Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells

The transactivating protein from human immunodeficiency virus type 1 (HIV-1), Tat, was found to bind to the nuclear matrix from uninfected and HIV-1-infected H9 cells. Addition of the Zn2+, Cd2+ and Cu2+ chelator o-phenanthroline destroyed the matrix fibrils and the binding affinity of Tat to the matrix. A sequential treatment of the matrix, first with o-phenanthroline and then with ZnCl2, partially restored the fibrillar-like matrix structure. Infection of H9 cells with HIV-1 resulted in a displacement of cellular mRNA by viral mRNA from the nuclear matrix. Both the matrix-bound host cell and HIV-1 mRNA were found to dissociate from the matrix in the presence of o-phenanthroline. This could be prevented by coincubation with Zn2+ or Cu2+ (but not Mg2+), which stabilize the mRNA containing nuclear matrix structure.     

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.     

Collagens as multidomain proteins

The number of proteins known to contain collagen-like triple helical domains is rapidly increasing. The functions of these domains are to provide molecular rods that separate spatially non-triple helical domains with varied properties and structures and to permit lateral interactions between molecules. Two-thirds of the amino acids of the triple helical domains have their side-chains at the surface of the protein. The triple helix is also a structure that is easily predictable from the primary structure. The structure of several recently discovered collagens are discussed in terms of domains and functions. The triple helical domains have sizes varying from 33 to over 1,000 amino acid residues. The longest uninterrupted triple helices are involved in the formation of the classical quarter-staggered fibrils. Other triple helical domains permit varied molecular aggregates. A very broad spectrum of non-triple helical or globular domains are interspersed by triple helices. Only those located at the extremities of the molecules are large in size, sometimes several hundred kDa, while the domains separating 2 triple helices are small (less than 50 amino acids) and provide the molecules with hinges, proteolytic cleavage sites or other specialized functions like a glycosaminoglycan attachment site. If the assembly of the 3 chains required for the triple helix formation can be controlled in vitro, collagen-like molecules offer an as yet unexploited potential for protein engineering.     

Iron status in the acute phase and six weeks after myocardial infarction

In a case-control study of 84 myocardial infarction patients and 84 population controls we investigated the association between iron status parameters and myocardial infarction during the acute phase and after six weeks. Immediately after the infarction mean ferritin levels were significantly higher, whereas iron levels and iron saturation of transferrin were significantly lower in cases than in controls. Six weeks after the infarction, serum iron levels were still significantly lower in cases than in controls. Neither serum ferritin levels nor serum iron levels did show a clear association with the size of the ischemic tissue damage as estimated by creatine phosphokinase levels. Our results indicate that serum ferritin and iron levels are influenced by the traumatic effects of the myocardial infarction. Possibly, these transient changes are an acute effect, as seen in infections. An increased uptake of iron in the reticulo-endothelial system for synthesis of ferritin, may account for the lowered serum iron level and the iron saturation of transferrin.     

The effect of gamma-linolenic acid, an in vitro cytostatic substance contained in evening primrose oil, on primary liver cancer. A double-blind placebo controlled trial

The cytostatic effects of essential fatty acid metabolic intermediates and of some prostaglandins and leukotrienes in vitro have been extensively documented. The essential fatty acids (EFAs) exhibit no side-effects when taken as a dietary supplement, even in large doses. Primary Liver Cancer (PLC) is a fatal disease in our area as it is always multifocal in nature. In vitro studies have shown a cytostatic effect of gamma-linolenic acid (GLA) on primary liver cancer cells. In a double-blind placebo controlled trial, using Evening Primrose Oil (as a source of GLA) as a dietary supplement in PLC patients, no statistically significant effect was observed on survival time or liver size. There was however a statistical significant beneficial effect on Gamma Glutamyl transferase values as a measure of liver function. No side-effects were observed. The large size of tumour and the low doses of GLA used in this trial probably explain the lack of significant effect on survival times.     

Identification of a new seventh complementation group of UV-sensitive mutants in Chinese hamster cells

The UV-sensitive mutant V-B11, isolated from the V79 Chinese hamster cell line (Zdzienicka and Simons, 1987) was further characterized. V-B11 has a slightly increased cross-sensitivity to 3me4NQO, whereas no increased sensitivity towards 4NQO was observed. A slightly increased sensitivity towards EMS and MMS was also found. The mutant shows a defect in the ability to perform the incision step of nucleotide-excision repair after UV irradiation: 2 h after UV exposure, the accumulation of incision breaks in V-B11, in the presence of HU and araC, was about 30% of that found in wild-type V79 cells. V-B11 was crossed to a panel of 6 UV-sensitive Chinese hamster ovary (CHO) cells, which represents all the previously identified 6 complementation groups of UV-sensitive Chinese hamster mutants. Since in all crosses complementation has been observed, V-B11 appears to be the first mutant of a new, 7th, complementation group.     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Summary Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproductbility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.