Robust and efficient synthetic method for forming DNA microarrays.

The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.

The external calcium-dependent NADPH dehydrogenase from Neurospora crassa mitochondria.

We have inactivated the nuclear gene coding for a putative NAD(P)H dehydrogenase from the inner membrane of Neurospora crassa mitochondria by repeat-induced point mutations. The respiratory rates of mitochondria from the resulting mutant (nde-1) were measured, using NADH or NADPH as substrates under different assay conditions. The results showed that the mutant lacks an external calcium-dependent NADPH dehydrogenase. The observation of NADH and NADPH oxidation by intact mitochondria from the nde-1 mutant suggests the existence of a second external NAD(P)H dehydrogenase. The topology of the NDE1 protein was further studied by protease accessibility, in vitro import experiments, and in silico analysis of the amino acid sequence. Taken together, it appears that most of the NDE1 protein extends into the intermembrane space in a tightly folded conformation and that it remains anchored to the inner mitochondrial membrane by an N-terminal transmembrane domain.

Acarbose partially inhibits microvascular retinopathy in the Zucker Diabetic Fatty rat (ZDF/Gmi-fa).

We compared quantitative capillary retinopathic changes between non-insulin-dependent diabetic Zucker Diabetic Fatty (ZDF) rats and heterozygous nondiabetic Zucker controls and evaluated the effect of an orally administered glucosidase inhibitor, acarbose, on retinopathy in these animals. Four groups of eight rats were analyzed: treated and untreated ZDF and treated and untreated Zuckers. Retinal capillary basement membrane thickening and retinal capillary cell density were determined from transmission electron microscopy and trypsin digestion preparations. ZDF rats had thicker basement membranes (p<0.0001) and more cells per unit capillary length (p=0.0003) compared to Zuckers. Acarbose treatment significantly reduced basement membrane thickening in the treated ZDF rats (p=0.001), but the effects on cell density showed only a favorable trend. Acarbose treatment has an ameliorative effect on the development of microvascular retinopathy in the ZDF rat, probably due to lessening of hyperglycemia.

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats.

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.

Expressed sequences from conidial, mycelial, and sexual stages of Neurospora crassa.

In the Neurospora Genome Project at the University of New Mexico, expressed sequence tags (ESTs) corresponding to three stages of the life cycle of the filamentous fungus Neurospora crassa are being analyzed. The results of a pilot project to identify expressed genes and determine their patterns of expression are presented. 1,865 partial complementary DNA (cDNA) sequences for 1,409 clones were determined using single-pass sequencing. Contig analysis allowed the identification of 838 unique ESTs and 156 ESTs present in multiple cDNA clones. For about 34% of the sequences, highly or moderately significant matches to sequences (of known and unknown function) in the NCBI database were detected. Approximately 56% of the ESTs showed no similarity to previously identified genes. Among genes with assigned function, about 43.3% were involved in metabolism, 32.9% in protein synthesis and 8.4% in RNA synthesis. Fewer were involved in defense (6%), cell signalling (3.4%), cell structure (3.4%) and cell division (2.6%).

Effect of reduced training and training cessation on insulin action and muscle GLUT-4.

This study examined the impact of a 50% reduction in training frequency or training cessation on insulin action and muscle GLUT-4 protein concentration. Middle-aged individuals were tested before and after 12 wk of exercise training (4 days/wk, 40-45 min/day). Subjects then either maintained training (n = 9), reduced training frequency by 50% (n = 11), or stopped exercising (n = 10) for the ensuing 2 wk. GLUT-4 protein concentration and insulin action (insulin sensitivity index, as determined by the minimal model) increased (P < or = 0.05) by an average of 1.6- and 1.9-fold, respectively, with the 12 wk of training. Insulin action and GLUT-4 did not increase further with the additional 2 wk of training in the maintained training group. Similarly, insulin sensitivity index and GLUT-4 concentration remained at trained levels when training frequency was reduced by 50% for 2 wk. GLUT-4 concentration and insulin action, however, were not different from sedentary values after 14 days of training cessation. These findings indicate that a 14-day 50% reduction in exercise frequency maintains the improvements in GLUT-4 protein concentration and insulin action gained with endurance training in moderately trained middle-aged adults; in contrast, these adaptations are largely lost with training cessation.

Differential effect of maturation on insulin- vs. contraction-stimulated glucose transport in Zucker rats.

Insulin-stimulated glucose transport has been shown to decline during maturation in lean rats. To determine whether this maturation-induced decrease occurred in the muscle of obese rats and whether the contraction-stimulated pathway for glucose transport was similarly affected, glucose transport rates were measured in insulin- and electrically stimulated skeletal muscle during hindlimb perfusion of 10- and 29-wk-old lean and obese male Zucker rats. Glucose transporter (GLUT-4) protein was also measured. Insulin-stimulated glucose transport rates were significantly decreased (36-56%) in 29-wk compared with 10-wk-old lean rats. There was no maturation-related decrease in GLUT-4. Insulin-mediated glucose transport was unaltered by maturation in skeletal muscle of obese rats. Differential effects of maturation on lean and obese rats caused the effect of obesity on maximally insulin-stimulated glucose transport to be much greater in 10- than 29-wk-old animals. Maturation had no effect on contraction-stimulated glucose transport rates in either lean or obese animals. The combined effect of maximal insulin plus contraction was not altered with maturity in lean animals but was significantly increased in 29- compared with 10-wk-old obese rats. Absence of a change in GLUT-4 content together with uncompromised contraction-stimulated glucose transport suggests that the maturation-induced decrease in insulin-stimulated glucose transport in lean rats is due to a defect in the insulin signaling pathway.

Skeletal muscle GLUT4 protein concentration and aging in humans.

The insulin resistance of aging has been attributed to a postreceptor defect in skeletal muscle. The present study examined whether a reduction in the concentration of the insulin-stimulated glucose transporter (GLUT4) in skeletal muscle was associated with advancing age in men (n = 55) and women (n = 29). Insulin sensitivity (minimal model) was negatively associated (P < 0.001) with age (range, 18-80 years) in men (r = -0.44) and women (r = -0.58). GLUT4 protein concentration in the vastus lateralis was also negatively associated (P < 0.05) with age (men, r = -0.28; women, r = -0.51). There was no relation (P > 0.15) between GLUT4 content in the gastrocnemius and age. GLUT4 concentration in the vastus lateralis was positively associated (P < 0.01) with insulin sensitivity in both sexes (r = 0.42); this relationship persisted in the men after adjusting for overall adiposity, regional adiposity, and cardiorespiratory fitness. These findings suggest that a decrement in GLUT4 protein concentration in skeletal muscle may at least partially contribute to the insulin resistance of aging in humans.

Exercise training increases GLUT-4 protein concentration in previously sedentary middle-aged men.

The purpose of this study was to determine if 14 wk of exercise training would increase insulin-sensitive glucose transporter protein (GLUT-4) concentration in skeletal muscle of previously sedentary middle-aged men (47.2 +/- 1.3 yr; n = 13). Muscle samples (lateral gastrocnemius) and insulin action [insulin sensitivity index (ISI), minimal model] were obtained in the sedentary condition and 48 h after the final training bout. GLUT-4 protein concentration increased (P < 0.001, 2,629 +/- 331 to 4,140 +/- 391 absorbance units/100 micrograms protein) with exercise training by 1.8-fold. ISI increased by twofold (P < 0.05, 2.1 +/- 0.5 to 3.4 +/- 0.7 SI x 10(5) min/pM) with training. The percentage of GLUT-4 rich type IIa muscle fibers increased by approximately 10% (P < 0.01), which may have contributed to the elevation in transporter protein. GLUT-4 concentration and citrate synthase activity (1.7-fold, P < 0.001) also increased by similar increments. These findings indicate that GLUT-4 protein concentration is elevated in middle-aged individuals with exercise training.

Role of transverse tubules in insulin stimulated muscle glucose transport.

Although the strongest evidence for recruitment of glucose transporters in response to insulin comes from studies with adipocytes, studies in muscle seem in general to confirm that glucose transporters are also translocated to the cell membrane in muscle in response to insulin. However, the observation that transverse tubule (T-tubule) membranes contain approximately five times more glucose transporter than sarcolemma raised a question as to where glucose transport occurs in muscle. The T-tubule membrane system is continuous with the surface sarcolemma and is a tubule system in which extracellular fluid is in proximity with the interior of the muscle fiber. The purpose of this Prospects article is to evaluate the possibility that the T-tubule membrane may represent a major site of glucose transport in skeletal muscle. Using immunocytochemical techniques we have located GLUT4 glucose transporters on the T-tubule membrane and in vesicles near T-tubules. Since T-tubules form channels into the interior of the muscle fiber, glucose could diffuse or be moved by some peristaltic-like pumping action into the transverse tubules and then be transported across the membrane deep into the interior of the muscle fiber. This mode of transport directly into the interior of the cell would be advantageous over transport across the sarcolemma and subsequent diffusion around the myofibrils to reach the interior of the muscle. Thus, in addition to the role of the T-tubule in ion fluxes and contraction, this unique membrane system can also provide a pathway for the delivery of substrates into the center of the muscle cell where many glycolytic enzymes and glycogen deposits are located.

Contractile activity restores insulin responsiveness in skeletal muscle of obese Zucker rats.

Both insulin and contraction stimulate glucose transport in skeletal muscle. Insulin-stimulated glucose transport is decreased in obese humans and rats. The aims of this study were (1) to determine if contraction-stimulated glucose transport was also compromised in skeletal muscle of genetically obese insulin-resistant Zucker rats, and (2) to determine whether the additive effects of insulin and contraction previously observed in muscle from lean subjects were evident in muscle from the obese animals. To measure glucose transport, hindlimbs from lean and obese Zucker rats were perfused under basal, insulin-stimulated (0.1 microM), contraction-stimulated (electrical stimulation of the sciatic nerve) and combined insulin-(+)contraction-stimulated conditions. One hindlimb was stimulated to contract while the contralateral leg served as an unstimulated control. 2-Deoxyglucose transport rates were measured in the white gastrocnemius, red gastrocnemius and extensor digitorum longus muscles. As expected, the insulin-stimulated glucose transport rate in each of the three muscles was significantly slower (P < 0.05) in obese rats when compared with lean animals. When expressed as fold stimulation over basal, there was no significant difference in contraction-induced muscle glucose transport rates between lean and obese animals. Insulin-(+)contraction-stimulation was additive in skeletal muscle of lean animals, but synergistic in skeletal muscle of obese animals. Prior contraction increased insulin responsiveness of glucose transport 2-5-fold in the obese rats, but had no effect on insulin responsiveness in the lean controls. This contraction-induced improvement in insulin responsiveness could be of clinical importance to obese subjects as a way to improve insulin-stimulated glucose uptake in resistant skeletal muscle.

Effect of aging and exercise on GLUT-4 glucose transporters in muscle.

This study was conducted to investigate whether changes in muscle glucose transporter GLUT-4 protein might be associated with a previously reported deterioration in glucose tolerance with aging, and, furthermore, to determine whether exercise training could increase GLUT-4 protein levels in older animals. GLUT-4 protein concentration was measured in soleus, gastrocnemius, and extensor digitorum longus muscles of trained (10 or 15 wk treadmill running) and untrained young (6-8 mo), middle-aged (15-17 mo), and old (27-29 mo) Fischer 344 rats. All GLUT-4 protein values were expressed as a percent of the mean for the young untrained group. Two-way analysis of variance demonstrated significant main effects of both training and aging in the gastrocnemius and soleus muscles. Exercise training produced significant increases in GLUT-4 protein in the soleus muscle of young (273 +/- 32.9 vs. 100 +/- 38.5%) and middle-aged rats (215 +/- 19.9 vs. 108 +/- 33.2%) compared with sedentary controls. Similar significant increases were also found in the gastrocnemius muscle of young (169 +/- 20.1 vs. 100 +/- 5.8%) and middle-aged rats (167 +/- 46.7 vs. 60 +/- 7.9%) with training. In the oldest rats, GLUT-4 was not significantly increased with training, but the trend toward an increase was apparent in all three muscle types. The main effect of aging was primarily due to a statistically significant difference between the old trained and young trained rats. A trend toward decreased GLUT-4 with aging was apparent in the untrained animals, but this was not statistically significant.

The longitudinal time course of QTc in early infancy. Preliminary results of a prospective sudden infant death syndrome surveillance program.

Eleven hundred one healthy neonates in Charleston County, SC, were enrolled in a prospective, serial measurement sudden infant death syndrome/QT surveillance program. Automated computer-enhanced ECGs were recorded at 1 day of age in the hospital nursery and again at 1 week and 1, 2, and 3 months in the participant's home. At 1 year, the families were contacted by phone or mail and questioned as to the health of the child. Validation studies demonstrated the computer-enhanced ECGs to be 96% accurate, whereas traditional ECG recording and measurement was 94% accurate. No systematic differences in the QTc according to race and sex were observed. There were parallel longitudinal time courses for each race and sex group with a significant (P less than .001) shortening of the QTc at 1 week. There was no evidence of tracking of the QTc during the first 3 months of life. In conclusion, (1) automated, enhanced ECG QTc intervals are superior to traditional electrocardiography while retaining the advantages of automation; (2) there is a significant shortening of the QTc during the first month of life; and (3) a home follow-up sudden infant death syndrome surveillance program is feasible and produces accurate, reliable information.

Melatonin acts in the brain to mediate seasonal steroid inhibition of luteinizing hormone secretion in the white-footed mouse (Peromyscus leucopus).

A melatonin-induced supersensitivity of the gonadotropin-secretory system to the negative feedback action of sex steroids is thought to be important to the timing of seasonal reproduction. However, little is known concerning this action of melatonin. In the present study the antigonadal action of melatonin in the anterior hypothalamus (AH) of the white-footed mouse, Peromyscus leucopus, was used to examine the neuroendocrine mechanism whereby melatonin enhances the sensitivity to sex steroid negative feedback. Mice received a melatonin-containing pellet in the AH for 14 weeks, at which time they were castrated and treated sc with a Silastic testosterone (T) capsule for 3 weeks. At the time of castration, weight of the testes and the concentration of T in the blood of mice with a melatonin pellet were greatly reduced compared to mice with a blank (melatonin-free) implant in the AH (P less than 0.01). In mice treated with melatonin the physiological dose of T significantly reduced the concentrations of LH in blood and pituitary (P less than 0.05). This dose of T, however, had little effect on LH in mice with a blank pellet in the AH. Melatonin in the AH markedly increased the content of gonadotropin-releasing hormone (GnRH) in the mediobasal hypothalamus (P less than 0.05) in mice treated with T; however, there was little effect of melatonin and/or T in any other region examined. Melatonin and T had little effect on the contents of immunoreactive beta-endorphin (B-EP) in the hypothalamus, but T alone increased the content of B-EP in the preoptic area. These results are evidence that melatonin and T act in concert to induce the reproductively-quiescent state by suppressing secretion of GnRH from the hypothalamus.