Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Fluctuation of body temperature and cuckoo brood parasitism

Body temperatures of 11 bird species, including cuckoos, were measured in an artificial meteorological room. Ratios of change in body temperature to that in air temperature were thereby obtained for each species. Cuckoos demonstrate a remarkably high value, indicating a particularly low ability to regulate body temperature. Viewed in this light, the cuckoo's parasitic behavior is very likely an adaptation to overcome a physiological disadvantage. This in turn might be expected to reinforce delay in evolution of temperature homeostasis.     

Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Release of Antifungal Phenolic Compounds from Cucumber Mosaic Virus-Infected and Noninfected Cowpea Protoplasts

Fungitoxic phenolic compounds were released from cucumber mosaic virus (CMV)-infected and noninfected cowpea protoplasts. These compounds were presumed by thin layer chromatography as similar compounds released into the leaf ambient fluids when CMV-infected cowpea leaves were incubated in water. Larger amounts of the compounds were released from CMV-infected cowpea protoplasts than from noninfected protoplasts.     

Chemical ionization and electron impact mass spectra of oligosaccharides derived from sphingoglycolipids

Chemical ionization (CI) mass spectra with isobutane and ammonia for the oligosaccharides obtained from sphingoglycolipids were compared with their electron impact (EI) mass spectra. The oligosaccahride moieties were liberated from the parent glycolipids and were further reduced with sodium borohydride. They were analyzed as their permethyl peracetyl and pertrimethylsilyl derivatives. In the CI spectra, peaks corresponding to QM+ and/or [M-59]+ were observed in all of the peracetylated oligosaccharides examined. In CI with ammonia as the reagent, H+ was transferred to nitrogen-containing saccharides to produce [MH]+ and NH4 was transferred to nitrogen-free saccharides to yield [M+NH4]+ as QM+. Non-reducing ends yielded very intense peaks in CI spectra. On the other hand, the reduced end, glucitol, produced rather prominent peaks in EI spectra. Fragment ions due to cleavage of glycosidic bonds were major ones under the CI conditions, and they could be used for elucidating the sugar sequence in the oligosaccharides. An additional characteristic feature in the CI spectra was that ions due to scission of hexosaminyl glycosidic linkages were observed with very high intensities.     

Experimental toxicoinfection in infant mice challenged with spores of Clostridium botulinum type E

Conventionally raised suckling mice were given 10(7) spores of a strain of Clostridium botulinum type E. Most but not all infant mice aged 8 through 19 days at the time of administration died after developing symptoms typical of botulism. However, none of the infant mice challenged with the spores at dose levels lower than 10(6) spores/mouse developed illness.     

REGIONAL BRAIN LEVELS OF γ-HYDROXYBUTYRATE IN HUNTINGTON''S DISEASE

A highly sensitive electron capture gas chromatographic method was developed for quantitation of γ-hydroxybutyrate (GHB) in tissue. This method involves an improved, extraction and purification procedure and a one-step derivatization of GHB to the methyl ester-O-heptafluorobutyrate. As low as 5 ng of GHB in tissue was accurately quantitated by this method. By means of this improved method, endogenous levels of GHB in several regions of brains obtained post-mortem from patients with Huntington's disease were determined, and compared with brain samples obtained post-mortem from non-neurological controls. The levels of GHB found in the caudate and substantia nigra obtained from Huntington's patients were significantly higher than the GHB levels found in similar regions of brain obtained from a non-neurological control group. The content of GABA in the same choreic and control brain samples was also determined. No significant correlation between changes in GHB and GABA levels was observed although there was a trend towards an inverse relationship. The high level of GHR in Huntington's disease may be related to the decrease in succinate:oxidoreductase (EC 1.3.99.1) activity reported by Stahl & Swanson (1974). In two subjects (one control and one Huntington patient) the zonal distribution of GHB in substantia nigra was also determined. The zona reticulata from choreic brain contained a substantially higher level of GHB, whereas the zona compacta contained an amount similar to the level found in control brain.     

Genetic structure of the IncN plasmid N3

N3, a plasmid of incompatibility group N (IncN) was mapped by cleavage with restriction endonucleases. The restriction fragments were cloned into vector plasmids. All of the genes unique to IncN plasmids such as specific replication machinery, a restriction-modification system, and repair functions were located on a large portion which had no cleavage sites for many of the site-specific six base-identifying restriction endonucleases tested.     

Interaction of basic oligo-L-amino acids with deoxyribonucleic acids. Oligo-L-arginines of various chain lengths and herring sperm DNA

The interaction of DNA and oligo-L-arginines having definite chain lengths of 1-17 residues was studied by precipitate formation and thermal denaturation of the complexes in order to obtain a better understanding of the roles of nuclear basic proteins. The results can be summarized as follows. 1. Those oligo-L-arginines, (Arg)n, in which n greater than or larger than 4 can bind with DNA irreversibly to form precipitates of the complexes. Among them, oligomers larger than (Arg)5 precipitate DNA completely in Arg/P input ratios below 1. The Arg/P ratios in the precipitates are between 0.6-0.8. 2. The thermal stability of the complexes depends on the method of complex formation, and complexes formed by the dialysis method are more stable than those formed by the mixing method. 3. The binding of (Arg)n to DNA was found to be reversible and in a equilibrium for n less than or equal to 6. In general, the longer the oligomer, the higher the stability of the complex at a definite Arg/P ratio. 4. For (Arg)7-10, three kinds of complexes with different stabilities are formed between DNA and oligopeptides. 5. For (Arg)14-17, only a restricted type of complexes can be formed between DNA and oligomers, as in the case with poly-L-arginine or protamines. 6. The interaction between basic nuclear proteins and DNA is discussed in the light of the basic region in protamine and histone molecules.