A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic

Carbohydrate-deficient glycoprotein syndrome (CDGS) represents a class of genetic diseases characterized by abnormal N-linked glycosylation. CDGS patients show a large number of glycoprotein abnormalities resulting in dysmorphy, encephalopathy, and other organ disorders. The majority of CDGSs described to date are related to an impaired biosynthesis of dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum. Recently, we identified in four related patients a novel type of CDGS characterized by an accumulation of dolichyl pyrophosphate-linked Man9GlcNAc2. Elaborating on the analogy of this finding with the phenotype of alg5 and alg6 Saccharomyces cerevisiae strains, we have cloned and analyzed the human orthologs to the ALG5 dolichyl phosphate glucosyltransferase and ALG6 dolichyl pyrophosphate Man9GlcNAc2 alpha1,3-glucosyltransferase in four novel CDGS patients. Although ALG5 was not altered in the patients, a C-->T transition was detected in ALG6 cDNA of all four CDGS patients. The mutation cosegregated with the disease in a Mendelian recessive manner. Expression of the human ALG5 and ALG6 cDNA could partially complement the respective S. cerevisiae alg5 and alg6 deficiency. By contrast, the mutant ALG6 cDNA of CDGS patients failed to revert the hypoglycosylation observed in alg6 yeasts, thereby proving a functional relationship between the alanine to valine substitution introduced by the C-->T transition and the CDGS phenotype. The mutation in the ALG6 alpha1,3-glucosyltransferase gene defines an additional type of CDGS, which we propose to refer to as CDGS type-Ic.

Syntrophin mutation associated with long QT syndrome through activation of the nNOS-SCN5A macromolecular complex.

Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for approximately 75-80% of cases (LQT1-11). Direct sequencing of SNTA1, the gene encoding alpha1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTS-susceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane Ca-ATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused S-nitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.

Hepatic and renal cytochrome p450 gene regulation during citrobacter rodentium infection in wild-type and toll-like receptor 4 mutant mice.

Citrobacter rodentium is the rodent equivalent of human enteropathogenic Escherichia coli infection. This study investigated regulation of hepatic and renal cytochrome P450 (P450) mRNAs, hepatic P450 proteins, cytokines, and acute phase proteins during C. rodentium infection. Female C3H/HeOuJ (HeOu) and C3H/HeJ (HeJ) mice [which lack functional toll-like receptor 4 (TLR4)] were infected with C. rodentium by oral gavage and sacrificed 6 days later. Hepatic CYP4A10 and 4A14 mRNAs were decreased in HeOu mice (<4% of control). CYP3A11, 2C29, 4F14, and 4F15 mRNAs were reduced to 16 to 55% of control levels, whereas CYP2A5, 4F16, and 4F18 mRNAs were induced (180, 190, and 600% of control, respectively). The pattern of P450 regulation in HeJ mice was similar to that in HeOu mice for most P450s, with the exception of the TLR4 dependence of CYP4F15. Hepatic CYP2C, 3A, and 4A proteins in both groups were decreased, whereas CYP2E protein was not. Renal CYP4A10 and 4A14 mRNAs were significantly down-regulated in HeOu mice, whereas other P450s were unaffected. Most renal P450 mRNAs in infected HeJ mice were increased, notably CYP4A10, 4A14, 4F18, 2A5, and 3A13. Hepatic levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNAs were significantly increased in infected HeOu mice, whereas only TNFalpha mRNA was significantly increased in HeJ mice. Hepatic alpha1-acid glycoprotein was induced in both groups, whereas alpha-fibrinogen and angiotensinogen were unchanged. These data indicate that hepatic inflammation induced by C. rodentium infection is mainly TLR4-independent and suggest that hepatic P450 down-regulation in this model may be cytokine-mediated.

A pharmacological investigation of noradrenergic receptor mechanisms in neophobia

The dorsal noradrenergic bundle (DB) is a major ascending pathway which originates in the locus coeruleus of the brainstem and projects to the forebrain. The behavioral role of the DB remains unclear, despite a great deal of effort. Selective attention and anxiety are two areas which have been the focus of recent research. Some studies of the DB utilize the neurotoxin 6-hydroxydopamine (6-OHDA), since 6-OHDA injection into this pathway results in greater than 90 percent depletion of cortical and hippocampal norepinephrine (NE). Neophobia, the fear of novelty, has been reported to be either increased or decreased by 6-OHDA lesions of the DB, depending on conditions. The selective attention hypothesis would be supported by increased neophobia after 6-OHDA lesions, while the anxiety hypothesis would be supported by decreased neophobia. We have examined the effects of 6-OHDA DB lesions on neophobia under conditions in which the test environment and/or the test food were novel. We found that the lesion attenuates neophobia, defined as an increased preference for novel food, when both the environment and food were novel. The lesion had no effect on neophobia when only the environment or food was novel.^ We examined the effects of chronic intraventricular NE infusions on behavior in our neophobia test, in sham and 6-OHDA DB lesioned animals. We found that chronic NE infusions into lesioned animals significantly reversed the lesion-induced attenuation of neophobia. Sham/NE infused animals demonstrated a 40 percent greater preference for familiar food compared to sham/saline infused animals. These data suggest that infusions of NE have an effect opposite to lesion-induced attenuation of neophobia. Chronic infusions of the alpha adrenoceptor agonists had no consistent effects on neophobia. The beta adrenoceptor agonist, isoproterenol reversed the lesion-induced attenuation of neophobia but not to a statistically significant degree. Isoproterenol increased neophobia in sham animals. Forskolin, an adenylate cyclase activator, mimicked the effects of NE infusion by significantly reversing the lesion-induced attenuation of neophobia, while increasing neophobia in sham animals. These results suggest that increased release of NE during stress increases neophobia in part by stimulating beta adrenoceptors which activate adenylate cyclase. ^

Molecular mechanisms of chondrocyte-specific expression on the mouse pro$\alpha$1(II) collagen gene

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro$\alpha1$(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a $\beta$-galactosidase reporter gene. A construction containing a 3000-bp promoter and a 3020-bp intron 1 fragment directed high levels of $\beta$-galactosidase expression specifically to chondrocytes. Successive deletions of intron 1 delineated a 48-bp fragment which targeted $\beta$-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. When the Col2a1 promoter was replaced with a minimal $\beta$-globin promoter, the 48-bp intron 1 sequence was still able to target expression of the transgene to chondrocytes, specifically. Therefore a 48-bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression to a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression. Nuclear proteins present selectively in mouse primary chondrocytes and rat chondrosarcoma cells bind to the three putative HMG (High-Mobility-Group) domain protein binding sites in this 48-bp sequence and the chondrocyte-specific proteins likely bind the DNA through minor groove. Together, my results indicate that a 48-bp sequence in Col2a1 intron 1 controls chondrocyte-specific expression in vivo and suggest that chondrocytes contain specific nuclear proteins involved in enhancer activity. ^

Use of Genetically Modified Glial Cells Overexpressing Laminin α1-Chain Peptides in Neurite Outgrowth Studies

1. C6 glioma cells were transfected with two constructs carrying C-terminal laminin alpha1-chain sequences of 117 and 114 bp length, respectively. These sequences are specifically known to code for peptides which have neurite-promoting activity. 2. The stable expression and secretion of the two peptides was detected by Northern and Western blot analysis. 3. Primary neuronal cultures derived from embryonic mouse forebrain were cocultured with these transfected cells and exhibited a substantial increase in neurite outgrowth and in survival time. Conditioned media from the transfected cells generated similar effects. 4. Organotypic cultures from embryonic mouse brain were used as a second system as being closer to the in vivo situation. Again, coculture of brain slices with transfected cells or treatment with laminin peptide-containing media increased neuronal outgrowth.

Can non-selective beta-blockers prevent hepatocellular carcinoma in patients with cirrhosis?

Hepatocellular carcinoma is the main liver-related cause of death in patients with compensated cirrhosis. The early phases are asymptomatic and the prognosis is poor, which makes prevention essential. We propose that non-selective beta-blockers decrease the incidence and growth of hepatocellular carcinoma via a reduction of the inflammatory load from the gut to the liver and inhibition of angiogenesis. Due to their effect on the portal pressure, non-selective beta-blockers are used for prevention of esophageal variceal bleeding. Recently, non-hemodynamic effects of beta-blockers have received increasing attention. Blockage of β-adrenoceptors in the intestinal mucosa and gut lymphatic tissue together with changes in type and virulence of the intestinal microbiota lead to reduced bacterial translocation and a subsequent decrease in the portal load of pathogen-associated molecular patterns. This may reduce hepatic inflammation. Blockage of β-adrenoceptors also decrease angiogenesis by inhibition of vascular endothelial growth factors. Because gut-derived inflammation and neo-angiogenesis are important in hepatic carcinogenesis, non-selective beta-blockers can potentially reduce the development and growth of hepatocellular carcinoma. Rodent and in vitro studies support the hypothesis, but clinical verification is needed. Different study designs may be considered. The feasibility of a randomized controlled trial is limited due to the necessary large number of patients and long follow-up. Observational studies carry a high risk of bias. The meta-analytic approach may be used if the incidence and mortality of hepatocellular carcinoma can be extracted from trials on variceal bleeding and if the combined sample size and follow up is sufficient.

Genomic cloning and expression of three murine UDP-galactose: beta-N-acetylglucosamine beta1,3-galactosyltransferase genes.

Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:beta-N-acetylglucosamine beta1, 3-galactosyltransferase (beta3GalT) genes from a mouse genomic library. The three genes, named beta3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three beta3GalT genes was contained in a single exon, which contrasts with the genomic organization of the beta1,4- and alpha1, 3-galactosyltransferase genes. The three beta3GalT genes were mainly expressed in brain tissue. The expression of the full-length murine genes as recombinant baculoviruses in insect cells revealed that the beta3GalT enzymes share the same acceptor specificity for beta-linked GlcNAc, although they differ in their Km for this acceptor and the donor UDP-Gal. The identification of beta3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.

Individual differences in inhibitory control - relationship between baseline activation in lateral PFC and an electrophysiological index of response inhibition

The capacity to inhibit inappropriate responses is crucial for goal-directed behavior. Inhibiting such responses seems to come more easily to some of us than others, however. From where do these individual differences originate? Here, we measured 263 participants' neural baseline activation using resting electroencephalogram. Then, we used this stable neural marker to predict a reliable electrophysiological index of response inhibition capacity in the cued Continuous Performance Test, the NoGo-Anteriorization (NGA). Using a source-localization technique, we found that resting delta, theta, and alpha1 activity in the left middle frontal gyrus and resting alpha1 activity in the right inferior frontal gyrus were negatively correlated with the NGA. As a larger NGA is thought to represent better response inhibition capacity, our findings demonstrate that lower levels of resting slow-wave oscillations in the lateral prefrontal cortex, bilaterally, are associated with a better response inhibition capacity.

Rivastigmine effects on EEG spectra and three-dimensional LORETA functional imaging in Alzheimer's disease

OBJECTIVE The objective of the study is to investigate the electrocortical and the global cognitive effects of 3 months rivastigmine medication in a group of mild to moderate Alzheimer's disease patients. MATERIALS AND METHODS Multichannel EEG and cognitive performances measured with the Mini Mental State Examination in a group of 16 patients with mild to moderate Alzheimer's Disease were collected before and 3 months after the onset of rivastigmine medication. RESULTS Spectral analysis of the EEG data showed a significant power decrease in the delta and theta frequency bands during rivastigmine medication, i.e., a shift of the power spectrum towards 'normalization'. Three-dimensional low resolution electromagnetic tomography (LORETA) functional imaging localized rivastigmine effects in a network that includes left fronto-parietal regions, posterior cingulate cortex, bilateral parahippocampal regions, and the hippocampus. Moreover, a correlation analysis between differences in the cognitive performances during the two recordings and LORETA-computed intracortical activity showed, in the alpha1 frequency band, better cognitive performance with increased cortical activity in the left insula. CONCLUSION The results point to a 'normalization' of the EEG power spectrum due to medication, and the intracortical localization of these effects showed an increase of cortical activity in frontal, parietal, and temporal regions that are well-known to be affected in Alzheimer's disease. The topographic convergence of the present results with the memory network proposed by Vincent et al. (J. Neurophysiol. 96:3517-3531, 2006) leads to the speculation that in our group of patients, rivastigmine specifically activates brain regions that are involved in memory functions, notably a key symptom in this degenerative disease.

Correlation between disease severity and brain electric LORETA tomography in Alzheimer's disease

OBJECTIVE To compare EEG power spectra and LORETA-computed intracortical activity between Alzheimer's disease (AD) patients and healthy controls, and to correlate the results with cognitive performance in the AD group. METHODS Nineteen channel resting EEG was recorded in 21 mild to moderate AD patients and in 23 controls. Power spectra and intracortical LORETA tomography were computed in seven frequency bands and compared between groups. In the AD patients, the EEG results were correlated with cognitive performance (Mini Mental State Examination, MMSE). RESULTS AD patients showed increased power in EEG delta and theta frequency bands, and decreased power in alpha2, beta1, beta2 and beta3. LORETA specified that increases and decreases of power affected different cortical areas while largely sparing prefrontal cortex. Delta power correlated negatively and alpha1 power positively with the AD patients' MMSE scores; LORETA tomography localized these correlations in left temporo-parietal cortex. CONCLUSIONS The non-invasive EEG method of LORETA localized pathological cortical activity in our mild to moderate AD patients in agreement with the literature, and yielded striking correlations between EEG delta and alpha1 activity and MMSE scores in left temporo-parietal cortex. SIGNIFICANCE The present data support the hypothesis of an asymmetrical progression of the Alzheimer's disease.

Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men

BACKGROUND: Mental stress reliably induces increases in salivary alpha amylase (sAA), a suggested surrogate marker for sympathetic nervous system (SNS) reactivity. While stress-induced sAA increases correlate with norepinephrine (NE) secretion, a potential mediating role of noradrenergic mechanisms remains unclear. In this study, we investigated for the first time in humans whether a NE-stress-reactivity mimicking NE-infusion with and without alpha-adrenergic blockade by phentolamine would induce changes in sAA. METHODS: In a single-blind placebo-controlled within-subjects design, 21 healthy men (29-66 years) took part in three different experimental trials varying in terms of substance infusion with a 1-min first infusion followed by a 15-min second infusion: saline-infusion (trial-1), NE-infusion (5 μg/min) without alpha-adrenergic blockade (trial-2), and with phentolamine-induced non-selective blockade of alpha1- and alpha2-adrenergic receptors (trial-3). Saliva samples were collected immediately before, during, and several times after substance infusion in addition to blood pressure and heart rate readings. RESULTS: Experimental trials significantly differed in sAA reactivity to substance-infusion (p=.001) with higher sAA reactivity following NE-infusion with (trial-3; p=.001) and without alpha-adrenergic-blockade (trial-2; p=.004) as compared to placebo-infusion (trial-1); sAA infusion reactivity did not differ between trial-2 and trial-3 (p=.29). Effective phentolamine application was verified by blood pressure and heart rate infusion reactivity. Salivary cortisol was not affected by NE, either with or without alpha-adrenergic-blockade. CONCLUSIONS: We found that NE-infusion stimulates sAA secretion, regardless of co-administered non-selective alpha-adrenergic blockade by phentolamine, suggesting that the mechanism underlying stress-induced sAA increases may involve NE.

Inflammatory, immunological, and intestinal disease biomarkers in Chinese Shar-Pei dogs with marked hypocobalaminemia.

Chinese Shar-Pei dogs have a high prevalence of hypocobalaminemia and are commonly presented with clinical signs suggestive of severe and long-standing gastrointestinal disease such as diarrhea, vomiting, and/or weight loss. The aim of the current study was to evaluate serum concentrations of inflammatory markers, markers for intestinal disease, and immunological markers in Shar-Peis with hypocobalaminemia or normocobalaminemia (serum cobalamin concentrations within the reference interval). Serum samples from Shar-Peis were collected from various parts of the United States. Serum concentrations of inflammatory markers (i.e., C-reactive protein [CRP], calprotectin [CP], and S100A12), hyaluronic acid (HA, a marker for cutaneous mucinosis), and analytes commonly altered in chronic intestinal diseases (i.e., albumin, zinc, alpha1-proteinease inhibitor [α1PI], immunoglobulin [Ig]A, and IgM) were compared between Shar-Peis with hypocobalaminemia and Shar-Peis with normocobalaminemia. Serum concentrations of CRP, CP, S100A12, HA, zinc, and cα1-PI concentrations did not differ between hypocobalaminemic and normocobalaminemic Shar-Peis (P > 0.05). Serum concentrations of albumin were significantly lower in hypocobalaminemic Shar-Peis (median: 2.5 g/dl) than in normocobalaminemic Shar-Peis (median: 2.9 g/dl; P < 0.0001). Higher serum IgA concentrations and lower serum IgM concentrations were observed in hypocobalaminemic Shar-Peis (median: 1.7 g/l and 0.8 g/l, respectively) than in normocobalaminemic Shar-Peis (median: 0.7 g/l and 1.9 g/l, respectively; both P < 0.0001). In conclusion, no difference was found in serum concentrations of CRP, CP, S100A12, and HA between hypocobalaminemic and normocobalaminemic Shar-Peis whereas some differences were observed in analytes (e.g., albumin, IgA, and IgM) that may be altered in patients with chronic enteropathies.

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na+/K+-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na+/K+-ATPase with the polyclonal antibody alpha (H-300) raised against the human alpha1-subunit of Na+/K+-ATPase. Na+/K+-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na+/K+-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na+/K+-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na+/K+-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 µmol/g FM/h) than in those of L. vulgaris (31.8 ± 3.3 µmol/g FM/h). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 µmol ATP/g FM/h, respectively. High concentrations of Na+/K+-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO2) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.

Dopamine inhibits mammalian photoreceptor Na+,K+-ATPase activity via a selective effect on the alpha3 isozyme.

The rat retina contains dopaminergic interplexiform cells that send processes to the outer plexiform layer where dopamine is released in a light-dependent manner. We report herein that physiologically relevant concentrations of dopamine inhibited ouabain-sensitive photoreceptor oxygen consumption in dark- and light-adapted rat retinas and inhibited Na+,K+-ATPase specific activity (EC 3.6.1.37) in a rat rod outer-inner segment preparation. Experiments with the selective D1 agonist fenoldopam or D2 agonist quinpirole and experiments with dopamine plus either the D1 antagonist SCH23390 or D2/D4 antagonist clozapine showed that the inhibition of oxygen consumption and enzyme activity were mediated by D2/D4-like receptors. The amphetamine-induced release of dopamine, monitored by the inhibition of oxygen consumption, was blocked by L-2-amino-4-phosphonobutyric acid and kynurenic acid. Pharmacological and biochemical experiments determined that the IC50 values of ouabain for the alpha1-low and alpha3-high ouabain affinity isozymes of photoreceptor Na+,K+-ATPase were approximately 10(-5) and approximately 10(-7) M, respectively, and that the D2/D4-like mediated inhibition of Na+,K+-ATPase was exclusively selective for the alpha3 isozyme. The dopamine-mediated inhibition of alpha3 first occurred at 5 nM, was maximal at 100 microM (-47%), had an IC50 value of 382 +/- 23 nM, and exhibited negative cooperativity (Hill coefficient, 0.27). Prior homogenization of the rod outer-inner segment completely prevented the long-lasting inhibition, suggesting that the effect was coupled to a second messenger. Although the physiological significance of our findings to photoreceptor function is unknown, we hypothesize that these results may have relevance for the temporal tuning properties of rods.