Changes in Blood-Brain Barrier Nutrient Transport in the Offspring of Iodine-Deficient Rats and Their Preventability

Thyroid hormones affect the structure and function of biological membranes. Whether or not they affect the Blood-Brain Barrier nutrient transport, the rate limiting membrane transport regulating nutrient supply to brain is to be established yet. That the impaired brain development and function seen in iodine deficiency could be due to such an effect has been assessed in situ by the brain uptake index (BUI) method in Wistar/NIN rat pups born to dams subjected to dietary iodine deficiency/rehabilitation for different times. Compared to controls (C), there was a significant decrease in the BUI values of 2-Deoxy-D-Glucose (2-DG) and L-leucine (Leu) in the pups (D1) born to dams chronically fed low iodine test (LIT) diet through their active growth and subsequent pregnancy and lactation. Surprisingly transport of L-Tyrosine (Tyr) and sucrose (the background marker) was not altered, nor was the BBB transport of all these nutrients affected by feeding LIT diet during the mothers' gestation (D2) and lactation (D3) only. The hypothyroidism in D1 pups was only moderate and preventable by rehabilitation of mothers with control diet from conception (R1) or parturition (R2), as were the changes in BBB nutrient transport. The results suggest that chronic material dietary iodine deficiency impairs BBB nutrient transport in the offspring and this could be prevented by their rehabilitation with iodine.     

Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect

The excluded volume effect (EVE) rules all life processes. It is created by macromolecules that occupy a given volume thereby confining other molecules to the remaining space with large consequences on reaction kinetics and molecular assembly. Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by procollagen C-proteinase (PCP/BMP-1) and proteolytic modification of its allosteric regulator, PCOLCE1. This led to a 20-30- and 3-6-fold increased collagen deposition in two- and three-dimensional cultures, respectively, and creation of crosslinked collagen footprints beneath cells. Important parameters correlating with accelerated deposition were hydrodynamic radius of macromolecules and their negative charge density.     

173 Role of free cysteines in leptin receptor

Leptin, a 16-kDa adipocytic peptide hormone (product of ob gene), is known to play a key role in the control of body weight and exerts its influence by binding to its long-form receptor (Ob-Rb). Ob-Rb belongs to class I cytokine receptor superfamily and consists of an extracellular, transmembrane, and an intracellular domain. Cysteines including free and disulphide-bonded are known to play a significant role in recognition of leptin by its receptor and are known to be highly conserved in different organisms including human, macaca, mouse, dog, sheep, zebrafish, and medaca. Recently, the crystal structure of leptin-binding domain of human leptin receptor has been determined (1). Using the structural data, we analyzed the role of free cysteines in leptin-binding domain of leptin receptor through docking studies using Rosettadock. The conserved free cysteines namely Cys-604 and Cys-613 were mutated to alanines and this resulted in drastic change in the binding orientation of leptin and its receptor. Based on computational analysis, we propose that cysteines either free or involved in disulphide bridges might play a crucial role during signaling and might be the primary determinant of leptin-receptor interactions, the details of which will be discussed. Currently, understanding the structural basis of leptin and its binding to leptin receptor gains much significance since it might pave the way for designing inhibitors that might be used in controlling obesity.     

Typing of clinical and environmental strains of Aeromonas spp. using two PCR based methods and whole cell protein analysis

Two PCR based typing methods i.e. random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR were evaluated for typing of 42 Aeromonas isolates from clinical and environmental sources and whole cell protein (WCP) profiles were analyzed. Both RAPD and ERIC-PCR showed a high level of genetic diversity. Numerical index of the discriminatory (D) values were 0.94 and 0.96 (>0.90) for RAPD and ERIC-PCR, respectively. No correlation in banding pattern and evidence of genetic similarity was found between Aeromonas isolates from environmental and clinical sources. Therefore these techniques are highly reproducible and sensitive methods for typing the Aeromonas isolate from different sources. WCP profile showed two major variable regions i.e. 20 kDa to 45 kDa region and 70 kDa to 85 kDa region. Though WCP profiling had less discriminatory power, use of this method in combination with other established typing methods such as RAPD and ERIC-PCR may be helpful for reliable typing of Aeromonas isolates or to identify new proteins with pathogenic potential.     

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Aims:  The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp.
Methods and Results:  The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli . Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila . Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations.
Conclusions:  The ompW -based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish.
Significance and Impact of the Study:  To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila . Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas . Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.     

Decreased extracellular deposition of fibrillin and decorin in neonatal Marfan syndrome fibroblasts

Abnormalities of the microfibrillar protein fibrillin (Fib) have been reported in Marfan syndrome (MFS). The so-called neonatal Marfan syndrome (nMFS) is a lethal phenotype displaying features that are not seen in classical MFS. We have therefore studied the biosynthesis and extracellular deposition of Fib and decorin in fibroblasts from a patient with nMFS and controls. Immunofluorescence of the patient's cell cultures showed an almost complete absence of Fib and a marked reduction of decorin in the extracellular matrix (ECM). The nMFS skin revealed Fib on subbasal microfibrillar bundles in the papillary dermis, and Fib associated with elastic fibers in the reticular dermis; the bundles and fibers were fragmented and thinner than normal. Pulse-chase labeling of cells with [³⁵S]Met/Cys revealed moderately reduced secretion, but a diminished deposition of Fib in the ECM; this was more apparent at a longer chase time. Fib mRNA and synthesis appeared to be normal, where-as both decorin mRNA and biosynthesis were reduced. We therefore assume a structural Fib defect in this patient causing reduced deposition into and/or enhanced removal from the ECM, whereas the reduced decorin biosynthesis may be a secondary regulatory phenomenon. The clinical relevance of this remains unclear. Our findings imply that Fib defects may be responsible for the severe, complex phenotype of nMFS.     

SSCP detection of a Gly565Val substitution in the proα(I) collagen chain resulting in osteogenesis imperfecta type II

A patient with perinatal lethal osteogenesis imperfecta (OI) type II has been studied in order to identify the causative mutation. By analysis of the type I collagen produced by cultured fibroblasts from the patient, the defect was mapped to 1 cyanogen bromide peptide 7, a region corresponding to 271 amino acid residues of either the 1(I) or 2(I) collagen chains. Polymerase chain reaction (PCR) amplification of the corresponding region of the 1(I) mRNA followed by single-strand conformation polymorphism analysis of restriction enzyme digestions of the PCR products allowed further mapping of the mutation to a small region of COL1A1. A heterozygous transversion of G to T within the last glycine codon of exon 32 was identified by DNA sequence analysis. This resulted in the substitution of glycine-565 by a valine residue, disrupting the repeating Gly-Xaa-Yaa sequence that is obligatory for correct formation of the collagen molecule. The mutation was shown to have occurred de novo and is thought to result in the OI phenotype.     

Modeling the Effect of Amino Acids and Copper on Monoclonal Antibody Productivity and Glycosylation: A Modular Approach

In manufacturing monoclonal antibodies (mAbs), it is crucial to be able to predict how process conditions and supplements affect productivity and quality attributes, especially glycosylation. Supplemental inputs, such as amino acids and trace metals in the media, are reported to affect cell metabolism and glycosylation; quantifying their effects is essential for effective process development. We aim to present and validate, through a commercially relevant cell culture process, a technique for modeling such effects efficiently. While existing models can predict mAb production or glycosylation dynamics under specific process configurations, adapting them to new processes remains challenging, because it involves modifying the model structure and often requires some mechanistic understanding. Here, a modular modeling technique for adapting an existing model for a fed-batch Chinese hamster ovary (CHO) cell culture process without structural modifications or mechanistic insight is presented. Instead, data is used, obtained from designed experimental perturbations in media supplementation, to train and validate a supplemental input effect model, which is used to “patch” the existing model. The combined model can be used for model-based process development to improve productivity and to meet product quality targets more efficiently. The methodology and analysis are generally applicable to other CHO cell lines and cell types.     

PEG mediated synthesis and pharmacological evaluation of some fluoro substituted pyrazoline derivatives as antiinflammatory and analgesic agents

A new series of fluoro substituted pyrazoline derivatives 5a–g and 6a–g were synthesized in good to excellent yield from the corresponding pyrazole chalcones, 4a–g, by using polyethylene glycol-400 (PEG-400) as an alternative reaction medium. The newly synthesized compounds were characterized and screened for their in vivo antiinflammatory and analgesic activity. Compounds 5g and 6g were found to be more potent than standard drug Diclofenac and six other compounds 5b, 5c, 5f, 6b, 6c and 6f showed significant antiinflammatory activity as compared to standard drug. Compounds 5c, 5d, 5e, 5f, 6c, 6d, 6e and 6f showed significant analgesic activity as compared to standard drug Aspirin.