Some rRNA operons in E. coli have tRNA genes at their distal ends.

We have previously isolated seven rRNA operons on plasmids or lambda transducing phages and identified various tRNAs encoded by these operons. Each of the seven operons has one of two different spacer tRNA gene arrangements between the genes for 16S and 23S rRNA: either tRNAGlu2 or both tRNAIle1 and tRNAAla1B genes. In addition, various tRNA genes are located at or near the distal ends of rRNA operons. In particular, genes for tRNATrp and tRNAAsp1 are located at the distal end of rrnC at 83 min on the E. coli chromosome. Experiments with various hybrid plasmids, some of which lack the rRNA promoter, have now demonstrated that this promoter is necessary for expression of the distal tRNA genes. Rifampicin run-out experiments have also provided evidence that the tRNATrp gene is located farther from its promoter than the spacer tRNA gene or the 5S RNA gene. These results confirm the localization of genes for tRNATrp and tRNAAsp1 at the distal end of rrnC and strongly suggest that they are co-transcribed with the genes for 16S, tRNAGlu2, 23S and 5S RNA. Other such distal tRNAs have been identified, and it is suggested that they too are part of rRNA operons.     

Quantitative analysis of the hemoglobin oxygenation state of rat brain in vivo by picosecond time-resolved spectrophotometry

Through the use of a picosecond laser pulse of near-infrared light at 1,064 nm, the temporal profile of the transmitted light through the anesthetized rat head has been investigated. The light intensity at a certain time after the input pulse was exponentially attenuated by the hemoglobin concentration with hematocrit values from 1.5 to 50%, although the transmitted pulse broadened markedly due to scattering by the cerebral tissue. The optical pathlength, which is required for quantitation of the absolute absorbance change, was directly determined, by the time of flight measurement of the light pulses, as the product of the velocity of light in tissue and time. The mean concentration of hemoglobin in the brain could be determined quantitatively by the use of this pathlength. The oxygen saturation of venous blood determined by our time of flight measurement was very close to that in the internal jugular vein determined directly with a gas analyzer. Thus, the picosecond laser technique is useful for quantifying the blood oxygenation in tissues.     

Purification and properties of human placental aminopeptidase B.

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.     

Self-Nonself Recognition Activity Extracted from Self-Sterile Eggs of the Ascidian, Ciona intestinalis

Eggs of the hermaphrodite, self-sterile ascidian, Ciona intestinalis , were washed with acid seawater (pH 3.2), and the washing solution was then adjusted to pH 8.2. This solution was found to inhibit only the binding of non-autologous sperm to the vitelline coat (VC) of eggs, indicating that it contained self-nonself recognition activity. This activity was heat-stable and insensitive to trypsin, but was destroyed by V-8 protease and α-glucosidase. Both the hydrophobic and hydrophilic components of a lyophilized powder of the extract showed allo-recognizing activity. On TLC, the hydrophobic components gave a major spot of glucose (Glc) and a peptide spot(s) containing mainly glutamic acid and/or glutamine (Glx). The glucosyl conjugate was purified by HPLC and shown to block sperm-egg binding to various extents. Individual peptide subfractions had no inhibitory activity, but in combination they showed inhibitory activity. These findings suggest that the acid extract of Ciona eggs contains a Glc-enriched nonspecific inhibitor of sperm-egg binding, which could be the primary effector of self-incompatibility, and Glx-enriched modulators, which serve as acceptors of allo-sperm. The cooperative interactions of these components may be responsible for the diversity of allo-recognition in Ciona gametes.     

Clinical significance of adrenal computed tomography in Addison's disease.

Adrenal computed tomographic (CT) scanning was conducted in twelve patients with Addison's disease during the clinical course. In tuberculous Addison's disease (n = 8), three of four patients examined during the first two years after disease onset had bilaterally enlarged adrenals, while one of four had a unilaterally enlarged one. At least one adrenal gland was enlarged after onset in all six patients examined during the first four years. Thereafter, the adrenal glands may atrophy bilaterally, in contrast to adrenal glands in idiopathic Addison's disease, which atrophy bilaterally from disease onset (n = 2). Adrenal calcification was a less sensitive clue in tracing pathogenesis, i.e., adrenal calcification was observed in five of eight patients with tuberculous Addison's disease, but not in idiopathic patients. Thus, adrenal CT scanning could show the etiology of Addison's disease (infection or autoimmunity) and the phase of Addison's disease secondary to tuberculosis, which may be clinically important for initiating antituberculous treatment.     

Expression of the human angiotensinogen gene in transgenic mice and transfected cells.

We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.     

Identification of a novel amino acid, o-bromo-L-phenylalanine, in egg-associated peptides that activate spermatozoa

Eight sperm-activating peptides containing a novel amino acid were isolated from the egg jelly of the sea urchin Tripneustes gratilla. Accurate mass measurement of the peptide in FAB mass spectrometry showed that the mass of the novel amino acid residue was 224.978. On the basis of the isotopic ion distribution and the degree of unsaturation, the mass value indicated that the elemental composition of the amino acid residue was C9H8O1N1Br1, suggesting that the novel amino acid was bromophenylalanine. Proton NMR spectroscopy, amino acid analysis, and RP-HPLC with three synthetic isomers of bromophenylalanine demonstrated that o-bromophenylalanine was the novel amino acid. Derivatization of the amino acid with Marfey's reagent, (1-fluoro-2,4-dinitrophen-5-yl)-L-alanine amide (FDAA), further indicated that the amino acid was the L-isomer. In other sperm-activating peptides isolated from the egg jelly of the sea urchin, both m- and p-bromophenylalanines were discovered. The presence of m-bromophenylalanine has not been previously reported in natural products, while p-bromophenylalanine is found in theonellamide F, an antifungal bicyclic peptide from a marine sponge.     

A new DNA profiling system for cell line identification for use in cell banks in Japan

Summary Using the polymorphic DNA probes, ChdTC-15, ChdTC-114, pYNH24, and λTM-18, a DNA profiling system was developed that verified identities of individual cultured cell lines collected in the Japanese cell banks, JCRB, RCB, and IFO. These highly polymorphic DNA probes include both VNTR (Variable Number of Tandem Repeats) sequences and substantial lengths of unique regions. In the mixed probe system, several distinct bands from four to eight can be used for cell line identification. These bands were widely spread in a range of molecular sizes, and were stable and reproducible under stringent conditions of Southern blot hybridization. Because the DNA profile was specific for each individual human cell line, it is useful not only to authenticate many existing cultured cell lines but also to monitor their identity during propagation in a laboratory, and to confirm newly established lines as unique.     

Mechanisms of somatic embryogenesis in cell cultures: Physiology,biochemistry, and molecular biology

Summary One of the most characteristic cell functions in plants is totipotency. Somatic embryogenesis can be regarded as a model system for the investigation of mechanisms of totipotency, because a high frequency and synchronous embryogenic system from single somatic cells has been established in carrot suspension cultures. Four phases are recognized in this process, and several molecular markers, viz. polypeptides, mRNAs, antigens against monoclonal antibodies, can be detected during the expression of totipotency, but they disappear during its loss. Four organ-specific genes have been isolated from hypocotyls and roots by differential screening. They were expressed preferentially after the globular-heart stages of embryogenesis, and were strongly suppressed by auxin. A CEM 1 gene was isolated by differential screening of embryogenic cell clusters. This gene was expressed strongly and transiently during the proglobular and globular stages. The sequence of CEM 1 was found to encode a polypeptide showing high homology to the elongation factor isolated from eucaryotic cells. Thus good progress is being made in understanding the basic mechanisms of somatic embryogenesis. Presented in the Session-in-Depth Developmental Biology of Embryogenesis at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

Restriction fragment length polymorphisms detected in N-ras-related sequences of rats and their linkage analyses

Novel restriction fragment length polymorphisms (RFLPs) in inbred rats were revealed with the human N-ras gene as probe. Three fragments hybridizing to the probe were detected by Southern blot hybridization under highly stringent conditions, and one of the fragments showed variation in inbred rat strains. Furthermore, on hybridization under low-stringency conditions, an additional fragment hybridizing to the probe was observed, and this fragment also showed interstrain variation. These two variant fragments showed different distributions in 27 inbred rat strains and segregated in backcross progeny as codominant alleles of independent single autosomal loci. Therefore, the loci for these RFLPs were named Nras-1 and Nras-2, respectively. Analyses of linkages between the RFLPs and 11 other loci revealed that the Nras-2 locus was closely linked to the c locus (3.7 +/- 2.6%), which belongs to rat linkage group I.