Overwhelming genetic heterogeneity and exhausting molecular diagnostic process in chronic and progressive ataxias: facing it up with an algorithm, a gene, a panel at a time.

Ataxias are one of the most frequent complaints in Neurogenetics units worldwide. Currently, more than 50 subtypes of spinocerebellar ataxias and more than 60 recessive ataxias are recognized. We conducted an 11-year prospective, observational, analytical study in order to estimate the frequency of pediatric and adult genetic ataxias in Argentina, to describe the phenotypes of this cohort and evaluate the diagnostic yield of the algorithm used in our unit. We included 334 ataxic patients. Our diagnostic approach was successful in one-third of the cohort. A final molecular diagnosis was reached in 113 subjects. This rate is significantly higher in the subgroup of patients with a positive family history, where the diagnostic yield increased to 55%. The most prevalent dominant and recessive ataxias in Argentina were SCA-2 (36% of dominant ataxias) and FA (62% of recessive ataxias), respectively. Next generation sequencing-based assays were diagnostic in the 65% of the patients requiring these tests. These results provide relevant epidemiological information, bringing a comprehensive knowledge of the most prevalent subtypes of genetic ataxias and their phenotypes in our territory and laying the groundwork for rationally implementing genetic diagnostic programs for these disorders in our country.

Normal table of embryonic development in the four-toed salamander, Hemidactylium scutatum.

We present a complete staging table of normal development for the lungless salamander, Hemidactylium scutatum (Caudata: Plethodontidae). Terrestrial egg clutches from naturally ovipositing females were collected and maintained at 15 °C in the laboratory. Observations, photographs, and time-lapse movies of embryos were taken throughout the 45-day embryonic period. The complete normal table of development for H. scutatum is divided into 28 stages and extends previous analyses of H. scutatum embryonic development (Bishop, 1920; Humphrey, 1928). Early embryonic stage classifications through neurulation reflect criteria described for Xenopus laevis, Ambystoma maculatum and other salamanders. Later embryonic stage assignments are based on unique features of H. scutatum embryos. Additionally, we provide morphological analysis of gastrulation and neurulation, as well as details on external aspects of eye, gill, limb, pigmentation, and tail development to support future research related to phylogeny, comparative embryology, and molecular mechanisms of development.

Modification of insulin resistance by diazoxide in obese Zucker rats.

Hyperinsulinism, insulin resistance, and decreased number of insulin receptors are characteristic of obesity in both humans and experimental animals. To assess the role of insulin in developing obesity, diazoxide (DZ), an inhibitor of glucose-stimulated insulin secretion, was administered for 8 weeks to 7-week-old female Zucker rats in two concentrations, 50 mg/kg.day (LD-DZ), and 100 mg/kg.day (HD-DZ). The obese and lean rats were divided into three subgroups: diazoxide (DZ), pair-fed (PF), and control (C) groups (n = 6 rats/subgroup-genotype). Diazoxide-treated obese and lean animals showed significantly lower postabsorptive plasma insulin concentrations (P < 0.005) than their respective obese and lean PF and C subgroups. HD-DZ obese rats consumed more calories (P < 0.001), yet gained less weight (P < 0.05) than PF and C rats. The plasma glucose concentrations in the postabsorptive state and during glucose tolerance tests in HD-DZ obese rats were significantly lower than those in PF and C rats (P < 0.01) despite a decrease in their plasma insulin concentrations (P < 0.01), whereas HD-DZ lean rats displayed a diabetic response (P < 0.01). The adipocyte-specific insulin receptor binding was dose-dependently increased in both lean and obese DZ animals (P < 0.01). DZ had a dual effect on insulin metabolism; it decreased insulin secretion and increased insulin receptor binding. This dual effect was associated with improved glucose tolerance and a decrease in weight gain in obese rats.

Taurine binding to the purified insulin receptor.

Taurine (2-aminoethanesulfonic acid) was shown to bind specifically and reversibly to the purified human insulin receptor. While insulin binding to the purified insulin receptor exhibited characteristic negative cooperativity and an apparent dissociation constant (Kd) of 1.2 X 10(-9) M, taurine binding was shown to exhibit positive cooperativity and had a lower affinity for the insulin receptor. The apparent Kd for taurine binding to the purified insulin receptor was calculated to be 130 X 10(-9) M and the maximum number of binding sites (Bmax) was 1.6 nmol/mg receptor protein. Chromatographic data demonstrated that taurine binds to the 138,000 molecular weight subunit of the insulin receptor. Taurine binding to the receptor protein was displaced by either taurine or insulin. Anti-human insulin receptor sera prevented insulin or taurine from binding to the receptor. Taurine binding to the protein was pH dependent, and sulfur-containing taurine analogues were able to displace taurine from the insulin receptor. These data supported our previous in vivo and in vitro observations that the hypoglycemic properties of taurine appear to be mediated through an interaction of taurine with the insulin receptor.

Distinct hydrodynamic forms of the insulin receptor: electrophoretic analysis of the RI and RII species.

In previous work, we identified two insulin receptor species, RI (KAV = 0.31) and RII (KAV = 0.53), that could be separated by gel filtration on Sepharose 6B. In the present study, we sought to establish that these two receptor species do represent larger (RI) and smaller (RII) oligomeric forms of the receptor, rather than representing receptor species separated from each other by differential adsorption to the Sepharose matrix. Receptor solubilized from isolated human placenta membranes was purified by lectin- and insulin-agarose chromatography and was radiolabeled with carrier-free 125I. The labeled receptor was separated by Sepharose 6B gel filtration into two fractions (peak I, KAV = 0.31; peak II, KAV = 0.53), was immunoprecipitated by anti-insulin receptor antibody, and was analysed by electrophoresis in nonreducing polyacrylamide slab gels. The autoradiograms of the gels indicated that peak I (KAV = 0.31, RI receptor form) contained a number of receptor species of 240 000 daltons or greater, whereas peak II (KAV = 0.53, RII receptor form) contained mainly receptor species of 210 000 daltons or smaller. In particular, large amounts of a 90 000 dalton species (presumably free receptor beta-subunit) were present in peak II. Incubation of the material obtained from peak I with insulin resulted in a change in the electrophoretic pattern, which became identical with that observed for material recovered from peak II.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptors for insulin and epidermal growth factor: interaction with organomercurial agarose.

The receptor for both insulin and epidermal growth factor (EGF) from human placental membranes, after crosslink labeling with 125I-labeled insulin and EGF, can be absorbed to an organomercurial-agarose derivative (Affi-Gel 501) and can be recovered from the gel by elution with dithiothreitol (DTT). Pretreatment of crosslink-labeled membranes with N-ethylmaleimide (NEM) blocks the ability of the receptor to react with the organomercurial column. NEM also abolishes the protein kinase activity of both receptors. Under appropriate conditions, insulin can promote the reaction of the insulin receptor with the organomercurial-agarose derivative. For both the insulin and EGF receptors, our results provide an avenue for the isolation of the sulfhydryl-containing receptor domains that may play a role in the control of receptor function.

Hypoglycemic properties of taurine: not mediated by enhanced insulin release.

Taurine (2-aminoethanesulfonic acid) has been shown to be a potent hypoglycemic agent in the Wistar Kyoto rat (WKY). Glucose and insulin levels were measured in serum at various times after glucose loading (400 mg/kg, i.p.). Pretreatment with taurine (200 mg/kg, i.p.) attenuated the rise in serum glucose levels at 0.5 hr after glucose administration. In addition, taurine also prevented the rise in serum immunoreactive insulin levels. The taurine analogue hypotaurine produced a similar inhibition in the rise of both serum glucose and insulin levels while beta-alanine, the carboxylic acid derivative of taurine, was totally ineffective. The enhanced glucose clearance can be explained by an increase in deoxyglucose accumulation in skeletal muscle and liver. In the liver, a 50% increase in glycogen synthesis was observed. A possible interrelationship between taurine and insulin receptor is discussed.

Endogenous isopropanol: forensic and biochemical implications.

Concomitant findings of isopropanol and acetone in biospecimens of decedents known not to have been exposed to the alcohol prompted a study to explain its origins. Mixtures of acetone, ADH, and NADH at pHs 7.3 and 8.8 were incubated at 37 degrees C for varying intervals. Reaction products were then analyzed by headspace GC and assured identification made by GC/MS. It was found that isopropanol is produced by reduction of acetone at pH 7.3 (to a lesser extent at pH 8.8), providing evidence for an alternate metabolic route for acetone. A mechanism for this reduction is proposed. Ranges for isopropanol (in mg/dL or mg/100 g) found in unexposed decedents were: blood 1-29; liver 7-59; brain 2-12; kidney 6-26. Thus, the forensic investigator must interpret isopropanol results cautiously, particularly when low concentrations are found.

Receptors for insulin and basic somatomedin: immunological and affinity-chromatographic cross-reactivity.

Human placental receptors for insulin and for human basic somatomedin (BSM, analogous to human insulinlike growth factor I (IGF-I] were selectively cross-link labelled with 125I-labelled insulin and BSM using disuccinimidyl suberate under conditions whereby cross-linking of 125I-labelled insulin to the BSM receptor and of 125I-labelled BSM to the insulin receptor was avoided. We have found that the cross-link-labelled receptors for insulin and BSM, present in equivalent amounts in human placenta membrane preparations, both cross-react with antibodies directed against purified rat liver insulin receptor. Compared with the human insulin receptor, the BSM receptor cross-reacted with the antireceptor antibody to a level of roughly 9%, indicating a limited sequence homology between the insulin and BSM receptors. Since both receptors are present in comparable amounts in solubilized placenta membrane preparations, we sought methods for the selective purification of both receptors from such extracts, so as to provide a basis for further comparative structural studies of the two receptors. We have observed that both receptors were adsorbed by affinity columns of insulin-agarose, in a manner that did not yield insulin receptor entirely free from the BSM receptor. As an alternative for receptor purification, we have found that immunoaffinity columns using antiligand antibody should provide a means for the selective isolation of cross-link labelled receptor from tissues in which both are present in equivalent amounts.

Insulin receptor: interaction with nonreceptor glycoprotein from liver cell membranes.

In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 A and 38 A. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 A) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 A to 72 A. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin.