Polyglutamine expansion inhibits respiration by increasing reactive oxygen species in isolated mitochondria.

Huntington's disease results from expansion of the polyglutamine (PolyQ) domain in the huntingtin protein. Although the cellular mechanism by which pathologic-length PolyQ protein causes neurodegeneration is unclear, mitochondria appear central in pathogenesis. We demonstrate in isolated mitochondria that pathologic-length PolyQ protein directly inhibits ADP-dependent (state 3) mitochondrial respiration. Inhibition of mitochondrial respiration by PolyQ protein is not due to reduction in the activities of electron transport chain complexes, mitochondrial ATP synthase, or the adenine nucleotide translocase. We show that pathologic-length PolyQ protein increases the production of reactive oxygen species in isolated mitochondria. Impairment of state 3 mitochondrial respiration by PolyQ protein is reversed by addition of the antioxidants N-acetyl-L-cysteine or cytochrome c. We propose a model in which pathologic-length PolyQ protein directly inhibits mitochondrial function by inducing oxidative stress.

Sodium valproate -- induced skeletal myopathy.

The authors report a case of skeletal myopathy in a four-year-old boy on long-term sodium valproate therapy for secondary epilepsy due to neurocysticercosis. He presented with clinical features of limb girdle weakness. EMG revealed features of myopathy. Carnitine deficiency due to sodium valproate was suspected and plasma carnitine levels were found to be low. Sodium valproate was withdrawn. L-carnitine supplementation resulted in marked clinical recovery as well as rise in plasma carnitine levels.

Sodium valproate - Induced skeletal myopathy.

The authors report a case of skeletal myopathy in a four-year-old boy on long-term sodium valproate therapy for secondary epilepsy due to neurocysticercosis. He presented with clinical features of limb girdle weakness. EMG revealed features of myopathy. Carnitine deficiency due to sodium valproate was suspected and plasma carnitine levels were found to be low. Sodium valproate was withdrawn. L-carnitine supplementation resulted in marked clinical recovery as well as rise in plasma carnitine levels.

Characterization of a new rat urinary metabolite of piperine by LC/NMR/MS studies.

Potential of piperine, an active alkaloid of black and long peppers, to increase the bioavailability of drugs in humans is of great clinical significance owing to its omnipresence in food. In an attempt to further study the reported differences in its metabolism in rats and humans, a new major urinary metabolite was detected in rat urine and plasma using HPLC. The metabolite was partially purified using reverse phase column chromatography on Sephadex((R))-LH 20 and characterized as 5-(3, 4-methylenedioxy phenyl)-2E,4E-pentadienoic acid-N-(3-yl propionic acid)-amide with the help of LC/NMR/positive ESI-MS studies. Complete mass fragmentation pattern could be assigned with MS/MS studies. The metabolite has a unique structure compared to the previously reported metabolites in that it retains methylenedioxy ring and conjugated double bonds while the piperidine ring is modified to form propionic acid group. Mechanism of formation of the metabolite by oxidation and cleavage of piperidine ring is proposed. Kidney appears to be the major excretion route for piperine metabolites in rats as no metabolite could be detected in feces.

Inflammatory myofibroblastic tumor.

Inflammatory myofibroblastic tumors are well described in lung and upper respiratory tract of young adults and children. Intra-abdominal forms of the disease are reported to occur most frequently in the liver, followed by stomach, bowel and spleen. A 13-year-old girl who had intermittent fever ranging from 99-101 degrees F of three months period and significant weight loss was referred as a case of pyrexia of unknown origin. The patient was subjected to laparotomy. A solid mass was found arising from the mesentery, four feet from the iliocaecal junction and was adherent to the wall of ileum. The patient became afebrile after the removal of the mass and has gained weight and remained well during the ten months since discharge.

Therapy and drug resistance in malaria.

The aim of this study was to highlight the high degree of clinical resistance of P. falciparum and also of P. vivax to chloroquine and also the importance of early diagnosis and prompt treatment. Ninety cases of smear positive malaria aged between 6 months to 14 years were studied with regards to clinical manifestations, management and outcome. Criteria for drug resistance were absence of clearance of parasitemia and/or persistance of fever after 72 hours of therapy. Chloroquin resistance was noted in 15 (62.5%) cases of falciparum malaria and 15 (51.7%) cases of vivax malaria. The resistant and complicated cases were treated with quinine. Four (6.6%) cases were resistant to quinine and responded to artemether. There was no mortality. Early reporting of cases, frequent sampling malarial parasite, prompt diagnosis of falciparum malaria, early institution of appropriate therapy and awareness of choloquine and/or quinine resistance helps in salvaging lives.

Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability.

Inactivation of DNA-mismatch repair underlies the genesis of microsatellite unstable (MSI) colon cancers. hPMS2 is one of several genes encoding components of the DNA-mismatch repair complex, and germline hPMS2 mutations have been found in a few kindreds with hereditary nonpolyposis colorectal carcinoma (HNPCC), in whom hereditary MSI colon cancers develop. However, mice bearing null hPMS2 genes do not develop colon cancers and hPMS2 mutations in sporadic human colon cancers have not been described. Here we report that in Vaco481 colon cancer the hPMS2 gene is inactivated by somatic mutations of both hPMS2 alleles. The cell line derived from this tumor is functionally deficient in DNA mismatch repair. This deficiency can be biochemically complemented by addition of a purified hMLH1-hPMS2 (hMutLalpha) complex. The hPMS2 deficient Vaco481 cancer cell line demonstrates microsatellite instability, an elevated HPRT gene mutation rate, and resistance to the cytotoxicity of the alkylator MNNG. We conclude that somatic inactivation of hPMS2 can play a role in development of sporadic MSI colon cancer expressing the full range of cancer phenotypes associated with inactivation of the mismatch repair system.

Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers.

Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI-positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism.

The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency.

Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/Thr sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.

Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors.

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa=Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa=Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

Central pontine myelinolysis.

A 9 year old boy presented with fever, drowsiness, quadriparesis and facial myokymia. MRI showed demyelination in the pontine region. A diagnosis of central pontine myelinolysis was made. Literature review revealed the rarity of uneventful recovery as has been seen in our case.

The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency.

N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.

The hydroxy amino acid in an Asn-X-Ser/Thr sequon can influence N-linked core glycosylation efficiency and the level of expression of a cell surface glycoprotein.

N-Linked glycosylation usually occurs at the sequon, Asn-X-Ser/Thr. In this sequon, the side chain of the hydroxy amino acid (Ser or Thr) may play a direct catalytic role in the enzymatic transfer of core oligosaccharides to the Asn residue. Using recombinant variants of rabies virus glycoprotein (RGP), we examined the influence of the hydroxy amino acid on core glycosylation efficiency. A variant of RGP containing a single Asn-X-Ser sequon at Asn37 was modified by site-directed mutagenesis to change the sequon to either Asn-X-Cys or Asn-X-Thr. The impact of these changes on core glycosylation efficiency was assessed by expressing the variants in a cell-free transcription/translation/glycosylation system and in transfected tissue culture cells. Substitution of Cys at position 39 blocks glycosylation, whereas substitution of Thr dramatically increases core glycosylation efficiency of Asn37 in both membrane-anchored and secreted forms of RGP. The substitution of Thr for Ser also dramatically enhances the level of expression and cell surface delivery of RGP when the sequon at Asn37 is the only sequon in the protein. Novel forms of membrane-anchored and secreted RGP which are fully glycosylated at all three sequons were also generated by substitution of Thr at position 39.

Retinoid regulation of human ectocervical epithelial cell transglutaminase activity and keratin gene expression.

Cornified envelope formation, the level of transglutaminase activity and the pattern of cytokeratin gene expression are important biochemical markers of cervical epithelial cell differentiation in vivo. In the present study we examine the effects of retinoid treatment on transglutaminase (TG) activity and keratin gene expression in cultured human ectocervical epithelial cells (ECE cells). All-trans-retinoic acid (RA) and a synthetic retinoid, Ro 13-6298, suppress TG activity by 85-90% with half-maximal inhibition at 0.1 nM Ro 13-6298 or 1 nM RA. In contrast, the predominant circulating retinoid, retinol, does not inhibit TG activity. The level of type I transglutaminase protein, measured using a type I TG-specific antibody, decreases in parallel with the decrease in activity as does the level of the TG RNA transcript. Cytokeratin K16 decreases more than 20-fold while the level of K7, K8 and K19 increase 5 to 10-fold in the presence of 10 nM RA. Studies using cDNAs encoding K5, K13, K16 and K19 indicate that the RNA transcript levels change in parallel with the change in keratin protein production. Thus, all-trans-retinoic acid suppresses ectocervical epithelial cell differentiation in vitro, a result that suggests an in vivo role for retinoids in regulating cervical cell differentiation.