A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

Sensitivity to camptothecin in Aspergillus nidulans identifies a novel gene, scaA + , related to the cellular DNA damage response

The anti-cancer drug camptothecin targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed between the catalytically active enzyme and DNA. We are interested in identifying factors, other than topoisomerase I, that are involved in mediating cellular sensitivity to camptothecin. To this end, we have isolated eighteen mutants that are sensitive to camptothecin (sca) in the filamentous fungus Aspergillus nidulans and characterised one of them, sca299. The mutant sca299 is hypersensitive to camptothecin, and sensitive to several different mutagenic agents and to actinomycin D. Using temperature-sensitive mutations in genes that are known to regulate the cell cycle, we showed that the camptothecin sensitivity of the mutant sca299 is not affected by a mitotic block. The abnormal nuclear morphology observed in the sca299 mutant strain suggests that the germlings might be undergoing mitosis in the presence of unrepaired DNA damage, which would result in mitotic catastrophe. The hypersensitivity of the sca299 mutant to camptothecin does not result from elevated levels of topoisomerase I mRNA or from alterations in enzyme activity. Using DNA-mediated complementation of the sca299 mutant phenotype, the scaA+ gene was cloned. This gene encodes a 594-amino acid product; moderate structural similarity suggests that the scaA gene product may be related to the human nibrin gene which encodes a product involved in DNA double-strand break repair. Strains disrupted in the scaA gene were sensitive to the anti-topoisomerase I agent berberine, the DNA crosslinking agents mitomycin C and cis-platinum, and also to t-butyl hydroperoxide, which is an inducer of oxidative stress.     

Genetic analyses of correlated solids, flavor, and health-enhancing traits in onion (Allium cepa L.)

Onion possesses organosulfur compounds and carbohydrates that provide unique flavor and health-enhancing characteristics. Significant phenotypic correlations have been reported among soluble solids content (SSC), total dry matter, pungency, and onion-induced in vitro antiplatelet activity. A genetic map and segregating F3M families derived from a cross between two inbred populations were used to identify and estimate the effects of quantitative trait loci (QTLs) controlling these traits at 30 and 90 days postharvest. In vitro antiplatelet activities among different onion populations were consistent across six human blood donors. Most of the populations showed in vitro antiplatelet activities; however, for some donors, one of the parental lines and two F3M families had pro-aggregatory effects under our experimental conditions. SSC, dry matter, pungency, and in vitro antiplatelet activity showed significant positive phenotypic and genetic correlations. A chromosome region on linkage group E accounted for a significant amount of the phenotypic variation for all of these traits. The correlations among these traits may be due to linkage or pleiotropy of genes controlling solids content. Our results indicate that it will be difficult to develop onion populations with lower pungency and high in vitro antiplatelet activity; however, the strong genetic and phenotypic correlations between high in vitro antiplatelet activity and high SSC are beneficial for the health functionality of onion.     

I(Ca(TTX)) channels are distinct from those generating the classical cardiac Na(+) current

The Na(+) current component I(Ca(TTX)) is functionally distinct from the main body of Na(+) current, I(Na). It was proposed that I(Ca(TTX)) channels are I(Na) channels that were altered by bathing media containing Ca(2+), but no, or very little, Na(+). It is known that Na(+)-free conditions are not required to demonstrate I(Ca(TTX).) We show here that Ca(2+) is also not required. Whole-cell, tetrodotoxin-blockable currents from fresh adult rat ventricular cells in 65 mm Cs(+) and no Ca(2+) were compared to those in 3 mM Ca(2+) and no Cs(+) (i.e., I(Ca(TTX))). I(Ca(TTX)) parameters were shifted to more positive voltages than those for Cs(+). The Cs(+) conductance-voltage curve slope factor (mean, -4.68 mV; range, -3.63 to -5.72 mV, eight cells) is indistinguishable from that reported for I(Ca(TTX)) (mean, -4.49 mV; range, -3.95 to -5.49 mV). Cs(+) current and I(Ca(TTX)) time courses were superimposable after accounting for the voltage shift. Inactivation time constants as functions of potential for the Cs(+) current and I(Ca(TTX)) also superimposed after voltage shifting, as did the inactivation curves. Neither of the proposed conditions for conversion of I(Na) into I(Ca(TTX)) channels is required to demonstrate I(Ca(TTX)). Moreover, we find that cardiac Na(+) (H1) channels expressed heterologously in HEK 293 cells are not converted to I(Ca(TTX)) channels by Na(+)-free, Ca(2+)-containing bathing media. The gating properties of the Na(+) current through H1 and those of Ca(2+) current through H1 are identical. All observations are consistent with two non-interconvertable Na(+) channel populations: a larger that expresses little Ca(2+) permeability and a smaller that is appreciably Ca(2+)-permeable.     

Mammalian photoperiodic system: formal properties and neuroendocrine mechanisms of photoperiodic time measurement

Photoperiodism is a process whereby organisms are able to use both absolute measures of day length and the direction of day length change as a basis for regulating seasonal changes in physiology and behavior. The use of day length cues allows organisms to essentially track time-of-year and to "anticipate" relatively predictable annual variations in important environmental parameters. Thus, adaptive types of seasonal biological changes can be molded through evolution to fit annual environmental cycles. Studies of the formal properties of photoperiodic mechanisms have revealed that most organisms use circadian oscillators to measure day length. Two types of paradigms, designated as the external and internal coincidence models, have been proposed to account for photoperiodic time measurement by a circadian mechanism. Both models postulate that the timing of light exposure, rather than the total amount of light, is critical to the organism's perception of day length. In mammals, a circadian oscillator(s) in the suprachiasmatic nucleus of the hypothalamus receives photic stimuli via the retinohypothalamic tract. The circadian system regulates the rhythmic secretion of the pineal hormone, melatonin. Melatonin is secreted at night, and the duration of secretion varies in inverse relation to day length; thus, photoperiod information is "encoded" in the melatonin signal. The melatonin signal is presumably "decoded" in melatonin target tissues that are involved in the regulation of a variety of seasonal responses. Variations in photoperiodic response are seen not only between species but also between breeding populations within a species and between individuals within single breeding populations. Sometimes these variations appear to be the result of differences in responsiveness to melatonin; in other cases, variations in photoperiod responsiveness may depend on differences in patterns of melatonin secretion related to circadian variation. Sites of action for melatonin in mammals are not yet well characterized, but potential targets of particular interest include the pars tuberalis of the pituitary gland and the suprachiasmatic nuclei. Both these sites exhibit uptake of radiolabeled melatonin in various species, and there is some evidence for direct action of melatonin at these sites. However, it appears that there are species differences with respect to the importance and specific functions of various melatonin target sites.     

The 300-kDa intermediate filament-associated protein (IFAP300) is a hamster plectin ortholog

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin.     

Evidence for mitochondrial DNA recombination in a human population of island Melanesia: correction

We recently presented evidence of mitochondrial DNA recombination in humans based on the observation of a rare mutation in several unrelated human lineages in Nguna, a small island in Vanuatu, island Melanesia. Since then, the mutation has been shown to be an artefact caused by misalignment of the DNA sequences. Our previous conclusion, that the presence of a rare mutation on different haplotypic backgrounds was a consequence of genetic recombination, is no longer tenable for these data.     

Distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5' -nucleotidase activity

Using a cytochemical assay we were able to show that the peritoneal macrophage population of normal nontreated mice (resident peritoneal macrophages) exhibits a heterogeneity with regard to the expression of the activity of the ecto-enzyme 5′-nucleotidase (5′-N). About 75% of the macrophages express high enzymic activity whereas the remaining 25% express low 5′-N activity. Macrophages accumulating in the peritoneum as a result of an inflammatory response are predominantly of the low activity type. In vitro activation of resident peritoneal macrophages by lymphokines does not result in a decrease in the number of macrophages expressing high enzymic activity though the level of the enzymic activity of these cells is reduced by about 36%. Bone marrow derived mononuclear phagocyte colonies developing in vitro, under liquid culture conditions, from bone marrows of normal mice can be divided into three types with respect to their expression of 5′-N activity: (1) high activity colonies–relatively small colonies in which all the cells express high 5′-N activity (about 20% of the colonies); (2) low activity colonies – relatively large colonies in which all the cells express low 5′-N activity (about 70% of the colonies); and (3) mixed colonies–relatively large colonies in which all the cells express low enzymic activity except for about 8% of cells located at the periphery of the colonies which express high enzymic activity (about 10% of the colonies). During an inflammatory response the frequency of the high activity colonies is significantly reduced. Our results provide evidence for distinct bone marrow precursors for mononuclear phagocytes expressing high and low 5′-N activity and suggest that (1) the resident macrophages derive from a subpopulation of bone marrow precursor cells developing in vitro into high 5′-N activity mononuclear phagocytes, and (2) during an inflammatory response there is a preferential expansion of clones of the low enzymic activity phenotype.     

Rapid assay for plasma chlorambucil and phenyl acetic mustard using reversed-phase liquid chromatography

A rapid assay for chlorambucil, a drug used for the treatment of chronic lymphocytic leukemia, and its major metabolite is described. Chromatographic behaviour of the two compounds on two different reversed-phase columns is discussed, as well as the kinetics of their hydrolysis in aqueous medium. The developed analysis can be applied to the determination of the plasma levels of the drug and its metabolite. No sample preparation is required and the spectrophotometric detection affords the sensitivity in the picomole range. Total analysis time is between 10 and 15 min.     

Of barn owls and bankers: a lush variety of alpha/beta hydrolases.

alpha/beta Hydrolase fold proteins are an important, diverse, widespread group of enzymes not yet fully exploited by structural biologists. We describe the current state of knowledge of this family, and suggest a smaller definition of the required core and some possible future avenues of exploration.