Effect of 30 kDa and above buffalo follicular fluid protein treatment and immunization on ovarian functions in goats (Capra hircus)

Information on the use of buffalo follicular fluid (buFF) in modulation of ovarian functions in farm animals is scanty compared to other species. This is an attempt to investigate the effect of direct administration and active immunization of 30 kDa and above buFF proteins on ovarian functions in goats. Treatment of goats (n = 6) with steroid free 30 kDa and above buFF protein fraction during late-luteal phase for 4 days (days 12 or 13 to days 15 or 16) of the natural cycle, delayed the onset of estrus by 24 h compared to control although the mean duration of estrus was unaffected. A 71% increase (P = 0.06) in mean ovulation number was also observed following treatment. However, the population of large (> or =5 mm diameter) follicle was not affected. The ovarian activity calculated as total of ovulation and large follicles increased (1.6 times) significantly (P = 0.02) in treated animals. Active immunization of goats (n = 5) against these proteins did not affect the onset and duration of estrus. Similarly, the ovulation rate, number of large follicles and the ovarian activity did not differ significantly between immunized and control groups. The study revealed that 30 kDa and above buffalo follicular fluid contains some factor(s) that cause delay in the onset of estrus in goats and increase the ovulation rate. Active immunization against these proteins in goat did not show any effect either on onset, duration of estrus or ovulation rate and large follicle population. Detailed study on these buffalo follicular fluid proteins may help to use them further for modulation of ovarian function in farm animals.     

Synthesis and SAR of novel oxazolidinones: discovery of ranbezolid

Novel oxazolidinones were synthesized containing a number of substituted five-membered heterocycles attached to the 'piperazinyl-phenyl-oxazolidinone' core of eperezolid. Further, the piperazine ring of the core was replaced by other diamino-heterocycles. These modifications led to several compounds with potent activity against a spectrum of resistant and susceptible gram-positive organisms, along with the identification of ranbezolid (RBx 7644) as a clinical candidate.     

Protonated structures of naturally occurring deoxyribonucleic acids and their interaction with berberine

Protonation-induced conformational changes in natural DNAs of diverse base composition under the influence of low pH, low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH3.40, 10mM [Na+], and at 5 degrees C, all natural DNAs irrespective of base composition adopted an unusual and stable conformation remarkably different from the canonical B-form conformation. This protonated conformation has been characterized to have unique absorption and circular dichroic spectral characteristics and exhibited cooperative thermal melting profiles with decreased thermal melting temperatures compared to their respective B-form counterparts. The nature of this protonated structure was further investigated by monitoring the interaction of the plant alkaloid, berberine that was previously shown from our laboratory to differentially bind to B-form and H(L)-form of poly[d(G-C)] [Bioorg. Med. Chem.2003, 11, 4861]. Binding of berberine to protonated conformation of natural DNAs resulted in intrinsic circular dichroic changes as well as generation of induced circular dichroic bands for the bound berberine molecule with opposite signs and magnitude compared with B-form structures. Nevertheless, the binding of the alkaloid to both the B and protonated forms was non-linear and non-cooperative as revealed from Scatchard plots derived from spectrophotometric titration data. Steady state fluorescence studies on the other hand showed remarkable increase of the rather weak intrinsic fluorescence of berberine on binding to the protonated structure compared to the B-form structure. Taken together, these results suggest that berberine can detect the formation of significant population of H(L)-form structures under the influence of protonation irrespective of heterogeneous base compositions in natural DNAs.     

Effect of glutamine analogue-acivicin on tumor induced angiogenesis in Ehrlich ascites carcinoma

The inhibition of tumor growth and tumor induced angiogenesis by the glutamine antimetabolite acivicin was evaluated in 6-7 weeks old male Swiss albino mice bearing Ehrlich ascites carcinoma (EAC) transplanted by intraperitoneal (ip) injections of EAC cells. Treatment involving ip injections with two different doses of acivicin (0.05 and 0.41microg/g body weight/day) in saline revealed decrease in tumor volumes and reduced number of blood vessels on peritoneal wall after 10 and 15 days of treatment when compared to control (i.e. injected with saline only). Vascular hyperpermeability was found to be lesser in the treated groups of mice than the control as indicated by the FITC- D and colloidal carbon assay. Serum VEGF level was found to decrease in the drug treated groups both after 10 and 15 days of treatment. The results thus suggest that acivicin may suppress tumoral angiogenesis through regulation of VEGF level.     

A topological model of biofeedback based on catecholamine interactions

Background  

The present paper describes a topological model of biofeedback. This model incorporates input from a sensory organ and a transduction phase mediated through catecholamine production in the feedback path. The transduction phase comprises both conservative and dissipative systems, from which the appropriate output is combined in a closed loop.     

Curcumin regulates expression and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer

Matrix metalloproteinases (MMPs) are suggested to play a critical role in extracellular matrix degradation and remodeling during inflammation and wound healing processes. However, the role of MMPs in indomethacin-induced gastric ulcer and its healing process are not clearly understood. This study is aimed at determining the regulation of MMP-9 and -2 activities in indomethacin-induced acute gastric ulceration and healing. Indomethacin-ulcerated stomach extracts exhibit significant up-regulation of pro-MMP-9 (92 kDa) activity and moderate reduction of MMP-2 activity, which strongly correlate with indomethacin dose and severity of ulcer. The anti-inflammatory and antioxidant properties of curcumin, an active component of turmeric, suggest that curcumin may exert antiulcer activity through scavenging reactive oxygen species, by regulating MMP activity, or both. To test these possibilities, the effect of curcumin in indomethacin-induced gastric ulcer is examined by biochemical and histological methods. The results show that curcumin exhibits potent antiulcer activity in acute ulcer in rat model by preventing glutathione depletion, lipid peroxidation, and protein oxidation. Denudation of epithelial cells during damage of gastric lumen is reversed by curcumin through re-epithelialization. Furthermore, both oral and intraperitoneal administration of curcumin blocks gastric ulceration in a dose-dependent manner. It accelerates the healing process and protects gastric ulcer through attenuation of MMP-9 activity and amelioration of MMP-2 activity. Omeprazole, an established antiulcer drug does not inhibit MMP-9 while protecting indomethacin-induced gastric ulcer. We conclude that antiulcer activity of curcumin is primarily attributed to MMP-9 inhibition, one of the major path-ways of ulcer healing.     

Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.     

Probing 2-dimensional protein-protein interactions on model membranes

This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in an orientated manner onto solid-supported lipid bilayers by using multivalent chelator lipids. Interaction between the ligand with the receptor subunits was probed by a combination of surface-sensitive spectroscopic detection techniques. Label-free detection by reflectance interferometry is used for following assembly of the membrane and tethering of the receptor subunits in quantitative terms. Total internal reflection spectroscopy is used for monitoring ligand binding to the membrane-anchored receptor, for monitoring ligand-receptor interactions by FRET and for monitoring ligand-exchange kinetics. These assays can be used for determining the affinities and stabilities of ligand-receptor complexes in plane of the membrane. The techniques described in this protocol can be established in 2-3 months.     

Synthesis of a multivalent chelator lipid for stably tethering histidine-tagged proteins onto membranes

This protocol describes the synthesis of a lipid-like molecule carrying a head group containing two nitrilotriacetic acid moieties. This multivalent chelator lipid can be incorporated into lipid membranes, to which histidine-tagged protein can then be tethered in an oriented fashion. Possible applications of this lipid are protein tethering to solid-supported membranes, to lipid vesicles or to live cells. As compared to conventional monovalent chelator lipids, this lipid can achieve highly stable tethering of proteins by the multivalent chelator head. The eight-step synthesis described in this protocol can be completed within 4-5 weeks.     

Disease state differentiation and identification of tuberculosis biomarkers via native antigen array profiling

A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional Western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy purified protein derivative-positive individuals demonstrated that human immunodeficiency virus (HIV)-negative cavitary and noncavitary tuberculosis (TB) patients' sera recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions of which five overlapped with those recognized by non-HIV TB patients' sera and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded 11 products recognized by both cavitary and noncavitary TB patients' sera and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary TB patients. Moreover four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.