Research centers on minority institutions (RCMI): II.

The goal of the Research Centers in Minority Institutions (RCMI) is to develop biomedical and behavioral research at institutions with 50% minority enrollment (African Americans, Hispanics, Native Hawaiians, Pacific Islanders, Native Americans and Alaska Natives) who have been underrepresented in the biomedical sciences. The program has made available resources vital to scientific development and progress. While these resources have included, equipment, personnel supplies, Core laboratories etc, important effective approaches to research also have been emerging.

Application of an ultraviolet (UV) spectroscopic method for measuring lipoprotein cholesterol in diabetic hypothyroid rats.

Methods for the isolation and quantitation of cholesterol involve time-consuming chromatographic procedures coupled with the development and measurement of color complexes. The intensity of most color complexes varies with time. This study was undertaken to develop a UV spectrophotometric method to measure cholesterol concentration in plasma lipoprotein fractions. The developed method compared favorably with other methods (chemical and enzymatic) and was then used to investigate the effects of diabetes and hypothyroidism on the lipoprotein cholesterol in rats. Lipoprotein fractions from fasted rats were obtained by gradient ultracentrifugation. Aliquots of the very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) layers were extracted into chloroform-methanol-water. Following hydrolysis of lipid extracts, the solvent was evaporated and the residue was dissolved in ethanol and read in the UV recording spectrophotometer. At 203 nm, a linear relationship between optical density and concentration of cholesterol was obtained. The results obtained with the new method were compared with those obtained using chemical and enzymatic methods and the statistical difference among the methods was insignificant (p>0.05). From the results of our comparative studies of the methods, it was concluded that the UV method is valid for measuring cholesterol in lipoprotein fractions. The method has the advantage being rapid, nondestructive and cost-effective.

Research Centers in Minority Institutions (RCMI).

The Research Centers in Minority Institutions (RCMI) Program was initiated in the United States of America in 1985 as a congressionally mandated program. The mission of the RCMI Program is to expand the national capacity for the conduct of biomedical and behavioral research by developing the research infrastructure at institutions granting doctoral degrees in health or health-related sciences, that have 50% or greater enrollment of minorities (African Americans, Hispanics, Native Hawaiians and Pacific Islanders, Native Americans and Alaska Natives) that are underrepresented in the biomedical sciences. The program administration is based in the National Center for Research Resources (NCRR), at the National Institutes of Health (NIH), an agency of the Department of Health and Human Services (DHHS). Since its inception, the program has provided critical resources (core research laboratories, equipment, personnel, supplies, etc.) at each of the RCMI-funded institutions. This article is intended to provide an overview of the RCMI Program, outline the research areas and list contact persons for additional information on research and core resources at each of the current RCMI sites.

The effect of regular alcohol use on the management of non-insulin diabetes mellitus.

This is a retrospective study, in which we investigated the impact of regular alcohol use on the clinical management of non-insulin dependent diabetes mellitus (NIDDM) patients from the outpatient clinic of the VA Medical Center in New Orleans, Louisiana. The study population included randomly selected NIDDM patients of which 40% used alcohol regularly. The fasting blood sugar (FBS) in non-users of alcohol stayed in the "normal" (< or = 140 mg/dl) and "acceptable " (< or = 175 mg/dl) range and that of regular users of alcohol remained at the "fair" (< or = 235 mg/dl) and "poor" (> 235 mg/dl) range. NIDDM patients who were regular users of alcohol had a higher frequency of dose adjustments than that of non-users of alcohol (96% vs 4%, respectively). The treatment failure was significantly higher among patients who regularly used alcohol than among those who abstained (90 vs 10%, respectively). On the basis of our findings, it was recommended that attending physician should routinely identify the regular alcohol users and monitor blood alcohol levels of ambulatory NIDDM patients during their follow-up visits. Also, complete cessation of alcohol consumption should be established prior to making dosage adjustment in situations where the oral hypoglycemic agent fails.

Effects of gamma-2-melanocyte-stimulating hormone on protein kinase C activity and expression in spontaneous hypertensive rats (SHR).

Administration of gamma-2-melanocyte stimulating hormone (gamma-2-MSH) to rats increases blood pressure, heart rate and natriuresis by acting through the nervous system and this response is more pronounced in spontaneous hypertensive rat (SHR). The molecular mechanisms underlying these effects are unknown, however, protein kinase C (PKC) activity is higher in SHR tissues and melanocortins are known to activate the phosphoinositide (PI) signaling pathway. In this study, we tested the hypothesis that gamma-2-MSH potentiation of PKC activation is increased in nerve terminals from SHR brain. Synaptosomes were isolated from SHR and age-matched control Wistar Kyoto (WKY) rats and incubated with gamma-2-MSH. Total particulate-fraction associated PKC activity was determined and the expression of individual isozymes analyzed by immunoblotting. Treatment with gamma-2-MSH resulted in an increase in particulate-associated PKC activity in hindbrain synaptosomes that was more prominent in SHR. The levels of membrane-associated PKC-alpha and beta-isozymes were considerably less than for PKC-gamma in these tissues as determined by immunoblotting. The novel PKC isozymes delta and epsilon were detected in total synaptosomes but not in membrane fractions. These data suggest that PKC-gamma is the major presynaptic PKC isozyme and that PKC may be an important mediator for gamma-2-MSH in neural tissues.

Expression and distribution of protein kinase C isozymes in brain tissue of spontaneous hypertensive rats.

Hypertension activates many endocrine, neuroendocrine and metabolic responses. How hypertension alters these functions remains unknown. Consequently the pathophysiology of hypertension related illnesses are incompletely understood. Protein kinase C (PKC) isoforms play an important role in cellular signal transduction and altered PKC activity has been reported in spontaneous hypertensive rats (SHR). In order to understand the role that PKC plays in hypertension, we hypothesized that PKC activity is significantly expressed in synaptosomal preparations from the brains of SHRs. In addition, the neuroanatomical distribution of this expression was mapped and compared to control animals. The brains were further studied for signs of neuropathology. Total PKC activity was significantly increased in synaptosomal samples isolated from the forebrain, midbrain, and hindbrain of SHR rats. Westem blot analysis identified PKC-alpha, -beta, -gamma, -delta, -epsilon and -zeta in all brain regions. Immunohistochemical analyses indicated that PKC-gamma was localized in cell bodies and processes in many autonomic cardiovascular control regions. These results suggest that PKC may be an important modulator of autonomic blood pressure control.

Isolation of cholinergic active ingredients in aqueous extracts of Mareya micrantha using the longitudinal muscle of isolated guinea-pig ileum as a pharmacological activity marker.

In our attempt to isolate the pharmacologically active ingredients in the aqueous extracts of Mareya micrantha, we have selected the contractions of the longitudinal muscle of the isolated guinea-pig ileum preparation as a pharmacological marker to monitor retention of pharmacological activity during the chromatographic separation. The aqueous extracts of Mareya micrantha elicited concentration-dependent contractions of the preparation. The maximum response elicited by the aqueous extracts was 50% of the maximum response elicited by the maximum dose of acetylcholine (ACh), 10(-7) M. Mepenzolate (10(-8)-10(-5) M), a specific muscarinic receptor antagonist, similarly antagonized contractions elicited by the aqueous extracts suggesting that the cholinergic ingredient(s) in the extract are acting at the muscarinic receptors of the preparation. Fraction 2-4, which was separated from the aqueous extracts by Sephadex gel chromatography, dose-dependently elicited contractions of the preparation. The maximum response was 80% of the maximum response elicited by the maximum dose of ACh suggesting that separation has enhanced the cholinergic activity of the content in the extract.

The use of isolated functional heart to pharmacologically characterize active ingredients in the aqueous extracts of Mareya micrantha.

Aqueous extracts of Mareya micrantha are used as folk medicine in West Africa. However, no systemic investigation directed to the identification of the active ingredients in M. micrantha has been done. Therefore, this study investigated the effects of M. micrantha on the cardiac contractility of the isolated frog heart. Also, two sequential fractions from M. micrantha were separated and their effects on cardiac contractility investigated. M. micrantha concentration-dependently suppressed cardiac contractility. Separation of the cardioactive components in series by column chromatography (Sephadex G-50, Column 2.5 x 30 cm and 1 x 20 cm) produced two fractions which facilitated a leftward shift of the dose-response curve of the cardiodepressant effects suggesting that column chromatograph is effective in the isolation of the cardioactive ingredients in M. micrantha. The data suggest that M. micrantha contains cardioactive components and that contractions of the isolated functional frog heart can be used as a pharmacological activity marker during the process of isolation of cardioactive ingredients in M. micrantha.

The effects of stress on lipoproteins and catecholamines in rats.

In order to investigate the effects of transportation stress on metabolic activities, we measured the changes in plasma lipoprotein and catecholamine levels in those rats that had just arrived in our Animal Facility and age-matched rats which had acclimatized in the Facility for at least 21 days. The acclimatized rats were considered as control, and the values from the newly arrived rats was done within 4-6 days of their arrival in the Facility. The cholesterol levels in very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were higher (71-84%) than the control levels. Also, the stressed animals had higher levels of norepinephrine (4.5-fold) and epinephrine (3-fold) than the control levels. However, dopamine levels was 34-fold lower than that of control. On the basis of the data, we concluded that the change in plasma levels of lipoprotein and catecholamines in response to the transportation stress is significant and may require at least three weeks after the transportation to establish a stable baseline for investigations in which the plasma levels of lipoproteins and catecholamines is a critical factor.

Autoradiographic demonstration of the distribution of diacylglycerol in the gastric muscularis muscle of Bufo marinus: is diacylglycerol one of the second messengers in the muscularis muscle?

The distribution of 1,2-diacylglycerol (DAG) in the muscularis muscle of Bufo marinus has been determined using autoradiographic techniques. Strips of the muscle from the body of the stomach were fixed in 4% paraformaldehyde/2.5% glutaraldehyde, postfixed, washed and longitudinal sections (15 microns) were cut on a cryotome and placed on gelatinized slides. The sections were then incubated in the presence and absence of agonist, acetylcholine (ACh) 10(-5) M or carbachol (CCh) 10(-5) M plus Li+ (10 mM) in the medium which causes a marked potentiation of agonist-stimulated formation of cytidine diphosphate-DAG (CDP-DAG). The sections were processed through an autoradiographic technique using [3H]-cytidine, which binds to 1,2-diacylglycerol within the tissue to form CDP-DAG. Analysis of the developed tissue slides indicated that the dose of ACh (10(-5) M) which had been predetermined to elicit automaticity of the preparation in vitro increased the density of CDP-DAG grains. When the muscle strips were pretreated with ACh (10(-5) M) and CCh (10(-5) M) in the presence of LiCl (10 mM), the density of the CDP-DAG grains were further enhanced. CDP-DAG grains were localized throughout the muscle fibers. The increase in the density of the grains which was induced by the conditions eliciting automaticity suggest that DAG is one of the second messengers in the manifestation of automaticity of the muscularis muscle of Bufo marinus.

Cassia alata and the preclinical search for therapeutic agents for the treatment of opportunistic infections in AIDS patients.

In our search for therapeutic agents from natural sources with potential for the treatment of opportunistic infections in patients afflicted with acquired immunodeficiency syndrome (AIDS), we investigated antibacterial and antifungal activities of water extracts of Cassia alata (C. alata). The extracts are traditionally used in Ivory Coast, West Africa to treat bacterial infections caused by Escherichia coli (E. coli), and fungal infections caused by Candida albicans (C. albicans) and dermatophytes. Our working hypothesis was that the extract contains active ingredient(s) which can be isolated, identified and developed into useful antibacterial/antifungal agents for the treatment of opportunistic infections in patients with AIDS. We used the broth dilution and agar dilution methods. Specifically, we focused on E. coli and C. albicans and the effectiveness of the extracts was evaluated relative to those of standard antibacterial agent chloramphenicol and antifungal agent amphotericin B. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the water extract of C. alata against E. coli were 1.6 mg/ml and 60 mg/ml, respectively; corresponding data for chloramphenicol were 2 micrograms/ml and 10 micrograms/ml. Similarly, the MIC and minimum fungicidal concentration (MFC) for the extract against C. albicans were 0.39 mg/ml and 60 mg/ml in contrast to 0.58 micrograms/ml and 0.98 micrograms/ml for amphotericin B. From the dose-response curve plots, the extract had an IC50 of 31 mg/ml for E. coli and 28 mg/ml for C. albicans. The data suggest that C. alata extracts contain agent(s) which have therapeutic potential and might be useful if isolated and developed for the treatment of opportunistic infections of AIDS patients.

Cassia alata and the preclinical search for therapeutic agents for the treatment of opportunistic infections in AIDS patients.

In our search for therapeutic agents from natural sources with potential for the treatment of opportunistic infections in patients afflicted with acquired immunodeficiency syndrome (AIDS), we investigated antibacterial and antifungal activities of water extracts of Cassia alata (C. alata). The extracts are traditionally used in Ivory Coast, West Africa to treat bacterial infections caused by Escherichia coli (E. coli), and fungal infections caused by Candida albicans (C. albicans) and dermatophytes. Our working hypothesis was that the extract contains active ingredient(s) which can be isolated, identified and developed into useful antimicrobial/antifungal agents for the treatment of opportunistic infections in patients with AIDS. We used the broth dilution and agar dilution methods. Specifically, we focused on E. coli and C. albicans and the effectiveness of the extracts was evaluated relative to those of standard antibacterial agent chloramphenicol and antifungal agent amphotericin B. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the water extract of C. alata against E. coli were 1.6 mg/ml and 60 mg/ml respectively; corresponding data for chloramphenicol were 2 ug/ml. Similarly, the MIC and minimum fungicidal concentration (MFC) for the extract against C. albicans were 0.39 mg/ml and 60 mg/ml in contrast to 0.58 ug/ml and 0.98 ug/ml for amphotericin B. From the dose-response curve plots, the extract had an IC50 of 31 mg/ml for E. coli and 28 mg/ml for C. albicans. The data suggest that C. alata extracts contain agent(s) which have therapeutic potential and might be useful if isolated and developed for the treatment of opportunistic infections of AIDS patients.

Pharmacological effects of berberine on the longitudinal muscle of the guinea-pig isolated ileum.

The effects of berberine on the contractions of the longitudinal muscle of the guinea-pig isolated ileum were investigated. Lower concentrations of berberine (less than or equal to 5 x 10(-5) M) induced a parallel rightward shift of the dose-response curve of acetylcholine, suggesting that berberine is antagonizing the actions of acetylcholine at the receptors competitively. At higher concentration, berberine (1 x 10(-4) M) facilitated a rightward shift of the dose-response curve of acetylcholine with a reduction of maximum response, indicating that the interactions of the two agents changed from competitive to noncompetitive antagonism. The competitive antagonism is due to the actions of acetylcholine at the muscarinic receptors, while the noncompetitive antagonism is probably due to the action of berberine at other sites in addition to the muscarinic receptor sites. Berberine dose-dependently antagonized the effects of muscarine, a specific muscarinic receptor agonist, in a fashion similar to the antagonism of acetylcholine, providing evidence that the site of action of berberine is at the muscarinic receptors. Hexamethonium (7.5 x 10(-4) M) did not influence the effect of berberine on the concentration-response curve of acetylcholine and berberine had no effects on the contractions of the preparation elicited by histamine, suggesting its specificity for muscarinic receptors. Berberine also concentration-dependently reduced electrically induced cholinergic contractions, corresponding with its effects at the muscarinic receptor sites. Berberine had no effects on the contractions elicited by KCl, which acts at postreceptor sites. The action of berberine was reversible and dependent on the duration of incubation with the preparation; the longer the time of incubation with the tissues, the slower the recovery. The results of this series of experiments support the hypothesis that berberine blocks muscarinic receptors and this might be part of the explanation of its efficacy in the reduction of intestinal motility and in the treatment of diarrhea.

Cardiovascular effects of doxorubicin.

1. Doxorubicin dose-dependently increased the cardiac contractility of isolated frog heart within the dose-range of 1.0 to 10.0 x 10(-7) M and dose-dependently increased the cardiac output of frog heart in situ with a dose-range between 10(-7) and 10(-5) M. 2. The results of the in situ investigation, using cardiac output as the index of cardiac contractility, were in agreement with the in vitro results. The positive inotropic effects of doxorubicin climaxed around 10(-5) M beyond which there was a dose-dependent decreased in contractility. 3. Haloperidol (10(-6) M), a dopaminergic receptor blocker, and propranolol (10(-8) M), a beta-adrenergic blocker, did not block the positive inotropic effects of doxorubicin. 4. These results provide sufficient basis to suggest that doxorubicin is acting on the isolated amphibian heart through a mechanism which is not associated with beta-adrenergic and/or dopaminergic receptors.

Low temperature and muscarinic receptor activities.

Lowering temperature from 37 degrees C to 22, 18, and 14 degrees C triggered automaticity of smooth longitudinal muscle of guinea pig isolated ileum. The amplitude of the hypothermia-induced automaticity was dependent on the degree of temperature drop: the greater the temperature drop, the greater the amplitude. However, when the preparation was initially prepared and maintained at 14 degrees C and then the temperature was raised at a similar rate to 18, 22, and 37 degrees C, the automaticity was not observed. This series of observations suggests that cooling rate may be the trigger and/or part of the triggering mechanism for the observed automaticity. Mepenzolate (1.0 x 10(-6) M), a specific muscarinic receptor antagonist, blocked the automaticity suggesting the involvement of muscarinic receptors in the pathogenesis and/or the manifestation of the automaticity. Verapamil (1.0 x 10(-7) M), a calcium channel blocker which inhibits the transmembrane Ca2+ influx into smooth muscle cells during excitation, blocked the automaticity suggesting that transmembrane Ca2+ influx plays a significant role in the pathogenesis and/or manifestation of the automaticity. A specific cytoplasmic calcium channel blocker, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (1.0 x 10(-6) M) blocked the automaticity, suggesting that cytoplasmic calcium also plays a significant role in the pathogenesis and/or manifestation of the automaticity. In order to characterize the temperature-induced changes in the muscarinic receptors, an attempt was made to use the classic method of Furchgott and Burstyn to determine the dissociation constants of acetylcholine at muscarinic receptors at different temperatures. However, the alkylation of muscarinic receptors with phenoxybenzamine at lower temperatures was erratic and the recovery from the occlusion was too rapid to apply the method of Furchgott and Burstyn. We concluded that the lack of reversibility of the effects of phenoxybenzamine at 37 degrees C is due to the predominance of covalent bonding of phenoxybenzamine with the receptors, whereas at lower temperatures like 24 degrees C, the blockade of the muscarinic receptors by phenoxybenzamine is mainly due to simple occlusion.

A comparative study of the effects of aging on the responsiveness of the cholinergic receptor of the isolated ileum of mouse and rat.

The responsiveness of the cholinergic receptor (ChR) of the isolated ileum of mouse and rat was characterized by determining the ED50 of acetylcholine (ACh) and pA2 of mepenzolate (MPZ) in tissues from animals of different ages. The ages (in months) of mice were 3, 6 and 18 and; 3, 6, 12 and 24 for rats respectively. The responsiveness of ChR in mice and rats decreased with age. There was no difference in the pA2 between the preparations derived from mice and rats nor was it affected by age. The results of this investigation suggest that (1) changes in the peripheral ChR with age may have potential in the understanding of central aging process and (2) the use of age rather than the weight of animal may provide reproducible data in studies using isolated preparations.

The effects of hypothermia on the blockade of muscarinic receptors by phenoxybenzamine.

To characterize the hypothermia-induced changes in the activity and kinetic constants for muscarinic receptors, we investigated the effects of hypothermia on the onset and offset of action of phenoxybenzamine from the muscarinic receptors of the longitudinal muscle of the guinea-pig isolated ileum. At 37 degrees C, the onset of phenoxybenzamine action was very rapid (less than 5 min) and there was no apparent recovery of the response to ACh 70 min after washing off the phenoxybenzamine. However, the onset at 24 degrees C was very slow (30 min) and there was a complete recovery of the response to ACh 40 min after washing off the unoccluded phenoxybenzamine. We concluded that the lack of reversibility of the effects of phenoxybenzamine at 37 degrees C is due to the predominance of covalent binding between the receptors and phenoxybenzamine whereas at 24 degrees C the blockade of the muscarinic receptor by phenoxybenzamine is mainly due to simple occlusion.

The effects of phenylbutazone on acetylcholine-initiated contractions of gastric muscularis muscle of Bufo marinus.

We have investigated the effects of phenylbutazone (PBZ), a potent prostaglandin (PG) synthesis inhibitor, on the acetylcholine (ACh)-initiated contractions of isolated gastric muscularis muscle of Bufo marinus. Two types of contractions were identified: tonic contractions and spontaneous contractions. The challenging dose of ACh (1.0 X 10(-4)M) was fixed at the level eliciting optimum contractions of the preparation while the dose of PBZ was varied between 0-5.0 X 10(-4)M. PBZ dose of 1.0 X 10(-4)M suppressed the spontaneous contractions but had no noticeable effect on the tonic contractions. To further elucidate the mechanism through which PBZ suppressed the spontaneous contractions, prostaglandin E2 (PGE2) (10(-7)M) was used to reverse the suppression induced by PBZ (5.0 X 10(-5)M). We concluded that PGE2 plays a part in the manifestation of the cholinergic-initiated contractions of the muscularis muscle of Bufo marinus and the effects of PBZ can be surmounted by administration of PGE2 exogenously.

The effects of aging on the responsiveness of the cholinergic receptor of the longitudinal muscle of guinea-pig isolated ileum.

The responsiveness of the cholinergic receptor (ChR) of the longitudinal muscle of guinea-pig isolated ileum was characterized by determining the ED50 of acetylcholine (ACh) and pA2 of mepenzolate (MPZ) in tissues from animals of different ages. The ages of guinea pig were 0.75, 3, 6 and 24 months respectively. According to our data, the sensitivity of ChR decreased with age peaking around 6 months after which time there was an increase in sensitivity. There was no difference in the pA2. The results of this investigation suggest that changes in the ChR with age may have potential in the understanding of aging process and the use of age rather than the weight of animal may provide more reproducible data in studies using isolated preparations.