Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.     

Optimization of Hydrogels for Transdermal Delivery of Lisinopril by Box–Behnken Statistical Design

The aim of this study was to investigate the combined influence of three independent variables on the permeation kinetics of lisinopril from hydrogels for transdermal delivery. A three-factor, three-level Box–Behnken design was used to optimize the independent variables, Carbopol 971 P (X ₁), menthol (X ₂), and propylene glycol (X ₃). Fifteen batches were prepared and evaluated for responses as dependent variables. The dependent variables selected were cumulative amount permeated across rat abdominal skin in 24 h (Q ₂₄; Y ₁), flux (Y ₂), and lag time (Y ₃). Aloe juice has been first time investigated as vehicle for hydrogel preparation. The ex vivo permeation study was conducted using Franz diffusion cells. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The regression equation generated for the cumulative permeation of LSP in 24 h (Q ₂₄) was Y ₁ = 1,443.3–602.59X ₁ + 93.24X ₂ + 91.75X ₃ − 18.95X ₁ X ₂ – 140.93X ₁ X ₃ – 4.43X ₂ X ₃ – 152.63X ₁ ² – 150.03X₂ ² − 213.9X ₃ ². The statistical validity of the polynomials was established, and optimized formulation factors were selected by feasibility and grid search. Validation of the optimization study with 15 confirmatory runs indicated high degree of prognostic ability of response surface methodology. The use of Box–Behnken design approach helped in identifying the critical formulation parameters in the transdermal delivery of lisinopril from hydrogels.     

Archaeal aIF2B Interacts with Eukaryotic Translation Initiation Factors eIF2α and eIF2Bα: Implications for aIF2B Function and eIF2B Regulation

Translation initiation is down-regulated in eukaryotes by phosphorylation of the α-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ?-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO₂ fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the α, β, and δ subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the α-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the α-subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2B?, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α.     

Stereoselective plasma protein binding of amlodipine

The binding of the (R)‐ and (S)‐enantiomers of amlodipine to bovine serum albumin (BSA), human serum albumin (HSA), α₁‐acid glycoprotein (AGP), and human plasma (HP) was studied by equilibrium dialysis over the concentration range of 75–200 μM at a protein concentration of 150 μM. Unbound drug concentrations were determined by enantioselective capillary electrophoresis using 50 mM phosphate buffer, pH 2.5, containing 18 mM α‐cyclodextrin as background electrolyte. Saturation of the protein binding sites was not observed over the concentration range tested. Upon application of racemic amlodipine besylate, (S)‐amlodipine was bound to a higher extend by HSA and HP compared with (R)‐amlodipine, whereas the opposite binding of the enantiomers was observed for BSA and AGP. Scatchard analysis was used to illustrate the different binding affinities of amlodipine besylate enantiomers to BSA, HSA and AGP. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Role of C-reactive protein in complement-mediated hemolysis in Malaria

Human C-reactive protein (CRP) is a clinically important classical acute phase protein. Although CRP has been reported to bind with many nucleated cells, the direct binding of CRP to erythrocytes in diseases remains largely unexplored. The main focus of the present study was to investigate the binding of disease-specific CRP to erythrocytes of same patients. Distinct molecular variant of disease-specific CRP was affinity purified from sera of malaria patients (CRP_Mal). This CRP showed strong binding with malaria erythrocytes (RBC_Mal) as confirmed by flow cytometric analysis (FACS), enzyme-linked immunosorbent assays (ELISA), and radio binding assays. Calcium and phosphoryl choline (PC) were found to be essential for this interaction. A 2.3-fold increased binding of induced CRP to RBC_Mal as compared to normal erythrocytes (RBC_N) confirmed disease-specificity. Preincubation of RBC_Mal with unconjugated CRP showed 3–5 fold inhibition. The association constant of CRP and RBC_Mal was 4.7 × 10⁶ cpm/μg with the corresponding number of receptors/cell being 4.3 × 10⁵. The effector function of CRP_Mal has been demonstrated by its potency to activate the complement pathway. An optimal dose of 10 μg/ml of CRP induced three-fold higher hemolysis of patient erythrocytes as compared to RBC_N. These studies provide direct evidence for an important phagocytic functional interaction of this acute-phase protein by triggering the CRP-complement pathway after the binding of CRP_Mal with RBC_Mal. Hemolysis as triggered by this pathway may be one of the causative factors of anemia, a common clinical manifestation of this disease.     

Mechanistics of formation and ultrastructural evaluation of hepatocyte spheroids

Summary Freshly harvested rat hepatocytes form spheroids on uncoated positively charged polystyrene surfaces. Time lapse microscopy revealed that cell movement and reorganization were involved in spheroid formation. Ultrastructural evaluation using scanning and transmission electron microscopy indicated polarized cellular morphology and extensive cell-cell communication within spheroids. Bile canalicular structures were observed to surround each individual hepatocyte, forming an intricate three-dimensional continuous network of channels that appeared to end as pores/holes on the surface of the spheroid. The maintenance of differentiated cellular morphology coincided with preservation of hepatocyte viability and enhanced levels of tissue specific functions in spheroids.     

Intronic gene conversion in the evolution of human X-linked color vision genes

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.