Heptapeptide toxin production during the batch culture of two Microcystis species (Cyanobacteria)

Changes in the content of cyclic heptapeptide hepatotoxins called microcystins were investigated during batch culture of two Microcystis species using high performance liquid chromatography. After adsorption to ODS-silica gel cartridges and elution with methanol, the toxins were analyzed and quantified by HPLC. 35 μg per 100 mg dry cells of microcystin-RR, 34 μg of -YR and 43 μg of -LR were present at the beginning of the exponential growth phase of M. viridis. Microcystin-RR increased markedly towards the end of the exponential phase with the maximum content of 112 μg per 100 mg cells was measured at the late stage of the exponential phase. A remarkable increase of microcystin-YR from 130 μg per 100 mg cells to 1020 μg was observed during the exponential phase of a highly toxic strain of M. aeruginosa. However no clear differences were found in the pattern of change among the three toxins during the growth course.     

Heterogeneity of mutations in argininosuccinate synthetase causing human citrullinemia

Citrullinemia is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase. In order to characterize mutations, RNA was isolated from cultured fibroblasts from 13 unrelated patients with neonatal citrullinemia. Ten mutations were identified by sequencing of amplified cDNA. Seven single base missense mutations were identified: Gly14----Ser, Ser180----Asn, Arg157----His, Arg304----Trp, Gly324----Ser, Arg363----Trp, and Gly390----Arg. Six of these missense mutations involved conversion of a CpG dinucleotide in the sense strand to TpG or CpA, and six of the seven mutations alter a restriction enzyme site in the cDNA. Two mutations were observed in which the sequences encoded by a single exon (exon 7 or 13) were absent from the cDNA. One mutation is a G----C substitution in the last position of intron 15 resulting in splicing to a cryptic splice site within exon 16. There is extreme heterogeneity of mutations causing citrulinemia. This heterogeneity may prove typical for less common autosomal recessive human genetic diseases.     

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.     

Close linkage of MEN2A with RBP3 locus in Japanese kindreds

Summary The gene responsible for multiple endocrine neoplasia type 2A (MEN2A) has recently been assigned to the pericentromeric region of chromsome 10 in European Caucasian kindreds by linkage analysis using a DNA marker, interstitial retinol-binding protein 3 (RBP3). We have found tight linkage between the MEN2A and RBP3 loci in Japanese MEN2A kindreds. The maximum lod score is 5.19 at a recombination fraction of 0.00. This result suggests that mutation of a certain gene close to RBP3 is responsible for MEN2A irrespective of ethnic backgrounds.     

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.