Molecular Genetics of Cystinuria: Identification of Four New Mutations and Seven Polymorphisms, and Evidence for Genetic Heterogeneity

A cystinuria disease gene (rBAT) has been recently identified, and some mutations causing the disease have been described. The frequency of these mutations has been investigated in a large sample of 51 Italian and Spanish cystinuric patients. In addition, to identify new mutated alleles, genomic DNA has been analyzed by an accurate and sensitive method able to detect nucleotide changes. Because of the lack of information available on the genomic structure of rBAT gene, the study was carried out using the sequence data so far obtained by us. More than 70% of the entire coding sequence and 8 intron-exon boundaries have been analyzed. Four new mutations and seven intragenic polymorphisms have been detected. All mutations so far identified in rBAT belong only to cystinuria type I alleles, accounting for ~44% of all type I cystinuric chromosomes. Mutation M467T is the most common mutated allele in the Italian and Spanish populations. After analysis of 70% of the rBAT coding region, we have detected normal sequences in cystinuria type II and type III chromosomes. The presence of rBAT mutated alleles only in type I chromosomes of homozygous (type I/I) and heterozygous (type I/III) patients provides evidence for genetic heterogeneity where rBAT would be responsible only for type I cystinuria and suggests a complementation mechanism to explain the intermediate type I/type III phenotype.     

Decreased lipolysis in peanuts by a pyridazinone bioregulator

Peanut,Arachis hypogaea, plants were treated in the field with the bioregulator BAS 105 00W, 4-chloro-5-dimethylamino-2-phenylpyridazin-3-one, a substituted pyridazinone, at different times of development. The seeds were harvested, dried, hand-shelled, and analyzed for lipoxygenase activity and conjugated diene hydroperoxide content. Reduced lipoxygenase activity occurred when the bioregulator was applied to the plants at flowering and pegging. The conjugated diene hydroperoxide content decreased the most in peanuts when the bioregulator was applied at pegging. The apparent K_m for lipoxygenase of treated peanuts with linoleic acid as substrate was the same as that for untreated peanuts.     

Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H⁺ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - NCCD N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide - Hepes 2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate - TMPD N,N,N,N-tetramethylphenylenediamine     

Blockade of alpha-and beta -adrenergic receptors can prevent stimulation of liver ornithine decarboxylase activity by glucocorticoid or laparatomy.

Rat liver ornithine decarboxylase induction by dexamethasone or laparatomy, which is dramatically impaired by catecholamine depletion, is not affected by alpha-and beta -adrenergic blockers administered simultaneously 1 h prior to steroid injection or operation. However, if blockade is maintained for 24 h, an effect comparable to that of catecholamine depletion is obtained. Reciprocally, the response of the decarboxylase to catecholamines is severely compromised in adrenalectomized rats. Under the same conditions, induction of tyrosine aminotransferase by dexamethasone is not significantly affected by catecholamine availability, which altogether demonstrates that rat liver ornithine decarboxylase activity is specifically governed by the interaction between glucocorticoids and catecholamines.     

In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death

Two hours after a single intraperitoneal injection of dexamethasone (20 micrograms/Kg b.w.) into adult male rats, a typical ladder of DNA fragments was detectable upon separation on agarose gels of DNA from thymocytes. This became maximally evident at 4 hours. Accumulation of sulfated glycoprotein-2 (SGP-2) mRNA, whose rate of expression has been associated to the processes of programmed cell death, preceded the appearance of DNA degradation, starting to increase as early as 30 min after steroid injection, and maintained higher than controls until 8 hrs; a different time course was shown by changes in the levels of beta-actin mRNA. In the spleen, under the same conditions, the SGP-2 message also increased at 30 min, prior to DNA fragmentation, but decreased thereafter below the control value.     

An improved detection system applied to the study of serum lipoproteins after single-step density gradient ultracentrifugation

Starting from a single-spin ultracentrifugation procedure described previously (Foreman et al., J. Lipid Res. 18, 759, 1977), we have improved the system for detection of the fractions eluted from the gradient by monitoring them continuously at 280 nm. A graphic display readily permits assessment of the distribution of the lipoproteins and their quantification with the aid of a computer program. By the use of appropriate factors, one can convert absorbance readings into actual lipoprotein values which correspond well (±7% for low-density lipoproteins and ±5% for high-density lipoproteins) to those obtained by means of chemical analyses. The examples provided indicate the versatility of the method and its sensitivity (down to 0.1 ml of serum).