Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis.

Telomere-associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non-targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.

All's well that ends well.

A recent Cold Spring Harbour meeting(*) reviewed the latest progress on telomeres (the specialized structures that form the ends of chromosomes) and telomerase (the enzyme primarily responsible for their replication). Among the many aspects of telomere biology covered were strong sessions elaborating telomere replication and length regulation, telomerase structure and function, end-binding and telomere-associated proteins, DNA-damage-response proteins and telomerase-independent telomere maintenance.

Acid soil indicators in forest soils of the Cherry River Watershed, West Virginia.

Declining forest health has been observed during the past several decades in several areas of the eastern USA, and some of this decline is attributed to acid deposition. Decreases in soil pH and increases in soil acidity are indicators of potential impacts on tree growth due to acid inputs and Al toxicity. The Cherry River watershed, which lies within the Monongahela National Forest in West Virginia, has some of the highest rates of acid deposition in Appalachia. East and West areas within the watershed, which showed differences in precipitation, stream chemistry, and vegetation composition, were compared to evaluate soil acidity conditions and to assess their degree of risk on tree growth. Thirty-one soil pits in the West area and 36 pits in the East area were dug and described, and soil samples from each horizon were analyzed for chemical parameters. In A horizons, East area soils averaged 3.7 pH with 9.4 cmol(c) kg(-1) of acidity compared to pH 4.0 and 6.2 cmol(c) kg(-1) of acidity in West area soils. Extractable cations (Ca, Mg, and Al) were significantly higher in the A, transition, and upper B horizons of East versus West soils. However, even with differences in cation concentrations, Ca/Al molar ratios were similar for East and West soils. For both sites using the Ca/Al ratio, a 50% risk of impaired tree growth was found for A horizons, while a 75% risk was found for deeper horizons. Low concentrations of base cations and high extractable Al in these soils translate into a high degree of risk for forest regeneration and tree growth after conventional tree harvesting.

Mitochondrial single-stranded DNA-binding protein is required for mitochondrial DNA replication and development in Drosophila melanogaster.

The discovery that several inherited human diseases are caused by mtDNA depletion has led to an increased interest in the replication and maintenance of mtDNA. We have isolated a new mutant in the lopo (low power) gene from Drosophila melanogaster affecting the mitochondrial single-stranded DNA-binding protein (mtSSB), which is one of the key components in mtDNA replication and maintenance. lopo(1) mutants die late in the third instar before completion of metamorphosis because of a failure in cell proliferation. Molecular, histochemical, and physiological experiments show a drastic decrease in mtDNA content that is coupled with the loss of respiration in these mutants. However, the number and morphology of mitochondria are not greatly affected. Immunocytochemical analysis shows that mtSSB is expressed in all tissues but is highly enriched in proliferating tissues and in the developing oocyte. lopo(1) is the first mtSSB mutant in higher eukaryotes, and its analysis demonstrates the essential function of this gene in development, providing an excellent model to study mitochondrial biogenesis in animals.

Birth plans and health facility based delivery in rural Uganda.

OBJECTIVES: To evaluate whether the completion of birth plans is associated with delivery in a health facility and the perceptual causes of birth plan completion and health facility based delivery were explored according to a well-tested health behaviour theory. DESIGN: A community survey. SETTING: Rakai and Luwero districts. SUBJECTS: A total of 415 (202 in Rakai and 213 in Luwero district) respondents were randomly selected and interviewed using a mixed survey questionnaire composed of open and close-ended questions. MAIN OUTCOME MEASURES: Health facility based delivery. RESULTS: The results demonstrate a statistically significant relationship between the completion of birth plans and delivery in a health facility (OR = 1.86, 95% CI =1.1, 3.1). The fear of consequences of delivering at home was found to be an important driving force in promoting the completion of birth plans, thereby indirectly influencing the likelihood of delivery in a health facility. CONCLUSION: Given the empirical evidence presented here, this study suggests that birth plans are an important tool in improving the rate of health facility based deliveries and thus essential in the fight against maternal mortality in Uganda. It is further recommended that campaigns market the use of birth plans as a way to reduce uncertainty and manage fear and the unknown about pregnancy.

Activated ras oncogenes in human thyroid cancers.

Human thyroid epithelial (follicular) cells give rise to two malignant tumors--"follicular" carcinomas, which metastasize almost exclusively via the bloodstream, and "papillary" carcinomas, which metastasize predominantly via lymphatics (Williams, E. D. In: W. Duncan (ed.), Recent Results in Cancer Research: Thyroid Cancer, pp. 47-55. Berlin: Springer-Verlag, 1980). We have investigated whether this contrast in biological behavior might be associated with different patterns of oncogene activation. DNA transfection analysis of five follicular and ten papillary cancers indeed showed a statistically significant difference in the pattern of genes responsible, activated ras oncogenes being found in 80% of follicular tumors but only 20% of papillary tumors. In addition, in follicular cancers we have found activation of all three ras oncogenes (H-ras, K-ras, and N-ras), the first time that this has been demonstrated in a primary human tumor type (as opposed to cell lines). We suggest therefore that ras activation may be an important determinant of metastatic capability in these epithelial cancers.

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

ras gene activation in a minor proportion of the blast population in acute myeloid leukemia.

DNAs from 22 acute myeloid leukaemia (AML) patients were screened for activated transforming genes using NIH3T3 transfection followed by assay for tumor formation in Nude mice. In four samples an activated N-ras oncogene, and in two samples an activated Ha-ras oncogene were detected in transfectants. Synthetic oligonucleotide probes were used to characterise the mutations in the ras genes. Three samples were found to be mutated to N-ras codon 12 ASP, one to N-ras codon 13 ASP and two to Ha-ras codon 12 VAL. When the corresponding AML DNAs were screened using direct gel hybridisation, the mutant ras genes were detectable in only one case. In two AML samples (82 and 84) with very low percentage blasts (3% and 19%), the absence of mutant ras signal from direct gel hybridisation may be due to the lack of sensitivity of this technique in detecting activated ras in such small fractions of the total DNA. These results illustrate the sensitivity of the in vivo tumour assay in detecting activated ras. When DNA from one of the remaining three AML DNAs was amplified using the polymerase chain reaction method, the mutation present in the transfectant was detected. These findings suggest that even in AMLs with high percentage blasts (40%, 70% and 90%), cells containing mutant ras may comprise only a minor proportion of the major leukaemic clone.

A study of ras gene mutations in colonic adenomas from familial polyposis coli patients.

Using an in vitro amplification step (polymerase chain reaction) followed by oligonucleotide dot blot analysis, DNA samples from 29 familial polyposis coli patients (75 polyp-derived and 26 'normal' colon samples with no epithelial atypia) were screened for the presence of K-, N-, and H-ras mutations. Only 5 polyps contained ras mutations (7%)--all in K-ras codon 12. In each case 'normal' colon DNA was available and found to be negative in this assay. We also report the detection of K-ras codon 12 mutations in a stably non-tumorigenic immortal adenoma-derived cell line, PC/AA, and in a tumorigenic colorectal carcinoma cell line, PC/JW. Both epithelial cell lines were derived from different FPC patients. An activated K-ras gene was also found in cell line S/AN, isolated from a sporadic villous adenoma. These results provide further evidence that there are common molecular events involved in sporadic and hereditary colorectal carcinogenesis and that K-ras mutations can precede the development of malignancy. To our knowledge PC/AA is the first reported example of a human cell line bearing a mutant ras gene that is not tumorigenic and shows that the presence of an activated ras gene even in an immortal human cell line is insufficient for malignancy.

RAS mutations in myelodysplasia detected by amplification, oligonucleotide hybridization, and transformation.

Members of the RAS gene family have been implicated in many neoplasms with activating mutations around amino acid positions 12 and 61. We have assessed the mutational activation of H, K, and NRAS in myelodysplasia (MDS) by polymerase chain reaction and hybridization with synthetic oligonucleotide probes. Using this method, point mutations in codons 12/13 and 61 of these RAS genes were detected in 20 of 50 patients including two with refractory anemia with ringed sideroblasts (RARS). Ten normal individuals had no detectable RAS mutations. In 11 instances, DNA from patients with detectable RAS mutations were shown to register in either NIH3T3 focus-forming or nude mouse tumorigenicity assays. In addition, one patient (RARS) was shown to have an activated NRAS gene detected by a tumorigenicity assay and Southern blot analyses. Two MDS patients had mutations detected in two different RAS genes. DNA from one of these patients was observed to give rise to transformants with activated N and HRAS. Two patients with detectable NRAS mutations in the MDS stage progressed to AML and DNA from the AML stage registered positively in a transformation assay with NRAS activation. These results show that RAS mutations can occur at early, as well as late, stages of leukemic progression. The incidence of RAS mutations appears to be significantly higher in CMML than in the other subgroups (p = 0.02).

Novel and known protein tyrosine kinases and their abnormal expression in human melanoma.

We have used the polymerase chain reaction and Northern blotting to identify protein tyrosine kinases that may play an important role in the process of melanoma initiation and progression. Degenerate primers from the conserved catalytic domain of tyrosine kinase genes were used to amplify and clone partial cDNA sequences from a human melanoma cell line (DX3-LT5.1) and normal human melanocytes. When the melanoma reaction products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named MEK (for melanocytic kinase). Of the remaining 12 known kinases, only two, ERB-B2 and IGF1-R, have previously been reported in pigment cells. Reaction products from melanocytes included only eight of these 13 sequences. To test for quantitative differences in tyrosine kinase expression between normal and malignant cells, a panel of eight melanoma lines and normal melanocytes was analyzed by Northern blotting. Two tyrosine kinases (JTK-14/TIE and TYRO-9) were detected in some melanomas but were not found in normal melanocytes, whereas others, including MEK, appeared to be overexpressed in some malignant lines. A minority of kinases showed either no change or a reduction in the level of mRNA. Expression of tyrosine kinases varied independently, and individual lines contained various combinations of these enzymes. Our findings are consistent with an increased overall expression of these putative growth factor receptors during melanoma development.

Protein tyrosine phosphatase genes downregulated in melanoma.

Phospho-tyrosine levels are increased in melanoma, apparently consistent with reports of elevated protein tyrosine kinase activity. Some protein tyrosine kinases are encoded by oncogenes and have been implicated in melanoma genesis. Decreased protein tyrosine phosphatase activity may also increase phospho-tyrosine. Protein tyrosine phosphatase genes are candidate tumor suppressors and loss of expression may contribute to melanoma genesis. Here we survey protein tyrosine phosphatase expression in pigment cells. Protein tyrosine phosphatase genes were cloned by reverse transcriptase polymerase chain reaction using degenerate primers based upon conserved sequences within the phosphatase catalytic domain. Reaction products were cloned and sequenced: 118 and 113 partial protein tyrosine phosphatase products were isolated from normal melanocytes and melanoma cells, respectively. Northern blotting analysis was used to study expression of 15 protein tyrosine phosphatase genes. Expression of PTP-kappa and PTP-pi was absent or downregulated in more than 20% of melanoma cell lines and in some unmanipulated melanoma biopsies. These closely related enzymes are members of the 2B receptor protein tyrosine phosphatase family previously implicated in contact inhibition. Loss of protein tyrosine phosphatase expression may contribute to the abnormal tyrosine phosphorylation seen in melanoma; these genes are candidate tumor suppressors.

Cloning and characterisation of the chicken gene encoding the telomeric protein TRF2.

The telomere-associated protein TRF2 binds as a homodimer to double-stranded (TTAGGG)n arrays in vitro and localises to chromosome ends in vivo. Inhibition of TRF2 in human cell lines and recent electron microscopy analyses suggest that TRF2 plays a crucial role in the maintenance of telomere integrity. To study the role of TRF2 in vertebrate telomere biology using an alternative model system, we report the isolation and characterisation of the chicken TRF2 locus. The TRF2 protein is highly conserved between mammals and birds, particularly within the dimerisation and myb-type DNA binding domains. However, the chicken ORF predicts an additional protein domain consisting of 15 copies of a degenerate 13 amino acid repeat. Indirect immunofluorescence reveals the localisation of a FLAG-tagged version of the chicken TRF2 protein at chromosome ends in both chicken and human cells suggesting that the protein is functionally conserved.

Hyaluronidase and its substrate hyaluronan: biochemistry, biological activities and therapeutic uses.

This is an overview of the biochemistry, biological function and therapeutic uses of hyaluronidase and its substrate, hyaluronate. We focus on the role of hyaluronate and its receptor CD44 in cell-cell and cell-matrix adhesion and cell activation as well as on the putative role of hyaluronate and hyaluronidase in morphogenesis. Variants of CD44 and their putative role in tumor metastasis are also included. Other topics that are discussed are the chemical and enzymatic nature of hyaluronidase, i.e. the mode of substrate degradation, pharmacodynamical and pharmacokinetic aspects of this enzyme and its role as spreading factor. Purification methods, possible contaminations and techniques of activity determinations are mentioned as well as the physiological role of hyaluronidase and tumor-associated alterations in serum and tissue enzyme levels. As far as therapeutic applications are concerned, we discuss uses of hyaluronidase in ophthalmology and regional anesthesia as well as pain management in osteoarthritis using hyaluronate.

Activation of Ha-ras in human chronic granulocytic and chronic myelomonocytic leukaemia.

DNAs from chronic granulocytic leukaemia (CGL) and chronic myelomonocytic leukaemia (CMML) were assayed for transforming genes by transfection into NIH 3T3 cells. Foci DNA was tagged with a geneticin-resistance cosmid, then followed through a drug selection and tumorigenicity assay. Activated Ha-ras genes, with point mutations at codon 12 (glycine to valine) were subsequently detected. The mutation was detected in the original samples by either MspI/HpaII digestion or polymerase chain reaction (PCR). Although mutations in the ras gene family may occur frequently in leukaemias, these are the first examples of Ha-ras mutations in CGL (blast crisis) and CMML.

ATP lowers the activation enthalpy barriers to DnaK-peptide complex formation and dissociation.

The role of nucleotide in controlling the pre-steady-state kinetics of peptide binding to the Escherichia coli 70-kDa molecular chaperone DnaK was investigated using stopped-flow fluorescence. The peptide used in this study, fVSV13 (representing amino acids 490-502 of the vesicular stomatitis virus glycoprotein), was dansylated specifically at its N-terminus. We found that (i) between 17 and 35 degrees C in the presence of ATP the second-order rate constant (k(on)) for fVSV13 binding to DnaK exhibited almost no dependence on temperature and did not deviate significantly from 3.8 x 10(5) M(-1) s(-1). In contrast, over the same temperature range in the presence of ADP, k(on) increased by a factor of 32 (7.3 x 10(4) to 2.3 x 10(6) M(-1) s(-1)); and (ii) ATP increased the apparent first-order rate constant for the release of fVSV13 from preformed DnaK-fVSV13 complexes by several orders of magnitude relative to ADP. The activation energy parameters for fVSV13 binding to and dissociation from DnaK are compared to the activation parameters for the binding of an unrelated peptide to DnaK and are also discussed in terms of an open-to-closed equilibrium in the polypeptide-binding domain. On the basis of this comparison, it is suggested that the activation entropy term deltaS++, which is related to the structure of the DnaK-bound peptide or the degree of solvation of the peptide, is a controlling factor in the kinetics of peptide binding to DnaK.

Absence of prenatal developmental toxicity from inhaled arsenic trioxide in rats.

A review of the literature revealed no published inhalational developmental toxicity studies of arsenic performed according to modern regulatory guidelines and with exposure throughout gestation. In the present study, inorganic arsenic, as arsenic trioxide (As(+3), As2O3), was administered via whole-body inhalational exposure to groups of twenty-five Crl:CD(SD)BR female rats for six h per day every day, beginning fourteen days prior to mating and continuing throughout mating and gestation. Exposures were begun prior to mating in order to achieve a biological steady state of As(+3) in the dams prior to embryonal-fetal development. In a preliminary exposure range-finding study, half of the females that had been exposed to arsenic trioxide at 25 mg/m3 died or were euthanized in extremis. In the definitive study, target exposure levels were 0.3, 3.0, and 10.0 mg/m3. Maternal toxicity, which was determined by the occurrence of rales, a decrease in net body weight gain, and a decrease in food intake during pre-mating and gestational exposure, was observed only at the 10 mg/m3 exposure level. Intrauterine parameters (mean numbers of corpora lutea, implantation sites, resorptions and viable fetuses, and mean fetal weights) were unaffected by treatment. No treatment-related malformations or developmental variations were noted at any exposure level. The no-observed-adverse-effect level (NOAEL) for maternal toxicity was 3.0 mg/m3; the NOAEL for developmental toxicity was greater than or equal to 10 mg/m3, 760 times both the time-weighted average threshold limit value (TLV) and the permissible exposure limit (PEL) for humans. Based on the results of this study, we conclude that arsenic trioxide, when administered via whole-body inhalation to pregnant rats, is not a developmental toxicant.

Epidermoid carcinoma of the mouth and pharynx at Memorial Sloan-Kettering Cancer Center, 1965 to 1969.

One thousand thirty-four cases of epidermoid carcinoma of the mouth and pharynx were seen by the Head and Neck Service of Memorial Hospital during a recent 5 year period, 1965 to 1969. The 656 determinate cases previously untreated offer a clear basis for evaluation of the 5 year results of treatment, which as surgical in 90 percent of the cases. Preoperative radiotherapy was used in 130 cases. Preoperative radiotherapy was used in 130 cases, matched with 126 untreated cases. Etiologic factors are discussed. The presence and degree of lymph node metastasis are of paramount significance. Indications for elective treatment of the clinically negative neck are discussed in terms of anatomic, logistic and statistical principles. An analysis of failure point toward a continuing effort at combined therapy in this regional disease.