Spectral evidence for non-calcium interactions of intracellular Indo-1

Indo-1 is widely used to measure intracellular free calcium, [Ca2+]i, by comparing the fluorescence emission at 2 or more wavelengths with the emissions, which are assumed to be known, of Indo-1 when it is fully calcium-bound and when it is fully calcium-free. Accurate quantitation requires that these "reference" values be obtained on intracellular dye, and the full spectra of this study show that the reason is a significant spectral shift of the calcium-free peak, but not the calcium-bound. A mathematical analysis shows that the new peak must be a new state of the Indo-1 molecule, since it cannot be simply due to residual calcium in the cell. When intracellular "reference" spectra were used in the data analysis, [Ca2+]i could be calculated from whole spectra or from the ratio of observations at two wavelengths with good agreement. When extracellular "reference" spectra were used, the value calculated by the ratio method depended on the choice of wavelengths.     

Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.     

Effect of micelle diameter on tryptophan dynamics in an amphipathic helical peptide in phosphatidylcholine

The effect of dimyristoylphosphatidylcholine (DMPC) on the conformation and environment of the single tryptophan residue of a model amphipathic helical polypeptide has been investigated by fluorescence quenching with a water-soluble, neutral quencher (acrylamide) and multiple-frequency phase fluorometry. The peptide H-Ser-Ser-Ala-Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Ly s-Glu- Ala-Phe-Ser-Ser-Ser-OH [18As; Kanellis, P., Romans, A.Y., Johnson, B.J., Kercret, H., Chiovetti, R., Jr., Allen, T.M., & Segrest, S.P. (1980) J. Biol. Chem. 255, 11464] was synthesized by solid-phase techniques. Peptide was incubated at 26 degrees C with DMPC at various peptide:lipid weight ratios. The diameter of the resulting disk-shaped micelles increases with increasing lipid concentration from 12.0 +/- 0.4 nm at a 1:1 weight ratio of peptide to lipid to a maximum of 48.7 +/- 1.0 nm at a 1:13 ratio. At a weight ratio of 1:5, the average diameter is 22.7 +/- 0.6 nm. Decreasing the peptide:lipid ratio of the micelle resulted in a blue-shift in the fluorescence emission maximum (from 337 nm at 1:1 to 334 nm at 1:5), an increase in the fluorescence lifetime of the tryptophan measured by the phase shift method at 18 MHz (from 3.12 ns at 1:1 to 3.61 ns at 1:5), a decrease in the rate of fluorescence quenching by acrylamide (from 0.87 x 10(9) M-1 s-1 at 1:1 to 0.42 x 10(9) M-1 s-1 at 1:5), and an increase in the activation energy for quenching (from 6.7 kcal/mol at 1:1 to 12.7 kcal/mol at 1:5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of energy storage in Pseudoboeckella poppei (Crustacea,copepoda) from two contrasting lakes on Signy Island,Antarctica

The copepod Pseudoboeckella poppei (Daday) (Calanoida, Centropagidae) was sampled from Sombre and Heywood Lakes on Signy Island, Antarctica (60° S, 45° W) between January 1984 and March 1985. Sombre Lake is clear and oligotrophic with little phytoplankton and a bottom sediment low in organic content. By contrast Heywood Lake is turbid and mesotrophic; a substantial phytoplankton develops in summer and the bottom sediments are comparatively rich in organics. Both lakes freeze over for much of the year, forcing the copepods to adopt a benthic feeding strategy over winter. Adult Pseudoboeckella feed on phytoplankton when this is available, but also on detritus, diatoms and short algal filaments stirred up from the sediment. In Heywood Lake, male copepods show a smooth seasonal trend in lipid content with lipid being synthesised in early summer and utilised in late summer and winter. The summer increase in lipid content is associated with an increase in dry weight. Female lipid contents show evidence of two peaks of egg production. In Sombre Lake both male and female copepods increase in size during summer and show a wider range of lipid contents than in Heywood Lake; it is likely that this is due to the poorer winter feeding conditions which necessitate the synthesis of a much larger store of reserves during the summer. In contrast to marine calanoid copepods, lipid stores are exclusively triacylglycerol with no trace of wax ester.     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

Measuring readmission rates.

OBJECTIVE--To assess the feasibility of extracting data on readmissions and readmission rates from Körner data for use as health service indicators. DESIGN--Retrospective analysis of inpatient Körner data for January 1988 to April 1989. SETTING--Three districts in North East Thames region. MAIN OUTCOME MEASURES--Number of readmissions after index discharge for all acute specialties combined and by specialty (general medicine, general surgery, gynaecology, trauma and orthopaedics, and geriatrics); readmission rates at 28 days after index discharge; and rates standardised for age group and sex by specialty and by consultant. RESULTS--All specialties showed an early peak in number of admissions, which levelled off by 28 days. Readmission rates at 28 days were appreciably lower in surgical specialties than in medical specialties (for example, general surgery 4.1% v geriatric medicine 15.1%). They were related to age and sex of the patient. Rates standardised for these variables did not significantly differ by district. Likewise, significant differences in standardised rates were not obtained for consultants within a specialty in one district. CONCLUSIONS--Readmission rates may be measured with Körner data. The pattern of readmissions with time means that readmission rates should be measured at not more than 28 days after the index discharge; the rates require standardisation for age and sex. Annual comparisons of standardised rates may be made among districts for combinations of specialties; those among individual consultants or specialties are unlikely to be statistically valid.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

Blood glucose discrimination training in insulin-dependent diabetes mellitus (IDDM) patients

Self-management of insulin-dependent diabetes mellitus (IDDM) is dependent on a negative feedback loop of blood glucose (BG) fluctuations, which in turn directs treatment decisions to maintain normal BG. Although this feedback is typically accomplished by self-monitoring of blood glucose (SMBG), SMBG has limitations, and patients often rely on what their BG "feels" like. Two studies were performed to evaluate whether patients could learn to more accurately "feel"/discriminate their BG on the basis of internal cues or internal plus external BG cues. In Study I, BG Awareness Training significantly improved pre- to posttreatment BG estimation accuracy, relative to a control group. Study II replicated BG Awareness Training efficacy in improving BG estimation accuracy. Improvement in estimation accuracy was related only to initial accuracy; those who were initially less accurate improved the most. This improvement was represented in a 31% reduction in dangerous BG estimation errors and a 9% increase in accurate estimates. Resulting estimations were, however, still significantly less accurate than SMBG at the end of training.     

DNA sequence analyses of the ribosomal spacer regions in theTriticeae

Two regions of the ribosomal DNA (rDNA) were sequenced from a range of species from the tribeTriticeae. One region, the central spacer, was found to be more divergent in sequence than the other, the 18 S-spacer junction. Both regions contained sequences 20–30 bp long which were more highly conserved than the remainder of the region and their possible significance in rDNA expression is discussed. Phenetic relationships based on the sequence data were generally consistent with the relationships based on other criteria. Species possessing the S, E, J₁J₂, D, and B genomes clustered together, with the H genome species being the most distinct of those examined. The R, P, and V genome species occupy an intermediate position in the overall pattern of relationships. Some relationships differed in detail from those established by other parameters, for example the position of the N genome species, and explanations for discrepancies of this type are discussed.