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Biomedical subjects

L A Johnson

Publications and source records attributed to L A Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1996

Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14.

The objective of these experiments was to determine the efficacy of the new membrane permeant nucleic acid stain, SYBR-14, for assessing boar sperm viability and to determine it's effect on fertilization and early embryonic development using the pig as a model. We examined the staining patterns of SYBR-14 and another vital stain, Hoechst 33342, both in combination with the dead cell stain, propidium iodide (PI), to quantify the proportion of living and dead spermatozoa in ejaculated and epididymal semen. Flow cytometry analyses of semen from 4 boars revealed significant differences among boars for the proportion of SYBR-14-stained spermatozoa in both epididymal and ejaculated samples, but not for Hoechst 33342 or PI stained spermatozoa. Gilts were inseminated with unstained spermatozoa or spermatozoa stained with 2 levels of SYBR-14 or 2 levels of the reference stain, Hoechst 33342. Embryos recovered at 42 to 48 h postinsemination were morphologically evaluated, and only 4 to 8-cell embryos were continued in culture. Overall, fluorescent staining of boar spermatozoa with SYBR-14 or Hoechst 33342 neither affected their ability to fertilize oocytes, nor the developmental competence of the resultant embryos.

Journal Article↗
PubMed1996

Comparison of seminal quality in Holstein bulls as yearlings and as mature sires.

Semen quality was compared in 5 Holstein bulls from samples collected as young sires (yearlings) and again as mature bulls after a mean interval of 1,265 d. At both sampling periods, the semen was examined for ejaculate volume, sperm numbers, post-thaw progressive motility and sperm viability. Sperm viability was assessed on cryopreserved samples with fluorescent SYBR-14 to stain living spermatozoa and propidium iodide (PI) to identify dead spermatozoa. The fluorescent populations of stained spermatozoa were quantified by flow cytometry. The percentages of living spermatozoa for the individual bulls, as determined by green fluorescence of SYBR-14, ranged from 44 +/- 3.1 to 54 +/- 0.3 for yearlings, and from 38 +/- 1.5 to 55 +/- 1.0 for mature sires. No differences in sperm viability were found between samples taken from yearling bulls and those of mature bulls. The percentage of spermatozoa stained with SYBR-14 was negatively correlated (r = -0.97; P = 0.0001) with the percentage of dead spermatozoa as indicated by PI staining. Comparisons of identical samples run on 2 different flow cytometers indicated that either flow instrument could be used to assess sperm viability. Although the individual bulls differed (P < 0.05) in ejaculate volume and sperm numbers as yearlings, they did not differ in these parameters as mature bulls. The average number of spermatozoa per ejaculate changed as a result of maturation, increasing from 6.2 +/- 1.0 to 10.7 +/- 1.1 x 10(9). Aging was significantly correlated with ejaculate volume (r = 0.76; P = 0.01) but not with the total number of spermatozoa per ejaculate (r = 0.51; P = 0.13). The maturational changes that occurred in the 5 bulls were minimal with the exception of the increased volume of the ejaculate and the number of spermatozoa per ejaculate.

Journal Article↗
PubMed1996

Flow cytometric sorting of sperm: influence on fertilization and embryo/fetal development in the rabbit.

Viable, intact rabbit sperm, prepared, processed, and flow cytometrically sorted, were used in this study to determine the influence of flow sorting on fertilization and embryo development. In experiment I, flow-sorted or control (unstained and unsorted) sperm were surgically inseminated into the uterine horn of hormonally primed does (10 to 12 does per time point). At 42 hr postsurgical insemination, flushed embryos were assessed for development. Fetal development was determined at day 7, day 14, and day 21 post-surgical insemination. Embryos resulting from does surgically inseminated with control sperm at 42 hr post-insemination were observed to be at the early morula stage of development (> 16 cell), whereas embryos from does inseminated with flow sorted sperm were at the 8- to 16-cell stage. No difference was observed between treatments at day 7, 14, or 21, however, there was a significant decrease in fetus number per doe inseminated with flow-sorted sperm over time. In experiment II, mature oocytes were flushed from the oviducts of superovulated does and coincubated in vitro (IVF) with flow-sorted or control rabbit sperm. Oocytes observed at 6 hr post-coincubation exhibited swollen sperm heads in the cytoplasm, demonstrating that fertilization had occurred (2 PN + T). There was a higher percentage of fertilized oocytes by 8 hr post-coincubation for both control (31%) and flow-sorted sperm (31%) when used for IVF. By 10 and 12 hr post-coincubation, little difference was observed in the number of fertilized oocytes between sperm treatments (52% and 66% for control vs. 57 and 54% for flow-sorted, respectively). These studies demonstrate that flow-sorted sperm are capable of fertilizing mature oocytes under in vitro conditions. In addition they show that flow sorting may not negatively influence fertilization events, but likely interferes during early embryonic and fetal development.

Animals↗
PubMed1996

Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis.

The only known and measurable difference between X- and Y-chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations. High resolution two-dimensional (2-D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2-D electrophoresis. Up to 1000 protein spots per gel could be detected and quantified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X- and Y-chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation.

Animals↗
PubMed1996

Binding of acrylonitrile to parvalbumin.

A previous study has shown that acrylonitrile (ACN) has a long half-life in rainbow trout muscle and that [14C]ACN appears to be bound to a 10,000-Da protein in muscle. The labeled protein was purified from muscle of trout exposed to [14C]ACN, separated on 20% SDS-PAGE, and digested for amino acid analysis and sequence analysis. These studies indicated that the labeled protein was the Ca(2+)-binding protein parvalbumin. Parvalbumin is an important calcium-binding protein thought to be involved in the regulation of calcium levels in various parts of the body ranging from neurons to fast-twitch muscle contractions. To study the reaction between parvalbumin and [14C]ACN, frog parvalbumin was incubated with [14C]ACN in vitro under various conditions. These studies indicated that the maximum labeling occurred at 1 nmol/nmol parvalbumin and at pH 7. Amino acid analysis of the labeled protein indicated that the labeled amino acid was probably histidine, and endoproteinase Glu-C (V-8) digestion studies revealed that the 14C was in the 1-81 amino acid segment of the protein, an area that contains two histidines.

Acrylonitrile↗
PubMed1996

Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia.

Expression of Hoxa7 and Hoxa9 is activated by proviral integration in BXH2 murine myeloid leukaemias. This result, combined with the mapping of the HOXA locus to human chromosome 7p15, suggested that one of the HOXA genes might be involved in the t(7;11)(p15;p15) translocation found in some human myeloid leukaemia patients. Here we show that in three patients with t(7;11), the chromosome rearrangement creates a genomic fusion between the HOXA9 gene and the nucleoporin gene NUP98 on chromosome 11p15. The translocation produces an invariant chimaeric NUP98/HOXA9 transcript containing the amino terminal half of NUP98 fused in frame to HOXA9. These studies identify HOXA9 as an important human myeloid leukaemia gene and suggest an important role for nucleoporins in human myeloid leukaemia given that a second nucleoporin, NUP214, has also been implicated in human myeloid leukaemia.

Acute Disease↗
PubMed1996

Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma.

Boar, bull and ram spermatozoa were examined after staining with the DNA-permeant Hoechst 33342 fluorochrome and flow cytometric sorting in the presence or absence of seminal plasma. Spermatozoa were assessed for viability with flow cytometry using the live cell nucleic acid stain SYBR-14 and propidium iodide (PI), and for membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum (FITC-PSA) and PI; motility and acrosome integrity were estimated by microscopy. Flow cytometric sorting was compared with pipette dilution of boar and bull spermatozoa into: (1) medium [boar: Test buffer containing 2% yolk (TY) or Beltsville thawing solution (BTS); bull: TY or HEPES buffer containing 0.1% bovine serum albumin (HEPES-BSA)] with or without 10% (v/v) seminal plasma; or (2) an empty tube containing no medium. Sorted spermatozoa were either not centrifuged or centrifuged before assessment during a 4-h holding period. The viability, motility and membrane integrity of boar, bull and ram spermatozoa centrifuged after sorting were also examined when seminal plasma was present or absent from the staining extender and/or the TY collection medium. The results indicate that the viability and membrane integrity of spermatozoa in vitro would be improved if: (1) seminal plasma (10%) was routinely included in the BTS and HEPES-BSA staining extenders for boar spermatozoa and ram spermatozoa, respectively, when used in preparation for flow cytometric sorting; and (2) 10% and 50% seminal plasma were included in the TY collection medium for boar or bull spermatozoa and ram spermatozoa respectively.

Animals↗
PubMed1996

A novel human homologue of yeast nucleosome assembly protein, 65 kb centromeric to the p57KIP2 gene, is biallelically expressed in fetal and adult tissues.

Three genes on 11p15.5 are known to undergo genomic imprinting. The gene for insulin-like growth factor II (IGF2) is normally expressed from the paternal allele, while H19 and p57KIP2, a cyclin-dependent kinase inhibitor, are expressed from the maternal allele. Five germline balanced chromosomal rearrangement breakpoints from patients with Beckwith-Wiedemann syndrome (BWS) have been mapped to 11p15.5 between p57KIP2 and IGF2, and all are derived from the maternal chromosome. By positional cloning from BWS breakpoints, we have isolated a gene 100 kb and 65 kb centromeric to the proximal end of this BWS breakpoint cluster and p57KIP2, respectively. This gene is homologous to yeast nucleosome assembly protein (NAP1) and to a human homologue of NAP1, and we designate it hNAP2 (human nucleosome assembly protein 2). hNAP2 diverges in its expression pattern from IGF2, H19, and p57KIP2, and it shows biallelic expression in all tissues tested. Thus, hNAP2 is functionally insulated from the imprinting domain of 11p15.

Adult↗
PubMed1996

Development of a culture medium (BECM-3) for porcine embryos: effects of bovine serum albumin and fetal bovine serum on embryo development.

Media are available that can deliver modest porcine embryonic development from a single-cell zygote to the blastocyst stage. However, few embryos develop to hatched blastocysts by Day 7 in vitro, indicating deficiencies in media that inhibit early embryonic development. A defined culture medium, Beltsville Embryo Culture Medium (BECM-3), was developed to support porcine zygote development to the blastocyst stage. When fetal bovine serum was added by late Day 5 (late morula/early blastocyst stage), 80% of total embryos cultured from Day 2 developed into hatched blastocysts by Day 8. There was also a significant increase in the mean cell number of blastocysts and hatched blastocysts when culture was performed in BECM-3-based media in the absence of BSA fraction V. These studies provide a chemically defined foundation for elucidating key developmental components of preimplantation pig embryos.

Animals↗
PubMed1996

Conflicts between managed care organizations and emergency departments in California.

To control costs, managed care organizations have begun to restrict the use of hospital emergency departments by their enrollees. They are doing this by educating enrollees, providing better access to 24-hour urgent care, denying preauthorizations for care for some patients who do present to emergency departments, and retrospectively denying payment for certain patients who use emergency services. Changing traditional use of emergency departments has resulted in conflicts between managed care organizations and these departments. Because federal law mandates access to emergency care for all persons, disagreements occur over the precise definition of an emergency medical condition. In addition, conflicts occur over the scope and payment for the medical screening examination required by federal law of persons presenting to an emergency department. Finally, issues arise related to the safety of patients who present to emergency departments and request care but are denied care because the managed care organization does not authorize the visit. Recent legislation in California has attempted to reconcile differences between managed care practices and federal and state laws; however, areas of continued conflict need to be resolved to prevent possible adverse consequences for patients actually needing emergency care.

Emergency Service, Hospital↗
PubMed1996

Pulmonary sarcoidosis: could mycoplasma-like organisms be a cause?

The etiology of sarcoidosis is unknown but an unusual bacterial agent is possible. Mycoplasma-Like Organisms [MLO] are obligate intracellular cell wall deficient bacteria with a distinctive ultrastructural appearance. MLO are a common cause of various transmissible plant diseases. Despite over 25 years of effort MLO remain uncultivated. Molecular biologic studies indicate MLO are only distantly related to extracellular cultivable mycoplasma. Diagnosis of MLO diseases is based chiefly on detection of the organisms in infected cells by electron microscopy. Recently MLO have been detected by electron microscopy within leucocytes in sterile inflamed aqueous and vitreous humor from patients with idiopathic chronic uveitis including sarcoidosis uveitis. Preliminary molecular biologic studies suggest that human MLO are quite closely related phylogenetically to plant MLO. Inoculation of human uveitis MLO into mouse eyelids produced chronic uveitis and lethal systemic granulomatous disease with MLO within leucocytes and endothelial cells in the disease sites. The MLO induced animal pulmonary disease resembled sarcoidosis. This report describes abnormal intracellular bodies consistent with MLO within leucocytes and endothelial cells adjacent to the granulomas in transbronchial biopsies from 9 corticosteroid untreated sarcoidosis patients versus none in 4 control lungs.

Adult↗
PubMed1995

Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments.

Beckwith-Wiedemann syndrome (BWS) involves fetal overgrowth and predisposition to a wide variety of embryonal tumors of childhood. We have previously found that BWS is genetically linked to 11p15 and that this same band shows loss of heterozygosity in the types of tumors to which children with BWS are susceptible. However, 11p15 contains > 20 megabases, and therefore, the BWS and tumor suppressor genes could be distinct. To determine the precise physical relationship between these loci, we isolated yeast artificial chromosomes, and cosmid libraries from them, within the region of loss of heterozygosity in embryonal tumors. Five germ-line balanced chromosomal rearrangement breakpoint sites from BWS patients, as well as a balanced chromosomal translocation breakpoint from a rhabdoid tumor, were isolated within a 295- to 320-kb cluster defined by a complete cosmid contig crossing these breakpoints. This breakpoint cluster terminated approximately 100 kb centromeric to the imprinted gene IGF2 and 100 kb telomeric to p57KIP2, an inhibitor of cyclin-dependent kinases, and was located within subchromosomal transferable fragments that suppressed the growth of embryonal tumor cells in genetic complementation experiments. We have identified 11 transcribed sequences in this BWS/tumor suppressor coincident region, one of which corresponded to p57KIP2. However, three additional BWS breakpoints were > 4 megabases centromeric to the other five breakpoints and were excluded from the tumor suppressor region defined by subchromosomal transferable fragments. Thus, multiple genetic loci define BWS and tumor suppression on 11p15.

Base Sequence↗
PubMed1995

Sex preselection by flow cytometric separation of X and Y chromosome-bearing sperm based on DNA difference: a review.

Recent research on the flow cytometry of sperm for the purpose of predetermining gender of offspring has led to a validated method to separate X from Y chromosome-bearing sperm for use with in vitro fertilization and embryo transfer, intratubal insemination or intracytoplasmic sperm injection. The basis for the method is the sex chromosome-specific marker, DNA, which is present in greater amounts in X-bearing sperm than in Y-bearing sperm of mammals. Sperm are exposed to the vital dye Hoechst 33342 which binds to the minor groove of the DNA helix. Flow cytometric sorting of the sperm using a laser as the excitation source results in populations of Y- or X-bearing sperm that are 85-90% pure. Several hundred offspring have been produced from swine, rabbits, sheep and cattle that confirm the predicted sex. The method is currently being applied to the commercial embryo market. The method is not likely to be used in conjunction with standard cattle or swine artificial insemination practice in its current form since only about 4 x 10(5) sorted sperm can be produced per hour of sorting. The technology has also been applied to human sperm for use by couples that are at risk to sex-linked disease expression in their offspring. Populations of human sperm have been sorted with X and Y purities of about 80% as confirmed by DNA probe technology and fluorescence in situ hybridization.

Cell Separation↗
PubMed1995

Atresia in follicles grown after ovulation in the pig: measurement of increased apoptosis in granulosa cells and reduced follicular fluid estradiol-17 beta.

The incidence of atresia in the first group of follicles grown after ovulation was investigated in the pig. At slaughter, 113 follicles 3-6 mm in diameter were dissected from the ovaries of four pregnant pigs per day on Days 5, 6, and 7 after the onset of estrus. Granulosa cells were isolated from each follicle. The percentage of granulosa cells containing sub-diploid amounts of DNA (%Ao cells), a measure of apoptosis, was determined for each follicle by DNA fluorescence flow cytometry of propidium iodide (PI) stained nuclei of ethanol-fixed cells. Granulosa cell DNA condition was used to classify follicles. Follicles with > or = 10% Ao cells (n = 33) were designated biochemically atretic (BA), and follicles with < 10% Ao cells (n = 80) were designated biochemically healthy (BH). Internucleosomal cleavage, also indicative of apoptosis, was determined by autoradiographic analysis of [32P]-3'-end-labeled DNA from granulosa cells. Densitometric analysis showed that optical density of [32P]-3'-end-labeled DNA fragments in the 0.18-20-kb size range was correlated with the %Ao cells (R > or = 0.90, N = 22, p < 0.001). The incidence of pigs with BA follicles was 2 of 4, 3 of 4, and 4 of 4 on Days 5, 6, and 7, respectively. The %BA follicles per pig (mean +/- SEM) increased (p < or = 0.01) between Days 5 and 7; values were 6.2 +/- 3.6, 28.1 +/- 13.5, and 50.0 +/- 7.1, respectively, on Days 5, 6, and 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗