MsSOCS expression indicates a potential role for JAK/STAT signalling in the early stages of Manduca sexta spermatogenesis.

Our understanding of the Drosophila melanogaster testis stem cell niche has identified the signalling pathways required to maintain stem cells and promote self-renewal. Here we present the first detailed examination of the testes stem cell niche in the lepidopteran Manduca sexta . We show that larval testes contain hub-like structures surrounded by mitotically active cells likely to represent a self-renewing stem cell population. In addition, we have cloned DNA fragments encoding parts of M. sexta Signal Transducer and Activators of Transcription (STAT) and Suppressor of Cytokine Signalling (SOCS) homologues and show that expression of MsSOCS is upregulated in hub-adjacent cells. Given the conservation of socs-like genes and their regulation as Janus Kinases/Signal Transducer and Activators Transcription (JAK/STAT) pathway targets, we suggest that increased expression within the testis stem cells indicates increased levels of JAK/STAT signalling and a conserved role for pathway signalling in testis stem cell maintenance.

Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family.

BACKGROUND: Primary pulmonary hypertension (PPH), resulting from occlusion of small pulmonary arteries, is a devastating condition. Mutations of the bone morphogenetic protein receptor type II gene (BMPR2), a component of the transforming growth factor beta (TGF-beta) family which plays a key role in cell growth, have recently been identified as causing familial PPH. We have searched for BMPR2 gene mutations in sporadic PPH patients to determine whether the same genetic defect underlies the more common form of the disorder. METHODS: We investigated 50 unrelated patients, with a clinical diagnosis of PPH and no identifiable family history of pulmonary hypertension, by direct sequencing of the entire coding region and intron/exon boundaries of the BMPR2 gene. DNA from available parent pairs (n=5) was used to assess the occurrence of spontaneous (de novo) mutations contributing to sporadic PPH. RESULTS: We found a total of 11 different heterozygous germline mutations of the BMPR2 gene in 13 of the 50 PPH patients studied, including missense (n=3), nonsense (n=3), and frameshift (n=5) mutations each predicted to alter the cell signalling response to specific ligands. Parental analysis showed three occurrences of paternal transmission and two of de novo mutation of the BMPR2 gene in sporadic PPH. CONCLUSION: The sporadic form of PPH is associated with germline mutations of the gene encoding the receptor protein BMPR-II in at least 26% of cases. A molecular classification of PPH, based upon the presence or absence of BMPR2 mutations, has important implications for patient management and screening of relatives.

Lack of complementation between xeroderma pigmentosum complementation groups D and H.

The construction of permanent hybrid cell lines between xeroderma pigmentosum (XP) cells from different complementation groups allows analysis not only of the degree of repair correction but also of the restoration of biological activity to the UV-irradiated cells. With use of an immortal human cell line (HD2) that expresses excision repair defects typical of XP group D, a series of permanent hybrid cells has been produced with XP cells from groups A to H. Excision repair, as measured by incision analysis and unscheduled DNA synthesis, is restored to normal or near normal levels in crosses involving HD2 and cells from XP groups A, B, C, E, F, G, and I. All these hybrids show complementation for the recovery of normal UV resistance. As expected, hybrids expressing poor incision and hypersensitivity to UV were produced in crosses between HD2 and XPD fibroblasts, but they were also produced without exception when XPH was the partner. In the permanent HD2 x XPD or XPH hybrids, analysis of incision capacity reveals abnormally low activity and therefore that there has been no complementation. The true hybrid nature of HD2 x XPH cells has been confirmed by HL-A and -B tissue typing; moreover, detailed kinetic analysis of incision in these cells shows that the XPH phenotype, rather than the XPD, is expressed, i.e. breaks accumulate at low UV fluence of 1 J/m2. To help confirm these findings, another immortal XPD cell line was used in fusions involving HD2, XPH, or XPI. Cells resistant to ultraviolet were produced only with XPI fibroblasts. These data are discussed in terms of whether XPD and H mutations are likely to be allelic with respect to incision.

DNA-repair reactions by purified HeLa DNA polymerases and exonucleases.

PM2 duplex DNA substrates containing small gaps were utilized to study DNA repair reactions of extensively purified HeLa DNase V (a bidirectional double strand DNA exonuclease) and DNA polymerases beta, gamma (mitochondrial and extramitochondrial), and alpha holoenzyme, and delta as a function of ionic strength. At 50 mM NaCl, DNase V carried out extensive exonucleolytic degradation, and beta-polymerase exhibited strand displacement synthesis. However, at 150 mM NaCl, the DNase appeared only to remove damaged nucleotides from DNA termini while beta-polymerase catalyzed only gap-filling synthesis. When present in equimolar amounts, beta-polymerase and DNase V (which can be isolated as a 1:1 complex) catalyzed more degradation than synthesis at 50 mM NaCl; however, at 150 mM NaCl a coupled very limited nick translation reaction ensued. At physiological ionic strength DNA polymerase alpha holoenzyme was not active upon these substrates. In 15 mM KCl it could fill small gaps and carry out limited nick translation with undamaged DNA, but it could not create a ligatable substrate from UV-irradiated DNA incised with T4 UV endonuclease. Mitochondrial DNA polymerase gamma was more active at 150 mM NaCl than at lower ionic strengths. It readily filled small gaps but was only marginally capable of strand-displacement synthesis. The extramitochondrial form of gamma-polymerase, conversely, was less sensitive to ionic strength; it too easily filled small gaps but was not effective in catalyzing strand displacement synthesis. Finally, DNA polymerase delta was able to fill gaps of several to 20 nucleotides in 0.05 M NaCl, but at higher NaCl concentrations there was little activity. DNA polymerases delta did not demonstrate strand displacement synthesis. Therefore, at physiological ionic strength, it appears that either DNA polymerase beta or extramitochondrial DNA polymerase gamma might aid in short patch DNA repair of nuclear (or transfecting) DNAs, whereas mitochondrial gamma-polymerase might fill small gaps in mitochondrial DNA.

Gender differences in self-consistency: Evidence from an investigation of self-concept structure.

This study investigates the relationship between two primary motivational components of the self-concept: self-esteem and self-consistency. Past research has shown that high self-esteem is associated with greater consistency. Developed from theories of the self-concept, a structural model relating these two constructs was posited such that (1) self-esteem is causally prior to self-consistency, and (2) the effect of self-esteem is (at least partially) mediated by other components of the self-concept (self-consciousness, the tendency to fantasize, and the tendency to present a false front by hiding one's feelings). Utilizing a structural equation model with unobserved variables, an analysis of covariance structures was applied simultaneously but separately to data from a sample of boys and girls (ages 8-19). Results showed that the direct effect of self-esteem on self-consistency was stronger for boys than for girls. Further, the mediational properties of the other self-concept components also varied across gender. These differences are interpreted in light of theories of gender socialization.

Slow DNA rejoining in ultraviolet-irradiated human diploid fibroblasts treated with the mitogens trypsin and insulin.

Normally in mammalian cells the postincision steps of UV-induced excision repair are much more rapid than the recognition of damage and incision. This means that at any one time the level of repair-generated single-stranded DNA breaks is very low. Here we report that detectable levels of DNA breaks accumulate in quiescent human fibroblasts which are UV irradiated a few hours after replating in conditions that stimulate progress through the cell cycle. Most DNA breaks accumulate in cultures trypsinized and seeded in medium supplemented with insulin, and irradiated in early G1. Because trypsin and insulin have no effect on UV-induced incision rates, as measured by DNA break accumulation in the presence of DNA synthesis inhibitors, we argue that our ability to detect incomplete repair-sites is due to a significant reduction in the rate of gap sealing indicative of a shift in the steady state of excision repair. Provision of DNA precursors prevents the enhancing effect of trypsin and insulin on the accumulation of DNA breaks, implying that these agents affect DNA precursor metabolism. Perturbation of the repair process, which leads to the accumulation of 1500-2000 DNA breaks/genome, is also associated with other effects including increased lethality, the appearance of double-strand breaks and the loss of NAD, the last effect presumably arising as a consequence of break-stimulated poly(ADPR) transferase activity. Addition of 3-amino-benzamide, an inhibitor of poly(ADPR) synthesis, completely blocks the decline in NAD levels, but does not change the rate of sealing of the accumulated DNA breaks. These results strongly suggest that ligation is largely, if not entirely, independent of ADP ribosylation in this system.

DNA repair under stress.

When the excision repair process of eukaryote cells is arrested by inhibitors of repair synthesis including hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (araC) or aphidicolin, major cellular changes follow the accumulation of repair-associated DNA breaks. These changes, each of which reflects more or less severe cellular stress, include cycle delay, chromosome behaviour, fall in NAD level, the development of double-stranded DNA breaks, rapid chromosome fragmentation and cell killing. Disruption of the repair process by agents such as araC after therapeutic DNA damage may, therefore, have some potential value in cancer treatment. The extreme cellular problems associated with the artificial arrest of repair may have their subtler counterparts elsewhere, and we discuss several systems where delays in the completion of excision repair in the absence of repair synthesis inhibitors have marked repercussions on cell viability. We also show that the average completion time of an excision repair patch varies according to the state of cell culture, and that completion time is extended after treatment with insulin or following trypsin detachment. Under certain growth conditions ultraviolet irradiation followed by mitogenic stimulation results in double-stranded DNA breakage and additional cell killing, and we discuss these data in the light of protocols that have been used successfully to transform human or rodent cells in vitro. Finally, we consider whether the rejoining of DNA breaks accumulated by repair synthesis inhibitors is a valid model system for studying ligation, and show that this protocol provides an extremely sensitive assay for most incision events and, thereby, a means for discriminating between normal human cells on the one hand, and Cockayne's Syndrome cells and their heterozygotes on the other.

Qualitative differences between replicative and repair synthesis of DNA in normal and transformed mouse cells as measured by precursor discrimination.

Inhibitors of DNA polymerase alpha such as aphidicolin (APC) or 1-beta-D-arabinofuranosyl-cytosine (araC) cause DNA-strand breaks to accumulate after UV-irradiation, at sites where repair resynthesis is inhibited. Transformed cells accumulate fewer such breaks than normal cells do; this may be due to differences in the extent, or the nature, of excision-repair synthesis in transformed and in normal cells. We have looked for differences in the nature of repair synthesis, comparing the labelling of DNA by deoxycytidine (dC) and araC through UV-induced repair in normal and transformed mouse cells. We have made parallel determinations of precursor discrimination in replicative synthesis, and find that normal cells discriminate better against araC in replicative synthesis than do transformed cells. But repair synthesis discriminates against araC less than normal replicative synthesis does, to a similar extent in both cell types. Thus, there are qualitative differences between the DNA polymerases engaged in UV excision repair and replication in normal and transformed mouse cells; but there is no evidence for a predominantly araC-insensitive repair synthesis in transformed cells, such as might account for the difference in break accumulation.

Analysis of DNA repair in XP-HeLa hybrids; lack of correlation between excision repair of u.v. damage and adenovirus reactivation in an XP(D)-like cell line.

Hybrids formed between HeLa cells and fibroblasts from xeroderma pigmentosum group D show either HeLa sensitivity or XPD-like hypersensitivity to u.v. radiation and corresponding high or low excision repair capability. Hybrids with low repair are presumed to have lost, via chromosome segregation, the HeLa wild type D alleles. In this paper we analyse the u.v. sensitivity and excision repair capability of another hybrid, HD1A, derived spontaneously from the normally sensitive hybrid HD1. While HD1A closely resembles the XPD phenotype in terms of u.v. sensitivity and excision repair it differs from XPD because of its ability to reactivate u.v.-irradiated adenovirus 2 to an extent similar to that of its HeLa parent. This capacity functionally dissociates excision repair of chromatin-based damage from damage in a viral environment. Moreover, on the basis of complementation studies the excision repair of genomic damage by HD1A is subtly different from that of a true XPD-like hybrid, HD2. The data are discussed in terms of a second change in the defective D allele of the HD1A cell.

Xeroderma pigmentosum D-HeLa hybrids with low and high ultraviolet sensitivity associated with normal and diminished DNA repair ability, respectively.

Fusion between HeLa and fibroblasts from complementation group D xeroderma pigmentosum (XPD) followed by challenge with small doses of ultraviolet light (u.v.) results in the production of hybrid cells expressing either HeLa (HD1) or XPD-like (HD2) sensitivity to u.v. and related repair capacity. Assays used included unscheduled DNA synthesis (UDS), DNA break accumulation in the presence of inhibitors of DNA repair synthesis and host cell reactivation of irradiated adenovirus. Complementation assay in heterokaryons reveals limited ability of HD2 to restore UDS in XPD nuclei. We believe this complementation is more apparent than real since proliferating hybrids of HD2 and XPD parentage are without exception u.v.-sensitive and express limited excision repair. On the other hand hybrids between HD2 and XPC, XPE or XPF fibroblasts show true complementation resulting in a return to normal u.v. sensitivity and elevated repair ability.

DNA repair in mouse embryo fibroblasts. II. Responses of nontransformed, preneoplastic and tumorigenic cells to ultraviolet irradiation.

Ultraviolet light-induced excision repair, as measured by single-strand DNA-break accumulation in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, undergoes an apparent decline concomitant with spontaneous transformation of mouse cells in vitro. This decline is seen in preneoplastic transformed cells as well as tumorigenic cells, suggesting that it is an early event in transformation. The difference between nontransformed and transformed mouse cells in apparent incision rates is greatest at short times after irradiation when nontransformed cells show a transient phase of rapid incision. No gross differences in the effects of UV on replicative DNA synthesis, bulk RNA synthesis, cell proliferation or clonal survival in nontransformed and transformed cells were seen, in spite of the reduced incision capacity of the latter. Taken together the results suggest that transformed cells are capable of growth in the presence of significantly increased amounts of DNA damage. A decreased ability of nontumorigenic cells to remove DNA lesions, coupled with unrestricted growth, may be responsible for genetic alterations which increase the probability of a cell becoming tumorigenic.

Influx and efflux of p in roots of intact maize plants : double-labeling with p and p.

Rates of P influx and efflux were determined in whole plants at ambient P concentrations comparable to those found in soil solutions. Maize (Zea mays L. var NC+59) seedlings were trimmed (endosperm and adventitious roots removed) and grown in a greenhouse in solution cultures at P concentrations of approximately 0.4 and 1.8 micromolar. Roots of intact plants previously exposed to (32)P-labeled solutions at 0.2 and 2.0 micromolar P for 48 hours were rinsed 10 minutes in P-free solution and exposed to (33)P solutions at 0.2 and 2.0 micromolar for 10 minutes. Net depletion of (33)P from and appearance of (32)P in the ambient solution were used to measure influx and efflux. The ration of (32)P efflux to (33)P influx was about 0.68 at 0.2 micromolar and 0.08 at 2.0 micromolar. When plants were allowed to deplete P from solutions, the P concentration in the medium dropped to about 0.15 micromolar within 24 hours and 0.05 micromolar within 60 hours. Results indicate that P efflux is a substantial component of net P accumulation at P concentrations normally found in soil solutions.

Dimensions of the self-concept: A source of further distinctions in the nature of self-consciousness.

This study distinguished between forms of self-consciousness (private self-consciousness and social anxiety) and investigated the effect of self-esteem, vulnerability to criticism, and the tendency to fantasize on each. Utilizing a sample of adolescents (age 12 to 19 years), a structural equation analysis using unobserved variables revealed that, as expected, vulnerability to others' criticism heightened both private self-consciousness and social anxiety. In contrast, the tendency to fantasize in everyday life increased only private self-consciousness, and low self-esteem led directly only to increased social anxiety. Further, results showed that self-esteem had an appreciable indirect effect on both private self-consciousness and social anxiety, as mediated by vulnerability and the tendency to fantasize, indicating that the effect of self-esteem (a primary motivator) was more complex than an analysis of direct effects would indicate. Implications for our understanding of self-consciousness and the self-concept are discussed.

DNA repair in mouse embryo fibroblasts. I. Decline in ultraviolet-induced incision rate accompanies cell transformation.

In this paper we examine DNA repair in the mouse. Using stringently applied inhibitors of DNA synthesis (hydroxyurea and 1-beta-D-arabinofuranosylcytosine) after ultraviolet irradiation we have quantitated an early step in the repair process - the frequency of single-strand DNA breaks, products of enzymic incision--in fibroblasts and epithelial cells grown in vitro from adults and embryos of different ages. We find that initial rates of incision are very similar in the earliest-passage fibroblasts, regardless of donor age, resembling the rates measured in other rodents while being considerably less than in diploid human fibroblasts. Consistently higher rates of incision are found in adult kidney epithelial cells. With increasing passage number the embryo cultures change with respect to growth rate and chromosome constitution, undergoing spontaneous transformation to established heteroploid cultures. This sequence is accompanied by characteristic changes in initial rates of inhibitor-sensitive incision. An early increase in incision rate, at a time when the cultures are enriched with tetraploid cells, is succeeded by progressive reduction in rate as the cultures become overtly transformed into permanent lines and heteroploidy is established. The lowest rate of incision occurs in a transformed line cloned from a late-passage culture. The decline in incision rate associated with transformation is accompanied by reduced unscheduled DNA synthesis (UDS). The sensitivity to inhibitors of UDS levels in the mouse cultures has been found to vary considerably; inhibitors are most effective in slowly proliferating cultures and apparently not at all when cultures are growing rapidly despite their ability to accumulate DNA breaks in these cells. Caution is needed, therefore, in the interpretation of repair data obtained in the presence of these commonly used inhibitors of DNA synthesis.

Self-esteem and self-presentation among the young as a function of age and gender.

This study investigated the effect of self-esteem on the tendency to convey a false impression to others by hiding one's feelings behind a facade. Utilizing a sample of youth (ages 8-19), a multiple regression analysis revealed that the lower the self-esteem, the greater the tendency to present a false front. In addition, other aspects of the self-concept, including vulnerability to criticism, self-consciousness, and the tendency to fantasize affect the tendency to present a false front. Furthermore, the nature of the self-esteem/ fabrication relationship is a joint function of age and gender. As age increases, the direct effect of self-esteem on fabrication loses statistical significance, but this loss occurs differently for boys and girls. For boys, the direct effect is significant in pre-adolescence (8-11), but not in early or late adolescence (12-14 and 15-19). For girls, the direct effect is significant in pre- and early adolescence (8-11 and 12-14), but not in late adolescence (15-19). Reasons for the differing patterns for boys and girls are discussed.