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Biomedical subjects

L A White

Publications and source records attributed to L A White.

At least 37 records · Page 2Linked to original sources
PubMed1994

Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons.

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T-antigen (T-ag), a neuronal cell line, RN46A, was cloned by serial dilution. At 33 degrees C, RN46A cells express nuclear T-ag immunoreactivity and divide with a doubling time of 9 hr. Undifferentiated RN46A cells express low levels of neuron-specific enolase (NSE) and low (NF-L)-and medium (NF-M)- but not high (NF-H)-molecular-weight neurofilament proteins. Under differentiation conditions, RN46A cells cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. Elevation of intracellular cAMP levels increases neurofilament protein expression, whereas activators of various other intracellular second messenger systems have no effect. Differentiated RN46A cells express low-affinity nerve growth factor (NGF) receptor (p75NGFR) and are immunoreactive using an antibody that recognizes the carboxy-terminal 13 amino acids of all three trk proteins (pan-trk). Both immunoreactivities could be potentiated by treatment with brain-derived neurotrophic factor (BDNF), NGF, and adrenocorticotropic hormone, fragment 4-10 (ACTH4-10). Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) immunoreactivity, which could be enhanced by treatment with ACTH4-10, BDNF, or NGF. Low levels of serotonin immunoreactivity are detected in differentiated RN46A cells, and this was potentiated by differentiating RN46A cells with BDNF for 8 d and 40 mM KCl for days 4-8. HPLC analysis confirmed these immunohistochemical data. RN46A cells should prove useful to elucidate intracellular mechanisms that control neurofilament assembly and 5-HT expression in differentiating raphe neurons.

Animals↗
PubMed1993

Target regulation of neuronal differentiation in a temperature-sensitive cell line derived from medullary raphe.

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen, a clonal cell line, RN33B, was isolated by serial dilution. At 33 degrees C, RN33B cells divide with a doubling time of 48 h and show T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities. RN33B cells are immortal, but not transformed, as they will not grow in soft agar. At non-permissive temperature (38.5 degrees C), T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes. Differentiated RN33B cells express enhanced neuronal-specific protein expression but do not synthesize astrocytic or oligodendrocytic-specific proteins. Moreover, differentiated RN33B cells returned to 33 degrees C re-express T antigen, but do not de-differentiate or begin dividing. Co-culture with embryonic hippocampus and cerebral cortex, but not medullary raphe or spinal cord, resulted in significantly greater survival, more complex neuronal morphology, and enhanced expression of neuronal-specific antigens. Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells. PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells. These data suggest that the observed target regulation of RN33B cell neuronal differentiation in co-culture may be mediated by neurotrophin(s).

Animals↗
PubMed1993

Induction of collagenase and stromelysin gene expression by mechanical injury in a vascular smooth muscle-derived cell line.

We describe a novel system for studying the production of matrix metalloproteinases types I and III (collagenase and stromelysin) by a vascular smooth muscle cell line (Rb-1 cells) in response to mechanical injury. Highly confluent Rb-1 cells are disrupted by passing a plastic comb around the plate to clear a series of circumferential paths, which are bordered by two ridges of displaced cells. Over the next 24 hours, cells migrate into the cleared areas. Northern blot analysis demonstrates the accumulation of mRNAs for collagenase and stromelysin during this process, although they are undetectable prior to injury. Cotreatment with all-trans-retinoic acid (10(-6) M) markedly decreases the level of mRNAs induced by injury, whereas dexamethasone (10(-7) M) causes only a slight reduction. In situ hybridization studies for stromelysin mRNA and immunohistochemical staining for collagenase protein on plates of injured cells showed the highest levels of stromelysin mRNA in cells in the ridges left by the injury; lower levels were observed in some cells migrating into the clear region. The same pattern of expression was observed when cells were stained with antiserum to collagenase protein. Nuclear run-on assays demonstrated increases in transcription of stromelysin and collagenase genes following injury. Transient transfection of cells with a vector containing the luciferase gene driven by a wild-type promoter comprising 1.8 kb of the 5'-flanking region of the rabbit collagenase gene showed increased activity associated with injury. We conclude that: (1) mechanical injury is associated with induction of mRNAs for the metalloproteinases collagenase and stromelysin, (2) retinoic acid effectively antagonizes this responses, and (3) the increase in steady-state mRNA levels is, at least in part, transcriptionally mediated. Thus our data suggest a role for mechanical forces in initiating the changes in gene expression in vascular smooth muscle cells following arterial injury in vivo.

Animals↗
PubMed1993

Gastro-intestinal bleeding in acquired von Willebrand's disease: efficacy of high-dose immuno-globulin where substitution treatments failed.

We report a case of acquired von Willebrand's disease with severe and persistent gastro-intestinal bleeding from multiple bleeding points. He received maximum substitution treatment including factor VIII concentrates, cryoprecipitate and platelets. He also received DDAVP. There was no change in the rate of blood loss. He was then treated with high-dose intravenous gamma-globulin and the blood loss stopped abruptly. He remained well for several months when bleeding recommenced and he again responded promptly to gamma-globulin. This is a useful maintenance treatment for patients with acquired von Willebrand's disease.

Aged↗
PubMed1992

Effects of deuterium oxide and temperature on heart rate in Drosophila melanogaster.

A non-intrusive optical technique has been developed to monitor heartbeat in late third-instar Drosophila larvae. Heartbeat in this insect is an oscillation that is not temperature compensated. Deuterium oxide lengthens the period of a number of high and low frequency oscillators and clocks in a variety of organisms. To determine whether deuterium affects heart rate, flies were raised on proteated and deuterated media and their heartbeat was monitored at four temperatures ranging from 18 to 33 degrees C. The rate of heartbeat increased linearly with increasing temperature, and decreased with increasing concentrations of deuterium. There was a significant interaction between temperature and deuterium: the higher the concentration of deuterium oxide the less temperature-sensitive was the heart rate. Raising temperatures also increased the amount of "noise" in the rhythm: signal-to-noise ratio, which characterizes the amount of power in a rhythmic signal, decreased with increasing temperatures. Deuterium oxide had no effect on signal-to-noise ratio.

Animals↗
PubMed1991

Acidic and basic fibroblast growth factor levels in spinal cord cultures are not regulated by alterations in heparan sulfate proteoglycan expression.

The present study was undertaken to assess both the levels of acidic and basic fibroblast growth factors in spinal cord cultures and to determine how they were presented to responsive cells. Western blots detected a single acidic fibroblast growth factor-like protein (17 kDa) and two (18 kDa, 24 kDa) basic fibroblast growth factor-immunoreactive proteins, the levels of which varied with the antibody used. Levels of all three proteins were unaltered in cultures grown in the presence of a mitotic inhibitor, which greatly reduced the number of astrocytes. Cell blots showed increased survival of spinal cord neurons at Mr that corresponded with the three proteins detected immunologically. Solubilized cultures separated on a P100 column showed mitogenic activity for NIH3T3 cells from 17-18 and 24 kDa fractions. Treatment of the cultures with heparitinase did not decrease the levels of acidic and basic fibroblast growth factors detected by Western blots, suggesting that these proteins were not associated with extracellular membrane heparan sulfate proteoglycans. The major fraction of both proteins appeared to be intracellular with a minor amount complexed with extracellular matrix proteins. An inhibitor of xylose-linked proteoglycan synthesis significantly altered heparan sulfate proteoglycan deposition into extracellular matrix, but did not alter the levels of acidic or basic fibroblast growth factors detected by Western blots, or the levels of choline acetyltransferase, glutamic acid decarboxylase, or aspartate aminotransferase activities. These results indicate that both acidic and basic fibroblast growth factors are stored predominantly intracellularly, with only a minor fraction complexed with extracellular proteins. We suggest that these intracellular proteins may be released following injury in the CNS and mediate a cascade of neuroprotective events.

Animals↗
PubMed1991

Differential effects of acidic and basic fibroblast growth factors on spinal cord cholinergic, GABAergic, and glutamatergic neurons.

When spinal cord cultures from embryonic day 12 rats were cultured at low density, both acidic and basic fibroblast growth factors significantly increased neuronal survival and neurite outgrowth in a dose-dependent manner. The effects of acidic fibroblast growth factor were independent of heparin, in contrast to its mitogenic effects on both NIH3T3 cells and cerebral cortical astrocytes. In high-density cultures, acidic fibroblast growth factor increased choline acetyltransferase activity by 57%, glutamic acid decarboxylase activity by 58%, and aspartate aminotransferase activity by 65%. Basic fibroblast growth factor increased choline acetyltransferase activity by 73% and glutamic acid decarboxylase activity by 200% but decreased aspartate aminotransferase activity by 40%. Growing these cultures in the presence of a mitotic inhibitor did not significantly alter the effect of acidic or basic fibroblast growth factor on these enzyme activities. These results demonstrate that acidic and basic fibroblast growth factors differentially affect neurotransmitter enzyme levels of multiple classes of neurons, rather than having effects on a single neuronal population.

Animals↗
PubMed1990

Inactivation and stability of viral diagnostic reagents treated by gamma radiation.

The objective of this study was to apply the pertinent findings from gamma inactivation of virus infectivity to the production of high quality diagnostic reagents. A Gammacell 220 (Atomic Energy of Canada, Ltd., Ottawa, Canada) was used to subject 38 viruses grown in either susceptible tissue cultures or embryonated chicken eggs to various doses of gamma radiation from a cobalt-60 source. The radiation required to reduce viral infectivity was 0.42 to 3.7 megarads (Mrad). The effect of gamma treatment on the antigenic reactivity of reagents for the complement fixation (CF), hemagglutination (HA) and neuraminadase assays was determined. Influenza antigens inactivated with 1.7 Mrad displayed comparable potency, sensitivity, specificity and stability to those inactivated by standard procedures with beta-propiolactone (BPL). Significant inactivation of influenza N1 and B neuraminidase occurred with greater than 2.4 Mrad radiation at temperatures above 4 degrees C. All 38 viruses were inactivated, and CF or HA antigens were prepared successfully. Antigenic potency remained stable with all antigens for 3 years and with 83% after 5 years storage. Influenza HA antigens evaluated after 9 years of storage demonstrated 86% stability. Gamma radiation is safer than chemical inactivation procedures and is reliable and effective replacement for BPL in preparing diagnostic reagents.

Animals↗
PubMed1990

Growth of Vero E-6 cells on microcarriers in a cell bioreactor.

Minimum essential medium supplemented with either 7% fetal bovine serum or 2% fetal bovine serum and 1.5% low protein serum replacement (Sigma Chemical Co., St. Louis, Mo.) factor was used to evaluate the growth of Vero 76 cells (E-6 clone) in a cell culture bioreactor. Three different microcarriers, collagen-coated polystyrene (Kontes Life Sciences Products, Vineland, N.J.), Gelibead (Hazelton Research Products, Inc., Lenexa, Kans.), and Cytodex 3 (Pharmacia Fine Chemicals, Piscataway, N.J.), were used to compare the propagation of these anchorage-dependent cells. The number of viable cells recovered during three harvests over an 11-day incubation period was evaluated. The Vero E-6 cells grew equally well with both media. When the collagen-coated dextran microcarriers (Cytodex 3) were used, better cell attachment and higher-density growth were obtained and 5.5 x 10(8) to 7.0 x 10(8) cells were recovered from the system.

Animals↗
PubMed1989

A follow-up study of three hepatitis B virus markers in personnel from the Canadian Armed Forces.

Prevalence rates for three hepatitis B virus (HBV) markers in two groups of personnel within the Canadian Armed forces (1848 incoming recruits and 210 crew members from a destroyer) were determined. Blood specimens for analysis were provided twice. The initial prevalence rates fell at the lower end of the spectrum when compared with those found in United States military personnel, Canadian military health personnel, and certain Canadian civilian populations. Eleven recruits and one destroyer crew member seroconverted for at least one of the markers between the first and the second testing. Their serological profiles are discussed in detail. No transmission of HBV between individuals in the group of recruits studied was established. However, evidence was found for a probable limited transmission of HBV between two crew members of HMCS Margaree. These findings combined with the high cost of the hepatitis B vaccine indicate that mass immunization for HBV in the Canadian Forces population cannot be justified on the basis of this study.

Adolescent↗
PubMed1989

Hepatitis A: a serological study in the Canadian Armed Forces.

Sera were collected from 3958 Canadian Forces personnel, including recruits, personnel posted to bases in Canada and on rotation with United Nations Peacekeeping forces, and the crew of a Canadian destroyer. All sera were tested for the presence of anti-hepatitis A antibodies by a competitive radioimmunoassay. The overall prevalence of anti-hepatitis A antibodies was found to be 23.5% (3.2% to 40.0%). The incidence of viral hepatitis A, as measured by seroconversion in paired sera, was found to be 0.17% (0.01% to 1.42%). The study results support the existence of a direct relationship between increasing age and an increased prevalence of anti-hepatitis A antibodies.

Adolescent↗
PubMed1987

Microtubule stability in severed axons.

We examined severed axons of cat sympathetic nerves and severed neurites of cultured chick sensory neurons for evidence of extensive microtubule depolymerization. Cat sympathetic fibres fixed at various times after severing were cross-sectioned for electron microscopy from both cut ends. The number density of microtubules was determined at various times after severing for matched proximal and distal regions equidistant from cut ends. These data show that the number density of microtubules was nearly identical in proximal and distal fragments at 10, 20 and 60 min and at distances between 10 and 50 micron from the cut ends. In chick sensory neurites the microtubule array was examined in longitudinal sections. In order to define objectively a normal microtubule array, the distance between 100 microtubule pairs was measured in seven control neurites, giving a mean distance (+/- S.D.) of 33 nm +/- 19 nm. A normal array of microtubules was defined as having microtubules within 52 nm of their nearest lateral neighbour. Among 11 neurites at times from 1 to 15 min after transection, the mean distance from the cut tip to the first microtubule was 1.3 micron in proximal fragments and 0.5 micron in distal fragments. The mean distance to the normal microtubule array was 2.8 micron in proximal fragments and 2.1 micron in distal fragments. There was no trend or pattern with respect to the times after severing that the neurite was fixed and the amount of microtubule depolymerization. Our results show no evidence for stabilization of axonal/neurite microtubules by capping structures at their ends. We conclude that microtubule instability is unlikely to play a role in the response of axons to axotomy.

Adrenergic Fibers↗
PubMed1987

Microtubule polarity reversal accompanies regrowth of amputated neurites.

Intact chicken sensory neurites have the same microtubule polarity reported for nongrowing axons in intact organisms. The assembly-favored or + ends of the microtubules are found at the distal terminal (growth cone) of the neuron. After amputation of chicken sensory neurites, the fragment removed from the cell body collapsed to a bead of axoplasm from which neurites rapidly regrew. In nine such regrown neurites analyzed for microtubule polarity, the + ends of microtubules faced the newly formed growth cones, i.e.,--ends of microtubules were now at the original distal terminal of the neuron. These results indicate that microtubules reorganized concomitant with neurite regrowth to form a uniformly polar microtubule array but with reversed polarity. This suggests that mechanisms within the neurite, independent of the cell body, are sufficient for organization of microtubule assembly during axonal elongation. Our data also indicate that microtubule + ends were correlated with growth cone formation in the following three experimental classes of neurites: normal, regrown, and amputated but extended. We speculate that + ends of microtubules are a requirement for growth cone formation and advance.

Animals↗
PubMed1987

Susceptibility of Aedes albopictus C6/36 cells to viral infection.

The susceptibility of the C6/36 clone of Aedes albopictus monolayer cell cultures was determined with 46 prototype viruses passed through three subcultures. Viral growth was confirmed by titration of the passage material in other susceptible host systems. Nineteen viruses demonstrated good growth in C6/36 cells: coxsackievirus group A type 10 and group B types 2, 3, 4, and 5; enterovirus 69; mumps virus; poliovirus types 1 to 3; reovirus types 1 to 3; vaccinia virus; dengue virus type 2; eastern equine encephalomyelitis virus; La Crosse virus; Rocio virus; and St. Louis encephalitis virus. Ten viruses did not adapt to growth in the C6/36 cultures. Seventeen other virus strains displayed only limited growth which was primarily restricted to the initial C6/36 passage or was detected by hemagglutinin reactions without observable cell degeneration. Of the 46 viruses, 33 (72%) were capable of initiating infection with a demonstrable cytopathic effect in the initial C6/36 passage. Hemagglutination or complement fixation titers or both were obtained with dengue virus type 2, eastern equine encephalomyelitis virus, La Crosse virus, mumps virus, reovirus types 1 to 3, and Rocio, St. Louis encephalitis, and vaccinia viruses.

Aedes↗
PubMed1986

In vitro effect of ascorbic acid on infectivity of herpesviruses and paramyxoviruses.

Suspensions of herpes simplex virus types 1 and 2, cytomegalovirus, and parainfluenzavirus type 2 were inactivated within 24 h when treated at 37 degrees C with 1 mg (5.05 mM) of copper-catalyzed sodium ascorbate per ml. The infectivity titer of respiratory syncytial virus was reduced substantially after 24 h but required 48 h for inactivation. Under these conditions, inactivation of these viruses was also successfully achieved with 5.68 mM catalyzed ascorbic acid. Copper (Cu2+), when added with the ascorbate solution at 5 micrograms/ml (0.022 mM), exhibited a catalytic effect on the inactivation of these viruses. The rate of inactivation was affected by the incubation temperature, time of exposure, and the virus concentration. Ascorbate concentrations as high as 10 mg/ml (50.5 mM) demonstrated only a minimum increase in effect on viral inactivation. The loss of infectivity did not alter either the hemagglutination or complement fixation qualities of the antigens.

Animals↗