Sources,fates, and impacts of nitrogen inputs to terrestrial ecosystems: review and synthesis

The relative importance of nitrogen inputs from atmospheric deposition and biological fixation is reviewed in a number of diverse, non-agricultural terrestrial ecosystems. Bulk precipitation inputs of N (l–l2 kg N ha^–1 yr^–1) are the same order of magnitude as, or frequently larger than, the usual range of inputs from nonsymbiotic fixation (< 1=" –=" 5=" kg=" n=">^–1 yr^–1), especially in areas influenced by industrial activity. Bulk precipitation measurements may underestimate total atmospheric deposition by 30–40% because they generally do not include all forms of wet and dry deposition. Symbiotic fixation generally ranges from 10–160 kg N ha^–1 yr^–1) in ecosystems where N-fixing species are present during early successional stages, and may exceed the range under unusual conditions.Rates of both symbiotic and nonsymbiotic fixation appear to be greater during early successional stages of forest development, where they have major impacts on nitrogen dynamics and ecosystem productivity. Fates and impacts of these nitrogen inputs are important considerations that are inadequately understood. These input processes are highly variable in space and time, and few sites have adequate comparative information on both nitrogen deposition and fixation.

The γ-crystallin gene families: Sequence and evolutionary patterns

Summary The -crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the -crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat -crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.     

Comparison of zona cutting and zona drilling as techniques for assisted fertilization in the mouse

Zona cutting and zona drilling of the mouse oocyte significantly increased the fertilization rate (3.8-90%) at low sperm concentrations (less than 200,000/ml) compared with zona-intact controls (0-45%). More oocytes were fertilized after zona drilling. Zona cutting was associated with a low loss of oocytes (less than 1%), no increase in polyspermy and normal development in vitro and in vivo after fertilization. There was a 4% oocyte loss rate after zona drilling, mostly due to extrusion of the oocyte from the zona during the procedure. Hatching of blastocysts occurred about 12 h earlier for zona-drilled than for zona-cut and zona-intact control oocytes. Zona drilling was associated with a higher, but not statistically significant, rate of polyspermy at all sperm concentrations tested. The proportion of zygotes developing to the blastocyst stage was not different between the techniques (zona cut, 77%; zona drilled, 66%; control, 71%). Similarly, no difference was found in the percentage of embryos implanting after blastocyst transfer to the uterine horns of pseudopregnant female mice (zona cut, 67%; zona drilled, 68%; control, 77%). Transmission electron microscopy demonstrated the induced defects in the zona with no damage to the oocyte or oolemma. Parthenogenetic activation was not seen after either of the micromanipulative techniques. Both techniques have promise for application to the human.     

Differential effects of largemouth and smallmouth bass on habitat use by stoneroller minnows in stream pools

Previous investigations of within-reach distribution patterns of the stoneroller minnow, Campostoma anomalum Rafinesque, suggest that this species behavioural response to piscivorous bass differs among streams with different bass species. This study compares the responses of Camposloma to two common piscivorous fishes, largemouth bass, Micropterus salmoides Lacepede, and smallmouth bass, M. dolomieui Lacepede. Field experiments in Brier Creek, Oklahoma, U.S.A. showed that Campostoma responded to the presence of adult largemouth bass by shifting to shallow water habitats (commonly < 20 cm), but responded weakly or not at all to adult smallmouth bass. The magnitude of the response of Campostoma was positively related to activity level of the predators. Differences in behaviour between these two predator species may contribute to the differences in spatial distribution patterns of Micropterus and Campostoma observed in earlier investigations, but differences in stream channel morphology and temperature regimes among streams may also be important.     

Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [³⁵S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS PAGE of the treated cells showed incorporation of label into a broad 97–121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97–121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5′-O-(3-thiotriphosphate). The 97–121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5′-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteoglycans.

The results demonstrate that chromaffin vesicle matrix proteins are rapidly and intensely thiophosphorylated in cultured chromaffin cells but not in isolated vesicles. The data suggest that phosphorylation must play an important role in the normal function of these vesicle proteins.     

Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice

Summary A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.