Gene Literature Dashboard

The 18th human chromosome is probably known mainly due to patients with Edwards syndrome, children dying usually during the first week of life, the syndrome being due to the presence of three chromosomes 18-trisomy. Some of its loci participate in oncogenesis either as cellular oncogenes (BCL2 in lymphomas), or antioncogenes (DCC--in colorectal carcinomas--Lynch I and Lynch II). It is assumed that some of the causes of maniac depression could be associated with chromosome no. 18.

The aim of this study was to investigate the interaction of tamoxifen (TAM) with the so-called Type II estrogen binding sites (Type II EBS) in both the cytosolic and the nuclear fraction of the ER-negative A 2780 human ovarian cancer cell line and in an ER-negative ovarian cancer tissue. Although cytosolic and nuclear Type II EBS in A 2780 cells showed substantially similar binding characteristics in terms of ligand affinity and specificity, TAM, while exhibiting the ability to displace [3H]estradiol from cytosolic Type II EBS failed to interact with nuclear Type II EBS. The ability of TAM to interact only with cytosolic Type II EBS seems also to be a characteristic of ovarian cancer tissue and to be shared by several TAM metabolites. The hypothesis that the interaction of TAM with cytosolic Type II EBS could mobilize the true endogenous ligand of Type II EBS which would become available for binding to nuclear Type II EBS was tested by incubating the nuclear fraction with the cytosolic fraction. In the presence of cytosol, TAM acquires the ability to displace the tracer from nuclear Type II EBS but when the cytosolic fraction was DCC, stripped in order to remove the endogenous ligand, the competing activity of TAM for nuclear Type II EBS was abolished. Our results suggest that TAM does not interact with nuclear Type II EBS, but can favor the nuclear binding of endogenous ligand by displacing it from cytosolic Type II EBS.

Synovial cell proliferation is one of the pathological bases of rheumatoid arthritis (RA). Several cytokines including IL-1 and IL-6 and growth factors have been shown to be involved in the synovial cell proliferation in RA. Thrombin is a multifunctional protease and acts as a mitogen for several cell types through its specific receptor. To assess whether thrombin is involved in overproliferation of rheumatoid synovial cells, we measured the concentration of thrombin-anti-thrombin III (ATIII) complex (TAT) in synovial fluid obtained from patients with RA or osteoarthritis (OA). We also examined the effect of thrombin or thrombin receptor agonist peptide (TRAP) on cell growth of synovial cell clones (SCCs) established from an RA patient. The concentrations of TAT in the synovial fluid from patients with RA were significantly higher than in those with OA. Moreover, both thrombin and TRAP enhanced proliferation of synovial cells in vitro. We also characterized the expression of thrombin receptor mRNA by reverse transcription-PCR. The expression of mRNA for thrombin receptor was up-regulated by thrombin or TRAP stimulation. Thrombin receptor antigen was also detected on both SCCs and synovial tissue from RA patients by immunostaining using a monoclonal antibody against thrombin receptor. These findings indicate that thrombin may act as a mitogen for synovial cells through thrombin receptor and may play some role in synovial overproliferation and remodeling in RA.

Urease (urea amidohydrolase; EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia and carbamate. The latter compound spontaneously decomposes to yield another molecule of ammonia and carbonic acid. The urease phenotype is widely distributed across the bacterial kingdom, and the gene clusters encoding this enzyme have been cloned from numerous bacterial species. The complete nucleotide sequence, ranging from 5.15 to 6.45 kb, has been determined for five species including Bacillus sp. strain TB-90, Klebsiella aerogenes, Proteus mirabilis, Helicobacter pylori, and Yersinia enterocolitica. Sequences for selected genes have been determined for at least 10 other bacterial species and the jack bean enzyme. Urease synthesis can be nitrogen regulated, urea inducible, or constitutive. The crystal structure of the K. aerogenes enzyme has been determined. When combined with chemical modification studies, biophysical and spectroscopic analyses, site-directed mutagenesis results, and kinetic inhibition experiments, the structure provides important insight into the mechanism of catalysis. Synthesis of active enzyme requires incorporation of both carbon dioxide and nickel ions into the protein. Accessory genes have been shown to be required for activation of urease apoprotein, and roles for the accessory proteins in metallocenter assembly have been proposed. Urease is central to the virulence of P. mirabilis and H. pylori. Urea hydrolysis by P. mirabilis in the urinary tract leads directly to urolithiasis (stone formation) and contributes to the development of acute pyelonephritis. The urease of H. pylori is necessary for colonization of the gastric mucosa in experimental animal models of gastritis and serves as the major antigen and diagnostic marker for gastritis and peptic ulcer disease in humans. In addition, the urease of Y. enterocolitica has been implicated as an arthritogenic factor in the development of infection-induced reactive arthritis. The significant progress in our understanding of the molecular biology of microbial ureases is reviewed.

We have cloned and sequenced a full-length cDNA for the HMG box-containing, SRY-related gene Sox6 from mouse. The deduced protein sequence of Sox6 has considerable homology with that of the previously determined Sox5 sequence. It seems likely that these genes have diverged more recently than other members of the SOX gene family, although the two genes map to different chromosomes in the mouse. In common with Sox5, Sox6 is highly expressed in the adult mouse testis and the HMG domains of both proteins bind to the sequence 5'-AACAAT-3'. This suggests that the two genes may have overlapping functions in the regulation of gene expression during spermatogenesis in the adult mouse. However, Sox6 may have an additional role in the mouse embryo, where it is specifically expressed in the developing nervous system.

Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.

The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntington. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cells from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.

The gene responsible for hereditary hemochromatosis has been shown to be closely linked to the HLA-A and D6S105 loci on the short arm of chromosome 6. Efforts at mapping the disease gene have been hindered, however, by a lack of informative recombinant in this region. We have identified two recombinant individuals in a single affected family and have confirmed recombination by analysis of 16 polymorphic markers located near HLA-A and D6S105. One of the recombinants provides evidence for the location of the hemochromatosis gene telomeric to HLA-F.

In previous studies, the prevalence of HLA-linked hemochromatosis, thought to be the most common genetic illness in whites, has been estimated by identifying homozygotes in the population. Because not all homozygotes express the disease phenotypically, the accuracy of these estimates is uncertain. We analyzed the distribution of transferrin saturation values in the second National Health and Nutrition Examination Survey to estimate the prevalence of hemochromatosis heterozygotes in the US population. After removing values for possible homozygotes, two populations were present (P < .01 for each gender). When weighted to reflect the US adult white male population as a whole, a proportion of 850 per 1,000 (95% confidence interval, 0.81 to 0.89) were included in a population with a lower mean saturation of 29.7% (29.1% to 30.3%), whereas 150 per 1,000 (0.11 to 0.19) comprised a population with a higher mean saturation of 47.0% (45.1% to 49.0%). Similar results were found for the female population. The gene frequencies were estimated to be 0.081 from the male population and 0.070 from the female population corresponding to prevalences of homozygotes of 6.6 and 4.8 per 1,000, respectively. Our results confirm that the gene for hemochromatosis is common.

Iron-responsive element-binding protein (IRE-BP) activity was studied in liver and intestinal samples of hemochromatosis and control patients using a short 32P-IRE-RNA probe on "retardation" nondenaturing polyacrylamide gels. IRE-BP activity was assessed in liver biopsy specimens in 36 patients--16 hemochromatosis homozygotes, 4 hemochromatosis heterozygotes, 6 patients with secondary iron overload, and 10 control patients with normal hepatic iron concentrations. Intestinal IRE-BP activity was assessed in 14 hemochromatosis homozygotes and 16 normal subjects. Endogenous IRE-BP activity was determined from 32P retarded on the gel, and total IRE-BP activity was assessed after reducing tissue samples with 2-mercaptoethanol. Hepatic endogenous IRE-BP activity was inversely related to hepatic iron concentration (r = .59, P < .0002). Mean hepatic endogenous IRE-BP activity in the hemochromatosis homozygotes, 0.25 +/- 0.04 pmol/mg protein, was significantly decreased compared with values in the normal controls, 0.45 +/- 0.06 pmol/mg protein, P < .05. Hepatic total IRE-BP was also significantly decreased in the hemochromatosis patients by gel retardation assay and Western blotting with anti-IRE-BP antibody. Intestinal endogenous IRE-BP activity, total IRE-BP activity, and iron concentration did not significantly differ between hemochromatosis patients and normal control subjects. This suggests that both endogenous IRE-BP activity and the total amount of the protein are downregulated in the liver by tissue iron. Intestinal IRE-BP activity that regulates intestinal transferrin receptor expression is normal in hemochromatosis and appropriate for the intracellular iron concentration.

alpha-, beta-, and gamma-tubulins are evolutionarily highly conserved members of the tubulin gene superfamily. While the abundant members, alpha- and beta-tubulins, constitute the building blocks of cellular microtubule polymers, gamma-tubulin is a low abundance protein which localized to the pericentriolar material and may play a role in microtubule assembly. To test whether gamma-tubulin mediates the nucleation of microtubule assembly in vivo, and co-assembles with alpha- and beta-tubulins into microtubules or self-assembles into macro-molecular structures, we experimentally elevated the expression of gamma-tubulin in the cell cytoplasm. In most cells, overexpression of gamma-tubulin causes a dramatic reorganization of the cellular microtubule network. Furthermore, we show that when overexpressed, gamma-tubulin causes ectopic nucleation of microtubules which are not associated with the centrosome. In a fraction of cells, gamma-tubulin self-assembles into novel tubular structures with a diameter of approximately 50 nm (named gamma-tubules). Furthermore, unlike microtubules, gamma-tubules are resistant to cold or drug induced depolymerization. These data provide evidence that gamma-tubulin can cause nucleation of microtubule assembly and can self-assemble into novel tubular structures.

The gene on chromosome 10 at band p12 (AF10), involved in the t(10;11) translocation in acute myeloid leukemia, has been identified and shown to contain conserved zinc finger and leucine zipper domains. These regions are highly homologous to the equivalent regions on AF17, the gene involved in the t(11;17) translocations. A series of adult, childhood, and infant leukemias with either simple or complex versions of the t(10;11) has been examined by Southern analysis and shown to involve rearrangement to the HRX locus. Reverse transcriptase-polymerase chain reaction from either bone marrow or peripheral blood cells showed that HRX sequence was fused to AF10 sequence in all 8 cases and subsequent sequence analysis showed an in-frame fusion between the HRX and AF10 sequence. A consistent feature of these fusions was the juxtaposition of the leucine dimerization motif of AF10 onto the NH2-terminal region of HRX. The published data suggest that a similar conclusion can be drawn about the t(11;17) translocation, implying a critical role for this motif in the chimaeric HRX protein.

Four kinds of calcium phosphate ceramics, beta-calcium pyrophosphate (Ca2P2O7), beta-tricalcium phosphate (Ca3(PO4)2), hydroxyapatite (Ca10(PO4)6(OH)2) and tetracalcium phosphate (Ca4(PO4)2O), were prepared. The calcium/phosphorus molar ratios were 1, 1.5, 1.66 and 2, respectively. Particles (150-300 microns) of these ceramics were packed into holes (diameter 2.5 mm) made in the tibial metaphysis of mature male rats. At 2 weeks, 4 weeks, 8 weeks and 6 months after the operation, undecalcified specimens were prepared. Transmission electron microscopy showed that the bone-bonding behaviour of calcium phosphate ceramics at the interface with bone did not vary with the calcium/phosphate molar ratio. Amorphous substances or needle-like microcrystals were observed on the surface of the ceramics at 2 weeks after implantation. The ceramics showed direct continuity with small crystallites of bone tissue at 4 weeks, 8 weeks and 6 months after implantation. The ceramics appeared to be getting smaller with time. Collagen fibres were not observed at the bone/ceramic interface. Neither chemical bonding nor mechanical bonding by interlocking between bone and ceramics was described by morphological observation using transmission electron microscopy.

Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17 beta, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [3H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [3H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [3H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [3H]thymidine and were unresponsive to hormones. Estradiol-17 beta increased [3H]thymidine incorporation slightly at 10(-10) mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [3H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17 beta. In the absence of estradiol, progesterone inhibited [3H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17 beta was present, progesterone stimulated [3H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [3H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth.

<h4>Background</h4>Tyr-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and Tyr-Tic-Ala were the first peptides with delta opioid antagonist activity lacking Phe, considered essential for opioid activity based on the N-terminal tripeptide sequence (Tyr-D-Xaa-Phe) of amphibian skin opioids. Analogs were then designed to restrain the rotational flexibility of Tyr by the substitution of 2,6-dimethyl-L-tyrosine (Dmt).<h4>Materials and methods</h4>Tyr and Dmt peptides were synthesized by solid phase and solution methods using Fmoc technology or condensing Boc-Dmt-OH or Boc-Tyr(But)-OH with H-L-Tic-OBut or H-D-Tic-OBut, respectively. Peptides were purified (> 99%) by HPLC and characteristics determined by 1H-NMR, FAB-MS, melting point, TLC, and amino acid analyses.<h4>Results</h4>H-Dmt-Tic-OH had high affinity (Ki delta = 0.022 nM) and extraordinary selectivity (Ki mu/Ki delta = 150,000); H-Dmt-Tic-Ala-OH had a Ki delta = 0.29 nM and delta selectivity = 20,000. Affinity and selectivity increased 8700- and 1000-fold relative to H-Tyr-Tic-OH, respectively. H-Dmt-Tic-OH and H-Dmt-Tic-NH2 fitted one-site receptor binding models (eta = 0.939-0.987), while H-Dmt-Tic-ol, H-Dmt-Tic-Ala-OH and H-Dmt-Tic-Ala-NH2 best fitted two-site models (eta = 0.708-0.801, F 18.9-26.0, p < 0.0001). Amidation increased mu affinity by 10- to 100-fold and acted synergistically with D-Tic2 to reverse selectivity (delta-->mu). Dmt-Tic di- and tripeptides exhibited delta antagonist bioactivity (Ke = 4-66 nM) with mouse vas deferens and lacked agonist mu activity (> 10 microM) in guinea-pig ileum preparations. Dmt-Tic analogs weakly interacted with kappa receptors in the 1 to > 20 microM range.<h4>Conclusions</h4>Dmt-Tic opioidmimetic peptides represent a highly potent class of opioid peptide antagonists with greater potency than the nonopioid delta antagonist naltrindole and have potential application as clinical and therapeutic compounds.

Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with 3H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components.

<h4>Background</h4>Iron overload in patients with thalassemia is a common feature which requires continuous chelation therapy and monitoring. Serum ferritin determination is widely accepted as a simple method for following iron load in patients with primary hemochromatosis; however, several reports on thalassemic patients emphasize that ferritinemia is not accurate and that other methods such as direct measurement of iron in the liver (HIC) and magnetic resonance imaging (MRI) are more precise.<h4>Materials and methods</h4>In order to contribute to the general understanding of iron load in thalassemia we used liver MRI to study 33 thalassemic patients, most of whom were also evaluated for iron content by liver biopsy. The data were then compared with serum ferritin levels.<h4>Results</h4>Ferritin levels ranged between 276 and 8031 ng/mL, and liver iron content ranged from 1.6 to 31.0 mg/g dry weight; grade III or IV liver siderosis was recorded in 23/33 patients, just as 23/33 patients were found to have severe or very severe siderosis at MRI. Significant correlations with ferritin levels were recorded between grade IV and grades III, II and I (p < 0.01, p = 0.02, and p = 0.03, respectively). Ferritinemia also showed significant linearity with liver iron content (r = 0.603, p = 0.001). No significant differences of levels were recorded, however, between patients found to have severe and those with mild iron load at MRI (p = 0.073).<h4>Conclusions</h4>Our study shows that serum ferritin levels exhibit a tendency to be significantly correlated with the true status of hemochromatosis in thalassemic patients; however, the discrepancies recorded in several patients and the scarce or total lack of correlation with MRI suggest exploring other approaches to this problem in order to make proper decisions about therapy.

In order to specifically modify the fatty acid composition of cell membrane phospholipids, we have developed an original method based on the transfer of pure phospholipid molecular species to membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) subclasses containing 18:2n-6 and 22:6n-3 at the sn-2 position were incorporated into human platelet membranes using the endogenous phosphatidylinositol/PC transfer protein (PI/PC-TP) and the phospholipid transfer protein from maize (L-TP), respectively. PI/PC-TP was shown to catalyze a strict exchange of phospholipids between platelet membranes and unilamellar vesicles containing 1,2-diacylglycerophosphocholine (diacyl-GPC; 16:0/18:2-GPC, or 16:0/22:6-GPC). The proportions of 18:2n-6 and 22:6n-3 in diacyl-GPC of platelet membranes were gradually increased from 10.7 to 16.9% and from 0.8 to 10.1%, respectively, whereas the PE and PI fatty acid compositions were not changed. The diacyl-GPC enrichment in 22:6n-3 and 18:2n-6 did not induce changes in membrane fluidity parameters measured by electron-spin resonance of 5- and 16-nitroxy stearic acids. Similarly, 18:2n-6 and 22:6n-3 esterified in 1,2-diacylglycerophosphoethanolamine (diacyl-GPE) have been incorporated in platelet membranes by an apparent exchange process under conditions where donor vesicles had a phospholipid composition equivalent to that of platelet membranes. The proportions of 18:2n-6 and 22:6n-3 were selectively and progressively increased from 6.0 to 21.2% and from 2.2 to 17.2%, respectively, in diacyl-GPE of platelet membranes. Thus, the L-TP- and PI/PC-TP-catalyzed enrichment can be used for studying the modulation of membrane biological activities by defined changes of fatty acid composition of specific phospholipid classes or subclasses.

An antiserum to peptide containing 15 amino acids corresponding to the region-D of human estrogen receptors (hERD) was obtained in mice by immunization with the peptide conjngated to KLH. Using this antiserum, the ER status of paraffin-embeded sections of 95 human breast carcinomas (in which, sections of 31 were both frozen and paraffin-embeded ones) were studied. The corresponding rate for determination of ER status between immunohistochemical staining (IHC) and dextran coated charcoal (DCC) assay was 89.5%. The concordance rate for semiquantitative gradings was 69.3%. In addition, in situ hybridization (ISH) of 15 frozen sections of the samples using digoxigenin labeled dUTP to identify the expression of ER mRNA was also done. The result of ISH was fully consistent with that of IHC (100%). The results show that the mouse antiserum to hERD obtained in this study is specific and sensitive for IHC assay of ER and IHC is a valuable adjunct and/or alternative to the biochemical method for determination of the ER status of breast cancer.