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Biomedical subjects

L A Johnson

Publications and source records attributed to L A Johnson.

At least 91 records · Page 5Linked to original sources
PubMed1995

Viability assessment of mammalian sperm using SYBR-14 and propidium iodide.

The proportion of living sperm in semen from six representative mammals was assessed by means of a dual staining technique using the stains SYBR-14 and propidium iodide (PI). SYBR-14, a newly developed fluorescent nucleic acid stain, maximally absorbs at 488 nm and emits at 518 nm when bound to DNA. Microscopic examination revealed that SYBR-14 stained the nuclei of living sperm bright green as determined by simultaneous examination of fluorescence and motility. Conversely, PI stained only nonmotile sperm that had lost their membrane integrity. Sperm from bulls, boars, rams, rabbits, mice, and men were stained and examined through use of fluorescence microscopy. The proportions of living and dead sperm were determined by first staining with SYBR-14 and PI and then assessing stain uptake by flow cytometry. Similar staining patterns were observed in all six mammalian species tested. Three populations of sperm were identified: living--SYBR-14 stained, dead--PI stained, and moribund--doubly stained. The SYBR-14 staining was replaced by PI staining as sperm progressed from living to moribund. The transition from green (SYBR-14) to red (PI) fluorescence started at the posterior region of the sperm head and proceeded anteriorly. The proportions of living and dead sperm in mammalian semen were readily identified through use of dual staining with SYBR-14 and PI and quantified through use of flow cytometry.

Animals↗
PubMed1995

Change in endometrial thickness in postmenopausal women undergoing hormone replacement therapy.

PURPOSE: To evaluate the effect over time of hormone replacement on endometrial thickness in asymptomatic postmenopausal women. MATERIALS AND METHODS: Endometrial thickness was measured on 644 sonograms of 120 postmenopausal volunteers receiving no hormones or one of three hormone regimens. Each subject underwent ultrasound (US) two to nine times. In addition, eight women using sequential hormones underwent US seven to 20 times over 2 months. The greatest endometrial thickness and change in endometrial thickness over time were evaluated. RESULTS: Endometrial thickness was statistically significantly greater in women using sequential hormones (8 mm) compared with that in control subjects (5 mm). Change in endometrial thickness (> 3-mm difference) was seen in 23 of 40 women using sequential hormones compared with six of 61 controls. Women using sequential hormones demonstrated maximum endometrial thickness on days 13-23 of the cycle. CONCLUSION: Women using sequential hormones show greater endometrial thickness than that in controls and show the most variation in measurements. They should undergo US either early or late in the hormone cycle to evaluate the endometrium at its thinnest.

Abdomen↗
PubMed1995

Insulin response of women athletes in relation to body fat quantified by magnetic resonance imaging.

We report the glucose and insulin response to a 300-min glucose tolerance test among 17 women athletes as compared with 11 normal, nonatheletic controls. Also reported is the relationship of the insulin area under the curve as a function of percentages of total fat (TF), subcutaneous fat, and internal fat of total volume (TV) quantified by magnetic resonance imaging overall and at six regional sites. Athletes had a more sensitive insulin response to the glucose tolerance test as compared with controls. The insulin area under the curve of athletes and controls was significantly (p = 0.05) related to their overall TF/TV%; athletes had significantly less TF/TV% compared with the controls.

Adipose Tissue↗
PubMed1995

Viability assessment of turkey sperm using fluorescent staining and flow cytometry.

Turkey sperm viability was evaluated using several fluorescent stains both singularly and in combination. Dilution curves and several extenders were used to determine optimal stain concentrations. Semen was collected from eight toms, pooled, diluted, stained, and evaluated microscopically within 2 h of collection. Replicates were assessed for both viable and nonviable sperm (green and red fluorescence, respectively) using flow cytometry. SYBR-14, which likely requires membrane potential for optimal fluorescence, or Calcein AM (CAL), which assesses the membrane integrity of cells (both green fluorescence), in combination with propidium iodide (PI) to stain the dead or degenerating cells (red fluorescence) provided optimal results. Sperm were killed by unprotected freeze-thawing to provide mixed aliquots containing known amounts of fresh:killed sperm. The percentage of viable sperm, as determined by SYBR-14 with PI or CAL with PI staining, were 76.6, 58.8, 39.3, 20.1, 8, and 73.5, 55.8, 36.0, 17.1, .4, respectively, for ratios of 100:0, 75: 25, 50:50, 25:75, and 0:100 of fresh:killed mixtures. Semen from 30 individual toms was collected on 2 d and examined in replicate using these staining combinations. The proportion of viable sperm, as indicated by uptake of SYBR-14 or CAL stain, ranged from 55.8 to 86.7 and 38.0 to 86.1, respectively. Staining combinations were effective in estimating the viability of turkey sperm and could be useful for monitoring sperm viability before and after storage.

Analysis of Variance↗
PubMed1995

Surgery in nonagenarians: morbidity, mortality, and functional outcome.

BACKGROUND: There are few studies that document the outcomes experienced by very old patients who undergo major surgery. METHODS: This is a case series and 7-year follow-up of 116 consecutive patients who were aged 90 years or older and underwent major surgery at a large university-affiliated community hospital. We describe the functional status, short- and long-term mortality, and predictors of mortality in this group of frail elders. RESULTS: The 116 nonagenarians in this study underwent 134 major operations. Sixty-three patients were admitted to the hospital from a nursing home. The most common surgical procedures were for hip fracture, lower extremity amputation, and abdominal problems. Nineteen patients died in the hospital following surgery, and 23 patients died within 30 days of operation. Follow-up at 7 years revealed that all but three patients had died. Survival was worse for patients admitted from nursing homes, those who were nonambulatory before surgery, and those with major or complete functional impairment. CONCLUSIONS: Major surgery in nonagenarians is associated with a 20% perioperative mortality. Functional status and ambulatory ability are maintained in most patients. Whether to operate on these frail elders is a complex decision.

Aged↗
PubMed1995

Quantifying the measurement of differential diagnosis.

Differential diagnosis is central to the work and training of all health care professionals. To develop solid differential diagnosis and skills, students required practice diagnosing numerous and varied patients. In the absence of real patients, patient simulations are commonly used to provide this range of diagnostic experiences. This study examined the benefits of interactive patient simulations on the diagnostic approaches of beginning dental students (novices) and practicing dentists (experts). The study tested the hypothesis that novices tend to use trial-and-error, while experts tend to use pattern recognition during differential diagnosis. a second goal of the study explored objective and subjective measures of a differential diagnosis approach. Seventy-five subjects comprised two treatment groups: a novice group and an expert group. Each group completed ten patient simulations and a case study test measured the diagnostic approach. A three factor MANOVA (p>0.05) was followed by univariate ANOVA's. The result indicated differences between the diagnostic approaches of experts and novices and that the subjective Ratings and the objective Maximum Decisions and Average Variation variables were the best measures of a differential diagnostic approach.

Analysis of Variance↗
PubMed1994

Cryopreservation of porcine embryos by vitrification: A study of in vitro development.

Until recently, attempts to preserve porcine embryos have been unsuccessful. Vitrification has been developed as a method of cryopreserving mammalian embryos by avoiding ice crystal formation, assuring a cryopreserved glass state during storage in liquid nitrogen. Vitrification may be a useful method of overcoming the deleterious effects of chilling injury when pig embryos are cryopreserved using conventional slow freezing procedures. In this study, we applied vitrification procedures for rodent and/or bovine embryos to cryopreserve porcine embryos. Following warming, survival was defined as normal development of embryos in culture, namely the formation or reexpansion of the blastocoelic cavity. Experiment 1 tested the relative toxicity of 3 vitrification procedures on Day-5, 6 and 7 porcine embryos. Embryos equilibrated in vitrification solution (VS3a) continued to develop in vitro at rates comparable to that of untreated control embryos. Experiment 2 was designed to evaluate embryonic development following cryopreservation by vitrification in VS3a. Day-5 porcine embryos did not survive cryopreservation while Day-6 and Day-7 embryos survived and continued development in vitro. In Experiment 3, we evaluated a period of culture prior to vitrification and its effect on cryosurvivability of porcine embryos. A 3-h culture period prior to vitrification had no effect on cryosurvivability over that of freshly recovered, immediately vitrified embryos. These studies indicate, for the first time, that porcine embryos can be successfully cryopreserved by vitrification based on morphology and subsequent development in vitro. However, survival following cryopreservation appears to depend upon embryonic age or stage of development.

Journal Article↗
PubMed1994

Successful gamete intrafallopian transfer (GIFT) in the porcine.

Gamete intrafallopian transfer (GIFT) was successfully established in the pig. In Experiment 1 (6 replicates) 234 oocytes (39 +/- 5.5 per recipient) plus spermatozoa (4000 to 8000 per oocyte) were transferred bilaterally into the oviducts of synchronized gilts, and embryos were recovered 48 h thereafter. The recovery rate was 50.4% and 50% of the recovered oocytes were fertilized. A total of 55 embryos was cultured in vitro in NCSU-medium for 48 h and 63.6% developed to morula or blastocyst stages. In Experiment 2 (5 replicates) 220 oocytes (44 +/- 4.9 per recipient) plus spermatozoa (4000 per oocyte) were transferred to 5 recipients which were allowed to go to term. Three gilts delivered 16 (n = 3, 5, 8) piglets. In Experiment 3 (5 replicates) 183 oocytes (36.6 +/- 1.2 per recipient) plus flow cytometry gender sorted spermatozoa (4000 per oocyte) were transferred to 5 recipients. The recovery rate was 47.8%, and 27.6% of the oocytes were fertilized. From all cleaved oocytes 45.8% developed to expanded blastocysts, with the number of blastomeres varying from 20 to 85 (38.3 +/- 22.5). These results indicate that the GIFT procedure can be used successfully in pigs, and can be a valuable tool for the study of gamete interaction as well as in the continued development of biotechnological procedures such as sex pre-determination.

Journal Article↗
PubMed1994

Flow cytometric determination of degraded deoxyribonucleic acid in granulosa cells to identify atretic follicles during preovulatory maturation in the pig.

Granulosa cells of individual follicles were analyzed by DNA fluorescence flow cytometry to determine how the percentage of cells with degraded DNA and the distribution of cells in the phases of the cell cycle (Go/G1, S1, G2/M) related to the incidence of morphological atresia and to changes in follicular steroid concentrations. Follicles were dissected from ovaries recovered at slaughter on Days 1, 3, 5, or 7 of altrenogest-synchronized preovulatory maturation. Twenty-one follicles with debris among their isolated granulosa cells were classified as morphologically atretic (MA); 92 follicles with debris-free granulosa cells were classified as morphologically healthy (MH). Granulosa cells were prepared for flow cytometry by fixation in 80% ethanol and staining with propidium iodide (PI) containing RNase. DNA fluorescence intensity was determined by use of the 488-nm line of an argon laser. A subpopulation of granulosa cells with degraded DNA (Ao cells), containing less fluorescence than the Go/G1 peak, was found in the DNA histogram of every follicle. The percentage of Ao cells ranged from 0.02 to 83.6% per follicle. The percentage of Ao cells was inversely related to the percentage of Go/G1 cells (r = -0.9611, p = 0.0001). The percentage of Ao cells (mean +/- SEM) was greater (p = 0.0001) in MA (45.9 +/- 6.3%) than in MH follicles (5.3 +/- 1.6%). Follicular estradiol-17 beta was less in MA than in MH follicles, but androstenedione or progesterone did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗
PubMed1994

A systematic evaluation of intraoral cameras.

Although intraoral cameras are being used with increasing frequency in dentistry, this is the first systematic evaluation of the technology. This paper summarizes the evaluation of five intraoral cameras and discusses techniques that practitioners can use in conducting their own evaluations. Additionally, issues specific to intraoral camera evaluations, including image quality, ease of use and the differences between analog and digital cameras, are summarized. Finally, a call is made for an ongoing series of intraoral camera evaluations that would provide practitioners with the information they need to make a purchase decision.

Dental Equipment↗
PubMed1993

Relaxation of imprinted genes in human cancer.

Genomic imprinting, or parental allele-specific expression of genes, has been demonstrated at the molecular level in insects and mice but not in man. Imprinting as a potential mechanism of human disease is suggested by paternal uniparental disomy of 11p15 in Beckwith-Wiedemann syndrome and by maternal uniparental disomy of 15q11-12 in Prader-Willi syndrome. Beckwith-Wiedemann syndrome is characterized by multiorgan overgrowth and predisposition to embryonal tumours such as Wilms' tumour of the kidney. A loss of heterozygosity of 11p15 is also frequently found in a wide variety of tumours, including Wilms' tumour and lung, bladder, ovarian, liver and breast cancers; 11p15 also directly suppresses tumour growth in vitro. Two genes in this band, H19 and insulin-like growth factor-II (IGF2) undergo reciprocal imprinting in the mouse, with maternal expression of H19 (ref. 13) and paternal expression of IGF2 (ref. 14). Here we find that both of these genes show monoallelic expression in human tissues and, as in mouse, H19 is expressed from the maternal allele and IGF2 from the paternal allele. In contrast, 69% of Wilms' tumours not undergoing loss of heterozygosity at 11p showed biallelic expression of one or both genes, suggesting that relaxation or loss of imprinting could represent a new epigenetic mutational mechanism in carcinogenesis.

Alleles↗
PubMed1993

Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from chromosome 11.

A fundamental problem in the identification and isolation of tumor suppressor and other growth-inhibiting genes is the loss of power of genetic complementation at the subchromosomal level. A direct genetic strategy was developed to isolate subchromosomal transferable fragments (STFs) from any chromosome, each containing a selectable marker within the human DNA, that could be transferred to any mammalian cell. As a test of the method, several overlapping STFs from 11p15 were shown to cause in vitro growth arrest of rhabdomyosarcoma cells. This activity mapped between the beta-globin and insulin genes.

Animals↗
PubMed1993

Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome-bearing porcine and bovine sperm.

Studies designed to answer the question whether or not H-Y antigen is preferentially expressed on Y chromosome bearing sperm have resulted in conflicting results. This is probably due to the absence of reliable methods for estimating the percentage of X and Y chromosome bearing sperm in fractions, enriched or depleted for H-Y antigen positive sperm. In recent years a reliable method for separating X and Y chromosome bearing sperm has been published. With this method, separation is achieved by using a flow cytometer/cell sorter, which detects differences in DNA content. This technique provided the first opportunity for testing anti-H-Y antibody binding to fractions enriched for X and Y chromosome bearing sperm, directly. A total of 7 anti-H-Y monoclonal antibodies were tested using sorted porcine sperm and in one experiment also sorted bovine sperm. All monoclonal antibodies bound only a fraction of the sperm (20 to 50%). However, no difference in binding to the X and Y sperm enriched fractions was found. Therefore, the present experiments do not yield evidence that H-Y antigen is preferentially expressed in Y chromosome bearing sperm.

Animals↗
PubMed1993

Aerobes isolated in fecal microflora of infants in the intensive care nursery: relationship to human milk use and systemic sepsis.

BACKGROUND: Staphylococcus epidermidis is a leading cause of nosocomial sepsis in the intensive care nursery. The relationship between rates of gastrointestinal colonization and the incidence of systemic sepsis with S. epidermidis in hospitalized neonates is under investigation. METHODS: In this study, we enrolled 46 infants consecutively admitted to the intensive care nursery (mean +/- standard deviation, birth weight 1300 +/- 337 gm, gestational age 29.4 +/- 2.2 weeks). At the time of enrollment, infants had been fed enterally for at least 1 week (28 were fed formula and 18 received their own mothers' frozen milk). Stool samples were collected when infants were 2 to 3 weeks of age (16.3 +/- 7.4 days). RESULTS: Aerobic stool flora were present in 65% of all patients. Human milk use was associated with a significant increase in the presence of aerobic stool flora (78% vs 46%, p = 0.035), as well as more frequent isolation of S. epidermidis. The incidence of S. epidermidis sepsis was 33% in those infants whose stool specimens grew S. epidermidis and 3.5% in those whose stool specimens did not (p < 0.01). CONCLUSIONS: These findings suggest the gastrointestinal tract as a possible site of entry for S. epidermidis in the hospitalized preterm infant. In addition, frozen human milk may be a vehicle for gastrointestinal S. epidermidis colonization.

Adult↗