PubMed Health⌕ Search

Biomedical subjects

A Garcia-Perez

Publications and source records attributed to A Garcia-Perez.

At least 19 recordsLinked to original sources
PubMed1992

Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein.

Kidney medullary cells in situ, as well as kidney-derived Madin-Darby canine kidney (MDCK) cells accumulate nonperturbing, small organic solutes (osmolytes), including myo-inositol, when bathed in hypertonic media. Accumulation of osmolytes balances the osmolality of extracellular fluid without raising intracellular salts that would perturb cellular functions. In hypertonic media, increased myo-inositol accumulation is the result of increased activity of a Na+/myo-inositol cotransporter. We have isolated a cDNA encoding a Na+/myo-inositol cotransporter from MDCK cells using expression in Xenopus oocytes. The cDNA sequence predicts a protein of 718 amino acids with a significant amino acid sequence similarity to the Na+/D-glucose cotransporters of absorbing epithelia. Transporter mRNA is present in kidney and brain and is markedly induced in MDCK cells by medium hypertonicity, demonstrating that adaptation to hypertonic stress involves up-regulation of transporter mRNA accumulation.

Amino Acid Sequence↗
PubMed1992

Cloning of a Na(+)- and Cl(-)-dependent betaine transporter that is regulated by hypertonicity.

Many hypertonic bacteria, plants, marine animals, and the mammalian renal medulla are protected from the deleterious effects of high intracellular concentrations of electrolytes by accumulating high concentrations of the nonperturbing osmolyte betaine. When kidney-derived Madin-Darby canine kidney (MDCK) cells are cultured in hypertonic medium, they accumulate betaine to 1,000 times its medium concentration. This results from induction by hypertonicity of high rates of betaine transport into cells. We have isolated a cDNA (BGT-1) encoding a renal betaine transporter by screening an MDCK cell cDNA library for expression of a betaine transporter in Xenopus oocytes. The cDNA encodes a single protein of 614 amino acids, with an estimated molecular weight of 69 kDa. The deduced amino acid sequence exhibits highly significant sequence and topographic similarity to brain gamma-amino-n-butyric acid (GABA) and noradrenaline transporters, suggesting that the renal BGT-1 is a member of the brain GABA/noradrenaline transporter gene family. Expression in oocytes indicates that the BGT-1 protein has both betaine and GABA transport activities that are Cl(-)- as well as Na(+)-dependent and functionally similar to betaine and GABA transport in MDCK cells. Northern hybridization indicates that transporter mRNA is localized to the kidney medulla and is induced in MDCK cells by hypertonicity.

Amino Acid Sequence↗
PubMed1992

Kidney aldose reductase gene transcription is osmotically regulated.

Cells generally adapt to long-term hypertonic stress by accumulating organic osmolytes. PAP-HT25 renal medullary cells in hypertonic medium accumulate sorbitol through a reaction catalyzed by aldose reductase and betaine through osmotically regulated transport. Hypertonicity increases aldose reductase protein synthesis rate by elevating its mRNA abundance. To test whether the rise in aldose reductase mRNA is due to enhanced transcription, PAP-HT25 cells adapted to isotonic medium were switched to hypertonic medium, and transcription rate was measured by nuclear run-on. Aldose reductase transcription rate peaked at 17-fold the isotonic level after 12 h of hypertonicity. Then, transcription fell as sorbitol and betaine accumulated. Transcription stabilized at fivefold the isotonic level within days. Aldose reductase mRNA stability was not significantly different between the hypertonic and isotonic steady states. Thus aldose reductase mRNA is osmotically regulated through changes in its transcription. The osmotically induced rise in aldose reductase transcription is blunted by the accumulation of intracellular betaine and is exaggerated and prolonged by preventing the accumulation of both sorbitol (by aldose reductase inhibition) and betaine (by removal from the medium). This study presents the first description of osmoregulated gene transcription in animal cells.

Aldehyde Reductase↗
PubMed1992

Time course of renal glutamate dehydrogenase induction during NH4Cl loading in rats.

To study mechanisms involved in renal glutamate dehydrogenase (GDH) regulation in response to systemic acid loading, we have measured blood pH, ammonium excretion, renal GDH mRNA levels, and GDH activity in rats. Acid intake (0.28 M NH4Cl in drinking water for 3 days) increased GDH mRNA levels in the renal cortex, but had no effect in the outer stripe of the outer medulla, inner stripe of the outer medulla, or the inner medulla. Rats were subjected to a step change in acid intake by alkali loading for 3 days (7.2 meq NaHCO3 per day in food slurry) and shifting to acid loading for up to 7 days (7.2 meq NH4Cl in food slurry). Ammonium excretion rose rapidly, increasing by 14-fold in the first 24-h period and 38-fold in the second 24-h period. Cortical GDH mRNA levels were increased relative to alkali-loaded values by 3.7-fold in 24 h, 4.3-fold in 4 days, but only 2.2-fold in 7 days. GDH activity was unchanged after 24 h of acid intake, but was significantly increased after 48 h. We concluded the following: 1) GDH mRNA is present in all regions of the kidney, but levels increase in response to acid loading only in the renal cortex; 2) GDH mRNA levels increase within 1 day after the initiation of acid loading, but the associated increase in functional enzyme activity takes 2 or more days; and 3) the large increases in ammonium excretion that occur in the first day after initiation of acid loading are not dependent on increased GDH activity.

Ammonium Chloride↗
PubMed1992

Age-related changes in alpha 1- and alpha 2-chain type IV collagen mRNAs in adult mouse glomeruli: competitive PCR.

Studies of age-related changes in glomerular extracellular matrix (ECM) synthesis in normal mice have been hampered by the difficulty of isolating sufficient numbers of intact glomeruli and by the inability to quantify different mRNA species. The purpose of this study was to identify and quantitate the individual mRNAs coding for alpha 1- and alpha 2-chains of type IV collagen in isolated, single glomeruli of normal mice at different ages. These data on normal ECM synthesis were necessary for the understanding of glomerulosclerosis, a condition characterized by excess deposition of collagen. Pools of freshly microdissected adult mouse glomeruli were reverse transcribed in situ, and alpha 1-IV and alpha 2-IV collagen mRNAs were individually amplified by means of specific primers and the polymerase chain reaction (PCR), according to a previously published method. A competitive PCR assay, based on utilization of mutated cDNAs, allowed the reproducible, quantitative, and separate determination of the absolute amounts of both alpha 1-IV and alpha 2-IV mRNAs measured, as their respective cDNAs, in one-tenth of one glomerulus. The levels of alpha 1-IV and alpha 2-IV collagen mRNA were 208 +/- 36.0 x 10(-4) and 161.2 +/- 18.6 x 10(-4) amol/glomerulus in 5-wk-old mice. There were no significant age-related differences at 8, 12, and 24 wk. The mean levels over this period were 60.2 +/- 4.9 x 10(-4) for alpha 1-IV collagen mRNA and 63.9 +/- 5.8 x 10(-4) amol/glomerulus for alpha 2-IV collagen mRNA. Two of three 24-wk-old mice had mild glomerulosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

Maturation of aldose reductase expression in the neonatal rat inner medulla.

Newborns are less able to concentrate urine than adults are. With development of the concentrating system and a hypertonic medullary interstitium, there is a need to generate intracellular osmolytes such as sorbitol, which is produced in a reaction catalyzed by the enzyme aldose reductase. We sought to discriminate between two possible mechanisms of aldose reductase induction during development: (a) a response to an osmotic stimulus generated by the concentrating mechanism; or (b) part of the genetic program for development of the kidney. We measured the change in aldose reductase mRNA and activity in terminal inner medullary collecting ducts (IMCDs) microdissected from Sprague-Dawley rats during the first month of life. Aldose reductase mRNA was assayed by Northern analysis of total RNA from inner medulla and by detection of the reverse transcription-polymerase chain reaction (RT-PCR) product obtained from single IMCDs using aldose reductase-specific primers. Aldose reductase activity was measured in IMCDs taken from the same rats using a fluorescent microassay. Newborn rat IMCDs had minimal aldose reductase mRNA or activity, however mRNA was readily detected in IMCDs from rats older than 3 d of age, with peak expression occurring at 1-3 wk of age before decreasing to adult levels. In contrast, the mRNA level for a housekeeping metabolic enzyme, malate dehydrogenase, did not change during maturation. Aldose reductase enzyme activity was readily detectable by 6 d of age, peaked at 20 d, then decreased to adult levels. Urine osmolality remained < 600 mosmol/kg until 16 d, then increased to > 1,100 mosmol/kg after 20 d. Thus, aldose reductase mRNA and activity increased before urinary osmolality reached 870 mosmol/kg. Because urine osmolality may not be indicative of inner medullary osmolality and because mother's milk may provide excessive free water to the pups under 3 wk of age, half of the animals in several litters were separated from their mothers for 1 d and inner medullary osmolality, in addition to urine osmolality, was measured by vapor pressure osmometry, while aldose reductase mRNA was assessed densitometrically in IMCDs after RT-PCR. Although fluid restriction resulted in a near doubling of urine osmolality and a tendency towards increased aldose reductase mRNA, there was no consistently significant increase in aldose reductase mRNA or inner medullary osmolality during the first 13 d of life compared to the suckling animals. On the other hand, 2-3-wk-old rats showed significant increases in aldose reductase mRNA, accompanied by increases in inner medullary osmolality, after fluid restriction. Thus, the dissociation between the increases in aldose reductase expression and inner medullary hyperosmolality indicates that the maturational induction of the aldose reductase gene is not a consequence of osmotic stimulation, but rather, part of the developmental program of the kidney.

Aldehyde Reductase↗
PubMed1992

How tonicity regulates gene expression.

The expression of a number of different mammalian genes is directly affected by hypertonicity. At present, the list of their products includes aldose reductase, heat shock proteins, early response factors, and transporters for betaine, inositol, and taurine. Hypertonicity increases the abundance of the mRNAs for all of them. Aldose reductase mRNA levels increase because of increased transcription with little change in the stability of its mRNA. Transcription of the betaine transporter also increases. The mechanisms by which hypertonicity increases the transcription of these mammalian genes remain speculative. However, the consideration of transcriptional control of betaine transport in bacteria and of heat shock proteins in many organisms provides interesting insight into this question.

Aldehyde Reductase↗
PubMed1991

Accumulation of glycerophosphocholine (GPC) by renal cells: osmotic regulation of GPC:choline phosphodiesterase.

Although GPC has long been recognized as a degradation product of phosphatidylcholine, only recently is there wide appreciation of its role as a compatible and counteracting osmolyte that protects cells from osmotic stress. GPC is osmotically regulated in renal cells. Its level varies directly with extracellular osmolality. Cells in the kidney medulla in vivo and in renal epithelial cell cultures (MDCK) accumulate large amounts of GPC when exposed to high concentrations of NaCl and urea. Osmotic regulation of GPC requires choline in the medium, presumably as a precursor for synthesis of GPC. Choline transport into the cells, however, is not osmoregulated. The purpose of the present studies was to use MDCK cell cultures as a defined model to distinguish whether osmotically induced accumulation of GPC results from increased GPC synthesis or decreased GPC disappearance. The rate of incorporation of 14C from [14C]choline into GPC, the steady-state GPC synthesis rate, and the activity of phospholipase A2 (which can catalyze a step in the synthesis of GPC from phosphatidylcholine) are not increased by high NaCl and urea. In fact all are decreased by approximately one-third. Therefore, we find no evidence that high NaCl and urea increases the GPC synthesis rate. On the other hand, the rate coefficient for cellular GPC disappearance and the activity of GPC:choline phosphodiesterase (EC 3.1.4.2), which catalyzes degradation of GPC, are decreased by approximately two-thirds by high NaCl and urea. We conclude that high NaCl and urea increase the level of GPC by inhibiting its enzymatic degradation.

Animals↗
PubMed1991

RT-PCR microlocalization of mRNA for guanylyl cyclase-coupled ANF receptor in rat kidney.

Microlocalization of mRNA coding for the guanylyl cyclase-coupled atrial natriuretic factor (ANF) receptor was carried out in the rat kidney. We used a combination of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, and vasa recta bundles. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P-labeled probe. Among renal tubule segments, the largest signal was found in the terminal inner medullary collecting duct (IMCD). Slightly smaller signals were found in the initial IMCD and in loop of Henle segments from the inner medulla. Readily detectable signals were also seen in the following segments (in descending order): cortical collecting duct, proximal convoluted tubule, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and outer medullary collecting duct. Large signals were also detected in glomeruli and in vasa recta bundles from the inner stripe of the outer medulla. Based on these results, we conclude that 1) renal microlocalization of specific mRNAs coding for hormone receptors is feasible through application of the RT-PCR procedure in microdissected renal tubules and vascular elements, and 2) the gene for the guanylyl cyclase-coupled ANF receptor is broadly expressed along the nephron, raising the possibility that multiple sites of ANF action are present.

Animals↗
PubMed1977

X-linked ichthyosis.

In the period 1962-1974, 36 patients with X-linked ichthyosis, belonging to 22 families, were selected for study. The frequency of this genodermatosis is higher in the province of Salamanca than in other countries. In most of the cases, the lesions were apparent at birth or shortly after. In 41% of the patients studied by slit-lamp microscopy, posterior embriotoxom was seen. Hyperkeratosis was seen in follicular and sweat duct orifices. Statistical analysis of the pedigrees showed a higher proportion of affected males among the offspring of carriers and also among the offspring of the ichthyotics' sisters. In addition, among the patients' descendants, the higher ratio of females is statistically significant, showing that there is likely to be selection in favour of the X chromosome.

Child↗
PubMed1977

Acral arteriovenous tumor.

A unique, superficial, lobulated, benign vascular tumor of the skin, found in the acral areas of adult males, is described. The combination of three different elements--arterial, venous, and transitional vascular channels--make up the predominant histologic features of this tumor. The transition of fibromuscular channels indicates a venous lesion, but the acral location of the lesions and the structure and staining characteristics suggest that the lesion is a hamartomatous proliferation of the Sucquet-Hoyer canal of the true glomus.

Adult↗
PubMed1976

Pipillon-Lefèvre syndrome: report of two familial cases.

Two cases with Papillon-Lefèvre Syndrome (PLS) are reported. They are two brothers belonging to a family which there is strong consanguinity. An autosomal recessive pattern of inheritance is suggested. Some pathological findings as congenital bronchiectasis observed in one of the patients as well as the PLS features are discussed and considered to be fortuitous defects. These observations are compared with those described in the literature.

Adolescent↗