Increased urinary phosphate excretion in pseudohypoparathyroidism type II with long-term treatment with phosphodiesterase inhibitor.

A 58-year-old woman was diagnosed to have pseudohypoparathyroidism (PHP) type II because of the absence of an increase of urinary phosphate secretion, despite a marked increase in urinary cAMP excretion on the Ellsworth-Howard test. We treated the patient with a cyclic-nucleotide phosphodiesterase inhibitor, theophylline, resulting in increased urinary phosphate and cAMP excretions. Dibutyl cAMP administration induced the increase in the urinary phosphate excretion. In this case, the unresponsiveness of the urinary phosphate secretion to cAMP was recovered by a high dose of cAMP or long-term administration of a phosphodiesterase inhibitor. These data imply that cAMP responsiveness to renal tubular phosphate reabsorption should be more strictly elucidated in the patient with PHP type II.     

Distribution and Diversity of Symbiotic Thermophiles, Symbiobacterium thermophilum and Related Bacteria, in Natural Environments

Symbiobacterium thermophilum is a tryptophanase-positive thermophile which shows normal growth only in coculture with its supporting bacteria. Analysis of the 16S rRNA gene (rDNA) indicated that the bacterium belongs to a novel phylogenetic branch at the outermost position of the gram-positive bacterial group without clustering to any other known genus. Here we describe the distribution and diversity of S. thermophilum and related bacteria in the environment. Thermostable tryptophanase activity and amplification of the specific 16S rDNA fragment were effectively employed to detect the presence of Symbiobacterium. Enrichment with kanamycin raised detection sensitivity. Mixed cultures of thermophiles containing Symbiobacterium species were frequently obtained from compost, soil, animal feces, and contents in the intestinal tracts, as well as feeds. Phylogenetic analysis and denaturing gradient gel electrophoresis of the specific 16S rDNA amplicons revealed a diversity of this group of bacteria in the environment.     

Transition from Caspase-dependent to Caspase-independent Mechanisms at the Onset of Apoptotic Execution

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal “committed stage” cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the “execution phase” extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.     

Characterization of the auxin-regulated par gene from tobacco mesophyll protoplasts.

The auxin-regulated par gene from tobacco mesophyll protoplasts was characterized in detail to deduce its possible function. An homology search of the par gene in the NBRF databases revealed that the par gene has homology to the stringent starvation protein (ssp) gene of Escherichia coli, which is induced under starved conditions and binds in an equimolar ratio to a holoenzyme of RNA polymerase. Hence, it is supposed that the par gene product could play a similar role to that of ssp. Although sequence homology of the par gene to the Gmhsp 26-A gene from soybean was observed, both genes were shown to respond differently to plant hormones and stresses. Gmhsp 26-A is induced by heat shock, 2,4-dichlorophenoxyacetic acid (2,4-D), cytokinin and abscisic acid (ABA), whereas the par gene was induced only by auxins. Furthermore, cycloheximide treatment prevents 2,4-D-mediated accumulation of Gmhsp 26-A mRNA, but not that of par mRNA. Both par and Gmhsp 26-A respond to CdCl2, but splicing of the par pre-mRNA proceeded in a normal way, whereas splicing off the Gmhsp 26-A pre-mRNA was inhibited. Hence, the par and Gmhsp 26-A genes should have a common ancestor, but have evolved in different directions. Detailed time-course experiments confirmed that the par gene was induced immediately after the addition of auxin and expressed upon the initiation of meristematic activity in tobacco mesophyll protoplasts. As the par gene was induced by the sole treatment of cycloheximide, it was proposed that the par gene belongs to a category of 'superinduction' genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

NEURONAL CELL BODY ENRICHED AND GLIAL CELL ENRICHED FRACTIONS FROM YOUNG AND ADULT RAT BRAINS: PREPARATION AND MORPHOLOGICAL AND BIOCHEMICAL PROPERTIES

—A technique for separating neuronal and glial cell fractions in bulk from rat brain has been developed which does not use any digestive enzymes. Carbonic anhydrase, S-100 protein and 2′,3′-cyclic nucleotide 3′-phosphohydrolase were associated with the glial fraction. Glutamate and aspartate were the most abundant free amino acids; the neuronal fraction was richer in GABA and glycine than the glial fraction. Both d -glutamate and potassium ions were actively accumulated in each fraction during aerobic incubation. Na,K-ATPase was higher in the glial fraction; its activity, especially in the glial fraction, increased rapidly from the 10th to the 20th day after birth. Lactate dehydrogenase showed higher activity in the neuronal than in the glial fraction during early development; its isozyme pattern in each fraction was similar to that of whole brain. Acetylcholine esterase had its maximum activity around the 15th day in both fractions. Limitations of this bulk separation technique are discussed.     

An amber dna mutant of Escherichia coli K12 affecting DNA ligase

We have isolated an amber mutant (dnaL321) of Escherichia coli K12, which affects DNA ligase and which is lethal unless it is suppressed. DNA is degraded under the restrictive conditions. The mutation also affects the sensitivity of the cell to ultraviolet light irradiation, and the capacity to support the growth of phage λ that is deficient in general recombination. This pleiotropy is considered to be due to a single mutation, and is suppressed by supD^?_Isu+ and by supF^?su_III⁺). The mutation is cotransducible with dapE(2%), and with ptsI(85%), by phage Plvir.