Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ～65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.     

Anatomical differences of poplar (<Emphasis Type="Italic">Populus</Emphasis> × <Emphasis Type="Italic">euramericana</Emphasis> clone I-214) roots exposed to zinc excess

Poplar is one of the suitable candidates for phytoremediation due to extensive root system, fast growth rate, easy propagation and high biomass production. Zinc (Zn) is an essential element, but at high concentration becomes toxic to plants, similarly like cadmium (Cd). In order to evaluate the effect of Zn on root tissue development we conducted experiments with poplar (Populus × euramericana clone I-214) grown in hydroponics. Plants were treated with low (control) and excess level of Zn (1 mM). Changes in the development of apoplasmic barriers — Casparian bands and suberin lamellae in endodermis, as well as lignification of xylem vessels have been investigated. We found that both apoplasmic barriers developed closer to the root apex in higher Zn-treated root when compared with control root. Similar changes were observed in lignification of xylem vessels. For localization of Zn within root tissues, cryo-SEM/EDXMA analyses were used. Most of Zn was localized in the cortical tissues and four-time less Zn was determined in the inner part of the root below the endodermis. This indicates that endodermis serves as efficient barrier of apoplasmic Zn transport across the poplar root.     

Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cytosine deaminase

Mesenchymal stromal cells (MSCs) are considered to be suitable vehicles for cellular therapy in various conditions. The expression of reporter and/or effector protein(s) enabled both the identification of MSCs within the organism and the exploitation in targeted tumor therapies. The aim of this study was to evaluate cellular changes induced by retrovirus-mediated transgene expression in MSCs in vitro. Human Adipose Tissue-derived MSCs (AT-MSCs) were transduced to express (i) the enhanced green fluorescent protein (EGFP) reporter transgene, (ii) the fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) enzyme along with the expression of dominant positive selection gene NeoR or (iii) the selection marker NeoR alone (MOCK). CDy::UPRT expression resulted in increased proliferation of CDy::UPRT-MSCs versus naïve AT-MSCs, MOCK-MSCs or EGFP-MSCs. Furthermore, CDy::UPRT-MSCs were significantly more sensitive to 5-fluorouracil (5FU), cisplatin, cyclophosphamide and cytosine arabinoside as determined by increased Caspase 3/7 activation and/or decreased relative proliferation. CDy::UPRT-MSCs in direct cocultures with breast cancer cells MDA-MB-231 increased tumor cell killing induced by low concentrations of 5FU. Our data demonstrated the changes in proliferation and chemoresistance in engineered MSCs expressing transgene with enzymatic function and suggested the possibilities for further augmentation of targeted MSC-mediated antitumor therapy.     

Is it possible to improve homologous recombination in Chlamydomonas reinhardtii?

Targeted modification of the genome has long been an aim of many geneticists and biotechnologists. Gene targeting is a main molecular tool to examine biological effects of genes in a controlled environment. Effective gene targeting depends on the frequency of homologous recombination that is indispensable for the insertion of foreign DNA into a specific sequence of the genome. The main problem associated with the development of an optimal procedure for gene targeting in a particular organism is the variability of homologous recombination (HR) in different species. Chlamydomonas reinhardtii is an attractive model system for the study of many cellular processes and is also an interesting object for the biotechnology industry. In spite of many advantages of this model system, C. reinhardtii does not readily express heterologous genes and does not allow targeted integration of foreign DNA into its genome easily. This paper compares data obtained from several different experiments designed for improving gene targeting in different organisms and reviews the suitability of particular techniques in C. reinhardtii cells. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Plasma membrane microdomains regulate turnover of transport proteins in yeast

In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins.     

Fast deprotection of synthetic oligodeoxyribonucleotides using standard reagents under microwave irradiation

Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz, dC Bz, dC Ac, dG ibu, dG PAC) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz, dC Bz, dG ibu) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170 degrees C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.     

Cryo-electron microscopy of an extremely halophilic microbe: technical aspects

Most halophilic Archaea of the class Halobacteriaceae depend on the presence of several molar sodium chloride for growth and cell integrity. This poses problems for structural studies, particularly for electron microscopy, where the high salt concentration results in diminished contrast. Since cryo-electron microscopy of intact cells provides new insights into the cellular and molecular organization under close-to-live conditions, we evaluated strategies and conditions to make halophilic microbes available for investigations in situ. Halobacterium salinarum, the test organism for this study, usually grows at 4.3 M NaCl. Adaptation to lower concentrations and subsequent NaCl reduction via dialysis led to still vital cells at 3 M salt. A comprehensive evaluation of vitrification parameters, thinning of frozen cells by focused-ion-beam micromachining, and cryo-electron microscopy revealed that structural studies under high salt conditions are possible in situ.

     

Genetic analysis of metabolic defects in the spontaneously hypertensive rat

Abnormalities in carbohydrate and lipid metabolism are common in patients with essential hypertension and in the spontaneously hypertensive rat (SHR). To identify chromosome regions contributing to this clustering of cardiovascular risk factors in the SHR, we searched for quantitative trait loci (QTL) associated with insulin resistance, glucose intolerance, and dyslipidemia by using the HXB/BXH recombinant inbred (RI) strains. Analysis of variance in RI strains suggested significant effects of genetic factors. A genome screening of the RI strains with more than 700 markers revealed QTL significantly associated with insulin resistance on Chromosomes (Chrs) 3 and 19. The Chr 19 QTL was confirmed by testing a previously derived SHR-19 congenic strain: transfer of a Chr 19 segment delineated by markers D19Rat57 and D19Mit7 from the Brown Norway (BN/Cr) strain onto the genetic background of the SHR/Ola was associated with decreased insulin and glucose concentrations and ameliorated insulin resistance at the tissue level. These findings suggest that closely linked genes on Chr 19, or perhaps even a single gene with pleiotropic effects, influence the clustering of metabolic disturbances in the SHR-BN model.