Cell-specific variation of X chromosome replication in the Syrian hamster (Mesocricetus auratus)

The sub-division of S-phase in Syrian hamsters, on the basis of BrdU/Hoechst 33258/Giemsa banding, has allowed a quantitative comparison of the replication of individual chromosome bands within defined subphases of S. This analysis has shown that in hamsters, as has been reported in humans, there are distinct patterns of early replication in vitro in the early X, the late X in fibroblasts, and the late X in lymphocytes. In addition, it has been possible to show that, although the pattern of replication of the late X in fibroblasts differs from that in lymphocytes, the time in S at which bands first appear on this chromosome is the same in the two cell types. — No significant heterogeneity can be ascribed to differences between individuals, adult or embryonic sources, culture media, or time of exposure to BrdU. — The absence of any detectable heterogeneity in the replication band frequencies in autosomal heterochromatic arms suggests that the cell-specific variability of the late-replicating X is a feature of facultative rather than constitutive heterochromatin.     

Molecular Properties of the ATP:D-Fructose-6-Phosphate 1-Phosphotransferase Isoenzymes from Cucumis sativus

The ATP:D-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.11 [EC] )isoenzymes from cucumber seeds were separated and purified.The calculated molecular weights of the two isoenzymes (approximately180,000) are similar and the isoenzymes are probably hetro-tetramers.The purified isoenzymes contained three polypeptides of 53.3,41.5 and 39.0 kDa for the plastid and 47.2, 42.4 and 40.4 forthe cytosolic isoenzyme, respectively. The purified phosphofructokinaseisoenzymes were used as the antigen for the production of polyclonalantibodies in rabbits. The obtained antisera clearly indicatedthat there is no immunological similarity between the two isoenzymes.The results also show that the phosphofructokinase isoenzymesin cucumber are not merely different stages of association ofthe same protein. (Received June 29, 1987; Accepted October 21, 1987)     

Evidence of deteriorating semen quality in the United Kingdom: birth cohort study in 577 men in Scotland over 11 years.

OBJECTIVE: To determine whether the quality of semen has changed in a group of over 500 Scottish men born between 1951 and 1973. DESIGN: Retrospective review of data on semen quality collected in a single laboratory over 11 years and according to World Health Organisation guidelines. SETTING: Programme of gamete biology research funded by Medical Research Council. SUBJECTS: 577 volunteer semen donors. Of these, 171 were born before 1959, 120 were born in 1960-4, 171 in 1965-9, and 115 in 1970-4. MAIN OUTCOME MEASURES: Conventional criteria of semen quality including semen volume (ml), sperm concentration (10(6)/ml), overall motility (% motile), total number of sperm in the ejaculate (10(6)), and total number of motile sperm in the ejaculate (10(6)). RESULTS: When the four birth cohort groups were compared a later year of birth was associated with a lower sperm concentration, a lower total number of sperm in the ejaculate, and a lower number of motile sperm in the ejaculate. The median sperm concentration fell from 98x10(6)/ml among donors born before 1959 to 78x10(6)/ml among donors born after 1970 (P=0.002). The total number of sperm in the ejaculate fell from 301x10(6) to 214x10(6) (P=0.0005), and the total number of motile sperm in the ejaculate fell from 169.7x10(6) to 129.0x10(6) (P=0.0065). CONCLUSION: This study provides direct evidence that semen quality is deteriorating, with a later year of birth being significantly associated with a reduced number of sperm in adult life.     

Development of RAPD and SCAR markers linked to the Russian wheat aphid resistance gene Dn2 in wheat

 RAPD (random amplified polymorphic DNA) analysis was used to identify molecular markers linked to the Dn2 gene conferring resistance to the Russian wheat aphid (Diuraphis noxia Mordvilko). A set of near-isogenic lines (NILs) was screened with 300 RAPD primers for polymorphisms linked to the Dn2 gene. A total of 2700 RAPD loci were screened for linkage to the resistance locus. Four polymorphic RAPD fragments, two in coupling phase and two in repulsion phase, were identified as putative RAPD markers for the Dn2 gene. Segregation analysis of these markers in an F₂ population segregating for the resistance gene revealed that all four markers were closely linked to the Dn2 locus. Linkage distances ranged from 3.3 cM to 4.4 cM. Southern analysis of the RAPD products using the cloned RAPD markers as probes confirmed the homology of the RAPD amplification products. The coupling-phase marker OPB10_880c and the repulsion-phase marker OPN1_400r were converted to sequence characterized amplified region (SCAR) markers. SCAR analysis of the F₂ population and other resistant and susceptible South African wheat cultivars corroborated the observed linkage of the RAPD markers to the Dn2 resistance locus. These markers will be useful for marker-assisted selection of the Dn2 gene for resistance breeding and gene pyramiding. Received: 1 July 1997 / Accepted: 20 October 1997     

The development of nuclear vacuoles during meiosis in plants

Vacuoles formed by the invagination of the inner membrane of the nuclear envelope have been observed during meiotic prophase in a wide range of plants. In the angiosperm Lycopersicon their formation was found to coincide with the completion of synaptonemal complex formation, and this timing is analogous to that observed during this stage in the silkworm Bombyx. The implications of this activity in relation to the process of chromosome movement are discussed. In the gymnosperm Pinus, the heterosporous fern Marsilea and homosporous ferns Pteridium and Dryopteris the formation of nuclear vacuoles begins much earlier, coinciding with the condensation of chromatin during leptotene. They enlarge and become more elaborate as meiosis proceeds, and may eventually become detached from the nuclear envelope. It is therefore thought unlikely that theyfulfil functions connected with chromosome movement in the manner proposed for the silkworm and the tomato. During diplotene/diakinesis they contain electron-opaque granules and fibrils, and the possible origin and significance of this material is discussed.     

Natural Killer Cells in Obesity: Impaired Function and Increased Susceptibility to the Effects of Cigarette Smoke

Background

Obese individuals who smoke have a 14 year reduction in life expectancy. Both obesity and smoking are independantly associated with increased risk of malignancy. Natural killer cells (NK) are critical mediators of anti-tumour immunity and are compromised in obese patients and smokers. We examined whether NK cell function was differentially affected by cigarette smoke in obese and lean subjects.

Methodology and Principal Findings

Clinical data and blood were collected from 40 severely obese subjects (BMI>40 kg/m²) and 20 lean healthy subjects. NK cell levels and function were assessed using flow cytometry and cytotoxicity assays. The effect of cigarette smoke on NK cell ability to kill K562 tumour cells was assessed in the presence or absence of the adipokines leptin and adiponectin. NK cell levels were significantly decreased in obese subjects compared to lean controls (7.6 vs 16.6%, p = 0.0008). NK function was also significantly compromised in obese patients (30% +/− 13% vs 42% +/−12%, p = 0.04). Cigarette smoke inhibited NK cell ability to kill tumour cell lines (p<0.0001). NK cells from obese subjects were even more susceptible to the inhibitory effects of smoke compared to lean subjects (33% vs 28%, p = 0.01). Cigarette smoke prevented NK cell activation, as well as perforin and interferon-gamma secretion upon tumour challenge. Adiponectin but not leptin partially reversed the effects of smoke on NK cell function in both obese (p = 0.002) and lean controls (p = 0.01).

Conclusions/Significance

Obese subjects have impaired NK cell activity that is more susceptible to the detrimental effects of cigarette smoke compared to lean subjects. This may play a role in the increase of cancer and infection seen in this population. Adiponectin is capable of restoring NK cell activity and may have therapeutic potential for immunity in obese subjects and smokers.     

The disparity between homologous chromosomes during DNA replication

When the patterns of replicating chromosome bands of homologous chromosomes within a diploid cell during DNA synthesis phase are compared, the frequency of disparity (i.e. a band present on only one homologue) is less than expected on the basis of chance. This could be taken as implying some “link” between homologues which constrains their programmes of replication to keep in step.This paper develops a model showing that the observed disparities can be accommodated within a framework of homologue independence. Differences between cells in the time of appearance of bands lead in any sample to the summation of an infinitude of binomial distributions and hence to over-dispersion.The model fits observed data for bands replicating in euchromatic and heterochromatic chromosome regions obtained from Syrian hamster fibroblast cells growing in vitro.     

Development of a novel mammalian display system for selection of antibodies against membrane proteins

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 10⁹ variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (K_D) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.