Germline mutations in the p16INK4a binding domain of CDK4 in familial melanoma.
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Base Sequence↗
Biomedical subjects
L Zuo
Publications and source records attributed to L Zuo.
Long-range sequence analysis in Xq28: thirteen known and six candidate genes in 219.4 kb of high GC DNA between the RCP/GCP and G6PD loci.
DNA comprising 219 447 bp was sequenced in nine cosmids and verified at > 99.9% precision. Of the standard repetitive elements, 187 Alus make up 20.6% of the sequence, but there were only 27 MERs (2.9%) and 17 L1 fragments (1.6%). This may be characteristic of such high GC (57%) regions. The sequence also includes an 11.3 kb tract duplicated with 99.2% identity at a distance of 38 kb. The region is 80-90% transcribed and 12.5% translated. Thirteen known genes and their exon-intron borders are all accurately predicted at least in part by GRAIL programs, as are six additional genes. From centromere to telomere, the orientation of transcription varies among the first eight genes, then runs centromeric to telomeric for the next five, and is in the opposite sense for the last six. Eighteen of the 19 genes are associated with CpG islands. Two islands are exact copies in the 11.3 kb repeat units, and could thus give rise to double dosage levels of an X-linked gene. Another island is associated with two genes transcribed in opposite directions. From the sequence data, three genes and their exon structure are inferred. One of them, previously associated with HEX2, is shown to be a different gene unrelated to hexokinases; a second gene, previously known by an EST, is plexin, from its 65.5% identity with the Xenopus analog; and a third is a subunit of a vacuolar H-ATPase, and is named VATPS1.
Base Sequence↗
Isolation and genomic structure of a human homolog of the yeast periodic tryptophan protein 2 (PWP2) gene mapping to 21q22.3.
As part of efforts to identify candidate genes for disease mapping to the 21q22.3 region, we have assembled a 770-kb cosmid and BAC contig containing eight tightly linked markers. These cosmids and BACs were restriction mapped using eight rare cutting enzymes, with the goal of identifying CpG-rich islands. One such island was identified by the clustering of NotI, EagI, SstII, and BssHII sites, and corresponded to the NotI linking clone LJ104 described previously. A 7.6-kb HindIII fragment containing this CpG-rich island was subcloned and partially sequenced. A homology search using the sequence obtained from either side of the NotI site identified an expressed sequence tag with homology to the yeast periodic tryptophan protein 2 (PWP2). Several cDNAs corresponding to the human PWP2 gene were identified and partially sequenced. Northern blot analysis revealed a 3.3-kb transcript that was well expressed in all tissues tested. A cDNA consensus of 3157 bp was obtained, and an open reading frame potentially encoding 919 amino acid residues was identified. The predicted protein shows 42% identity and 57% similarity at the amino acid level to the yeast PWP2 protein, which is a member of the WD-repeat containing superfamily, and potentially encodes a G-protein beta subunit. The PWP2 gene is split into 21 exons, ranging in size from 53 to 516 bp, and spans an estimated 25 kb. The gene is transcribed in a 21cen-->21qter direction, with its 5' end mapping approximately 195 kb proximal to the 5' end of the phosphofructokinase-liver isoform gene. Four single base-pair polymorphisms were identified using single-stranded conformation polymorphism analysis. Possible functions of the protein based on homology to other members of the WD-repeat-containing family are discussed.
Amino Acid Sequence↗
[Reconstitution of Hu-SCID mice transplanted with human tonsillar lymphocytes immunized with dengue virus-2 in vitro and its immune response].
OBJECTIVE: To study the measure for the reconstitution of severe combined immunodeficient disease mice (SCID mice) transplanted with human lymphocytes immunized with specific antigen in vitro. METHODS: Hu-SCID mice were constructed by intraperitoneal (i.p.) transfer of human tonsillar lymphocytes (TLC) first immunized with dengue virus-2 (DV2) in vitro, then boostered with different doses of DV2 antigen in vivo. RESULTS: Human immunoglobulin (Ig) and specific human anti-DV2 antibodies could be detected in sera of the hu-SCID mice by ELISA. Two weeks after transplantion of the in vitro-immunized hu-TLC, the highest human IgG concentration was 412.86 micrograms/ml, two or four weeks later specific human anti-DV2 IgM or IgG and the neutralizing antibody could found in these sera, respectively, the highest titer was 1:6400. The levels of human IgG and human anti-DV2 antibodies were related to the dose of DV2 antigen. The presence of human B cells (CD20+) and T/cells (CD3+, CD4+, CD8+) were shown in the sleen, lung, intestine and mesentery of the hu-SCID mice as determined by immunoperoxidase staining, and the cells were distributed correspondingly with the lymphocyte cells in proper human tissue. Human anti-DV2 IgG could also be detected in the spleen cell culture obtained from the SCID mice. CONCLUSION: By co-immunized in vitro and in vivo the SCID mice deduced stronger immune response for specific antigen, and the reconstitution of the human lymphocytes could be established successfully.
Animals↗
Differential expression of intestinal and splenic cytokines after parenteral nutrition.
OBJECTIVE: To determine the effect of parenteral nutrition (PN) on the expression of message for inflammatory cytokines in the spleen and different segments of the intestine. DESIGN: Randomized controlled trial. PARTICIPANTS: Eleven adult male Sprague-Dawley rats weighing 250 to 300 g. INTERVENTIONS: All rats underwent central venous cannulation and were randomized to two groups. Group 1 (n = 6) received saline solution infusion and chow ad libitum; group 2 (n = 5) received lipid-free PN with no oral feeding. After 7 days, the animals were killed and the spleens and segments of small and large intestine were removed. MAIN OUTCOME MEASURES: The expression of message for tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1 in the spleen and intestine was determined using a semiquantitative reverse transcription polymerase reaction. Splenic macrophages were isolated and cultured for 24 hours with and without lipopolysaccharide. Production of TNF-alpha and IL-6 was determined by bioassay followed by enzyme-linked immunosorbent assay. RESULTS: After 7 days of infusion, messenger RNA (mRNA) expression for TNF-alpha, IL-1, and IL-6 was increased in the jejunum (P < .05), and TNF-alpha mRNA and IL-6 mRNA expression was decreased in the spleen (P < .01) of PN-fed animals when compared with saline/chow controls. In addition, TNF-alpha mRNA expression was increased in the cecum (P < .05), IL-1 mRNA expression was increased in the ileum (P < .05), and IL-6 mRNA expression was increased in the cecum (P < .05) and Peyer's patches (P < .007) in the PN-fed animals. Production of TNF-alpha and IL-6 by splenic macrophages was decreased following PN infusion in both lipopolysaccharide-treated and untreated cultures (P < .05). CONCLUSIONS: Infusion of lipid-free PN induces a differential mRNA expression for inflammatory cytokines in the spleen and intestine with an overall up-regulation of the expression of inflammatory cytokines in the intestine and a down-regulation in the spleen. These data provide evidence that the regulatory mechanisms for cytokine production are different in the intestine and the spleen. Further study is needed to elaborate the mechanism of this differential expression following lipid-free PN infusion.
Animals↗
Quantitation of P53 protein expression in gastrointestinal smooth muscle tumors. Clinicopathological correlation and prognostic significance.
Quantitative analysis of P53 protein expression was performed on paraffin-embedded tissues from 55 smooth muscle tumors of the gastrointestinal tract, using immunofluorescence and flow cytometry. No positive expression was found in normal smooth muscle tissues of the gastrointestinal tract. Over-expression of P53 gene was found in a significantly higher proportion in leiomyosarcomas (90%) and potentially malignant smooth muscle tumors (75%) as compared to leiomyomas (14%) (P < 0.005). The quantitation of P53 expression was found to be progressively enhanced in the sequence from leiomyoma through potentially malignant smooth muscle tumor to leiomyosarcoma (P < 0.005). It was markedly over-expressed when the mitotic counts ranged from one to more than one per 10 high power fields (P < 0.005) or the mild cytologic atypia was found (P < 0.005). The five-year survival rate was significantly higher in patients with low-expression of P53 than in those with over-expression of P53 (P < 0.005). It was suggested that P53 over-expression might be associated with the transformation of leiomyoma into leiomyosarcoma and could be used as an objective parameter in distinguishing the malignant from the benign and predicting the prognosis of patients with smooth muscle tumors of the gastrointestinal tract.
Adolescent↗
Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients of Chinese descent and identification of new base substitutions in the human G6PD gene.
The underlying DNA changes associated with glucose-6-phosphate dehydrogenase (G6PD)-deficient Asians have not been extensively investigated. To fill this gap, we sequenced the G6PD gene of 43 G6PD-deficient Chinese whose G6PD was well characterized biochemically. DNA samples were obtained from peripheral blood of these individuals for sequencing using a direct polymerase chain reaction (PCR) sequencing procedure. From these 43 samples, we have identified five different types of nucleotide substitutions in the G6PD gene: at cDNA 1388 from G to A (Arg to His); at cDNA 1376 from G to T (Arg to Leu); at cDNA 1024 from C to T (Leu to Phe); at cDNA 392 from G to T (Gly to Val); at cDNA 95 from A to G (His to Arg). These five nucleotide substitutions account for over 83% of our 43 G6PD-deficient samples and these substitutions have not been reported in non-Asians. The substitutions found at cDNA 392 and cDNA 1024 are new findings. The substitutions at cDNA 1376 and 1388 account for over 50% of the 43 samples examined indicating a high prevalence of these two alleles among G6PD-deficient Chinese. Our findings add support to the notion that diverse point mutations may account largely for much of the phenotypic heterogeneity of G6PD deficiency.
Asian↗
Central administration of interleukin-1 beta increases norepinephrine turnover in the spleen.
We have previously shown that intracerebroventricular (ICV) injection of interleukin-1 beta (IL-1 beta) suppressed splenic macrophage function. Sympathetic noradrenergic innervation of the spleen was implicated as a mediator of this IL-1 beta signal as surgical sympathectomy ablated the macrophage suppression. In this study, we have determined whether ICV administration of IL-1 beta has an effect on sympathetic outflow and norepinephrine (NE) turnover in the spleen. Adult male rats were injected with 5 ng of IL-1 or saline, and NE turnover in the spleen was determined using the rate of decline of NE content in the spleen after synthesis inhibition. The splenic NE turnover rate was increased significantly from 69.52 ng/g/h in saline-treated animals to 111.05 ng/g/h in IL-1-treated animals. In addition, serum corticosterone and ACTH were significantly elevated in IL-1 beta-treated animals 4 h postinjection. These data indicate that central administration of IL-1 beta increases both sympathetic outflow to the spleen and activates the hypothalamic-pituitary-adrenal axis during the period when IL-1 beta induces immunosuppression.
Adrenocorticotropic Hormone↗
Regulation of alveolar macrophage transforming growth factor-beta secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.
In a model of pulmonary inflammation and fibrosis induced by the antineoplastic antibiotic, bleomycin, we previously demonstrated that TGF-beta was markedly elevated within 7 d of bleomycin administration. At the time of maximal TGF-beta production, TGF-beta 1 was localized by immunohistochemistry to be present almost exclusively in alveolar macrophages. In this study, we have demonstrated that alveolar macrophages stimulated by bleomycin-induced injury secrete large quantities of biologically active TGF-beta 1 when explanted into tissue culture. However, alveolar macrophages from normal saline-treated rats secrete small quantities of biologically inactive TGF-beta. In contrast, splenic macrophages secrete large quantities of inactive TGF-beta and are unaffected by the intratracheal bleomycin treatment. High doses of the corticosteroid methylprednisolone given intramuscularly before and concomitantly with bleomycin administration prevented the influx of alveolar macrophages into the lungs, diminishing both the number of macrophages present in the alveoli and the total lung content of TGF-beta. However, the rate of secretion of TGF-beta by alveolar macrophages recovered from the alveoli was unchanged after corticosteroid treatment. When activated alveolar macrophages were cultured in the presence of several concentrations of dexamethasone that completely suppressed IL-1 secretion, little effect on TGF-beta secretion was observed. The findings in this study demonstrate that during bleomycin-induced injury, alveolar macrophages not only secrete large quantities of active TGF-beta 1, but are a predominant source of the enhanced TGF-beta response seen in this model. Furthermore, the alveolar macrophage secretion of TGF-beta is not inhibited by the presence of high concentrations of corticosteroids.
Animals↗
A new mutation responsible for severe G6PD deficiency in two ethnic Chinese with different clinical presentations: determination by a direct PCR sequencing technique.
To test the hypothesis that clinical manifestation in G6PD deficiency correlates with a molecular lesion, we investigated the G6PD gene of two Chinese Americans both of whom had G6PD deficiency, but who manifested different clinical presentations. In this study, we have developed a direct PCR sequencing protocol to examine the human G6PD gene. By using optimized PCR conditions with internal primers, we were able to amplify a 4.2 kb DNA fragment (covering exon 3 through 13 of the G6PD gene) of consistently high quality. From this we were then able to generate high quality single-stranded DNA templates by asymmetric PCR for subsequent sequencing. We also overcame the crossband problem by using internal primers, high temperature reaction with Taq I DNA polymerase, and/or sequencing with gene 32 protein. We could consistently amplify exons 1 and 2 despite their high G/C content by substituting 75% of dGTP with deoxy-7-deaza-guanosine triphosphate. By using this novel approach, we have identified a new mutation at cDNA position 1376 from G to T, which causes substitution of Leu for Arg at amino acid position 459. This mutation has not been reported in other ethnic groups. It is the only genetic defect in the coding regions of the G6PD gene of these two G6PD deficient individuals. We speculate that in addition to a defect in the G6PD gene, other factors also play a role in the clinical manifestation of G6PD deficiency.
Base Sequence↗
A to G substitution identified in exon 2 of the G6PD gene among G6PD deficient Chinese.
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Asian People↗
Two commonly occurring nucleotide base substitutions in Chinese G6PD variants.
Using a direct PCR sequencing technique, we have identified two DNA base substitutions in 8 different biochemical G6PD variants of Chinese origin. Neither one of these abnormalities has been reported in other ethnic groups. An abnormality (C1) of G to T substitution at cDNA 1376 causing an amino acid change from Arg to Leu has been found in 3 variants. Another abnormality (C2) of G to A substitution at cDNA 1388 causing an amino acid change from Arg to His has been found in 5 variants. Both C1 and C2 are located in exon 12 of the G6PD gene and are only 12 base pairs apart. However, C1 is associated with a significant increase in the deamino-NADP utilization rate, whereas C2 is not. Taken together, our data suggest that C1 and C2 are very common among Chinese with a G6PD deficiency and exon 12 may define an important functional domain of the human G6PD.
Base Sequence↗
NADPH, not glutathione, status modulates oxidant sensitivity in normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is characterized by the loss of NADPH and enhanced erythrocyte oxidant sensitivity. Historically, it has been theorized that the elevated oxidant sensitivity of G6PD-deficient erythrocytes arises as the direct consequence of decreased intracellular glutathione (GSH) concentrations. To directly investigate the basis of G6PD deficiency oxidant sensitivity, the effects of altered GSH and NADPH concentrations were examined in normal and G6PD-deficient erythrocytes. The results of this study demonstrated that GSH depletion, by 1-chloro-2,4-dinitrobenzene (CDNB), had no effect on hemoglobin oxidation in response to hydrogen peroxide (H2O2) generating systems (phenazine methosulfate and menadione bisulfite) in either normal or G6PD-deficient cells. Furthermore, a fourfold to sixfold increase in intracellular GSH concentration also did not protect against H2O2-generating systems in the normal or G6PD-deficient erythrocytes. Conversely, introduction of an NADPH-generating system (purified G6PD) into G6PD-deficient cells resulted in a significant decrease in oxidant sensitivity and an ability to cycle GSH. Further experiments demonstrated that the reduced oxidant sensitivity of the G6PD-reconstituted erythrocytes was not due to the maintenance of GSH levels because CDNB-mediated depletion of GSH did not alter this protective effect. Analysis of these results demonstrated a direct correlation between NADPH, but not GSH, concentration and hemoglobin oxidant sensitivity.
Dinitrochlorobenzene↗
Recognition signals for phagocytic removal of favic, malaria-infected and sickled erythrocytes.
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Anemia, Sickle Cell↗
Conditioned taste aversion but not adrenal activity develops to ICV administration of interleukin-1 in rats.
In a previous investigation with mice, the paired presentation of either odor or taste cues with the peripheral (IP) administration of the immunoactive peptide interleukin-1 (IL-1) led to the conditioned enhancement of glucocorticoid production. The present study found that an initial central infusion of IL-1 in the presence of saccharin cues produced a robust taste aversion but not a conditioned elevation of either ACTH or corticosterone production. These results indicate that the glucocorticoid response induced by centrally administered IL-1 in rats is independent of the behaviorally aversive properties of this cytokine which are conditionable. The differential effects of IP versus ICV administration of IL-1 on glucocorticoid conditioning requires a clearer specification of the respective signaling mechanisms and pathways activated by these two routes of administration.
Adrenocorticotropic Hormone↗
[Advances in the study of membrane lipid fluidity of erythrocyte].
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Animals↗
Erythrocyte defense against hydrogen peroxide: preeminent importance of catalase.
To investigate the relative importance of catalase and glutathione in erythrocyte oxidant defense, human and mouse (normal and acatalasemic) erythrocytes were reversibly lysed and resealed in the presence of exogenous catalase or glutathione. This resulted in an increase in intracellular catalase activity or glutathione concentration in the resealed erythrocytes while normal cellular structure, hemoglobin concentration, cell volume, cellular deformability, and adenosine triphosphate concentration were maintained. Resealing alone had no effect on oxidant sensitivity. In human cells, a threefold increase in catalase activity resulted in the maintenance of glutathione levels in response to hydrogen peroxide (H2O2) challenge. Reconstitution of congenitally acatalasemic mouse erythrocytes, which were extremely sensitive to even micromolar concentrations of H2O2 with purified catalase resulted in complete protection against H2O2. Indeed, the catalase-reconstituted acatalasemic cells were less sensitive to H2O2-mediated damage than were normal, catalase-replete mouse cells. In contrast, alteration of the glutathione status of human and mouse (normal and acatalasemic) cells had no significant effect on oxidant sensitivity. Even a five-fold increase in intracellular glutathione concentration (greater than 30 micromoles glutathione per gram of hemoglobin) in normal or catalase-deficient (azide-treated or acatalasemic) red blood cells had no protective effect against H2O2-mediated lipid peroxidation or methemoglobin generation. Similarly, depletion of glutathione by 1-chloro-2,4-dinitrobenzene also had no effect on erythrocyte H2O2 sensitivity. These results suggest an important role for catalase in protection against H2O2-mediated damage at physiologic levels and that catalase is as at least as important as glutathione in cellular defense against H2O2.
Animals↗