Characterization of chromosome replication during S-phase with bromodeoxyuridine labelling in Chinese hamster ovary and HeLa cells

The same C-banded human polymorphic chromosomes were observed in the light microscope (LM) and then in the scanning electron microscope (SEM) to investigate the structural changes produced by the C-banding technique. C-banded regions, which stained positively in LM, were highly condensed with tightly packed chromatin fibres, resembling non-banded chromosomes. In striking contrast, adjacent non-C-banded regions were represented by loosely arranged fibres, resembling G-banded chromosomes. The significance of these observations in relation to current theories on the effects of C-banding on chromosome structure is discussed.     

Chloroplast DNA synthesis in light and dark grown cultured Nicotiana tabacum cells as determined by molecular hybridization

A simple method using molecular hybridization was devised to quantitatively measure chloroplast DNA synthesis in vivo. Total cellular DNA isolated from Nicotiana tabacum suspension cells, labeled with ³H-thymidine, was hybridized to nitrocellulose membrane-bound cloned chloroplast DNA (ct DNA) fragments. Colorless, dark grown N. tabacum cells were found to contain approximately 3300–4800 chloroplast genome copies per cell, whereas light grown, green cells contain about 9500–12000 chloroplast genomes per cell. This difference in ct DNA levels suggests that the chloroplast genome is somewhat amplified during growth of the cells in the light. The hybridization technique was also used to measure the efficiency of hybridization between cloned spinach ct DNA and tobacco ct DNA. The two DNAs were found to cross-hybridize with an efficiency of 69–75%.     

Identification of novel allosteric inhibitors of mutant isocitrate dehydrogenase 1 by cross docking-based virtual screening

IDH1 mutation (mIDH1) occurs in 20–30% of gliomas and is a promising target for the cancer therapy. In this article, a cross docking-based virtual screening was employed to identify seven small molecules for the allosteric site of mIDH1. Compounds ZX01, ZX05 and ZX06 exhibited the potent inhibitory activity and the high selectivity against WT-IDH1, providing a good starting point for the further development of highly selective mIDH1 inhibitors. Importantly, the parallel artificial membrane permeation assay of the blood-brain barrier (PAMPA-BBB) identified ZX06 with a good ability to penetrate BBB. These findings indicate that ZX06 deserves further optimization as a lead compound for the treatment of patients with IDH1 mutated brain cancers.     

Programs,databases, and expert systems for human geneticists — a survey

We present an overview of the variety of databases and programs that offer substantial aid to medical and molecular geneticists. Databases and expert systems for genetic diseases and birth defects, programs for segregation and linkage analysis, certain DNA and protein sequence databases, and information resources in general for molecular biology are addressed. These systems cannot be used effectively without the newly developed techniques of information exchange based on international computer networks. A short introduction is given to the Internet and to European institutions and organizations that offer help with the aquisition and use of bioinformatic resources.     

Inheritance of the S113L mutation within an inbred family with carnitine palmitoyltransferase enzyme deficiency

Deficiency of carnitine palmitoyltransferase type II (CPT II) is a clinically heterogeneous autosomal recessive disorder of lipid metabolism. The most common mutation in the CPT 11 gene is the S113L mutation, which substitutes leucine for serine at amino acid position 113. We studied an inbred family with three affected cousins with CPT II deficiency and found the S113L mutation to be present in a homozygous state in all three patients. Pedigree analysis traced the S 113L mutation back to one common ancestor. Although the patients in this family have an identical genotype at the CPT II locus, their clinical picture ranges from asymptomatic to lethal.     

A ZYGOTE-SPECIFIC PROTEIN WITH HYDROXYPROLINE-RICH GLYCOPROTEIN DOMAINS AND LECTIN-LIKE DOMAINS INVOLVED IN THE ASSEMBLY OF THE CELL WALL OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

The cell wall of Chlamydomonas reinhardtii zygotes, which forms rapidly after the fusion of wall-free gametes, provides a tractable system for studying the properties and assembly of hydroxyproline-rich glycoproteins, the major proteinaceous components of green algal and plant cell walls. We report the cloning of the zsp2 gene and the analysis of its ZSP-2 product, a 58.9 kDa polypeptide that is synthesized exclusively by zygotes. The protein contains two (SP)_x repeats, establishing it as a member of the cell wall hydroxyproline-rich glycoproteins family. It also contains a 4-fold iteration of an amino acid sequence centered around cysteine residues, a configuration found in both plant and animal lectins. Furthermore, we report four observations on pellicle composition and production. First, cell-free preparations of the pellicle matrix are rich in hydroxyproline, arabinose, and galactose and contain bundles of very long fibrils. Second, glutathione blocks pellicle formation and results in the accumulation of long fibrils in the growth medium. Third, antibody to ZSP-2 also blocks pellicle formation. Fourth, ZSP-2 immunolocalizes to the boundary between the outer layers of the wall proper and the pellicle matrix. These observations are consistent with the possibility that the Cys-rich (glutathione-sensitive) lectin-like domains of ZSP-2 may bind to sugar residues on the long fibrils and anchor them to the cell wall, thereby initiating and maintaining pellicle formation.     

Profiling gene expression in whole blood samples following an in-vitro challenge.

Genomics tools (gene- and protein-expression studies) can be used to find possible target genes involved in a quantifiable trait or disease state. However in many instances, cells and tissues directly involved in the trait's expression, for example, brain tissue, are not amenable for gene expression analysis. Whole blood cells share a molecular make-up for cellular communication and gene regulation systems with many other cell types, for example, neuronal cells, and have the advantage of being very accessible for gene profiling. We investigated the feasibility of nationwide blood sample collection for lymphocyte RNA isolation and real-time PCR analysis to quantify genomic responses. We tested several designs for blood collection and storage: blood sampling in PAXgene blood collection tubes and storage at -20 degrees C, blood sampling in heparin tubes and decanting the samples (with or without in-vitro stimulus) into either PAXgene blood collection tubes and storage at -20 degrees C, or polypropylene tubes followed by snap-freezing and storage at -80 degrees C. The latter procedure is the best cost-wise when only small amounts of total RNA are needed for downstream applications. Lymphocyte gene expression studies are most likely hampered by the quality of isolated RNA rather than the sampling method. We show that large-scale nationwide sample collections did not alter RNA quality or gene expression levels when compared to sampling and processing in a more controlled way. To this end, we present an optimized protocol for easy and standardized isolation of high quality RNA using the PAXgene isolation kit. Based on these results, we suggest that whole blood genomic data can be used as a genomic probe in experimental and clinical research.