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D Weil

Publications and source records attributed to D Weil.

At least 19 recordsLinked to original sources
PubMed1991

Endemic and epidemic aspergillosis associated with in-hospital replication of Aspergillus organisms.

When a new hospital opened in 1983, environmental culturing for Aspergillus organisms and surveillance for nosocomial aspergillosis cases were begun to characterize the relationship between environmental contamination and infection. Monthly air sampling demonstrated increasing concentrations of Aspergillus flavus and Aspergillus fumigatus to mean levels greater than 1 cfu/m3 during 1986-1987, accompanied by a progressive increase in incidence of aspergillosis to 1.2% in immunocompromised patients. This prompted an inspection that revealed heavy growth of Aspergillus organisms on air filters. Subsequent inspections of hospital wards showed small foci of A. flavus growth on other materials. Removal of the contaminated filters and improved environmental maintenance were associated with reduction in A. flavus and A. fumigatus to 0.01 cfu/m3 and a fourfold decline in aspergillosis incidence during the next 2 years. These findings, together with laboratory studies that showed aspergilli could proliferate on common hospital materials when moistened, indicate a need for careful environmental maintenance.

Air Microbiology↗
PubMed1990

Structural and functional characterization of a splicing mutation in the pro-alpha 2(I) collagen gene of an Ehlers-Danlos type VII patient.

A splicing mutation in the pro-alpha 2(I) collagen gene of a patient with Ehlers-Danlos syndrome type VII has been characterized. Protein microsequencing of peptides prepared from the patient's dermal collagen identified an interstitial deletion of 18 residues. The deleted segment corresponds to the amino-terminal telopeptide junction domain encoded by the sixth exon of the pro-alpha 2(I) collagen gene. Sequencing of specifically primed cDNA clones confirmed the presence of two distinct populations of pro-alpha 2(I) mRNAs, a normal one and another which lacks the sequences of exon 6. Limited sequencing of genomic clones showed that one of the pro-alpha 2(I) alleles displays a conservative change in the seventh codon of exon 6 (GAC for GAT), and a base substitution at position +1 of intron 6 (A for G). Since the normal transcript contains the GAT codon, the intronic change was associated with the allele that gives rise to the shortened pro-alpha 2(I) collagen mRNA. The two allelic fragments were subcloned into an expression vector and the pattern of splice-site selection for exons 5-8 was assessed for each of the constructs after transfection into COS cells. This documented skipping of exon 6 sequences only in transcripts of the minigene construct that harbors the G to A transition. Expression of allelic cross-constructs confirmed that the single-base substitution at position +1 of intron 6 is the mutation responsible for the abnormal joining of exons 5 and 7 sequences in the patient's shortened pro-alpha 2(I)mRNA.

Alleles↗
PubMed1990

RNA processing is a limiting step for murine tumor necrosis factor beta expression in response to interleukin-2.

We have previously reported that tumor necrosis factor beta (TNF beta) expression is induced by interleukin-2 (IL-2) in the murine lymphocytic T-cell line CTLL-2. In this study, we have characterized the nuclear and cytoplasmic TNF beta transcript and assessed their role in TNF beta gene expression. A unique feature of TNF beta expression was the accumulation of nuclear precursors, which reflected a slow nuclear RNA processing. As a consequence, there was a delay in the appearance of cytoplasmic messengers after the transcriptional induction of TNF beta by IL-2. We also found that two messengers, the fully spliced messenger and an intron 3-retaining messenger, were exported to the cytoplasm and actively translated. The same pattern of expression was observed in concanavalin A-stimulated splenocytes, although the level of expression was much lower than in CTLL-2 cells. The simple genetic structure and the high level of accumulation of nuclear precursors make TNF beta a particularly attractive model system to use for studies of RNA processing and cytoplasmic transport of partially spliced messengers.

Amino Acid Sequence↗
PubMed1990

Secretion of tumor necrosis factor-alpha by fresh human acute nonlymphoblastic leukemic cells: role in the disappearance of normal CFU-GM progenitors.

The disappearance of normal hematopoiesis during acute nonlymphoblastic leukemia (ANLL) is poorly understood. Several reports indicate that conditioned medium obtained from leukemic cells might inhibit the formation of normal hematopoietic progenitors. However, these blast-conditioned medium (BCM) inhibitory activities are not well characterized. In order to evaluate whether BCM might contain an activity inhibiting the growth of normal marrow progenitors, BCM from 13 consecutive patients with ANLL were tested on normal bone marrow in methylcellulose assays. In all the cases, a significant inhibition of the growth of granulocyte-macrophage colony-forming unit (CFU-GM) progenitors was observed, whereas erythroid burst-forming unit (BFU-E) progenitors were not affected. Further characterization of the BCM inhibitory activity showed using both a biological assay and RIA, the presence of tumor necrosis factor-alpha (TNF-alpha) in 10 out of 13 BCM. Northern blot analysis performed in six patients showed a correlation between the expression of TNF-alpha mRNA by leukemic cells and the presence of TNF-alpha in BCM. Moreover, the BCM inhibitory activity could be neutralized with an anti-TNF-alpha antiserum. These data indicate that leukemic cells express and release frequently TNF-alpha, which may therefore play an important role in the inhibition of granulopoiesis during leukemia.

Adult↗
PubMed1990

[Prenatal diagnosis of sacro-coccygeal teratomas].

Seventeen sacrococcygeal teratomas with prenatal diagnosis are studied. The diagnosis was made at 27 weeks. Three fetuses died in utero, two of them from the evolution of the tumor. Two died post-natally from hemorrhage and one post natally because of his prematurity. From the eleven long term survivors, one developed a recurrence and five sequelae. This study confirms the poor prognosis of fetal sacrococcygeal teratomas. The 2 major prognosis factors are the growth of the tumor and the occurrence of fetal congestive heart failure. That congestive heart failure is diagnosed by Doppler echocardiography before hydrops and placentomegaly who are of very poor prognosis. The extraction of the fetus is mandatory if these features appeared after 30 weeks. Before this term fetal surgery is the only effective treatment. At a long term, the risk of recurrence is low but implies a long term surveillance of the level of alfa foeto protein.

Female↗
PubMed1990

Falling among the sensorially impaired elderly.

Recent studies of the healthy elderly indicate that accidental falls account for many lengthy hospital stays and permanent disabilities. An ongoing investigation by our group is examining the contribution of neuromuscular feedback and the possible substitution of visual-perceptual feedback as a major contributor to falling in this population. This combination of decreased weighting of neuromuscular factors and increased weighting of the visual-perceptual factors led to the following hypothesis: Those persons who cannot see should show less change in frequency of falling as they age than sighted cohorts. The deaf, who rely more than others on visual perceptual stimuli, are less likely to fall as they age than the general elderly population, who first begin to rely more heavily on visual stimuli when they age. Thus, the deaf and blind constitute a population whose change in frequency of falls with increased age should differ markedly from change in the frequency of falls among the nonblind and nondeaf segments of the population. Our results support the hypothesis. The blind demonstrated a higher rate of falls than the deaf or nonimpaired population. However, the more elderly blind failed to show the increase in falling demonstrated by the deaf and nonimpaired. If these findings are confirmed in subsequent studies, the nonimpaired and deaf elderly could be trained to focus on visual feedback. The blind may be able to reduce the frequency of falls by enhancing musculoskeletal feedback and strength via exercise.

Accidental Falls↗
PubMed1989

Temperature-dependent expression of a collagen splicing defect in the fibroblasts of a patient with Ehlers-Danlos syndrome type VII.

In this article we report the characterization of the molecular lesion in a patient with Ehlers-Danlos syndrome Type VII and provide evidence that a de novo substitution of the last nucleotide of exon 6 in one allele of the pro-alpha 2(I) collagen gene produces normally spliced mRNA and transcripts from which exon 6 sequences have been outspliced as well. Unexpectedly, the expression of the alternative splicing was found to be temperature-dependent, for missplicing in cellula is effectively abolished at 31 degrees C and gradually increases to 100% at 39 degrees C. In contrast, in a similar patient harboring a substitution in the obligatory GT dinucleotide of the 5' splice site of intron 6, complete outsplicing of exon 6 sequences was found at all temperatures.

Amino Acid Sequence↗
PubMed1989

Cloning and analysis of the 5' portion of the human type-III procollagen gene (COL3A1).

We have isolated overlapping clones containing the 5'-terminal portion of the human pro-alpha 1(III) collagen gene (COL3A1). This has enabled us to extend our previous studies and thus generate a restriction map of nearly 64 kb of DNA encompassing all of COL3A1 and more than 20 kb of flanking sequences. Aside from the complete nucleotide and amino acid sequences of type-III N-pre-propeptide, this study has established the number of the corresponding exons, whose relative organization deviates from the pattern observed in the analogous regions of type-I procollagen genes, COL1A1 and COL1A2. Moreover, we have sequenced 1628 bp of the 5'-flanking region of COL3A1, from the transcription start point (tsp) to an AluI repetitive element. Pairwise comparison with the analogous segment of the mouse gene has showed 78% sequence similarity in nearly 270 bp immediately preceding the tsp and including the TATA element and a presumptive NF-1 binding site. Relatively close to the tsp, but upstream from the region of homology with the murine gene, a potential AP-1 binding site has also been identified.

Amino Acid Sequence↗
PubMed1989

A base substitution in the exon of a collagen gene causes alternative splicing and generates a structurally abnormal polypeptide in a patient with Ehlers-Danlos syndrome type VII.

An unusual splicing mutation has been characterized in the pro alpha 1(I) collagen gene of a sporadic case of Ehlers-Danlos Syndrome Type VII. Cloning of primer extended cDNA in conjunction with R-looping experiments established that nearly half of the pro alpha 1(I) collagen gene transcripts are abnormally spliced, for they lack exon 6 sequences. Analysis of cloned genomic fragments revealed that one of the proband's alleles displays the substitution of an A for a G in the last nucleotide of exon 6. The change converts the normal Met (ATG) codon to Ile (ATA) and, in addition, obliterates a NcoI restriction site. The latter event was exploited to demonstrate the de novo nature of the mutation since DNA from the unaffected parents was fully digested with the enzyme, after in vitro amplification by the polymerase chain reaction. Further confirmation of the missplicing was obtained by transient expression into animal cells of allelic minigene constructs. Finally, Western blot analysis of cyanogen bromide cleaved collagen and nucleotide sequencing of appropriately selected cDNA clones demonstrated the production of relatively low amounts of correctly spliced molecules harboring the Ile substitution, as well.

Amino Acid Sequence↗
PubMed1989

Human nidogen: cDNA cloning, cellular expression, and mapping of the gene to chromosome Iq43.

A human placental lambda gt11 expression cDNA library was screened for nidogen cDNAs by hybridizations with a heterologous mouse nidogen cDNA. A total of four positive overlapping clones were identified, and the sizes of the inserts were shown to vary from 0.8 to 2.8 kb. Nucleotide sequencing of the human cDNAs revealed that the largest clone, cHPN-16, contained both a 5' open reading frame encoding 582 amino acids and a 3' untranslated region of 1,063 nucleotides. Comparison of human cDNA sequences with mouse nidogen sequences revealed 84% identity on the nucleotide level and 88% identity with the deduced amino acid sequence. The deduced amino acid sequence of the human cDNAs revealed the presence of cysteine-rich epidermal growth factor-like repeats and the sequence Arg-Gly-Asp (RGD), a potential cell binding site, two features previously identified in mouse nidogen. The sequence Asn-Pro-Ser, a consensus sequence for N-linked glycosylation, was also noted. The newly isolated human cDNAs were utilized to analyze the expression of the nidogen gene by cultured human cells. Northern hybridizations revealed a single mRNA transcript of approximately 6.0 kb in human skin fibroblast and in HT 1080 fibrosarcoma cell cultures. However, the human choriocarcinoma cell line JEG-3, which expressed laminin genes, did not contain detectable levels of nidogen mRNAs. Quantitation of the relative nidogen mRNA abundance in HT 1080 fibrosarcoma cells indicated that nidogen mRNA levels were approximately the same as those for the laminin B2 chain. Finally, one of the nidogen cDNAs was used to map the nidogen gene onto locus q43 of chromosome 1.

Amino Acid Sequence↗
PubMed1988

Regulation of pim and myb mRNA accumulation by interleukin 2 and interleukin 3 in murine hematopoietic cell lines.

We have studied the mRNA accumulation of pim and myb genes in two interleukin 2- (IL2-)dependent, CTLL-2 and B6.1, and one IL3-dependent, FDC-P2, murine hematopoietic cell lines. To be able to dissociate the IL2 response from the phenomenon of lymphocyte activation, we used cell lines constitutively expressing the high affinity IL2 receptor. Deprivation of IL2 for 16 h led to an accumulation of CTLL-2 cells in G0/G1, and stimulation with IL2 induced a progression in S phase after 10 h. An increased accumulation of pim mRNA was observed in all cases in response to IL2 or IL3. This regulation did not require de novo protein synthesis and was, in CTLL-2 cells, mostly at the transcriptional level. Expression of myb was more complex: in CTLL-2 and FDC-P2 it is high and constitutive, while in B6.1 it is low and induced by IL2. This difference in myb regulation correlates with the higher level of myb expression in immature cells, as only B6.1 is functionally mature. Furthermore, it shows that transcription of myb does not affect the control of the cell cycle by the growth factors IL2 and IL3. These studies demonstrate that pim belongs to the small group of protooncogenes that can be induced during the primary response to growth factors (fos, myc, and myb) and that constitutive expression of myb, at least at the RNA level, is not sufficient to abrogate the growth factor requirement of hematopoietic cell lines.

Animals↗
PubMed1988

Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant.

Recent biochemical studies have shown that the fibroblasts from a patient with Ehlers-Danlos Syndrome Type VIIB produce nearly equal amounts of normal and shortened pro-alpha 2(I) collagen chains (Wirtz, M.K., Glanville, R. W., Steinmann, B., Rao, V. H., and Hollister, D. (1987) J. Biol. Chem. 262, 16376-16385). Compositional and sequencing studies of the abnormal pro-alpha 2(I) chain identified an interstitial deletion of 18 residues corresponding to the N-telopeptide of the collagen molecule. Since this region is encoded by a 54-base pair exon, number 6, the protein defect could have been caused by gene deletion, abnormal pre-mRNA splicing, or both. Here, in order to elucidate the molecular nature of this mutation we have analyzed the sequences of pro-alpha 2(I) collagen cDNA and genomic clones obtained from RNA and DNA of the patient's fibroblasts. Using oligomer-specific cloning we identified a cDNA that contains a 54-base pair deletion corresponding precisely to the sequence of exon 6. Identification of the normal gene was based on the finding of an identical sequence polymorphism in a normal cDNA and in the genomic clone derived from one of the two collagen alleles. The other gene, instead, displayed a base substitution (T to C) in the obligatory GT dinucleotide of the 5' splice-site sequence of intron 6. Analysis of nearly 100 base pairs immediately 5' to exons 5, 6, and 7, and 3' to exons 5 and 7 did not reveal any additional change. Therefore, the data strongly suggest that the observed GT-to-GC transition at the splice donor site of intron 6 generates an abnormally spliced mRNA in which the sequence of exon 5 is joined to the sequence of exon 7. Since skipping of exon 6 does not interfere with the coding frame of the mRNA, the resulting shortened polypeptide, albeit utilized in the assembly of a procollagen trimer, ultimately causes the Ehlers-Danlos Syndrome Type VII phenotype.

Alleles↗
PubMed1988

cDNA cloning, expression and mapping of human laminin B2 gene to chromosome 1q31.

A laminin B2 chain cDNA clone was isolated from a human lung cDNA library by screening with antibody against mouse laminin. The authenticity of the human cDNA clone was established by comparison of the nucleotide and deduced amino acid sequences of the cDNA insert with those of the previously reported mouse laminin cDNA clones. The human clone (LC7) contained an insert of 0.75 kb (kilobase pair) that corresponded to the last 232 amino acid residues in the carboxyl terminus of the B2 chain. Northern blot analyses with the LC7 probe detected two mRNA transcripts of 8.2 and 5.6 kb in both normal human skin fibroblasts and three human tumor cell lines. The cDNA probe was also used in Southern blot analysis of DNA from human rodent somatic cell hybrids to localize the gene to human chromosome 1. In situ hybridization of the cDNA with metaphase chromosome spreads confirmed the assignment and further mapped the human laminin B2 chain gene to the long arm of chromosome 1 in the band q31.

Amino Acid Sequence↗
PubMed1988

Genetic distance of two fibrillar collagen loci, COL3A1 and COL5A2, located on the long arm of human chromosome 2.

Two of the human fibrillar collagen genes, proa1(III) (COL3A1) and proa2(V) (COL5A2), map to the same region of the long arm of chromosome 2. To establish the genetic distance between the two loci, we analyzed the segregation of COL3A1 and COL5A2 RFLPs in five families informative for the two loci specific markers. We found that the maximum lod score was 9.33 at a recombination frequency of theta = 0.00. The data therefore provide strong evidence for tight linkage between two evolutionarily related fibrillar collagen genes on the 2q14----2q32 segment of chromosome 2.

Amino Acid Sequence↗
PubMed1988

Growth and phenotypic characteristics of human nevus cells in culture.

Nevus cells were isolated from the three cutaneous components, epidermis, basal layer, and dermis, of nonmalignant pigmented lesions and were cultured separately in the presence or absence of the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate in medium that supports the rapid proliferation of melanocytic cells. The separation procedure used provided cultures that were essentially free from normal melanocytes (dermis) or fibroblasts (epidermis). In short term culture, nevus cells of all skin compartments expressed markers associated with differentiated melanocytes, such as presence of premelanosomes and melanosomes and elevated tyrosinase levels. Nevus cells also expressed melanoma-associated antigens, such as NGF-receptor, transferrin-related p97, proteoglycan, and HLA-DR as detected with monoclonal antibodies. After several subpassages, cells showed a decreased expression of melanoma-associated antigens, decreased tyrrosinase levels, and melanosomes could no longer be detected. Morphologically, these cells were similar to fibroblasts. The disappearance of melanoma-associated cell surface antigens was concomitant with the appearance of a melanocyte-associated 145 kd protein that might serve as a marker of fibroblast-like differentiation in nevus cells and normal melanocytes. Nevus cell cultures grown in the presence of 12-0-tetradecanoyl phorbol-13-acetate maintained a stable differentiated phenotype throughout their lifespan. As reported earlier, nevus cells in culture, irrespective of the presence or absence of 12-0-tetradecanoyl phorbol-13-acetate, have a finite lifespan in vitro, grow anchorage-independent in soft agar, but do not form tumors when xenografted to nude mice. These studies demonstrate that nevus cells isolated from the epidermal, basal layer, and dermal components of lesional skin can serve as models to characterize the initial steps of tumor progression in a human cell system.

Adolescent↗
PubMed1988

Induction of tumor necrosis factor-alpha and -beta and interferon-gamma mRNA by interleukin 2 in murine lymphocytic cell lines.

We have investigated genes regulated by interleukin-2 (IL2) in the murine lymphocytic cell line CTLL-2 by differential screening of a cDNA library. One of the clones we have identified is a partial cDNA of tumor necrosis factor-beta, (TNF-beta), a gene previously shown to be expressed in response to lymphocyte activation. This led us to investigate the regulation by IL2 of TNF-alpha, TNF-beta, and interferon-gamma (IFN-gamma) mRNA. In two murine lymphocytic cell lines all three genes are induced during the primary response to IL2. This regulation of lymphokine expression by IL2 is correlated with cell proliferation and not with the cytotoxic activity. As activated T lymphocytes express receptors to both TNF and IFN-gamma, our observation raises the possibility that these lymphokines are involved in an autocrine regulation of cytotoxic lymphocytes proliferation.

Amino Acid Sequence↗
PubMed1988

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.

Amino Acid Sequence↗