Method for determination of nucleotide sequence homology between viral genomes by DNA reassociation kinetics.

A model and appropriate equations were derived for the quantitative estimation of nucleotide sequence homology between two partially related viral genomes by measurement of the initial rate of reassociation of one labeled DNA in the presence of a second unlabeled DNA. The validity and usefulness of this procedure were demonstrated by the analysis of the reassociation kinetics of labeled adenovirus 7 DNA in the presence of unlabeled adenovirus 2 DNA. Based on DNA reassociation, the extent of homology between adenovirus 2 and 7 genomes was found to be 10 to 12%. The duplex formed between adenovirus 2 and 7 DNA had the appropriate thermal stability for a well-matched DNA-DNA hybrid.     

Ca2+ influx mediated through the GPIIb/IIIa complex during platelet activation

When aequorin-loaded platelets were stimulated with thrombin, the luminescence signal of aequorin showed two peaks. From experiments with 1 mM external Ca2+ or EGTA, both one-half of the first peak and the entire second peak reflected the influx of Ca2+ from the external medium, and the remaining half of the first peak reflected the mobilization of Ca2+ from its storage site. A monoclonal antibody (TM83) that recognizes the glycoprotein IIb/IIIa (GPIIb/IIIa) complex which has binding sites for fibrinogen and the synthetic peptide GRGDSP are known to inhibit fibrinogen binding and platelet aggregation. Both eliminated the second peak of intracellular free calcium ([Ca2+]i). Similar effects were observed during activation by collagen, but not during PMA activation. It was concluded that the GPIIb/IIIa complex was intimately related to a part of the Ca2+ influx during the activation of platelets.     

Purification of caldesmon and myosin light chain (MLC) kinase from arterial smooth muscle: comparisons with gizzard caldesmon and MLC kinase

We have developed a simple and conventional purification method for caldesmon and MLC kinase from bovine arterial smooth muscle, and compared the arterial and gizzard proteins. Arterial caldesmon shares the alternative binding to calmodulin or F-actin in a Ca2+-dependent manner and the antigenic determinants with the gizzard protein. Both caldesmons have the same association constant with F-actin (1.3-1.7 X 10(7) M-1) and the same maximum binding (1 caldesmon per 12-14 actins). However, the molecular weight of arterial caldesmon (dimer of a 148 kDa polypeptides) was slightly different from that of gizzard caldesmon (heterodimer of 150/147 kDa polypeptides). The molecular weight of arterial MLC kinase (160 kDa) was much larger than that of the gizzard enzyme (135 kDa). The enzyme activities of both MLC kinases were comparable (Km = 9.5 microM, Vmax = 12.5 mumol/min X mg). The association constant of the arterial enzyme to F-actin (5.1 X 10(6) M-1) was much larger than that of the gizzard enzyme (9.0 X 10(5) M-1) but the maximum binding was the same (1 enzyme per 12-13 actins). Immunocytochemical examinations showed that caldesmon and MLC kinase in cultured arterial cells have a restricted localization along the stress fibers, suggesting functional linkages between both proteins and actin filaments in vivo.     

Effect of pH on Absorption Spectra of Pea 114 and 121 Kilodalton Phytochromes during and after Red-Light Irradiation

Absorption spectra of pea 114 and 121 kDa phytochromes weremeasured at pH 6.8, 7.8 and 8.8 using a custom-made transientmultichannel spectrum analyzer. The absorption spectra of 114kDa phytochrome as P_R and P_FR were least affected by mediumpH. The absorption spectra at photostationary state under redlight, however, were different under the three different pHconditions, and were different from those obtained 55 s afterred-light irradiation, owing to rapid pH-dependent absorbanceincrease in both red and far-red regions in the dark. In contrast,the absorption spectra of 121 kDa phytochrome were significantlyless affected by medium pH. The absorption spectra measuredat the photostationary state showed a lower P_FR peak at higherpH. The absorption spectra obtained 55 s after the irradiationwere similar under the three pH conditions since the rapid absorbanceincrease in the far-red region in the dark was small. Possibleaccumulation of 114 kDa phytochrome population(s) with low absorbanceat red-light-induced photostationary state at pH 8.8, and theprotective role of the 7 kDa polypeptide at the amino terminusagainst the pH effect in 121 kDa phytochrome are discussed. (Received February 1, 1986; Accepted April 1, 1986)     

Adsorption of aspartase onto hydrophobic cloth for conversion of ammonium fumarate

Summary A simple aspartase assay was developed. Aspartase fromEscherichia coli Crooks strain was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme did not desorb in 1 M ammonium fumarate. The adsorbed enzyme exhibited the same pH vs. activity curve as free enzyme and had a half life of approx. 40 weeks. A column packed with the adsorbed aspartase showed 100% conversion of 1 M ammonium fumarate at a space velocity of approx. 2.     

Gene Flow in Natural Populations of DROSOPHILA MELANOGASTER with Special Reference to Lethal Allelism Rates and Protein Variation

A simultaneous survey of 14 protein loci, together with frequencies and within- and between-population allelism rates of lethal chromosomes, was carried out in five (four Japanese and one Korean) natural populations and one cage population of Drosophila melanogaster. It was found that lethal allelism rates decrease rapidly as geographic distance between two populations increases, while variation at protein loci shows a remarkable similarity over all populations examined. These findings suggest that there are very high levels of gene flow in these natural populations and that selection at protein loci which can maintain substantial geographic variation, if present, is overshadowed by gene flow. There is no indication that invasion of D. melanogaster to the Far East occurred so recently that the frequencies of lethal chromosomes are still in nonequilibrium.     

Proposal of standardized methods and reference for assaying recombinant human tumor necrosis factor

Two assay methods for recombinant human tumor necrosis factor (rH-TNF) were developed, one a biological L-cell assay and the other an enzyme-linked immunosorbent assay. The accuracy and reproducibility of each and the correlation between the two were studied. As a result of this investigation, the two assay methods were found appropriate for standardization of rH-TNF. A freeze-dried reference was prepared, and examination of its potency and stability showed it to be suitable for use as a reference standard for rH-TNF assays.     

Separation and Characterization of Prolamellar Bodies and Prothylakoids from Squash Etioplasts

Intact etioplasts of squash cotyledons, which had been preparedby Percoll density gradient centrifugation, were ruptured hypotonicallyin the presence of deoxyribonuclease I then fractionated intoprolamellar bodies and prothylakoids by differential and Percolldensity gradient centrifugations. This procedure provided ahighly purified prolamellar body fraction that was composedmainly of a 36,000-dalton protein. This protein was identifiedas NADPH:protochIorophyllideoxidoreductase [Ikeuchi and Murakami(1982) Plant & Cell Physiol. 23: 1089]. The fraction alsohad a high content of protochlorophyllide that absorbed at 648nm and its NADPH:protochlorophyllide oxidoreductase had highactivity. When the fraction was illuminated, a chlorophyllidethat absorbed at 684–685 nm formed. In contrast, the prothylakoid fraction, which showed high activityfor the Ca²⁺-dependent ATPase of coupling factor 1, containedonly a small amount of the 36,000-dalton protein and showedvery low NADPH:protochlorophyllide oxidoreductase activity.The protochlorophyllide content of this fraction also was low,and the ratio of protochlorophyll to protochlorophyll(ide) high.The absorption peak in the prothylakoids was at 633–635nm, and after a brief illumination a chlorophyllide that absorbedat 672–673 nm formed. These results indicate that thephotoactive protochlorophyllide-NADPHreductase complex in etioplastsis concentrated in the prolamellar body and that the physicalstate of protochlorophyll(ide) in the prolamellar body differsfrom that of the prothylakoid. (Received April 28, 1982; Accepted November 15, 1982)     

A radioimmunoassay for a highly active luteinizing hormone-releasing hormone analogue and relation between the serum level of the analogue and that of gonadotropin

Antisera to an LH-RH analogue, des-Gly10-[D-Leu6]-LH-RH-ethylamide (TAP-144) were produced in 10 rabbits. By using these antisera, a specific and sensitive radioimmunoassay for TAP-144 was established. Sensitivity of the radioimmunoassay ranged from 5 to 100 per assay tube with these antisera. Lh, FSH, TRH, LH-RH, and the 1-6 fragment of TAP-144 did not practically cross-react with LH-RH analogues, though the degree of the cross-reactivity differed among individual sera. The average cross-reactivity of the 10 antisera showed low specificities to TAP-144 analogues which are altered at positions 2, 3, 4, 5, and 6, but showed high specificities to those altered at positions 8 and 9. The antisera also showed low cross-reactivities to LH-RH analogues replaced at position 6 by D-amino acids and those altered at position 10 by alkylamines, but they showed fairly high cross-reactivities to analogues which are altered simultaneously at both positions 6 and 10. When TAP-144 was administered intraperitoneally to rats on the diestrous day, serum concentrations of TAP-144 increased dose-dependently but maximal serum concentrations of both LH and FSH were attained in response to higher doses of TAP-144. The peak LH and FSH concentrations appeared 70 to 110 min after the peak TAP-144 concentration had been reached. Similar delays in reaching the peak LH and FSH levels were also observed when TAP-144 was administered intravenously, subcutaneously and intramuscularly. When TAP-144 was administered intravaginally, a low but constant serum level of TAP-144 was maintained from 5 to 300 min after the administration, but serum LH and FSH levels declined to a low level from 180 min after the TAP-144 administration.