Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Regulation of S-adenosylmethionine decarboxylase activity in rat liver and prostate

Two methods were used for the quantitation of S-adenosylmethionine decarboxylase protein. The first involved titrating the active site of the enzyme by reduction of the Schiff base between 3H-decarboxylated S-adenosylmethionine and the pyruvate prosthetic group with sodium cyanoborohydride. The second method was radioimmunoassay with rabbit antiserum which was used to determine the total immunoreactive enzyme protein. It was found that the increased S-adenosylmethionine decarboxylase activity produced in rat prostate by treatment with alpha-difluoromethylornithine and in both prostate and liver by methylglyoxal bis(guanylhydrazone) were due entirely to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either method) remained constant in rats treated with the drugs. Treatment with 2% alpha-difluoromethylornithine in the drinking water for 3 days increased prostatic S-adenosylmethionine decarboxylase protein by 5-fold. A substantial part, but not all, of this increase could be accounted for by a slowing of the rate of degradation of the enzyme. The half-life for loss of activity and titratable protein after inhibition of protein synthesis by cycloheximide was increased from 35 to 108 min by treatment with alpha-difluoromethylornithine. However, the half-life for loss of immunoreactive protein which was considerably longer was only increased from 139 to 213 min. The molecular weight of the S-adenosylmethionine decarboxylase subunit determined by immunoblotting was 32,000, and no smaller immunoreactive fragments were detected. These results indicate that spermidine depletion produced by alpha-difluoromethylornithine affects the degradation of S-adenosylmethionine decarboxylase at an early step involving the loss of the active site without substantial breakdown of the protein.     

Strain differences of interferon-generating capacity and resistance in toxoplasma-infected mice

To explore a possible correlation between susceptibility to Toxoplasma and interferon (IFN)-generating capacity in mice, we compared the levels of serum IFN induced by stimulation with Toxoplasma lysate antigen (TLA) in different strains of Toxoplasma-infected and uninfected mice. Injection of TLA into five strains of mice with chronic Toxoplasma infection resulted in the release of considerable amounts of IFN into the circulation. Most of these IFN activities were acid labile and not neutralized by sheep antiserum against mouse IFN-alpha/beta, indicating that IFN-gamma was the dominant form produced in this system. In contrast, the majority of IFN induced in uninfected mice was characterized as IFN-alpha/beta by their acid stability and antigenicity. The response of IFN production in Toxoplasma-infected and uninfected mice varied quantitatively depending on the mouse strains examined. C57BL/6 mice were found to be the best producers of both IFN-alpha/beta and IFN-gamma, while BALB/c mice were consistently poor producers of both IFN populations. A/J, DBA/2, and C3H/He mice could be roughly classified as intermediate producers of both IFN populations. C57BL/6 and C3H/He mice showed a significant prolongation of mean survival time following primary or secondary infection with Toxoplasma compared to that of BALB/c mice. However, there was no direct correlation between the susceptibility to Toxoplasma and the levels of serum IFN.     

Further cholinergic aspects of carotid body chemotransduction of hypoxia in cats

Fitzgerald, Robert S., Machiko Shirahata, and Tohru Ide.Further cholinergic aspects of carotid body chemotransduction ofhypoxia in cats. J. Appl. Physiol.82(3): 819-827, 1997.From the 1930s into the 1970s, the role ofacetylcholine (ACh) in the carotid body's chemotransduction of hypoxiawas debated. Since the late 1970s, the issue has been pursued onlyintermittently or not at all. The purpose of this study was to testagain with a new preparation the hypothesis that ACh is an excitatoryneurotransmitter in the cat carotid body's chemotransduction ofhypoxia. We tested the effect of the specific nicotinic blockermecamylamine and the muscarinic blocker of all five muscarinicreceptors, atropine. We further tested the effects ofM₁ andM₂ muscarinic-receptor blockers.The carotid body region was selectively perfused with hypoxicKrebs-Ringer bicarbonate (KRB) solutions that were blocker free orcontained varying doses of the blockers. Both mecamylamine and atropinereduced the response to hypoxic KRB in a dose-related manner. TheM₂ muscarinic-receptor blockersgallamine and AFDX 116 increased the response to hypoxic KRB, whereasthe M₁ muscarinic-receptor blockerpirenzepine reduced the response to hypoxic KRB. These data areconsistent with an excitatory role for ACh in the carotid bodychemotransduction of hypoxia in the cat.

     

Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene

The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.     

Peripheral chemoreceptor modulation of the pulmonary vasculature in the cat.

The present study was undertaken to determine whether stimulation of the carotid and aortic bodies (cb and ab) could affect the pulmonary vasculature. Our hypothesis was that each promoted vasodilation and thus could modulate the pulmonary vasoconstrictor response to hypoxia. The experimental design of the first set of experiments took advantage of the facts that 1) the ab, but not the cb, increases its neural output in response to CO, whereas both respond to a decreased arterial PO2 (hypoxic hypoxia, HH) and 2) the aortic nerves in cats are easily transected. Hence, both cb and ab sent neural activity to the brain stem when the intact cat was exposed to 10% O2 in N2. Only the ab sent information during CO hypoxia (COH intact). Only the cb did so during HH in the cat in which the aortic nerves had been transected, removing the aortic body (HH abr); neither ab nor cb did so during COH abr. Fifteen anesthetized paralyzed artificially ventilated cats were fit with catheters in the femoral artery and vein, right and left atria, left ventricle, and pulmonary artery and with an aortic flow probe. In the HH intact and HH abr conditions, there was a significant rise in cardiac output, whereas pulmonary arterial pressure (Ppa) rose initially but then leveled off while cardiac output continued to rise. During the 15-min exposure to HH, pulmonary vascular resistance [PVR = (Ppa - Pla)/cardiac output, where Pla is left atrial pressure] rose initially and then decreased significantly at 2-3 min. In response to COH, PVR showed only a significant decrease. In the second set of experiments, seven cats were instrumented as above and had loops placed in the common carotid arteries for selectively perfusing the cbs. In response to a brief infusion of venous blood mixed with 0.3-0.5 micrograms NaCN, which selectively stimulated only the cb, aortic flow remained relatively constant while heart rate and Ppa - alveolar pressure difference decreased significantly; so also did PVR. These data are consistent with the hypothesis that stimulation of the ab and cb singly or together can provoke a significant pulmonary vasodilation in the anesthetized paralyzed artificially ventilated cat.     

Effects of immune lymphocyte products and serum antibody on the multiplication of Toxoplasma in murine peritoneal macrophages.

In vitro studies on cell-mediated immunity against Toxoplasma infection were carried out by estimating the ability of antigenically stimulated lymphocytes. Splenic lymphocytes from normal mice and from hyper-immunized mice were cultured in the presence or absence of Toxoplasma lysate antigen. The cell-free supernatant fluids from the lymphocyte cultures were assassed for their ability to alter the functional capacities of normal macrophages. When glycogen-induced peritoneal macrophages were incubated with the culture supernatant from immune lymphocytes reacting with specific angigen, the intracellular multiplication of the organisms was inhibited remarkably. In contrast, the addition of antitoxoplasma antibody to culture medium had no effect on the enhancement of phagocytic activity of culture macrophages. However, when extra-cellular organisms were exposed to fresh or heat-inactivated immune serum just before infection of the monolayers, the intracellular multiplication of Toxoplasmas was inhibited significantly by either activated or normal macrophages.     

Subtractive screening of genes involved in cellular senescence

We attempted to identify the genes involved in cellularsenescence, telomere maintenance and telomerase regulationthrough subtractive screening of cDNA libraries prepared froma human lung adenocarcinoma cell line A549 and its sublinesnamed A5DC7, CK and AST-9. Cell phenotypes of A5DC7, CK andAST-9 are normal cell-like, cancer cell-like and intermediate,respectively. These cell lines have different phenotypes interms of telomerase activity and telomere maintenance, andthus are thought to be useful for identifying the genesinvolved in cellular senescence and telomerase regulation. In this study, we identified 86 independent cDNA clones bysubtractive screening. Among these cDNA clones, subtractingA5DC7 cDNAs from A549 cDNAs and CK cDNAs gave 7 and 3 cDNAclones which highly and specifically expressed in tester celllines. Genes corresponding to these 10 cDNA clones mightparticipate in maintaining cancer-cell phenotypes. As aresult of database searching, each four of A549 specific cDNAclones are found to correspond to known cDNAs. Each two ofA549 specific and two of CK specific cDNA clones have highhomology to independent ESTs. Sequences having homology toeach one of A549 specific and one of CK specific cDNA cloneshave not been deposited in the Genbank database, indicatingthat these two cDNA clones are part of novel genes. Weanticipate that their involvement in telomerase regulationand/or senescence program can be clarified by functionalanalysis using each full-length cDNA.