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D R Smith

Publications and source records attributed to D R Smith.

At least 163 records · Page 9Linked to original sources
PubMed1994

Purification, characterization, gene sequence, and significance of a bacterioferritin from Mycobacterium leprae.

The study of tissue-derived Mycobacterium leprae provides insights to the immunopathology of leprosy and helps identify broad molecular features necessary for mycobacterial parasitism. A major membrane protein (MMP-II) of in vivo-derived M. leprae previously recognized (Hunter, S.W., B. Rivoire, V. Mehra, B.R. Bloom, and P.J. Brennan. 1990. J. Biol. Chem. 265:14065) was purified from extracts of the organism and partial amino acid sequence obtained. This information allowed recognition, within one of the cosmids that encompass the entire M. leprae genome, of a complete gene, bfr, encoding a protein of subunit size 18.2 kD. The amino acid sequence deduced from the major membrane protein II (MMP-II) gene revealed considerable homology to several bacterioferritins. Analysis of the native protein demonstrated the iron content, absorption spectrum, and large native molecular mass (380 kD) of several known bacterioferritins. The ferroxidase-center residues typical of ferritins were conserved in the M. leprae product. Oligonucleotides derived from the amino acid sequence of M. leprae bacterioferritin enabled amplification of much of the MMP-II gene and the detection of homologous sequences in Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. The role of this iron-rich protein in the virulence of M. leprae is discussed.

Amino Acid Sequence↗
PubMed1994

Inhibition of interleukin 8 attenuates angiogenesis in bronchogenic carcinoma.

We investigated the role of interleukin 8 (IL-8) in mediating angiogenesis in human bronchogenic carcinoma. Increased quantities of IL-8 were detected in tumor tissue as compared with normal lung tissue. Immunohistochemical staining of tumors revealed primary localization of IL-8 to individual tumor cells and demonstrated the capacity of tumor to elaborate IL-8. Functional studies that used tissue homogenates of tumors demonstrated the induction of both in vitro endothelial cell chemotaxis and in vivo corneal neovascularization. It is important to note that the addition of neutralizing antisera to IL-8 to these assays resulted in the marked and specific attenuation of these responses. Our observations definitively establish IL-8 as a primary mediator of angiogenesis in bronchogenic carcinoma and offer a potential target for immunotherapies against solid malignancies.

Animals↗
PubMed1994

High frequency of allelic deletion on chromosome 17p in advanced colorectal cancer.

BACKGROUND: Colorectal cancers often show allelic loss of chromosomes 5q and 17p, regions where the tumor suppressor genes p53 and adenomatous polyposis coli are known to reside. Currently, the inactivation of tumor suppressor genes and the activation of oncogenes are considered major events involved in tumor development. According to a recent genetic model, ras gene mutations and allelic deletion of chromosome 5q are early changes, whereas chromosome 17p and 18q deletions are late changes in colorectal tumorigenesis. It has been shown that 17p and 18q deletions are associated with an increased tendency of disease dissemination in colorectal cancer. Most of the studies on allelic deletion in colorectal cancer were undertaken with Western population cohorts. The authors examined the association of chromosomes 5q and 17p deletions with clinical parameters, including metastasis in a predominantly Chinese population with a high incidence of colorectal cancer. METHOD: Allelic deletion was studied with the restriction fragment length polymorphism technique in tumors from 102 and 100 sporadic colorectal cancer cases for chromosomes 5q and 17p, respectively. Probes pi 227 and ECB27 were used for chromosome 5q, and probe YNZ22.1 was used for chromosome 17p. RESULTS: 5q Deletion was found in 33% of informative cases, whereas 17p deletion was seen in 69% of informative cases. 17p Allelic loss showed significant association with Dukes' Stage D as well as the presence of distant metastasis, whereas 5q deletion showed no such association. CONCLUSION: Allelic loss on chromosome 17p may be a useful prognostic marker in cases of colorectal cancer.

Adult↗
PubMed1994

The effects of succimer on the absorption of lead in adults determined by using the stable isotope 204Pb.

The chelating agent succimer (meso-2,3-dimercaptosuccinic acid) is orally effective at inducing a urinary lead diuresis and a decrease in blood lead levels in lead poisoned children and adults. However, there are concerns that succimer may increase the absorption of lead from the gastrointestinal (GI) tract during treatment, particularly in cases of continuing lead exposure, which would compromise its effectiveness in reducing whole body lead stores. This preliminary study investigated the effects of succimer on the absorption of lead in adults using a stable lead isotopic tracer (204Pb). Twelve male subjects were divided into control (no succimer), 10, and 30 mg succimer/kg body wt treatment groups of 4 individuals each. All subjects ingested a single tracer dose (200 micrograms) of 204Pb, followed by a single oral dose of placebo (control) or succimer. Whole blood was collected at intervals of 0, 2, 4, and approximately 26 hr following ingestion of the 204Pb tracer, and composite urine and feces samples were collected over the duration of the study (approximately 26 hr). Mean intestinal excretion of 204Pb was reduced in the succimer-treated groups compared to the control (placebo), whereas urinary diuresis of 204Pb was higher in the succimer groups. The amount of lead 204Pb tracer accounted for at the end of the study was lower in the succimer-treated groups. These results suggest that GI lead absorption was enhanced by succimer and that succimer mediated the redistribution of lead from the circulation to other tissues. However, none of the differences between treatment groups were statistically significant (P < 0.05, t test) because of the relatively large within-group variability. This study demonstrates the utility of microgram doses of a stable lead isotopic tracer to assess the efficacy of clinical chelating agents in humans. Future studies are necessary to further clarify the effects of succimer on the absorption and retention of lead in adults and children. Although, based upon these preliminary data, it appears advisable that patients be maintained in a lead-safe environment while being treated with succimer.

Adult↗
PubMed1994

Territorial interactions between two Myxococcus Species.

It is unusual to find fruiting bodies of different myxobacteria occupying the same territory on natural samples. We were thus interested in determining whether myxobacteria establish territorial dominance and, if so, what the mechanism of that interaction is. We had previously observed that vegetative swarms of Myxococcus xanthus and Stigmatella aurantiaca placed close to each other on an agar surface initially merged but eventually separated. Further studies indicated that these two species also formed separate fruiting bodies when mixed together on developmental agar (unpublished observation). We examined the interactions between two more closely related myxobacteria, M. xanthus and M. virescens, in greater detail. When mixtures of a kanamycin-resistant strain of M. xanthus and a kanamycin-sensitive strain of M. virescens were placed together under developmental conditions, the cells sorted themselves out and established separate fruiting body territories. In addition, differential viable counts of a mixture of the two species during development indicated that each strain was producing an extracellular component that inhibited the growth and development of the other. Nevertheless, finally, M. virescens invariably outcompeted M. xanthus at all input ratios of M. xanthus/M. virescens tested. This is consistent with the observation that M. virescens is by far the more commonly encountered of the two species. The properties of the inhibitory substance from M. virescens are consistent with the possibility that it is a bacteriocin. Our working hypothesis is that the bacteriocin plays a role in the establishment of myxobacterial territoriality. If so, this is an example of an ecological function of bacteriocins.

Bacteriocins↗
PubMed1994

Role of lead-binding proteins in renal cancer.

High-dose lead exposure in rodents has been shown to produce pathognomonic lead intranuclear inclusion bodies and to result in an increased incidence of renal adenocarcinomas. Studies from this laboratory and others have demonstrated the presence of high-affinity renal lead-binding proteins in rat kidneys which act as tissue sinks for lead at low dose levels. Cell-free nuclear translocation studies have shown that these molecules are capable of facilitating the intranuclear movement of lead and that they are associated with chromatin. These data suggest that renal lead-binding proteins may play a role in mediating known alterations in renal gene expression associated with formation of intranuclear inclusion bodies. More recent studies from this laboratory have demonstrated the presence of chemically similar lead-binding proteins in kidneys of both monkeys and humans. Such observations suggest that a similar mechanism may be operating in primates since lead intranuclear inclusion bodies are also observed in these species. These data provide a testable mechanistic approach for assessing the possible role(s) of lead-binding proteins in mediating the intranuclear movement of lead and lead-induced renal cancer in primate species.

Animals↗
PubMed1994

A detailed study of p53 mutations in colorectal cancer in Singapore.

The p53 tumour suppressor gene plays a pivotal role in colorectal cancer formation. Understanding the mechanisms by which p53 is altered in tumour cells could lead to a better understanding of this common cancer. Unfortunately, there is no published information on Chinese patients. We have therefore conducted a series of studies on the p53 tumour suppressor gene in Singaporean colorectal cancer patients and have since demonstrated that 69% of patients have lost one copy of the p53 gene by allelic deletion of chromosome 17p, 55% of patients have a point mutation in one copy of the p53 gene, and that these changes are associated with tumour site, DNA aneuploidy and the pattern of disease dissemination. We present here a further study conducted on the specific nature and frequency of p53 mutations. DNA sequence analysis of 38 tumours from Chinese colorectal cancer patients with p53 mutations showed that 97% (37/38) of the mutations consisted of base substitutions, leading to the incorporation of alternate amino-acids in the p53 protein. The remaining mutation consisted of a single base insertion leading to the generation of a stop-codon, leading to the truncation of the protein some 14 amino-acids downstream from the site of the mutation. Sixty-eight percent of the mutants consisted of guanine to adenine (G-->A) transitions and 58% occurred at CpG dinucleotides. Eighty-seven percent of all mutations occurred at evolutionarily highly conserved regions, with 50% of mutations occurring in codons 175, 248 and 282.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1994

Reducing healthcare costs through co-op care.

The crisis of rising consumer costs in healthcare has its roots in the history of medicine combined with the public's perceived right to healthcare. To reduce costs, many institutions have reduced staff which, according to some critics, threatens quality of care. A true solution may be quite complex, requiring movement from the current paternalistic approaches typical of acute-care settings toward a cooperative model of illness care with an emphasis on wellness behavior. The concepts of efficacy and negotiated levels of health in the cooperative model care triad have been shown to reduce the cost of illness care while increasing satisfaction, client compliance and quality of care.

Delivery of Health Care↗
PubMed1994

Production of interleukin-10 by human bronchogenic carcinoma.

Interleukin-10 (IL-10) is a recently characterized cytokine with suppressive activity against various aspects of the cellular immune response. Our laboratory has previously demonstrated that another anti-inflammatory cytokine, IL-1 receptor antagonist (IRAP) is produced and secreted by human bronchogenic carcinomas. We speculated that tumor production of IRAP may mitigate host responses and confer increased tumor viability. In this study, we investigated the capacity of human bronchogenic tumors to produce IL-10 as another possible mechanism to attenuate host defenses. We found increased levels of antigenic IL-10 in tissue homogenates of human bronchogenic carcinomas compared with normal lung tissue (13.69 +/- 2.87 versus 5.84 +/- 0.84 ng/mg total protein). Immunohistochemical staining of tumors illustrate primary localization of antigenic IL-10 to individual tumor cells. Analysis of supernatants of several unstimulated human bronchogenic cell lines in vitro demonstrated the ability of tumor cells to constitutively produce IL-10. Functional studies of mononuclear cells, cultured in the presence of conditioned medium from a bronchogenic cell line, demonstrated their increased tumor necrosis factor and IL-6 production with the addition of neutralizing antibodies to IL-10. These findings demonstrate that human bronchogenic carcinomas elaborate functional IL-10, which may significantly impair immune effector cell function and enable the tumor to evade host defenses.

Carcinoma, Bronchogenic↗
PubMed1993

Structure of an interleukin-1 beta mutant with reduced bioactivity shows multiple subtle changes in conformation that affect protein-protein recognition.

Site-specific mutagenesis was used to obtain the human interleukin-1 beta mutant protein with glycine substituted for threonine at position 9 (IL-1 beta Thr9Gly). The mutant maintains receptor binding but exhibits significantly reduced biological activity. The crystal structure of IL-1 beta Thr9Gly has been determined at 2.4-A resolution by molecular replacement techniques and refined to a crystallographic R-factor of 19.0%. IL-1 beta Thr9Gly crystallizes in a different space group (P6(5)22) than does native IL-1 beta (P4(3)); thus the molecules pack differently. Their overall structure is similar, nevertheless, with both composed of 153 amino acids which form 12 antiparallel beta-strands. However, significant conformational differences both close to and far from the site of the mutation may explain the mutant's altered properties.

Amino Acid Sequence↗
PubMed1993

A human genome YAC library in a selectable high-copy-number vector.

An experimental yeast artificial chromosome (YAC) library consisting of one genome equivalent of human DNA was prepared in a selectable high-copy-number (hcn) YAC vector. Screening for unique loci was accomplished by PCR of successively smaller DNA pools and by hybridization to high-density microcolony blots. Inserts averaged 200 kb in size, but several YACs with inserts averaging about 650 kb were obtained when polyamines were added prior to yeast transformation. YACs were identified for 17 out of 29 sequence-tagged sites (STS) screened by a PCR-based approach. All YACs in the size range of 100-600 kb that were examined could be obtained at significantly elevated copy numbers following growth of the clones in methotrexate/sulfanilamide/thymidine-supplemented medium. The hcn YACs could also be selected during growth in microtiter dishes, and the resulting clones were used to prepare high-density microcolony DNA blots for hybridization with radiolabeled PCR products. DNA pools for the PCR-based screening of this experimental library are available to investigators interested in applications of hcn YACs.

Base Sequence↗
PubMed1993

Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination.

We have developed a pair of vectors for exchanging yeast artificial chromosome (YAC) arms by targeted homologous recombination. These conversion vectors allow the introduction of copy-number control elements into YACs constructed with pYAC4 or related vectors. YACs modified in this way provide an enriched source of DNA for genetic or biochemical studies. A LYS2 gene on the conversion vector provides a genetic selection for the modified YACs after transformation with appropriately prepared vector. A background of Lys+ clones that do not contain modified YACs is also present. However, clones with converted YACs can be distinguished from this background by counter-screening for loss of the original p YAC4 TRP1 arm (Trp- phenotype). The elimination of yeast replication origins (ARS elements) from the conversion vectors increased the frequency of Lys+ Trp- clones, but resulted in weaker amplification. Several YACs have been converted with these vectors, and the fate of the transformed DNA and of the resident YAC DNA has been systematically investigated.

Chromosomes, Fungal↗
PubMed1993

Over-expression of the c-myc proto-oncogene in colorectal carcinoma.

Alterations in the c-myc proto-oncogene in colorectal cancer were studied at the level of RNA expression, gene amplification and rearrangements. One hundred cases of colorectal cancer, stratified by Dukes' stage were examined. The level of messenger RNA expression was measured in tumours and matched normal mucosa from the same patient. Between 5 and 400 fold over-expression was found in 66% of tumours. Neither the presence nor the level of over-expression correlated with tumour staging. A significant correlation (P < 0.01) was found between over-expression of c-myc in tumours and the presence of synchronous adenomas elsewhere in the colon. In contrast to other tumours, no rearrangements of the gene were found on Southern analysis of colorectal cancers. Similarly, amplification of the gene was not found in the cancers examined.

Actins↗
PubMed1993

Ten nucleotide differences, five of which cause amino acid changes, are associated with the Ah receptor locus polymorphism of C57BL/6 and DBA/2 mice.

We have analysed by heteroduplex formation (HF), single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), and nucleotide sequencing the cDNAs of the Ahrb-1 and Ahrd allelic forms of the aromatic hydrocarbon receptor (AhR) present in inbred strains of mice. The Ahrb-1 allele, found in the C57BL and C57BR strains, encodes a 95 kDa receptor with an affinity for ligand 15-20 times higher than the affinity of the 104 kDa receptor encoded by the Ahrd allele, found in the DBA/2 strain. Five overlapping fragments of the AhR coding sequence were obtained from liver RNA by reverse transcriptase synthesis of a cDNA first strand, followed by polymerase chain reaction amplification of these cDNA sequences (RT-PCR). Analysis by HF and SSCP revealed the presence of sequence differences in three of the five fragments. When the complete nucleotide sequence of the coding regions was determined by PCR sequencing, we found a total of ten nucleotide differences between the two alleles, nine of which localized to the three fragments where differences were detected by HF and SSCP. Five of the differences are silent. Of the other five, one changes the opal termination codon in Ahrb-1 to the codon for Arg in Ahrd, extending translation of the mRNA by 43 amino acids and accounting for the larger size of the AhR peptide in DBA/2 mice. One of the four remaining differences causes the replacement of a leucine residue in Ahrb-1 by a proline residue in Ahrd, and breaks a potential alpha-helix near the AhR Q-rich region; it is likely that structural changes associated with this amino acid change are responsible for the differences in agonist affinity observed between the Ah receptors of these two strains of mice.

Amino Acid Sequence↗
PubMed1993

Native and baculovirus-expressed forms of the immuno-protective protein BM86 from Boophilus microplus are anchored to the cell membrane by a glycosyl-phosphatidyl inositol linkage.

A glycoprotein (BM86) from the gut cells of the cattle tick Boophilus microplus, when used to vaccinate cattle, has been shown to protect cattle from tick infestation. A recombinant BM86 protein is the principal component of a novel tick vaccine currently under development. The nature of the anchorage of BM86 to tick gut epithelial cells has been investigated using BM86 from B. microplus and recombinant BM86 proteins expressed in insect cells using the baculovirus expression system. BM86 from B. microplus and a full length recombinant BM86 are shown to be anchored to the extracellular surface of tick gut epithelial cells and baculovirus-infected insect cells, respectively by a glycosyl-phosphatidyl inositol membrane anchor. A recombinant BM86 truncated by the removal of a hydrophobic region coding for thirty amino acids at the carboxy-terminal end was secreted from baculovirus-infected Sf9 cells. This secreted form of recombinant BM86 showed strong protective activity against ticks in cattle vaccinated with this protein.

Animals↗