A single cytotoxic T lymphocyte (CTL) is capable of performing the two most fundamental functions of an immune response, recognition and elimination of foreign antigens. It is now clear that in a CTL these two functions are linked via the antigen-specific, heterodimeric receptor. We review here some experimental approaches that justify this conclusion and provide the means for further examination of the mechanisms by which CTLs lyse their target cells. When antireceptor antibodies serving as antigen substitutes are attached to various cells, they trigger the lytic activity of particular CTLs, which results in lysis of the antibody-modified cell. In the process, a novel serine esterase, which is located within cytolytic granules of the CTL, is released. The presence of this enzyme and a complement-like protein, perforin, in granules of a CTL has led to the suggestion that CTLs and complement have similar cytolytic mechanisms. However, the resistance of some CTLs to lysis by other CTLs, but not to lysis by antibody-activated complement, suggests fundamental differences between cytolytic mechanisms of CTLs and complement.
Previous studies have shown that the chicken λ immunoglobulin light chain gene undergoes a single rearrangement that results in functional VJ joining of the unique variable (Vλ1) and joining (Jλ) coding regions. The immunologic repertoire of λ genes is created through extensive sequence diversification within the rearranged locus during B cell development in the bursa of Fabricius. This sequence diversification was detected only at the rearranged Vλ1 segment and not within the 5′ leader sequence, the Jλ segment, or the unrearranged Vλ1 segment. The selective diversification of the rearranged Vλ1 segment was associated with unique DNAase I-hypersensitive sites on the rearranged allele. While probes for Vλ1 sequences detect multiple homologous Vλ segments, probes for both the 5′ leader and Jλ segments fail to detect homologous sequences. Taken together, these results suggest that a highly selective process, possibly gene conversion, operates during B cell ontogeny to generate diversity within the λ gene.
Xanthine (X) and xanthine oxidase (XO) were injected intratracheally (IT) in hamsters at Day 0 (38 mg X, 100 micrograms XO) and Day 5 (38 mg X, 250 micrograms XO). Control hamsters received saline or X (38 mg) plus boiled XO (100, 250 micrograms). Cytoplasmic superoxide dismutase (SOD) activity increased from control of 286 to 337 and 335 units/lung at Days 12 and 19, respectively, but decreased to 228 units/lung at Day 33; mitochondrial SOD activity increased at Day 12 from control of 57 to 71 units/lung and then decreased at Days 26 and 33 to 42 and 33 units/lung, respectively. Glutathione peroxidase (GP) and glutathione reductase (GR) activities rose from their control values of 1161 and 1151 to 1561 and 2287 units/lung at Day 12, respectively; thereafter, GR activity decreased to 512 and 462 units/lung at Days 19 and 26, respectively. Glutathione transferase declined at Day 12 but increased at Day 26 after initial treatment. Glucose-6-phosphate dehydrogenase activity declined from control of 1071 to 693 units/lung at Day 2 and returned to control thereafter. Catalase activity remained unaffected. Hydroxyproline was increased from 903 micrograms/lung in control to 1080, 1301, 1195, and 1148 micrograms/lung at Days 12, 19, 26, and 33, respectively. Malonaldehyde increased from 40 nmole/lung in control to 70 and 113 nmole/lung at Days 12 and 33, respectively. The ratio of right ventricle to left ventricle and septum increased significantly from control of 0.277 to 0.318 at Day 33. Histopathology at Days 2 and 4 revealed peribronchiolar and arteriolar inflammation, and diffuse alveolitis. By Day 12 there were thickened alveolar septa and foci of fibrotic consolidation.
Spectrum analysis of the Doppler signals was performed 0.5 tube diameters downstream from an axisymmetric constriction with an area reduction of 80 percent in steady flow at a jet Reynolds number of 2840. Both pulsed and continuous wave (CW) Doppler spectra showed significant reverse flow components in the separated flow. The pulsed Doppler spectra exhibited sudden changes when the sample volume crossed the shear layer between the center jet and the separated flow. A power spectrum equation was theoretically derived from continuity of flow to define the Doppler shift frequency for the shear layer velocity. The CW Doppler spectrum showed a minimum spectrum density at a frequency which equalled the shear layer Doppler shift frequency derived from the equation. The pulsed spectra exhibited the sudden changes at the same frequency as well.
Glutathione S-transferase (GST) isoenzymes isolated from human tissues and rat liver are differentially inhibited by quercetin, alizarin, purpurogallin and ellagic acid. Rat liver GST isoenzymes are far more sensitive to these compounds as compared to the human GST isoenzymes. Among human GST, the anionic isoenzymes containing C type and A' type subunits are inhibited to a greater extent as compared to the cationic isoenzymes containing A and B type subunits. The anionic GST isoenzymes of human erythrocytes and placenta are differentially inhibited by these plant phenols indicating that the placental and erythrocyte isoenzymes may be distinct proteins.
1. Patch clamp studies on colonic tumor cell line T84 show the presence of chloride channels. 2. The channels are activated by forskolin, PGE2, or 8-Br-cAMP. 3. Single channel conductance was ca 40 pS at the reversal potential, increasing to 70 pS at +80 mV and decreasing to 25 pS at -80 mV. 4. Relative permeabilities were I greater than Br greater than Cl greater than F.
A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta.
Intermittent delivery of hyperbaric O2 protects animals from pulmonary and central nervous system toxicity: more total O2 time can be tolerated if interrupted by short periods of low O2. Little is known about the mechanisms or optimization of systematically varied intermittency. Survival time was recorded in groups of 16 awake guinea pigs (239 ± 23(SD) g) exposed to continuous O2 at 2.8 ATA or to one of six different schedules of O2 delivered with periodic air (PO2 = 0.588 ATA) interruptions. The survival curves had a lag time (11–14 hr of O2 time depending on the intermittency schedule) with a rapid loss of animals thereafter. Data were analyzed with risk models linking the probability of death to the accumulation of a putative toxic substance, X1. A model in which X1 accumulated in proportion to the PO2 and disappeared by first-order decay during periods of low O2 exposure was modified to include an effective rate constant for changes in X1: dX1dt=a·PO2+K1·(O2−OS·X1· First-order kinetics operated when PO2 was below the oxygen set point (Os), but the rate constant reversed sign to become a self-amplifying system when PO2 was above Os. This model achieved an excellent fit as judged by goodness-of-fit statistics while a simpler one did not. Our analysis suggests that the accumulation of toxicity does not correspond to a stable linear toxic process, but requires one in which a toxic process grows autocatalytically.
A mixture of cholesterol autoxidation products, prepared from an aged sample of cholesterol by recrystallization from methanol, inhibits calmodulin irreversibly in a Ca2+-dependent reaction. Inhibitory activity is lost after treatment with NaBH4, NaCNBH3, or NaI, from which we conclude that calmodulin inhibition is due to one or more cholesterol hydroperoxides. Partially purified cholesterol hydroperoxides, with or without cholesterol, were fed to young adult white rabbits. Cholesterol in the diet caused extensive atheroma formation in the aortas, but the addition of cholesterol hydroperoxides markedly reduced lesion formation. A cholesterol hydroperoxide preparation that was reduced by treatment with NaI was not effective in preventing atheroma formation. Cholesterol hydroperoxides did not lower cholesterol concentrations in blood plasma, liver, or heart.
After a change in cardiac output, the magnitude of potential blood volume redistribution was investigated in 10 dogs anesthetized with chloralose. All of the venous return was pumped into a reservoir, using servocontrolled pumps to maintain fixed superior and inferior vena cava pressures. The cardiac output was set at various levels by pumping from the reservoir into the right atrium. Changes in reservoir volume were assumed to reflect the changes in vascular blood volume. After measuring the control responses, cardiovascular reflexes were blocked with hexamethonium. Reducing the cardiac output, for example, from 110 to 80 ml/(min.kg) with reflexes intact, caused a 9.2-ml/kg transfer of blood from the dog to the reservoir. With reflexes blocked, the same change in cardiac output caused 6.8 ml/kg of the blood to be transferred. Under the control conditions, throughout the range of 50-140 ml/(min.kg), an increase or decrease of cardiac output of 1 ml/(min.kg) elicited a 0.304 +/- 0.086 (mean +/- SD) ml/kg change in dog blood volume; with reflexes blocked, the flow sensitivity was 0.239 +/- 0.062 ml/kg. Thus, only 21% of the total blood volume redistribution was attributable to active reflex responses. Deterioration of the preparation may have attenuated the magnitude of active reflex activity. Neither the systemic vascular compliance of 1.80 +/- 0.35 ml/mm Hg.kg nor the fraction of venous return from the superior vena cava of 26.5 +/- 4.6% was significantly changed by reflex blockade.(ABSTRACT TRUNCATED AT 250 WORDS)
Existing mechanical models of chronic obstructive lung disease have failed to explain a number of experimental findings of airway obstruction, e.g., the varying manners of frequency dependence of resistance (FDR). Departing from the parallelunit concept and attempting to account for the “check-valve” mechanism in the emphysematous lung, we proposed a single-compartment lung model with a nonlinear pressure-flow relationship:\(P + P* = L\ddot V + K_1 \dot V + K_3 (\dot V \pm \dot V*)^3 + V/c\), where\(P* = K_3 \dot V*, \dot V*\) is a constant. The plus and minus signs in the cubic term indicate the expiratory and inspiratory check valves, respectively. The choice of an asymmetric\(P - \dot V\) relation reflects several properties of emphysematous lungs such as airflow limitation and higher expiratory resistance. Implementation of the above equation using sine wave, white noise, and step inputs resulted in various forms of FDR at frequencies between 0 and 40 Hz depending on the type of input used. Resistance was most sensitive to changes in input pressure amplitude. The model's results suggest that the\(P - \dot V\) nonlinearity can have a significant influence on the impedance construct in obstructed lung disease.
Opioid peptides and their precursors of the proenkephalin family are found in the chromaffin cells of the rat adrenal medulla in low quantities. However, if the gland is denervated, there is a 10 to 20-fold increase in enkephalin-containing (EC) peptides consisting mostly of the precursor proenkephalin. The denervation-induced rise in medullary EC peptides is blocked by hypophysectomy, and partially reinstated by corticosterone, dexamethasone or ACTH treatment. In the intact rat, intermediate doses of corticosterone or dexamethasone reduce the denervation-induced increase in EC peptides, while a high dose of dexamethasone restores this response. These results indicate that glucocorticoids exert a permissive effect in vivo on the denervation-induced stimulation of EC peptide biosynthesis.
1.1. The immunological relatedness of several annelid extracellular hemoglobins and chlorocruorins was investigated using ELISAs and Western blotting to determine the binding of purine polyclonal and monoclonal antibodies to Lumbricus terrestris hemoglobin with the hemoglobins of Tubifex tubifex, Tylorrhynchus heterochaetus, Arenicola marina and Macrobdella decora and the chlorocruoins of Myxicola infundibulum and Eudistylia vancouverii.2.2. Polyclonal antibodies to Lumbricus terrestris hemoglobin bound to all the other hemoglobins and chlorocruorins. However, the titers were in all cases one to several orders of magnitude smaller than with Lumbricus terrestris hemoglobin.3.3. Polyclonal antibodies to Eudistylia vancouverii chlorocruorin bound to the hemoglobins of Lumbricus terrestris, Tubifex tubifex, Arenicola marina, Tylorrhynchus heterochaetus and Macrobdella decora.4.4. Of the nine monoclonal antibodies to Lumbricus terrestris hemoglobin isolated, two (No. 24 and No. 26) bound to the other hemoglobins and to Myxicola chlorocruorin, but the binding was again weaker than with Lumbricus hemoglobin. Antibody No. 26 also bound to Eudistylia chlorocruorin. Although antibody No. 24 appears to recognize a conformation-dependent epitope, antibody No. 26 recognizes a common epitope in each of the four subunits M, DI, D2, and T of unreduced Lumbricus hemoglobin.5.4. An additional two monoclonal antibodies to Lumbricus hemoglobin (No. 21 and No. 25) bound also only to Tubifex hemoglobin. Antibody No. 21 recognizes subunits D1 and M of Lumbricus hemoglobin and No. 25 appears to recognize a conformation-dependent epitope.
Glycogen phosphorylase, a dimer of identical subunits, is activated by phosphorylase kinase-catalyzed phosphorylation of one serine residue in each subunit. In this paper, the effect of the phosphorylation of one subunit on the phosphorylation of the other subunit was examined. The three forms of phosphorylase, phosphorylase b (nonphosphorylated), phosphorylase ab (one subunit phosphorylated), and phosphorylase a (both subunits phosphorylated), were separated by anion-exchange high-performance liquid chromatography (HPLC). Purified phosphorylase ab was found to be stable under the conditions of the phosphorylase kinase assay. Initial rate kinetics showed that phosphorylase kinase had a lower KM for phosphorylase ab (3.9 +/- 0.24 microM) than for phosphorylase b (14.9 +/- 2.6 microM). Using the HPLC separation as a simultaneous assay for the three forms of phosphorylase during the phosphorylase kinase reaction, it was found that the pseudo-first-order rate constant for the second phosphorylation step (k2) was 3.7 times greater than that for the first step (k1). The activator AMP reduced the ratio k2/k1 from 3.7 without AMP to 1.4. When the monomeric gamma delta complex of phosphorylase kinase subunits was used as the enzyme, the ratio k2/k1 was 2.1, compared to 3.7 with the multimeric holophosphorylase kinase. One explanation for these data is that phosphorylation of one subunit of phosphorylase b causes conformational changes that make the other subunit a better substrate for the kinase. In this context, the effect of AMP is to reduce the conformational differences between phosphorylases b and ab, and the gamma delta complex is less sensitive to the conformational differences between the two forms of phosphorylase.
An enzyme-linked immunosorbent assay (ELISA) was used to measured IgG antiboody titers againt a synthetic peptide whose sequence was derived from the glycine-alanine repeating region of Epstein-Barr virus nuclear associated antigen 1 (EBNA-1). Antibody titers were determined in sera from 15 normal subjects, sera from 21 normal male siblings of X-linked lymphoproliferative syndrome (XLP) patients, from 20 XLP patients comprising a total of 42 samples, and ten samples before and ten samples after gamma-globulin therapy in ten patients with XLP. Data analysis demonstrated that while there are differences between the ELISA and ACIF, they appear to measure a similar response as demonstrated by their correlation coefficient (0.77) and the GMT to EBNA observed by both methods. No cross-reactivity of cytomegalovirus antibodies to the EBNA-1 peptide was observed by immunobv using adsorption against AD-169 infected MRC-5 cells.. However, non-specific binding was observed if samples were not pre-incubated in a 10% goat serum PBS-Tween 20 solution. This pre-treatment removed the non-specific binding that falsely elevated GMT in approximately 15% of both normal and XLP samples in ELISA. The ELISA system appears to be a sensitive, reproducible and objective test that may be useful for assessing the antibody responses of patients to the EBNA-1 protein.
Gold compounds are used clinically in rheumatoid arthritis therapy. Acute renal toxicity is observed in some patients receiving chrysotherapy. The present study addresses morphofunctional and biochemical changes in rat kidneys during the first 8 days following a single ip injection of gold sodium thiomalate (Au TM), one of the gold compounds presently in clinical use. Compared to controls, AuTM pretreatment resulted in increased urine output and elevated serum creatinine and urea nitrogen concentrations. Also, by Day 8, treated rats had decreased body weights and increased kidney weights. Postmortem examination on Day 1 showed pale and mottled kidneys and diffusely pale inner cortex. Microscopically, there was severe coagulative necrosis of the proximal tubular epithelium. Epithelial regeneration was prominent by Day 4 and was nearly complete by Day 8. The regenerating epithelium was hyperplastic with basophilic cytoplasm and pleomorphic nuclei. Alterations in renal heme biosynthesis and drug metabolism paralleled the morphologic changes. The activity of δ-aminolevulinic acid dehydratase and benzo[a]pyrene hydroxylase were inhibited on Days 1, 2, and 4 following AuTM administration. Decreases in monooxygenase activity were accompanied by decreases in renal cytochrome P-450 levels. In contrast, renal microsomal heme oxygenase activity was elevated 9.5-fold on Day 1 and 2.5-fold on Day 2. By Day 8, all renal enzymatic activities assayed for were similar to those obtained with untreated rats.
A method has been developed to determine the association constant for a heterogeneous association of the type A + B ⇌ AB. This method requires knowledge of the two initial concentrations and of the resulting weight-average molecular weight for each data point. Computer simulations using Gaussian-distributed error on the measured parameters show that the researcher can readily determine whether the particular concentration range chosen is appropriate for the strength of binding and therefore how reliable the calculated constant might be. It is also shown that errors in measuring molecular weight have, in general, a more profound effect than do errors in concentration.
1.1. Calf thymus histone H3 bound irreversibly to the isolated rat liver nuclei.2.2. The rate and extent of binding was a function of the incubation period and the concentration of both H3 and nuclei, but independent of the temperature.3.3. The binding was saturable and was inhibited by simultaneous presence of various histones.4.4. Approximately 94% of the bound H3 was associated with nuclear membrane fraction.
