Chromosomal Characteristics of Ribosomal DNA in the Primitive Semionotiform Fish, Longnose Gar Lepisosteus osseus

The chromosomes of longnose gar, Lepisosteus osseus, an extant representative of early radiation of actinopterygian fishes, were studied using conventional Giemsa-staining, Ag-staining, CMA₃-fluorescence and fluorescence in-situ hybridization (FISH). The diploid chromosome number was 2n = 56 and the karyotype contained 11 pairs of metacentric, 6 pairs of submetacentric, 3 pairs of subtelocentric macrochromosomes and 16 microchromosomes. Nearly all macrochromosomes showed large CMA₃-positive regions resembling the R-bands of higher vertebrates, indicating extensive distribution of GC-rich DNA along chromosomes. The nucleolar organizer regions (NORs) were located on the end of the short arm of a single small metacentric macrochromosomal pair. These sites were strongly CMA₃-positive, suggesting that ribosomal sites are associated with GC- rich DNA. In-situ hybridization (FISH) with a rDNA probe gave consistently positive signals in the same regions detected by Ag- staining and CMA₃-fluorescence. The evolutionary conservation of positive CMA₃-fluorescence of ribosomal sites in holostean and teleostean fishes is discussed.     

Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to –60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 M) and amiloride (500 M) and was insensitive to -conotoxin (-CgTx) (4 M) and to the Ca agonist Bay K 8644 (5 M). The high-voltage-activated (HVA) Ca current turned on positive to –40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. CgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the -CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) -CgTx-sensitive and Bay-K-8644-insensitive (-S/BK-I) and (ii) -CgTx-insensitive and Bay-K-8644-sensitive (-I/BK-S), the latter displaying a stronger Cadependent inactivation.     

Gene assignment of α-fucosidase and glucose dehydrogenase to the p21→pter region of chromosome 1 in man

Assignment of human genes coding for -fucosidase (FUC) and glucose dehydrogenase (GDH) to chromosome 1 has been confirmed and a location in the p21pter region demonstrated using man-mouse somatic cell hybrids. The regional location af FUC andGDH was established in cell hybrids using human cells possessing 1/2 translocation chromosomes [46,XX,t(1;2)(p21;q37)]. Hybrids which retained the 2q⁺ chromosome carrying the 1p211pter region concordantly expressed FUC, GDH, and the short-arm markers ENO₁, AK₂, and PGM₁. Hybrids which retained the 1p211qter region only expressed human PEPC and FH. Data obtained from hybrids in which spontaneous breaks in chromosome 1 had occurred indicate that the gene order in 1p21ar1pter is (ENO₁,GDH)-FUC-AK₂-PGM₁.     

Avian genomes: different karyotypes but a similar distribution of the GC-richest chromosome regions at interphase

The chicken karyotype, like that of the vast majority of avian species, shows a large number of dot-shaped microchromosomes that are characterized, like most telomeric regions of the macrochromosomes, by the highest GC levels and the highest gene densities. In interphase nuclei, these gene-dense regions are centrally located, and are characterized by an open chromatin structure (a similar situation also exists in mammals). Avian species belonging to the Accipitridae family (diurnal raptors) show a karyotype with no very large chromosomes, and with only a very small number of microchromosomes. To identify the GC-rich (and gene-rich) regions of the chromosomes and nuclei from Accipitridae, we performed heterologous in-situ hybridizations using chicken GC-richest isochores as probes. Our results clearly show that the gene-rich regions are prevalently located in the few microchromosome pairs and in the telomeric regions of the middle-sized chromosomes, as well as in the interior of the interphase nuclei. This result is consistent with a common organization of the genome in the nuclei of warm-blooded vertebrates. Indeed, in spite of the different size and morphology of the chromosomes, the gene-dense regions are always located in the interior of the nuclei.     

Generation of an ilv bradytrophic phenocopy in yeast by antisense RNA

Summary We report for the first time on the regulation of gene expression in yeast by antisense RNA. Chimaeric genes were constructed containing the 5 upstream and partial coding sequence of SMR1 — a sulfometuron methyl resistant allele of the ILV2 locus. Such fragments were placed 5 to 3 and 3 to 5 under control of the GAL10 promoter and CYCl terminator in a high copy YEp plasmid. Following galactose induction only transformants containing antisense RNA genes showed biological activity against SMR1 gene expression. Antisense RNA inhibited synthesis of the SMR1 gene product acetolactate synthase and thus repressed cellular growth which resulted in a bradytrophic auxotroph revertable by addition of isoleucine and valine. Antisense RNA inhibition was enhanced in galactose medium containing sulfometuron methyl and in gcn4 cells deficient for positive regulation of the ILV2 locus. This system can be used to study factors that interfere with antisense RNA function and to assign biological function to randomly cloned DNA fragments.     

Structural and immunocytochemical studies on α-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminidase deficiency (Schindler/Kanzaki disease)

-N-Acetylgalactosaminidase (-NAGA) deficiency (Schindler/Kanzaki disease) is a clinically and pathologically heterogeneous genetic disease with a wide spectrum including an early onset neuroaxonal dystrophy (Schindler disease) and late onset angiokeratoma corporis diffusum (Kanzaki disease). In -NAGA deficiency, there are discrepancies between the genotype and phenotype, and also between urinary excretion products (sialyl glycoconjugates) and a theoretical accumulated material (Tn-antigen; Gal NAc1--Ser/Thr) resulting from a defect in -NAGA. As for the former issue, previously reported genetic, biochemical and pathological data raise the question whether or not E325K mutation found in Schindler disease patients really leads to the severe phenotype of -NAGA deficiency. The latter issue leads to the question of whether -NAGA deficiency is associated with the basic pathogenesis of this disease. To clarify the pathogenesis of this disease, we performed structural and immunocytochemical studies. The structure of human -NAGA deduced on homology modeling is composed of two domains, domain I, including the active site, and domain II. R329W/Q, identified in patients with Kanzaki disease have been deduced to cause drastic changes at the interface between domains I and II. The structural change caused by E325K found in patients with Schindler disease is localized on the N-terminal side of the tenth -strand in domain II and is smaller than those caused by R329W/Q. Immunocytochemical analysis revealed that the main lysosomal accumulated material in cultured fibroblasts from patients with Kanzaki disease is Tn-antigen. These data suggest that a prototype of -NAGA deficiency in Kanzaki disease and factors other than the defect of -NAGA may contribute to severe neurological disorders, and Kanzaki disease is thought to be caused by a single enzyme deficiency.     

Diameter and flow velocity changes of feline small pulmonary vessels in response to sympathetic nerve stimulation

Using an X-ray television system, we measured directly changes in the internal diameter (ID), flow velocity, and volume flow of the small pulmonary vessels (100–500 m ID) in response to electrical sympathetic nerve stimulation (SNS) in anaesthetized cats before and after adrenergic receptor blockade. Flow velocity was obtained by measuring the distance that the leading edge of the contrast medium moved per 0.1 s in the small arteries. Volume flow was obtained from the product of flow velocity and cross-sectional area calculated from the ID of the small arteries. SNS was accolmplished with 10- to 15-V square-wave pulses of 2-ms duration at 20–30 Hz for 20-s periods. In response to SNS, arterial ID decreased significantly by 8–13% in the 200- to 500-m vessels but not in the 100- to 200-m vessels. In the veins, on the other hand, there was no significant ID decrease in any of the 100- to 500-m vessels. After -receptor blockade (phentolamine, 2 mg/kg i.V.), there were significant ID increases (4–9%) in the 100- to 500-m arteries in response to SNS, the maximum increases being in the 100- to 200-m arteries. After -blockade (propranolol, 2 mg/kg i.V.), the ID decrease due to SNS in the 200- to 500-m arteries was enhanced (24–27%) and, in addition, the 100- to 200-m arteries exhibited a significant ID decrease (18%). Combined and -blockade completely abolished the ID decrease due to SNS. In the veins, on the other hand, no ID change occurred even after - or -blockade. The results indicate that SNS selectively constricts 200- to 500-m arteries. The data suggests that SNS has -mediated vasoconstrictor and -mediated vasodilator effects on the 100- to 500-m arteries and that the ID response pattern to SNS depends chiefly on the balance between -mediated vasoconstriction and -mediated vasodilation. Associated with the ID decrease due to SNS, flow velocity was increased by 21%. However, SNS did not affect volume flow, because the increase in velocity was compensated by the reduction in the cross-sectional area (due to the decreased ID).     

Caenorhabditis elegans Ce-rdh-1/rad-51 functions after double-strand break formation of meiotic recombination

During meiotic prophase 1, homologous recombination is accompanied by dynamic chromosomal changes. The Ce-rdh-1/rad-51 gene is the only bacterial recA-like gene in the nematode C. elegans genome. Upon depletion of Ce-rdh-1/rad-51 using the RNA interference method, abnormal kinked chromosomes can be observed in mature oocytes at diakinesis, whereas synapsis between homologous chromosomes during the pachytene stage is normal. Following fertilization, Ce-rdh-1/rad-51-depleted embryos die early in embryogenesis, and their nuclei exhibit abnormal chromosome fragments and bridges. From epistasis analyses with Ce-spo-11 defective mutant and ionizing radiation, it is indicated that Ce-rdh-1/rad-51 functions after double-strand break (DSB) formation of meiotic recombination. Under the Ce-chk-2 defective condition, whose meiotic synapsis and meiotic recombination between homologous chromosomes are completely inhibited, the Ce-rdh-1/rad51 is normally expressed in the gonadal cells. Moreover, it seems that exogenous DSBs in the Ce-chk-2 defective nuclei at the pachytene stage can be repaired between sister chromatids in a Ce-rdh-1/rad-51-dependent manner. These results indicate that Ce-rdh-1/rad51 functions after both endogenous and exogenous DSB formation during meiosis, but not as pairing centers for meiotic synapsis.     

Direction of DNA sequences within chromatids determined using strand-specific FISH

The 5 to 3 direction of DNA strands within chromatids of metaphase chromosomes can be determined by using simultaneous hybridization of a single strand of the telomere probe and a single strand of a repetitive sequence to slides pretreated for strand-specific hybridization. The telomere probe identifies the direction of the DNA helical strand remaining in each chromatid of the metaphase chromosomes. The direction of the repetitive sequence is then determined from the direction of the strand to which it hybridizes. This method was used to determine the 5 to 3 direction of three repetitive DNA sequences, each for a different human repeat family.     

TRP channels: a TR(I)P through a world of multifunctional cation channels

The transient receptor potential (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed from yeast to man. On the basis of structural homology, the TRP family can be subdivided in to seven main subfamilies: the TRPC (Canonical) group, the TRPV (Vanilloid) group, the TRPM (Melastatin) group, the TRPP (Polycystin), the TRPML (Mucolipin), the TRPA (Ankyrin) and the TRPN (NOMP) family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of cell types, tissues and species. This paper briefly reviews the TRP superfamily and the basic properties of its many members as a readers guide in this Special Issue. Hopefully, a better understanding of TRP channel physiology will provide important insight into the relationship between TRP channel dysfunction and human diseases.     

Direct induction of tetraploids or homozygous diploids in the industrial yeast Saccharomyces cerevisiae by hydrostatic pressure

Summary Hydrostatic pressure and a dye plate method were used to investigate the direct induction of tetraploids or homozygous diploids from the industrial diploid or haploid yeast Saccharomyces cerevisiae. Above 200 MPa, hydrostatic pressure greatly inactivated the strains HF399s1 ( haploid), P-540 (a/ diploid), and P-544 (a/ diploid). At the same time, when pressure-treated cells of these strains were spread on a dye plate, some of the visible colonies were stained red/blue or dark blue (variant colonies); the rest stained violet, similar to colonies originating from diploid cells or haploid cells that were not pressure-treated. In addition, above 100 MPa, the formation of variant colonies increased with increasing pressure, and maximized (1x10^-1) at 200 and 250 MPa, respectively. The size of almost all variant cells from P-544, P-540, and HF399s1 was visibly increased compared with that of untreated cells and the measured cellular DNA content of P-540 and HF399s1 was double that of untreated cells. Furthermore, based on random spore analysis and mass-matings, induced variants in the diploid strains were found to be tetraploid with an a/a// genotype at the mating-type locus or, in the haploid strains, homozygous diploid with an / genotype. From these results we conclude that pressure treatment in combination with a dye plate is a useful method for strain improvement by direct induction of tetraploids or homozygous diploids from industrial strains whether diploid or haploids.     

3′-Deoxy-3′-fluoroinosine as a potent antileishmanial agent

We studied the antileishmanial activity of 3-deoxy-3-fluoroinosine (3-FI) againstLeishmania tropica andL. donovani. In in vitro cultivation, the EC₅₀ values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3-FI against the promastigotes ofL. tropica andL. donovani were 2.3×10^–7 and 1.0×10^–6 M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC₅₀ value was 1.9×10^–4 M. Leishmania promastigote metabolized 3-FI to 3-deoxy-3-fluoroadenosine 5-triphosphate (3-FATP) but FM3A cells did not. 3-FI was effective againstL. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3-FI (50 mg/kg, given i.v.)showed a cytotoxic effect against the amastigotes ofL. donovani in mice.     

Molecular Cloning and Functional Characterization of Human MIP-1δ, a New C–C Chemokine Related to Mouse CCF-18 and C10

We have isolated a novel human C–C chemokine, MIP-1, from a human fetal spleen cDNA library. The human MIP-1 cDNA has an unusually long 400-bp 5-prime untranslated region and a predicted 113-amino acid protein of 10 kDa. The coding sequence contains a signal peptide of 21 amino acids, indicating that the mature protein has 92 amino acids (8 kDa). Recombinant human MIP-1 produced by transfected human embryonic kidney 293 cells produced an 8-kDa protein, which confirmed the presence of a signal peptide. Compared with other human C–C chemokines, human MIP-1 shows the highest homology with human HCC-1, CK-8, murine C10, and CCF18 (MIP-1). The human MIP-1 gene is localized on chromosome 17 where most of the C–C chemokine superfamily is located. Human MIP-1 is expressed in T and B lymphocytes, NK cells, monocytes, and monocyte-derived dendritic cells, but not in bone marrow-derived dendritic cells. Its expression can be induced by other proinflammatory cytokines in monocytes and dendritic cells. Human MIP-1 is chemotactic for T cells and monocytes, but not for neutrophils, eosinophils, or B cells. Human MIP-1 induced calcium flux in human CCR1-transfected cells.     

Characterization of internal DNA-binding and C-terminal dimerization domains of human centromere/kinetochore autoantigen CENP-C in vitro: role of DNA-binding and self-associating activities in kinetochore organization

Human centromere protein C (CENP-C), a chromosomal component of the inner plate of kinetochores, was originally identified as one of the centromere auto-antigens. In a previous study, we showed that it possesses DNA-binding activity in vitro. Recently, centromere-binding activity was suggested at the C-terminal region in vivo. However, little is known about the role of CENP-C in kinetochore organization. Here, to characterize its biochemical properties, three separate antigenic regions of human CENP-C were expressed in Escherichia coli, affinity purified and used in South-western blotting and chemical cross-linking analyses. We found that the internal DNA-binding domain was composed of two kinds of elements: the core and two flanking stabilizing elements that support the activity. When cross-linked with disuccinimidyl suberate (DSS), the N-terminal region produced the ladder bands of dimerand tetramer: the C-terminal region exclusively produced the dimer band, whereas the internal region was not affected at all. Dimer formation at the C-terminus in the native state was also indicated by gel filtration and the presence of conformation-specific autoantibodies in the patient's sera. These results suggest that human CENP-C consists of three functional units required for kinetochore assembly: a putative N-terminal oligomerization domain, an internal DNA-binding domain and a C-terminal dimerization domain.This revised version was published online in November 2005 with corrections to the Cover Date.     

Correlations between in vitro effects of preparations of interferon and its inducers on blood cells in patients with multiple sclerosis

We studied in vitro production of interferon- and interferon- by peripheral blood leukocytes from 15 patients with multiple sclerosis. The priming effects of interferon preparations weakly correlated with interferon- production by leukocytes from patients with multiple sclerosis, but negatively correlated with interferon- production. The effects of interferon inducers in most cases positively correlated with its spontaneous production. We found a weak positive correlation between the priming effect of natural interferon- and the effect of recombinant interferons. There were positive or strong positive correlations between the effects of recombinant interferons on leukocytes from patients with multiple sclerosis. The relationship between the effects of medicinal interferon inducers and interferon preparation varied from negative to strong positive correlations. These data suggest that correlation analysis can be used for dynamic control and elaboration of methods for combined immunotherapy of multiple sclerosis with various interferon preparations or interferon and its inducers.     

Erythromycin and cycloheximide sensitivities of protein and RNA Synthesis in sporulating cells of Saccharomyces cerevisiae: Environmentally induced modifications controlled by chromosomal and mitochondrial genes

Summary The purpose of the experiments reported below was to examine the response in sporulation medium of the three diploid cell types MAT MAT, MAT MAT (asporogenic diploids) and MAT MAT (sporogenic diploid) to erythromycin, a specific inhibitor of mitochondrial protein synthesis (MPS) in vegetative cultures, and cycloheximide, a specific inhibitor of cytosol protein synthesis (CPS) in vegetative cultures. When MAT MAT diploids are transferred to sporulation medium a significant fraction of total protein synthesis (CPS + MPS) becomes sensitive to erythromycin in contrast to the behavior of MATa MATa and MAT MAT diploids in which the resistance of CPS to erythromycin is maintained. The decompartmentalization of erythromycin sensitivity is thus cell type specific. Erythromycin stimulates total RNA synthesis of MAT MAT cells in sporulation medium but not of MAT MAT and MAT MAT cells. Cycloheximide inhibits protein synthesis and stimulates RNA synthesis in all three diploid cell types. An erythromycin resistant mutant, shown to be due to a mutation of the mitochondrial genome, exhibited only partial resistance of CPS to erythromycin in sporulation medium in the background of the MAT MAT mating type genotype. Total RNA synthesis in this mutant was not stimulated. The results reported indicate that mitochondrial functions during sporulation are not restricted to those involving respiratory metabolism.     

Assignment of RFLP,RAPD and isoenzyme markers to Aspergillus nidulans chromosomes,using chromosome-substituted segregants of a hybrid of A. nidulans and A. quadrilineatus

Chromosome-substituted haploid segregants were selected from among the benomyl-induced progeny of an interspecific hybrid produced by polyethylene-glycol-induced fusion of protoplasts of an Aspergillus nidulans master strain and an A. quadrilineatus auxotrophic mutant. These segregants were examined by RFLP, RAPD, and isoenzyme analysis. The A. nidulans ribosomal repeat unit was assigned to chromosome V, while the benA and the pyrG genes were assigned to linkage groups VIII and I, respectively, of A. nidulans. None of the other cloned genes tested (gdhA, amdS and 25s rRNA) showed polymorphism between the two parents. The method was also used to assign RAPD markers and isoenzyme bands of -arylesterase, phosphatases, NAD-dependent malate dehydrogenase, and cellulase, to A. nidulans chromosomes and/or to their A. quadrilineatus equivalents. The isoenzyme and DNA sequences assigned to chromosomes could be used to saturate the genetic map of A. nidulans, or could serve as starting points for the construction of a genetic map of A. quadrilineatus. No method affording the same possibilities has been described so far in Aspergilli. This chromosome-assay method may be a useful alternative to pulsed-field-gel electrophoretic procedures for the assignment of molecular markers to chromosomes.     

Peripheral nerve stimulation increases fos immunoreactivity without affecting type II Ca2+/calmodulin-dependent protein kinase, glutamic acid decarboxylase, or GABAA receptor gene expression in cat spinal cord

Expression patterns of the immediate early gene c-fos and of other genes including those for the -subunit of type II Ca²⁺/calmodulin-dependent protein kinase (CaMKII), 67-kDa glutamic acid decarboxylase (GAD), and the 1-, 2-, and 2-subunits of the GABA_A receptor were described in the spinal cord of normal cats and following peripheral nerve stimulation. As revealed by in situ hybridization histochemistry, CaMKII messenger RNA (mRNA) is normally distributed only in cells of Rexed's laminae I–IV, whereas GAD mRNA is expressed by subpopulations of cells in all laminae, with the heaviest hybridization signal found in laminae I–III and medial parts of laminae V and VI. The three GABA_A receptor subunits have varying expression patterns in the laminae. All of them are expressed by many cells located in the base of the dorsal horn and the intermediate zone, but only the 2-subunit is intensely expressed by motoneurons. Single-pulse, electrical stimulation of the sciatic or median and ulnar nerves of anesthetized cats at a pulse rate of 1/s for 6–8 h failed to induce observable changes in gene expression for CaMKII, GAD, or for the three subunits of the GABA_A receptor; although immunoreactivity for the protein products of c-fos (or c-fos-related genes) was markedly upregulated in some neurons of the dorsal horn and the intermediate zone. Therefore, under the present experimental conditions, upregulation of the immediate early gene c-fos (or c-fos-related genes) is not associated with changes in expression of late-effector genes potentially involved in central nervous system plasticity.     

Examination of a purified A′ influenza virus preparation by potentiometry

Summary The potentiometric titration of a purified influenza A virus preparation revealed 100.7×10^–4 M base and 68.8 × 10^–4 M acid-binding capacity per g. of virus protein N. The titration curve was characterized by the following fourpK values:pK ₁ = 3.37;pK ₂ = 4.50;pK ₃ = 6.37, andpK ₄ = 9.75. The isoionic point was at pH 5.43.Attempt was made to identify the dissociating groups and it was found that the carboxylic groups (pK ₁ andpK ₂) may either be glutamyl or aspartyl groups, while the cationic groups are probably the imidazolium of histidine (pK ₃) and the -amino residues of lysine (pK ₄).Inaotivation of the hemagglutinating activity of the virus preparation by mild treatment with formaldehyde at pH 8.0 resulted in a simultaneous disappearence of the -amino groups of lysine (pK ₄). The same treatment at pH 9.0 resulted in the loss of all the cationic groups previously demonstrable.The possible role of the stable positive charges on the surface of the virus at physiological pH is discussed from the point of view of the physico-chemistry of the hemagglutination.     

A Neurospora crassa heat-shocked cell lysate translates homologous and heterologous messenger RNA efficiently,without preference for heat shock messages

Summary Cell-free protein synthesis systems were prepared from normally-grown (N-lysate) and heat-shocked (HS-lysate) Neurospora crassa mycelium. Although both lysates translated homologous mRNA, the HS-lysate was more active, yielding a higher incorporation of [³⁵S]-methionine into hot TCA-insoluble material and a vastly superior protein synthesis profile. The optimal temperature for translation by both lysates was 21 °C; the HS-lysate did not translate heat-shock mRNA preferentially at any temperature tested. Fortuitously, heterologous messenger RNAs from diverse eukaryotic and viral sources — Drosophila, dog pancreas, rabbit globin mRNA, brome mosaic virus, tobacco mosaic virus — were translated by the HS-lysate with an efficiency comparable to that of the commercial rabbit reticulocyte system and superior to the wheat germ system. The cap analogues, m⁷G(5)ppp(5)G and m⁷G(5)ppp(5)Gm, inhibited translation significantly.