Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na+/K+/Cl- cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 microM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener 86Rb+ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of 86Rb+ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359-367) as being mediated by a Na+/K+/Cl- cotransporter. Inhibition by 10 microM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of 86Rb+ influx, mediated by Na+/K+-ATPase. After 3 h of incubation with 10 microM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [3H]TPMP+, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K+ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumor activity, did not inhibit 86Rb+ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 +/- 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na+/K+/Cl- cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.     

Common variants of ACE contribute to variable age-at-onset of Alzheimer’s disease

Studies on the role that genetic variation may play in a complex human disease can be empowered by an assessment of both disease risk in case-control or family models and of quantitative traits that reflect elements of disease etiology. An excellent example of this can be found for the 4 allele of APOE in relation to Alzheimers disease (AD) for which association with both risk and age-at-onset (AAO) is evident. Following a recent demonstration that variants of the gene encoding angiotensin I converting enzyme (ACE) contribute to AD risk, we have explored the potential influence of ACE upon AAO in AD. A total of 2861 individuals from three European populations, including six independent AD samples, have been examined in this study. Three single nucleotide polymorphisms (SNPs) previously demonstrated to have maximum effects upon ACE plasma levels and that span the ACE locus were genotyped in these materials. A strong effect upon AAO was observed for marker rs4343 in exon 17 (P<0.0001), but evidence was also obtained indicating a possible independent effect of marker rs4291 (P=0.0095) located in the ACE promoter. Effects were consistent with data from previous studies suggesting association with AD in case-control models, whereby alleles demonstrated to confer risk to disease also appear to reduce AAO. Equivalent effects were evident regardless of APOE 4 carrier status and in both males and females. These results provide an important complement to existing AD risk data, confirming that ACE harbors sequence variants that contribute to aspects of AD pathology.     

Care Seeking for Neonatal Illness in Low- and Middle-Income Countries: A Systematic Review

Background

Despite recent achievements to reduce child mortality, neonatal deaths continue to remain high, accounting for 41% of all deaths in children under five years of age worldwide, of which over 90% occur in low- and middle-income countries (LMICs). Infections are a leading cause of death and limitations in care seeking for ill neonates contribute to high mortality rates. As estimates for care-seeking behaviors in LMICs have not been studied, this review describes care seeking for neonatal illnesses in LMICs, with particular attention to type of care sought.

Methods and Findings

We conducted a systematic literature review of studies that reported the proportion of caregivers that sought care for ill or suspected ill neonates in LMICs. The initial search yielded 784 studies, of which 22 studies described relevant data from community household surveys, facility-based surveys, and intervention trials. The majority of studies were from South Asia (n = 17/22), set in rural areas (n = 17/22), and published within the last 4 years (n = 18/22). Of the 9,098 neonates who were ill or suspected to be ill, 4,320 caregivers sought some type of care, including care from a health facility (n = 370) or provider (n = 1,813). Care seeking ranged between 10% and 100% among caregivers with a median of 59%. Care seeking from a health care provider yielded a similar range and median, while care seeking at a health care facility ranged between 1% and 100%, with a median of 20%. Care-seeking estimates were limited by the few studies conducted in urban settings and regions other than South Asia. There was a lack of consistency regarding illness, care-seeking, and care provider definitions.

Conclusions

There is a paucity of data regarding newborn care-seeking behaviors; in South Asia, care seeking is low for newborn illness, especially in terms of care sought from health care facilities and medically trained providers. There is a need for representative data to describe care-seeking patterns in different geographic regions and better understand mechanisms to enhance care seeking during this vulnerable time period. Please see later in the article for the Editors'' Summary     

Storage protein profiles in Spanish and runner market type peanuts and potential markers

Background  

Proteomic analysis has proven to be the most powerful method for describing plant species and lines, and for identification of proteins in complex mixtures. The strength of this method resides in high resolving power of two-dimensional electrophoresis (2-DE), coupled with highly sensitive mass spectrometry (MS), and sequence homology search. By using this method, we might find polymorphic markers to differentiate peanut subspecies.     

Fruit set, nectar reward, and rarity in the Orchidaceae

A review of comparative levels of reproductive success among nectariferous and nectarless orchids worldwide was compiled from a comprehensive survey of fruit set from 117 orchid species in the literature and from our own field studies. It confirms the hypothesis that nectariferous orchids are more successful in setting fruit than are nectarless species. Overall fruit set figures for nectarless and nectariferous orchids were 19.5 and 49.3% for North America, 27.7 and 63.1% for Europe, 41.4 and 74.4% for the temperate southern hemisphere, and 11.5 and 24.9% for the tropics, demonstrating that the dichotomy is consistent across all geographical areas. On average, the provision of nectar doubles the probability of fruit set in both temperate and tropical areas, but tropical orchids are remarkable in that all (whether nectarless or nectariferous, or terrestrial or epiphytic) display low fruit productivity (<50%). Fruiting failure in the tropics may be balanced by higher productivity per capsule, since tropical orchid fruits contain on average 150 times more seeds than temperate ones. Hybridization occurs more frequently among nectarless orchids in Britain and Europe than among nectariferous ones, and there is a significant positive association between orchid rarity and lack of nectar reward in the British Isles. Sexual reproduction in the Orchidaceae is predominantly pollinator dependent, but this can sometimes be successfully circumvented by asexual seed production (agamospermy) or, more frequently, by automatic self-pollination (autogamy). The proportion of highly successful nectarless orchids from all geographic areas is very low and comparable with that of orchids offering rewards other than nectar (～14% of species in each case) emphasizing that high reproductive success is only associated with nectar reward (53% of species). It is suggested that the evolution of nectar production within the family has been the most frequent means of escaping the reproductive limitations of low pollinator visitation frequencies.     

Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4

We have employed a direct radiolabel binding assay to investigate the interaction between3H-heparin and recombinant envelope glycoproteins, rgp120s, derived from several different isolates of HIV-1. Comparable dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN and SF-2. Under identical experimental conditions the binding of3H- heparin to a recombinant soluble form of the cellular receptor for gp120, CD4, is negligible. The binding of3H-heparin to rgp120 is competed for by excess unlabeled heparin and certain other, but not all, glycosaminoglycan and chemically modified heparins. Of a range of such polysaccharides tested, ability to compete with3H-heparin for binding was strictly correlated with inhibition of HIV-1 replication in vitro. Those possessing potent anti-HIV-1 activity were effective competitors, whereas those having no or little anti-HIV-1 activity were poor competitors. Scatchard analysis indicates that the K d of the interaction between heparin and rgp120 is 10 nM. Binding studies conducted in increasing salt concentrations confirm that the interaction is ionic in nature. Synthetic 33-35 amino acid peptides based on the sequence of the V3 loop of gp120 also bind to heparin with high affinity. V3 loop peptides that are cyclized due to terminal cysteine residues show more selective binding than their uncyclized counterparts. Overall, these data demonstrate further that heparin exerts its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1, rather than its cellular receptor, CD4. This study confirms that the V3 loop of gp120 is the site at which heparin exerts its anti- HIV-1 activity. Moreover, it reveals that high affinity binding to heparin is shared by all four rgp120s examined, despite amino acid substitutions within the V3 loop.      

Vaccinia virus F12L protein is required for actin tail formation, normal plaque size, and virulence

Vaccinia virus gene F12L is shown to encode a 65-kDa protein that is synthesized early and late during infection and that is not modified by glycosylation. Computational sequence comparison revealed that related proteins are encoded by all sequenced chordopoxviruses. A virus deletion mutant lacking the F12L gene (vDeltaF12L) and a revertant virus with the F12L gene reinserted into the deletion mutant (vF12L-rev) were constructed and analyzed. A version of the F12L gene with a C-terminal amino acid tag derived from the influenza virus hemagglutinin and that is recognized by a monoclonal antibody was also inserted into the F12L locus of vDeltaF12L. Loss of the F12L protein reduced the formation of IMV 2-fold, but there was a dramatic (99.5%) reduction in actin tail formation, and the levels of cell-associated enveloped virus and extracellular enveloped virus were reduced 8- to 11-fold and 7-fold, respectively. Consistent with the lack of actin tail formation, vDeltaF12L produced a very small plaque. The vDeltaF12L virus was severely attenuated in vivo, such that a dose of vDeltaF12L 10,000-fold greater than the dose of wild-type virus that induced severe disease was unable to induce disease in mice infected intranasally.     

Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord

Despite great insight into the molecular mechanisms that specify neuronal cell type in the spinal cord, cell behaviour underlying neuron production in this tissue is largely unknown. In other neuroepithelia, divisions with a perpendicular cleavage plane at the apical surface generate symmetrical cell fates, whereas a parallel cleavage plane generates asymmetric daughters, a neuron and a progenitor in a stem cell mode, and has been linked to the acquisition of neuron-generating ability. Using a novel long-term imaging assay, we have monitored single cells in chick spinal cord as they transit mitosis and daughter cells become neurons or divide again. We reveal new morphologies accompanying neuron birth and show that neurons are generated concurrently by asymmetric and terminal symmetric divisions. Strikingly, divisions that generate two progenitors or a progenitor and a neuron both exhibit a wide range of cleavage plane orientations and only divisions that produce two neurons have an exclusively perpendicular orientation. Neuron-generating progenitors are also distinguished by lengthening cell cycle times, a finding supported by cell cycle acceleration on exposure to fibroblast growth factor (FGF), an inhibitor of neuronal differentiation. This study provides a novel, dynamic view of spinal cord neurogenesis and supports a model in which cleavage plane orientation/mitotic spindle position does not assign neuron-generating ability, but functions subsequent to this step to distinguish stem cell and terminal modes of neuron production.