Translation of bacteriophage Q messenger RNA in a murine Krebs 2 ascites tumor cell-free system.

Qbeta is a small bacterial virus whose three genes are encoded in a single-stranded molecule of RNA. This RNA serves directly as the Qbeta message. Here we describe conditions under which RNA corresponding to the coat cistron of this bacterial virus is translated in a system derived from mammalian cells. Translation of the bacterial virus messenger RNA is less effective than that of mammalian globin messenger RNA, but is somewhat enhanced by mild alkali treatment of the messenger. The synthesized product when subjected to electrophoresis migrates with authentic Qbeta coat protein and yields tryptic peptides that correspond to those derived from the Qbeta coat protein.

Shortened telomere length in white blood cells of patients with IDDM.

IDDM is a polygenic and autoimmune disorder in which subsets of white blood cells (WBCs) are engaged in the destruction of beta-cells of the pancreas. The mechanisms that account for the abnormal behavior of these cells in IDDM are not fully understood. By measuring the mean length of telomeres of WBCs from patients with IDDM, we tested the concept that telomeres might play a role in IDDM. We examined the lengths of the terminal restriction fragments (TRFs) of DNA of WBCs from 234 white men comprising 54 patients with IDDM, 74 patients with NIDDM, and 106 control subjects. When adjusted for age, the TRF length from WBCs of patients with IDDM was significantly shorter than that of nondiabetic control subjects (mean +/- SE: 8.6 +/- 0.1 vs. 9.2 +/- 0.1, P = 0.002). No significant difference was observed between the TRF length from WBCs of patients with NIDDM versus nondiabetic subjects. Neither the duration nor the complications of IDDM (i.e., nephropathy and hypertension) had an effect on the TRF length of WBCs from patients with IDDM. The shortened TRF length of WBCs of patients with IDDM likely reflects a marked reduction in the TRF length of subsets of WBCs that play a role in the pathogenesis of IDDM.

Inhibition of lactogenic activities of ovine prolactin and human growth hormone (hGH) by a novel form of a modified recombinant hGH.

A recombinant analog of human GH (hGH) lacking 13 amino acids at the amino-terminus (Met14hGH) inhibited the hGH- or ovine PRL (oPRL)-stimulated proliferation of Nb2 lymphoma cells and bovine PRL-stimulated fat synthesis and alpha-lactalbumin secretion in explants from bovine lactating mammary gland. The inhibition was competitive in nature, and in Nb2 cells could be abolished by an excess of hGH or oPRL. Inhibition of oPRL-stimulated proliferation of Nb2 cells by Met14hGH could also be specifically abolished by anti-hGH monoclonal antibodies. Met14hGH had no growth-stimulating activity in Nb2 cells and was not cytotoxic. It also did not affect glucose uptake by the mammary gland explants. Met14hGH competed with [125I]hGH for binding to intact Nb2 cells, IM-9 lymphocytes, solubilized microsomal fraction from lactating bovine mammary gland, and microsomal fraction from the liver of female virgin rats, but its affinity for those receptors was 2 orders of magnitude lower than the affinity of hGH. Since Met14hGH used in most experiments contained about 25% impurities and degradation products, a small amount of it was further purified by immunoaffinity chromatography. Two purified fractions, one consisting of a single 20K protein and the other accompanied by a small amount of 25K protein, were obtained. Both fractions exhibited increased inhibition of hGH- or oPRL-stimulated proliferation of Nb2 cells, thus indicating that the inhibitory activity results from the intact Met14hGH molecule. To the best of our knowledge, this is the first report describing the inhibition of lactogenic hormone activities by a modified hGH.

Reflections on telomeres, growth, aging, and essential hypertension.

Here we review the "telomere hypothesis of cellular aging." We propose that this hypothesis is relevant to our understanding of the roles of genetics as well as growth and development in the etiology of essential hypertension and its cardiovascular complications. Elements of this hypothesis and the speculations that we make can be directly tested using tissues (cells) obtained from human beings.

Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis.

Little is known about the turnover rate (i.e. the rate of replication and death) of cells in the intima and media of human arteries as a function of age and atherosclerosis. One indicator of the replicative history of cells is telomere length. In this work we explored the rate of telomere attrition as a function of age and atherosclerosis in cells of the human abdominal aorta. Telomere length, measured by the terminal restriction fragment using Southern analysis, was determined in the intima and media of the distal (infrarenal) versus proximal (suprarenal) segments of the abdominal aorta. Telomere length was then correlated with age and atherosclerotic grade. The rate of age-dependent telomere attrition was higher in both the intima and media of the distal versus proximal abdominal aorta. In addition, telomere length was negatively correlated with atherosclerotic grade. However, after adjustment for age, this relationship was not statistically significant. The high rate of age-dependent telomere attrition in the distal abdominal aorta probably reflects enhanced cellular turnover rate due to local factors such as an increase in shear wall stress in this vascular segment.

Age dependent aneuploidy and telomere length of the human vascular endothelium.

RATIONALE: Aneuploidy and telomere length are two major parameters that have been associated with cellular senescence in vitro. In order to explore the role of aneuploidy and telomere length in aging of the human vasculature, we studied these two parameters in direct preparations of endothelial cells of the human abdominal aorta. METHODS: Using fluorescent in situ hybridization on 'touch prep' slides, we evaluated aneuploidy of two autosomes (chromosomes 6 and 16) and sex chromosomes in non cultured endothelial cells of the abdominal aorta as a function of the donor's age. RESULTS: We found that the frequency of aneuploidy of vascular endothelial cells significantly increased with age. This was expressed by age-dependent tetrasomy (r(s)=0.56, P=0.006 for chromosome 6; and r(s)=0.54, P=0.008 for chromosome 16), and age dependent loss of the Y chromosome (r(s)=0.85, P=0.0003). In addition, we found that telomere length was inversely correlated with age (r=-0.38, P=0.008). DATA INTERPRETATION: These findings suggest that indicators of cellular senescence, earlier observed in vitro, are also expressed in the human vascular endothelium in vivo. Aneuploidy and telomere attrition might thus play a role in the aging of the human vasculature.

Evaluation of immunological methods for detection of bovine growth hormone (BGH) produced in E. coli.

The use of several immunological methods for studies on synthesis of bovine growth hormone (BGH) by E. coli is described here. The ELISA procedure was shown to be the least sensitive and unfit for assaying BGH in E. coli extracts. The solid-phase radioimmunoassay (RIA) proved to be highly sensitive, but since E. coli extract itself (not containing BGH) interfered with the immunological reaction, its use for measuring BGH was practically limited. The best adequate procedure proved to be radioimmunoassay in solution, which was not adversely affected by the E. coli extract and was sufficiently sensitive to detect nanogram quantities of BGH. The size of the BGH produced by normal bacterial cells was investigated by protein fractionation, transfer to nitrocellulose paper and detection by anti-BGH serum. This method was also served for semi-quantitative determination of BGH in the bacterial extract.

Familial transmission of a deletion of chromosome 21 derived from a translocation between chromosome 21 and an inverted chromosome 22.

Chromosome analysis of a newborn boy with Down syndrome resulted in the identification of a family with an unusual derivative chromosome 22. The child has 46 chromosomes, including two chromosomes 21, one normal chromosome 22, and a derivative chromosome 22. Giemsa banding and fluorescent in situ hybridization (FISH) studies show that the derivative chromosome is chromosome 22 with evidence of both paracentric and pericentric inversions, joined to the long arm of chromosome 21 from 21q21.2 to qter. The rearrangement results in partial trisomy 21 extending from 21q21.2 to 21q terminus in the patient. The child's mother, brother, maternal aunt, and maternal grandmother are all carriers of the derivative chromosome. All have 45 chromosomes, with one normal chromosome 21, one normal chromosome 22, and the derivative chromosome 22. The rearrangement results in the absence of the short arm, the centromere, and the proximal long arm of chromosome 21 (del 21pter-21q21.2) in carriers. Carriers of the derivative chromosome in this family have normal physical appearance, mild learning disabilities and poor social adjustment.

Sex chromosome mosaicism in gonads of a fetus with cystic hygroma and deletion of the short arm of Y chromosome including loss of SRY.

The SRY gene on the short arm of the Y chromosome is necessary for male development. Without SRY, patients with 46,XY karyotype develop as females, fail to achieve normal puberty and have dysgenic gonads and a high incidence of gonadoblastoma. Here we report a female fetus, aborted at 17 weeks of pregnancy, with a non-mosaic 46,X,del(Y)(p11.2).ish del(Y)(SRY-) karyotype diagnosed by classical cytogenetics and fluorescence in situ hybridization (FISH). Ovarian tissue was full of oocytes and mitotic figures. FISH studies of ovarian tissues with X and Y centromere probes revealed extensive sex chromosome mosaicism, manifested by loss of the Y chromosome and polysomy of the X chromosome. We propose that X chromosome polysomy is a post-zygotic event that arises to facilitate gonadal differentiation in the absence of all factors necessary for normal gonadal development.

Interstitial insertion of Y-specific DNA sequences including SRY into chromosome 4 in a 45,X male child.

A 45,X chromosome complement was found in the lymphocytes and skin fibroblast cultures of a male infant with minor facial anomalies and gastrointestinal abnormalities. Fluorescence in situ hybridization (FISH) studies with DNA probes specific for the entire Y chromosome (painting) and SRY identified insertion of a short piece of Y chromosome DNA, including the SRY region, into a der(4) chromosome at 4p15. FISH studies with DNA probes specific for Wolf-Hirschhorn syndrome (WHS) and telomere of 4p indicated that these 2 regions were intact and that the insertion of Y DNA had occurred proximal to the WHS region. High-resolution chromosome analysis performed after FISH studies showed an altered banding pattern of 4p at the region of insertion. The typical Giemsa dark band of 4p15 was consistently replaced by a gray band; this probably indicates deletion of the distal part of 4p15. The consequences of the double-chromosome anomaly in this patient were discussed in relation to his phenotype.

Telomeres and essential hypertension.

The dynamics of telomere attrition in human beings might shape the course of age-dependent, complex genetic traits. One of these traits is essential hypertension. Age-dependent telomere attrition could lead to critically shortened telomeres and aneuploidy (ie, the loss or gain of chromosomes) with a resultant mosaicism that will be variably expressed in different cells and tissues. The chromosomal instability and loss of heterozygosity resulting from this process would promote an age-dependent expression of variant genes that harbor susceptibility for essential hypertension or genes that accelerate the process of aging.

Gentamicin bioautography assay vs. the microbiological disk test.

The use of bioautography for quantitative measurement of gentamicin concentrations was compared with the disk test. Following chromatographic separation and bioautography, gentamicin produced inhibition zones, 2 approximately 7 times larger than the inhibition zones formed by the same amounts of gentamicin in the disk test. Bioautography, therefore, is a more sensitive assay method.

Alveolar soft part sarcoma--reciprocal translocation between chromosome 17q25 and Xp11. Report of a case with metastases at presentation and review of the literature.

The molecular pathogenesis of alveolar soft part sarcoma, a rare tumor with uncertain histogenesis, was elucidated recently and was shown to be due to a translocation between chromosome 17q25 and Xp11 resulting in a fusion product between TFE3 (a transcription factor gene) at chromosome Xp11 and a novel gene designated as ASPL at chromosome 17q25. This results in the transcriptional dysregulation in the pathogenesis of this neoplasm. Of the 12 cases reported so far, the translocation was due to non-reciprocal translocation in 11 cases with only one case demonstrating a reciprocal translocation with respective fusion products. We report yet another case with reciprocal translocation between chromosomes 17q25 and Xp11 with TFE3/ASPL fusion product who presented with metastatic disease. A standard cytogenetic analysis of primary tumor cells with G-banding revealed an abnormal karyotype: 46, X, t(X;17)(p11;q25)[15]/46,XX[5]. PCR analysis of the frozen tumor tissue revealed a type 1 fusion product as described in the literature. We demonstrate a rare cytogenetic abnormality in ASPS, namely reciprocal translocation between chromosomes 17q25 and Xp11 with demonstration of molecular fusion product between TFE3 and ASPL in a patient who initially presented with pulmonary metastases.

Radioimmunoassay of gentamicin in Micromonospora medium extracts.

Medium extracts of Micromonospora adversely affected a radioimmunoassay which was used for the measurement of gentamicin in the medium. An overestmation or an underestimation resulted, as judged by the addition of extracts to a gentamicin standard sample.

Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose.

A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.

Quantitation of labeled globin messenger RNA by hybridization with excess complementary DNA covalently bound to cellulose.

A method is described to quantitate labeled globin mRNA by hybridization with excess cDNA which was enzymatically polymerized on oligo(dT)-cellulose. In a large excess of cDNA-cellulose the rate of RNA hybridization was dependent on DNA concentration and not on RNA concentration. Nonhybridized RNA can be digested by RNase and washed from the cDNA which is covalently bound to cellulose. This enables the detection of labeled globin mRNA even when present in a porportion as low as 0.02-0.03% of the total RNA.

Preferential synthesis of viral late RNA by nuclei isolated from SV40 lytically infected cells.

Nuclei from SV40-infected monkey cells were isolated late in lytic infection and their cell-free transcriptional activity was characterized. 3H-RNA synthesized in vitro was hybridized to excess quantities of separated SV40 DNA strands which were each covalently bound to Sepharose. It was found that 3-5% of the newly synthesized RNA is virus-specific and that the plus-strand DNA, coding for late RNA sequences, is transcribed at a rate about 15 times higher than that of the minus-strand DNA, which codes for early RNA sequences. This indicates that transcriptional control has a major role in determining the relative abundancy of early and late RNA classes in lytically infected cells.