Effects of in utero and in vitro incubation on the lipid-bound fatty acids and sterols of porcine spermatozoa

Sperm-rich semen and washed porcine spermatozoa were incubated for up to 2 hr either in utero in the presence of oviduct fluid or in vitro at 37°C. Sperm lipids were extracted and separated into phospholid and neutral lipid fractions. Eleven phospholipid and five neutral lipid fatty acids were identified and quantified using GC and GC-MS. The percentage of 22:5n6, the major phospholipid fatty acid, decreased slightly but significantly during 1.5 hr of in utero incubation (41.2–38.0%), but after 2.0 hr of in utero incubation no significant difference was observed (40.0%). None of the phospholipid fatty acids changed in concentration during in vitro incubation. The mole ratio of phospholipid to phospholipid fatty acid (1.00:1.27) did not change during incubation. The levels of neutral lipid-bound 14:0 decreased (43.5% to 31.8%) and that of 18:0 increased (11.1% to 18.2%) during in utero incubation. Similar but less pronounced changes were observed during in vitro incubation. (43.5% to 36.0%; and 11.1% to 15.8%, respectively). Two major sterols, cholestrol (73%) and desmosterol (27%) were identified by gas chromatography–mass spectrometry. The mole ratio of phospholipid to sterol (2.47:1:00) did not change during incubation.     

The role of ovarian sympathetic innervation in the control of estrous responsiveness in the rat

This study examined the contribution of the superior ovarian nerve (SON) to estrous responsiveness and ovarian function in cycling rats. Section of the SON was carried out at 1100 on proestrus, and lordotic responsiveness was measured at 1500, 1700, and 2100 on that day and at 0900, 1200, and 1500 on the day of estrus. SON section decreased lordosis intensity significantly at 1500 and 1700 on proestrus and at 0900 on estrus. Pacing of sexual contacts with males was decreased at 2100 in nerve-sectioned (Nervx) animals when compared with sham-operated controls (SHAM). Serum progesterone (P) concentrations were significantly lower in Nervx animals than in Sham animals 30 min after surgery, but were not different between groups at 4.5 hr. Serum estradiol (E2) concentrations did not differ between groups at either time. In addition, Nervx and Sham groups did not differ on measures of pregnancy/pseudopregnancy initiation or on measures of ovarian function 10 days after surgery. These data suggest that the integrity of the SON is necessary for the display of full estrous responsiveness in cycling rats, and suggest that the acute decreases in serum P occurring as a consequence of SON section may be responsible for the deficits seen in Nervx animals.     

Whole-cell and membrane lipids of the methylotrophic bacterium Methylosinus trichosporium.

The lipid composition of the methylotrophic bacterium Methylosinus trichosporium was examined. Whole-cell lipid distribution was 39.1% neutral lipids, 34.5% polar lipids, and 26.4% poly-beta-hydroxybutyric acid. Membrane lipids were 83% phospholipids, with phosphatidylethanolamine and phosphatidylglycerol accounting for over 94% of the total. All the phospholipids had similar fatty acid compositions, with 18:1 accounting for about 87% of the total and most of the rest consisting of 16:1. Similarities between the lipid composition of this bacterium and other bacteria are discussed.     

N-wasp and the arp2/3 complex are critical regulators of actin in the development of dendritic spines and synapses

Changes in the number, size, and shape of dendritic spines are associated with synaptic plasticity, which underlies cognitive functions such as learning and memory. This plasticity is attributed to reorganization of actin, but the molecular signals that regulate this process are poorly understood. In this study, we show neural Wiskott-Aldrich syndrome protein (N-WASP) regulates the formation of dendritic spines and synapses in hippocampal neurons. N-WASP localized to spines and active, functional synapses as shown by loading with FM4-64 dye. Knock down of endogenous N-WASP expression by RNA interference or inhibition of its activity by treatment with a specific inhibitor, wiskostatin, caused a significant decrease in the number of spines and excitatory synapses. Deletion of the C-terminal VCA region of N-WASP, which binds and activates the actin-related protein 2/3 (Arp2/3) complex, dramatically decreased the number of spines and synapses, suggesting activation of the Arp2/3 complex is critical for spine and synapse formation. Consistent with this, Arp3, like N-WASP, was enriched in spines and excitatory synapses and knock down of Arp3 expression impaired spine and synapse formation. A similar defect in spine and synapse formation was observed when expression of an N-WASP activator, Cdc42, was knocked down. Thus, activation of N-WASP and, subsequently, the Arp2/3 complex appears to be an important molecular signal for regulating spines and synapses. Arp2/3-mediated branching of actin could be a mechanism by which dendritic spine heads enlarge and subsequently mature. Collectively, our results point to a critical role for N-WASP and the Arp2/3 complex in spine and synapse formation.     

Crystal structure of a beta-catenin/Tcf complex

The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear beta-catenin/Tcf complex, activating Wnt target genes. A crystal structure of beta-catenin bound to the beta-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to beta-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in beta-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies.     

Effects of endothelin-1 and homologous trout endothelin on cardiovascular function in rainbow trout

The cardiovascular effects of endothelin (ET)-1 and the recently sequenced homologous trout ET were examined in unanesthetized trout, and vascular capacitance curves were constructed to evaluate the responsiveness of the venous system to ET-1. A bolus dose of 667 pmol/kg ET-1 doubled ventral aortic pressure; produced a triphasic pressor-depressor-pressor response in dorsal aortic pressure (P(DA)); increased central venous pressure, gill resistance, and systemic resistance; and decreased cardiac output, heart rate, and stroke volume. These responses were dose dependent. Bolus injection of trout ET (333 or 1,000 pmol/kg) produced essentially identical, dose-dependent cardiovascular responses as ET-1. Dorsal aortic infusion of 1 and 3 pmol. kg(-1). min(-1) ET-1 and central venous infusion into the ductus Cuvier of 0.3 and 1 pmol. kg(-1). min(-1) produced similar dose-dependent cardiovascular responses, although the increase in P(DA) became monophasic. The heightened sensitivity to central venous infusion was presumably due to the more immediate exposure of the branchial vasculature to the peptide. Infusion of 1 pmol. kg(-1). min(-1) ET-1 decreased vascular compliance but had no effect on unstressed blood volume. These results show that ETs affect a variety of cardiovascular functions in trout and that branchial vascular resistance and venous compliance are especially sensitive. The multiplicity of effectors stimulated by ET suggests that this peptide was extensively integrated into cardiovascular function early on in vertebrate phylogeny.     

Defective in vitro T cell colony formation in the acquired immunodeficiency syndrome

Depressed T cell immunity is a universal characteristic of the acquired immunodeficiency syndrome (AIDS). In the present study, 25 patients with AIDS and opportunistic infections, 22 individuals with AIDS-related complex (ARC, or chronic lymphadenopathy syndrome), and 20 healthy homosexuals were evaluated by means of the T cell colony assay. Forty-seven healthy heterosexual controls showed an average of 3964 +/- 319 colonies/7.5 X 10(5) cells, with a range of 880 to 9340. The mean in the 20 healthy homosexuals (3173 +/- 483) did not differ significantly from the controls; in this group, only three patients had values less than 1000 colonies/plate. By contrast, all AIDS patients and 14 ARC patients had colony counts less than 1000. The mean value for the AIDS patients was only 24 +/- 15 (p less than 0.0005 compared with either controls or healthy homosexuals); values in the ARC group were intermediate (1180 +/- 360). The addition of interleukin 2 to the plates promoted correction of the proliferative abnormality in ARC patients. This interleukin increased colony scores in the AIDS group, but the mean value was still significantly less than controls. Comparison indicated that the colony assay is a more sensitive indicator for detecting proliferative abnormalities than responses to PHA, Con A, or pokeweed mitogen in suspension cultures.     

Proteolysis of the matricellular protein hevin by matrix metalloproteinase-3 produces a SPARC-like fragment (SLF) associated with neovasculature in a murine glioma model

The matricellular SPARC-family member hevin (Sparc-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. Based on sequence similarity, we hypothesized that proteolytic digestion of hevin would result in SPARC-like fragments (SLF) that affect the activity and/or location of these proteins. Incubation of hevin with matrix metalloproteinase-3 (MMP-3), a protease known to cleave SPARC, produced a limited number of peptides. Sequencing revealed the major proteolytic products to be SPARC-like in primary structure. In gliomas implanted into murine brain, a SLF was associated with SPARC in the neovasculature but not with hevin, the latter prominent in the astrocytes encompassed by infiltrating tumor. In this model of invasive glioma that involves MMP-3 activity, host-derived SLF was not observed in the extracellular matrix adjacent to tumor cells. In contrast, it occurred with its homolog SPARC in the angiogenic response to the tumor. We conclude that MMP-3-derived SLF is a marker of neovessels in glioma, where it could influence the activity of SPARC.     

The Evolution of Duplicate Glyceraldehyde-3-Phosphate Dehydrogenase Genes in Drosophila

In Drosophila melanogaster there are two genes which encode the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Gapdh-43E and Gapdh-13F. We have shown that Gapdh-43E codes for the GAPDH subunit with an apparently larger molecular weight while Gapdh-13F encodes the GAPDH subunit having an apparently smaller molecular weight. Immunoblots of sodium dodecyl sulfate gels were used to survey species from throughout the genus and results indicated that two classes of GAPDH subunits are present only in Drosophila species of the melanogaster and takahashi subgroups of the melanogaster group. Only the smaller subunit is found in species of the obscura group while all other species have only a large subunit. Drosophila hydei was analyzed at the DNA level as a representative species of the subgenus Drosophila. The genome of this species has a single Gapdh gene which is localized at a cytogenetic position likely to be homologous to Gapdh-43 E of D. melanogaster. Comparison of its sequence with the sequence of the D. melanogaster Gapdh genes indicates that the two genes of D. melanogaster are more similar to one another than either is to the gene from D. hydei. The Gapdh gene from D. hydei contains an intron following codon 29. Neither Gapdh gene of D. melanogaster has an intron within the coding region. Southern blots of genomic DNA were used to determine which species have duplicate Gapdh genomic sequences. Gene amplification was used to determine which species have a Gapdh gene that is interrupted by an intron. Species of the subgenus Drosophila have a single Gapdh gene with an intron. Species of the willistoni and saltans groups have a single Gapdh gene that does not contain an intron.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequential unfolding of the hemolysin two‐partner secretion domain from Proteus mirabilis

Protein secretion is a major contributor to Gram‐negative bacterial virulence. Type Vb or two‐partner secretion (TPS) pathways utilize a membrane bound β‐barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C‐proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N‐proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site‐specific mutants of HpmA265. By correlating the effect of individual site‐specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C‐terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C‐terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N‐proximal polar core subdomain.