The effect of pH on incorporation of galactose by a normal human cell line and cell lines from patients with defective galactose metabolism.

Incorporation of radioactive galactose into TCA-insoluble material of galactosemic fibroblasts is more sensitive to low pH than is the incorporation by normal human fibroblasts. This study was undertaken to determine (1) whether there was any pH which could correct or counteract the galactosemic defect relative to galactose incorporation, and (2) whether the low pH effect was specific for galactose metabolism or whether general cellular metabolism in galactosemic cells was more sensitive to low pH than that in normal cells. The pH dependencies of incorporation of radioactive galactose and glucose into cellular macromolecules were investigated in galactosemic and normal cells. Normal cells have a biphasic curve with respect to galactose incorporation with peaks at pH 7.0 and 8.5. Galactosemic cells have only the high pH peak. The maximum incorporation by galactosemic cells was never more than about 30% that seen by normal cells under the conditions of these experiments. Thus manipulation of the pH alone cannot correct the galactosemic defect. The rate of incorporation of radioactive galactose was studied in normal, galactosemic and galactokinase deficient cells, at pH 7.2 and at pH 6.3. At pH 7.2, galactosemic cells incorporate galactose at a linear rate which is 30 to 40% that of normal cells while incorporation by kinase-deficient cells is between 5 and 10% of normal. At pH 6.3, the incorporation is also linear. However, galactosemic cells now exhibit the same rate as kinase-deficient cells in which the low level of incorporation is unaffected by pH. These results suggest that incorporation of galactose by galactosemic cells at low pH is not due to metabolic death of the cells, but may be due to the inhibition of some specific step or steps along a metabolic route of galactose metabolism other than the Leloir pathway.     

Galactose-1-phosphate uridyl transferase in density-fractionated erythrocytes

Summary Galactose-1-phosphate uridyl transferase (GALT), the deficient enzyme in classical galactosemia, was studied by Percoll-gradient age-fractionation of erythrocytes. For normal GALT, a rapid and substantial decrease in GALT activity and loss of most of two isozymes was found to occur in the reticulocyte fractions. The loss of activity was then followed by relative stabilization of both GALT-specific activity and microheterogeneity in mature and aging erythrocytes. When applied to the study of mutant GALT from galactosemic patients, the Percoll-gradient fractionation method permitted detection in the reticulocyte-enriched fractions of up to 5% of normal GALT-specific activity and an isoelectric focusing pattern essentially the same as that of normal GALT. Percoll-gradient fractionation of erythrocytes offers a simple and direct method to study characteristics of GALT activity and microheterogeneity in normal and galactosemic human erythrocytes.     

Galactose and glucose metabolism in galactokinase deficient, galactose-1-P-uridyl transferase deficient and normal human fibroblasts.

Despite the genetic interruption of the Leloir pathway both galactosemic patients and galactosemic fibroblasts can convert galactose to CO2 and TCA precipitable products, although at less than the normal rate. These observations stimulated investigations into the identity of the alternative metabolic routes which allows for galactose metabolism in the absence of in vitro galactose-1-P-uridyl transferase. Four lines of galactosemic cells, each without detectable gal-transferase, produced 14CO2 from [1-14C]-galactose (0.094 mumoles in 20 cc of medium) at approximately 39% +/- 16% the rate of transferase positive cells over a 48-hour period. However, galactokinase deficient fibroblasts produced 14CO2 and TCA precipitable products from [1-14C]-galactose or [U-14C]-galactose at only 3% to 9% the rate of normal fibroblasts. Therefore it seems likely that gal-transferase deficient fibroblasts must first synthesize galactose-1-P for further metabolism of galactose.     

Characterization of normal and abnormal variants of galactose-1-phosphate uridylyltransferase (EC 2.7.7.12) by isoelectric focusing

Isoelectric focusing (IEF) in polyacrylamide gels has been used to study the isozymes of human galactose-1-phosphate uridylyltransferase (GALT) in erythrocytes and fibroblasts. In addition to the usefulness of IEF in differentiating normal, Duarte variant, and galactosemic homozygotes and heterozygotes, the ability of IEF to distinguish the residual GALT activity in two different galactosemic fibroblast lines and in revertants from them is demonstrated.     

Combination of glycemic and oxidative stress in lens: implications in augmentation of cataract formation in diabetes

It is well known that the incidence of cataract is higher in diabetics as compared to non-diabetics. Its rate of maturation is also faster in the diabetics. The precise mechanism of this acceleration is not clearly understood. It is hypothesized that this could be a result of the combination of the metabolic and oxidative stress induced by glycemia itself with the age-associated increase in ambient generation of oxyradical species. In the current studies, we have investigated this possibility using the galactose cataract model. Galactosemia was induced by feeding rats a 50% galactose diet. The increased susceptibility of the glycemic lenses to physiological damage by reactive oxygen species (ROS) was studied by incubating them in Tyrode in the absence and presence of menadione. The resulting physiological damage to the lens was assessed initially in terms of its ability to maintain Na⁺-K⁺ ATPase dependent active transport of potassium ions, as represented by the uptake of rubidium ions. Subsequently, the level of ATP, indexing the general metabolic status, and the level of glutathione (GSH), indexing the status of antioxidant reserve, were also determined. The uptake of rubidium in the normal lenses incubated in the presence of the quinone was depressed to more than 50% of the controls run in the basal medium. A similar depression existed in the galactosemic lenses in comparison to the normal lenses. However, in the presence of menadione, the inhibition of the uptake was accentuated further in the case of galactosemic lenses, the uptake here being only 20% of the normal controls. Similarly, the galactosemic lenses were also more susceptible to menadione dependent decrease in ATP and GSH.     

Characterization of Oxidative Stress in Various Tissues of Diabetic and Galactose-fed Rats

Rats fed a galactose-rich diet have been used for several years as a model for diabetes to study, particularly in the eye, the effects of excess blood hexoses. This study sought to determine the utility of galactosemia as a model for oxidative stress in extraocular tissues by examining biomarkers of oxidative stress in galactose-fed rats and experimentally-induced diabetic rats. Sprague-Dawley rats were divided into four groups: experimental control; streptozotocin-induced diabetic; insulin-treated diabetic; and galactose-fed. The rats were maintained on these regimens for 30 days, at which point the activities of catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase, as well as levels of lipid peroxidation and reduced and oxidized glutathione were determined in heart, liver, and kidney. This study indicates that while there are some similarities between galactosemic and diabetic rats in these measured indices of oxidative stress (hepatic catalase activity levels and hepatic and renal levels of oxidized glutathione in both diabetic and galactosemic rats were significantly decreased when compared to normal), overall the galactosemic rat model is not closely parallel to the diabetic rat model in extra-ocular tissues. In addition, several effects of diabetes (increased hepatic glutathione peroxidase activity, increased superoxide dismutase activity in kidney and heart, decreased renal and increased cardiac catalase activity) were not mimicked in galactosemic rats, and glutathione concentration in both liver and heart was affected in opposite ways in diabetic rats and galactose- fed rats. Insulin treatment reversed/prevented the activity changes in renal and cardiac superoxide dismutase, renal and cardiac catalase, and hepatic glutathione peroxidase as well as the hepatic changes in lipid peroxidation and reduced and oxidized glutathione, and the increase in cardiac glutathione. Thus, prudence should be exercised in the use of experimentally galactosemic rats as a model for diabetes until the correspondence of the models has been more fully characterized.     

Inhibitor of apoptosis proteins and ovarian dysfunction in galactosemic rats

Galactosemia is a genetic disease with deficiency of galactose-1-uridyltransferase, resulting in the accumulation of galactose or galactose-1-phosphate in the blood and tissues. Rats were fed with normal rat chow and with a high-galactose diet for 4 weeks to give control and galactosemic groups, and their ovarian function was studied. The two groups of rats were injected with pregnant mare's serum gonadotrophin (PMSG) and were killed at different time points after human chorionic gonadotrophin (hCG) injection. The number of oocytes ovulated in the controls was significantly higher than in the galactosemic group. Morphometric studies of the ovaries also showed a higher number of corpora lutea in the controls. Western blot analysis of granulosa cells showed that the overall expressions of Fas and FasL were lower in the control group and their expressions of inhibitor of apoptosis proteins (IAPs) were higher than in the galactosemic group, especially at 8 h post hCG injection. TDT-mediated dUTP-biotin nick end-labeling (TUNEL) and immunohistochemical staining of ovarian sections with Ki-67 and IAPs showed more apoptotic granulosa cells in the galactosemic group and the expressions of IAPs in granulosa cells also confirmed the result of the Western blot. These findings support our hypothesis that ovarian dysfunction in galactosemic rats is due to increased apoptosis in granulosa cells of maturing follicles.     

The phenotypic range of hemophilia A carriers.

We have described the study of a small kindred with X-linked hemophilia A. It was ascertained through a clinically affected female, the daughter of a man with moderately severe hemophilia. The pedigree and the proband's phenotype suggest that she may be a heterozygote in whom most of the normal alleles at the VIII-1 locus are not active. She has two sisters, also obligatory carriers. The three sisters exhibit the three phenotypes possible for heterozygous females: clinically affected, clinically normal but phenotypically abnormal as determined by laboratory tests, and clinically and phenotypically normal.     

Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes

Classic galactosemia, an inborn error of human galactose metabolism, is characterized by a deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). The current model for the pathophysiology of this disease ascribes most of its symptoms to the toxicity of intracellular galactose-1-phosphate (Gal-1-P), one of the substrates of GALT which accumulates in the untreated disease state. Recently, a reduction in the intracellular concentration of UDP-Gal (uridine diphosphogalactose), one of the products of GALT, has been described in treated galactosemic patients. We investigated whether galactosemic patients might also have reduced amounts of those macromolecules that depend on UDP-Gal for their biosynthesis. We report a reduction in glycolipids that contain either galactose or its derivative N-acetylgalactosamine and an accumulation of the precursors to these compounds in the brain of a neonate with galactosemia. We also found an imbalance in glycolipids in galactosemic lymphoblasts. This novel biochemical abnormality observed in galactosemic patients is not addressed by dietary galactose-restriction therapy and could explain some of the chronic neurologic and other complications of galactosemia.     

Role of nonenzymatic glycosylation in experimental cataract formation

The relevance of nonenzymatic glycosylation of lens proteins to cataract formation was studied in rats on a normal and high galactose diet, treated with and without sorbinil, an aldose reductase inhibitor. All galactosemic rats not receiving sorbinil had cataracts; none receiving sorbinil had cataracts. Lens homogenate was treated with a 200 fold molar excess of [³H]-borohydride and the extent of glycosylation was estimated from radioactivity incorporation and quantitation of hexitol-lysine adduct after extensive dialysis. We found no differences in the radioactivity uptake nor the amounts of hexitol-lysine in the lenses of galactosemic rats treated with and without sorbinil. Thus, nonenzymatic glycosylation was not responsible for the sugar-induced cataracts.     

Ultrastructural localization of blood-retinal barrier breakdown in diabetic and galactosemic rats

Breakdown of the blood-retinal barrier (BRB) is an early event in diabetic and galactosemic rats, but the location and nature of the specific defect(s) are controversial. Using an electron microscopic immunocytochemical technique, the retinas of normal, diabetic, and galactosemic rats were immunostained for endogenous albumin. Normal rats showed little evidence of BRB breakdown at either the inner barrier (retinal vasculature) or the outer barrier (retinal pigment epithelium) (RPE). In diabetic and galactosemic rats, as was true in human diabetics, BRB breakdown occurred predominantly at the inner BRB, but in some cases at the outer barrier as well. Treatment with the aldose reductase inhibitor sorbinil largely prevented BRB failure in galactosemic rats. In the inner retina of diabetic and galactosemic rats, albumin was frequently demonstrated on the abluminal side of the retinal capillary endothelium (RCE) in intercellular spaces, basal laminae, pericytes, ganglion cells, astrocytes, and the perinuclear cytoplasm of cells in the inner nuclear layer. Albumin did not appear to cross RCE cell junctions; however, it was occasionally seen in RCE cytoplasm of galactosemic rats. In the outer retina, albumin was frequently detected in the subretinal space, in the intercellular space between photoreceptors, and in the perinuclear cytoplasm of photoreceptor cells, but was only infrequently found in the RPE cells constituting the barrier. Albumin derived from the choroidal vasculature did not appear to cross the tight junctions of the RPE. These findings suggest that specific sites of BRB compromise are infrequent but that once albumin has crossed the RCE or RPE it freely permeates the retinal tissue by filling intercellular spaces and permeating the membranes of cells not implicated in BRB formation. The diffuse cytoplasmic staining of some RCE and RPE cells suggests that the predominant means of BRB breakdown in diabetes and galactosemia involves increased focal permeability of the surface membranes of the RCE and RPE cells rather than defective tight junctions or vesicular transport.     

Defective galactosylation of serum transferrin in galactosemia

The glycosylation of serum transferrin from galactosemic patients with a deficiency of galactose-1-phosphate uridyl transferase (EC 2. 7.7 12) is abnormal but becomes normal after treatment with a galactose-free diet. To understand the structural and biochemical basis of the abnormal glycosylation, transferrin was purified from the serum of untreated and treated galactosemic patients and normal controls and the N-linked glycans analyzed by HPLC. The glycans from normal transferrin consisted predominantly (86%) of the disialylated biantennary complex type. The glycans from untreated galactosemic patients were more heterogeneous and contained four major truncated glycans in addition to a smaller amount (13%) of the disialylated biantennary complex type. The truncated glycans were deficient in galactose and sialic acid and their structures were consistent with a decrease in galactosyltransferase activity in hepatocytes, the probable cells of origin of the transferrin. This is postulated to be due to direct inhibition of the galactosyltransferase activity by the accumulated galactose-1-phosphate or to an effect on the formation of UDP- galactose, the donor substrate in the reaction. After treatment the proportion of the truncated glycans decreased and the proportion of the disialylated biantennary complex type increased, returning almost but never completely to normal, even after prolonged treatment in some cases. There was no clear relationship between the length of treatment and the normalization of glycosylation and the level of galactose-1- phosphate in red blood cells, the usual parameter for monitoring the treatment of galactosemics. It is suggested that the persistence of abnormally glycosylated proteins may contribute to the long-term complications in galactosemia.      

Genetic variation in rice

Completion of the genomic sequencing of rice has enhanced the discovery of new genes. Wild rice relatives are good sources for extending the genetic variation of cultivated rice. Reproductive barriers are commonly found in distant crosses of rice and are attracting attention. The combination of genetic analyses and molecular tools has greatly facilitated the molecular cloning of rice genes based on the classical approach and enabled the tracking of dissemination of the alleles for domestication. Basic information for population genetics study in rice is still being collected and is expected to provide an alternative approach for finding new genes. The wide genetic variation available in wild rice relatives and the combination of various genetic approaches will allow the analysis and understanding of genetic variation at the nucleotide sequence level, as well as the discovery of novel alleles by sequence-based approaches.     

Apparent regression of the CGG repeat in FMR1 to an allele of normal size

The fragile X syndrome is the result of amplification of a CGG trinucleotide repeat in the FMR1 gene and anticipation in this disease is caused by an intergenerational expansion of this repeat. Although regression of a CGG repeat in the premutation range is not uncommon, regression from a full premutation (>200 repeats) or premutation range (50–200 repeats) to a repeat of normal size (<50 repeats) has not yet been documented. We present here a family in which the number of repeats apparently regressed from approximately 110 in the mother to 44 in her daughter. Although the CGG repeat of the daughter is in the normal range, she is a carrier of the fragile X mutation based upon the segregation pattern of Xq27 markers flanking FMR1. It is unclear, however, whether this allele of 44 repeats will be stably transmitted, as the daughter has as yet no progeny. Nevertheless, the size range between normal alleles and premutation alleles overlap, a factor that complicates genetic counseling.     

Cranial nerve dysfunction in conscious galactosemic rats as measured by the auditory-evoked brainstem response

The purpose of this study was to extend our previous work with the auditory-evoked brainstem response and determine whether galactosemia would produce a functional neuropathy similar to that previously seen in streptozocin-induced diabetic rats. Sprague-Dawley male rats implanted with cortical electrodes received either normal chow (n = 17) or a 50% galactose diet (n = 17) for 5 weeks. Peak II latency of the auditory-evoked brainstem response, interpreted as a functional measure of the auditory nerve (VIII cranial) in rats, was significantly prolonged in galactose-fed rats relative to controls (P less than 0.05). These results demonstrate a functional deficit in the auditory nerves of galactosemic rats. The deficit in the auditory-evoked brainstem response of galactosemic rats is similar to our previous finding in streptozocin-induced diabetic rats.     

Higher nervous activity in rats with symptoms of hereditary galactosemia]

The patterns of behaviour and neural processes in rats with symptoms of hereditary galactosemia were investigated. Certain impairment of neural processes, particularly of the internal inhibition process in galactosemic rats was established. This is evidenced by low conditioning rate and lower level of responding in 2-way shuttle-box avoidance achieved by galactosemic rats in comparison with galactose-resistant rat substrain. Significant changes in motor activity and emotionality level in the course of repeated open-field testings were not found in galactosemic rats. The pecularities of the active avoidance acquisition, an analysis of the capacity to the retention of acquired task demonstrated an impaired mechanism of long-term memory storage in galactosemic animals.     

Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia.

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosemic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylysine, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonenzymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated with DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot account for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

Characterisation of Major Histocompatibility Complex Class I in the Australian Cane Toad,Rhinella marina

The Major Histocompatibility Complex (MHC) class I is a highly variable gene family that encodes cell-surface receptors vital for recognition of intracellular pathogens and initiation of immune responses. The MHC class I has yet to be characterised in bufonid toads (Order: Anura; Suborder: Neobatrachia; Family: Bufonidae), a large and diverse family of anurans. Here we describe the characterisation of a classical MHC class I gene in the Australian cane toad, Rhinella marina. From 25 individuals sampled from the Australian population, we found only 3 alleles at this classical class I locus. We also found large number of class I alpha 1 alleles, implying an expansion of class I loci in this species. The low classical class I genetic diversity is likely the result of repeated bottleneck events, which arose as a result of the cane toad''s complex history of introductions as a biocontrol agent and its subsequent invasion across Australia.     

Early explorations of the pathways of uridine diphosphate galactose in man and in microorganisms

Thirty years ago, a number of human inborn errors in carbohydrate metabolism were explored with specific enzymatic tests on blood samples (hemolysates). Hereditary galactosemia was the first example. When the inoperative step in galactose metabolism was specified, the basis for the diet therapy used on the galactosemic infants, namely galactose-free diet, could be shown to be securely founded. As far as galactose metabolism is concerned, the cells of the infant are faced with two problems: (i) the conversion of dietary lactose (galactosyl glucose) to glucose and its catabolites involved in energy metabolism, and (ii) the conversion of dietary glucose or lactose to galactosyl units of glycolipids and glycoprotein cell structures. Subsequent studies on microorganisms revealed several types of hereditary defect in galactose metabolism. One type which permits the bacteria to develop a normal carbohydrate pattern in their cell walls includes an enzyme defect, like that described in the cells of the galactosemic infant. Two other types, with the inability to synthesize UDPGlc or UDPGal from glucose, do not permit the bacteria to build the fabric of the normal bacterial cell wall. This is the subject for discussion.