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1.  Nucleoside salvage pathway for NAD biosynthesis in Salmonella typhimurium. 
Journal of Bacteriology  1982;152(3):1111-1116.
A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.
PMCID: PMC221616  PMID: 6216244
2.  The Computer-Aided Diagnosis of Different Histological Types of Primary Bronchogenic Carcinoma from Radiologic Signs 
On the basis of examining a number of resected specimens the gross types of the primary lung cancer were stated and the relationship between histologic types and gross types was studied. Futhermore the comparative study among X-ray films, resected specimens of tumor and pathologic examinations upon cases was made and some radiographic signs were extracted. After determining the gross types by radiologists using the sequential Bayes' model the computer-aided diagnosis was made. The accuracy of the computer diagnosis was significantly higher than that of film-reading by radiologistsb.
PMCID: PMC2580182
3.  Modulation of the Plasminogen Activator Activity of a Transformed Cell Line by Cell Density 
Molecular and Cellular Biology  1982;2(11):1410-1416.
The effects of variations in cell density on the expression of the plasminogen activator activity of a tumorigenic rat cell line were analyzed. At low cell densities, the plasminogen activator activity per cell was high and independent of cell density. As the cell density increased, the plasminogen activator activity per cell decreased until it eventually became inversely proportional to cell density. Inhibition of the plasminogen activator activity per cell by increases in cell density was not the result of the presence of a soluble inhibitor but seemed to require cell-to-cell contact. The Vmax per cell for the activation of plasminogen changed at high cell densities, but the Km did not change. This change in the Vmax per cell was in part the result of a change in the catalytic rate constant for the conversion of plasminogen to plasmin. This was inferred from studies on the kinetics of inhibition of plasminogen activator activity by diisopropyl fluorophosphate as a function of cell density. For cells growing at high densities, the rate of inhibition was constant, exhibiting a second-order rate constant of 2.6 × 10−2M−1 s−1. For cells growing at low densities, the plasminogen activator activity was inhibited at two different rates, one exhibiting a second-order rate constant of 2.6 × 10−2M−1 s−1 and the other exhibiting a second-order rate constant of 9.4 × 10−2M−1 s−1. We discuss the importance of cell density in assays of the plasminogen activator activity of cells, the use of this cell line to study the biochemical basis of the density dependence of plasminogen activator activity, and the density-dependent role of plasminogen activator activity in tumor formation and metastasis.
PMCID: PMC369945  PMID: 6819454
4.  Molecular Defect of Spectrin in Hereditary Pyropoikilocytosis 
Journal of Clinical Investigation  1982;70(5):1019-1030.
In hereditary pyropoikilocytosis (HPP) the erythrocyte membrane skeleton exhibits mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To detect structural changes in the functional domains of HPP spectrin we have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from two families that contain three HPP patients. Limited tryptic digestion of all three HPP patients revealed a similar and reproducible decrease in the staining intensity of an 80,000-, and 22,000-, and an 88,000-dalton polypeptide with a concomitant increase in a 74,000- and a 90,000-dalton polypeptide as compared with controls. Only changes in the 80,000-, and 74,000-, and 22,000-dalton polypeptides could be correlated to defective spectrin self-association and the amount of spectrin dimers in 0°C extracts of the HPP patients and their affected kindred. Similar results were obtained when the tryptic digests were analyzed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the affected 74,000- and 80,000-dalton polypeptides focusing into multiple spots ranging in isoelectric point from 5.3-5.4. When HPP spectrin dimers and tetramers were separated and subjected to trypsin digestion, changes in the 80,000-, 74,000-, and 22,000-dalton polypeptides were found predominantly in the spectrin dimer pool. Similar results were obtained for spectrin from two of the probands' mother, whom we have identified as an HPP carrier. We conclude that these HPP patients contain a population of normal, (principally tetrameric) and mutant (principally dimeric) spectrin. The latter is characterized by a defective spectrin dimer self-association due to conformational changes that affect the 80,000-dalton domain.
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PMCID: PMC370314  PMID: 7130392
5.  DNA clone of avian Fujinami sarcoma virus with temperature-sensitive transforming function in mammalian cells. 
Journal of Virology  1982;44(1):401-412.
We have molecularly cloned an integrated proviral DNA of Fujinami sarcoma virus (FSV) into a lambda phage vector and further subcloned it into plasmid pBR322. The source of provirus was a quail nonproducer cell clone transformed by FSV. The FSV strain used is temperature sensitive in the maintenance of transformation of avian cells. The recombinant plasmid was shown to contain an entire FSV genome by fingerprinting the hybrids formed with 32P-labeled FSV RNA. This analysis also revealed a previously undetected env-related sequence in FSV which represents the 3' end of the gp85 env gene. A physical map of cloned FSV DNA identifying sites of several restriction enzymes is described. Upon transfection, FSV DNA cloned in pBR322 transformed mouse NIH-3T3 cells, which proved to be temperature sensitive in maintaining transformation. Phosphorylation but not synthesis of p140, the only known gene product of FSV, was also temperature sensitive in these cells. The correlation between transformation and phosphorylation of p140 suggests that phosphorylation of p140 is necessary for transformation of mouse cells, as was shown previously for avian cells. These results provide direct genetic evidence that the mechanisms for maintaining transformation of mammalian and avian cells involve the same FSV gene product, p140. Homology was detected by hybridization between transformation-specific sequences of FSV DNA and certain restriction endonuclease-resistant fragments of cellular DNA of two avian species, chicken and quail. Under the same conditions homology was also detected with DNA of non-avian species, although apparently to a lower degree than with avian cells.
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PMCID: PMC256275  PMID: 6292501
6.  Commensalistic Interaction Between Lactobacillus acidophilus and Propionibacterium shermanii 
Propionibacterium shermanii and Lactobacillus acidophilus were grown in batch mixed culture in a 5-liter fermenter under controlled conditions of pH 5.8 and 35°C on a semisynthetic medium with glucose as an energy source. Cellular efficiencies and fermentation balances were developed for this pair and compared with P. shermanii grown in pure culture on glucose, lactate, and a mixture of these substrates and with L. acidophilus grown on glucose. P. shermanii had ATP yield coefficient values of 17 for each substrate alone but had an average value of 30 for substrate mixtures. Growth rates were similar for P. shermanii on glucose or lactate but higher cell yields were observed for glucose. P. shermanii used both lactate and glucose in mixed substrate until lactate was exhausted, and growth rates slowed thereafter. L. acidophilus had a similar ATP yield coefficient of 15 but produced lower cell yields than did P. shermanii on glucose. Mixed culture of both microorganisms on glucose resulted in much faster and nearly equal growth rates for both and no lactate accumulation in the medium. Acetic acid production rates per generation were lower in mixed culture, suggesting use by the growing culture. The cause of the synergistic effect was not determined but may be due to the rapid production and removal of lactate or CO2 enhancement in mixed culture.
PMCID: PMC242081  PMID: 16346098
7.  RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes. 
Nucleic Acids Research  1982;10(12):3591-3604.
The expression of the gene for variant surface glycoprotein (VSG) 118 in Trypanosoma brucei is activated by transposing a DNA segment containing the gene and 1-2 kb in front of it to an expression site elsewhere in the genome. By S1 nuclease protection and RNA blotting experiments we show here the presence of several minor transcripts in trypanosomes synthesizing VSG 118, one of which covers the entire transposed segment. Comparison of the sequence of the 5' terminal segment of VSG 118 messenger RNA (mRNA), determined by primed reverse transcription, and the corresponding region of the 118 VSG gene, shows that the 5' terminal 34 nucleotides of the mRNA are not encoded in the 118 VSG gene contiguous with the remainder of the mRNA. We conclude that synthesis of a VSG mRNA involves splicing of a much longer primary transcript, which may start outside the transposed segment.
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PMCID: PMC320737  PMID: 6287414
9.  Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA. 
Nucleic Acids Research  1982;10(8):2565-2576.
Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.
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PMCID: PMC320634  PMID: 6281736
10.  Occurrence of temperature-sensitive influenza A viruses in nature. 
Journal of Virology  1982;41(2):353-359.
The origin and characteristics of the first naturally occurring temperature-sensitive (ts) strain of influenza A virus identified in 1973, Xia-ts, are described. Natural ts strains were found to occur in the early egg passage material of all influenza A subtypes examined, but the proportion of ts virus varied from 8.3% for old H1N1 virus (1949 to 1957) to 82.4% for recent H3N2 virus (1979 to 1980). A number of strains were found to be composed of a mixture of ts and wild-type (ts+) particles. Six natural ts strains with different shutoff temperatures and one ts+ strain of the H1N1 subtype were tested in antibody-free volunteers. Strains with a shutoff temperature of 38 degrees C or lower caused very mild symptoms, whereas those with a shutoff temperature of 39 degrees C and the ts+ strain were much more reactogenic. By complementation tests against a set of prototype WSN ts mutants with a defined genetic lesion, the ts lesion of two H3N2 viruses (HK/8/68 and Xia-ts) was located on the NP gene and that of two H1N1 viruses (Tianjin/78/77 and Beijing/1/79) was located on the M protein gene. The present study demonstrates the widespread occurrence in nature of influenza viruses of different degrees of temperature sensitivity and presumably of different degrees of virulence.
PMCID: PMC256765  PMID: 7077746
11.  Bluetongue virus: comparative evaluation of enzyme-linked immunosorbent assay, immunodiffusion, and serum neutralization for detection of viral antibodies. 
Journal of Clinical Microbiology  1982;15(1):159-162.
Comparative studies on the detection of bovine serum immunoglobulin G antibodies to bluetongue virus with an enzyme-linked immunosorbent assay, an immunodiffusion method, and a serum neutralization assay demonstrated complete concordance between the enzyme-linked immunosorbent assay and the serum neutralization assay results. However, the immunodiffusion method failed to detect bluetongue virus antibody in a substantial number of sera found to possess bluetongue virus immunoglobulin G with the enzyme-linked immunosorbent assay.
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PMCID: PMC272042  PMID: 6313740

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