alpha-Fetoprotein production and erythropoiesis in cell cultures of human fetal liver.

alpha-Fetoprotein and the synthesis of heme associated with hemoglobin were measured simultaneously in short-term cultures of human fetal liver cells to correlate the relationship of alpha-fetoprotein to erythroid cell function. Both synthetic processes decreased exponentially during the first 5 days of culture. The use of media supplemented with different batches of fetal calf serum and porcine portal vein serum indicated that the optimal conditions for the production of alpha-fetoprotein were different from those required for the synthesis of heme associated with hemoglobin. Moreover, the alpha-fetoprotein-producing cells could be separated from erythroid cells after velocity sedimentation in Ficoll gradients. Although it is well known that erythropoiesis and alpha-fetoprotein production occur simultaneously during ontogenesis, alpha-fetoprotein itself (0.01-100 micron g/ml) did not stimulate heme synthesis in liver erythroid cells. Erythropoietin did not stimulate alpha-fetoprotein production. It is concluded that there is no cause-effect relationship between alpha-fetoprotein production and erythroid cell fuction in human fetal liver cells and that the two processes occur independently in different cell types.     

Density-functional investigation on the mechanism of H-atom abstraction by lipoxygenase

Using experimentally calibrated density functional calculations on models of the active site of soybean lipoxygenase 1 (SLO-1), insight has been obtained into the coordination flexibility of the iron active site and its molecular mechanism of catalysis. The ferrous form of SLO-1 shows a variation in coordination number in solution that is related to a weakly coordinating Asn694 ligand. From the calculations it is determined that the weak Fe-O(694) bond associated with this coordination flexibility is due to a sideways tilted geometry of Asn694 that is imposed on the site by the protein. Release of this constraint (by altering the hydrogen bonding network) leads to a pure six-coordinate site. In contrast, the ferric form of the enzyme stays five-coordinate. In this case, deprotonation of a coordinated water gives a strong hydroxo donor in the cis position to Asn694, weakening the Fe-O(694) bond. Hence, Asn694 is a stronger ligand to the reduced relative to the oxidized site. Using these experimentally calibrated models, the reaction energy for H-atom transfer in SLO-1 has been calculated to be about -18 kcal/mol. The observed change in coordination number going from five-coordinate in ferric to six-coordinate in ferrous SLO-1 increases the reduction potential of the iron active site. Hence, the protein adjusts the active site for optimal reactivity. Analysis of the electronic structure along the reaction coordinate shows that the H-atom transfer in SLO-1 actually corresponds to a proton-coupled electron transfer (PCET). The transferred electron does not localize on the proton, but tunnels directly from the substrate to the ferric active site in a concerted proton tunneling-electron tunneling (PTET) process. The covalently linked Fe-O-H-C bridge in the transition state lowers the energy barrier and provides an efficient superexchange pathway for this tunneling. The thermal barrier for the PTET process is estimated from the calculations to be about +15 kcal/mol including zero-point energy corrections. This corresponds to a thermal reaction rate of k(therm) approximately 1 s(-1). In comparison, the rate of proton tunneling can be as high as 2 x 10(9) s(-1) under these conditions.     

Salmon stock and recruitment, and stock enhancement

The precise nature of the stock and recruitment relationship in natural populations is of vital importance to any attempt at stock enhancement by removing or by-passing a limiting factor, or at management by manipulation of spawning stock. In this paper the evidence for and against a dome-shaped stock and recruitment curve is reviewed, and consideration given to several factors which complicate the conventional, simplistic interpretation of density-dependent mortality. These include environmentally mediated changes in 'carrying capacity', distribution of spawning, and the need to consider whole river systems. The implications of these factors for fisheries management and stock enhancement are discussed.     

Soluble benzodiazepine receptors: Gabaergic regulation

Benzodiazepine receptor binding has been measured in soluble brain extracts with ³H-flunitrazepam as a ligand. Binding to soluble receptors is enhanced by GABAergic agonists with potencies and maximal augmentation essentially the same as on membrane bound benzodiazepine receptors. The GABA induced increase of binding to soluble receptors is reversed by the GABA antagonist bicuculline.     

Reaching providers is not enough to increase IUD use: a factorial experiment of 'academic detailing' in Kenya

Although the IUD is an extremely effective and low-cost contraceptive method, its use has declined sharply in Kenya in the past 20 years. A study tested the effectiveness of an outreach intervention to family planning providers and community-based distribution (CBD) agents in promoting use of the IUD in western Kenya. Forty-five public health clinics were randomized to receive the intervention for providers only, for CBD agents only, for both providers and CBD agents, or no detailing at all. The intervention is based on pharmaceutical companies' "detailing" models and included education/motivation visits to providers and CBD programmes, as well as provision of educational and promotional materials. District health supervisors were given updates on contraceptives, including the IUD, and were trained in communication and message development prior to making their detailing visits. Detailing only modestly increased the provision of IUDs, and only when both providers and CBD agents were targeted. The two detailing visits do not appear sufficient to sustain the effect of the intervention or to address poor provider attitudes and lack of technical skills. The cost per 3.5 years of pregnancy protection was US$49.57 for the detailing intervention including the cost of the IUD, compared with US$15.19 for the commodity costs of the current standard of care--provision of the injectable contraceptive depot-medroxyprogesterone acetate (DMPA). The effectiveness of provider-based activities is amplified when concurrent demand creation activities are carried out. However, the cost of the detailing in comparison to the small number of IUDs inserted indicates that this intervention is not cost-effective.     

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 μg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.     

Distribution of Y chromosomes among native North Americans: a study of Athapaskan population history

In this study, 231 Y chromosomes from 12 populations were typed for four diagnostic single nucleotide polymorphisms (SNPs) to determine haplogroup membership and 43 Y chromosomes from three of these populations were typed for eight short tandem repeats (STRs) to determine haplotypes. These data were combined with previously published data, amounting to 724 Y chromosomes from 26 populations in North America, and analyzed to investigate the geographic distribution of Y chromosomes among native North Americans and to test the Southern Athapaskan migration hypothesis. The results suggest that European admixture has significantly altered the distribution of Y chromosomes in North America and because of this caution should be taken when inferring prehistoric population events in North America using Y chromosome data alone. However, consistent with studies of other genetic systems, we are still able to identify close relationships among Y chromosomes in Athapaskans from the Subarctic and the Southwest, suggesting that a small number of proto-Apachean migrants from the Subarctic founded the Southwest Athapaskan populations.