Spectrum of spontaneous missense mutations causing cyclic AMP-resistance phenotypes in cultured S49 mouse lymphoma cells differs markedly from those of mutations induced by alkylating mutagens

Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mutagen-induced isolates by sequence analysis of amplified cDNAs. Twenty-nine single basepair substitutions in 19 codons produced selectable phenotypes. The spontaneous mutant spectrum was dominated by a CpG transition hotspot in the codon for Arg334. This and other nearby CpG sites were found to be methylated in genomic S49 cell DNA by restriction enzyme analyses. Most of the remaining spontaneous mutants had either G-CC-G or T-AG-C transversions, which have been associated with damage caused by oxygen radicals. In contrast, the majority of mutants induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine had G-CA-T mutations at non-CpG sites; in addition, EMS induced several A-TG-C, A-TT-A, and G-CT-A substitutions. A single ICR191-induced mutant analyzed had a unique A-TG-C lesion. A number of spontaneous and mutagen-induced isolates had closely linked double or triple substitutions, and two isolates had tandem triple substitutions.     

The role of acidic residues in the “fusion segment” of influenza A virus hemagglutinin in low-pH-dependent membrane fusion

Summary To clarify the role of acidic amino acid residues in the fusion segment of hemagglutinin (HA) of influenza A virus (H1N1) in pH-dependent membrane fusion, we have constructed and expressed five mutant HA cDNAs in CV-1 cells by SV40-HA virus vectors (SVHA). Fusion activities of the five mutant HAs were examined by lipid mixing and polykaryon formation assays. In spite of the substitution of Gly and Lys for the acidic residues, all the mutants were found to retain their low-pH-dependent fusion activity by lipid mixing assay. Although SVHA-G19(HA₂19D G), –K11 (HA₂11E K) and –K19(HA₂19D K) induced polykaryon formation at low pH as wild type HA did, SVHA-G11(HA₂11E G) induced limited polykaryon formation and SVHA-G11,19 (HA₂11E G, 19D G) did not. The substitution of Gly for Glu at position 11 inhibited widening of the initial fusion pore. However, Lys mutants induced the formation of an initial fusion pore and widened it at low pH where Lys residues might have positive charges. These results suggest that the neutralization of the charges on acidic residues in the fusion segment at low pH is not important for interaction of the fusion segment with the target lipid bilayer or for triggering the membrane fusion.     

A Matrix Associated Region Localizes the Human SOCS-1 Gene to Chromosome 16p13.13

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3 end of the human PRM1 PRM2 TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3 of the spermatid-expressed PRM1 PRM2 TNP2 domain at position 16p13.13.     

Cognitive mediators of the social influence-exercise adherence relationship: A test of the theory of planned behavior

The purpose of this study was to examine cognitive constructs from the theory of planned behavior (i.e., attitude, perceived behavioral control, and intention) as potential mediators of the relationship between selected social influence constructs (i.e., subjective norm, social support, and cohesion) and adherence to structured exercise classes. Sixty-two participants completed self-administered questionnaires during the fourth week (social influence constructs) and eighth week (cognitive constructs) of a 12-week exercise program. Exercise adherence was monitored during weeks 9 through 12 using perceived intensity and attendance. Pearson correlations indicated that social support correlated with perceived behavioral control, whereas cohesion correlated with attitude. Path analysis supported two distinct paths from social influence to exercise adherence: (a) social support perceived behavioral control intention excersise adherence, and (b) cohesion attitude intention exercise adherence. Discussion focuses on the theoretical importance of these findings, conceptual and measurement issues regarding subjective norm, and suggestions for future research.     

Six novel UDP-glucuronosyltransferase (UGT1A3) polymorphisms with varying activity

Human UDP-glucuronosyltransferase (UGT) is a part of a major excretion pathway for endobiotics and xenobiotics. The UGT family of genes is highly polymorphic, and our aim is to describe novel polymorphisms at the UGT1A3 locus and determine how they alter substrate metabolism and drug reactions. One hundred healthy Japanese adults volunteered for the present study. We sequenced PCR-amplified fragments of the gene directly, and calculated the frequency of the genetic variations detected. To measure variant enzyme activity, we constructed five expression models and used estrone as the substrate in the assays. We identified six novel single nucleotide polymorphisms (SNPs). Of these, four caused amino acid substitutions (17AG: Q6R, 31TC: W11R, 133CT: R45W, and 140TC: V47A) and the remaining two were silent (81GA: E27E and 447AG: A159A). We found five types of alleles having differing SNP combinations: wild type (frequency=0.61), W11R-E27E-A159A (0.10), Q6A-W11R-E27E-A159A (0.055), W11R-E27E-V47A-A159A (0.125), and R45W (0.11). Expression studies found that the variants changed the enzyme efficiencies (K _m/V _max) to 121% of the wild type for W11R, 86% for Q6R-W11R, 369% for W11R-V47A, and 70% for R45W. Several UGT 1A3 polymorphisms exist in the Japanese population, having different levels of activity. These polymorphisms are capable of affecting the steady state levels of estrogens, and may increase sensitivity to adverse drug effects.The accession numbers of new nucleotides: AB120359, AB120360, AB120361, AB120362.     

Intrachromosomal gene mapping in man: The gene for tryptophanyl-tRNA synthetase maps in region q21→qter of chromosome 14

A gene for tryptophanyl-tRNA synthetase (EC 6.1.1.2), the enzyme which attaches tryptophan to its tRNA, has previously been assigned to human chromosome 14 by analysis of man-mouse somatic cell hybrids. We report here a method for the electrophoretic separation of Chinese hamster and human tryptophanyl-tRNA synthetases and its application to a series of independently derived Chinese hamster-human hybrids in which part of the human chromosome 14 has been translocated to the human X chromosome. When this derivative der (X),t(X;14) (Xqter Xp22::14q21 14qter) chromosome carrying the human gene for hypoxanthine-guanine phosphoribosyltransferase was selected for and against in cell hybrid lines by the appropriate selective conditions, the human tryptophanyl-tRNA synthetase activity was found to segregate concordantly. These results provide additional confirmation for the assignment of the tryptophanyl-tRNA synthetase gene to human chromosome 14 and define its intrachromosomal location in the region 14q21 14qter. Our findings indicate that the genes for tryptophanyl-tRNA synthetase and for ribosomal RNA are not closely linked on chromosome 14.     

Spatial properties of the visual detectability of moving spatial white noise

Summary We determined the spatial parameters that describe the visual detection of spatio-temporal correlation in moving two-dimensional noise patterns. The target field (5.21×5.31 deg of visual angle) was divided into horizontal stripes of equal width D. Adjacent bars alternately contained noise patterns moving with velocity v₁ and v₂. We varied D, v₁ and v₂.Roughly three different percepts occurred. If the stripes were very broad the different movements in alternate stripes were perceived together with the division of the field into stripes. If the stripes were very narrow the division into stripes was not seen, but the moving noise patterns with velocities v₁ and v₂ were perceived as transparent sheets moving through each other. For intermediate stripe widths the target field looked incoherent and the subject was not clear about the percept. In this region the subject found it difficult and sometimes impossible to discriminate these patterns from a completely uncorrelated spatiotemporal white noise pattern (snow).To quantify the detectability, the patterns were masked with snow (spatio-temporal white noise). The r.m.s. contrast of the total stimulus was kept at a constant value, whereas the subject set the signal-to-noise ratio (SNR) to a threshold value. At certain barwidths the thresholds reached a maximum value. These critical barwidths depended on the velocities v₁ and v₂.These critical barwidths were interpreted in terms of a simple general model for the detection of spatiotemporal correlation. In these terms the span of the elementary correlators rose monotonically with the velocity to which the correlator is most sensitive.Supported in part by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)     

Tumour necrosis factor α production by rat blood and itsex vivo pharmacological modulation

Rat blood was investigated as a suitable test system for the discovery of inhibitors of tumour necrosis factor (TNF) biosynthesis. Lipopolysaccharide (LPS) caused a concentration- and time-dependent stimulation of TNF production by heparinised rat blood with peak levels (1000–5000 U/ml; L929 bioassay) at 6h. Bioactive material was neutralised with a polyclonal rabbit anti-murine TNF antibody which cross-reacts with rat TNF. Dexamethasone, pentoxifylline and denbufylline inhibited TNF production with IC₅₀s of 6.0±2.0 nM, 20.6±8.00 M and 138.0 nM, respectively. When rats were dosed p.o. with dexamethasone or pentoxifylline or i.p. with denbufylline and 1.5h later TNF production was assessedex vivo by LPS-stimulated blood, a dose-related inhibition of TNF production occurred with ID₅₀s of approximately 0.08, 250.0 and 5.0 mg/kg, respectively. These results demonstrate that rat blood provides a useful test system for the detection andex vivo evaluation of inhibitors of TNF biosynthesis.     

Association of polymorphisms of paraoxonase 1 and 2 genes,alone or in combination,with bone mineral density in community-dwelling Japanese

Oxidative stress may affect cellular functions in various pathological conditions, including osteoporosis. Paraoxonase 1 confers antioxidant properties on high-density lipoprotein, with which it is associated, by reducing the accumulation of lipid peroxidation products. We have now examined whether the 584AG (Gln192Arg) and 172TA (Leu55Met) polymorphisms of the paraoxonase 1 gene and the 959GC (Cys311Ser) polymorphism of the paraoxonase 2 gene are associated with bone mineral density (BMD) in community-dwelling Japanese (1,087–1,094 women and 1,112–1,125 men). The subjects were aged 40 –79 years and were randomly recruited to a population-based prospective cohort study of aging and age-related diseases. BMD for the lumbar spine and right femoral neck was measured by dual-energy X-ray absorptiometry. Genotypes were determined with a fluorescence- or colorimetry-based allele-specific DNA primer-probe assay system. The 584AG and 172TA polymorphisms of the paraoxonase 1 gene and the 959GC polymorphism of the paraoxonase 2 gene were associated with BMD for the lumbar spine or femoral neck in postmenopausal women, with the 584GG, 172TT, and 959CC genotypes representing risk factors for reduced bone mass. None of these three polymorphisms was associated with BMD in premenopausal women or in men. Our results suggest that the paraoxonase 1 and 2 genes are candidate loci for reduced bone mass in postmenopausal Japanese women.     

Counts of Stromal Precursor Cells in Heterotopic Splenic Transplants in CBA Mice of Different Age

The content of stromal precursor cells in heterotopic splenic transplants from old and young mice changed appreciably after cross transplantation to old and young animals. The content of CFC-F in the youngold transplants decreased almost 1.5 times in comparison with the youngyoung transplants, the counts of CFC-F in oldold transplants were minimum in comparison with all other groups (2.5±0.1), while in the old young group transplants this value increased almost 8-fold (to 19.0±1.3) and surpassed the control level. Age-associated shifts in the splenic stromal tissue were determined by regulatory influences of the host, rather than by decreased count of stromal precursor cells in the tissue.__________Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 139, No. 2, pp. 196–198, February, 2005     

Polymerase chain reaction-single strand conformation polymorphism analysis of the p53 gene in paraffin-embedded surgical material from human renal cell carcinomas

p53 tumour suppressor gene mutations were studied in 118 renal cell carcinomas using paraffin-embedded surgical material. Optimal results were obtained with analysis of exon lengths between 150 and 200 base pairs for polymerase chain reaction. Single strand conformation polymorphism and sequencing analysis revealed only two point mutations (2/118, 2%): one involving codon 135; TGCTTC (cysteinephenylalanine) and the other codon 175; CGCCAC (argininehistidine). Both of these cases were classified as granular cell subtype on microscopic observation. The data suggest that the p53 tumour suppressor gene is not related to tumour initiation, promotion, or progression of renal cell carcinomas. However, there is the possibility that granular cell type carcinomas may have a different genetic background from clear cell type renal neoplasms.     

Temporal properties of the visual detectability of moving spatial white noise

Summary We obtained movement detection thresholds for two-dimensional random speck-patterns (Julesz patterns) homogeneously moving over the whole target field (5.21×5.31 degrees of visual angle). We alternated between two uncorrelated but otherwise similar patterns, one moving with velocity v₁, the other with velocity v₂, such that each pattern was on for T ms. We masked this pattern (signal) with spatio-temporal white noise (snow). The total r.m.s. contrast was kept constant, whereas the ratio of the r.m.s. contrasts of signal and noise was varied. The square of this ratio was designated SNR.At low SNR values the pattern was not perceptually different from the snow alone. At high SNR values the subject detected spatio-temporal correlation (e.g., movement). In these experiments we determined the threshold SNR values as a measure of the detectability of spatio-temporal correlation as a function of the parameters T, v₁ and v₂.When v₁ and v₂ were sufficiently dissimilar one of three percepts occurred: for very large T the alternation could be followed, for very small T two transparent, simultaneously moving sheets of noise-pattern with different velocities could be seen. For intermediate T-values no systematic movement at all could be observed. At these T-values the threshold SNR was maximal. This critical T-value decreased with increasing velocity.We found that it was possible to have more than one percept of uniform smooth movement at a single location in the visual field if these movements had velocity vectors with an angular difference of at least 30 deg or if their magnitudes differed by at least a factor of 4.Supported in part by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)     

Die s?ulenchromatographische Fraktionierung der sauren Mucopolysaccharide im Harn

Zusammenfassung Es wurde eine säulenchromatographische Methode beschrieben, die es gestattet, Heparitinsulfat (Heparansulfat) (HMS), Chondroitinsulfat A und C [Chondroitin-4-Sulfat (CSA), Chondroitin-6-Sulfat (CSC)], Chondroitinsulfat B (Dermatansulfat) (CSB) und Keratosulfat (Keratansulfat) (KS) deutlich voneinander zu trennen und einzeln colorimetrisch (z. B. mit der Carbazol- und/oder Anthron-Reaktion) zu bestimmen. Als Trägersubstanz diente Dowex 1 × 2. Die Elution erfolgte mit NaCl-Lösungen steigender Konzentration: 0,5 m, 1,25 m (HMS); 1,5 m (CSA, CSC); 2,0 m (CSB); 3,0 m (KS).Die Vollständigkeit der Trennung wurde durch Bausteinanalyse der erhaltenen Elutionsgipfel (Bestimmung des Hexuronsäure-, Hexosamin-, Neutralzukkergehaltes) und Papierchromatographie geprüft und mit gereinigten sauren Mucopolysacchariden (SMP) verglichen.Für die Anwendung der Dowex-Säulenchromatographie zur Ermittlung des SMP-Musters im menschlichen Harn wurde folgender Arbeitsgang vorgeschlagen: Dialyse des 24 h-Harnes gegen 0,9%ige NaCl-Lösung Fällung der Gesamt-SMP mittels Cetyltrimethylammoniumbromid Waschen des Präcipitates mit NaCl-gesättigten 95%igen Äthanol DowexSäulenchromatographie colorimetrische Bestimmung des Hexuronsäure- und Hexosegehaltes jeder Fraktion.Bei Normalpersonen konnte mit dieser Methode HMS, CSA, CSB und KS im Harn nachgewiesen werden. CSA stellte die Hauptmenge dar.

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Summary A column chromatographic method using Dowex 1 × 2 is described which clearly separates heparitin sulfate (HMS), chondroitin sulfate A and C (CSA and CSC), chondroitin sulfate B (CSB) and keratosulfate (KS) from a mixture of acid mucopolysaccharides (AMPS). Subsequently, these compounds are individually measured by colorimetric assays.The elution of AMPS is achieved by NaCl-solutions of increasing concentrations: 0,5 M, 1,25 M (HMS); 1,5 M (CSA, CSC); 2,0 M (CSB); 3,0 M (KS).In order to examine the completeness of chromatographic separation aliquots of each peak were subjected to paper chromatography. The contents of hexuronic acid, hexosamine and neutral sugar were determined by appropriate color reactions and compared to values obtained with purified AMPS.For the determination of AMPS patterns in normal human urine on the basis of the described Dowex column chromatography the following procedure was used: dialysis of 24h urine against normal saline precipitation of total AMPS material with cetyltrimethylammoniumbromide washing of precipitate with 95% ethanol saturated with NaCl column chromatography on Dowex 1 × 2 colorimetric determination of hexuronic acid and neutral sugar contents of each fraction.Normal persons reveled an urinary excretion of HMS, CSA, CSB and KS, with CSA being the main compound.

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Mit dankenswerter Unterstützung der Deutschen Forschungsgemeinschaft, Bad Godesberg.Auszugsweise vorgetragen auf der 3. Arbeitstagung für pädiatrische Forschung, Marburg a. d. Lahn, 25./26. Februar 1966.     

Influence of central neuronal histamine on the pituitary-adrenocortical activity stimulated by neurotransmitters

The effect of brain neuronal histramine and its receptors on monoaminergic stimulation of the hypothalamic-pituitary-adrenal (HPA) activity, measured indirectly through corticosterone secretion, was investigated in conscious rats. Pretreatment with -fluoromethylhistidine, (-FMH) a histamine synthesis inhibitor, did not markedly affect the increase in serum corticosterone levels induced by intracerebroventricular (icv) injection of muscimol, a GABA_A receptor agonist and noradrenaline, an - and -adrenergic agonist, and slightly diminished the corticosterone response to isoprenaline, a -adrenergic agonist. -FMH totally abolished the increase in serum corticosterone induced by carbachol, a cholinergic muscarinic receptor agonist and significantly diminished the rise in corticosterone levels induced by clonidine, an ₂-adrenergic agent. Pretreatment with the histamine receptor antagonists mepyramine and cimetidine also considerably reduced the carbachol-induced corticosterone response and the response induced by clonidine.These results indicate that brain neuronal histamine is considerably involved in stimulation of the HPA axis by cholinergic muscarinic and ₂-adrenergic agonists, but not by GABA_A and ₁- and -adrenergic agonists.     

Identification of genetic alterations associated with the process of human experimental colon cancer liver metastasis in the nude mouse

Understanding the genetic elements controlling the process of tumor metastasis to distant organ sites such as the liver may be the key to improving survivorship from colon cancer. By using standard cytogenetic techniques in combination with comparative genomic hybridization, multiple genetic imbalances within three human colon cancer cell lines previously selected for differences in liver-metastatic behavior were identified. The entire genome of one poorly metastatic cell line (KM12C) was compared directly with that of two highly metastatic cell lines (KM12SM, KM12L4A) derived from it. A number of chromosomal gains (8q, 12g15, 20q11.2) and losses (5p13, 6p21.3, 18) were common to all three cell lines and are likely related to early tumor development rather than to the selection process used to generate cell lines of increased metastatic potential. Chromosomal imbalances detected only in the highly metastatic cell lines were also observed. KM12SM showed losses of portions of 2p22, 2824.32q32.2, 4p15.3cen, 4q24 without the 13q and 15q22.3 gains noted for KM12C. Both gains (1p31.31p21, 28222q33, 3cen3q26.2, 5q145q23, 6cen6q23) and losses (16p, 17p, 17q, 19p, 19q, 22q) were observed for KM12L4A but not for the other two cell lines. Identification of these alterations provides valuable insight into the process of experimental liver metastasis and is a first step towards mapping genes linked to the terminal phases of human colon cancer progression.     

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

Summary We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3 termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I fromEscherichia coli, which has 35 exonuclease and replacement activities as well as its main 53 polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.     

The effects of thyroid status on some properties of rat fast-twitch muscle

Summary The effects of different thyroid states on some histochemical and biochemical properties of fast-twitch muscle were studied using rat extensor digitorum longus (EDL) muscle. This muscle was found to be much less responsive to thyroidal influence than the slow-twitch soleus muscle. In EDL muscles of hypothyroid rats, fast slow conversions were observed in fibre type composition, myosin ATPase activity and light chain pattern, and in the subunit composition of lactate dehydrogenase, while the only significant slow fast conversion observed in thyrotoxicosis was a decrease in the proportion of slow-oxidative fibres. Denervation of the hypothyroid muscle produced the highest degree of fast slow transformation. These findings support the view that denervation and dysthyreosis alter gene expression in muscle by independent mechanisms.