Abnormally spliced messenger RNA in erythroid cells from patients with β+ thalassemia and monkey cells expressing a cloned β+-thalassemic gene

The reduced β-globin synthesis characterizing the β⁺ thalassemia phenotype has been shown to be caused by anomalous processing within the small Intervening sequence (IVS1) of the β-globin mRNA precursor. The β-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3′ junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3′-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with β⁺ thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned β⁺-thalassemic gene preferentially generated the anomalously spliced RNA when expressed In monkey kidney cells. The anomalously spliced RNA constituted approximately 80%–90%, and normal β RNA approximately 10%–20%, of the total β mRNA. In contrast, the small amount of β mRNA present in reticulocytes from such patients consisted predominantly of normal β mRNA. These results suggest that the reduced amount of normally functioning β mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent Instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.     

The selection of cultured plant cell lines producing high levels of biotin

We have found that biotin is synthesized in many species of cultured plant cells, e.g. Lavandula vera Labiatae), Nicotiana tabacum (Solanaceae) and Glycine max Leguminosae). Cultured green L. vera cells grown under light contained the greatest amounts of free biotin of the cells studied although the specific amounts varied among the cell lines. Cell lines were selected after their free biotin contents had been analysed. Cells containing large amounts of free biotin were cultured repeatedly, analysed and reselected. Lines with high levels of free biotin were obtained from cells which survived on a medium containing pimelic acid and l-alanine or from gamma irradiated cells. One L. vera cell line obtained from irradiated cells contained seven times the amount of free biotin found in the original unselected cultured cells and four and a half times that found in the leaves.     

Abscisic acid from Pinus densiflora pollen

(+)-Abscisic acid was isolated as the methyl ester from Pinus densiflora pollen and identified spectroscopically.     

Novel p-coumaric acid esters from Pinus densiflora pollen

Solvent-extractable lipids in Pinus densiflora pollen were investigated. The cis- and trans-isomers of 1,16-dioxo-, 1-hydroxy-16-oxo- and 1, 16-dihydroxyhexadecan-7-yl p-coumarates were identified.     

13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.     

A trial designed to obtain a specific pathogen free Syrian hamster colony by administration of chemicals]

Conventional Syrian hamsters, contaminated with Giardia spp., Spironucleus muris, Trichomonas spp., Pasteurella pneumotropica and Pseudomonas aeruginosa were treated with chemicals in order to obtain specific pathogen free animals. Hamsters kept in the laminar flow rack were treated orally with metronidazole several times to obtain a flagellate-free colony. After all flagellates had been eradicated, one pair of animals were kept in an isolator and mating was allowed to occur. When their offspring reached the age of seven weeks, they were intramuscularly injected daily with netilmicin sulfate for 10 consecutive days. Following these treatments, all of the hamsters were free of Pasteurella and Pseudomonas. Further breeding of these animals was continued in isolators. To confirm the absence of selected pathogens, they were placed in a barrier room for further breeding as specific pathogen free animals.     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

Production of tropane alkaloids in cultured cells of Hyoscyamus niger

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS.Abbreviations NAA 1-Naphthaleneacetic acid - BA Benzyladenine - BSA N,O-Bis(trimethylsilyl) acetamide