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Here we review the zebrafish hemostatic system, its relevance to mammalian hemostasis, and its efficacy as a vertebrate genetic model to further the understanding of hemostasis and thrombosis.

Human serum high-density lipoprotein (HDL) is necessary and sufficient for the short-term maintenance of Plasmodium falciparum in in vitro culture. However, at high concentrations it is toxic to the parasite. A heat-labile component is apparently responsible for the stage-specific toxicity to parasites within infected erythrocytes 12–42 h after invasion, i.e. during trophozoite maturation. The effects of HDL on parasite metabolism (as determined by nucleic acid synthesis) are evident at about 30 h after invasion. Parasites treated with HDL show gross abnormalities by light and electron microscopy.

The acid rock drainage-affected stream of St. Kevin Gulch recharges the Quaternary sand and gravel aquifer of Tennessee Park, near Leadville, Colorado, lowering pH and contributing iron, cadmium, copper, zinc and sulphate to the groundwater system. Dissolved metal mobility is controlled by the seasonal spring runoff as well as oxidation/reduction (redox) reactions in the aquifer. Oxidizing conditions occur in the unconfined portions of the aquifer whilst sulphate-reducing conditions are found down gradient where semi-confined groundwater flow occurs beneath a natural wetland. Iron-reducing conditions occur in the transition from unconfined to semi-confined groundwater flow. Dissolved iron concentrations are low to not detectable in the alluvial fan recharge zone and increase in a down gradient direction. The effects of low-pH, metal-rich recharge are pronounced during low-flow in the fall when there is a defined area of low pH groundwater with elevated concentrations of dissolved zinc, cadmium, copper and sulphate adjacent to St. Kevin Gulch. Dissolved metal and sulphate concentrations in the recharge zone are diluted during spring runoff, although the maximum concentrations of dissolved zinc, cadmium, copper and sulphate occur at selected down gradient locations during high flow. Dissolved zinc, cadmium and copper concentrations are low to not detectable, whereas dissolved iron concentrations are greatest, in groundwater samples from the sulphate-reducing zone. Attenuation of zinc, cadmium and copper beneath the wetland suggests sulphide mineral precipitation is occurring in the semi-confined aquifer, in agreement with previous site investigations and saturation index calculations. Adsorption of dissolved zinc, cadmium and copper onto iron hydroxides is a minor attenuation process due to the low pH of the groundwater system.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

The burden and duration of asymptomatic malaria infections were measured in residents of the malaria endemic village of Gonoa, Madang Province, Papua New Guinea. Plasmodium falciparum, P. vivax and P. malariae infections in people aged 4 years to adulthood were compared. Frequent sampling at 3-day intervals for up to 61 days allowed assessment of individual episodes of infection. Statistical assessment of P. falciparum detection revealed a periodicity consistent with synchronous replication of this species over periods up to 27 days. The duration of P. falciparum episodes was longer across all age groups than that of P. vivax and P. malariae. A trend for decreasing duration with age was also noted in data from each species. This was most prominent in P. falciparum infections: median duration in 4-year-olds was > 48 days compared with a median between 9 and 15 days in older children and adults. The results are consistent with the slow acquisition of immunity to antigenically diverse Plasmodium populations and suggest a faster rate of acquisition to P. vivax and P. malariae than to P. falciparum.

We describe the dynamics of co-infections of Plasmodium falciparum and P. vivax in 28 asymptomatic children by genotyping these species using the polymorphic loci Msp2 and Msp3α, respectively. The total number of Plasmodium spp. infections detected using 3 day sampling over 61 days varied between 1 and 14 (mean 6·6). The dynamics of P. falciparum and P. vivax genotypes varied greatly both within and amongst children. Periodicity in the detection of P. falciparum infections is consistent with the synchronous replication of individual genotypes. Replication synchrony of multiple co- infecting genotypes was not detected. In 4-year-old children P. falciparum genotype complexity was reduced and episodes lasted significantly longer (median duration > 60 days) when compared to children aged 5–14 years (median duration 9 days). P. vivax genotype complexity was not correlated with age but the episode duration was also longer for this species in 4-year-olds than in older children but was not as long as P. falciparum episodes. Recurrence of P. falciparum and P. vivax genotypes over weeks was observed. We interpret these major fluctuations in the density of genotypes over time as the result of the mechanism of antigenic variation thought to be present in these Plasmodium species.

Polymerase chain reaction (PCR)-based genotyping of oocysts dissected from mosquito midguts has previously been used to investigate overall levels of inbreeding within malaria parasite populations. We present a re-analysis of the population structure of Plasmodium falciparum malaria using diploid genotypes at three antigen-encoding loci in 118 oocysts dissected from 34 mosquitoes. We use these data to ask whether mating is occurring at random within the mosquito midgut, as is generally assumed. We observe a highly significant deficit of heterozygous oocysts within mosquitoes at all three loci, suggesting that fusion of gametes occurs non-randomly in the mosquito gut. A variety of biological explanations, such as interrupted feeding of mosquitoes, positive assortative mating and outcrossing depression, could account for this observation. However, an alternative artefactual explanation – the presence of non-amplifying or null alleles – can account for the observed data equally well, without the need to invoke non-random mating. To evaluate this explanation further, we estimate the frequencies of null alleles within the oocyst population using maximum likelihood, by making the assumption that non-amplifying oocysts at any of the three loci are homozygous for null alleles. Observed levels of visible heterozygotes fit closely with those expected under random mating when non-amplifying oocysts are accounted for. Other lines of evidence also support the artefactual explanation. Overall inbreeding coefficients have been recalculated in the light of this analysis, and may be considerably lower than those estimated previously. In conclusion, we suggest that the deficit of heterozygotes observed is unlikely to indicate non-random mating within the mosquito gut and is better explained by misscoring of heterozygotes as homozygotes.

A potential target of hormone action during prostate and mammary involution is the intercellular junction of adjacent secretory epithelium. This is supported by the long-standing observation that one of the first visible stages of prostate and mammary involution is the disruption of interepithelial adhesion prior to the onset of apoptosis. In a previous study addressing this aspect of involution, we acquired compelling evidence indicating that the disruption of E-cadherin-dependent adhesion initiates apoptotic programs during prostate and mammary involution. In cultured prostate and mammary epithelial cells, inhibition of E-cadherin-dependent aggregation resulted in cell death following apoptotic stimuli. Loss of cell-cell adhesion in the nonaggregated population appeared to result from the rapid truncation within the cytosolic domain of the mature, 120-kDa species of E-cadherin (E-cad(120)). Immunoprecipitations from cell culture and involuting mammary gland demonstrated that this truncation removed the beta-catenin binding domain from the cytoplasmic tail of E-cadherin, resulting in a non-beta-catenin binding, membrane-bound 97-kDa species (E-cad(97)) and a free cytoplasmic 35-kDa form (E-cad(35)) that is bound to beta-catenin. Examination of E-cadherin expression and cellular distribution during prostate and mammary involution revealed a dramatic reduction in junctional membrane staining that correlated with a similar reduction in E-cad(120) and accumulation of E-cad(97) and E-cad(35). The observation that E-cadherin was truncated during involution suggested that hormone depletion activated the same apoptotic pathway in vivo as observed in vitro. Based on these findings, we hypothesize that truncation of E-cadherin results in the loss of beta-catenin binding and cellular dissociation that may signal epithelial apoptosis during prostate and mammary involution. Thus, E-cadherin may be central to homeostatic regulation in these tissues by coordinating adhesion-dependent survival and dissociation-induced apoptosis.

Why there are so few gametocytes (the transmission stage of malaria) in the blood of humans infected with Plasmodium spp. is intriguing. This may be due either to reproductive restraint by the parasite or to unidentified gametocyte-specific immune-mediated clearance mechanisms. We propose another mechanism, a cross-stage immunity to Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1). This molecule is expressed on the surface of the erythrocyte infected with either trophozoite or early gametocyte parasites. Immunoglobulin G antibodies to PfEMP-1, expressed on both life cycle stages, were measured in residents from an area where malaria is endemic, Papua New Guinea. Anti-PfEMP-1 prevalence increased with age, mirroring the decline in both the prevalence and the density of asexual and transmission stages in erythrocytes. These data led us to propose that immunity to PfEMP-1 may influence malaria transmission by regulation of the production of gametocytes. This regulation may be achieved in two ways: (i) by controlling asexual proliferation and density and (ii) by affecting gametocyte maturation.

Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0·21 to 0·87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

E-cadherin and the retinoblastoma tumor suppressor (Rb) are traditionally associated with diverse regulatory aspects of cell growth and differentiation. However, we have discovered new evidence, which suggests that these proteins are functionally linked in a physiologic pathway required for cell survival and programmed cell death. Pharmacological activation of protein kinase C (PKC) or inducible overexpression and activation of the alpha isozyme of PKC (PKCalpha) resulted in approximately 60% apoptosis of mammary and prostate epithelial cells. Interestingly, the surviving cells had undergone dramatic aggregation concurrent with increased E-cadherin expression. When aggregation was inhibited by the addition of an E-cadherin-blocking antibody, apoptosis increased synergistically. We hypothesized that survival of the aggregated population was associated with contact-inhibited growth and that apoptosis might result from aberrant growth regulatory signals in non-aggregated, cycling cells. This hypothesis was confirmed by experiments that demonstrated that E-cadherin-dependent aggregation resulted in Rb-mediated G1 arrest and survival. Immunoblot analysis and flow cytometry revealed that hypophosphorylated Rb was present in non-aggregated, S phase cultures concurrent with synergistic cell death. We have also determined that the loss of membrane E-cadherin and subsequent hypophosphorylation of Rb in luminal epithelial cells preceded apoptosis induced by castration. These findings provide compelling evidence that suggests that E-cadherin-mediated aggregation results in Rb activation and G1 arrest that is critical for survival of prostate and mammary epithelial cells. These data also indicate that Rb can initiate a fatal growth signal conflict in non-aggregated, cycling cells when the protein is hypophosphorylated as these epithelial cells enter S phase.

The paper describes a computer program which calculates the amounts of each of 39 nutrients in a food, presented to it as a list of ingredients. Allowances can be made for losses of water and vitamins on cooking. The recipe can be compared with other foods already listed in the food tables. Other comparisons can also be made in terms of percent recommended daily intake. The new food may be retained as an addition to the food tables.

Epithelial cells are dependent upon adhesion to extracellular matrix for survival. We show that loss of beta1 integrin receptor contact with extracellular matrix signals the inhibition of G1 cyclin-dependent kinase activity. This loss of cyclin-dependent kinase activity leads to accumulation of the hypophosphorylated (active) form of the retinoblastoma tumor suppressor protein (Rb). We present evidence that in epithelial cells deprived of matrix contact, the growth suppression signal elicited by hypophosphorylated Rb opposes stimulatory signals from serum growth factors, leading to a cell cycle conflict that triggers apoptosis. This apoptotic pathway is modulated by Bcl-2 through a novel mechanism that regulates Rb phosphorylation. We present evidence that the Rb-dependent apoptotic pathway functions in vivo in the apoptosis of the prostate glandular epithelium following castration.

Both protein kinase C and the retinoblastoma tumor suppressor protein have been linked to the regulation of cell growth and cell death, suggesting the differential roles these factors play in mediating cell fate. In some cells, protein kinase C-induced activation of the retinoblastoma protein results in G1 arrest. However, inducible overexpression and activation of the protein kinase Calpha isozyme or the addition of 12-O-tetradecanoylphorbol-13-acetate in the prostate epithelial cell line, LNCaP, resulted in apoptosis preceded by induction of p21 and dephosphorylation of the retinoblastoma protein. Consistent with a role for the retinoblastoma growth suppressor protein in protein kinase C-induced apoptosis, DU145 cells, which do not express functional retinoblastoma protein or LNCaP cells, which have been transfected with the retinoblastoma inhibitor, E1a, were resistant to apoptosis. LNCaP apoptosis was initiated by a unique conflict between the growth-suppressive activity of the retinoblastoma protein and growth-promoting mitogenic signals. Thus, when this conflict was prevented by serum depletion, apoptosis was suppressed. The caspase family of cysteine proteases is believed to encompass the execution machinery of mammalian apoptosis, and addition of the cell-permeable caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, afforded nearly total protection from protein kinase C-signaled apoptosis. This protection correlated with the total loss of caspase activity as measured by the proteolytic cleavage of nuclear poly(ADP-ribose) polymerase. On the basis of these results, we propose that protein kinase C regulates a novel cell death pathway that is initiated by a cellular conflict between retinoblastoma growth-suppressive signals and serum mitogenic signals in proliferating prostate epithelial cells and that is executed by the caspase family of cysteine proteases.

Some molecular characteristics of Plasmodium falciparumlines which do not produce gametocytes are described. Parasites carrying a subtelomerically deleted chromosome 9 cannot form even the earliest forms of gametocytes, detectable with antibodies against the gametocyte-specific antigen Pfg27, In a parasite culture of clone HB3, in which both intact and deleted forms of chromosome 9 are present, full-length chromosome 9 molecules are retained mainly in gametocytes. These data suggest that the subtelomeric portion of chromosome 9 is required at an early stage of gametocytogenesis. Parasite subclones derived from gametocyte producing clone 3D7, which completely lost ability to produce gametocytes, are also described. Unlike the previous gametocyteless lines, these parasites stably maintain a full-length chromosome 9 and the ability to cytoadhere to C32 melanoma cells after prolonged asexual propagation, Their defect in sexual development is therefore genetically and functionally distinct from that of parasites carrying a deleted chromosome 9. Gametocyteless subclones derived from 3D7 do not produce any Pfg27 mRNA, while this gene is anomalously expressed in asexual stage parasites of two lines of a different genetic background, 1776se18 and C10, one able and the other unable to produce gametocytes.

Let $ {T_n}(a) = ({a_{i - j}})_{i,j = 0}^n$ $ {\sigma _n} = \{ {\lambda _{n0}}, \ldots ,{\lambda _{nn}}\} $. Define a sequence of measures $ {\alpha _n},{\alpha _n}(E) = {(n + 1)^{ - 1}}{\Sigma _{{\lambda _{ni}} \in E}}1,{\lambda _{ni}} \in {\sigma _n}$ a set in the $ \lambda $ of the measures $ {\alpha _n}$ which give rise to atoms are identified.

Let $ {T_n}(f) = ({a_{i - j}})_{i,j = 0}^n$ $ \det ({T_n}(f) - \lambda )$ is a point or consists of a finite number of analytic arcs.

Gives advice on how to execute a backhand slice crosscourt return from service; Pictures the execution of the return; Key points to developing a good return; Strategy.