Wood duck hatch date: relationship to pairing chronology, plasma luteinizing hormone, and steroid hormones during autumn and winter

We examined variation in courtship activity and hormone levels of male and female wood ducks (Aix sponsa) in relation to hatch date. Young wood ducks were assigned in groups of 8 (4 males and 4 females) to 4 experimental pens; 2 pens contained early-hatched ducks (3-12 April) and 2 pens contained late-hatched ducks (7-16 June). Courtship behaviors occurred less frequently in October and November than in December and January-February for both early- and late-hatched groups. Early-hatched wood ducks participated in courtship more frequently and formed pair bonds sooner than late-hatched individuals. Testosterone (T) and androstenedione (AD) levels of males did not differ between treatment groups; however, average levels of luteinizing hormone (LH) were greater for early-hatched males. Differences in LH occurred in 2 of 12 samples (6 October and 3 November); otherwise, LH did not vary by treatment. Levels of T and LH in males varied temporally, but there was no significant temporal variation in AE levels. Temporal variation in hormone levels was similar for early- and late-hatched males. Estradiol (E), progesterone (P), and LH levels of females did not differ between treatment groups. There was temporal variation in levels of E and LH, but not of P; this variation was similar for early- and late-hatched females. These data indicate that behavioral differences occurring temporally and between early- and late-hatched wood ducks were not related to corresponding differences in hormone levels.     

The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.     

Relation of transition probabilities in the Leslie model to those from experimental cumulative distributions

We explore the relationship between transition probabilities in the Leslie model and those derived from experimental cumulative distributions. The nature of the two kinds of probabilities are discussed, and a formula derived for converting from one to the other. A numerical example is given to illustrate the differences.     

Unusual effects of benzodiazepines and cyclodiene insecticides on an expressed invertebrate GABAA receptor.

We have previously reported [(1991) EMBO J. 10, 3239-3245] the sequence of an invertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor polypeptide which forms homo-oligomeric GABA-gated, bicuculline-sensitive, chloride-ion channels upon heterologous expression. We now demonstrate that the benzodiazepines Ro5-4864 (4'-chlorodiazepam) and diazepam, that are active at mammalian peripheral benzodiazepine sites, and not those benzodiazepines specific for central sites, directly active the homo-oligomeric receptor and evoke larger maximal responses than those elicited by GABA. In addition, members of the cyclodiene class of insecticides block the channel of the receptor in a manner indistinguishable from that of picrotoxin.     

Immunoregulation in cancer-bearing hosts. Down-regulation of gene expression and cytotoxic function in CD8+ T cells.

The causes of the decreased immune responsiveness in tumor-bearing hosts are incompletely understood. The impact of a decreased immune response in cancer patients on the clinical response in immunotherapy trials has not been evaluated. The present report demonstrates a marked decrease in the therapeutic efficacy of adoptively transferred T lymphocytes obtained from murine hosts bearing tumor for greater than 30 days [late tumor-bearing mice (TBM)] as compared with normal mice and mice bearing tumor for less than 21 days (early TBM). In vitro analysis of the functions of the T lymphocytes from late TBM showed an apparently normal proliferative response to anti-CD3 and IL-2 with adequate lymphokine production from CD4+ cells, but a significant decrease in the cytotoxic function of CD8+ cells. The decreased cytotoxicity was not because of cell-mediated suppression. The expression of granzyme B mRNA was significantly delayed and decreased in magnitude in CD8+ cells from late TBM. Culture supernatants from two unrelated tumor cell lines were able to inhibit the cytotoxic activity of normal CD8+ cells in vitro. The tumor-derived suppressive factor is not transforming growth factor-beta (TGF-beta), but it has not been further characterized. The data suggest that one potential mechanism responsible for immunologic defects in patients with large tumor burdens is a tumor-induced defect that compromises the function of CD8+ effector T cells.     

The low affinity binding site for the cocaine analog, WIN 35,428 is an artifact of freezing caudate tissue.

Binding of the cocaine analog [3H] WIN 35,428 was investigated in rat and rabbit caudate. In membranes prepared from fresh tissue, [3H] WIN 35,428 binding was characterized by a single high affinity site with a Kd of 2.5 nM for the rabbit and 5.3 nM for the rat. In contrast, [3H] WIN 35,428 binding to membranes prepared from frozen tissue (stored at -70 degrees C) revealed two binding sites, a high affinity site similar to the one observed in membranes from fresh tissue and a low affinity site with a Kd of 39 nM for the rabbit and 65 nM for the rat. The low affinity WIN 35,428 binding site was observed only in membranes derived from frozen tissue, suggesting that it was an artifact produced by the freezing/thawing process.     

Delineation of the functional site of a snake venom cardiotoxin: preparation, structure, and function of monoacetylated derivatives

Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.     

Phospholipid lamellae are cholesterol carriers in human bile

Cholesterol solubility and precipitation in bile are major factors in the pathogenesis of cholesterol gallstones. At present, mixed micelles and phospholipid vesicles are considered to be the only cholesterol carriers in bile. In this study we present evidence showing that phospholipid lamellae are major cholesterol carriers in human bile. Lamellae are a known aggregational form in pure phospholipid model systems. In the present study, lamellae were demonstrated by electron microscopy after negative staining and by small-angle X-ray diffraction in all human gallbladder bile samples examined. During diffraction experiments, cholesterol was found to crystallize from these lamellae. Cholesterol carriers in bile were separated by high-resolution chromatography and by prolonged ultracentrifugation. Lamellae were shown to solubilize most of the biliary cholesterol; vesicles solubilized a lesser amount; while micelles solubilized only a minor portion. Our data suggest that phospholipid aggregates are the main cholesterol carriers in bile. Bile salts may control the equilibrium between the various aggregational forms of cholesterol-carrying phospholipids.     

Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans

The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.     

An antibody to a synthetic peptide that recognises SV40 small-t antigen.

A peptide Tyr.Arg.Asp.Leu.Lys.Leu corresponding to the carboxy-terminal six amino acids of small-t antigen predicted from the DNA sequence of SV40 was synthesised, coupled to bovine serum albumin and to ovalbumin and used to raise antibody in rabbits. The sera obtained immunoprecipitated [125I]peptide. It also recognised SV40 small-t that was synthesised in vitro from SV40 mRNA or extracted from SV40 infected monkey cells. The immunoprecipitation of small-t was inhibited by added peptide. To demonstrate that the determinant was present at the carboxy-terminal end of the molecule, truncated versions of small-t coded for by 0.54-0.59 deletion mutants were tested. dl 890 small-t, which contains an in-phase deletion removing nine amino acids but leaving the carboxy-terminal sequences intact, was recognised by the antipeptide serum. By contrast dl 885 small-t, which has an out-of-phase deletion leading to an altered carboxy terminus coded in an alternative reading frame, was not recognised. The data confirm the location and specificity of the determinant recognised on small-t by the antipeptide serum.