Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques.

Pulsed-field gel electrophoresis techniques were used to study chromosome-sized DNA molecules of C. albicans. Chromosome-sized DNA of two strains of Candida albicans has been resolved into 8 bands by orthogonal-field-alternation gel electrophoresis (OFAGE). Six bands were observed in chromosomal preparations of C. albicans using field-inversion gel electrophoresis (FIGE). Differences in the electrophoretic mobilities of bands of the strains of C. albicans examined suggests that chromosome-length polymorphisms exist and make it difficult to correlate the banding patterns among strains. These correlations were facilitated, however, by assignment of C. albicans chromosomes by hybridization using a collection of cloned DNA probes specific for each of the 8 observed bands. Southern blotting showed that the 6 FIGE bands consisted of 4 singlets and 2 comigrating doublets, accounting for the 8 bands observed by OFAGE analysis. The agreement between OFAGE and FIGE analysis suggests that the C. albicans haploid genome contains a minimum of 8 chromosomes.     

Metabolism of exogenous galactosylceramide in the twitcher mouse brain

The in vivo metabolism of galactosylceramide (gal-cer) in normal mice and in twitcher mice, a model of human GLD, was examined following intracerebral administration of gal-cer containing [1-¹⁴C]stearic acid. In normal mice, gal-cer was hydrolyzed to ceramide within 6 hours and ceramide was hydrolyzed to sphingosine and fatty acid. Most of the released fatty acid was immediately incorporated into other lipids. About 75% of injected gal-cer was hydrolyzed 80 hours after the injection, while in the twitcher mouse, only 17% of gal-cer was hydrolyzed. These results show that degradation of gal-cer is impaired in the twitcher mouse brain, but contradict to the fact that there was no evidence of any accumulation of gal-cer in the brain. This discrepancy may be due to the different sorting routes of biosynthesized and exogenously-administered gal-cer in the mouse brain. Most of the biosynthesized gal-cer is incorporated into myelin, while the injected gal-cer is incorporated into lysosomes.     

Primary structure of rat insulin-like growth factor-I and its biological activities

Rat insulin-like growth factor-I (IGF-I), a serum polypeptide with growth promoting activity, was isolated from rat serum by a combination of acid/ethanol extraction, affinity chromatography, and a series of reversed phase high performance liquid chromatography, cation exchange, and reversed phase. All peptide fragments produced by chymotrypsin digestion of reduced and carboxymethylated rat IGF-I were amino acid sequenced and compared with the sequence of human IGF-I. Three out of 70 of the rat amino acid residues differed from those of human IGF-I as follows: Asp20----Pro, Ser35----Ile and Ala67----Thr. Purified rat IGF-I cross-reacted with polyclonal anti-human IGF-I antibody 75% as compared to human IGF-I, but it cross-reacted only 3% with monoclonal anti-human IGF-I antibody. Thus, it is possible to monitor the metabolic fate of human IGF-I, when injected into rats, without interference by endogenous rat IGF-I. Rat IGF-I showed 65% activity in the radioreceptor, 28.6% activity in the lipogenesis and 22.5% activity in the free fatty acid release inhibition assays as compared to human IGF-I on a protein quantity basis.     

Cloning and sequencing of the gene for Tetrahymena calcium-binding 25-kDa protein (TCBP-25)

With the intention of studying calcium-dependent ciliary reversal in Tetrahymena, we isolated a Tetrahymena calcium-binding protein of 10 kDa (TCBP-10) which was not calmodulin and reported its properties (Ohnishi, K., and Watanabe, Y. (1983) J. Biol. Chem. 258, 13978-13985). However, immunoblotting with an antiserum against TCBP-10 and sequencing of the cDNAs and partial genomic DNAs for this calcium-binding protein prove that this previously reported TCBP-10 is the degraded product of a 25-kDa calcium-binding protein. Thus, we correct the name of the protein from TCBP-10 to TCBP-25. From the analysis of the cDNA for TCBP-25, it is shown to be composed of 218 amino acid residues and its molecular weight is estimated to be 24,702. This protein is predicted to contain four EF-hand-type calcium binding domains and to be a member of the calmodulin family. Little sequence homology with other proteins was shown by a computer search, except in the EF-hand regions. The special feature of TCBP-25 is that the distance between calcium-binding domains II and III is extraordinarily long for a calmodulin family protein having four calcium-binding domains. The genomic DNA for TCBP-25 contains two introns situated at short distances before calcium-binding domains I and III, implying gene duplication in genealogy.     

Effects of a New Plant Growth Regulator Prohexadione Calcium (BX-112) on Shoot Elongation Caused by Exogenously Applied Gibberellins in Rice (Oryza sativa L.) Seedlings

The effects of a novel plant growth regulator (PGR) prohexadionecalcium (BX-112; calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate)on shoot elongation caused by exogenously applied GA₁, GA₃,GA₄₎ GA₁₉ and GA₂₀ were investigated in rice (Oryza sativa L.cv. Nihonbare and cv. Tan-ginbozu) seedling test. Dependingon the dose, BX-112 reduced shoot elongation in both cultivarscaused by GA₁₉ and GA₂₀, but not by GA₁. When a high dose ofBX-112 (e.g. 250 ng/plant and over) was applied with GA₁, orGA₄, shoot elongation was even promoted. This promotive effect,however, was not observed with GA₃. These results suggest thatBX-112 inhibits gibberellin (GA) biosynthesis in the rice plantat the 3ß- and 2ß-hydroxylation of GAs,namely steps of activation and inactivation, respectively. (Received September 6, 1989; Accepted November 27, 1989)     

Isolation and characterization of a calcium-binding protein derived from mRNA termed p9Ka, pEL-98, 18A2, or 42A by the newly synthesized vasorelaxant W-66 affinity chromatography.

W-66 (N-(2-aminoethyl)-N-[2-(4-chlorocinnamylamino) ethyl]-5-isoquinolinesulfonamide), a newly synthesized isoquinolinesulfonamide, was shown to have a potent vasodilatory action and calmodulin (CaM)-antagonizing action. Using the W-66 affinity chromatographic technique, we purified two Ca(2+)-binding proteins from the EGTA-soluble fraction of bovine aorta. One was CaM and the other was an acidic protein with a molecular mass of 11 kDa. It was tentatively named "calvasculin." Calvasculin was a dimeric protein. Equilibrium dialysis showed that 1 mol of calvasculin (dimer) bound to 1.98 +/- 0.30 mol of Ca2+ in the presence of 10(-3) M Ca2+. Calvasculin failed to activate Ca2+/CaM-dependent enzymes such as myosin light chain kinase, Ca2+/CaM-dependent phosphodiesterase, or Ca2+/CaM-dependent protein kinase II and to inhibit the CaM stimulation of these enzymes. The partial amino acid sequence of calvasculin revealed a high homology to the predicted protein derived from mRNA, named pEL-98, 18A2, 42A, or p9Ka. We also examined the physicochemical and biochemical properties of calvasculin. Using the antibody specific for calvasculin, we obtained evidence that calvasculin is present in abundance in bovine aorta but not in brain, lung, heart, or testis.