A comparison of two procedures used for complexing Fe(III) with human apotransferrin. II. Uptake of Fe(III) by K-562 cells from 55Fe . transferrins and Fe . [3H]transferrins

Samples of human apotransferrin (apo . HTr) were saturated with Fe(III) by two different techniques, a method employing excess trisodium citrate to chelate Fe(III) and a nonchelating approach which involves the ferroxidase activity of ceruloplasmin to convert Fe(II)----Fe(III). The samples were radiolabelled with either 55Fe or 3H. Using an initial molar Fe/apo . HTr ratio of 2.0-2.1, preparations of human transferrin with bound Fe (Fe . HTr) using the citrate method invariably contained 2.2-2.4 atoms Fe/molecule, whereas Fe . HTr (ceruloplasmin method) contained 2.0 atoms/molecule as shown by spectrophotometric and radioactivity measurements. Uptake of Fe from these Fe . HTr preparations by K-562 cells grown in a serum-free medium was marginally, but consistently, more rapid from 55Fe . HTr (citrate) than from 55Fe . HTr (ceruloplasmin). Taking account of the different Fe contents of the Fe . HTr preparations, the rate measured over a 2-h period amounted to approximately 12,700 and 16,100 Fe atoms/(cell . min) for Fe . HTr (ceruloplasmin) and Fe . HTr (citrate), respectively. However, cell binding by the two Fe . [3H]HTr preparations did not differ significantly over the 8-h incubation period. Furthermore, from the 3H distribution, the quantities of Fe . HTr bound reversibly at the cell surface and contained within the cell were similar for the two Fe . HTr preparations. The results indicate that apo . HTr may bind Fe in different ways depending on the method of Fe presentation and that the Fe . HTr product can donate Fe to K-562 cells at a rate which may reflect the method used for Fe-complex formation.     

The effect of coastline changes on mangrove community structure,Portuguese Island,Mozambique

Changes in coastline configuration and sand movements on Portuguese Island, a small island lying close to Inhaca Island 35 km east of Maputo, Mozambique have resulted in large areas of the Island being eroded away and consequent restricted tidal recharge of the mangrove. Measurements made during 1989 show that flooding of the mangrove occurred on only three occasions. Percentage mortality of the dominant species,Ceriops tagal (Perr.) C. B. Robinson., is high. However, at the time of the study, the co-dominant,Bruguiera gymnorrhiza (L.) Lam., showed no signs of stress. Halophytic conditions are indicated by an invasive spread of salt-tolerant herbs. All species of crustaceans and molluscs have disappeared from the mangrove.Although mortality ofC. tagal is high, the regeneration status of this species is also high, comparable to that in a regularly recharged mangrove on Inhaca Island. It is suggested thatC. tagal propagules were shed under conditions when seedling establishment was favorable i.e. flooding. These seedlings may represent the last cohort which may also suffer stress and death unless tidal recharge is re-established.     

Assessment of the impact of the Lower Kihansi Hydropower Project on the forests of Kihansi Gorge, Tanzania

Construction of a hydropower project on the Kihansi river in Southern Tanzania will substantially alter the river's flow. On the basis of both qualitative and quantitative botanical surveys, forest in the Kihansi gorge below the dam site was found to be mostly typical species and endemic-rich Eastern Arc forest. An unusual forest type, dominated almost entirely by Filicium decipiens was also present. Eastern Arc forests are of both global and national importance for biodiversity conservation, and measures to mitigate impact of the dam on the forest are suggested. Further environmental monitoring is recommended in order to evaluate the choice of mitigation measures.     

On the PAM matrix model of protein evolution

The internal consistency of the PAM matrix model of protein evolution is here investigated. The 1 PAM matrix has been constructed from amino acid replacements observed in closely related sequences. Such replacements are of two types, those that do not require an intermediate amino acid replacement and those that do. The second type of replacement must generally be produced by a repetition of the first. This allows data on the first type to be used in predicting data on the second type so that some elements of the 1 PAM matrix may be used to predict others. A discrepancy of more than two orders of magnitude is found between the predictions and the data when this is carried out. This is partly accounted for by an error in constructing the matrix. However, it also seems necessary that the basic model be modified. Several possibilities are considered. One of these is to incorporate a site-dependent spectrum of mutabilities associated with each amino acid.      

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Litter type control on soil C and N stabilization dynamics in a temperate forest

While plant litters are the main source of soil organic matter (SOM) in forests, the controllers and pathways to stable SOM formation remain unclear. Here, we address how litter type (¹³C/¹⁵N‐labeled needles vs. fine roots) and placement‐depth (O vs. A horizon) affect in situ C and N dynamics in a temperate forest soil after 5 years. Litter type rather than placement‐depth controlled soil C and N retention after 5 years in situ, with belowground fine root inputs greatly enhancing soil C (x1.4) and N (x1.2) retention compared with aboveground needles. While the proportions of added needle and fine root‐derived C and N recovered into stable SOM fractions were similar, they followed different transformation pathways into stable SOM fractions: fine root transfer was slower than for needles, but proportionally more of the remaining needle‐derived C and N was transferred into stable SOM fractions. The stoichiometry of litter‐derived C vs. N within individual SOM fractions revealed the presence at least two pools of different turnover times (per SOM fraction) and emphasized the role of N‐rich compounds for long‐term persistence. Finally, a regression approach suggested that models may underestimate soil C retention from litter with fast decomposition rates.     

Metabolism of DMSP, DMS and DMSO by the cultivable bacterial community associated with the DMSP-producing dinoflagellate Scrippsiella trochoidea

Bacterial species associated with the dimethylsulfoniopropionate (DMSP)-producing phytoplankton Scrippsiella trochoidea were cultured and identified, with the aim of establishing their ability to metabolise DMSP, dimethylsulfide (DMS) and dimethylsulfoxide (DMSO). Results demonstrate that of the cultivable bacteria only α-Proteobacteria were capable of producing DMS from DMSP. The concentration of DMSP was shown to affect the amount of DMS produced. Lower DMSP concentrations (1.5?μmol?dm^?3) were completely assimilated, whereas higher concentrations (10?μmol?dm^?3) resulted in increasing amounts of DMS being produced. By contrast to the restricted set of bacteria that metabolised DMSP,?~?70% of the bacterial isolates were able to ‘consume’ DMS. However, 98-100% of the DMS removed was accounted for as DMSO. Notably, a number of these bacteria would only oxidise DMS in the presence of glucose, including members of the γ-Proteobacteria and Bacteroidetes. The observations from this study, coupled with published field data, identify DMS oxidation to DMSO as a major transformation pathway for DMS, and we speculate that the fate of DMS and DMSP in the field are tightly coupled to the available carbon produced by phytoplankton.     

A Distinct Smoothened Mutation Causes Severe Cerebellar Developmental Defects and Medulloblastoma in a Novel Transgenic Mouse Model

Deregulated developmental processes in the cerebellum cause medulloblastoma, the most common pediatric brain malignancy. About 25 to 30% of cases are caused by mutations increasing the activity of the Sonic hedgehog (Shh) pathway, a critical mitogen in cerebellar development. The proto-oncogene Smoothened (Smo) is a key transducer of the Shh pathway. Activating mutations in Smo that lead to constitutive activity of the Shh pathway have been identified in human medulloblastoma. To understand the developmental and oncogenic effects of two closely positioned point mutations in Smo, we characterized NeuroD2-SmoA2 mice and compared them to NeuroD2-SmoA1 mice. While both SmoA1 and SmoA2 transgenes cause medulloblastoma with similar frequencies and timing, SmoA2 mice have severe aberrations in cerebellar development, whereas SmoA1 mice are largely normal during development. Intriguingly, neurologic function, as measured by specific tests, is normal in the SmoA2 mice despite extensive cerebellar dysplasia. We demonstrate how two nearly contiguous point mutations in the same domain of the encoded Smo protein can produce striking phenotypic differences in cerebellar development and organization in mice.     

Autofeedback effects of progressively rising oxytocin concentrations on supraoptic oxytocin neuronal activity in slices from lactating rats

Suckling stimuli induce somatodendritic oxytocin (OT) release from supraoptic nucleus (SON) neurons, which raises intranuclear OT concentrations and contributes to the effectiveness of the milk-ejection reflex. To clarify how such changes in OT concentrations modulate the activity of OT neurons, we examined OT effects using whole cell patch-clamp recordings from SON neurons in slices from lactating rats. Progressive increases from extremely low OT concentrations (0.1-10 fM) to high concentrations (0.1-10 nM) induced excitation and subsequent spike frequency reduction (SFR) in OT neurons. Significant effects of OT on firing rates were observed starting at 1 fM, reached peak level from 1 fM to 1 pM before SFR occurred in most neurons. The buildup of OT concentrations progressively promoted depolarization of membrane potential, spike broadening, decreases in spike amplitude, and increases in the rise time of spike afterhyperpolarizations, which were unrelated to firing rate. However, intermittent application of OT (1 fM, 1 pM, and 1 nM, each for 5 min) evoked dose-dependent excitation but not the SFR. Application of 1 pM OT for 40 min simulated the effects of progressively increasing OT concentrations. Vasopressin neurons were also activated by OT but did not show SFR. Consistent with presynaptic loci of OT action, ionotropic glutamate receptor antagonists reduced OT effects on firing rate, whereas bicuculline did not change the excitatory effects. These results suggest that the specific autoregulatory effects of OT, and perhaps other neuropeptides as well, are time and concentration dependent.     

A 60-kilodalton protein component of the counting factor complex regulates group size in Dictyostelium discoideum

Much remains to be understood about how a group of cells or a tissue senses and regulates its size. Dictyostelium discoideum cells sense and regulate the size of groups and fruiting bodies using a secreted 450-kDa complex of proteins called counting factor (CF). Low levels of CF result in large groups, and high levels of CF result in small groups. We previously found three components of CF (D. A. Brock and R. H. Gomer, Genes Dev. 13:1960-1969, 1999; D. A. Brock, R. D. Hatton, D.-V. Giurgiutiu, B. Scott, R. Ammann, and R. H. Gomer, Development 129:3657-3668, 2002; and D. A. Brock, R. D. Hatton, D.-V. Giurgiutiu, B. Scott, W. Jang, R. Ammann, and R. H. Gomer, Eukaryot. Cell 2:788-797, 2003). We describe here a fourth component, CF60. CF60 has similarity to acid phosphatases, although it has very little, if any, acid phosphatase activity. CF60 is secreted by starving cells and is lost from the 450-kDa CF when a different CF component, CF50, is absent. Although we were unable to obtain cells lacking CF60, decreasing CF60 levels by antisense resulted in large groups, and overexpressing CF60 resulted in small groups. When added to wild-type cells, conditioned starvation medium from CF60 overexpressor cells as well as recombinant CF60 caused the formation of small groups. The ability of recombinant CF60 to decrease group size did not require the presence of the CF component CF45-1 or countin but did require the presence of CF50. Recombinant CF60 does not have acid phosphatase activity, indicating that the CF60 bioactivity is not due to a phosphatase activity. Together, the data suggest that CF60 is a component of CF, and thus this secreted signal has four different protein components.