Monogamy in black vultures: genetic evidence from DNA fingerprinting

Recent research has indicated that avian mating systems maycommonly deviate from monogamy due to extrapair fertilizations(EPFs). Because the majority of avian species have long beenconsidered monogamous, accurate measurement of the frequencyof EPFs in a variety of species is important to enhance understandingof the evolution of avian mating systems. We used DNA fingerprintingto investigate the apparently monogamous mating system of blackvultures (Coragyps airaius) by assaying parentage within severalnuclear families. Monogamy is suggested in black vultures becausemated pairs exhibit long-term pair bonding and year-round association,and share incubation and nestling feeding duties equally. Thirtytwobreeders and 36 nestlings representing 16 complete nuclear familieswere tagged for individual identification and sampled for DNAanalysis using 2 restriction enzymes and 3 probes for hypervariableregions. Putative parents were assigned parentage in all cases.We empirically examined the probability of detecting EPFs bycomparing nestlings' fingerprints to those of a putative parentand another randomly chosen adult. All putative parents couldbe assigned with 95%confidence and all outside adults couldbe similarly excluded. There is therefore no evidence for successfulEPFs in this population, indicating a mating system that doesnot deviate from strict monogamy. The complex social behaviorof black vultures may eliminate the opportunity for EPFs dueto the prohibition of copulations in the presence of relatives.     

Purification of denatured epidermal growth factor-receptor from A431 human epidermoid carcinoma cells

A rapid and simple method was developed for isolating denatured epidermal growth factor (EGF)-receptor suitable for use in preparation of polyclonal antisera. Membranes from A431 cells (which possess unusually high numbers of EGF-receptors) were phosphorylated in vitro with [gamma-32P]ATP and run on preparative sodium dodecyl sulfate (SDS)-polyacrylamide gels. The Mr 170,000 major phosphorylated region was excised from the gels, eluted, and protein chromatographed on SDS-hydroxylapatite. Fractions containing the Mr 170,000 tyrosine-phosphorylated protein were pooled, concentrated, and rerun on preparative SDS gels. The protein eluted from these gels was judged to be highly purified, based on peptide mapping and on comparison of proteins immunoprecipitated by monoclonal antibody against the EGF-receptor with proteins precipitated by polyclonal antibody prepared against the Mr 170,000 protein described here. The polyclonal antiserum recognized native and denatured EGF-receptor from human, rat, and mouse cells and should prove useful in studying EGF-receptor synthesis and function.     

Use of chemically modified activated carbon as a support for immobilized enzymes

Loading and activity assays of the enzymes alpha-chymotrypsin, alpha-chymotrypsinogen, and glucose oxidase covalently bound to an activated carbon support are presented. The activated carbon support material was pretreated using either a radio-frequency oxygen plasma or an electrochemical oxidation to maximize the enzyme attachment. Cyanuric chloride or water-soluble carbodiimide linking reactions were used to covalently attach the enzymes to the carbon support. Discussion of the relative merits of each reaction scheme is presented.     

The relationship of mechanical properties to morphology in patellar tendon autografts after posterior cruciate ligament replacement in sheep.

In a sheep model the posterior cruciate ligament (PCL) was replaced by a patellar tendon autograft (PTAG) using the central one-third of the ipsilateral patellar tendon (PT). The sheep were sacrificed at 16, 26, 52 and 104 weeks postoperation. The PTAG, and, as controls, the contralateral PCL and PT were harvested. These were examined using biomechanical testing as well as light and transmission electron microscopy, including immunohistological techniques. The material properties (maximum stress, elastic modulus) were compared to the morphological features. The cellular distribution, the distribution of glycosaminoglycans (GAGs), the collagen fibril diameter and the occurrence of Type III collagen were studied. Prior to transplantation, the PTAG was shown to be superior in maximum stress (57.2 +/- 5.5 MPa vs 41.3 +/- 1.9 MPa) and elastic modulus (368.8 +/- 49.3 MPa vs 172.3 +/- 14.6 MPa) to the PCL. The early decline in material properties of the PTAG (maximum stress 22% and elastic modulus 42% of the control) after free grafting paralleled a cell- and capillary-rich PTAG tissue with remnants of necrosis and a poorly organized extracellular matrix. Two years after implantation, with progressive alignment of the tissue matrix, maximum stress and elastic modulus acquired approximately 60 and 70% of the control, respectively. However, there was also an evidence of degenerative changes characterized by acellular areas, loss of the normal bundling pattern of collagen fibers and abnormal accumulation of GAGs. Ultrastructurally, there was a predominant shift to thin collagen fibrils in the PTAG compared to PCL and PT, both consisting of thick and thin collagen fibrils. Thin fibrils were demonstrated to be, in part, split thick fibrils as well as newly formed fibrils. Most of these thin fibrils revealed a positive reaction with antibodies to Type III collagen.     

Peripheral T cell receptor gamma delta variable gene repertoire maps to the T cell receptor loci and is influenced by positive selection.

Although the mechanisms that determine TCR-alpha beta V gene repertoire are well studied, the genetic influences involved in TCR-gamma delta repertoire development are unclear. Unlike the TCR-gamma delta populations that localize in epithelial tissues, the circulating peripheral TCR-gamma delta V region repertoire is quite diverse. Previous studies have shown that three TCR-gamma chains and at least six TCR-V delta genes are expressed by splenic TCR-gamma delta cells. However, the relative frequency of individual gamma delta subsets among genetically diverse mice has not been determined. Therefore, the repertoire of TCR-gamma delta cells was examined using anti-TCR V region specific mAb against V gamma 2 and V delta 4 on TCR-gamma delta + cells from total splenocytes. We found that there was a strain-specific variation in TCR-gamma delta usage. The frequency of V gamma 2 expression in different strains varied from 54 to 12%, and the frequency of V delta 4 expression in different strains varied from 38 to 10%. However, the level of V delta 4 and V gamma 2 expression for an individual strain was highly consistent from experiment to experiment. F1 analysis between parental strains that differed in relative frequency of either V gamma 2+ or V delta 4+ cells revealed that high expression was genetically dominant, suggesting that positive selection events play a major role in the peripheral gamma delta repertoire. Variations in the levels of V gamma 2+ cells and V delta 4+ cells was not associated with Mls or MHC haplotype. Analysis of recombinant inbred strains revealed that high V delta 4 expression mapped to the TCR-gamma locus, while high V gamma 2 expression was influenced by the TCR-delta locus. Back-cross analysis confirmed that the TCR loci dominantly influenced the level of V delta 4+ cells and V gamma 2+ cells; however, there was clear evidence that multiple genes affect the TCR-gamma delta repertoire.     

Occurrence of 28- and 27-Carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant acromyrmex octospinosus (reich) (hymenoptera,formicidae: Attini)

The major ecdysteroids in large worker pupae of the leaf-cutting ant Acromyrmex octospinosus were characterized at the peak ecdysteroid concentration by using high-performance liquid chromatography, enzyme immunoassay, and mass spectrometry. In decreasing amounts, they were determined to be makisterone A, an unidentified C₂₈ ecdysteroid bearing a molecular weight of 494, 20-hydroxyecdysone (ratio of 1 to 6 as compared to makisterone A), and putative but negligible ecdysone. The presence of both C₂₈ and C₂₇ ecdysteroids is discussed in relation to the content of 4-desmethylsterols determined by gas chromatography and mass spectrometry to be ergosta-5,7,24 (28)-trien-3β-ol, ergosterol, ergosta-5,7-dien-3β-ol and ergosta-7,24(28)-dien-3β-ol for the main sterols, and with a small amount of cholesterol.     

Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.