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A Wyrobek

Publications and source records attributed to A Wyrobek.

10 recordsLinked to original sources
PubMed2003

Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development.

A quasistatic nanoindentation technique, enhanced by scanning probe microscopy, was used to measure cuticle stiffness of live Drosophila melanogaster during its larval, pupal, and early adult development in vivo. Stiffness was defined as the reduced elastic modulus (E(r)), which is a material property related to the elastic modulus. E(r) was measured at the local contact while indenting the live sample at a constant loading rate using a spherical tip. E(r) was derived from the resultant force-displacement curves. Insect cuticle exhibits viscoelastic behavior. Constant loading rate quasistatic measurements were used so that the effects of viscosity and contact force adhesion introduced systematic measurement effects. E(r) values were as follows: larvae, mean (SE), 0.39 (0.01) MPa; the puparium without evidence of adult structures 15.43 (1.78) MPa; and the adult, measured in the puparium at the completion of metamorphosis, 4.37 (0.31) MPa. Thus, as expected, the puparium and adult cuticle were very much stiffer than larval cuticle. Results also indicated stiffness variation that related to developmental events. This study has shown that this quasistatic nanoindentation-scanning probe microscopy approach is a suitable method for analyzing live biological samples.

Animals↗
PubMed1996

Aneuploid epididymal sperm detected in chromosomally normal and Robertsonian translocation-bearing mice using a new three-chromosome FISH method.

We present a new method to detect epididymal sperm aneuploidy (ESA) in mice using simultaneous fluorescence in situ hybridization (FISH) with DNA probes specific for mouse chromosomes X, Y and 8. The method was applied to Robertsonian (Rb) translocation (8.14) heterozygotes and homozygotes as well as the chromosomally normal B6C3F1. The sex ratios of sperm did not differ from the expected 1:1 and the hybridization efficiencies were approximately 99.7% for over 60 000 sperm analyzed. Mice heterozygous for Rb (8.14) produced about tenfold higher rates of sperm with chromosome 8 hyperhaploidy than did Rb (8.14) homozygotes or chromosomally normal mice, while frequencies of sperm with hyperhaploidies for chromosomes X and Y were unaffected in all three lines of mice. Hyperhaploid frequencies obtained with the ESA method were consistent with those of the previous testicular FISH method and were validated by published data obtained by conventional cytogenetic analyses (meiotic metaphase II and first cleavage). Thus, the mouse three-chromosome ESA assay together with the previously developed aneuploidy assay for human sperm constitute a promising pair of interspecific biomarkers for comparative studies of the genetic and physiologic mechanisms of the induction and persistence of aneuploidy in male germ cells.

Aneuploidy↗
PubMed1995

Aneuploidy in late-step spermatids of mice detected by two-chromosome fluorescence in situ hybridization.

A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late-step spermatids in mice using DNA probes specific for repetitive sequences near the centromeres of chromosomes 8 and X. These probes were nick-translated with biotin- or digoxigenin-labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late-step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in approximately 50% and > 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of > 80,000 late-step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, approximately 3/10,000 spermatids had fluorescence phenotypes indicative of X-X or 8-8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of "bridging biomarkers" between human and animal studies. They have promising applications for investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin.

Aneuploidy↗
PubMed1993

Identification of the binding site of two monoclonal antibodies to human protamine.

We have previously developed a number of monoclonal antibodies (Mabs) that bind to protamine. One of these antibodies, Hup1N, binds to human protamine 1 but not to protamine 2. In contrast, Mab HupA binds both protamine 1 and protamine 2. The epitopes for these two Mabs were observed to overlap, and were localized to the evolutionarily conservative amino-terminal region of protamine 1. This assignment is based on antibody binding to protamine from different species in which the protamine sequence is known, as well as analysis of antibody binding to synthetic peptides and synthetic peptides with specific amino acid substitutions.

Amino Acid Sequence↗
PubMed1992

Immunological evidence for a P2 protamine precursor in mature rat sperm.

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.

Animals↗
PubMed1987

Monoclonal antibodies to human protamines.

Nine monoclonal antibodies to human protamine, hup1a, 1b, 1c, 1d, 1e, 2a, 2b, A, and B, have been isolated and partially characterized. Enzyme-linked immunoabsorption assay analyses with HPLC-separated human protamine 1 and protamine 2+3 mixture identified five of these antibodies as specific for human protamine 1, two antibodies specific for protamine 2+3 mixture and two monoclonal antibodies reactive with all three human protamines. These findings were confirmed by immunoblotting. None of the antibodies reacted with poly-arginine or somatic histone proteins. Additional analyses with bull, boar, and ram protamines indicated that all of the monoclonal antibodies except hupA are specific for human protamine. HupA reacted with protamines from all of the species tested. These studies suggest that each of the antibodies recognizes one of at least four distinct epitopes on protamine.

Animals↗
PubMed1987

High-performance liquid chromatographic separation and partial characterization of human protamines 1, 2, and 3.

A method for separating the three human protamines by HPLC of underivatized, total protamine extracts on a Nucleosil RP-C18 column is described. The identities of the three proteins have been confirmed by a combination of disc gel electrophoresis, amino acid composition, and primary sequence analysis. The results show that human protamine 3 elutes first, closely followed by protamine 2. Protamine 1 elutes later. The amino acid compositions and partial amino terminal sequences of human protamines 2 and 3 indicate that these two proteins are very closely related and suggest that they differ only by three amino-terminal amino acids.

Amino Acids↗
PubMed1984

Chromosomal abnormalities in human sperm: comparisons among four healthy men.

We have used the human-sperm/hamster-egg system to compare the frequencies of structural and numerical chromosomal aberrations in 909 sperm karyotypes from four normal healthy men. The frequency of structural aberrations was 1.3, 4.8, 9.0, and 10.4% respectively in the four donors. Certain specific breakpoints were seen twice or even three times in three of the donors. The incidence of aneuploidy was 1.3, 1.4, 1.4, and 1.9%. In three donors the frequencies of structural aberrations were significantly higher in sperm than in lymphocytes from the same man. X-to-Y ratios did not differ significantly from the expected 50:50.

Adult↗