PURPOSE: Determining which patients are at risk for the development of diabetic retinopathy is expected to greatly improve existing prevention and treatment options. In this study, using an animal model of diabetic retinopathy, the hypothesis was tested that magnetic resonance imaging (MRI) and a carbogen inhalation challenge provides important diagnostic information regarding the risk of developing diabetic retinopathy. METHODS: MRI was used to measure noninvasively the change in oxygen tension along the entire inner retina (i.e., from superior ora serrata to inferior ora serrata) during a carbogen (95% O2/5% CO2) inhalation challenge (IOVS 1996;37:2089). Two animal groups were examined by this MRI method at two time points: (1) rats fed either normal rat chow (n = 20) or a 50% galactose diet (n = 20) for 3.5 months (i.e., before the appearance of extensive retinal lesions) or (2) rats fed either normal rat chow (n = 3) for 15 months or a 30% galactose diet (n = 4) for 15 to 18 months (i.e., when lesions are present). Retinal biochemical and morphometric measurements were also obtained. RESULTS: After 3.5 months of galactosemia, before the appearance of extensive retinal morphologic lesions, a significant (P < 0.05) reduction in the panretinal oxygenation response was observed in the galactosemic group compared with its age-matched control. These galactose-fed animals also displayed a significantly (P < 0.05) larger oxygenation response in the inferior hemiretina than in the superior hemiretina. After 15 to 18 months of galactosemia, during the period when lesions are present, the panretinal oxygenation response remained significantly (P < 0.05) lower in the galactose-fed animals than in their age-matched controls. In contrast to the 3.5-month results, the oxygenation response in galactosemic animals at 15 to 18 months was significantly (P < 0.05) larger in the superior than in the inferior hemiretina. Hemiretinal oxygenation responses were not different in normal controls at either duration. CONCLUSIONS: MRI measurement of the retinal oxygenation response to a carbogen challenge appears to be a powerful new and noninvasive approach that may be useful for assessing aspects of pathophysiology underlying the development of diabetic retinopathy in galactosemic rats. These results support our working hypothesis and suggest that further research into the diagnostic potential of this MRI approach for predicting the development of diabetic retinopathy is warranted. (+info)
A mouse model of galactose-induced cataracts.
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Galactokinase (GK; EC 2.7.1.6) is the first enzyme in the metabolism of galactose. In humans, GK deficiency results in congenital cataracts due to an accumulation of galactitol within the lens. In an attempt to make a galactosemic animal model, we cloned the mouse GK gene (Glk1) and disrupted it by gene targeting. As expected, galactose was very poorly metabolized in GK-deficient mice. In addition, both galactose and galactitol accumulated in tissues of GK-deficient mice. Surprisingly, the GK-deficient animals did not form cataracts even when fed a high galactose diet. However, the introduction of a human aldose reductase transgene into a GK-deficient background resulted in cataract formation within the first postnatal day. This mouse represents the first mouse model for congenital galactosemic cataract. (+info)
The morphological identification of pathogenic yeasts using carbohydrate media.
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Eight isolates of C. albicans were used to determine the frequency with which germ tube formation occurred: on rice extract -Tween 80 agar, on its components, and on 1% bactopeptone agar after three hr at 37 degrees C; in 0.5% aqueous solution of various carbohydrates; in various concentrations of glucose; on 0.5 and 0.1% glucose agar and on various types of agar alone. Subsequently 250 isolates of yeast of the genera Candida, Torulopsis, Trichosporon, Cryptococcus, and Saccharomyces, which were obtained from a clinical laboratory, were spread on rice extract -Tween 80 agar and on 0.1% glucose agar and covered with coverslips. Direct microscopic examination after incubation for three hours at 37 degrees C demonstrated germ tube formation by all 140 isolates of C. albicans, but by none of the other yeasts. The characteristic features of the pseudomycelia of isolates of Candida and Trichosporon were evident on reexamination after a further 45 to 69 hours at room temperature (22 degrees C). These morphological observations suggested the identity of the isolates of Torulopsis, Cryptococcus, and Saccharomyces but identified virtually all (98.2%) of those of the genera which formed pseudomycelia. Of the latter group only four isolates required fermentation and assimilation tests to determine whether they were C. parapsilosis (1) or C. guilliermondii (3). (+info)
Transmissible substrate-utilizing ability in enterobacteria.
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Three of 152 strains of Escherichia coli transmitted their ability to utilize sucrose (Sac+) to other strains by conjugation. The transfer factor of one of them and of a Sac+ Salmonella thompson strain was thermosensitive. The raffinose-utilizing ability of 27 of 163 E. coli strains was also transmissible. Transmissible raffinose-utilizing ability was a feature of porcine enterpathogenic strains possessing the K88 antigen. The determinants controlling raffinose utilization (Raf) and K88 antigen production were commonly transmitted together from these strains; so also was the determinant controlling enterotoxin production, but to a lesser extent. It was not possible to transfer lactose-utilizing ability from 320 strains of E. coli, salicin-utilizing ability from 12 strains of E. coli or dulcitol-utilizing ability from 99 strains of E. coli and 88 strains of salmonellae. Sucrose- and raffinose-utilizing ability were transmitted separately to several Salmonella sp., including Salm. typhi, to Shigella flexneri and Sh. sonnei and to a variety of strains of E. coli. A strain of Salm. typhimurium in which Sac had been established and a strain of E. coli in which Raf had been established survived less well in the alimentary tract of chickens than their Sac minus or Raf minus parent strains. (+info)
Riboflavin and mouse hepatic cell structure and function. Mitochondrial oxidative metabolism in severe deficiency states.
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Weanling mice were fed a riboflavin-deficient diet or the same diet with added galactoflavin. Both diets produced changes in hepatic mitochondrial morphology, the most striking of which was the development of giant mitochondria. The livers from these animals were fractionated, and the nuclear and mitochondrial fractions were examined by electron microscopy. The nuclear fraction contained giant mitochondria; the mitochondrial fraction contained the remaining normal to moderately enlarged mitochondria. Oxidative studies were carried out on the mitochondrial fractions. It was found that both experimental diets resulted in a marked reduction in fatty acid oxidation by the mitochondria. In addition, the mitochondria of mice with advanced riboflavin deficiency (induced simply by a riboflavin-free diet) showed a severely decreased state 3 (ADP-stimulated) respiration and depressed respiratory control ratios, but normal ADP/O ratios. In contrast, mitochondrial performance (aside from fatty acid oxidation) in galactoflavin-supplemented, riboflavin-deficient mice was related to the gross appearance, i.e., color, of the liver from which these organelles were derived. In mice fed this diet, the livers were either red or yellow. Mitochondria from yellow livers showed normal oxidative phosphorylation. Mitochondria from red livers showed a serious reduction in state 3 oxidation. This study demonstrates that in the mouse, riboflavin deficiency, however produced, not only results in altered mitochondrial morphology but also results in significantly impaired mitochondrial function. (+info)
Pasteurella multocida subsp. multocida and P. multocida subsp. septica differentiation by PCR fingerprinting and alpha-glucosidase activity.
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Pasteurella multocida is composed of three subspecies that are often differentiated by fermentation of sorbitol and dulcitol. We studied 35 dulcitol-negative P. multocida isolates from infected dog and cat bite wounds, 16 of which yielded weak and/or conflicting fermentation reactions in Andrades sorbitol, thus making it difficult to distinguish between the two dulcitol-negative subspecies of P. multocida, i.e., P. multocida subsp. multocida and P. multocida subsp. septica. All isolates and two control strains were further analyzed using a PCR fingerprinting technique with a single primer (M13 core) and assessed for alpha-glucosidase (alpha-Glu) activity. Although the PCR fingerprint patterns and alpha-Glu activity did not correlate well with the sorbitol fermentation reactions, they did correlate well with each other. All strains identified as P. multocida subsp. septica were positive for alpha-Glu activity and exhibited the group I PCR fingerprint profile. All strains categorized as P. multocida subsp. multocida displayed either the group II or group III PCR fingerprint profile; 9 of 11 of these isolates were alpha-Glu negative. These data suggest that both PCR fingerprinting and alpha-Glu activity provide reliable means for differentiating P. multocida subsp. multocida from P. multocida subsp. septica, particularly in strains that produce weak and/or discrepant sorbitol fermentation reactions. (+info)
Osmoregulatory alterations in taurine uptake by cultured human and bovine lens epithelial cells.
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PURPOSE: Comparative assessment of cultured human lens epithelial cells (HLECs) and bovine lens epithelial cells (BLECs) established the nature of the relationship between taurine-concentrating capability and intracellular polyol accumulation or extracellular hypertonicity. METHODS: The kinetic characteristics of active taurine accumulation based on the measurement of in vitro [3H]-taurine uptake were resolved by side-to-side review of cultured HLECs and BLECs pre-exposed to either galactose-supplemented medium or extracellular hypertonicity. Competitive RT-PCR was used to appraise variation in taurine transporter (TauT) mRNA abundance from cells maintained in hyperosmotic medium over a 72-hour exposure period. RESULTS: The capacity to accumulate [3H]-taurine was significantly lowered after prolonged (20-hour) incubation of cultured BLECs in 40 mM galactose in contrast to HLECs, the latter cells' velocity curve being indistinguishable from control cells in physiological medium. Inhibition of the intracellular taurine transport site appeared to be noncompetitive, in that there was a marked reduction in the V(max) without significant alteration in the K(m) to a high-affinity transport site. Galactitol content in BLECs exceeded five times that found in HLECs. The coadministration of the aldose reductase inhibitor, sorbinil, with 40 mM galactose completely prevented the inhibitory effect of galactose on [3H]-taurine uptake. Acute exposure (3 hours) of HLECs and BLECs to a range of 10 to 40 mM galactitol or 10 to 40 mM galactose plus sorbinil-supplemented medium suggested by Dixon plot that neither galactitol nor galactose interacted with the extracellular taurine transport site. In contrast, [3H]-taurine accumulation was markedly elevated in both HLECs and BLECs after prolonged exposure to galactose-free medium made hyperosmotic by supplementation with sodium chloride. The enhanced taurine uptake capacity involved increase in peak velocity (V(max)) without significant change in Michaelis-Menten constant (K(m)). Cultured HLECs and BLECs responded to hypertonicity with an inducible but transitory upregulation of TauT mRNA. CONCLUSIONS: These results demonstrate that lens epithelial cells express a high-affinity TauT protein capable of active uptake, but predisposed to inhibition by intracellular galactitol when the sugar alcohol is present in sufficiently high concentration to interfere with cell metabolism. Furthermore, lens epithelial cells respond to hypertonic stress by raising taurine transport activity. The increase in taurine uptake is due to an increase in the number of high-affinity TauTs expressed as a result of an increase in the manifestation of taurine mRNA stemming from exposure to hypertonic medium. (+info)
Relationship between hepatic mitochondrial oxidative metabolism and morphology during riboflavin deficiency and recovery in mice.
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Changes in hepatic mitochondrial oxidative metabolism were examined during the development of severe riboflavin deficiency in mice, and during recovery from this deficiency. There was a marked reduction in oxidative rates for all substrates tested, with the decline being most pronounced with palmitoyl-1-carnitine. These effects were not enhanced by addition of galactoflavin to the riboflavin-deficient diet. Treatment of the deficient mice with riboflavin restored hepatic mitochondrial oxidation to normal within 24 hours in those mice fed a simple riboflavin-deficient diet, but required 72 hours in galactoflavin-supplemented mice. These metabolic changes in hepatic mitochondria appear to be temporally independent of the striking morphological changes occurring in these organelles during ariboflavinosis and recovery. (+info)