Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes

Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24h after UVB light with only one minimal erythemal dose. Since UVB generates also H(2)O(2), we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1x10(-3)M), the V(max) for enzyme activity increased 4-fold by H(2)O(2) (>2.0x10(-3)M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp(120) residue to 5-OH-Trp(120) by H(2)O(2) causing a conformational change in the regulatory domain. PAH was still activated by H(2)O(2) in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H(2)O(2) by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis.     

Prokaryotic expression, purification, and polyclonal antibody production against a novel drug resistance gene of Leishmania donovani clinical isolate

Diseases produced by protozoan parasites are one of the main causes of morbidity and mortality around the world, affecting millions of people. Among these, leishmaniasis has become the second most common cause of death and the problem is further complicated by the expansion of parasite resistance to the conventional drugs. The high rate of therapeutic failure thus calls for new rational approaches to develop alternative drugs. Understanding resistance mechanisms may help identify new targets for drug development. So we present here the cloning, expression, purification, and antibody production of a gene implicated in imparting resistance to pentavalent antimony (SbV) in clinical isolates of kala azar with a view to gain insight into the novel mechanism of its drug resistance.     

In vitro antibacterial activity in seed extracts of Manilkara zapota, Anona squamosa, and Tamarindus indica

Extracts prepared from seeds of Manilkara zapota, Anona squamosa, and Tamarindus indica were screened for their antibacterial activity by disc diffusion and broth dilution methods. Acetone and methanol extracts of T. indica seeds were found active against both gram-positive and gram-negative organisms. MIC values of potent extracts against susceptible organisms ranged from 53-380 μg/mL. Methanol extract of T. indica and acetone extract of M. zapota seeds were found to be bactericidal.     

Binding of a cationic phenazinium dye in anionic liposomal membrane: a spectacular modification in the photophysics

Interaction of a cationic phenazinium dye, phenosafranin (PSF), with the anionic liposomal vesicle/bilayer of dimyristoyl-l-α-phosphatidylglycerol (DMPG) has been demonstrated using steady state and time resolved fluorescence and fluorescence anisotropy techniques. The charge transfer emission spectrum of PSF shows a dramatic modification in terms of fluorescence yield together with an appreciable hypsochromic shift in the lipid environment. The blue shift indicates a lowering in polarity inside the vesicle as compared to that in bulk water. The fluorescence and fluorescence quenching studies and micropolarity determination reveal that the cationic fluorophore has a profound binding interaction with the anionic DMPG membrane. Anisotropy study indicates the imposition of a motional restriction on the probe inside the bilayer. The electrostatic interaction between the cationic dye and the anionic lipid membrane has been argued to be the reason behind all these observations. The results could be useful in analyzing membrane organization and heterogeneity in natural membranes exploiting PSF or alike compounds as fluorescent probes.     

Curcumin enhances the polyglutamine-expanded truncated N-terminal huntingtin-induced cell death by promoting proteasomal malfunction

Formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various proteasome components is the major hallmark of the polyglutamine diseases. Curcumin is a polyphenolic compound having anti-inflammatory, anti-tumor, and anti-oxidative properties. Recently, curcumin has been reported to suppress the amyloid-beta accumulation, oxidative damage, and inflammation in the transgenic mice model of Alzheimer's disease. Here, we found that the treatment of curcumin increases the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-dependent cell death. Curcumin also causes rapid proteasomal malfunction in the mutant huntingtin expressing cells in comparison with normal glutamine repeat expressing cells. Finally, we show that N-acetyl cysteine (NAC), a potent antioxidant, reverted the curcumin-induced mutant huntingtin aggregation and proteasomal malfunction in the mutant huntingtin expressing cells. NAC also protects curcumin-induced cell death. Our result suggests that curcumin promotes mutant huntingtin-induced cell death by mimicking proteasomal dysfunction.     

Development of multiple embryos in polyembryonic insertional mutant OsPE of rice

A T-DNA insertional mutant OsPE of rice gives twin and triplet seedlings in up to 20?% of the seeds. Detailed cytological and histological analysis of OsPE indicated normal male and female gametogenesis in the OsPE mutant. Confocal laser scanning microscopic (CLSM) analysis of the developing seeds of OsPE showed multiple embryo development in up to 60?% of the ovules. The multiple embryos, mostly twins and triplets, and rarely quadruplets, developed through sequential cleavage from a single zygotic embryo in each ovule. The reduced number of multiple seedlings compared with multiple embryos observed in CLSM study may be attributed to their inability to develop further due to competition in a single embryo sac. Key message Multiple seedlings in the OsPE mutant are due to sequential proliferation and cleavage of the zygotic embryos. The nucellar tissue was not involved in multiple embryo development.     

The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3's LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly.     

Role of insulin-like growth factors (IGFs), their receptors and genetic regulation in the chondrogenesis and growth of the mandibular condylar cartilage

Growth of the mandibular condylar cartilage (MCC) is reviewed as a function of genetic and epigenetic factors. The growth centers around the differential spatial concentration of the chondrocytes, influence of growth factors like TGF-β and heterogeneity in the number of IGF receptors, control the action of IGF. Besides these factors, growth of the mandibular condyle is influenced by differential response of chondrocytes as a function of their source/ageing, which in turn is regulated by TGF-β, BMPs and IGFs. While IGF-1 promotes proteoglycan synthesis and survival of the chondrocytes to maintain cartilage homeostasis, TGF-β synergistically catalysed the effect of IGF-1, while BMPs catalysed proteolysis as and when physiologically needed. To understand these processes, role of IGF-1 and its six receptors is at the center to a number of physiological processes being regulated by its mode of application for the growth and differentiation. Probing deeper, biological functions of IGFs seemed to depend on their level of free status rather than bound status to respective IGF-binding proteins (IGF-BPs), considered prerequisite to modulate their biological functions. Genetic regulation of their secretion has thrown light on their insulin-like structural homology, level and response in osteo-arthritis (OA), rheumatic arthritis (RA) and diabetes type-II. Biochemistry and spatial distribution of IGF receptors in different domains exerts control on IGF-1 activities. In ultimate analysis, IGF-axis conserved during the evolution to regulate cell growth and proliferation affect nearly every organ in the body as judged from the techniques determining skeletal maturity and decision making dependent on it for orthodontic, orthognathic/orthopedic and dental implant applications.     

Inhibitory effect of antibodies against human chorionic gonadotropin on the growth of colorectal tumour cells

Human chorionic gonadotropin (hCG) was initially believed to be secreted exclusively by the embryo with its primary function being "rescue" of the corpus luteum. However, recently it has been found that the hormone (or its individual subunits) is also secreted by many cancers and that in many cases secretion is associated with poor patient prognosis. In this study, we assessed the presence of hCG in colorectal cancer cells (CCL-253) and evaluated the anti-tumour effects of anti-hCG antibodies in vitro and in vivo. Anti-hCG antibodies were reactive with CCL-253, as revealed by confocal immunoflourescence microscopy; both cell surface and intracellular expression were observed. Western blot analysis showed that antibodies appeared to interact with several moieties, indicating a level of cross-reactivity. Anti-hCG antiserum specifically reduced the viability of tumor cells and the addition of complement increased in vitro anti-tumor effects. In nude mice implanted with CCL-253 cells, administration of anti-hCG antiserum caused a significant reduction in tumor volume; all treated animals survived, while mortality was observed in control animals. Results suggest that anti-hCG antibodies can mediate significant anti-tumor activity both in vitro and in vivo and lend support to the rationale of anti-hCG immunization in the therapy of gonadotropin- sensitive cancers.