Biomedical subjects
L S Lin
Publications and source records attributed to L S Lin.
Recombinant tumor necrosis factor causes activation of human granulocytes.
We have tested the hypothesis that tumor necrosis factor (TNF), by binding to and activating granulocytes, may contribute to the pathogenesis of gram-negative sepsis and the adult respiratory distress syndrome (ARDS). Buffy coat granulocytes incubated with as little as 0.5 ng/mL of recombinant TNF (rTNF) showed a dose-related increase in nitroblue tetrazolium dye reduction, in granulocyte polarization, in superoxide anion release, and in visually apparent aggregation. Purified lipopolysaccharide (1 microgram/mL) caused polymorphonuclear (PMN) aggregation and activation that was neutralized by polymyxin B. The release of superoxide was augmented by preincubation of the PMNs with gamma-interferon. The effect of TNF was neutralized by TNF-specific murine monoclonal antibodies but not by polymyxin B. Scatchard analysis of 125I-rTNF binding to granulocytes revealed about 1,200 receptors per cell with a Kd of 4.9 X 10(-10) mol/L. These results suggest that the release of TNF by mononuclear phagocytes contributes to granulocyte activation and aggregation during inflammation.
Purification of recombinant human interferon beta expressed in Escherichia coli.
Explore the source record for details and available documents.
Directly repeated, 20-bp sequence of plasmid R1162 DNA is required for replication, expression of incompatibility, and copy-number control.
DNA required in cis for the replication of the broad-host-range plasmid R1162 is located on two contiguous HpaII fragments of 210 and 370 bp. The latter of these contains three and one-half, perfectly conserved, 20-bp directly repeated sequences. The significance of these for plasmid replication, incompatibility, and copy-number control was examined by generating deletions into these repeats and testing the properties of the remaining DNA. We conclude from the results that the direct repeats are essential for expression of incompatibility and for the decrease in copy number observed when the directly repeated DNA is cloned into R1162. Little, if any, additional DNA is required from the ori region for these properties. Moreover, deletions of intermediate size result in an intermediate level of incompatibility, indicating the importance of the periodic structure of the direct repeats. The directly repeated DNA is also required for an active origin of replication, as are additional, nonrepeated sequences adjacent to this DNA. The properties of the direct repeats are discussed with respect to their possible role in the replication of R1162 DNA.
Mode of action of abscisic Acid in barley aleurone layers : induction of new proteins by abscisic Acid.
As part of a continuing effort to elucidate the mode of action of abscisic acid (ABA) in barley (Hordeum vulgare L. cv Himalaya) aleurone layers, we have investigated the induction of several polypeptides by ABA in this tissue. There were nine ABA-induced polypeptides as observed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and considerably more (at least 16 spots) on a two-dimensional gel. These proteins started to show enhanced synthesis 2 to 4 hours after ABA treatment, and their synthesis continued for at least 48 hours. In vitro translation using total RNA isolated from ABA-treated aleurone layers indicated that translatable mRNA levels of these proteins essentially paralleled the levels of in vivo synthesized proteins. The most abundant of the ABA-induced proteins was a 29 kilodalton polypeptide which was also synthesized in tissue incubated without ABA. In vivo synthesis of this protein declined as ABA concentration was decreased, with 1 nanomolar ABA approaching control level. Cell fractionation experiments located the 29 kilodalton major ABA-induced protein in 1,000g and 13,000g pellets; most other induced proteins were in the 80,000g supernatant. The 29 kilodalton protein appeared to be sensitive to degradation by sulfhydryl type proteases. As expected, the induction of these proteins by ABA was suppressed by gibberellic acid. Phaseic acid, the first stable metabolite of ABA, suppressed the gibberellic acid-enhanced alpha-amylase synthesis but was unable to induce the ABA-induced proteins. None of the ABA-induced proteins were secreted into the incubation medium. A 36 kilodalton ABA-induced protein showed cross-reactivity with antibody against a barley lectin specific for glucosamine, galactosamine, and mannosamine.
Alterations in physiological functions and in brain monoamine content in the sympathectomized rats.
In the present study, we conducted pre-ganglionic decentralization (or sympathetic trunk resection) of the superior cervical ganglia and observed alterations in several physiological functions and in the monoamine content of different brain regions. Over an ambient temperature range of 8-30 degrees C, these sympathectomized rats maintained their rectal temperatures within a normal limit displayed by the intact controls. These sympathectomized animals, although showing no change in the level of spontaneous pain threshold or motor activity, did display an increased sensitivity of analgesic responses to morphine administration or locomotor stimulant responses to amphetamine administration. Biochemical examination revealed that these sympathectomized animals had a higher level of norepinephrine, dopamine or 5-hydroxytryptamine in the hypothalamus, as well as a higher level of dopamine in the corpus striatum. However, in the brainstem, these sympathectomized animals had a unaltered monoamine level. The data indicate that, in a sympathectomized condition, changes in the monoamine content of different brain regions may be correlated with the above-mentioned alterations in somatosensory and motor neural functions.
Evaluation of the pharmacodynamics and pharmacokinetics of ritodrine when administered as a loading dose. On establishing a potentially useful drug administration regimen in cases of fetal distress.
Inhibition of labor during the intrapartum period has been suggested as a method of managing acute fetal distress. In such cases, rapid tocolysis is desirable but, in high doses, beta-adrenergic-receptor agonists, such as ritodrine, may cause severe maternal hypotension that could aggravate the existing fetal distress. We undertook the present study to establish a safe infusion protocol for ritodrine that achieves high plasma concentration rapidly. Twelve nonpregnant female volunteers received, on separate days, three infusions of ritodrine, that is, 1, 2, and 3 mg, during a 2-minute period. The peak plasma concentration measured by high-performance liquid chromatography with electrochemical detection averaged 37, 74, and 100 ng/ml after the 1, 2, and 3 mg doses, respectively. Ritodrine concentrations decreased rapidly and with the 3 mg dose the ritodrine concentration was only 14 ng/ml after 15 minutes. The elimination phase half-life of ritodrine averaged 6.11 hours. None of the doses significantly affected systolic blood pressure but ritodrine increased heart rate and the plasma glucose level and decreased diastolic blood pressure and the plasma potassium concentration. Even at the highest infusion rate, the maximal changes in cardiovascular and metabolic variables were short-lived and clinically modest; heart rate increased 29 bpm, diastolic blood pressure decreased 8 mm Hg, glucose level increased 26 mg/dl, and potassium concentration decreased 0.6 mEq/L. These data indicate that high plasma concentrations of ritodrine can be achieved rapidly without serious side effects.
Molecular cloning of the complementary DNA for human tumor necrosis factor.
Tumor necrosis factor (TNF) is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF was purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and showed cytotoxic and cytostatic activities against various human tumor cell lines. The amino acid sequence was determined for the amino terminal end of the purified protein, and oligodeoxyribonucleotide probes were synthesized on the basis of this sequence. Complementary DNA (cDNA) encoding human TNF was cloned from induced HL-60 messenger RNA and was confirmed by hybrid-selection assay, direct expression in COS-7 cells, and nucleotide sequence analysis. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. Expression of high levels of human TNF in Escherichia coli was accomplished under control of the bacteriophage lambda PL promoter and gene N ribosome binding site.
Broad host-range plasmid R1162: replication, incompatibility, and copy-number control.
Explore the source record for details and available documents.
Treatment of central diabetes insipidus with DDAVP.
Explore the source record for details and available documents.
Analysis of ritodrine in serum by high-performance liquid chromatography with electrochemical detection.
A sensitive and specific assay for ritodrine in serum was developed using high-performance liquid chromatography (HPLC) with electrochemical detection. Serum samples were alkalinized to pH 9.4 by the addition of a sodium carbonate buffer and extracted with ethyl acetate. The extracts were evaporated to dryness and the residues were reconstituted in the HPLC mobile phase and chromatographed on a octadecylsilane reverse-phase column. The detection of ritodrine was achieved by an electrochemical detector with a glassy carbon electrode. The sensitivity was 0.2 ng for on-column injection. The extraction efficiency was 80%.
Nucleotide sequence and functional properties of DNA encoding incompatibility in the broad host-range plasmid R1162.
A 370 base pair (bp) fragment of R1162 DNA encoding the incompatibility determinant has been cloned and sequenced. The DNA is located between 6.1 and 6.5 on the R1162 map, near the origin of replication. The sequence contains three perfectly conserved 20 bp direct repeats, with 11 bp of this sequence repeated a fourth time. The direct repeat unit shows some homology with that of another, unrelated broad host-range plasmid, RK2. The cloned DNA has two other properties: it lowers the copy number of R1162 when cloned into this plasmid, and it is required in cis for replication of R1162 satellite plasmids.
Site-specific mutagenesis of the human fibroblast interferon gene.
Human fibroblast interferon has three cysteine residues, located at amino acid positions 17, 31, and 141. Using the technique of site-specific mutagenesis with a synthetic oligonucleotide primer, we changed the codon for cysteine-17 to a codon for serine. The resulting interferon, IFN-beta Ser-17, retains the antiviral, natural killer cell activation, and antiproliferative activities of native fibroblast interferon. The purified IFN-beta Ser-17 protein has an antiviral specific activity of 2 X 10(8) units/mg, similar to that of purified native fibroblast interferon. In addition, the purified protein is stable to long-term storage at -70 degrees C.
Pharmacodynamics of ritodrine in pregnant women during preterm labor.
We evaluated the relationship of ritodrine concentration to several maternal variables and to fetal heart rate in 17 women who received the drug for inhibition of preterm labor. Ritodrine was measured by high-performance liquid chromatography with electrochemical detection. Ritodrine increased maternal and fetal heart rate and decreased serum potassium in a dose-related manner, but wide variability was noted between patients and within individual patients. Tachyphylaxis of the maternal heart rate response to continuing treatment with ritodrine was seen in at least seven women. Maternal blood pressure, serum glucose concentration, and frequency of uterine contractions were changed by ritodrine treatment, but the changes in these variables were not closely correlated to the concentration of ritodrine (r less than or equal to 0.30 in all cases). The maximal infusion rate and the concentration of ritodrine in maternal serum after 4 hours of treatment were significantly (p less than 0.001) correlated with the frequency of uterine contractions prior to treatment. Successful inhibition of labor was achieved with serum concentrations of 15 to 31 ng/ml in 10 of 17 women; in six of the other seven women, labor could not be inhibited in spite of serum concentrations of 90 to 146 ng/ml. Side effects, such as hypotension, vomiting, chest discomfort, and shortness of breath, were most commonly observed when the infusion rate and concentration of ritodrine were increasing.
Characteristics of spontaneously produced and of virus-induced human LuKII cell interferons.
Several human lymphoblastoid cell lines produced significant levels of interferon (IFN) activity in the absence of any IFN induces. We have partially purified spontaneous IFN (SpIFN) from LuKII cells and compared it with the IFN activity produced by the same cells following Sendai virus induction. Virus-induced LuKII IFN reached maximum titers of 1000-6000 reference units/ml at 12 to 15 houts post-induction and produced a heterogeneous electrophoretic profile with major (18,500 dalton) and minor (23,500 dalton) species. Spontaneous LuKII IFN was produced very slowly over several days and reached maximum titers of 100-1000 reference units/ml. Crude SpIFN (25-100 ref. units/ml) was purified to 1-6 X 10(5) ref. units/ml with a 53-80 percent overall recovery, and it consistently migrated as a homogeneous 20,000 dalton band upon SDS gel electrophoresis. Although spontaneously produced and virus-induced lymphoblastoid IFNs differed in their ability to be neutralized by anti-mouse IFN antiserum (18), both types of IFN were only 1 percent cross-reactive on heterologous mouse L cells. We conclude that IFN definitely can be produced by many human lymphoblastoid cell lines in the absence of inducers, and that LuKII SpIFN has different characteristics than the IFNs produced by the same cell line following viral induction. Since it is now known that there are many IFN-alpha genes (3, 10), it seems likely that these cell lines may provide a system for studying the selective expression of one or more of these genes.
Metabolism of 7,12-dimethylbenz[a]anthracene by Cunninghamella elegans.
The metabolites of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, in cultures of Cunninghamella elegans were isolated by high-pressure liquid chromatography and characterized by UV spectroscopy and mass spectrometry. The major metabolites were DMBA-trans-8,9-dihydrodiol and DMBA-trans-3,4-dihydrodiol. The 7-hydroxymethyl and the 12-hydroxymethyl derivatives of these dihydrodiol metabolites were also formed. The metabolic profile described in this report contrasts with those obtained in our earlier experiments in which the incubation of DMBA with Pseudomonas aeruginosa and Penicillium notatum produced no dihydrodiol metabolites but only methyl-hydroxylated metabolites.
[Rupture of arteriovenous malformations during pregnancy--report of 3 cases].
Explore the source record for details and available documents.
Size characteristics of human leucocyte interferon under reducing and non-reducing conditions.
Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was used to characterize human leucocyte interferon (HuIFN-alpha) under reducing and non-reducing conditions. Under non-reducing conditions HuIFN-alpha possesses two size forms, but under reducing conditions (r-HuIFN-alpha) only one is observed. The apparent molecular weight of this one form varies with the concentration of 2-mercaptoethanol used. When r-HuIFN-alpha is permitted to reoxidize the bimodal configuration of HuIFN-alpha is restored. The size heterogeneity of native HuIFN-alpha can be eliminated by mild treatment with NaIO4 [HuIFN-alpha/IO4; Stewart II, Lin, Wiranowska-Stewart & Cantell (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4200-4204]. The size of the HuIFN-alpha/IO4 increases after treatment with 2-mercaptoethanol (r-HuIFN-alpha/IO4) and the apparent molecular weight of this component also varies with the concentration of 2-mercaptoethanol used. In the case of r-HuIFN-alpha the single peak observed apparently originates from both the higher- and lower-molecular-weight components.