Immunoselection of B cell hybridomas with anti-idiotypic antisera to study the role of heavy and light chains for idiotype expression and antibody activity.

Conventional reconstitution experiments with heavy (H) and light (L) offins of hybridoma anti-poly-(Glu60Ala30Tyr10) (anti-GAT) antibodies demonstrated that H and L chains are required for efficient expression of anti-GAT activity and the previously defined common CGAT idiotype. On the basis of this observation, we treated a CGAT+ hybridoma cell line (F9.195.6), which was produced by fusion with an MOPC 21 (gamma 1, K) secretor line, with anti-CGAT idiotypic antibodies plus complement to select CGAT-variants. Such treatment greatly enhanced the frequency of isolating hybridoma variants. Molecules secreted from these variants bear anti-GAT mu-chain and MOPC 21 L chain (as judged by serology and Southern blot studies), lack GAT binding activity and CGAT idiotype, and exhibit a "new" private idiotype (E8). Anti-private idiotypic sera were successfully used to select a series of CGAT+, E8- clones that secrete molecules bearing only the appropriate anti-GAT mu- and K chains. From E8- hybridoma supernatants, CGAT- molecules bearing MOPC 21 H chain and anti-GAT L chain could be obtained. The data indicate that immunoselection of hybridoma clones with anti-idiotypic antibodies is a convenient means of preparing reconstituted antibody molecules. Furthermore, the data demonstrate that CGAT idiotype determinants are expressed on antibody molecules composed of appropriate H and L chains.

Biochemical correlates of progesterone-induced plasma membrane depolarization during the first meiotic division in Rana oocytes.

Changes in protein synthesis, protein phosphorylation and lipid phosphorylation in the amphibian oocyte plasma membrane have been correlated with electrical changes following steroid induction of the completion of the first meiotic division. The oocyte first depolarizes from about -60 mV (inside negative) to about -25 mV 1 to 2 hr before breakdown of the large nucleus followed by a further depolarization beginning 3 to 6 hr after nuclear breakdown. The initial depolarization is associated with appearance of previously described cycloheximide-sensitive cytoplasmic factor(s) which induce both nuclear breakdown and plasma membrane depolarization. We found a similar ED50 (0.4 microM) for cycloheximide inhibition of nuclear breakdown, membrane depolarization, and [3H]-leucine incorporation. Emetine (1 nM to 1 mM) was inactive. The period of cycloheximide sensitivity (first 5 hr) is essentially the same for plasma membrane depolarization and nuclear breakdown. The onset of the second depolarization phase following nuclear breakdown is associated with a marked increase in the rate of [3H]-leucine and [32PO4] incorporation into membrane protein and lipid. Polyacrylamide gel electrophoresis of membrane protein and lipoprotein indicated that a major newly synthesized membrane component is proteolipid. An increase in [32PO4] incorporation into membrane phosphatidylserine and phosphatidylethanolamine (with a decrease in phosphatidylcholine [32PO4] begins during the second depolarization phase and coincides with the appearance of excitability in the oocyte plasma membrane. In toto, the bulk of the biochemical changes (proteins, phosphoproteins, proteolipids, phospholipids) appear to be associated with plasma membrane components and coincide with stepwise changes in membrane permeability to specific ions (e.g. Cl-).

Effect of cannabinoids on estrous cycle, ovulation and reproductive capacity of female A/J mice.

Virgin A/J female mice were intubated daily for 8 days (short term) or 70 days (long term) with 0, 1, 5, or 25 mg/kg delta 9-tetrahydrocannabinol (delta 9-THC) or 0, 3, 15, or 75 mg/kg crude marihuana extract (CME) in a sesame oil:polysorbate 80:saline vehicle. These dosages approximate light, moderate, and heavy human usage. Short-term exposure to CME has no significant effect on PMS-HCG-induced ovulation but appears to: (1) delay entry into proestrus at all dose levels; (2) depress serum progesterone during the luteal phase at the highest CME level used (75 mg/kg), and (3) inhibit female receptivity to males at least at the highest dosage. Long-term oral administration of CME or delta 9-thc had no significant effect on length of estrous cycles or mating (plug formation) but term pregnancies were reduced by 32 and 68% for medium and high dosages, respectively. After a 30-day recovery period, 80% of those females that failed to have successful pregnancies now became pregnant.