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M J Howard

Publications and source records attributed to M J Howard.

At least 55 records · Page 3Linked to original sources
PubMed1993

Role of growth factors in catecholaminergic expression by neural crest cells: in vitro effects of transforming growth factor beta 1.

The differentiation of neural crest cells into catecholaminergic neurons is dependent upon both intrinsic properties and signals from the embryonic microenvironment. In tissue culture, the development of catecholaminergic traits is dependent upon factors present in chick embryo extract (CEE). This dependency suggests that soluble growth factors affect catecholaminergic differentiation in vivo. We have studied the role of CEE-derived factors and the potentially related influence of characterized growth factors on catecholaminergic phenotypic expression in avian neural crest cells. In this report, we show that CEE-derived factors and transforming growth factor beta1 (TGF-beta 1) differentially influence catecholaminergic phenotypic expression as well as melanogenesis. TGF-beta 1 substituted for CEE-derived factors and supported the in vitro differentiation of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) immunoreactivities, as well as catecholamine biosynthesis and storage. Differentiation of catecholaminergic cells was dependent on factors present in 10% CEE during the first 1-4 days in culture suggesting an initial critical period for exposure. One day of initial exposure to either CEE-derived factors or TGF-beta 1 was sufficient to support the subsequent expression of catecholaminergic phenotypic characteristics. The time course of responsiveness to TGF-beta 1 was different than for CEE-derived factors. Neural crest cells remain responsive to TGF-beta 1 for at least 5 days, which is past the critical period for CEE-derived factors. Bioassay of CEE shows that endogenous levels of TGF-beta are less than or equal to 0.5 ng/ml. Immunoprecipitation of TGF-beta from CEE or blockade by neutralizing antibodies did not result in a loss of catecholaminergic differentiation by neural crest cells. Although CEE supports melanogenesis under all of the growth conditions tested, TGF-beta 1 was found to be inhibitory.

Animals↗
PubMed1993

Virulence of Naegleria fowleri affected by axenic cultivation and passage in mice.

The virulence of Naegleria fowleri decreases with prolonged axenic cultivation. The decline in virulence of highly virulent, mouse-passaged LEE strain amebae was monitored during 5 years of continuous axenic cultivation. The most rapid decrease in virulence occurred during the first 2 years. Virulence could be restored to original levels by 3 serial passages in mice. The composition of axenic media also affected the virulence of N. fowleri, with a more enriched medium appearing to restore some of the virulence of a weakly virulent strain.

Animals↗
PubMed1991

Direction of gaze during vibrotactile choice reaction time tasks.

A vibrotactile choice reaction time (RT) task was used, with the hands in their own hemispace (arms uncrossed), and in their opposite hemispace (arms crossed). Gaze was directed at the stimulated and responding hand, away from it at the other (inactive) hand, or at a central fixation point (a neutral control). Responses were slower in the crossed than the uncrossed condition. Further, in the crossed condition, responses were faster when subjects looked at the stimulated and responding hand, rather than at the inactive hand or at the central fixation point. As RT in the latter two conditions did not differ, there is a benefit when subjects look at the stimulated and responding hand, rather than a cost when they look at the inactive hand. In the look at condition, visual or attentional factors may reduce the response coding conflict which occurs when arms are crossed.

Analysis of Variance↗
PubMed1991

Detection of immobilised Murray Valley encephalitis virus RNA using oligonucleotide probes with varying degrees of mismatch.

The design of oligonucleotides used for hybridisation studies often utilises available sequence information of the type strain of a particular virus. If hybridisation studies, using such oligonucleotides, are carried out with field isolates of the same virus, the problem of base pair mismatches and consequent difficulties in detection may arise. This study examined the effect of base pair mismatches on the hybridisation between membrane-bound Murray Valley encephalitis virus (MVE) RNA derived from various strains and deliberately mismatched oligonucleotide probes. Under conditions of very low stringency, probes containing up to 5 mismatches were able to detect MVE RNA, but not yeast RNA. Under washing conditions of increased stringency, hybridisation could be detected between MVE virus RNA and probes with only 3 to 4 mismatches. However, the extent of this interaction was dependent on the number and type of mismatches and their relative sequence position.

Animals↗
PubMed1991

Decreased beta 2-adrenergic receptor mRNA expression in receptor-deficient S49 lymphoma cells.

Many variants of the S49 mouse lymphoma cell have been isolated along the pathway of cyclic AMP generation and response. Two such variants, beta p and beta d, were isolated by Johnson and colleagues and described in 1979 [Mol. Pharmacol. 15:16-27 (1979)]. The beta p and beta d variants express one half and one quarter, respectively, of the wild-type number of beta 2-adrenergic receptors. This observation has now been extended through the use of DNA-excess solution hybridization. Using this exquisitely sensitive technique for quantitation of gene and mRNA, we have been able to demonstrate that the beta 2-adrenergic receptor-deficient variant cells contain the same quantity of the beta 2-adrenergic receptor gene as the wild-type cells. In contrast, the beta 2-adrenergic receptor-deficient variant cells express reduced quantities of beta 2-adrenergic receptor-specific mRNA. The amount of beta 2-adrenergic receptor-specific mRNA correlates very well with the reduction in receptor expression in these cells. Both gene and mRNA in the wild-type and variant cells appear to be the same size, as judged by Southern and Northern analysis. Thus, the diminution of beta 2-adrenergic receptors in the beta p and beta d variants appears to reflect primarily the relative paucity of gene transcripts in the variant cells. These data imply that variations in cellular content of beta 2-adrenergic receptor mRNA, which may occur among closely related cells, is one explanation for differences in receptor number.

Animals↗
PubMed1990

Identification of hydroxyalkenals formed from omega-3 fatty acids.

The highly toxic lipid peroxidation product, 4-hydroxynonenal, is formed from the decomposition of hydroperoxides of omega-6 fatty acids. In this study the analogous hydroxyalkenals formed from the decomposition of hydroperoxides of omega-3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid) were isolated and identified using TLC densitometry, HPLC and GC/Mass Spectrometry. The major hydroxyalkenal formed from both fatty acids was a diene analog of 4-hydroxynonenal, 4-hydroxynona(2,6)dienal, while 4-hydroxyhexanal was a minor product. Measurement of specific omega-3 lipid peroxidation products may be important in studies using dietary fish oil.

Aldehydes↗
PubMed1990

Elevated extracellular K+ enhances arachidonic acid release in MDCK-D1 cells.

Depolarization can alter the expression of membrane receptors and change certain receptor-mediated events, but previous studies have not assessed the impact of depolarization on generation of arachidonic acid and its metabolites (AA) in nonexcitable tissues. We assessed AA generation in Madin-Darby canine kidney (MDCK) cells grown for 3 days in increased extracellular [K+], which is known to acutely depolarize these cells. Growth under these conditions resulted in decreases in the number of alpha 1-adrenergic receptors (alpha 1 AR), a small decrease in receptor-mediated phosphoinositide hydrolysis, but increases in alpha 1 AR-mediated prostaglandin E2 formation and AA release. Calcium ionophore (A23187)-, melittin-, and bradykinin-stimulated AA release were also enhanced. The reduction in alpha 1 AR number and increased AA release were substantially reduced or eliminated when K(+)-treated cells were grown in the absence of extracellular calcium. The results provide evidence that hormone-stimulated AA release and prostaglandin production can be enhanced by chronic exposure to elevated extracellular K+ concentration, perhaps as a consequence of a depolarization-induced enhancement in phospholipase A2 activity. The results provide evidence for the parallel and independent regulation of the pathways for receptor-mediated phosphoinositide hydrolysis (phospholipase C activation) and AA release (phospholipase A2 activation) in MDCK cells.

Animals↗
PubMed1990

Networks strengthen ties, status of rural hospitals.

Rural hospitals are a integral part of the health care system in this country. But a lack of funds and a shrinking patient-base are leaving many of them without the resources to provide state-of-the-art care in their communities. In the following article, the authors discuss a network model that brings urban hospitals to the financial and service aid of rural facilities.

Hospital Bed Capacity, 300 to 499↗
PubMed1988

What is the perception of the CFO: manager or technician?

This survey indicates that while both CEOs and CFOs feel that the ideal CFO should be more management oriented, both still place an emphasis on the technical characteristics of the job. This technical emphasis is seen when both CFOs and CEOs were asked to list the most and least important characteristics of the role of the ideal CFO. And while many CEOs view their CFOs as being viable candidates for possible CEO positions in the future, twice as many CFOs view themselves as possible CEOs in the future.

Attitude of Health Personnel↗
PubMed1987

DNA sequences of human papillomavirus types 11, 16 or 18 in invasive cervical carcinoma of Western Australian women.

The presence of human papillomavirus (HPV) types 11, 16 and 18 in 77 biopsies of cervical intraepithelial neoplasia (dysplasia) and carcinoma of the uterine cervix of a sample of women from Western Australia was examined using "Southern" blot hybridisation. HPV-DNA was found in 17 of the 23 dysplasias and 43 of the 54 invasive carcinomas examined but not in the 5 biopsies obtained from areas assessed as normal by colposcopy and histology. Five of 11 biopsies of mild to moderate dysplasias contained HPV type 11 (HPV-11), 2 HPV-16 and 1 HPV-18. Of 12 severe dysplasias/carcinoma in situ, 2 contained HPV-11, 4 HPV-16 and 2 HPV-18. One biopsy contained both HPV-11 and HPV-16. Of 45 squamous cell carcinomas examined for HPV-DNA, 24 contained HPV-16, 5 HPV-11 and 1 HPV-18. Both HPV-11 and HPV-16 were found in 6 of the squamous cell carcinomas and 2 contained both HPV-16 and HPV-18. Of 6 adenosquamous carcinomas examined, 3 contained HPV-DNA, 2 with HPV-16 and 1 with HPV-11. HPV types 16 or 18 were also found in 2 of 3 adenocarcinomas. This study shows a strong association between the papillomavirus and uterine cervical cancer in a sample of women from Western Australia. HPV-16 was more frequently associated with severe dysplasia and cancer than with mild or moderate dysplasia supporting the view that this HPV genotype may have a greater oncogenic potential than HPV-11.

Adenocarcinoma↗
PubMed1987

Amiloride interacts with renal alpha- and beta-adrenergic receptors.

We have used radioligand binding techniques to assess whether amiloride and certain analogues of amiloride (ethylisopropyl amiloride and benzamil) can bind to adrenergic receptors in the kidney. We found that amiloride could compete for [3H]rauwolscine (alpha 2-adrenergic receptors), [3H]prazosin (alpha 1-adrenergic receptors), and [125I]iodocyanopindolol (beta-adrenergic receptors) binding in rat renal cortical membranes with inhibitor constants of 13.6 +/- 5.7, 24.4 +/- 7.4, and 83.6 +/- 13.5 microM, respectively. Ethylisopropyl amiloride and benzamil were from 2- to 25-fold more potent than amiloride in competing for radioligand binding sites in studies with these membranes. In addition, amiloride and the two analogues competed for [3H]prazosin sites on intact Madin-Darby canine kidney cells and amiloride blocked epinephrine-stimulated prostaglandin E2 production in these cells. We conclude that amiloride competes for binding to several classes of renal adrenergic receptors with a rank order of potency of alpha 2 greater than alpha 1 greater than beta. Binding to, and antagonism of, adrenergic receptors occurs at concentrations of amiloride that are lower than previously observed "nonspecific" interactions of this agent.

Amiloride↗
PubMed1987

Alpha 2 adrenergic agonists stimulate Na+-H+ antiport activity in the rabbit renal proximal tubule.

The role of adrenergic agents in augmenting proximal tubular salt and water flux, was studied in a preparation of freshly isolated rabbit renal proximal tubular cells in suspension. Norepinephrine (NE, 10(-5) M) increased sodium influx (JNa) 60 +/- 5% above control value. The alpha adrenergic antagonist, phentolamine (10(-5) M), inhibited the NE-induced enhanced JNa by 90 +/- 2%, while the beta adrenergic antagonist, propranolol, had a minimal inhibitory effect (10 +/- 2%). The alpha adrenergic subtype was further defined. Yohimbine (10(-5) M), an alpha2 adrenergic antagonist but not prazosin (10(-5) M), an alpha1 adrenergic antagonist completely blocked the NE induced increase in JNa. Clonidine, a partial alpha2 adrenergic agonist, increased JNa by 58 +/- 2% comparable to that observed with NE (10(-5) M). Yohimbine, but not prazosin, inhibited the clonidine-induced increase in JNa, confirming that alpha2 adrenergic receptors were involved. Additional alpha2 adrenergic agents, notably p-amino clonidine and alpha-methyl-norepinephrine, imparted a similar increase in JNa. The clonidine-induced increase in JNa could be completely blocked by the amiloride analogue, ethylisopropyl amiloride (EIPA, 10(-5) M). The transport pathway blocked by EIPA was partially inhibited by Li and cis H+, but stimulated by trans H+, consistent with Na+-H+ antiport. Radioligand binding studies using [3H]prazosin (alpha1 adrenergic antagonist) and [3H]rauwolscine (alpha2 adrenergic antagonist) were performed to complement the flux studies. Binding of [3H]prazosin to the cells was negligible. In contrast, [3H]rauwolscine showed saturable binding to a single class of sites, with Bmax 1678 +/- 143 binding sites/cell and KD 5.4 +/- 1.4 nM. In summary, in the isolated rabbit renal proximal tubular cell preparation, alpha2 adrenergic receptors are the predominant expression of alpha adreno-receptors, and in the absence of organic Na+-cotransported solutes, alpha2 adrenergic agonists enhance 22Na influx into the cell by stimulating the brush border membrane Na+-H+ exchange pathway.

Adrenergic alpha-Agonists↗
PubMed1987

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Central Supply, Hospital↗
PubMed1987

Interactions of amiloride with alpha- and beta-adrenergic receptors: amiloride reveals an allosteric site on alpha 2-adrenergic receptors.

Interaction of amiloride with adrenergic receptors was studied using radioligand binding techniques. Amiloride competed for [3H]prazosin binding to alpha 1-adrenergic receptors on rat renal cortical membranes and BC3H-1 muscle cell membranes. Non-linear regression analysis of radioligand binding isotherms showed that amiloride increased Kd without a change in Bmax, suggesting the drug binds competitively in a mutually exclusive manner with the radioligand at the receptor-binding site. Similarly, amiloride competitively blocked [125I]iodocyanopindolol binding to beta-adrenergic receptors on both tissues. The addition of guanylyl 5'-imidodiphosphate or sodium chloride did not alter the interaction of amiloride with alpha 1- or beta-adrenergic receptors. The interaction of amiloride with alpha 2-adrenergic receptors was more complex and revealed an allosteric site. In both rat renal cortical membranes and intact human platelets, amiloride increased the Kd for [3H]rauwolscine binding, as well as decreasing the apparent Bmax. In binding experiments where amiloride competed for [3H]rauwolscine-binding sites, pseudo-Hill slopes of less than 1.0 were obtained for both platelet and renal alpha 2 receptors. In addition, amiloride increased the rate of [3H]rauwolscine dissociation from renal alpha 2 receptors. In the presence of 100-120 mM sodium chloride, the Ki for amiloride competition was decreased an average of 54% in renal membranes; in contrast, sodium increased the Kd of the agonist epinephrine. Taken together, these data support the hypothesis that alpha 2-adrenergic receptors, but not alpha 1- or beta-adrenergic receptors, have an allosteric site to which amiloride binds and which we propose to be a cation-binding site.

Allosteric Regulation↗
PubMed1987

Compartmentation of alpha 2-adrenergic receptors in human erythroleukemia (HEL) cells.

We have identified alpha 2-adrenergic receptors on human erythroleukemia (HEL) cells, a suspension-grown cell line related to human platelets. properties of receptors were assessed in intact cells by binding of the antagonist [3H]yohimbine and by inhibition of cAMP accumulation. [3H]Yohimbine labeled 5900 +/- 2100 receptors/cell with a Kd of 3.6 +/- 0.9 nM (n = 7). alpha 2-Adrenergic receptors were potently coupled to inhibition of adenylate cyclase, with EC50 values for epinephrine, UK-14,304, and p-aminoclonidine in the low nM range. Treatment of cells with pertussis toxin abolished this response. In radioligand binding studies with membrane preparations [3H]yohimbine and [3H]UK-14,304 bound to the same number of sites (71 versus 69 fmol/mg of protein), and epinephrine competed for [3H]yohimbine binding in a biphasic manner. After addition of GTP, no high affinity [3H]UK-14,304 binding was detected, and epinephrine competed for [3H]yohimbine binding with lower affinity at both 4 degrees and 37 degrees. In studies with intact cells, we detected no specific binding of [3H]UK-14,304 at either 37 degrees or 4 degrees. At 37 degrees, epinephrine competed for all [3H]yohimbine binding sites with a low apparent affinity (Ki = 21 microM), whereas at 4 degrees epinephrine (up to 1 mM) was able to compete for only 59 +/- 13% of [3H]yohimbine-binding sites. The potency of epinephrine in competing for [3H]yohimbine sites in intact cells at 4 degrees was greater than at 37 degrees (Ki = 1 microM) and was similar to that observed with membranes in the presence of GTP. We hypothesize that sites not detectable by epinephrine at 4 degrees are sequestered within the cell. Treatment of HEL cells with pertussis toxin reduced the proportion of receptors on the surface from 51 +/- 12% to 23 +/- 7% (n = 3, p less than 0.05) of the total sites. Treatment of HEL cells with epinephrine (100 microM, 1 hr) reduced the cell surface component to 25 +/- 8% (n = 3) of the total sites. This treatment was not accompanied by significant desensitization of the ability of epinephrine to inhibit cAMP accumulation. We conclude that alpha 2-adrenergic receptors exist in more than one compartment in HEL cells and that interaction of receptors with a guanine nucleotide-binding protein or with agonist may regulate this compartmentation. These cells provide a new model system for the study of expression and metabolism of alpha 2-adrenergic receptors.

Adenylyl Cyclases↗
PubMed1986

Neural tube-derived factors influence differentiation of neural crest cells in vitro: effects on activity of neurotransmitter biosynthetic enzymes.

Previously, we have demonstrated that a factor present in chick embryo extract or medium conditioned by neural tube cells supports adrenergic differentiation of some neural crest cells in vitro. These studies have been extended here to examine the effects of this factor(s) on the development of enzymes involved in neurotransmitter biosynthesis. The time course of expression of choline acetyltransferase (ChAT), a marker for cholinergic cells, and dopamine-beta-hydroxylase (DBH), a marker for adrenergic cells, was examined in neural crest cell cultures grown under three conditions: in medium containing 10% embryo extract, in medium containing 2% embryo extract, and in medium containing 2% embryo extract that was conditioned by neural tube cells (NTCM). Significant levels of DBH activity were measured in neural crest cell cultures grown in 10% embryo extract containing medium or in NTCM, while only low levels were present in cultures grown in medium containing 2% embryo extract. In contrast, ChAT activity was inhibited by NTCM in comparison to levels in both 10 and 2% embryo extract containing medium. As a preliminary characterization of the factor(s) present in chick embryo extract, we have fractionated embryo extract and find that a pool of 10 kDa or less can support adrenergic differentiation of some neural crest cells. These results suggest that low molecular weight factors present in embryo extract and NTCM support adrenergic expression of neural crest cells, whereas NTCM suppresses cholinergic expression.

Animals↗