Assignment of genes to the human X chromosome by the two-dimensional electrophoretic analysis of total cell proteins from rodent-human somatic cell hybrids.

The technique of two-dimensional (2-D) gel electrophoresis was sued to identify five human X-linked gene products in crude cell extracts of mouse-human and Chinese hamster-human somatic cell hybrids. The human origin of these five polypeptides was demonstrated by their comigration with human fibroblast proteins and their failure to comigrate with polypeptides in extracts from the mouse or hamster parental cells. All five polypeptides were present in extracts of rodent-human hybrids that contained a human X chromosome, but were not found in extracts of cells that lacked a human X chromosome. Chromosome analysis of the hybrid clones revealed that the human X chromosome is both necessary and sufficient for the expression of the five polypeptides, designated pX-24, pX-27, pX-37, pX-40, and pX-56. pX-56 can be identified as the human X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) (E.C.1.1.1.49), while polypeptides pX-24, pX-27, pX-37 and pX-40 have molecular properties unlike those of known human X-linked gene products. pX-24 appears to be a membrane-bound protein that maps to the distal portion of the long arm of the human X chromosome, while pX-27, pX-37, and pX-40 are soluble proteins that map to the proximal long arm or to the short arm of the human X chromosome. 2-D gel electrophoretic analysis of extracts from somatic cell hybrids provides a general method for identifying polypeptides in crude cell extracts coded for by any specific chromosome and can be used to study primary gene products not previously amenable to genetic analysis.     

Nucleotide sequence of 7 S RNA. Homology to Alu DNA and La 4.5 S RNA

7 S RNA, a component of normal higher eukaryotic cells and several oncornaviruses, was shown to be conserved in evolution (Erikson, E., Erikson, R. L., Henry, B., and Pace, N. R. (1973) Virology 53, 40-46). Recently, 7 S RNA was shown to be partially complementary to Alu family DNA sequences (Weiner, A. (1980) Cell 22, 209-218). In the present study the nucleotide sequence of Novikoff hepatoma 7 S RNA was determined to be: (formula, see text) Comparison of 7 S RNA, Alu and B1 family DNA, and La 4.5 S RNA sequences for homologies showed that 1) one-third of 7 S RNA, mainly the 5'-end, was homologous to Alu and B1 family sequences; 2) one 300-nucleotide long Alu family sequence contained two binding sites for 7 S RNA; and 3) the 5'-ends of 7 S RNA and La 4.5 S RNA also had extensive (60%) homologies. A model for the secondary structure of 7 S RNA based on maximal base pairing and preferential nuclease cleavage sites is also presented.     

Analysis of the effect of periodate oxidation and phenol extraction on the antitumour properties of C. parvum

Summary The antitumour activity of C. parvum and its relationship to spleen weight increase have been analysed in a tumour model using syngeneic M₄ fibrosarcoma cells. It was found that treatment of C. parvum with low concentrations of periodate (2 mM) produced a minor decrease in the ability to abrogate tumour growth, following intratumour injection, although the capacity to increase spleen weight after IP injection was abolished. Higher concentrations of periodate (20 mM) produced complete inactivation of the bacteria. A phenol extract of C. parvum was weakly active in the intratumour test but amounts up to 1 mg injected IP were unable to increase the spleen weight. The data support the hypothesis that a surface carbohydrate is responsible for the increase in spleen weight. It is suggested that the antitumour activity may reside in a molecule that also reacts with periodate but at a slower rate, perhaps because of a more complex structure or a different location in the cell, for instance in the cytoplasm. The phenol extract was soluble in chloroform and at least two major components were glycolipids, so it is conceivable that a molecule of this type, which could be degraded by periodate, might be responsible for the antitumour activity of C. parvum. The precise biochemical nature of the active component is, as yet, undetermined.     

A mouse cell line, which is unprotected by interferon against lytic virus infection, lacks ribonuclease F activity

A mouse cell line, NIH 3T3, does not respond to some of the activities of interferon. Even after treatment with high concentrations of interferon the replication of lytic viruses, such as encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) is not inhibited in these cells. In contrast, interferon treatment of these same cells results in the inhibition of Moloney murine leukemia virus (MMuLV) production. We have analyzed enzymatic pathways which are induced by interferon in these cells. After interferon treatment, the level of the (2'-5')oligoadenylate [(2'-5)An] synthetase activity and the phosphorylation of the 67000-dalton protein (P1) are enhanced in NIH 3T3 cells to approximately the same level as interferon-sensitive mouse L-cells. Moreover, NIH 3T3 and L-cells, contain approximately the same levels of enzymes which inactivate (2'-5')An. Both exogenously added (2'-5')A3 or double-stranded RNA (dsRNA) failed to inhibit protein synthesis in NIH 3T3 extracts even though they were potent inhibitors of L-cell extract-directed protein synthesis. Direct measurements of the (2'-5')An-dependent ribonuclease F (RNase F) failed to detect such activity in NIH 3T3 cells. Our results, therefore, suggest that the presence of RNase F activity is necessary for the interferon-induced antiviral activity against EMCV and against VSV. The induction of protein kinase activity by interferon treatment of NIH 3T3 cells appears to have no direct effect on EMCV and VSV replication.     

Increased sodium intake is somehow induced in rats by intravenous angiotensin II

Adult male rats maintained on dry food, water, and 3% NaCl solution received continuous infusion of angiotensin II (A II) via right atrial catheters. A II was delivered in 0.315 ml/hr at doses of 15, 30, and 60 ng/min/rat for 3 to 5 days. At the higher doses mean daily salt solution intake rose from very low preinfusion levels to 18.7 and 19.6 ml, respectively. Daily water intakes increased in some animals and decreased in others on the first day of infusion but were double preinfusion levels by the second day. Persistence of the sodium appetite after the end of A II infusion was seen in most of the rats which received the highest dose. The mechanisms which might underlie the effect of blood-borne A II on sodium intake are discussed and three possibilities considered: (1) that A II may act on the brain to stimulate sodium appetite, (2) that A II may act via the adrenal cortex, and that the sodium appetite may arise as a result of increased plasma levels of adrenal steroids, and (3) that A II may act via the kidney, producing a natriuresis. According to this view, sodium appetite would arise as a result of loss of body sodium and/or blood volume.     

T-lymphocyte function and sensitivity to interferon in trisomy 21

Previous studies have shown that cells from subjects with trisomy 21 have enhanced sensitivity to the antiviral effects of interferon, presumably because of the location of the gene, IfRec, coding for the species-specific response to interferon on chromosome 21. Interferon is also known to have many other effects including the ability to inhibit the proliferation of many types of cells. To determine whether proliferating trisomic lymphocytes are more sensitive to the antiproliferative effect of interferon we have investigated, using healthy noninstitutionalized subjects with trisomy 21, the ability of interferon to inhibit the proliferation of lymphocytes stimulated with phytohemagglutinin P(PHA), concanavalin A (Con A), and tetanus toxoid. The trisomic subjects had normal numbers of peripheral blood leukocytes, and normal numbers and proportions of T and B lymphocytes. The production of interferon by PHA-stimulated trisomic T lymphocytes was normal. Trisomic lymphocytes also had normal proliferative responses to PHA and Con A. There were no differences between the inhibitory effects of interferon on the proliferation of PHA-stimulated trisomic and normal lymphocytes. However, trisomic lymphocytes stimulated with low doses of Con A did display significantly enhanced sensitivity to the antiproliferative effects of interferon. In contrast to normal lymphocytes, trisomic lymphocytes were not stimulated to proliferate by tetanus toxoid, and exposure to interferon resulted in enhancement, rather than inhibition, of DNA synthesis.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Synthesis of LDH-1 during mamalian oogenesis and early development.

Nuclear multiplication stage embryos were punctured in either the anterior, midlateral, or posterior regions. Both embryos and adults were examined for defects, and the defects were correlated with whether there had been any leakage of cytoplasmic material from the egg at the time of puncturing. Embryonic defects were found, correlated to the site of damage, in all three regions. A number of embryos was followed through development and it was found that 15.1% of the embryos which leaked cytoplasm hatched into larvae, compared to 82.3% of those which did not leak any cytoplasm. Morphological defects arising as a result of lateral puncture only were observed in adults. Many sterile adults were obtained from eggs in which the posterior region had been punctured. The results show that nuclear multiplication embryos are well able to tolerate the disturbance of the cortical cytoplasm created by puncture, but only rarely are they able to compensate for the actual loss of material by regulation. The results were similar to those observed after puncturing Drosophila embryos at the cellular blastoderm stage.