Evaluation of visual motion perception ability in mice with knockout of the dyslexia candidate susceptibility gene Dcdc2.

Developmental dyslexia is a heritable disability characterized by difficulties in learning to read and write. The neurobiological and genetic mechanisms underlying dyslexia remain poorly understood; however, several dyslexia candidate risk genes have been identified. One of these candidate risk genes-doublecortin domain containing 2 (DCDC2)-has been shown to play a role in neuronal migration and cilia function. At a behavioral level, variants of DCDC2 have been associated with impairments in phonological processing, working memory and reading speed. Additionally, a specific mutation in DCDC2 has been strongly linked to deficits in motion perception-a skill subserving reading abilities. To further explore the relationship between DCDC2 and dyslexia, a genetic knockout (KO) of the rodent homolog of DCDC2 (Dcdc2) was created. Initial studies showed that Dcdc2 KOs display deficits in auditory processing and working memory. The current study was designed to evaluate the association between DCDC2 and motion perception, as these skills have not yet been assessed in the Dcdc2 KO mouse model. We developed a novel motion perception task, utilizing touchscreen technology and operant conditioning. Dcdc2 KOs displayed deficits on the Pairwise Discrimination task specifically as motion was added to visual stimuli. Following behavioral assessment, brains were histologically prepared for neuroanatomical analysis of the lateral geniculate nucleus (LGN). The cumulative distribution showed that Dcdc2 KOs exhibited more small neurons and fewer larger neurons in the LGN. Results compliment findings that DCDC2 genetic alteration results in anomalies in visual motion pathways in a subpopulation of dyslexic patients.

High-performance liquid chromatographic assay for two rifamycin-derived hypocholesterolemic agents in liver and biological fluids.

CGP 43371 (compound I), a mono-pivaloyl oxazole derivative of a 3-piperazino-rifamycin, has been in clinical trials as a potential hypocholesterolemic agent. A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed using a C18 column and a gradient solvent system of methanol-0.1 M sodium acetate, pH 4.5, at a flow-rate of 1 ml/min. The compound and internal standard (rifampicin) were detected by their ultraviolet absorption at 254 nm. Isolation of the compounds from plasma and liver homogenates was accomplished by precipitation of proteins with acetonitrile, followed by evaporation under nitrogen and reconstitution in methanol. Bile, lymph and urine were injected onto the HPLC column without pretreatment. Calibration curves were linear (r > 0.999) over the concentration range 0.25-20.0 micrograms/ml. The assay procedure was also applicable to other rifamycin derivatives and was able to distinguish between molecular species containing small differences in functionality.

Prinomide tromethamine pharmacokinetics: mutually dependent saturable and competitive protein binding between prinomide and its own metabolite.

Prinomide tromethamine, a nonsteroidal antiinflammatory drug, was orally administered at doses of 250, 500, and 1000 mg every 12 hr for 28 days to healthy male volunteers. The pharmacokinetic behavior of prinomide and its primary plasma metabolite displayed nonlinear characteristics, while those of free prinomide and its metabolite were dose proportional. The nonlinear pharmacokinetic behavior of total prinomide and p-hydroxy metabolite was found to be caused by both saturable and mutually dependent competitive Langmuir-type plasma protein binding between prinomide and its p-hydroxy metabolite. The extent of the protein interaction displayed at steady state was due to the extensive accumulation of the p-hydroxy metabolite. While ligand-protein interactions are known for xenobiotic competitors, the characteristic behavior of prinomide is the first known example to be reported for a competitive protein interaction between a xenobiotic and its own in vivo generated metabolite. The findings of this study may have implications regarding the disposition of other extensively bound nonsteroidal antiinflammatory drugs with long-lived metabolites.

Acetylcholine formation from glucose following acute choline supplementation.

The effects of choline administration on acetylcholine metabolism in the central nervous system are controversial. Although choline supplementation may elevate acetylcholine (ACh) content in brain, turnover studies with labelled choline precursors suggest that systemic choline administration either has no effect or actually diminishes brain ACh synthesis. Since choline supplementation elevates brain choline levels, the apparent decreases in previous turnover studies may reflect dilution of the labelled choline precursor pool rather than altered ACh formation. Therefore, brain ACh formation from [U-14C]glucose was determined after choline supplementation. A two to three fold elevation of brain choline did not alter ACh levels or [U-14C]glucose incorporation into ACh in the cortex, hippocampus or striatum. Although atropine stimulated ACh formation from [U-14C]glucose in hippocampus, two to three fold increases in brain choline did not augment ACh synthesis or content in atropine pretreated animals. Atropine depressed brain regional glucose utilization and this effect was not reversed by choline treatment. These results suggest that short-term elevation of brain choline does not enhance ACh formation from [U-14C]glucose, and argue against enhanced presynaptic cholinergic function after acute, systemic choline administration.

Biochemical epidemiology of colon cancer: effect of types of dietary fiber on fecal mutagens, acid, and neutral sterols in healthy subjects.

Several epidemiological studies suggest an inverse relationship between fiber intake and colon cancer risk. Animal model studies indicate that this inhibitory effect depends on the source of dietary fiber. Because of the potential significance of certain colonic mutagens and secondary bile acids in the pathogenesis of colon cancer, the effect of types of supplemental fiber on fecal mutagens and bile acids was studied in human volunteers. Seventy-two healthy individuals consuming high-fat/moderately low-fiber diets were screened for fecal mutagenic activity using the Ames Salmonella typhimurium/microsomal assay system. Twenty-one of them were found to excrete high levels of mutagens, and 19 of them were recruited for the diet intervention study. All participants provided two 24-h stool specimens and a 4-day food record while consuming their normal (control) diet. They were then asked to consume the control diet plus 10 g of dietary fiber from wheat bran, oat fiber, or cellulose for 5 wk. After each fiber period, they were asked to consume their control diet. At the end of each fiber and control diet period, each subject provided two 24-h stool specimens. Stool samples were analyzed for bile acids and mutagens using the Ames strains TA98 and TA100 with or without S9 (microsomal) activation. The concentrations of fecal secondary bile acids (deoxycholic acid, lithocholic acid, and 12-ketolithocholic acid) and of fecal mutagenic activity in TA98 and TA100 with and without S9 activation were significantly lower during the wheat bran and cellulose supplementation periods. Oat fiber supplementation had no such effect on these fecal constituents. Thus, the increased fiber intake in the form of wheat bran or cellulose may reduce the production and/or excretion of mutagens in the stools and decrease the concentration of fecal secondary bile acids in humans.

Reduced activities of thiamine-dependent enzymes in the brains and peripheral tissues of patients with Alzheimer's disease.

A report of cell loss in the nucleus basalis of Meynert in patients with Wernicke-Korsakoff disease prompted the examination of thiamine pyrophosphate (TPP)-dependent enzymes in the brain and peripheral tissues of patients with Alzheimer's disease. In these brains, the activities of the 2-ketoglutarate dehydrogenase complex were reduced more than 75% and those of transketolase more than 45%. Decreases occurred in histologically damaged and in relatively undamaged areas. Small but statistically significant abnormalities of transketolase, but not of 2-ketoglutarate dehydrogenase complex, were identified in red blood cells and cultured fibroblasts. Previous studies have shown deficiencies in the brain and variable effects in peripheral tissues on another TPP-dependent enzyme--the pyruvate dehydrogenase complex. Activities of TPP-dependent enzymes appear to be deficient in the brain and perhaps in some peripheral tissues in patients with Alzheimer's disease.

In vivo brain calcium homeostasis during aging.

Since in vitro experiments suggest that brain calcium metabolism is altered with aging, the estimated rate of calcium uptake by the brain in vivo was determined with senescence. Calcium-45 incorporation into cortex, striatum, hippocampus, cerebellum, forebrain, midbrain and brainstem was determined in 3-, 10- and 30-month-old mice at 5 h after either an intravenous or intraperitoneal injection. Calcium uptake (brain dpm/mg protein divided by blood specific activity at 5 h) into these regions declined 19-33% at 10 months and 41-51% at 30 months. Subcellular fractionation of the cortex revealed that the decrease was similar in P1 (myelin, nuclei and tissue debris), P2 (synaptosomes, mitochondria and myelin) and S2 (microsomes, ribosomes and cytosol). Brain calcium concentrations declined with age in brain stem (-62%) and midbrain (-48%) but did not significantly vary with age in the other regions. These results support the suggestion that alterations in calcium homeostasis may underlie age-related changes in neurotransmitter metabolism.

Functional abnormalities of islets of Langerhans of obese hyperglycemic mouse.

Glucose-induced insulin release was studied in vitro with isolated islets of Langerhans obtained from obese hyperglycemic C57Bl/6J-ob/ob (ob/ob) and lean C57Bl/6J-+/+ (control) mice. The threshold concentrations of glucose for insulin release were determined. In addition, the effect of total fast and of chronic food restriction on in vitro insulin release were studied. The following was observed: 1) with fasting, islet volume decreased. Islets obtained from ob/ob mice were larger than control islets, except for the chronic food restricted group. 2) Ob/ob islets were more sensitive to glucose than were controls in that the threshold for glucose-induced insulin release occured at lower glucose concentrations. 3) Fasting for 48 h completely abolished glucose-induced insulin release in control islets, whereas glucose-induced insulin release was maintained in 48-h and 7-day fasted ob/ob islets. 4) The increased glucose sensitivity of the ob/ob islets was maintained despite chronic food restriction.

Reversal of somatostatin inhibition of insulin and glucagon secretion.

These studies were designed to elucidate the mechanism of inhibitory action of somatostatin (SRIF) on glucagon (IRG) and insulin (IRI) secretion. Studies were carried out in the unrecirculated isolated rat pancreas perfusion with arginine 19.2 mM and glucose 5.5 mM as stimulus primarily for IRG but also IRI secretion. The effects of excess Ca++ (15.2 mEq./L.) and excess K+ (12.8 mEq./L.) on IRG, IRI, and the SRIF-inhibited pancreas were studied. Ca++ excess in five perfusions strikingly stimulated IRG secretion (+92 per cent) but only stabilized IRI secretion compared with control perfusions. K+ excess (in seven perfusions) markedly inhibited IRG secretion (-39 per cent) while stimulating IRI secretion (+16 per cent). Restoration of normal concentrations of K+ resulted in a rebound of IRG to levels 120 per cent that of controls. SRIF, at concentrations from 0.1-20 ng./ml., produced inhibition of both IRG and IRI. In 11 perfusions, with SRIF at 10 ng./ml., IRG decreased more than IRI (-75.2 per cent IRG and -46.9 per cent IRI). In five perfusions, addition of Ca++ (15.2 mEq./L.) 10 minutes after SRIF was started resulted in a reversal of IRG inhibition to 69.4 per cent and IRI to 73.2 per cent of the arginine controls. The reversal by Ca++ of SRIF effect on IRG was greater at higher concentrations of Ca++, suggesting some form of competition. In four perfusions, excess K+ reversed SRIF-induced IRI inhibition to 79.6 per cent that of controls but had no effect on IRG inhibition. Studies in vitro with isolated islets revealed that SRIF (2 mug./ml.) inhibited 45Ca uptake of islets as did epinephrine (10(-5) M). It was concluded that SRIF-induced inhibition of hormone release appears related to an action on Ca++ uptake.

Plasma immunoreactive glucagon fractions in four cases of glucagonoma: increased "large glucagon-immunoreactivity".

Immunoreactive glucagon (IRG) fractions from plasma of 8 normal subjects and 4 patients with glucagon secreting tumors were studied by gel filtration techniques on Bio Gel P--30 and Sephadex G--50 columns. The pancreatic glucagon specific anti serum (30K) of Unger was utilized to measure IRG. Columns were calibrated with labelled albumin, proinsulin, insulin and glucagon. Four peaks were defined in normal and tumor bearing patients: peak I (greater than 20 000 mol. wt.), peak II (primarily 9000 mol. wt.), peak III pancreatic glucagon (3500 mol. wt.) and peak IV small gucagon (less than 3500 mol. wt.). Glucagonoma patients differed from our normal and reported normal subjects in that peak II contained most of the circulating IRG. The percent of IRG associated with peak II was 9.5--31.5% in normals and 39.1--61.2% in glucagonomas. Glucagon-like biological activity in an isolated hepatocyte system was demonstrated for all peaks. However, relative to immunoreactivity, peak II showed reduced activity (25--33%). Immunoassay of dilutions of all peaks revealed the probability of immuno determinants identical with procine pancreatic glucagon. The presence of heterogenous IRG peaks with biological glucagon-like activity suggest that the larger molecules may be prohormones. Further, it is possible that specific elevation of peak II may be a diagnostic feature of glucagonomas.

Elevated plasma proglucagon-like component with a glucagon-secreting tumor. Effect of streptozotocin.

To determine the character of the glucagon secretion and its modification by streptozotocin, were studied the plasma of a patient with recurrent pancreatic alpha-cell carcinoma. The plasma immunoreactive glucagon level before treatment of 4.80 ng per milliliter. Biogel column separation of the plasma immunoreactive glucagon revealed four components; the predominant component had a molecular weight of 9000 daltons and was designated as proglucagon-like. This fraction constituted 60 to 90 per cent of the total circulating immunoreactive glucagon, and had a biologic activity of 32 percent of that of an immunoequivalent amount of normal (porcine) pancreatic glucagon. After treatment with streptozocin (1.5 g per square meter) the plasma immunoreactive glucagon level decreased to 0.24 ng per milliliter. Treatment was accompanied by a marked reduction in the proglucagon-like component and the appearance of pancreatic glucagon (molecular weight of 3500 daltons) as the major post-therapy fraction. These findings support the use of streptozotocin in the management of unresectable glucagon-secreting tumors.

The effect of fasting on tissue cyclic cAMP and plasma glucagon in the obese hyperglycemic mouse.

The effect of 48 h of fasting in C57B1/6J-ob/ob and +/+ mice on body weight (BW), blood glucose (BG), serum immunreactive insulin (IRI), plasma immunoreactive glucagon (IRG) and on tissue levels of cyclic adenosine monophosphate (cAMP) were studied. Both groups of mice lost weight and demonstrated a decrease in BG and IRI with fasting. However, the BG and IRI of the ob/ob animals were initially highter and remained higher than those of the 2% of their initial weight while the +/+ lost 14 %. The +/+ mice exhibited an increase in cAMP levels in skeletal muscle, fat and liver with fasting, while the ob/ob mice had increased levels of cAMP in fat, but not in muscle. They also had a paradoxical decrease in liver cAMP levels with fasting, and associated with this was the lack of stimulation of glycogenolysis. Glycogenolysis was significant in the livers of fasted +/+ mice. The plasma IRG levels of the fed ob/ob mice were significantly higher (1.8) times) than those of the fed +/+ mice. Islet cAMP levels were decreased with fasting in ob/ob mice. However, the levels were significantly higher in 48-h faster ob/ob mice compared to the fasted +/+ group. The apparent paradoxical response to fasting observed in the livers of the ob/ob mice remains unexplained.

Hormone receptors, 5. Binding of glucagon and insulin to human circulating mononuclear cells in diabetes mellitus.

Preparations of human peripheral mononuclear leukocytes, containing over 85% lymphocytes, specifically bound glucagon and insulin. Binding of physiological concentrations of both hormones was seen to be substantially diminished when cells were derived from either adult-type diabetics or individuals with a unilateral genetic history of diabetes.