Biomedical subjects

R J Rodriguez

Publications and source records attributed to R J Rodriguez.

At least 37 records · Page 2Linked to original sources

PubMed1996

Inhibition of mitochondrial function in isolated rate liver mitochondria by azole antifungals.

Ketoconazole is an imidazole oral antifungal agent with a broad spectrum of activity. Ketoconazole has been reported to cause liver damage, but the mechanism is unknown. However, ketoconazole and a related rug, miconazole, have been shown to have inhibitory effects on oxidative phosphorylation in fungi. Fluconazole, another orally administered antifungal azole, has also been reported to cause liver damage despite its supposedly low toxicity profile. The primary objective of this study was to evaluate the metabolic integrity of adult rat liver mitochondria after exposure to ketoconazole, miconazole, fluconazole, and the deacetylated metabolite of ketoconazole by measuring ADP-dependent oxygen uptake polarographically and succinate dehydrogenase activity spectrophotometrically. Ketoconazole, N-deacetyl ketoconazole, and miconazole inhibited glutamate-malate oxidation in a dose-dependent manner such that the 50% inhibitory concentration (I50) was 32,300, and 110 microM, respectively. In addition, the effect of ketoconazole, miconazole, and fluconazole on phosphorylation coupled to the oxidation of pyruvate/malate, ornithine/malate, arginine/malate, and succinate was evaluated. The results demonstrated that ketoconazole and miconazole produced a dose-dependent inhibition of NADH oxidase in which ketoconazole was the most potent inhibitor. Fluconazole had minimal inhibitory effects on NADH oxidase and succinate dehydrogenase, whereas higher concentrations of ketoconazole were required to inhibit the activity of succinate dehydrogenase. N-deacetylated ketoconazole inhibited succinate dehydrogenase with an I50 of 350 microM. In addition, the reduction of ferricyanide by succinate catalyzed by succinate dehydrogenase demonstrated that ketoconazole caused a dose-dependent inhibition of succinate activity (I50 of 74 microM). In summary, ketoconazole appears to be the more potent mitochondrial inhibitor of the azoles studied; complex I of the respiratory chain is the apparent target of the drug's action.

Animals↗

PubMed1995

Comparison of ketoconazole- and fluconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes.

Ketoconazole (KT) and fluconazole (FLU) are azole antifungal agents with a broad spectrum of activity against both superficial and systemic mycoses. KT is also an anticancer agent in the treatment of advanced prostate cancer. In many clinical and retrospective studies, KT has been reported to cause liver damage, i.e. chemical hepatitis. Histologic analysis of KT induced hepatotoxicity shows massive centrilobular necrosis in which the hepatotoxicity was not thought to be mediated through an immunoallergic mechanism. According to the medical literature, the pattern of hepatic injury appears to be primarily of the hepatocellular type. Because of the documented reports of KT and FLU hepatotoxicity, a cytotoxicity comparison of KT and FLU was implemented. The objective of this comparison was to evaluate the cytotoxicity of these azoles such that future mechanistic investigations of hepatotoxicity could be performed. The relative hepatotoxicity of KT and FLU was evaluated using primary cultures of postnatal rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium; by assessing mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT); by assessing lysosomal uptake of neutral red (NR); and by gross morphology (phase contrast microscopy). The cultures were exposed to various concentrations of KT (56-188 microM) for 0.5-4 h and to various concentrations of FLU (50 microM to 1.0 mM) for 0.5-6 h. There was a significant increase (P < 0.05) in LDH leakage and a large decrease in MTT reduction and lysosomal uptake of NR at 4 h for KT. One millimolar FLU had minimal effects on the LDH leakage and MTT reduction. These results demonstrate that KT is a more potent cytotoxicant than FLU; and its toxicity was expressed in a dose- and time-dependent manner.

Analysis of Variance↗

PubMed1994

Maturation of the cholinergic response of tracheal smooth muscle in the piglet.

Maturational changes of the contractile properties of airway smooth muscle have been suggested to occur during the late prenatal and early postnatal period in the piglet. However, the timing and mechanisms involved remain elusive. Therefore, we studied isometric contraction of tracheal smooth muscle (TSM) to evaluate the effects of maturation on 1) the response of TSM to the muscarinic agonist acetylcholine (Ach); and 2) the regulatory activity of acetylcholinesterase (AChE) during the early postnatal period in the piglet. Studies were performed in 1-5-day-old (n = 6) and 14-22-day-old (n = 8) anesthetized, mechanically ventilated piglets. Changes in tracheal tension were measured in vivo from an open tracheal segment and normalized for both longitudinal length of the tracheal segment and cross-sectional area. Dose-response curves to locally applied Ach were obtained before and after the intravenous administration of the AChE inhibitor physostigmine (Phys). Ach-induced changes in tracheal tension significantly increased with maturation during the first 3 weeks of life (P < 0.01), regardless of normalization method. The addition of Phys significantly increased the cholinergic responses of TSM in both age groups (P < 0.02). However, this increase was comparable between age groups. There was also no difference in AChE activity measured in tracheal smooth muscle homogenates between the 1-5 and 14-22-day-old animals. These data indicate that maturation significantly enhances the cholinergic responses of tracheal smooth muscle in the early postnatal period in the piglet.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1993

Identification of a whitefly species by genomic and behavioral studies.

An introduced whitefly species, responsible for over a half billion dollars in damage to U.S. agricultural production in 1991, is morphologically indistinguishable from Bemisia tabaci (Gennadius). However, with the use of polymerase chain reaction-based DNA differentiation tests, allozymic frequency analyses, crossing experiments, and mating behavior studies, the introduced whitefly is found to be a distinct species. Recognition of this new species, the silverleaf whitefly, is critical in the search for management options.

Alleles↗

PubMed1993

Polyphosphate present in DNA preparations from filamentous fungal species of Colletotrichum inhibits restriction endonucleases and other enzymes.

During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, O.C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25 degrees C and to temperatures as high as 100 degrees C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.

DNA Ligases↗

PubMed1991

[Complete autotransplantation of the upper eyelid for basal cell carcinoma].

Because many surgical alternatives exist, and because full invasion of the entire eyelid is rare, we described a new way of surgery: we performed transposition of the whole eyelid, including the part of the elevator muscle of the eyelid, through the rotation of a part of frontal skin. The lacrimal lubrication of the eye was kept intact.

Aged↗

PubMed1990

Physiologic peripheral pulmonic stenosis in infancy.

We studied 14 premature infants with the clinical diagnosis of peripheral pulmonic stenosis (PPS) and 15 normal full-term neonates by echocardiographic Doppler examinations. The PPS group had an average main pulmonary artery (PA) diameter similar to the control group (0.91 vs 0.96 cm, difference not significant), but had smaller branch PA diameters: right PA = 0.41 vs 0.50 cm, p less than 0.001, and left PA = 0.41 vs 0.49 cm, p less than 0.001. The PPS group also had greater peak velocities in the main PA (76 vs 63 cm/s, p less than 0.05), right PA (193 vs 118 cm/s, p less than 0.001) and left PA (187 vs 123 cm/s, p less than 0.001). Similarly, the ratio of peak velocity in the branch/main PA was greater for the PPS group: right/main PA peak velocity = 2.91 vs 1.92, p less than 0.01, and left/main PA peak velocity = 2.73 vs 1.99, p less than 0.05. The calculated right ventricular output for the PPS group was more than the control group: 437 vs 261 ml/min/kg, p less than 0.001. Hematocrits were not done on the control group, but the PPS group had an average hematocrit which was low (34%). It is concluded that patients with PPS have mild underdevelopment of the PA branches, with consequent increased flow velocity and turbulent flow. This turbulent flow may be contributed to by increased cardiac output and mild anemia.

Blood Flow Velocity↗

PubMed1990

Electromyographic analysis of rowing stroke biomechanics.

Electromyography was used to study muscle activity in the upper and lower extremities and torso during the rowing stroke. Five experienced male rowers were tested while rowing on a Concept II ergometer. In order to fully evaluate the rowing stroke, six phases were utilized to identify the transition points where individual muscles become more active due to body angle or nature of the motion. The results indicate that the strength of an individual muscle is most likely not as important to the rowing stroke as the combined activity of two or more groups of muscles. Thus, in addition to strength, it is of great value to the oarsman to develop technical skill to coordinate his upper and lower body reactions.

Adult↗

PubMed1987

Relationship between intracellular sterol content and sterol esterification and hydrolysis in Saccharomyces cerevisiae.

The relationship between the supply of free sterol and the synthesis of steryl esters by an auxotroph of Saccharomyces cerevisiae has been examined in order to understand the role of cellular free sterol content in the regulatory interactions of sterol esterification. Our results show that the yeast cells must maintain an essential, low level of free sterol that is critical for growth. An additional, expandable pool of free sterol is maintained by the cells, provided there is adequate available sterol. As the quantity of sterol in the expandable pool increases, there is a progressively increasing rate of sterol esterification, which is consistent with the results from in vitro assays of acyl-Co A:ergosterol acyltransferase. Some increases in acyltransferase activity were insensitive to the protein synthetic inhibitor, cycloheximide. The data support the conclusion that sterol interconversion between the free and esterified forms is directed toward maintaining the essential amount of free sterol and that the activity of the relevant sterol enzymes in this organism are modulated in response to intracellular sterol content.

Acyltransferases↗

PubMed1987

Selectable genes for transformation of the fungal plant pathogen Glomerella cingulata f. sp. phaseoli (Colletotrichum lindemuthianum).

Glomerella cingulata f. sp. phaseoli (Gcp) was transformed using either of two selectable markers: the amdS + gene of Aspergillus nidulans, which encodes acetamidase and permits growth on acetamide as the sole nitrogen source and the hygBR gene of Escherichia coli which encodes hygromycin B (Hy) phosphotransferase and permits growth in the presence of the antibiotic Hy. The amdS+ gene functioned in Gcp under control of A. nidulans regulatory signals and hygBR was expressed after fusion to a promoter from Cochliobolus heterostrophus, another filamentous ascomycete. Protoplasts to be transformed were generated with the digestive enzyme complex Novozym 234 and then were exposed to plasmid DNA in the presence of 10 mM CaCl2 and polyethylene glycol. Transformation occurred by integration of single or multiple copies of either the amdS+ or hygBR plasmid into the fungal genome. There was no evidence of autonomous plasmid replication. Transformants were mitotically stable on selective and nonselective media. However, transforming DNA in hygBR transformants was observed to occasionally rearrange during nonselective growth, resulting in fewer copies of the plasmid per genome. These transformants were capable of infecting bean (Phaseolus vulgaris), the Gcp host plant, and after recovery from infected tissue were found to have retained both the transforming DNA unrearranged in their genomes and the Hy resistance phenotype. All single-conidial cultures derived from both amdS+ and hygBR transformants had the transplanted phenotype, suggesting that transformants were homokaryons.

Amidohydrolases↗

PubMed1986

An essential fungal growth factor derived from ergosterol: a new end product of sterol biosynthesis in fungi?

Concentrations of ergosterol as low as 1.2 mM are effective in satisfying the essential high specificity microrequirement for C5,6 unsaturated sterol in yeast. In a sterol auxotroph supplemented with saturated sterol (cholestanol), aqueous extracts of yeast provide a growth factor that eliminates the ergosterol microrequirement. The factor is easily differentiated from ergosterol by solubility, thermostability, and thin layer and liquid chromatography and is functional at levels equivalent to those of vitamins, hormones and pheromones.

Cholestanols↗

PubMed1986

Characteristics of sterol uptake in Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sterol auxotroph, FY3 (alpha hem1 erg7 ura), was used to probe the characteristics of sterol uptake in S. cerevisiae. The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol. When cultured on 1 microgram of sterol ml-1, the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were termed sterol depleted. When cultured on 11 micrograms of sterol ml-1 or more, the cells contained a maximal cellular free-cholesterol concentration of 6.8 nmol/mg (dry weight) and were termed free sterol saturated. Cells with free-sterol concentrations below the maximal level were capable of accumulating free sterol from the medium. The capacity of the cells for cholesterol uptake was inversely proportional to the initial intracellular concentration. The uptake of sterol was shown to be a nonactive process that is independent of cellular energy sources or viability. The intracellular transport of sterol for esterification is not sensitive to anti-microtubule agents.

Biological Transport↗

PubMed1985

Multiple functions for sterols in Saccharomyces cerevisiae.

Analyses with a yeast sterol auxotroph indicated that there are at least four different levels of function for sterol which have been designated sparking, critical domain, domain and bulk. Growth of yeast sterol auxotrophs on cholestanol is precluded unless minute amounts of ergosterol are available. We have designated this phenomenon the sparking of growth, in which cholestanol satisfies an overall membrane sterol requirement and ergosterol fulfills a high specificity sparking function. The critical domain role for sterol is observed under conditions of lanosterol supplementation where low levels of ergosterol (10-times those necessary for sparking on cholestanol) are required for growth. The sterol functions designated domain and bulk are illustrated by assessing cellular free sterol levels and plasma membrane properties of a sterol auxotroph after growth on different concentrations of exogenously supplied sterol. Plasma membranes isolated from auxotrophs grown on domain or bulk levels of sterol underwent no lipid thermotropic transitions, while plasma membranes from cells grown on critical domain levels of sterol underwent a lipid thermotropic transition, when analyzed by steady-state fluorescence anisotropy.

Fluorescence Polarization↗

PubMed1985

Modulation of yeast plasma membrane composition of a yeast sterol auxotroph as a function of exogenous sterol.

Plasma membranes isolated from a yeast sterol auxotroph (RD5-R) grown on 1, 5, and 15 micrograms ml-1 exogenous concentrations of sterol showed no discontinuity in plots of steady-state fluorescence anisotropy. Liposomes constructed from phospholipid and sterol extracted from RD5-R grown on different sterols indicated that exogenously supplied sterol modulated cellular phospholipids such that lipid-phase transitions were avoided. Liposomes derived from sterol and phospholipid extracted from the same culture exhibited no lipid-phase transitions. However, when phospholipid extracted from a culture grown on a specific sterol was mixed with sterol extracted from a heterologous culture grown on a different sterol to form liposomes, discontinuities were detected in the anisotropy measurements of the liposomes produced. Quantitative analyses revealed that the exogenously supplied sterol coordinately regulated specific phospholipid species, fatty acid composition, and sterol to phospholipid ratios in yeast auxotrophs.

Cell Membrane↗

PubMed1985

Influence of sterol structure on yeast plasma membrane properties.

Fluorescence anisotropy measurements indicated that physical changes occurred in the lipids of plasma membranes of yeast sterol mutants but not in the plasma membrane of an ergosterol wild-type. Parallel experiments with model membrane liposomes verified that the physical changes in lipids observed in the sterol mutants are dependent on the sterol present and not the phospholipid composition. In addition, the physical changes in lipids observed in liposomes derived from wild-type phospholipids were eliminated by addition of ergosterol but persisted in the presence of cholesterol, cholestanol, ergostanol, or sterols from the sterol mutants. No physical changes in lipids were observed, however, in plasma membranes from a sterol auxotroph, even when the auxotroph was grown on cholesterol or cholestanol. The lack of physical changes in lipids in the sterol auxotroph may reflect the ability of the auxotroph to modify its phospholipid composition with respect to its sterol composition. These results indicate that high specificity 'sparking' sterol is not required for the regulation of overall bulk lipid properties of the plasma membrane.

Cell Membrane↗

PubMed1985

High-performance liquid chromatography of sterols: yeast sterols.

It is evident that the high-pressure liquid chromatograph is an excellent tool for studying sterol metabolism. As noted in the text, the individual effects of unsaturations and alkyl groups on reverse phase elution volumes cannot be extrapolated to predict quantitative effects of multiple functional groups. The mechanism(s) of retention seems more complex than can be explained simply by polarity or hydrophobicity. Since the molecular location of these functional moieties seems critical, retention and separation of sterols may involve specific structural configurations and hence specific interactions with the stationary phase material. The association we have drawn between polarity and HPLC elution may indeed be a secondary effect of another phenomenon. Future studies may unveil the true mechanism(s) of HPLC retention and separation, and allow for the construction of HPLC systems which will separate all isomeric combinations of sterols at the analytical level. The simplicity, rapidity, and reproducibility of these methods make the coupled technique very useful for investigating sterol metabolism. Application of this technique to analyzing putative sterol mutants, purifying sterols for auxotrophic feeding, and analyzing the metabolism of supplemented sterols in auxotrophs provides for significant advances in membrane physiology.

Chromatography, High Pressure Liquid↗

PubMed1985

Yeast sterols: yeast mutants as tools for the study of sterol metabolism.

Yeast mutants defective in ergosterol synthesis are valuable tools for investigating sterol metabolism. Both sterol mutants and sterol auxotrophs have been utilized in determining what sterol structural features are required for yeast cell viability. Both types of mutants can also be studied to ascertain how changes in sterol structure affect membrane properties. Other aspects of sterol metabolism, such as the specificity of sterol esterification, have been elucidated by the sterol auxotrophs. In broader applications, interrelationships between sterol metabolism and other cellular functions (e.g., heme metabolism) may also be examined with these mutants. By analyzing the lipid composition of the sterol mutants, on the other hand, much of the ergosterol biosynthetic pathway has been delineated. The unusual sterols of the mutants can also be obtained to develop assays for the enzymes involved in ergosterol synthesis. Thus, by utilizing mutants, the simple eukaryotic system of yeast may be extended to explore the entire field of sterol metabolism and its relationship to cellular physiology.

Chromatography, High Pressure Liquid↗

PubMed1985

Recovery of Saccharomyces cerevisiae from ethanol-induced growth inhibition.

Ethanol caused altered mobility of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene in plasma membrane preparations of Saccharomyces cerevisiae. Because lipids had been shown to protect yeast cells against ethanol toxicity, sterols, fatty acids, proteins, and combinations of these were tested; however, protection from growth inhibition was not seen. Ethanol-induced, prolonged lag periods and diminished growth rates in S. cerevisiae were reduced by an autoconditioning of the medium by the inoculum.

Adaptation, Physiological↗