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.     

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.     

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     

Imbalance of purine nucleotides in alanosine-resistant baby hamster kidney cells

Human DNA was used to transform adenosine kinase (AK)-deficient BHK cells followed by selection of AK⁺ cells in medium containing alanosine, adenosine, and uridine (AAV medium). Twenty AAU^r isolates were analyzed, and none of them contained AK activity. Several purine salvage enzymes were, however, found to be affected in these cells. The levels of hypoxanthine-guanine phosphoribosyltransferase and adenylosuccinate synthetase activities were elevated, while the adenylosuccinase activity was reduced. AAU-resistance may be explained by elevated activity of adenylosuccinate synthetase to overcome the alanosine block; thus AAU^r cells were able to convert exogenous adenosine inosine hypoxanthine IMP AMPS AMP. Moreover, these AAU^r cells required exogenous purines for growth. HPLC analyses of endogenous nucleotide pools of AAU^r cells showed that the levels of adenine nucleotides have diminished to less than 10% of the parental levels. These results suggest that the AAU-resistant mutation, which elicits pleiotropic phenotypes in BHK cells, affects an important component in the regulation of adenine nucleotide synthesis. By including erthyro-9-(2-hydroxy-3-nonyl)adenine in the AAU medium (renamed as AAUE medium) to block deamination of adenosine, AK⁺ BHK cells were isolated.     

Spontaneous mutations ataprt locus in a mammalian cell line defective in mismatch recognition

Clone B is a CHO cell line that showns a moderate mutator phenotype as a consequence of a defect in mismatch recognition. To identify the classes of mutation that accumulate spontaneously in a functional gene, we isolated and sequenced 54 clone B spontaneous mutants at the adenine phosphoribosyltransferase gene. This spectrum was compared to 42 mutants collected in the parental cells. Rates of ATTA transversions and frameshifts were strikingly increased in clone B (almost eight- and sixfold, respectively). Minor increases were also observed for GCTA transversions and GCAT transition rates. Frameshifts occurred in repeated sequences, and a large proportion were losses of 2 bases occurring in dinucleotide runs of a type similar to microsatellite sequences. ATTA transversions clustered in regions of secondary structure and their formation might be explained by slippage-mediated mechanisms. These data indicate that an important function of mismatch recognition is in repair of extrahelical bases generated by misalignment during DNA replication.This work was partially supported by a grant from the CNR/ACRO.     

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.)     

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.     

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.     

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.     

Different attenuated phenotypes of GM2 gangliosidosis variant B in Japanese patients with <Emphasis Type="Italic">HEXA</Emphasis> mutations at codon 499, and five novel mutations responsible for infantile acute form

Eight mutations of the subunit of -hexosaminidase A gene (HEXA) were identified in eight patients with GM2 gangliosidosis variant B. They were five missense mutations, two splice-site mutations, and one two-base deletion. Five of them, R252L (CGTCTT), N295S (AATAAC), W420C (TGGTGT), IVS 13, +2AC, and del 265–266AC (exon 2), were novel mutations responsible for infantile acute form of GM2 gangliosidosis. Two missense mutations, R499H and R499C, were found in one allele of two patients with attenuated phenotypes. The patient with R499C showed a late infantile form, and the other patient with R499H showed a juvenile form. These two mutations have been reported previously in the patients of other ethnic groups, and they have been known to cause attenuated phenotypes. The milder phenotypes of GM2 gangliosidosis variant B, different from the infantile acute form, have not been reported so far in Japan, and this is the first report of Japanese patients with attenuated phenotypes and their molecular analysis.This work was supported by the Foundation of Osaka Fellowship (AT, 2001) from Osaka City, Japan, and the grant AT-11557060 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

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.     

Altered expression of the steroid bioconverting pathway in pAN 7-1 transformants of Cochliobolus lunatus

Summary The filamentous fungus C. lunatus converts progesterone mainly to its 11-hydroxy derivative. C. lunatus transformed with the plasmid pAN 7-1, which contains the E. coli hph gene expressed under the control of the A. nidulans gpd and trpC expression signals, lacks this activity, but exhibits acetyl side chain degradation of progesterone through the reaction scheme progesterone20-hydroxy-progesterone ⁴-androstene-3,17-dione testolactone+testosterone. The main partof this metabolic pathway is not expressed in the non-transformed strain. It was determined that the site-specific integration of the plasmid into the genome directly influences the expression of genes involved in the bioconversion of steroids.     

Distribution of glycoconjugates in the optic vesicle and optic cup

Summary Lectin histochemical methods and immunohistochemical techniques have been utilized to investigate and partially characterize glycoconjugates in the developing eye. Peanut-lectin-binding sites associated with radial glial cells were found in the diencephalon. In the optic primordia, binding sites associated with radial glia were masked by terminal sialic acid, and only reacted with peanut lectin when pretreated with sialidase. This finding indicates that glycoconjugates associated with diencephalic radial glia contain terminal galactose--(13)N-acetyl galactosamine, but glycoconjugates associated with radial glia in the optic primordia contain sialic acidgalactose-(13)N-acetyl galactosamine. The selective distribution of galactose, N-acetyl galactosamine and fucose associated with radial glial cells has also been demonstrated. We postulate that these distributions mediate the shaping of the developing eye.This work was supported by NIH Grants # NS 11066 and # DE 05832     

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.)     

Partial fast-to-slow conversion of regenerating rat fast-twitch muscle by chronic low-frequency stimulation

Chronic low-frequency stimulation (CLFS) of rat fast-twitch muscles induces sequential transitions in myosin heavy chain (MHC) expression from MHCIIb MHCIId/x MHCIIa. However, the final step of the fast-to-slow transition, i.e., the upregulation of MHCI, has been observed only after extremely long stimulation periods. Assuming that fibre degeneration/regeneration might be involved in the upregulation of slow myosin, we investigated the effects of CLFS on extensor digitorum longus (EDL) muscles regenerating after bupivacaine-induced fibre necrosis. Normal, non-regenerating muscles responded to both 30- and 60-day CLFS with fast MHC isoform transitions (MHCIIb MHCIId MHCIIa) and only slight increases in MHCI. CLFS of regenerating EDL muscles caused similar transitions among the fast isoforms but, in addition, caused significant increases in MHCI (to 30% relative concentration). Stimulation periods of 30 and 60 days induced similar changes in the regenerating bupivacaine-treated muscles, indicating that the upregulation of slow myosin was restricted to regenerating fibres, but only during an early stage of regeneration. These results suggest that satellite cells and/or regenerating fast rat muscle fibres are capable of switching directly to a slow program under the influence of CLFS and, therefore, appear to be more malleable than adult fibres.     

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.     

Macrophage Stimulator β-(1→3)-D-Carboxymethylglucan Improves the Efficiency of Chemotherapy of Lewis Lung Carcinoma

We studied the effect of macrophage stimulator water-soluble -(13)-D-carboxymethylglucan on the efficiency of cyclophosphamide chemotherapy in Lewis lung carcinoma. Cyclophosphamide inhibited the growth of primary tumor nodes by 57%. The preparation possessed pronounced antimetastatic activity: metastases were found in 40.9% animals. Combination therapy with cyclophosphamide and (13)-beta;-D-glucan inhibited the growth of intramuscular tumors by 75-89% and reduced the incidence of metastases into the lungs by 92-94%. The therapeutic effect was most pronounced after simultaneous administration of these preparations: tumor growth was suppressed by 89.3% and metastases were found in only 7.5% animals (vs. 100% in the control). The potentiating effect of -(13)-D-carboxymethylglucan is related to accumulation of cysteine proteinase inhibitors in the tumor tissue and plasma, but not to changes in blood cell composition.     

Sequence specificity of mutations induced by benzo[a]pyrene-7,8-diol-9,10-epoxide at endogenousaprt gene in CHO cells

We have determined the spectrum of mutations induced, by±trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE) at the endogenous aprt locus in an hemizigous Chinese hamster ovary cell line exposed to 0.7 M BPDE. Southern analysis of 59 independent mutants revealed no major genomic alterations, indicating that gene inactivation was the result of a point mutation. This conclusion was confirmed by the cloning and sequencing of 21 of these mutants. The predominant mutation, the GCTTA transversion, comprised 62% of the spectrum, but other base pair substitutions and frameshifts were recovered. An examination of the target sequences for BPDE mutation revealed that mutations were localized within runs of GC base pairs. However, approximately half of these GC runs involved a particular sequence—a run of guanines flanked by adenine residues. Of seven such sites within the coding sequence ofaprt, mutations were clustered within five of them. This class of sequence occurs at codon 61 of the human C-Ha-ras1 protooncogene and may account for the selective activation of this codon by BPDE.     

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.