Influence of somatotropin and nutrition on bovine oocyte retrieval and in vitro development

The objective of this study was to determine the effects of supplemental bovine somatotropin (bST) and limit feeding on follicular growth and oocyte competence in yearling beef heifers. Sixteen growing heifers (424+/-4 kg) were randomly assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement, with main effects of bST (0 or 33 microg/kg BW/d) and feeding regimen (ad libitum or 0.75 ad libitum intake). Animals were treated for 100 d prior to follicular aspiration, and treatments continued for the 42-d period that follicles were aspirated. Follicles were observed ultrasonically then aspirated, and recovered oocytes were matured, fertilized and developed in vitro. The number of follicles observed ultrasonically was greater with bST treatment (P<0.01) but was unchanged by plane of nutrition. The number and quality of recovered oocytes were similar among treatments, as was the number of oocytes resulting in blastocyst formation.     

Topology of catalytic portion of prostaglandin I(2) synthase: identification by molecular modeling-guided site-specific antibodies

Prostaglandin I(2) synthase (PGIS) is an eicosanoid-synthesizing cytochrome P450, located in the endoplasmic reticulum (ER) membrane. The membrane topology of the catalytic portion of PGIS is still unknown. General models of the membrane topology of microsomal P450s have been proposed in two forms: (a) large part of the polypeptide exposed on the cytoplasmic side with an NH(2)-terminal membrane anchor to the ER membrane and (b) deep immersion of the polypeptide in the membrane, as described by J. P. Miller et al. (1996, Biochemistry 35, 1466-1474). We have characterized the membrane topology of catalytic portion of PGIS using molecular modeling-guided site-specific antibodies. A 3D working model of PGIS was constructed by homology modeling using P450(BM-3) crystal structure as a template (S. K. Shyue et al., 1997, J. Biol. Chem. 272, 3657-3662). Three hydrophilic peptides corresponding to different regions of the surface portion of PGIS with residues 109-127 (P109-127), 353-368 (P353-368), and 411-431 (P411-431) predicted from the model and an NH(2)-terminal hydrophobic peptide (residues 1-28, P1-28) were synthesized and used to prepare site-specific antibodies. All three of the hydrophilic peptide antibodies have high titer and are specifically recognized human PGIS, as shown by binding assays and Western blot analysis. In contrast, the hydrophobic NH(2)-terminal peptide has a much lower titer binding to the PGIS protein. The overall arrangement of the PGIS polypeptide with respect to the endoplasmic reticulum (ER) membrane was examined by immunocytochemistry techniques in transiently transfected COS-1 cells with recombinant human PGIS cDNA and in ECV cells expressing endogenous PGIS. The immunofluorescence staining for the cells with selective permeabilization of the plasma membrane using streptolysin O indicated that all three of the hydrophilic peptide antibodies bound to the cytoplasmic surface of the ER membrane. These results provide direct experimental evidence supporting the predicted 3D protein topological model in which the segments are located on the protein surface and the membrane topological model in which PGIS is largely exposed on the cytoplasmic side of the ER membrane. It also led us to conclude that the PGIS substrate, prostaglandin H(2) (PGH(2)), produced by prostaglandin H(2) synthase (PGHS) in the ER lumenal side must pass through the ER membrane barrier to the catalytic site of the PGIS in the cytoplasmic side of the ER membrane.     

Three-dimensional crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution

Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption.     

Effect of gender on the development of hypocapnic apnea/hypopnea during NREM sleep.

We hypothesized that a decreased susceptibility to the development of hypocapnic central apnea during non-rapid eye movement (NREM) sleep in women compared with men could be an explanation for the gender difference in the sleep apnea/hypopnea syndrome. We studied eight men (age 25-35 yr) and eight women in the midluteal phase of the menstrual cycle (age 21-43 yr); we repeated studies in six women during the midfollicular phase. Hypocapnia was induced via nasal mechanical ventilation for 3 min, with respiratory frequency matched to eupneic frequency. Tidal volume (VT) was increased between 110 and 200% of eupneic control. Cessation of mechanical ventilation resulted in hypocapnic central apnea or hypopnea, depending on the magnitude of hypocapnia. Nadir minute ventilation in the recovery period was plotted against the change in end-tidal PCO(2) (PET(CO(2))) per trial; minute ventilation was given a value of 0 during central apnea. The apneic threshold was defined as the x-intercept of the linear regression line. In women, induction of a central apnea required an increase in VT to 155 +/- 29% (mean +/- SD) and a reduction of PET(CO(2)) by -4.72 +/- 0.57 Torr. In men, induction of a central apnea required an increase in VT to 142 +/- 13% and a reduction of PET(CO(2)) by -3.54 +/- 0.31 Torr (P = 0.002). There was no difference in the apneic threshold between the follicular and the luteal phase in women. Premenopausal women are less susceptible to hypocapnic disfacilitation during NREM sleep than men. This effect was not explained by progesterone. Preservation of ventilatory motor output during hypocapnia may explain the gender difference in sleep apnea.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.     

Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide

The Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) has been registered and is commercially produced in China as a biopesticide to control the bollworm in cotton. However, the virus has a relatively slow speed of action. To improve its efficacy, recombinant HearNPVs were generated by deleting the ecdysteroid UDP-glucosyltransferase (egt) gene (HaCXW1 and HaLM2) or by inserting the insect-specific toxin gene AaIT in the egt locus (HaCXW2) of HearNPV using conventional recombination strategies in insect cell culture. The various recombinants remained genetically stable when cultured in HzAM1 insect cells. Bioassay data showed a significant reduction in the time required for all HearNPV recombinants to kill second instar H. armigera larvae. The LT(50) of the egt deletion recombinants HaCXW1 and HaLM2 was about 27% faster than that of wild-type HearNPV. The largest reduction in LT(50) was achieved by inserting the gene for the insect-specific neurotoxin, AaIT, in the egt locus, giving a reduction in LT(50) of 32% compared to wild-type HearNPV. The ability to genetically improve the properties of HearNPV as a biopesticide provides a further opportunity to develop this virus into a commercially viable product to control the bollworm in China.     

IL-1 beta attenuates IFN-alpha beta-induced antiviral activity and STAT1 activation in the liver: involvement of proteasome-dependent pathway

IFN-alphabeta is the only established treatment for viral hepatitis; however, more than 60% of patients are poorly responsive. Because viral hepatitis is associated with inflammation, we hypothesized that inflammation may attenuate the efficacy of IFN therapy. To test this hypothesis, the effect of IL-1beta, one of the major proinflammatory cytokines, on IFN signaling pathway in the liver was examined. Administration of IL-1beta in vivo attenuated IFN-alphabeta-induced STAT1 tyrosine phosphorylation in the liver but not in the spleen. The inhibitory action of IL-1beta in vivo was not affected by depleting hepatic Kupffer cells, suggesting that IL-1beta may directly target IFN-alphabeta signaling in hepatocytes. Indeed, pretreatment of human hepatocellular carcinoma HepG2 cells with IL-1beta suppressed IFN-alphabeta-induced antiviral activity and antiviral protein MxA mRNA expression. Furthermore, IL-1beta attenuated IFN-alphabeta-induced STAT1 binding and tyrosine phosphorylation without affecting the level of STAT1 protein. This inhibitory effect can be reversed by pretreatment with either proteasome inhibitors or transfection of dominant negative NF-kappaB inducing kinase mutants. Taken together, these findings suggest that IL-1beta attenuates IFN-alphabeta-induced STAT1 activation by a proteasome-dependent mechanism. In view of high levels of IL-1beta in the serum or within the liver of patients with chronic liver diseases, attenuation of IFN-alphabeta signaling in the liver by IL-1beta could be one of the mechanisms underlying the resistance to IFN therapy in chronic hepatitis C, and IL-1beta could be a potential therapeutic target for improving the efficacy of IFN therapy.     

Melatonin reduces rat hepatic macromolecular damage due to oxidative stress caused by delta-aminolevulinic acid

Delta-aminolevulinic acid, precursor of heme, accumulates in a number of organs, especially in the liver, of patients with acute intermittent porphyria. The potential protective effect of melatonin against oxidative damage to nuclear DNA and microsomal and mitochondrial membranes in rat liver, caused by delta-aminolevulinic acid, was examined. Changes in 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, an index of DNA damage, and alterations in membrane fluidity (the inverse of membrane rigidity) and lipid peroxidation in microsomal and mitochondrial membranes, as indices of damage to lipid and protein molecules in membranes, were estimated. Measurements were made in rat liver after a 2 week treatment with delta-aminolevulinic acid (40 mg/kg b.w., every other day). To test the potential protective effects of melatonin, the indole was injected (i.p. 10 mg/kg b.w.) 3 times daily for 2 weeks. 8-OHdG levels and lipid peroxidation in microsomal membranes increased significantly whereas microsomal and mitochondrial membrane fluidity decreased as a consequence of delta-aminolevulinic acid treatment. Melatonin completely counteracted the effects of delta-aminolevulinic acid. Melatonin was highly effective in protecting against oxidative damage to DNA as well as to microsomal and mitochondrial membranes in rat liver and it may be useful as a cotreatment in patients with acute intermittent porphyria.     

Hybrid vector designs to control the delivery, fate and expression of transgenes

One of the greatest challenges to gene therapy is the targetting of gene delivery selectively to the sites of disease and regulation of transgene expression without adverse effects. Ultimately, the successful realization of these goals is dependent upon improvements in vector design. Over the years, viral vector design has progressed from various types of replication-defective viral mutants to replication-conditioned viruses and, more recently, to 'gutted' and hybrid vectors, which have, respectively, eliminated expression of non-relevant or toxic viral genes and incorporated desired elements of different viruses so as to increase the efficacy of gene delivery in vivo. This review will focus on the different viral and cellular elements which have been incorporated into virus vectors to: improve transduction efficiencies; alter the entry specificity of virions; control the fate of transgenes in the host cells; and regulate transgene expression.     

Weight stability masks sarcopenia in elderly men and women

Skeletal muscle loss or sarcopenia in aging has been suggested in cross-sectional studies but has not been shown in elderly subjects using appropriate measurement techniques combined with a longitudinal study design. Longitudinal skeletal muscle mass changes after age 60 yr were investigated in independently living, healthy men (n = 24) and women (n = 54; mean age 73 yr) with a mean +/- SD follow-up time of 4.7 +/- 2.3 yr. Measurements included regional skeletal muscle mass, four additional lean components (fat-free body mass, body cell mass, total body water, and bone mineral), and total body fat. Total appendicular skeletal muscle (TSM) mass decreased in men (-0.8 +/- 1.2 kg, P = 0.002), consisting of leg skeletal muscle (LSM) loss (-0.7 +/- 0.8 kg, P = 0.001) and a trend toward loss of arm skeletal muscle (ASM; -0.2 +/- 0.4 kg, P = 0.06). In women, TSM mass decreased (-0.4 +/- 1.2 kg, P = 0.006) and consisted of LSM loss (-0.3 +/- 0.8 kg, P = 0.005) and a tendency for a loss of ASM (-0.1 +/- 0.6 kg, P = 0.20). Multiple regression modeling indicates greater rates of LSM loss in men. Body weight in men at follow-up did not change significantly (-0.5 +/- 3.0 kg, P = 0.44) and fat mass increased (+1.2 +/- 2.4 kg, P = 0.03). Body weight and fat mass in women were nonsignificantly reduced (-0.8 +/- 3.9 kg, P = 0.15 and -0.8 +/- 3.5 kg, P = 0.12). These observations suggest that sarcopenia is a progressive process, particularly in elderly men, and occurs even in healthy independently living older adults who may not manifest weight loss.