein involved in the processes required for fungal adaptation and its survival in the intracellular hostile conditions of the macrophages. A 210-bp fragment of the Hcp100 marker has proved to be an excellent tool for H. capsulatum molecular detection in clinical samples. The potential use of this gene as a therapeutic target in Plasmodium falciparum has been explored through the inhibition of both, the gene and the protein p100 of the parasite, by blocking its growth.MethodsBased on the above mentioned antecedents, we believe that the Hcp100 has an important role in the development and maintenance of the H. capsulatum yeast cells within macrophages.Results and conclusionsTo study the probable function of Hcp100 in the yeast-phase of this fungal pathogen is relevant to understand its activity and to propose it as a therapeutic target for histoplasmosis treatment.     

Prognostic Impact of del(17p) and del(22q) as Assessed by Interphase FISH in Sporadic Colorectal Carcinomas

Background

Most sporadic colorectal cancer (sCRC) deaths are caused by metastatic dissemination of the primary tumor. New advances in genetic profiling of sCRC suggest that the primary tumor may contain a cell population with metastatic potential. Here we compare the cytogenetic profile of primary tumors from liver metastatic versus non-metastatic sCRC.

Methodology/Principal Findings

We prospectively analyzed the frequency of numerical/structural abnormalities of chromosomes 1, 7, 8, 13, 14, 17, 18, 20, and 22 by iFISH in 58 sCRC patients: thirty-one non-metastatic (54%) vs. 27 metastatic (46%) disease. From a total of 18 probes, significant differences emerged only for the 17p11.2 and 22q11.2 chromosomal regions. Patients with liver metastatic sCRC showed an increased frequency of del(17p11.2) (10% vs. 67%;p<.001) and del(22q11.2) (0% vs. 22%;p = .02) versusnon-metastatic cases. Multivariate analysis of prognostic factors for overall survival (OS) showed that the only clinical and cytogenetic parameters that had an independent adverse impact on patient outcome were the presence of del(17p) with a 17p11.2 breakpoint and del(22q11.2). Based on these two cytogenetic variables, patients were classified into three groups: low- (no adverse features), intermediate- (one adverse feature) and high-risk (two adverse features)- with significantly different OS rates at 5-years (p<.001): 92%, 53% and 0%, respectively.

Conclusions/Significance

Our results unravel the potential implication of del(17p11.2) in sCRC patients with liver metastasis as this cytogenetic alteration appears to be intrinsically related to an increased metastatic potential and a poor outcome, providing additional prognostic information to that associated with other cytogenetic alterations such as del(22q11.2). Additional prospective studies in larger series of patients would be required to confirm the clinical utility of the new prognostic markers identified.     

Molecular markers reveal no genetic differentiation between Myrica rivas-martinezii and M. faya (Myricaceae)

Background and Aims

Myrica rivas-martinezii is a critically endangered endemic of the laurel forest of the Canary Islands and co-occurs very close to M. faya. Some authors suggest that M. rivas-martinezii and M. faya are two morphs of the same species, so molecular markers were used to estimate the levels and structuring of genetic variation within and among natural populations in order to evaluate genetic relationships between these two congeners.

Methods

Six polymorphic microsatellite (simple sequence repeat, SSR) markers were used to determine the genetic diversity and the genetic relationship between both Myrica species.

Key Results

Most of the natural populations analysed were in Hardy–Weinberg equilibrium for both taxa. Analysis of molecular variance (AMOVA) for both species revealed that most of the genetic variability detected was contained within populations (92·48 and 85·91 % for M. faya and M. rivas-martinezii, respectively), which it is consistent with outcrossing and dioecious plants. Estimates of interpopulation genetic variation, calculated from F_ST and G′_ST, were quite low in the two taxa, and these values did not increase substantially when M. rivas-martinezii and M. faya populations were compared. The UPGMA dendrogram based on Nei''s genetic distance clustered the populations by their island origin, independently of taxon. In fact, the mixture of individuals of both taxa did not appreciably disrupt the intrapopulational genetic cohesion, and only 3·76 % variation existed between species.

Conclusions

All the results obtained using molecular markers indicate clearly that both taxa share the same genetic pool, and they are probably the same taxa. Considering that M. rivas-martinezii is classified as at risk of extinction, there should be a change of focus of the current management actions for the conservation of this putatively endangered Canarian endemic.Key words: Canary Islands, conservation genetics, microsatellites, Myrica rivas-martinezii, Myrica faya, plant conservation