Unfolding of Novel Independent Missense Mutations in VAMP2 and AGRN and Their Collective Role in Global Developmental Delay: A Case Report.

Vesicle-associated membrane protein 2 (VAMP2) and Agrin (AGRN) are crucial proteins in neurotransmission. VAMP2 is a vesicular protein that facilitates the exocytosis of neurotransmitters. At the same time, AGRN plays a critical role in the maintenance and function of neuromuscular junctions. Mutations in the signaling pathway of VAMP2 and AGRN impair proper signaling between the presynaptic and postsynaptic neurons, and can result in neurodevelopmental conditions known as global developmental delay (GDD). This study highlights a presentation of GDD in a patient with concurrent mutations in VAMP2 and AGRN. A three-year-old female child presented with GDD characterized by hypotonia, intellectual disability, and dysphagia. Physical exam exhibited signs of developmental delay and severe muscle weakness. EEG findings were suggestive of a hypsarrhythmia pattern. The ophthalmological evaluation showed partial optic atrophy bilaterally. Therapeutic interventions included Keppra and Topamax, which proved ineffective. The patient's outcome was inconclusive as care was transferred to another facility. This case study reports the novel appearance of two concurrent mutations: p.Gln76Pro associated with VAMP2 and p.Gln970Glu associated with AGRN. Mutations in VAMP2 lead to a dysfunctional SNARE complex and inhibit exocytosis of neurotransmitters into the synaptic cleft. Mutations in AGRN impair the ability to form and activate postsynaptic nicotinic acetylcholine receptors. Improper signaling between presynaptic and postsynaptic neurons is an important determinant of GDD. We hope that accounting for this mutational pattern will contribute to understanding synapse assembly and help unravel the complex interplay of factors involved in the pathology of neuromuscular disorders and GDD.

Entecavir competitively inhibits deoxyguanosine and deoxyadenosine phosphorylation in isolated mitochondria and the perfused rat heart.

Deoxyguanosine kinase (dGK) is reported responsible for the phosphorylation of deoxyadenosine (dA) and deoxyguanosine (dG) in the mitochondrial purine salvage pathway. Antiviral nucleoside analogs known as nucleoside reverse transcriptase inhibitors (NRTIs) must be phosphorylated by host enzymes for the analog to become active. We address the possibility that NRTI purine analogs may be competitive inhibitors of dGK. From a group of such analogs, we demonstrate that entecavir (ETV) competitively inhibited the phosphorylation of dG and dA in rat mitochondria. Mitochondria from the brain, heart, kidney, and liver showed a marked preference for phosphorylation of dG over dA (10-30-fold) and ETV over dA (2.5-4-fold). We found that ETV inhibited the phosphorylation of dG with an IC50 of 15.3 ± 2.2 µM and that ETV and dG were both potent inhibitors of dA phosphorylation with IC50s of 0.034 ± 0.007 and 0.028 ± 0.006 µM, respectively. In addition, the phosphorylation of dG and ETV followed Michaelis-Menten kinetics and each competitively inhibited the phosphorylation of the other. We observed that the kinetics of dA phosphorylation were strikingly different from those of dG phosphorylation, with an exponentially lower affinity for dGK and no effect of dA on dG or ETV phosphorylation. Finally, in an isolated heart perfusion model, we demonstrated that dG, dA, and ETV were phosphorylated and dG phosphorylation was inhibited by ETV. Taken together, these data demonstrate that dGK is inhibited by ETV and that the primary role of dGK is in the phosphorylation of dG rather than dA.

Hypotonia-cystinuria 2p21 deletion syndrome: Intrafamilial variability of clinical expression.

Two siblings presented similarly with congenital hypotonia, lactic acidosis, and failure to thrive. Later in childhood, the brother developed cystinuria and nephrolithiasis whereas the older sister suffered from cystinuria and chronic neurobehavioral disturbances. Biopsied muscle studies demonstrated deficient cytochrome c oxidase activities consistent with a mitochondrial disease. Whole exome sequencing (WES), however, revealed a homozygous 2p21 deletion involving two contiquous genes, SLC3A1 (deletion of exons 2-10) and PREPL (deletion of exons 2-14). The molecular findings were consistent with the hypotonia-cystinuria 2p21 deletion syndrome, presenting similarly in infancy with mitochondrial dysfunction but diverging later in childhood and displaying intrafamilial phenotypic variability.

Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

Congenital Parvovirus B19 Infection: Persistent Viremia and Red Blood Cell Aplasia.

We describe a case of fetal parvovirus B19 infection resulting in preterm birth and leading to hydrops fetalis requiring multiple in utero transfusions. The infant developed chronic postnatal anemia responsive to intravenous immunoglobulin therapy. Serum viral load decreased after immunoglobulin treatment but remained detectable for over 1 year.

Mitochondrial complex I defect and increased fatty acid oxidation enhance protein lysine acetylation in the diabetic heart.

AIMS: Cardiomyopathy is a major complication of diabetes. Our study was aimed to identify the sites of mitochondrial dysfunction and delineate its consequences on mitochondrial metabolism in a model of type 1 diabetes. METHODS AND RESULTS: Diabetes was induced by streptozotocin injection to male Lewis rats. We found a decrease in mitochondrial biogenesis pathway and electron transport chain complex assembly that targets Complex I. Oxidation of Complex II and long-chain fatty acid substrates support the electron leak and superoxide production. Mitochondrial defects do not limit fatty acid oxidation as the heart's preferred energy source indicating that the diabetic heart has a significant reserve in Complex I- and II-supported ATP production. Both mitochondrial fatty acid oxidation and Complex I defect are responsible for increased protein lysine acetylation despite an unchanged amount of the NAD(+)-dependent mitochondrial deacetylase sirt3. We quantitatively analysed mitochondrial lysine acetylation post-translational modifications and identified that the extent of lysine acetylation on 54 sites in 22 mitochondrial proteins is higher in diabetes compared with the same sites in the control. The increased lysine acetylation of the mitochondrial trifunctional protein subunit α may be responsible for the increased fatty acid oxidation in the diabetic heart. CONCLUSION: We identified the specific defective sites in the electron transport chain responsible for the decreased mitochondrial oxidative phosphorylation in the diabetic heart. Mitochondrial protein lysine acetylation is the common consequence of both increased fatty acid oxidation and mitochondrial Complex I defect, and may be responsible for the metabolic inflexibility of the diabetic heart.

Heart mitochondrial TTP synthesis and the compartmentalization of TMP.

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.

New platelet glycoprotein polymorphisms causing maternal immunization and neonatal alloimmune thrombocytopenia.

BACKGROUND: Maternal immunization against low-frequency, platelet (PLT)-specific antigens is being recognized with increasing frequency as a cause of neonatal alloimmune thrombocytopenia (NAIT). STUDY DESIGN AND METHODS: Serologic and molecular studies were performed on PLTs and DNA from two families in which an infant was born with severe thrombocytopenia not attributable to maternal immunization against known PLT-specific alloantigens. RESULTS: Antibodies reactive only with paternal PLTs were identified in each mother using flow cytometry and solid-phase assays. Unique mutations encoding amino acid substitutions K164T in glycoprotein (GP)IIb (Case 1) and R622W in GPIIIa (Case 2) were identified in paternal DNA and in DNA from the affected infants. Each maternal antibody recognized recombinant GPIIb/IIIa mutated to contain the polymorphisms identified in the corresponding father. None of 100 unselected normal subjects possessed these paternal mutations. CONCLUSIONS: Severe NAIT observed in the affected infants was caused by maternal immunization against previously unrecognized, low-frequency antigens created by amino acid substitutions in GPIIb/IIIa (α(IIb) /ß(3) integrin). A search should be conducted for novel paternal antigens in cases of apparent NAIT not explained on the basis of maternal-fetal incompatibility for known human PLT antigens.

Regulation of glucose- and mitochondrial fuel-induced insulin secretion by a cytosolic protein histidine phosphatase in pancreatic beta-cells.

We report localization of a cytosolic protein histidine phosphatase (PHP; approximately 16 kDa) in INS 832/13 cells, normal rat islets, and human islets. siRNA-mediated knockdown of PHP markedly reduced glucose- or mitochondrial fuel-induced but not KCl-induced insulin secretion. siRNA-mediated knockdown of PHP also attenuated mastoparan-induced insulin secretion, suggesting its participation in G protein-sensitive signaling steps, leading to insulin secretion. Functional assays revealed that the beta-cell PHP catalyzes the dephosphorylation of ATP-citrate lyase (ACL). Silencing of PHP expression markedly reduced ACL activity, suggesting functional regulation of ACL by PHP in beta-cells. Coimmunoprecipitation studies revealed modest effects of glucose on the interaction between PHP and ACL. Confocal microscopic evidence indicated that glucose promotes association between ACL and nm23-H1, a known kinase histidine kinase, but not between PHP and ACL. Furthermore, metabolic viability of INS 832/13 cells was resistant to siRNA-PHP, suggesting no regulatory roles of PHP in cell viability. Finally, long-term exposure (24 h) of INS 832/13 cells or rat islets to high glucose (30 mM) increased the expression of PHP. Such increases in PHP expression were also seen in islets derived from the Zucker diabetic fatty rat compared with islets from the lean control animals. Together, these data implicate regulatory roles for PHP in a G protein-sensitive step involved in nutrient-induced insulin secretion. In light of the current debate on putative regulatory roles of ACL in insulin secretion, additional studies are needed to precisely identify the phosphoprotein substrate(s) for PHP in the cascade of events leading to nutrient-induced insulin secretion.

Protein farnesylation-dependent Raf/extracellular signal-related kinase signaling links to cytoskeletal remodeling to facilitate glucose-induced insulin secretion in pancreatic beta-cells.

OBJECTIVE: Posttranslational prenylation (e.g., farnesylation) of small G-proteins is felt to be requisite for cytoskeletal remodeling and fusion of secretory vesicles with the plasma membrane. Here, we investigated roles of protein farnesylation in the signaling steps involved in Raf-1/extracellular signal-related kinase (ERK1/2) signaling pathway in glucose-induced Rac1 activation and insulin secretion in the pancreatic beta-cell. RESEARCH DESIGN AND METHODS: These studies were carried out in INS 832/13 cells and normal rat islets. Molecular biological (e.g., overexpression or small interfering RNA [siRNA]-mediated knockdown) and pharmacologic approaches were used to determine roles for farnesylation in glucose-mediated activation of ERK1/2, Rac1, and insulin secretion. Activation of ERK1/2 was determined by Western blotting. Rac1 activation (i.e., Rac1.GTP) was quantitated by p21-activated kinase pull-down assay. Insulin release was quantitated by enzyme-linked immunosorbent assay. RESULTS: Coprovision of structure-specific inhibitors of farnesyl transferase (FTase; e.g., FTI-277 or FTI-2628) or siRNA-mediated knockdown of FTase beta-subunit resulted in a significant inhibition of glucose-stimulated ERK1/2 and Rac1 activation and insulin secretion. Pharmacologic inhibition of Raf-1 kinase using GW-5074 markedly reduced the stimulatory effects of glucose on ERK1/2 phosphorylation, Rac1 activation, and insulin secretion, suggesting that Raf-1 kinase activation may be upstream to ERK1/2 and Rac1 activation leading to glucose-induced insulin release. Lastly, siRNA-mediated silencing of endogenous expression of ERK1/2 markedly attenuated glucose-induced Rac1 activation and insulin secretion. CONCLUSIONS: Together, our findings provide the first evidence of a role for protein farnesylation in glucose-mediated regulation of the Raf/ERK signaling pathway culminating in the activation of Rac1, which has been shown to be necessary for cytoskeletal reorganization and exocytotic secretion of insulin.

Glucose activates prenyltransferases in pancreatic islet beta-cells.

A growing body of evidence implicates small G-proteins [e.g., Cdc42 and Rac1] in glucose-stimulated insulin secretion [GSIS] in the islet beta-cell. These signaling proteins undergo post-translational modifications [e.g., prenylation] at their C-terminal cysteine residue and appear to be essential for the transport and fusion of insulin-containing secretory granules with the plasma membrane and the exocytotic secretion of insulin. However, potential regulation of the prenylating enzymes by physiological insulin secretogues [e.g., glucose] has not been investigated thus far. Herein, we report immunological localization, sub-cellular distribution and regulation of farnesyltransferases [FTases] and geranylgeranyltransferase [GGTase] by glucose in insulin-secreting INS 832/13 beta-cells and normal rat islets. Our findings suggest that an insulinotropic concentration of glucose [20mM] markedly stimulated the expression of the alpha-subunits of FTase/GGTase-1, but not the beta-subunits of FTase or GGTase-1 without significantly affecting the predominantly cytosolic distribution of these holoenzymes in INS 832/13 cells and rodent islets. Under these conditions, glucose significantly stimulated [2.5- to 4.0-fold over basal] the activities of both FTase and GGTase-1 in both cell types. Together, these findings provide the first evidence to suggest that GSIS involves activation of the endogenous islet prenyltransferases by glucose, culminating in the activation of their respective G-protein substrates, which is necessary for cytoskeletal rearrangement, vesicular transport, fusion and secretion of insulin.

Protective effect of potato peel extract against carbon tetrachloride-induced liver injury in rats.

Our earlier studies have shown that extracts derived from potato peel (PPE) are rich in polyphenols and possess strong antioxidant activity both in vitro and in vivo. The objective of the present study was to investigate its potential to offer protection against acute liver injury in rats. Rats pretreated with PPE (oral, 100mg/kgb.w./day for 7 days) were administered a single oral dose carbon tetrachloride (CCl(4), 3ml/kg b.w., 1:1 in groundnut oil) and sacrificed 8h of post-treatment. Hepatic damage was assessed by employing biochemical parameters (transaminase enzyme levels in plasma and liver [AST-aspartate transaminase; ALT-alanine transaminase, LDH-lactate dehydrogenase]). Further, markers of hepatic oxidative damage were measured in terms of malondialdehyde (MDA), enzymic antioxidants (CAT, SOT, GST, GPX) and GSH (reduced glutathione) levels. In addition, the CCl(4)-induced pathological changes in liver were evaluated by histopathological studies. Our results demonstrated that pretreatment of rats with PPE significantly prevented the increased activities of AST and ALT in serum, prevented the elevation of hepatic MDA formation as well as protected the liver from GSH depletion. PPE pretreatment also restored CCl(4)-induced altered antioxidant enzyme activities to control levels. The protective effect of PPE was further evident through the decreased histological alterations in liver. Our findings provide evidences to demonstrate that PPE pretreatment significantly offsets CCl(4)-induced liver injury in rats, which may be attributable to its strong antioxidant propensity.

Altered glucose homeostasis and oxidative impairment in pancreas of rats subjected to dimethoate intoxication.

The primary objective of this study was to investigate the effect of repeated sublethal doses of dimethoate (DM), an organophosphorus insecticide on glucose homeostasis, oxidative stress induction in pancreas and pancreatic damage in adult rats. Daily oral administration of DM (20 and 40 mg/kg b.w.) for 30 days induced a significant increase in blood glucose levels which was associated with impaired glucose tolerance. DM treatment resulted in elevated levels of pancreatic tissue specific markers such as activities of amylase and lipase in serum and pancreatic tissue indicating pancreatic dysfunction. Further, the activities of DT-diaphorase and NADPH-diaphorase in pancreas of DM treated rats were also found to be elevated. Interestingly, these biochemical dysfunctions were accompanied by a marked dose-related enhancement of lipid peroxidation and ROS levels in the pancreatic tissue indicating significant induction of oxidative damage. Additional evidence such as depletion in reduced glutathione levels and significant alterations in enzymic antioxidant defenses in pancreas among DM treated rats suggested induction of oxidative stress. Taken together, these findings provide experimental evidence that dimethoate at subchronic oral doses has the propensity to impair glucose homeostasis, induce significant pancreatic damage and also provide an account of the associated oxidative damage to pancreatic tissue in adult rats.

Angiotensin converting enzyme inhibitory activity of amino acid esters of carbohydrates.

L-alanyl-D-glucose, L-valyl-D-glucose, L-phenylalanyl-D-glucose and L-phenylalanyl-lactose esters were synthesized enzymatically using two lipases viz., Rhizomucor miehei lipase (RML) and porcine pancreas lipase (PPL) and tested for their potential as inhibitors of angiotensin converting enzyme (ACE) in vitro. The esters exhibited concentration related ACE inhibitory activity. The potency of the various esters measured in terms of IC50 values were as follows: L-phenylalanyl-D-glucose, IC50-0.121 mM (mixture of five diastereomeric esters: 6-O-24.1%; 3-O-23.3%; 2-O-19.2%; 2,6-di-O-16.6% and 3,6-di-O-16.8% from the total yield of 92.4%); L-phenylalanyl-lactose, IC50-0.229 mM (mixture of three diastereomeric esters: 6-O-42.1%; 6'-O-30.9%; and 6,6'-di-O-27.0% from the total yield of 50.58%); alanyl-D-glucose, IC50-0.23 mM (mixture of five diastereomeric esters: 6-O-46.7%; 3-O-11.5%; 2-O-19.9%; 2,6-di-O-6.6% and 3,6-di-O-15.3% from the total yield of 26.5%) and L-valyl-D-glucose, IC50-0.396 mM (mixture of five diastereomeric esters: 6-O-32.4%; 3-O-26.5%; 2-O-26.4%; 2,6-di-O-8.8% and 3,6-di-O-5.9% from the total yield of 68.2%). These in vitro data suggest a potential therapeutic role for the aminoesters of carbohydrates as inhibibitors of ACE.

Asymmetric crying facies: a possible marker for congenital malformations.

Asymmetric crying facies (ACF) is caused by agenesis or hypoplasia of the depressor anguli oris muscle on one side of the mouth. Though it is an isolated finding in most cases, ACF can be associated with other congenital malformations especially of the cardiovascular system. We report a case of ACF that was subsequently diagnosed as Cayler syndrome based on associated tetralogy of Fallot (TOF) and deletion of chromosome 22q11.

Protective effect of potato peel powder in ameliorating oxidative stress in streptozotocin diabetic rats.

The potential of dietary potato peel (PP) powder in ameliorating oxidative stress (OS) and hyperglycemia was investigated in streptozotocin (STZ)-induced diabetic rats. In a 4-week feeding trial, incorporation of potato peel powder (5 and 10%) in the diet of diabetic rats was found to significantly reduce the plasma glucose level and also reduce drastically the polyuria of STZ diabetic rats. The total food intake was significantly reduced in the diabetic rats fed 10% PP powder compared to the control diabetic rats. However, the body weight gain over 28 days was nearly four times greater in PP powder supplemented diabetic rats (both at 5 and 10%) compared to the control diabetic rats. PP powder in the diet also decreased the elevated activities of serum transaminases (ALT and AST) and nearly normalized the hepatic MDA and GSH levels as well as the activities of specific antioxidant enzymes in liver of diabetic rats. The result of these studies clearly establishes the modulatory propensity of PP against diabetes induced alterations. Considering that potato peels are discarded as waste and not effectively utilized, these results suggest the possibility that PP waste could be effectively used as an ingredient in health and functional food to ameliorate certain disease states such as diabetes.

Attenuation of hyperglycemia and associated biochemical parameters in STZ-induced diabetic rats by dietary supplementation of potato peel powder.

BACKGROUND: Both dietary fiber and polyphenols have been reported to exert antihyperglycemic effect. Potato peel (PP), a waste by product of potato processing, is found to be a good source of both dietary fiber and polyphenols. The current study examined the attenuating influence of dietary potato peel (PP) powder on hyperglycemia and various oxidative stress-associated biochemical parameters in diabetic rats. METHODS: Streptozotocin (STZ)-induced diabetic male Wistar rats were used as experimental models. The rats were divided into nondiabetic (control), diabetic, potato-peel-supplemented (5% and 10%) and diabetic-PP (5% and 10%)-supplemented groups and were maintained for 4 weeks on the experimental regime. The modulatory role of PP was assessed by determining its effect on blood glucose, urine output, body weight gain, lipid peroxidation, reduced glutathione, serum aminotransferases, lipid profiles, selected antioxidant enzymes in liver/kidney and selected eye lens parameters. RESULTS: Diabetic rats fed with PP-powder-supplemented diet for 4 weeks showed a significant decrease in blood glucose levels. Incorporation of PP powder reduced significantly the hypertrophy of both liver and kidney of STZ-diabetic rats and also normalized the activities of serum ALT and AST, hepatic and renal MDA and GSH, as well as activities of various antioxidant enzymes in liver and kidney of diabetic rats. Furthermore, PP powder in the diet also appeared to attenuate the eye lens damage associated with the diabetic condition. CONCLUSION: Potato peel powder supplementation in diet was found to effectively attenuate diabetic alterations in rats.