Detection of HTLV-I in peripheral blood lymphocytes from patients with chronic HTLV-I-associated myelopathy/tropical spastic paraparesis and asymptomatic carriers by PCR-in situ hybridization

Less than 5% of people infected with human T-lymphotropic virus type I (HTLV-I) develop HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive neurologic disease. A number of factors have been implicated in the development of HAM/TSP including heterogeneity of viral sequences, host-genetic background, viral-specific cellular immune responses and viral load. This study examined the presence of HTLV-Itax DNA in peripheral blood lymphocytes (PBL) from 2 chronic HAM/TSP patients and 2 asymptomatic HTLV-I carriers by using PCR-in situ hybridization (PCR-ISH) for the in situ presence of proviral HTLV-Itax DNA. By this technique, rare PBL from these HTLV-I-infected individuals contained HTLV-I DNA. PCR-ISH did not detect any difference in the number of infected cells between HAM/TSP patients and asymptomatic carriers.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Insulin biosynthesis in the bullhead, Ictalurus nebulosus, and the effect of temperature

Insulin biosynthesis in the brown bullhead, Ictalurus nebulosus (Le Sueur), was studied by measuring the incorporation in vitro of [(3)H]leucine into proteins of the principal islet. The tissue was incubated for 6-15h in Krebs-Ringer bicarbonate buffer with [(3)H]leucine, supplemented with amino acids and glucose. Proteins, precipitated with trichloroacetic acid and extracted with acid ethanol, were separated by gel-filtration on Biogel P-30 in 3m-acetic acid. Three major components were found after incubation of the islets at 22 degrees C. On the basis of the results of sulphitolysis, biological activity and the demonstrated precursor-product relationship, components I and II were identified as proinsulin and insulin respectively. The third component was not identified. At 12 degrees C, [(3)H]leucine was incorporated only into proinsulin. No radioactivity was found in insulin or the unidentified component III at 12 degrees C as was found after incubation at 22 degrees C. When the temperature was lowered from 22 degrees to 12 degrees C after 3h of a 15h incubation, decreased conversion of proinsulin into insulin resulted at the lower temperature compared with the control tissue maintained at 22 degrees C. When the temperature was raised from 12 degrees to 22 degrees C at 3h of a 15h incubation, conversion of proinsulin into insulin occurred. No conversion occurred in the control tissue with the temperature maintained at 12 degrees C. No qualitative difference in the incorporation of [(3)H]leucine into proinsulin and its conversion into insulin at 12 degrees and 22 degrees C could be demonstrated between islet tissue from fish acclimated to less than 12 degrees C or to 22 degrees C. The results suggest that the enzyme(s) responsible for converting proinsulin into insulin in the bullhead may be temperature sensitive with low activity at 12 degrees C.     

Studies of the Cultural Physiology of the Lichen Alga Trebouxia

The pholosynthetic growth of three species of Trebouxia was so slow as to preclude the usual inorganic methods of algal cultivation. Heterotrophic growth was much stronger — as many as 0.4 doublings per day were obtained in Bold's solution with glucose and amino acid hydrolysates. Ammonium salts of inorganic acids could not be used in the culture medium because they caused excessive reductions in pH. Although the organisms showed variations in their growth response to amino acids, no specific requirement was found. T. erici produced significantly lower quantities of photosynthetic pigments in the dark. Of the nineteen carbohydrates tested, only glucose, mannitol, and galactosc stimulated growth of the algae. Preliminary studies showed that Trebouxia incorporated ¹⁴CO₂ more slowly than Chlorella pyrenoidosa. However, the carbon fixed by Trebouxia remained soluble in 80% ethanol longer than Chlorella. The carbon incorporated as CO₂ in short term experiments was greater than would be expected from the growth rates of the organisms studied.     

The Fibrinogen Molecule: Its Size, Shape, and Mode of Polymerization

Improved electron micrographs of the shadow-cast bovine fibrinogen molecule have been obtained establishing its general morphology and dimensions in the dry state. It consists of a linear array of 3 nodules held together by a very thin thread which is estimated to have a diameter of from 8 to 15 A, though it is not clearly resolved. The two end nodules are alike but the center one is slightly smaller. Measurements of shadow lengths indicate that nodule diameters are in the range 50 to 70 A. The length of the dried molecule is 475 ± 25 A. Adopting the molecular volume from previous physical chemical data and the general morphological features and length from electron microscopy, we calculate the diameters of the end nodules to be 65 A and the center one as 50 A. The model of the molecule so obtained is consistent with the electron microscopical observations and the data from physical chemistry. The intermediate polymers formed when fibrinogen is activated with thrombin were also examined and found to be end-to-end aggregates of altered fibrinogen molecules which shrink in length during the process. Intermediate polymer lengths are from 1000 to 5000 A. The nodular nature of fibrinogen, its shrinkage and end-to-end aggregation on polymerization permits us to deduce an explanation for the system of cross-bands previously observed in stained fibrin fibrils.     

Purification and Characterization of Two Endoxylanases from Clostridium acetobutylicum ATCC 824

Two endoxylanases produced by C. acetobutylicum ATCC 824 were purified to homogeneity by column chromatography. Xylanase A, which has a molecular weight of 65,000, hydrolyzed larchwood xylan randomly, yielding xylohexaose, xylopentaose, xylotetraose, xylotriose, and xylobiose as end products. Xylanase B, which has a molecular weight of 29,000, also hydrolyzed xylan randomly, giving xylotriose and xylobiose as end products. Xylanase A hydrolyzed carboxymethyl cellulose with a higher specific activity than xylan. It also exhibited high activity on acid-swollen cellulose. Xylanase B showed practically no activity against either cellulose or carboxymethyl cellulose but was able to hydrolyze lichenan with a specific activity similar to that for xylan. Both xylanases had no aryl-β-xylosidase activity. The smallest oligosaccharides degraded by xylanases A and B were xylohexaose and xylotetraose, respectively. The two xylanases demonstrated similar K_m and V_max values but had different pH optima and isoelectric points. Ouchterlony immunodiffusion tests showed that xylanases A and B lacked antigenic similarity.