Haemocyanin oxygen binding and the physiological ecology of a range of talitroidean amphipods (Crustacea) II. Effect of freezing,inorganic ions,and urate on O2 binding in vitro

Summary The effect of variations in [K], [Ca], [Mg], [NaCl], and [urate] on the in vitro O₂ binding properties of haemocyanin (Hc) from three talitroidean species, viz. the aquatic Apohyale pugettensis, the semi-terrestrial Megalorchestia californiana, and the semi-/euterrestrial Traskorchestia traskiana were studied. Freezing altered the cooperativity of Hc from A. pugettensis and M. californiana but not T. traskiana. Variations in [NaCl], [K], and [Mg] had no effect on cither O₂ affinity or cooperativity of the Hc except for A. pugettensis Hc where an increase in [Mg] resulted in an increase in both O₂ affinity and cooperativity. Increasing [Ca] or [urate] increased O₂ affinity of both A. pugettensis and M. californiana but not T. traskiana Hc. These effects were most marked in A. pugettensis. The results suggest a negative correlation between sensitivity to Hc effectors and the degree of terrestrial adaptation of a particular amphipod species.Abbreviations Hc haemocyanin - STR Stock traskorchestia ringer     

The 44P subunit of the T4 DNA polymerase accessory protein complex catalyzes ATP hydrolysis

The genes encoding all three T4 DNA polymerase accessory proteins have been cloned into overexpression plasmids. Induction of cells harboring these plasmids results in the synthesis of each accessory protein at levels that approach 10% of the total cellular protein. The solubility of the accessory proteins after induction at 42 degrees C ranges from about 60% to greater than 95%. A plasmid that allows overexpression of the 44P/62P complex has been manipulated further to overexpress selectively the 44P subunit without 62P, permitting us to assess how each subunit contributes to the properties of the 44P/62P complex. A comparison of 44P and 44P/62P by conventional hydrodynamic techniques shows that 44P forms a subcomplex nearly as large as the 44P/62P complex. In addition, 44P catalyzes DNA-dependent ATP hydrolysis with a specific activity similar to that of the 44P/62P ATPase. However, unlike the 44P/62P complex, the ATPase activity of 44P alone is only slightly stimulated by 45P. This suggests that one role of the 62P subunit is to facilitate a productive interaction of 44P and 45P.     

Ionic components of secretory cell surfaces in relation to secretory function

Synopsis Rat pancreas was examined by ultrastructural cytochemical methods for localizing cations and anions, as well as polyanions and, more specifically, sulphated mucosubstance. Exceptionally abundant antimonate-precipitable cation was demonstrated between pancreatic acinar cells and at the base of the centro-acinar and other duct epithelial cells. Precipitates of nuclear heterochromatin appeared lighter whereas those of mitochondria and cytoplasm were coarser and more conspicuous in acinar that duct cells. Stimulation with synthetic secretin at a low level diminished antimonate reactivity of nuclei as well as the precipitation at the basement membrane of centro-acinar cells. At a higher dose, secretin selectively eliminated precipitation between and below centro-acinar and other duct cells while inducing increased antimonate-reactive cation in centro-acinar cells and the acinar lumens. Pancreozymin stimulation elimated antimonate-precipitable cations between acinar cells and, to a much lesser extent, those between duct cells and increased cytoplasmic precipitates on granular reticulum of acinar cells.Silver-precipitable anions were localized on the luminal surface of the apical plasma lemma and the outer surface of the latero-basal plasmalemma of centro-acinar cells but not on acinar cell surfaces. Silver precipitates also occurred on junctional complexes of acinar and duct epithelial cells and at tight junctions of acinar cells and on the inner face of the lateral plasmalemma of acinar cells.Dialysed iron staining demonstrated the most number of sites of acid mucosubstance on the luminal surface of the plasmalemma of acinar cells. Lateral and basal plasmalemmas of centro-acinar and more distal duct cells stained lightly with dialysed iron but those of acinar cells did not. Dialysed iron visualized acid mucosubstance in the lamina lucida of the basement membrane of duct but not of acinar cells. Dialysed iron staining of the plasma membranes succumbed to prior sialidase treatment whereas that of basement membrane resisted digestion. High iron diamine staining demonstrated sulphated mucosubstance in the lamina lucida of the duct basement membrane exclusively. The cytochemical results implicate centro-acinar cells as primarily responsible for contributing fluid and electrolytes to pancreatic secretion.     

T4 gene 32 protein trypsin-generated fragments. Fluorescence measurement of DNA-binding parameters.

The intrinsic fluorescence of the T4 helix-destabilizing protein specified by gene 32 (32P) is not altered by the proteolytic removal of either the 6200-dalton COOH-terminal "A" region (32P*-A) or both the A and the 2300-dalton NH2-terminal "B" region (32P*-(A + B)). The intrinsic fluorescence of 32P, 32P*-A, and 32P*-(A + B) is decreased 23% by the addition of d(pT)8 and 34% by the addition of poly(dT). Saturation binding curves of the percentage of change in protein fluorescence as a function of nucleotide concentration show that the intact 32P as well as the two proteolysis-generated fragments all have association constants of approximately 10(6) M-1 for d(pT)8. This demonstrates that the DNA binding site is not contained within either the A or B regions of 32P. Both 32P and 32P*-A bind cooperatively to poly(dT) as evidenced by a 400- to 1000-fold increase in association constant for poly(dT) compared to d(pT)8. Since within the limits of our measurements 32P and 32P*-A bind equally well to poly(dT) (Kassoc approximately 5 . 10(8) M-1), the enhanced helix-destabilizing properties previously reported for 32P*-A cannot be accounted for by a significant increase in binding affinity of 32P*-A for single-stranded DNA. The binding constant for the 32P*-(A + B):poly(dT) complex is only 3-fold higher than that for the 32P*-(A + B):d(pT)8 complex, which confirms our proposal that the B region is essential for cooperative 32P:32P protein interactions.     

Effect of days after calving on insulin-like growth factor-I, insulin-like growth factor binding proteins, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant follicles of postpartum beef cows

The effect of days after calving on IGF-I, IGFBP, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant ovarian follicles (DF) was evaluated in Angus × Hereford cows. Growth of DF (>9 mm) was monitored daily by ultrasonography and fluid from DF was collected in vivo at either 30 ± 2 d or 47 ± 2 d postpartum. Follicular fluid (FF) was also aspirated from DF of contemporary ovulatory cows at proestrus. Estrous behavior was monitored continuously using the HeatWatch system, and progesterone in plasma collected twice weekly was used to assess luteal activity. Anovulatory DF aspirated 30 and 47 d postpartum had similar concentrations of IGF-I, IGFBP, progesterone, estradiol and androstenedione in FF and IGF-I and IGFBP in plasma. The intervals from aspiration to estrus were similar for cows aspirated 30 and 47 d postpartum. Proestrous follicles had greater (P < 0.01) estradiol (435 ± 79 ng/mL) than DF at 30 d (107 ± 63 ng/mL) or 47 d (68 ± 53 ng/mL) after calving. Concentrations of androstenedione in FF were also greater (P < 0.01) in proestrous follicles than in DF aspirated at 30 or 47 d after calving. Concentrations of insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding proteins (IGFBP) in FF and plasma, and aromatase mRNA in granulosa cells were similar for anovulatory and proestrous cows. In conclusion, estradiol production by DF of postpartum anovulatory cows may be limited by inadequate production of androstenedione during the postpartum anovulatory interval and this may influence follicular maturation. Concentrations of IGF-I and IGFBP were similar in anovulatory and proestrous cows, an indication that alterations in the IGF-I system in the DF at 30–47 d after calving are not associated with delayed follicular development in postpartum beef cows.     

Differential sensitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay.

Both the V79 and CHO cell lines are routinely used in the in vitro MN screening assay for the detection of possible genotoxicants. The CHO cell line is the predominant cell line currently used in the genetic toxicology testing industry. However, some laboratories routinely utilize the V79 cell line since the in vitro MN screening assay was initially developed using V79 cells. Our laboratory has historically used the CHO cell line. Therefore, our laboratory was interested in comparing the two cell lines with regard to possible similarities or differences in MN induction sensitivity after exposure to cyclophosphamide (CPA) and mitomycin C (MMC), the two standard positive control chemicals routinely used in this assay. Three exposure conditions in the presence of CPA and MMC were examined in both cell lines. Replicate cultures of CHO cells in McCoy's 5A and V79 cells in both McCoy's 5A and E-MEM were established and treated with 5 microg CPA/ml (4h exposure with S9), 0.5 microg MMC (4h exposure without S9) and 0.5 microg MMC (24h exposure without S9). A total of 400 cytochalasin B-blocked binucleated cells and 200 consecutive cells were analyzed from each culture for MN and cell cycle kinetics, respectively. Analysis of the data demonstrated that CHO cells were up to approximately five-fold more sensitive to the induction of CPA- and MMC-induced MN than V79 cells. Both cell lines exhibited similar average generation times among identical exposure groups. Therefore, the difference in MN sensitivity cannot be attributed to possible differences in cell cycle kinetics and is possibly related to inherent cellular differences in the processing of and/or repair of CPA- and MMC-induced damage by V79 and CHO cells.     

Is southern Africa different? An investigation of the relationship between leaf physiognomy and climate in southern African mesic vegetation

Leaves from 24 South African vegetation sites, including 3 Fynbos sites, exhibiting high levels of endemism, were assessed by both LMA (Leaf Margin Analysis) and CLAMP (Climate Leaf Multivariate Program) to determine the effect of endemism on these palaeoclimate proxies. We examined whether existing calibrations using either locally recorded climate data or globally gridded climate data are appropriate for South Africa, or whether new calibrations specific to the region provide more accurate results. The results suggest that calibrations using gridded data yield slightly cooler estimates using LMA when the percentage of entire margined species is over 25. Overall, however, the differences are small and both gridded and local climate station calibration data can be used with equal accuracy. Xeric sites differ from mesic sites regarding the relationship between leaf margin proportion (LMP) and MAT, but with the exception of Fynbos sites, the differences are small when the percentage of entire margined species is < 65. Fynbos sites plot up to 8 °C cooler for a given leaf margin percentage (LMP) than do other sites. LMA was also sensitive to the number of taxa scored and in general should not be attempted on less than 15 leaf morphotypes per site. For CLAMP, the African non-Fynbos sites plotted within the parameters characterized by the existing PHYSG3BRC data set, indicating that they had a leaf physiognomic response to climate consistent with that of the rest of the world. However, the African non-Fynbos sites did fill a previously unoccupied void within that space, indicating the existence of a regional variance from the global pattern. This suggests that endemism per se does not prevent CLAMP from yielding reliable climate predictions. The inclusion of African non-Fynbos sites into the calibration improved the ability of CLAMP to predict Fynbos site climate, although this remained poor for non-coastal xeromorphic Fynbos vegetation. The addition of the African non-Fynbos vegetation did not degrade significantly the PHYSG3BRC calibration, particularly regarding key climatic variables such as enthalpy. Enthalpy appears to be a particularly robust variable with which to test climate model performance against CLAMP palaeoclimate predictions.     

Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II)

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.