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Biomedical subjects

L A Johnson

Publications and source records attributed to L A Johnson.

At least 55 records · Page 3Linked to original sources
PubMed1998

Birth of piglets preselected for gender following in vitro fertilization of in vitro matured pig oocytes by X and Y chromosome bearing spermatozoa sorted by high speed flow cytometry.

The present study examined the ability to establish pregnancies after transfer of pig embryos derived from in vitro fertilization (IVF) of in vitro matured (IVM) oocytes by X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Cumulus-oocyte complexes (COC) were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/mL), epidermal growth factor (10 ng/mL), LH (0.5 microgram/mL) and FSH (0.5 microgram/mL) for 22 h, then the oocytes were cultured without hormonal supplements for an additional 22 h. Boar semen was collected and prepared by flow cytometry sorting of X and Y chromosome bearing spermatozoa. After IVM, cumulus-free oocytes were co-incubated with sorted X or Y spermatozoa (2 x 10(4)/mL) for 6 to 7 h in modified Tris-buffered medium containing 2.5 mM caffeine and 0.4% BSA. After IVF, putative embryos were transferred to NCSU-23 medium containing 0.4% BSA for culture. A portion of the oocytes was fixed 12 h after IVF, the remainder were cultured up to 96 h. At 96 h after IVF, 8-cell to morula stage embryos (n = 30 to 35) from each gender were surgically transferred to the uterus of recipient gilts. Insemination of IVM pig oocytes with X- or Y-bearing sperm cells did not influence the rate of penetration (67 vs 80%), polyspermy (40 vs 53%), male pronuclear formation (95 vs 96%), or mean number of spermatozoa per oocyte (1.6 vs 1.6), respectively. Furthermore, no difference was observed between cleavage rates at 48 h after IVF (X, 49 vs Y, 45%). Transfer of embryos derived from X-bearing spermatozoa to 18 recipients resulted in 5 pregnancies and delivery of 23 females and 1 male piglet. Similarly, transfer of embryos derived from Y-bearing sperm cells to 10 recipients resulted in 3 pregnancies, with 9 male piglets delivered. The results show that X- and Y-bearing spermatozoa sorted using USDA sperm sexing technology can be successfully used in an IVM-IVF system to obtain piglets of a predetermined sex.

Animals↗
PubMed1998

The diversity of lipases from psychrotrophic strains of Pseudomonas: a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens.

Strains of Pseudomonas fluorescens and Ps. fragi are the predominant psychrotrophs found in raw milk and may cause spoilage due to the secretion of hydrolytic enzymes such as lipase and protease. The diversity of lipases has been examined in Pseudomonas isolates from raw milk which represent different taxonomic groups (phenons). Significant diversity was found using both DNA hybridization and immunoblotting techniques, which has implications for the development of a diagnostic test. The lipase-encoding gene (lipA) was cloned from one strain, C9, of Ps. fluorescens biovar V. In contrast to previously reported lipase sequences from Ps. fluorescens, the gene encodes a lipase of M(r) 33 kDa. Alignment of all known Pseudomonas and Burkholderia lipase amino acid sequences indicates the existence of two major groups, one of M(r) approximately 30 kDa comprising sequences from Ps. fragi, Ps. aeruginosa, Ps. fluorescens C9 and Burkholderia, and one of approximately 50 kDa comprising Ps. fluorescens lipases. The lipase from C9 does not contain a signal peptide and is presumed to be secreted via a signal peptide-independent pathway. The lipA gene of strain C9 was disrupted by insertional mutagenesis. The mutant retained its lipolytic phenotype, strongly suggesting the presence of a second lipase in this strain.

Amino Acid Sequence↗
PubMed1998

The relationship between membrane status and fertility of boar spermatozoa after flow cytometric sorting in the presence or absence of seminal plasma.

The motility, viability (percent live), capacitation status and in vitro fertility of boar spermatozoa were examined, after staining with Hoechst 33,342 and flow cytometric sorting in the absence or presence of seminal plasma. Viability was higher in unstained controls and when seminal plasma was present in the medium used to collect spermatozoa from the cell sorter than when seminal plasma was absent or in the staining extender only, but motility was highest when seminal plasma was included in the extender only, compared with the controls and other treatments. The proportions of capacitated spermatozoa were increased by sorting, but were lower when seminal plasma was present, rather than absent, from the staining extender and the collection medium. Compared with unstained controls, extension and staining without sorting only increased the proportion of capacitated spermatozoa after washing in preparation for in vitro fertilization. The percentages of polyspermic, penetrated and cleaved oocytes were lower when inseminated with unsorted (stained) than control (unstained) spermatozoa, regardless of the presence or absence of seminal plasma. These parameters were higher for sorted than for control spermatozoa in the absence of seminal plasma, but in its presence penetration and cleavage were substantially lower. The proportions of capacitated spermatozoa were lower when seminal plasma was present in the collection medium only than in the staining extender or when it was absent altogether, but the former treatment substantially reduced the proportions of polyspermic, penetrated and cleaved oocytes, and the proportion of blastocysts. These findings indicate that sperm capacitation associated with flow cytometric sorting can be reduced by the inclusion of seminal plasma in the collection medium, but this treatment reduces the ability of spermatozoa to fertilize oocytes in vitro under these conditions.

Animals↗
PubMed1997

A 2.5-Mb transcript map of a tumor-suppressing subchromosomal transferable fragment from 11p15.5, and isolation and sequence analysis of three novel genes.

11p15.5 is an important tumor-suppressor gene region, showing loss of heterozygosity in Wilms tumor, rhabdomyosarcoma, adrenocortical carcinoma, and lung, ovarian, and breast cancer. We previously mapped directly by genetic complementation a subtransferable fragment (STF) harboring an embryonal tumor-suppressor gene and spanning about 2.5 Mb. We have now mapped the centromeric end of this STF between D11S988 and D11S12 and its telomeric end between D11S1318 and TH. We have isolated a complete contig of PAC, P1, BAC, and cosmid genomic clones spanning the entire 2.5-Mb region defined by this STF, as well as more than 200 exons from these genomic clones using exon trapping. We have isolated genes in this region by directly screening DNA libraries as well as by database searching for ESTs. Nine of these genes have been reported previously by us and by others. However, the initial mapping of most of those genes was based on FISH or somatic cell hybrid analysis, and here we precisely define their physical location. These genes include RRM1, GOK (D11S4896E), Nup98, CARS, hNAP2 (NAP1L4), p57KIP2 (CDKN1C), KVLQT1 (KCNA9), TAPA-1, and ASCL2. In addition, we have identified several novel genes in this region, three of which, termed TSSC1, TSSC2, and TSSC3, are reported here. TSSC1 shows homology to Rb-associated protein p48 and chromatin assembly factor CAF1, and it is located between GOK and Nup98. TSSC2 is homologous to Caenorhabditis elegans beta-mannosyl transferase, and it lies between Nup98 and CARS. TSSC3 shows homology to mouse TDAG51, which is implicated in FasL-mediated apoptosis, and it is located between hNAP2 and p57KIP2. Thus, these genes may play a role in malignancies that involve this region.

Amino Acid Sequence↗
PubMed1997

Membrane status of boar spermatozoa after cooling or cryopreservation.

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.

Journal Article↗
PubMed1997

Comparative viability of bovine sperm frozen on a cryomicroscope or in straws.

The accuracy and repeatability of freezing rates and effects of evaporation were examined using a new cryomicroscope system to establish its usefulness in assessing the development of cryopreservation protocols for bovine semen. Post-thaw sperm plasma membrane integrity, as assessed by using combinations of fluorescent stains and flow cytometry, was used in evaluating protocols for freezing spermatozoa on the cryomicroscope. Semen was diluted in Test-yolk (20%) extender containing 7% glycerol and frozen in 0.5-ml straws, 0.25-ml straws (over liquid nitrogen for 8 min) or in a quartz crucible using a Linkam BCS 196 cryomicroscope. Thawed samples were diluted with Hepes buffered medium containing 0.1% bovine serum albumin (BSA) and stained with either carboxymethylfluorescein diacetate (CMFDA) or SYBR-14 each in combination with propidium iodide (PI). Flow cytometry analysis of the samples revealed 2 major populations: 1) spermatozoa with intense green fluorescence (stained with CMFDA or SYBR-14), which were classified as plasma membrane-intact and 2) spermatozoa with intense red fluorescence, (stained with PI), which were classified as plasma membrane-damaged. Samples frozen using the cryomicroscope contained 29 and 26 % plasma membrane-intact (PMI) sperm cells, as assessed by CMFDA and SYBR-14, respectively. Cryopreservation of spermatozoa in 0.5-ml straws resulted in 22 and 20% plasma membrane- intact sperm cells, while spermatozoa frozen in 0.25-ml straws resulted in 34 and 31% PMI sperm cells for CMFDA and SYBR-14, respectively. No significant difference was observed (P > 0.05) for PMI spermatozoa stained with either CMFDA or SYBR-14. In addition, the ability to recover spermatozoa after freezing on the cryomicroscope establishes the Linkam BCS 196 as a useful tool for the study of sperm cell cryopreservation.

Journal Article↗
PubMed1997

Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation.

In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F (uncapacitated), B (capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 microg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F-pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B-pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38 degrees C, 4 hr), flow sorting, cooling (to 15 or 5 degrees C) and freezing reduced the proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C (30.4, 48.5, 21.1%) than in those cooled to 15 degrees C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively).

Animals↗
PubMed1997

Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry.

In vivo-matured porcine oocytes were fertilized in vitro with X and Y chromosome-bearing spermatozoa, and sorted for sex on the basis of DNA content by flow cytometry. Developmental competence of the sexed embryos was determined through established pregnancies after embryo transfer. Spermatozoa were stained with Hoechst 33342 and sorted using a flow cytometry cell sorter. Purity of sorting was 83% for Y spermatozoa and 92% for X spermatozoa. A total of 387 mature cumulus-oocyte-complexes (COC) was collected from 18 superovulated prepuberal gilts shortly before ovulation. In vitro fertilization with sorted spermatozoa was performed in 4 replicates. After 18 h of sperm- oocyte co-culture at 39 degrees C, the zygotes were placed into culture medium (NCSU-23) for another 24 h. The average cleavage rate was 56.2%. Ninety-two embryos produced from X-sorted sperm cells were transferred surgically into the uterus of 2 recipients. Two gilts farrowed and delivered 6 and 4 healthy female piglets, respectively. Additionally, 2 gilts were inseminated intratubally via surgical laparotomy with either X or Y sorted spermatozoa (2 x 10(5)) per oviduct. The 2 sows farrowed producing 15 piglets. Thirteen of the 15 piglets were of the predicted gender (85%).

Journal Article↗
PubMed1997

A computer program to aid in visual concept development in dentistry.

Beginning dental students normally receive their first exposure to the study of tooth forms (morphology) through a dental anatomy laboratory course in which they are required to reproduce tooth morphology, usually with wax. The fabrication of a tooth in wax requires proper visual recognition skills and fine eye-hand coordination. Many students struggle with one or both of these. A computer program, designed to teach recognition concepts, was delivered to three groups of beginning freshman dental students in conjunction with their dental anatomy laboratory course while a group of their classmates served as the controls. This study investigated (1) instructional design and interface improvement and (2) the best method to implement the computer program. Experimental and control groups all received normal daily critiques of their course project work. After completion of the computer program, all groups were tested with a recognition-based examination as well as with a practical examination, requiring the reproduction of a tooth in wax. All experimental groups scored better than the control group on both examinations. Results indicated that computer-based instruction may be a useful means to foster visual concept development. An expanded program, using better graphics, animation and movies is currently under development.

Anatomy↗
PubMed1997

Improving cardiopulmonary resuscitation skills retention: effect of two checklists designed to prompt correct performance.

Previous research has shown that regardless of an individual's experience, life support skills such as cardiopulmonary resuscitation (CPR) are poorly performed as soon as 1 month following training. The purpose of this study was to compare the effects of two checklists designed to prompt correct CPR performance. We compared the performance of 169 undergraduate students, at the time of course assessment, with retention testing that occurred 2 months following the course assessment. Students were randomly assigned to a control group, a short version of a CPR checklist and a longer more detailed version. Two groups of variables were created: procedural and compression-ventilation variables. In addition, an overall-performance variable was created, summarizing performance on the procedural variables. Binary variables were assessed with chi 2-tests of independence. One-way ANOVAs, using 'group' as the between-subjects factor, were used to assess each continuous variable. Comparisons between groups yielded significant differences of P < 0.05. The long checklist generally led to superior performance on the procedural variables. The results support the hypothesis that remembering the steps of CPR is too complex for some. Though preliminary, the findings of this study indicate that the detailed checklist was an effective strategy to improve the post-course performance of CPR.

Cardiopulmonary Resuscitation↗
PubMed1997

Human KVLQT1 gene shows tissue-specific imprinting and encompasses Beckwith-Wiedemann syndrome chromosomal rearrangements.

Genomic imprinting is an epigenetic chromosomal modification in the gamete or zygote causing preferential expression of a specific parental allele in somatic cells of the offspring. We and others have identified three imprinted human genes on 11p15.5, IGF2, H19, and p57KIP2, although the latter gene is separated by 700 kb from the other two, and it is unclear whether there are other imprinted genes within this large interval. We previously mapped an embryonal tumour suppressor gene to this region, as well as five balanced germline chromosomal rearrangement breakpoints from patients with Beckwith-Wiedemann syndrome (BWS), a condition characterized by prenatal overgrowth and cancer. We isolated the upstream exons of the previously identified gene KVLQT1, which causes the familial cardiac defect long-QT (LQT) syndrome. We found that KVLQT1 spans much of the interval between p57KIP2 and IGF2, and that it is also imprinted. We demonstrated that the gene is disrupted by chromosomal rearrangements in BWS patients, as well as by a balanced chromosomal translocation in an embryonal rhabdoid tumour. Furthermore, the lack of parent-of-origin effect in LQT syndrome appears to be due to relative lack of imprinting in the affected tissue, cardiac muscle, representing a novel mechanism for variable penetrance of a human disease gene.

Amino Acid Sequence↗
PubMed1997

Effectiveness of computer-aided removable partial denture design.

PURPOSE: Computer programs have been developed for removable partial denture (RPD) design, but their educational impact has never been investigated. This study measured the effectiveness of computer-based RPD design simulations in a traditional RPD design course for second-year dental students. MATERIALS AND METHODS: Thirty-six students were randomly assigned to four groups, two simulation and two control groups, at the beginning of the RPD design module. A cross-over design compared the two strategies during laboratory exercises. First, the simulation group worked with the simulation, while the control group met in small-group seminars with faculty. Post-test 1 was given, and then students switched teaching methods and post-test 2 was given. Each post-test had two parts. Part 1 involved designing an RPD based on specific patient criteria. Part 2 involved choosing the more correct of the two designs. RESULTS: Results showed a significant difference (p = .0072) in two of four test groups. Although it was an initial evaluation, the computer simulation was shown to be equivalent to faculty-led small-group seminars. Students also responded positively to a questionnaire on their perceived effectiveness of the simulations. CONCLUSIONS: The results suggest that RPD design can be taught as effectively with computer-based simulations as with faculty-led seminars.

Computer-Aided Design↗
PubMed1997

Geriatric patient simulations for dental hygiene.

The rapidly increasing number of this country's elderly requires that dental hygiene students practice the clinical problem-solving skills of information gathering, assessment, and treatment applied to geriatric patients. Computer-based simulations are purported to provide this experience, but little research has been completed with simulations in the education of dental hygienists. This paper summarizes the process used to design, develop, and evaluate a series of eighteen computer-based geriatric simulated patients. It contains a brief description of the simulations and a description of the design, validation, authoring, and formative evaluation phases. The paper also describes the summative evaluation, provides implementation suggestions, and summarizes future directions. The summative evaluation, conducted at four institutions, suggests that computer-based simulations are an effective instructional method as measured by pre/post-tests. The results suggest that simulations can provide a standardized set of geriatric patient experiences. These simulations may prove especially valuable at institutions that are unable to provide clinical geriatric experiences or lack the expertise to conduct a didactic course in geriatrics.

Aged↗
PubMed1997

Advances in gender preselection in swine.

Gender preselection using isolated populations of X- and Y-chromosome bearing spermatozoa that have been separated on the basis of DNA content is currently possible in swine and other farm animals, as well as in humans. Semen from most livestock species can now be successfully separated into predominantly X or Y sperm populations before their use for intra-tubal insemination, deep-uterine insemination or for in vitro fertilization (IVF) to produce sexed offspring. Birth of progeny of the desired sex in cattle, sheep and swine under semi-practical conditions has successfully validated the sexing technology. Spermatozoa are separated on the basis of inherent differences in DNA content in the X- and Y-chromosome bearing sperm population using modified flow cytometry/cell sorting technology. Spermatozoa are stained with Hoechst 33342 which binds to the DNA in an amount proportional to the amount of DNA present in the individual spermatozoa. Over 300 animals from various species at several locations have been born using the USDA-Beltsville Sperm Sexing Technology for separating X and Y spermatozoa. Sex ratios are shifted from the normal 50:50 to 85 to 90% of one sex or the other. In swine, offspring have been born as the result of surgical intratubal insemination of separated spermatozoa and also from IVF and embryo transfer. At the present time, standard swine artificial insemination techniques are not optimized for use with the small numbers of flow cytometrically separated X or Y sperm populations. Cattle, swine and rabbit offspring have been reproduced through the second generation with normal morphology and reproductive function. Numerous improvements have been made in the sexing technology since it was first reported in 1989. Increasing the speed of the sexing process to make the application of the technology available to a larger segment of the livestock industry is paramount, even with insemination technology designed for small numbers of spermatozoa.

Animals↗
PubMed1996

Slit-scan flow cytometry for consistent high resolution DNA analysis of X- and Y-chromosome bearing sperm.

This paper describes the application of slit-scan flow cytometry for accurate DNA analysis of X- and Y-chromosome bearing sperm. The introduction of the slit-scanning technique was initiated to improve the consistency in resolution of the X and Y population from donor to donor. An optimal resolution is essential for high purity sorting of X and Y sperm, as the difference in DNA content is small (3-4%) in most mammals. This difference is the discriminatory parameter for the flow cytometric sorting of the two populations. Our approach was to focus on the role of the sperm tail in the detection process. Slit-scan flow cytometric analysis allows the whole sperm to be spatially analyzed along the direction of flow. Sperm were stained with Dansyl Lysine, a UV excitable fluorescent membrane dye, which stained the head, midpiece, and principal piece. Analysis of these stained sperm showed that there was no difference between the relative number of sperm that travel headfirst or tailfirst through the detection zone of the flow cytometer. The influence of sperm with coiled tails on DNA analysis was also investigated. The proportion of sperm with coiled tails influences semen quality. The standard X-Y separation procedure uses Hoechst 33342, which stains all intact sperm, both living and dead. Propidium iodide was added to discriminate the dead sperm population. Slit-scan analysis showed that measurement of a sample containing a high proportion of living sperm with coiled tails results in an inferior DNA histogram and reduced X-Y resolution. Sperm with coiled tails can result in a lower detected fluorescence intensity, but the reason for this is unclear. Slit-scan flow cytometry allows exclusion of sperm with coiled tails from the analysis, resulting in a restoration of high resolution of X- and Y-chromosome bearing sperm populations.

Animals↗