Simultaneous addition of EGF prolongs the increase in cytosolic free calcium seen in response to bradykinin in NRK-49F cells

The calcium-sensitive fluorescent indicator fura-2 and a microscope equipped for rapidly changing excitation wavelengths were used to look at the effects of growth factors on cytosolic free calcium ([Ca²⁺]_i,) in NRK-49F cells. In these cells bradykinin induced a rapid increase in [Ca²⁺]_i, which generally decayed to near basal [Ca²⁺]_i within 3 minutes. The initial rise in [Ca²⁺]_i in response to bradykinin was relatively independent of extracellular calcium; however, the decay to basal [Ca²⁺]_i was more rapid in the absence of extracellular calcium. Measurements made on individual cells showed a heterogeneity in the response to bradykinin. Epidermal growth factor (EGF) had no effect on [Ca²⁺]_i in NRK-49F cells when added alone in the presence of extracellular calcium. Simultaneous addition of bradykinin and EGF produced a more prolonged increase in [Ca²⁺]_i than bradykinin alone. The prolongation was dependent on the presence of extracellular calcium and did not occur in its absence. Transient increases in [Ca²⁺]_i occurring after the initial peak were occasionally seen in these cells. Our results indicate that there is rapid interaction between the signaling mechanisms for bradykinin and EGF. When this occurs, one effect is the transport of calcium into the cell from the extracellular environment, causing a more prolonged rise in [Ca²⁺]_i. This effect occurs within 1 minute after combined addition of bradykinin and EGF.     

The ecosystem approach to environmental assessment: moving from theory to practice

The ecosystem approach to environmental management is viewed by many as being fundamental to the development of appropriate management strategies. While this approach represents a major advance in the way researchers view environmental assessment, the approach in itself does not provide practical information as to what questions to ask and what tools to use in assessing and managing ecosystems. Similarly, the concept of ecosystem health, as it is usually defined, has little practical value for ecosystem managers. We suggest the next stage in environmental assessment will be the development of specific frameworks designed to assess individual ecosystems. Of primary importance is the need to consider the basic structure and function of the ecosystem itself. Such consideration, together with explicit identification of anthropogenic stresses particular to the system, serves to identify those components most at risk and those issues most deserving of attention. Researchers should explore critical linkages between environmental stressors and their observable, measurable and predictable effects on ecological parameters and use this understanding to develop a management strategy that incorporates appropriate ecological indicators. The importance of these considerations will be illustrated using examples from the Northern River Basins Study.     

DOES RECOMBINATION CONSTRAIN NEUTRAL DIVERGENCE AMONG BACTERIAL TAXA?

A coalescence model for predicting the fate of neutral divergence among closely related taxa distinguishable as separate DNA sequence clusters is presented here. The model simulates iteratively the positive feedback between sequence divergence and sexual isolation among taxa, where increases in sequence divergence result in reduced recombination, and reduced recombination results in increased sequence divergence. Iteration of this feedback is continued until sequence divergence either converges on a steady state or reaches a runaway process. The eventual outcome of sequence divergence was shown to depend on four estimable population-genetic parameters: the expected intrataxon sequence diversity, the baseline rate of intertaxon recombination, the sensitivity of the recombination rate to sequence divergence, and the neutral mutation rate. The model can be used to determine whether neutral divergence among actual taxa is destined to stop at an equilibrium level, or whether neutral divergence will reach a runaway process. Application of the model to the group of taxa containing Bacillus subtilis and its closest relatives showed these taxa to be on a trajectory of unbounded neutral divergence from one another.     

RAPD分析─鉴定柑桔体细胞杂种的快速方法

本文利用改进的DNA提取方法,从Volkamer柠檬(Citrus volkameriana Ten. and Pasq.)和酸橙(C. aurantium L.)及其原生质体杂种植株的叶片中抽提总DNA,进行RAPD(Random Amplified Polymorphic DNA)分析。结果表明: 在随机选取的15种引物中,有10种可单独或与其它引物一道鉴定这一组合的体细胞杂种。与形态学性状观察、同工酶及ONA杂交分析等方法比较,RAPD分析是一种可在试管苗期即可直接、准确、快速鉴定柑桔体细胞杂种的方法。     

The relationship between insulin and vanadium metabolism in insulin target tissues

Vanadium (V) is an orally effective treatment for diabetes, but relatively little is known about the mechanisms controlling its normal metabolism nor the long term pharmacokinetics of oral administration. We have examined the accumulation of V in various organs from rats fed liquid diet for up to 18 days, containing no additional V, 1.6, 80, or 160 mole/kg/day as either sodium orthovanadate (SOV) or vanadyl sulfate (VS). V content was assayed using a sensitive neutron activation analysis method. The organs of the nonsupplemented animals contained widely varying concentrations (ng of V/g dry tissue weight) with brain < fat < blood < heart < muscle < lung < liver < testes < spleen < kidney. All organs accumulated V in a dose dependent manner. Not all organs showed steady state amount of V at 18 days, so additional rats were fed SOV or VS, switched to control diet, and assayed at 0, 4 and 8 days. From this data we calculated organ half lives of V. Insulin sensitive tissue tissues, such as liver and fat, had shorter half-lives than tissues that are relatively less insulin sensitive, such as spleen, brain and testes. SOV and VS fed rats showed similar patterns, but VS had somewhat shorter t1/2's. Additional studies of old and young rats fed control diet for 45 days show accumulation of V in spleen and testes. These results indicate that vanadium metabolism varies widely among different organs, and that insulin, either directly or indirectly has effects on the retention of vanadium. This may have impact on the therapeutic use of vanadium in Type I diabetics with no insulin, or Type II patients who may be relatively hyperinsulinemic.     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Vinculin Proteolysis Unmasks an ActA Homolog for Actin-based Shigella Motility

In polarized Madin-Darby canine kidney (MDCK) cells, the transferrin receptor (TR) is selectively delivered to the basolateral surface, where it internalizes transferrin via clathrin-coated pits and recycles back to the basolateral border. Mutant tailless receptors are sorted randomly in both the biosynthetic and endocytic pathways, indicating that the basolateral sorting of TR is dependent upon a signal located within the 61–amino acid cytoplasmic domain. To identify the basolateral sorting signal of TR, we have analyzed a series of mutant human TR expressed in MDCK cells. We find that residues 19–41 are sufficient for basolateral sorting from both the biosynthetic and endocytic pathways and that this is the only region of the TR cytoplasmic tail containing basolateral sorting information. The basolateral sorting signal is distinct from the YTRF internalization signal contained within this region and is not tyrosine based. Detailed functional analyses of the mutant TR indicate that residues 29–35 are the most important for basolateral sorting from the biosynthetic pathway. The structural requirements for basolateral sorting of internalized receptors from the endocytic pathway are not identical. The most striking difference is that alteration of G₃₁DNS₃₄ to YTRF impairs basolateral sorting of newly synthesized receptors from the biosynthetic pathway but not internalized receptors from the endocytic pathway. Also, mutations have been identified that selectively impair basolateral sorting of internalized TRs from the endocytic pathway without affecting basolateral sorting of newly synthesized receptors. These results imply that there are subtle differences in the recognition of the TR basolateral sorting signal by separate sorting machinery located within the biosynthetic and endocytic pathways.