Synthesis and in vitro bioactivity of C-terminal deleted analogs of human growth hormone-releasing factor

A series of C-terminal deleted analogs of human growth hormone-releasing factor (hGRF) with either an amidated or a free carboxylic acid C-terminus were synthesized by solid phase methodology. Their capacity to release growth hormone was tested on rat anterior pituitary cells in monolayer culture. A gradual decrease of bioactivity down to 23% relative to hGRF was noted when the C-terminal amino acids were deleted to hGRF (1-34)OH. Further deletions, however, did not decrease the bioactivity because the potencies of the fragments, hGRF(1-31)NH2, (1-30)NH2 and (1-29)NH2 remained at about 50% of that of hGRF. Continual deletion of residues to hGRF(1-23)NH2, (1-22)NH2 and (1-21)NH2 still yielded bioactive fragments with full intrinsic activity despite very low potency. Only with the deletion down to hGRF(1-19)NH2 did the bioactivity completely disappear. Thus, together with the data published in a previous paper (1), the minimal biologically active core of hGRF with full intrinsic activity comprises the fragment (3-21).     

Identification of distinct P-glycoprotein gene sequences in rat.

In higher vertebrates, P-glycoprotein is usually encoded by a small family of genes. We have determined that the rat contains three P-glycoprotein genes and have cloned distinct genomic fragments containing the putative 3' untranslated regions of these P-glycoprotein genes. Sequence analysis indicates that the rat P-glycoprotein genes belong to the three P-glycoprotein classes identified in mammals. These cloned sequences will be useful for delineating the expression of P-glycoprotein genes in the rat. We have also isolated a fourth clone which contains only a short, but highly conserved P-glycoprotein domain. This clone appears not be a member of the P-glycoprotein gene family, and its relationship to P-glycoprotein is unknown.     

Ultrastructure of interstitial cells of Cajal at the gastro-oesophageal junction of the monkey (Macaca fascicularis)

The ultrastructure of the interstitial cells of Cajal (ICC) in the oesophagus of the monkey resembled that described in the oesophagus of other mammalian species but differed in their paucity and almost lack of smooth endoplasmic reticulum, caveolae and filaments. The plasmalemma of the ICC was in close contact (20- to 30-nm gaps) with that of smooth muscle cells. This may occasionally take the form of a desmosome, but gap junctions have not been observed. Vesiculated axon profiles, containing large granular or agranular vesicles were in close contact (20- to 30-nm gaps) with the plasmalemma of ICC. In a few vesiculated profiles a presynaptic density could be recognized. The intercalation of the ICC between the vesiculated axon profiles and the smooth muscle cells suggest a role in oesophageal motility. Between 3 and 21 days following bilateral vagotomy some ICC showed regressive changes such as increased electron density and shrinkage of the cytoplasm, crowding of the organelles and dissolution of the nuclear chromatin material. Axon profiles in the vicinity of the affected ICC contained glycogen granules suggesting injury. In late stages, the number of ICC and smooth muscle contacts was reduced. The results suggest that the vagus nerves exert a trophic influence on the ICC and that the intercellular relationships between ICC and smooth muscle cells possess a degree of plasticity. It is tentatively suggested that these vagal effects may be mediated via the oesophageal myenteric ganglia.     

Zoogeography of Intertidal Communities in the West Indian Ocean as Determined by Ocean Circulation Systems: Patterns from the Tetraclita Barnacles

The Indian Ocean is the least known ocean in the world with the biogeography of marine species in the West Indian Ocean (WIO) understudied. The hydrography of WIO is characterized by four distinct oceanographic systems and there were few glacial refugia formations in the WIO during the Pleistocene. We used the widely distributed intertidal barnacle Tetraclita to test the hypothesis that the distribution and connectivity of intertidal animals in the WIO are determined by the major oceanographic regime but less influenced by historical events such as Pleistocene glaciations. Tetraclita were studied from 32 locations in the WIO. The diversity and distribution of Tetraclita species in the Indian Ocean were examined based on morphological examination and sequence divergence of two mitochondrial genes (12S rDNA and COI) and one nuclear gene (histone 3, H3). Divergence in DNA sequences revealed the presence of seven evolutionarily significant units (ESUs) of Tetraclita in WIO, with most of them recognized as valid species. The distribution of these ESUs is closely tied to the major oceanographic circulation systems. T. rufotincta is distributed in the Monsoonal Gyre. T. ehsani is present in the Gulf of Oman and NW India. Tetraclita sp. nov. is associated with the Hydrochemical Front at 10°S latitude. T. reni is confined to southern Madagascan and Mauritian waters, influenced by the West Wind Drift. The endemic T. achituvi is restricted to the Red Sea. Tetraclita serrata consists of two ESUs (based on mtDNA analysis) along the east to west coast of South Africa. The two ESUs could not be distinguished from morphological analysis and nuclear H3 sequences. Our results support that intertidal species in the West Indian Ocean are associated with each of the major oceanographic circulation systems which determine gene flow. Geographical distribution is, however, less influenced by the geological history of the region.     

ERp57 is up‐regulated in free fatty acids‐induced steatotic L‐02 cells and human nonalcoholic fatty livers

Pathogenesis of nonalcoholic fatty liver disease (NAFLD) is not clear. In this study we aimed to identify proteins involved in NAFLD development in free fatty acids (FFA)‐induced hepatosteatotic cells and in human liver biopsies. Steatosis was induced by incubating a normal human hepatocyte‐derived cell line L‐02 with FFA. Differentially expressed proteins in the steatotic cells were analyzed by two‐dimensional gel electrophoresis‐based proteomics. Involvement of one of the up‐regulated proteins in steatosis was characterized using the RNA interference approach with the steatotic cells. Protein expression levels in liver biopsies of patients with NAFLD were assessed by immunohistochemistry. Proteomic analysis of L‐02 steatotic cells revealed the up‐regulation of ERp57, a condition not previously implicated in NAFLD. Knockdown of ERp57 expression with siRNA significantly reduced fat accumulation in the steatotic cells. ERp57 expression was detected in 16 out of 17 patient biopsies and correlated with inflammation grades or fibrosis stages, while in 5 normal biopsies ERp57 expression was not detectable in hepatocytes. In conclusion, ERp57 was up‐regulated in FFA‐induced steatotic hepatic cells and in NAFLD patient livers and demonstrated steatotic properties in cultured cells. Further investigations are warranted to verify the involvement of ERp57 in NAFLD development. J. Cell. Biochem. 110: 1447–1456, 2010. © 2010 Wiley‐Liss, Inc.     

Design,synthesis, biological evaluation,and molecular modeling studies of chalcone-rivastigmine hybrids as cholinesterase inhibitors

A series of novel chalcone-rivastigmine hybrids were designed, synthesized, and tested in vitro for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase. Most of the target compounds showed hBChE selective activity in the micro- and submicromolar ranges. The most potent compound 3 exhibited comparable IC₅₀ to the commercially available drug (rivastigmine). To better understand their structure activity relationships (SAR) and mechanisms of enzyme-inhibitor interactions, kinetic and molecular modeling studies including molecular docking and molecular dynamics (MD) simulations were carried out. Furthermore, compound 3 blocks the formation of reactive oxygen species (ROS) in SH-SY5Y cells and shows the required druggability and low cytotoxicity, suggesting this hybrid is a promising multifunctional drug candidate for Alzheimer’s disease (AD) treatment.     

Identification of Key Factors Involved in the Biosorption of Patulin by Inactivated Lactic Acid Bacteria (LAB) Cells

The purpose of this study was to identify the key factors involved in patulin adsorption by heat-inactivated lactic acid bacteria (LAB) cells. For preventing bacterial contamination, a sterilization process was involved in the adsorption process. The effects of various physical, chemical, and enzymatic pre-treatments, simultaneous treatments, and post-treatments on the patulin adsorption performances of six LAB strains were evaluated. The pre-treated cells were characterized by scanning electron microscopy (SEM). Results showed that the removal of patulin by viable cells was mainly based on adsorption or degradation, depending on the specific strain. The adsorption abilities were widely increased by NaOH and esterification pre-treatments, and reduced by trypsin, lipase, iodate, and periodate pre-treatments. Additionally, the adsorption abilities were almost maintained at pH 2.2–4.0, and enhanced significantly at pH 4.0–6.0. The effects of sodium and magnesium ions on the adsorption abilities at pH 4 were slight and strain-specific. A lower proportion of patulin was released from the strain with higher adsorption ability. Analyses revealed that the physical structure of peptidoglycan was not a principal factor. Vicinal OH and carboxyl groups were not involved in patulin adsorption, while alkaline amino acids, thiol and ester compounds were important for patulin adsorption. Additionally, besides hydrophobic interaction, electrostatic interaction also participated in patulin adsorption, which was enhanced with the increase in pH (4.0–6.0).