Aspects of common marmoset basic biology and life history important for biomedical research

While common marmosets are increasingly used as alternative primate models in biomedical research, their life history, specialized behavior and unique physiology are not well known. This paper describes important marmoset attributes that are particularly relevant for biomedical research, including reproduction, neurobiology, immunology, endocrine signaling, obesity and aging, in addition to fetal and postnatal development. While common marmosets exhibit characteristic anthropoid primate traits, they clearly differ from Old World primates and humans in a variety of functions, including reproduction, endocrine signaling and immunology. These differences, however, permit the use of common marmosets in unconventional research strategies targeted on human pathology.     

High-level expression of Aliciclobacillus acidocaldarius thioredoxin in Pichia pastoris and Bacillus subtilis

Thioredoxins are ubiquitous proteins which catalyze the reduction of disulfide bridges on target proteins and are involved in many cellular reactions. In a previous work, a thioredoxin from the thermophilic organism Aliciclobacillus acidocaldarius (Alitrx) was purified, characterized, and its gene expressed in Escherichia coli. In order to produce larger quantities of Alitrx, the protein has been expressed in the methylotrophic yeast Pichia pastoris and in the gram positive bacteria Bacillus subtilis. The growth conditions of strains showing high-level expression of Alitrx were optimized for both systems in shake-flask cultures. Active proteins were secreted in the culture media at a level of approximately 0.9 and 0.5 g/l, respectively, for P. pastoris and B. subtilis. The proteins were purified almost to homogeneity by a thermal precipitation procedure, with a 90-fold and 50-fold higher total yield with respect to that obtained with the same protein expressed in E. coli. The results indicate that either of these two systems could be utilized as a host for large-scale production of recombinant Alitrx.     

Studies on chromium(VI) adsorption-desorption using immobilized fungal biomass

The aim of this study was to investigate the Cr(VI) biosorption potential of immobilized Rhizopus nigricans and to screen a variety of non-toxic desorbing agents, in order to find out possible application in multiple sorption-desorption cycles. The biomass was immobilized by various mechanisms and evaluated for removal of Cr(VI) from aqueous solution, mechanical stability to desorbents, and reuse in successive cycles. The finely powdered biomass, entrapped in five different polymeric matrices viz. calcium alginate, polyvinyl alcohol (PVA), polyacrylamide, polyisoprene, and polysulfone was compared for biosorption efficiency and stability to desorbents. Physical immobilization to polyurethane foam and coir fiber was less efficient than polymer entrapment methods. Of the different combinations (%, w/v) of biomass dose compared for each matrix, 8% (calcium alginate), 6% (polyacrylamide and PVA), 12% (polyisoprene), and 10% (polysulfone) were found to be the optimum. The Cr sorption capacity (mg Cr/g sorbent) of all immobilized biomass was lesser than the native, powdered biomass. The Cr sorption capacity decreased in the order of free biomass (119.2) > polysulfone entrapped (101.5) > polyisoprene immobilized (98.76) > PVA immobilized (96.69) > calcium alginate entrapped (84.29) > polyacrylamide (45.56), at 500 mg/l concentration of Cr(VI). The degree of mechanical stability and chemical resistance of the immobilized systems were in the order of polysulfone > polyisoprene > PVA > polyacrylamide > calcium alginate. The bound Cr(VI) could be eluted successfully using 0.01 N NaOH, NaHCO3, and Na2CO3. The adsorption data for the native and the immobilized biomass was evaluated by the Freundlich isotherm model. The successive sorption-desorption studies employing polysulfone entrapped biomass indicated that the biomass beads could be regenerated and reused in more than 25 cycles and the regeneration efficiency was 75-78%.     

Peptide profiling in epithelial tumor plasma by the emerging proteomic techniques

The plasma peptide component (PPC) from ten melanoma (Mel), breast cancer (BC) and healthy individuals was examined by a combination of RP-HPLC, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and tandem mass spectrometry. A three peak pattern (2023, 2039, 2053.5 m/z) was primarily observed in melanoma. Two peaks (2236.1 and of 2356.3 m/z) were found only in BC samples. Fibrinogen alpha and inter-alpha-trypsin inhibitor heavy chain H4 fragments were absent in both tumor samples.     

Continuous adsorption and recovery of Cr(VI) in different types of reactors

This study reports the results of experiments on continuous adsorption and desorption of Cr(VI) ions by a chemically modified and polysulfone-immobilized biomass of the fungus Rhizopus nigricans. A fixed quantity of polymer-entrapped biomass beads corresponding to 2 g of dry biomass powder was employed in packed bed, fluidized bed, and stirred tank reactor for monitoring the continuous removal and recovery of Cr(VI) ions from aqueous solution and synthetic chrome plating effluent. Parameters such as flow rate (5, 10 and 15 mL/min), inlet concentration of Cr(VI) ions (50, 100, 150 and 250 mg/L) and the depth of biosorbent packing (22.8, 11.2 and 4.9 cm) were evaluated for the packed bed reactor. The breakthrough time and the adsorption rates in the packed bed column were found to decrease with increasing flow rate and higher Cr inlet concentrations and to increase with higher depths of sorbent packing. To have a comparative analysis of Cr adsorption efficiency in different types of reactors, the fluidized bed reactor and stirred tank reactor were operated using the same quantities of biosorbent material. For the fluidized bed reactor, Cr(VI) solution of 100 mg/L was pumped at 5 mL/min and fluidized by compressed air at a flow rate of 0.5 kg/cm.(2) The stirred tank reactor had a working volume of 200 mL capacity and the inlet/outlet flow rate was 5 mL/min. The maximum removal efficiency (mg Cr/g biomass) was obtained for the stirred tank reactor (159.26), followed by the fluidized reactor (153.04) and packed bed reactor (123.33). In comparison to the adsorption rate from pure chromate solution, approximately 16% reduction was monitored for synthetic chrome plating effluent in the packed bed. Continuous desorption of bound Cr ions from the reactors was effective with 0.01 N Na(2)CO(3) and nearly 80-94% recoveries have been obtained for all the reactors.     

Comparative analysis of HIV-1 Tat variants

HIV-1 Tat protein is a crucial element for viral replication; therefore, its inhibition might be exploited against the AIDS infection. To gain insights on the natural variability of this protein, we present a comparative investigation on the relationship between the primary sequences and the experimentally available three-dimensional structures from the HIV-1 Tat variants Z2, BRU, and MAL. Our computational tools include sequence conservation algorithms, structural analysis, electrostatic modeling, and molecular dynamics (MD) simulations. We find that two regions located between residues 10-18 and 41-52 display the highest primary sequence conservation, while the most conserved region among the available structures corresponds approximately to the segment between positions approximately 44 and 50. Furthermore, in spite of their large structural divergence, Tat variants share a common mode for long-range intramolecular interactions. Finally, the flexibility of the Z2, BRU, and MAL variants, as emerging from multinanosecond MD simulations, is rather similar. Based on this work, we conclude that the turnlike region between amino acids 44 and 50 is structurally most conserved, emerging as an important motif for pharmaceutical targeting aimed toward inhibiting Tat action.     

A plant polyphenol-rich extract restores the suppressed functions of phagocytes in influenza virus-infected mice

Influenza infection was induced in white ICR mice by intranasal (i.n.) inoculation of the virus A/Aichi/2/68 (H3N2). The number, migration and phagocyte indices of alveolar and peritoneal macrophages (pM?) and of blood polymorphonuclear leukocytes (PMNs), as well as the inhibition of the PMN adherence in the presence of a specific antigen were followed for 9 days after infection. The effect of the i.n. application of a polyphenol-rich extract, designated as polyphenolic complex (PC), isolated from the medicinal plant Geranium sanguineum L., on the inspected immune parameters was studied in parallel with the virological parameters of the infection, e.g. rate of mortality, mean survival time (MST), infectious lung virus titre and consolidation of the lungs. It was found that the application of PC induced a continuous 2- to 2.5-fold rise in the number of both peritoneal and alveolar macrophages (aM?) in the infected and healthy controls. The migration of both peritoneal and aM? increased 1.5- to 2-fold in the group of infected PC-treated animals and four to fivefold in the control group, the maximum being on day 9. PC stimulated phagocyte activities of blood PMNs in both infected and healthy mice. The leukocyte adherence inhibition (LAI) index decreased in the infected and PC-treated animals. The restoration of the suppressed functions of phagocytes in influenza virus-infected mice (VIM) was consistent with a prolongation of MST and reduction in mortality rate, infectious virus titre and lung consolidation. The immunoenhancing properties of PC apparently contribute to the overall protective effect of the plant preparation in the lethal murine experimental influenza A/Aichi infection.     

Merosin-deficient congenital muscular dystrophy: neuropathology case reports

The aims of our study were: to present cases of congenital muscular dystrophy (CMD) with deficiency in merosin and the importance of immunohistochemistry in the diagnosis of merosin-deficient CMD. In four years (1997-2000), we found three patients with merosin-deficient CMD, one of them having an unusual clinical and pathological manifestation of the disease. Muscle biopsies of gastrocnemius or quadriceps muscles were investigated. In addition with the conventional HE staining, indirect immunohistochemistry for merosin, dystrophin, utrophin and for the proteins of the dystrophin associated complex (α,β, γ- sarcoglycans; β-dystroglycan) was performed on cryosections. The findings suggest that there is no correlation between the clinical and histological picture of the disease and the expression of merosin in skeletal muscles. The degree of muscle involvment (assessed by histology) is parallel with the clinical neuromotor deficiency, but not with expression of merosin, which can be absent even in mild cases. The clinical investigations as well as current morphological techniques, only together with immunohistochemistry can differentiate between merosin - deficient CMD and other muscular dystrophy forms.     

The two thrombospondin type I repeat domains of the heparin-binding growth-associated molecule bind to heparin/heparan sulfate and regulate neurite extension and plasticity in hippocampal neurons

HB-GAM (heparin-binding growth-associated molecule, also designated as pleiotrophin) and midkine form a two-member family of extracellular matrix proteins that bind tightly to sulfated carbohydrate structures such as heparan sulfate. These proteins are used by developing neurons as extracellular cues in axonal growth and guidance. HB-GAM was recently reported to enhance differentiation of neural stem cells. Based on the solution structure of HB-GAM, we have recently shown that HB-GAM consists of two beta-sheet domains flanked by flexible lysine-rich N- and C-terminal tails with no apparent structure. These domains are homologous to thrombospondin type I repeats present in numerous extracellular proteins that interact with the cell surface. Our findings showed that the two beta-sheet domains fold independently. We showed that the domains (but not the lysine-rich tails) in HB-GAM are required and sufficient for interaction with hippocampal neurons. The individual domains bind heparan sulfate weakly and fail to produce significant biological effects in neurite outgrowth and long term potentiation assays. The amino acids in the linker region joining the two domains may be replaced with glycines with no effect on protein function. These results suggest a co-operative action of the two beta-sheet domains in the biologically relevant interaction with neuron surface heparan sulfate.