Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides

All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.     

First report of Perkinsus beihaiensis in Crassostrea madrasensis from the Indian subcontinent

Protozoan parasites of the genus Perkinsus are considered important pathogens responsible for mass mortalities in many wild and farmed bivalve populations. The present study was initiated to screen populations of the Indian edible oyster Crassostrea madrasensis, a promising candidate for aquaculture along the Indian coasts, for the presence of Perkinsus spp. The study reports the presence of P. beihaiensis for the first time in C. madrasensis populations from the Indian subcontinent and south Asia. Samples collected from the east and west coasts of India were subjected to Ray's fluid thioglycollate medium (RFTM) culture and histology which indicated the presence of Perkinsus spp. PCR screening of the tissues using specific primers amplified the product specific to the genus Perkinsus. The taxonomic affinities of the parasites were determined by sequencing both internal transcribed spacer (ITS) and actin genes followed by basic local alignment search tool (BLAST) analysis. Analysis based on the ITS sequences showed 98 to 100% identity to Perkinsus spp. (P. beihaiensis and Brazilian Perkinsus sp.). The pairwise genetic distance values and phylogenetic analysis confirmed that 2 of the present samples belonged to the P. beihaiensis clade while the other 4 showed close affinities with the Brazilian Perkinsus sp. clade. The genetic divergence data, close affinity with the Brazilian Perkinsus sp., and co-existence with P. beihaiensis in the same host species in the same habitat show that the remaining 4 samples exhibit some degree of variation from P. beihaiensis. As expected, the sequencing of actin genes did not show any divergence among the samples studied. They probably could be intraspecific variants of P. beihaiensis having a separate lineage in the process of evolution.     

Studies on the Mode of Membrane Interaction of C-terminally Amidated Brevinin1 HYba1 and 2 Peptides Against Bacteria

In this study, we investigated the possible mode of action of two C-terminally amidated novel peptides identified from the frog skin on both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio cholerae). The results indicate that the peptides initially induce membrane depolarization followed by pore formation in a concentration-dependent manner. A microscopic examination revealed the fact that the peptides are capable of destroying bacterial cells physically. The activity of these peptides against Gram-negative bacteria was dependent on the presence of divalent cations (Ca²⁺ and Mg²⁺) but not in Gram-positive bacteria. This study also emphasizes that the endemic frogs of the Western Ghats may provide a valuable source of its skin peptides having the potential for further improvement as lead drug molecules.     

Size-spectra as indicators of the effects of fishing on coral reef fish assemblages

The data requirements and resources needed to develop multispecies indicators of fishing impacts are often lacking and this is particularly true for coral reef fisheries. Size-spectra, relationships between abundance and body-size class, regardless of taxonomy, can be calculated from simple sizeabundance data. Both the slope and the mid-point height of the relationship can be compared at different fishing intensities. Here, we develop size-spectra for reef fish assemblages using body size- abundance data collected by underwater visual census in each of ten fishing grounds across a known gradient of fishing intensity in the Kadavu Island group, Fiji. Slopes of the size-spectra became steeper (F_9,69=3.20, p<0.01) and the height declined (F_9,69=15.78, p<0.001) with increasing fishing intensity. Regressions of numbers of individuals per size class across grounds were negative for all size classes, although the slope was almost zero for the smallest size class. Response to exploitation of each size class category was greatest for larger fish. Steepening of the slope with increasing fishing intensity largely resulted from reductions in the relative abundance of large fish and not from the ecological release of small fish following depletion of their predators. The slope and height of the size-spectrum appear to be good indicators of fishing effects on reef fish assemblages.     

Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

Introduction

Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.

Methods

Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.

Results

We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.

Conclusion

We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.     

Combined evidence indicates that <Emphasis Type="Italic">Perna indica</Emphasis> Kuriakose and Nair 1976 is <Emphasis Type="Italic">Perna perna</Emphasis> (Linnaeus, 1758) from the Oman region introduced into southern India more than 100 years ago

The specific status of the brown mussel, Perna indica, in southern India and northern Sri Lanka has long been questioned. Its limited geographical distribution within the extensive range of the Asian green mussel, P. viridis, and its morphometric similarity to the African brown mussel, P. perna, have led several authorities to suggest that P. indica is not a distinct species but is in fact introduced P. perna. Analysis of DNA sequences for nuclear ITS and mitochondrial COI sequence data from newly collected mussels from southern India examined in the context of data from GenBank for mussels of the genus Perna reveals that P. indica is indeed P. perna from the Oman region. A literature review indicates that P. indica (now P. perna) has been established in southern India for at least 100 years. Reports of ecological interactions between the native P. viridis and the introduced P. perna reveal that P. perna grows to a larger size than P. viridis and outcompetes the native mussel. P. perna forms high density populations that have long been fished by local coastal communities and recently the fishery for P. perna has expanded to markets beyond the coastal area. Eradication of the introduced P. perna is not feasible and is not desirable given its important role as a source of protein and revenue for the local community. Because no monitoring of the range of P. perna in southern India occurs it is not possible to know if this species is increasing its area of distribution, but a qualitative assessment of ongoing range expansion is made based on limited evidence from published reports. The possibility exists for inter-specific hybridisation between P. viridis and P. perna and we recommend that testing commence to check for this. This research highlights the value of a detailed understanding of the genetic structure (four different clades) of the genus Perna, without which the status of P. indica as P. perna from Oman in southern India could not have been made.