Modulation of post-thaw sperm functions with oviductal proteins in buffaloes

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.     

Sodium selenite stimulates neurobehavior and neurochemical activities in rats

The effect of 0.05, 0.1, and 0.2 mg sodium selenite/kg body weight ip on the activities of neurobehavioral, acetyl cholinesterase, monoamine oxidase, and the content of dopamine and its metabolites in circadian rhythm centers of male Wistar rats was studied after 7 d of treatment. The results show an appreciable increase in locomotion, stereo-events, distance traveled, and average speed at the dose of 0.1 and 0.2 mg sodium selenite/kg. The data have shown hyperactivity of animals with various doses of sodium selenite, and it was significant and dose-dependent after 3 d of treatment. The activity of acetylcholinesterase (AChE) was inhibited dose dependently, and it was significant in preoptic area with 0.1 or 0.2 mg sodium selenite/kg. Conversely, in the posterior hypothalamus its activity was significantly elevated with the dose of 0.2 mg sodium selenite/kg, but its alteration in brain stem was not significant. Monoamine oxidase (MAO) activity was increased in preoptic area with the dose of 0.1 mg sodium selenite/kg, but its alteration in posterior hypothalamus and brain stem was not significant. The content of dopamine (DA), 3,4-dihydroxyphenyl acetic acid (DOPAC), and homovanilic acid (HVA) was elevated dose dependently and it was significant with the doss of 0.1 and 0.2 mg sodium selenite/kg, but the content of DOPAC and HVA in posterior hypothalamus was not significant with the dose of 0.1 mg sodium selenite/kg.     

Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid

Mycobacterium tuberculosis cell envelope is a treasure house of biologically active lipids of fascinating molecular architecture. Although genetic studies have alluded to an array of genes in biosynthesis of complex lipids, their mechanistic, structural, and biochemical principles have not been investigated. Here, we have dissected the molecular logic underlying the biosynthesis of a virulence lipid phthiocerol dimycocerosate (PDIM). Cell-free reconstitution studies demonstrate that polyketide synthases, which are usually involved in the biosynthesis of secondary metabolites, are responsible for generating complex lipids in mycobacteria. We show that PapA5 protein directly transfers the protein bound mycocerosic acid analogs on phthiocerol to catalyze the final esterification step. Based on precise identification of biological functions of proteins from Pps cluster, we have rationally produced a nonmethylated variant of mycocerosate esters. Apart from elucidating mechanisms that generate chemical heterogeneity with PDIMs, this study also presents an attractive approach to explore host-pathogen interactions by altering mycobacterial surface coat.     

Syntheses and biological activities of chalcone and 1,5-benzothiazepine derivatives: promising new free-radical scavengers, and esterase, urease, and alpha-glucosidase inhibitors

A series of 2,4-diaryl-2,3,4,5-tetrahydro- (36-40) and 2,4-diaryl-2,3-dihydro-1,5-benzothiazepines (25-35) have been synthesized from the corresponding chalcones 1-24. Both the benzothiazepines and chalcones were evaluated as DPPH free-radical scavengers and as inhibitors of cholinesterases, urease, and alpha-glucosidase. Compounds 2, 5, 6, 7, 10, 13, 18, 21, 36a, 37a, 37b, and 39a showed significant cholinesterase inhibiting activities. Among the 15 dihydro-1,5-benzothiazepines, 26, 32, and 35 exhibited significant radical-scavenging activities; and six tetrahydro-1,5-benzothiazepines (35, 36a, 36b, 37a, 37b, and 39a) were found to be inhibitors of AChE and BChE. Compounds 22, 25, 26, 33, 35, 36a, 37b, and 39a inhibited urease, and 25 and 27-31 were found to be potent inhibitors of alpha-glucosidase.     

Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.     

Chemical xenobiotics and mitochondrial autoantigens in primary biliary cirrhosis: identification of antibodies against a common environmental, cosmetic, and food additive, 2-octynoic acid

Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.     

Expansion of bone marrow IFN-alpha-producing dendritic cells in New Zealand Black (NZB) mice: high level expression of TLR9 and secretion of IFN-alpha in NZB bone marrow

Patients with systemic lupus erythematosus have elevated IFN-alpha production. Furthermore, sera IFN-alpha levels correlate with disease activity. We have focused our attention on whether this phenotype is also seen in the New Zealand Black (NZB) mice and simultaneously addressed the underlying mechanisms. Specifically, we analyzed: 1) levels of sera IFN-alpha after type A CpG ODN 2216 injection in autoimmunity-prone NZB and control mice, and 2) levels of IFN-alpha synthesized by IFN-alpha-producing dendritic cells (IPDCs) using highly enriched populations of CD11c+B220+ IPDCs derived from NZB and control mice; IPDCs are divided into two subpopulations (CD4+CD11c+B220+ and CD4-CD11c+B220+). Our data demonstrate that NZB mice produced higher levels of sera IFN-alpha after type A CpG ODN 2216 injection when compared with control mice (p < 0.01). In addition, the cell numbers, frequency, and TLR9 mRNA levels of CD4+ and CD4- IPDC were markedly increased in the bone marrow (BM) of NZB mice. Upon in vitro stimulation with TLR9 ligand-CpG ODN 2216, higher levels of IFN-alpha were synthesized by IPDCs from the BM of NZB. The major contributor of IFN-alpha was the CD4-CD11c+B220+ IPDC subpopulation. Furthermore, NZB BM IPDCs manifest impaired expression of homing chemokine CCR7 and CD62L, and IL-12 production. These data on the functional characteristics of the IPDC lineages explain in part the mechanism of hyper-IFN-alpha production and help clarify the mechanism for the expansion of NZB BM IPDCs.     

Selection of a highly virulent fungal isolate, Metarhizium anisopliae CLO 53, for controlling Hoplia philanthus

The June beetle, Hoplia philanthus Füessly (Coleoptera: Scarabaeidae), has become a widespread and destructive insect pest of lawns, sport turf, pastures, and horticultural crops in Belgium. The virulence of 34 entomopathogenic fungal isolates from the genera Metarhizium, Beauveria, and Paecilomyces to third-instar H. philanthus was tested in bioassays by dipping larvae in 10(7)conidia/ml suspensions. Two isolates of Metarhizium anisopliae (CLO 53 and CLO 54) caused maximally 90% mortality 10 weeks post-inoculation while other isolates only caused mortalities between 10 and 62%. The virulence of M. anisopliae CLO 53 was further tested by exposing H. philanthus larvae to conidial serial concentrations of 10(4)-10(9)conidia/g sandy soil for up to 11 weeks at 15, 20 or 25 degrees C. Mortality was dependant on the fungal concentration, exposure time, and temperature. Eleven weeks after inoculation, the LC50 values for this isolate ranged from 1.3 to 4.0 x 10(6), 1.0 to 3.2 x 10(5), and 2.5 x 10(4) to 10(5)conidia/g soil at 15, 20, and 25 degrees C, respectively. The LT50 values for this isolate ranged from 3.5 to 21.7, 2.4 to 18.7, and 2.9 to 16.1 weeks at concentrations of 10(9) and 10(4)conidia/g soil at 15, 20, and 25 degrees C, respectively. In glasshouse pot experiment with perennial ryegrass (Lolium perenne L.), the isolate CLO 53 caused mortalities of 50 and 88% of H. philanthus larvae 10 weeks after application of 10(4) and 10(6)conidia/cm(2) soil surface, respectively. The present results suggest that the Belgian isolate CLO 53 has excellent potential for biological control of H. philanthus.