Induction of blindness by formoguanamine hydrochloride in adult male roseringed parakeets (Psittacula krameri)

Formoguanamine (2,4-diamino-s-triazine) was known to be an effective chemical agent in inducing blindness in poultry chicks, but not in adult birds. The present study was undertaken to demonstrate the influences, if any, of this chemical on the visual performance and retinal histology in an adult sub-tropical wild bird, the roseringed parakeet (Psittacula krameri). Formoguanamine (FG) hydrochloride was subcutaneously injected into adult parakeets at a dosage of 25 mg (dissolved in 0.75 ml physiological saline)/100 g body weight/day, for two consecutive days while the control birds were injected only with a placebo. The effects were studied after 10, 20 and 30 days of the last treatment of FG. Within 24 h of the treatment of FG, about 90% of the total birds exhibited lack of visual responses to any light stimulus and even absence of pupillary light reactions. The remaining birds became totally blind on the day following the last injection of FG and remained so till the end of investigation. At the microscopic level, conspicuous degenerative changes were noted in the outer pigmented epithelium and the photoreceptive layer of rods and cones in the retinas of FG treated birds. A significant reduction in the thickness of the outer nuclear layer was also found in the retinas of FG treated parakeets, compared to that in the control birds. However, the inner cell layers of the retina in the control and FG administered parakeets were almost identical. It deserves special mention that the effects of FG, noted after 30 days of last treatment, were not very different from those noted just after 10 days of treatment. Collectively, the results of the present investigation demonstrate that FG can be used as a potent pharmacological agent for inducing irreversible blindness through selective damage in retinal tissue even in the adult wild bird, thereby making FG treatment an alternative euthanasic device to a cumbersome, stressful, surgical method of enucleation of the ocular system for laboratory studies.     

Bone grafts and bone morphogenetic proteins in spine fusion

Spinal fusions are being performed for various pathologies of the spine. Stabilizing vertebral segments by eliminating motion across those segments becomes critical in dealing with pathologies of the spine that lead to instability. The use of autograft has been the gold standard for spine fusion. However, due to complications such as donor site morbidity, increased operating time, and limited supply, the use of allograft as a graft extender has become an acceptable practice especially in fusions spanning multiple segments. The discovery and isolation of novel proteins (i.e., bone morphogenetic proteins, BMPs), which initiate the molecular cascade of bone formation, have experimentally been shown in numerous animal studies to be as effective as autografts. Although the use of BMPs has exciting applications in spine surgery, long-term clinical studies must be evaluated for its efficacy in various applications in humans. The use of biomimetic materials such as hydroxyapatite (HA), or tricalcium phosphate (TCP) has also been examined in several animal models as bone graft substitutes or carriers. Although these materials have shown some promise in specific site applications, more work remains in elucidating an efficacious combination of these materials and BMPs that can be as effective as autografts. This review will present the status of bone grafts, bone morphogenetic proteins, gene therapy, and work that has been done to facilitate spinal fusion and simultaneously eliminate the need for bone graft. This revised version was published online in July 2006 with corrections to the Cover Date.     

First synthesis of phosphonobile acids and preliminary studies on their aggregation properties

The synthesis of three novel phosphonobile acids from natural bile acids is reported. The CMC of phosphonodeoxycholic acid (PDCA) at pH 8.2 was found to be lower than that of the parent deoxycholic acid (DCA). PDCA micelles were also found to have higher microviscosity compared to DCA micelles, suggesting higher hydrophobicity and tighter packing in the interior of PDCA micelles. PDCA aggregated further to form an aqueous gel at pH 4.     

A high-throughput amenable colorimetric assay for enantioselective screening of nitrilase-producing microorganisms using pH sensitive indicators

Based on the color change of an indicator due to the release of hydrogen ion from a nitrilase-catalyzed reaction, a rapid colorimetric method was established for the enantioselective screening of nitrilase-producing microorganisms. The formation of acids due to the nitrilase-mediated hydrolysis of nitriles causes a drop in the pH, which in turn results in a change of color of the solution (containing indicator) that can be observed visually. The buffer (0.01 M phosphate, pH 7.2) and indicator (Bromothymol blue, 0.01%) were selected in such a way that both have the same affinity for the released protons. The enantioselectivity of nitrilases was estimated by comparing the hydrolysis of (R)-mandelonitrile with that of racemate under the same conditions. The method was used to screen a library of nitrilase-producing microorganisms, isolated in the authors' laboratory for their ability to enantioselectively hydrolyze mandelonitrile to mandelic acid, an important chiral building block.     

Heterologous expression of a mammalian protein tyrosine phosphatase gene in Leishmania: effect on differentiation

Leishmania is a protozoan pathogen which is transmitted to humans through the bite of an infected sandfly. This infection results in a spectrum of diseases throughout the developing world, collectively known as leishmaniasis. During its life cycle, Leishmania differentiates from the promastigote stage in the sandfly vector into the amastigote stage in the mammalian host where it multiplies exclusively in macrophage phagolysosomes. Although differentiation of Leishmania is essential for its survival and pathogenesis in the mammalian host, this process is poorly understood. In higher eukaryotic cells, protein tyrosine phosphorylation plays a central role in cell proliferation, differentiation and overall function. We have therefore investigated the role of protein tyrosine phosphorylation in Leishmania differentiation by undertaking complementary approaches to mediate protein tyrosine dephosphorylation in vivo. In the present study, L. donovani were engineered to express a mammalian protein tyrosine phosphatase, or were treated with inhibitors of protein tyrosine kinases, and the resulting phenotype was examined. Both approaches resulted in a partial differentiation from promastigotes to amastigotes including the expression of the amastigote specific A2 protein, morphological change and increased virulence. These data provide support for the involvement of tyrosine phosphorylation in the differentiation of Leishmania.     

Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro.     

Interaction of ATP with a Small Heat Shock Protein from Mycobacterium leprae: Effect on Its Structure and Function

Adenosine-5’-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of “HSP18-ATP” interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.     

Neurons under viral attack: Victims or warriors?

When the central nervous system (CNS) is under viral attack, defensive antiviral responses must necessarily arise from the CNS itself to rapidly and efficiently curb infections with minimal collateral damage to the sensitive, specialized and non-regenerating neural tissue. This presents a unique challenge because an intact blood–brain barrier (BBB) and lack of proper lymphatic drainage keeps the CNS virtually outside the radar of circulating immune cells that are at constant vigilance for antigens in peripheral tissues. Limited antigen presentation skills of CNS cells in comparison to peripheral tissues is because of a total lack of dendritic cells and feeble expression of major histocompatibility complex (MHC) proteins in neurons and glia. However, research over the past two decades has identified immune effector mechanisms intrinsic to the CNS for immediate tackling, attenuating and clearing of viral infections, with assistance pouring in from peripheral circulation in the form of neutralizing antibodies and cytotoxic T cells at a later stage. Specialized CNS cells, microglia and astrocytes, were regarded as sole sentinels of the brain for containing a viral onslaught but neurons held little recognition as a potential candidate for protecting itself from the proliferation and pathogenesis of neurotropic viruses. Accumulating evidence however indicates that extracellular insult causes neurons to express immune factors characteristic of lymphoid tissues. This article aims to comprehensively analyze current research on this conditional alteration in the protein expression repertoire of neurons and the role it plays in CNS innate immune response to counter viral infections.     

Epigenetic modulation of host: new insights into immune evasion by viruses

Viruses have evolved with their hosts, which include all living species. This has been partly responsible for the development of highly advanced immune systems in the hosts. However, viruses too have evolved ways to regulate and evade the host’s immune defence. In addition to mutational mechanisms that viruses employ to mimic the host genome and undergo latency to evade the host’s recognition of the pathogen, they have also developed epigenetic mechanisms by which they can render the host’s immune responses inactive to their antigens. The epigenetic regulation of gene expression is intrinsically active inside the host and is involved in regulating gene expression and cellular differentiation. Viral immune evasion strategies are an area of major concern in modern biomedical research. Immune evasion strategies may involve interference with the host antigen presentation machinery or host immune gene expression capabilities, and viruses, in these manners, introduce and propagate infection. The aim of this review is to elucidate the various epigenetic changes that viruses are capable of bringing about in their host in order to enhance their own survivability and pathogenesis.