Further studies on the thymocyte stimulating factor.

The thymocyte-stimulating activity produced by spleen cells cultured in the presence of PHA (cell. Immunol.17, 495, 1975) was further investigated. It was found that this activity is caused by a factor having the chemical properties of a protein with a molecular weight of 30,000–32,000 that, at high concentration, probably dimerizes to a molecule of molecular weight about 55,000 as measured by Sephadex gel filtration. This factor is produced also in mixed lymphocyte cultures of CBA and C57 spleen cells. The observation that the factor produced in the presence of PHA and the factor produced in the mixed lymphocyte cultures have the same molecular weight and the same rate of denaturation at two different temperatures suggests that they are the same, or very similar, substance. The fact that spleen cells of nu/nu mice do not produce thymocyte-stimulating factor suggests that thymus-dependent cells are involved in the production of this factor. This assumption is consistent with the observation that spleen cells of mice less than two weeks old (which show a very small response to PHA) do not produce significant amounts of thymocyte-stimulating factor. On the other hand, results of experiments with hydrocortisone-injected mice suggest that the target cell for thymocyte-stimulating factor is the immature, cortisone-sensitive thymocyte. The properties of thymocyte-stimulating factor are discussed and compared to those of factors with similar actions reported in the literature.     

Further characterization of the inhibitory effect of monensin on adrenal steroidogenesis

We have previously reported that treatment of cultured mouse adrenal tumor cells with 0.6-1.2 microM monensin, a monovalent carboxylic ionophore, results in disruption of the organized structure of the Golgi complex. This is associated with an inhibition of adrenocorticotropic hormone (ACTH) or dibutyryl cAMP-stimulated steroidogenesis and impairment of mitochondrial cholesterol side-chain cleavage activity. The present report describes further investigations regarding possible mechanisms for the inhibition. Monensin inhibits both synthesis of fluorogenic steroids and incorporation of [14C]acetate into the end-product steroid 11 beta,20 alpha-dihydroxy-4-pregnen-3-one. Supplementation of monensin-treated cells with 25-hydroxycholesterol, a readily available substrate for steroidogenesis, does not reverse the inhibitory effect on the reaction. The incorporation of L-[35S]methionine into trichloroacetic acid precipitable proteins in the isolated mitochondria of monensin-treated cells is inhibited approximately by 40%, whereas the inhibitory effect on the proteins in the cell homogenate is marginal. These findings suggest that a deficiency of newly synthesized proteins in mitochondria, rather than the availability of the substrate cholesterol, may be the primary factor causing impairment of steroidogenesis.     

Further evidence for the inhibitory action of baclofen on a prolactin-releasing factor

The mechanism of action of a specific gamma-aminobutyric acid B receptor agonist, beta-p-chlorophenyl-gamma-aminobutyric acid or baclofen, in its inhibitory action on prolactin release, was studied. Dose-response studies of the effect of baclofen on prolactin (PRL) secretion were performed in stressed male rats. Furthermore, the action of the drug was evaluated in (i) rats treated with haloperidol or alpha-methyl-p-tyrosine, (ii) stressed or suckled rats pretreated with sulpiride, and (iii) animals treated with serotonin, alone, or with alpha-methyl-p-tyrosine. Baclofen showed a clear dose-dependent inhibition of prolactin secretion in males under stress. The drug was unable to inhibit the prolactin release induced by haloperidol or alpha-methyl-p-tyrosine, although it reduced the PRL secretion induced by serotonin. It also inhibited PRL release in sulpiride-pretreated stressed or suckled rats. These results suggest that the dose-dependent effect of baclofen on PRL secretion is the consequence of an inhibition exerted on the prolactin-releasing factor component of the neuroendocrine responses evoked by stress or suckling, possibly acting at the serotonergic system.     

Immune response to chemically modified flagellin. IV. Further studies on the relationship between humoral and cell-mediated immunity

Acetoacetylation converts flagellin from an antigen which preferentially induces humoral antibodies to an antigen which exclusively provokes cell-mediated immunity and, under certain circumstances, induces antibody tolerance. Studies reported in this paper revealed that the acetoacetylated flagellins expressed similar immunological properties in flagellin primed rats as in normal rats. Thus, on the one hand, acetoacetylation destroyed the capacity of flagellin to trigger a secondary antibody response, but on the other hand, the acetoacetyl-flagellins very effectively induced delayed-type hypersensitivity reactions in flagellin primed animals. It was concluded from these results that humoral and cell-mediated immunity may be opposing immunological processes in both unprimed and primed animals.Acetoacetylated flagellin induced antibody tolerance in both strain W (low responder) and J (high responder) Wistar rats. Maximum tolerance was induced 12 hr after injection of antigen, but in strain J animals the tolerance had disappeared by 48 hr, whereas in strain W rats tolerance persisted for >28 days. The potential to recover from tolerance in strain J rats appeared to coincide with the level of delayed hypersensitivity at the time of challenge. However, this delayed hypersensitivity disappeared when breaking of tolerance occurred. These results suggest that the T cells which participate in delayed hypersensitivity reactions may also act as “helper” cells in antibody responses. On the other hand, it was found that priming for a secondary antibody response by flagellin appeared to coincide with development of primary antibodies rather than with induction of delayed-type hypersensitivity. The relative importance of specific T and B cells in these phenomena is discussed.     

Further studies on elastase and trypsin inhibitory activities in mammalian lenses

Elastase and trypsin inhibitory capacities increased significantly on heat treatment of the lens extract for 15 min at 60 degrees C in human infant (mean increase 290 and 335%), human adult (130 and 245%), ovine (90 and 140%), and bovine (70 and 90%) lenses. No increase was observed in human cataractous lenses. Preincubation with target enzymes in the absence of substrate abolished the antitryptic activity in lenses whereas antielastase activity was more resistant. No decrease in antielastase activity in human adult and cataractous lenses was observed on 15-min preincubation whereas about 50% of activity was abolished in human infant lenses. The differences were attributed to the changes in the levels of endogenous proteinases and proenzymes during cataractogenesis and aging.     

Further studies on bovine serum amylase--the effect of gel buffer pH.

This paper describes the effect of gel buffer pH on the resolution of bovine serum amylase (Amylase I) isozymes in starch gel and the consequences for the understanding of the genetics of this locus. The two main findings are: (1) the existence of a satellite isozyme E to isozyme C which at pH 7.3 has the same mobility as the B isozyme but which at pH 8.0 migrates slower than B, and (2) the finding of three alleles Aml A, Aml B and Aml C in British cattle populations previously reported as having only Aml B and Aml C.     

Further studies of the effect of L-canavanine on the tobacco hornworm, Manduca sexta.

Fifth instar Manduca sexta growth response to injected doses of canavanine was concentration-dependent over a range of 0·5 to 2·0 mg/g body weight. Twenty-four hr after injection of ¹⁴C-guanidinooxy-d,l-canavanine, M. sexta larvae incorporated approximately 3·6% of the labelled l-canavanine into protein of non-gut tissue. Adult M. sexta mortality was related to the level of injected canavanine over a range of 2 to 8 mg/g body weight. Injection of as little as 2 mg canavanine/g body weight caused hyperactivity in adult M. sexta. Arginine, able to negate the toxic effects of canavanine during larval growth, was only marginally capable of overcoming canavanine effects on larval-pupal ecdysis.     

Further studies on the deoxyribonucleic acid helping effect during transformation

Summary Transformation studies of the Challis strain of the H group of streptococcus were performed to further investigate the molecular basis of the deoxyribonucleic acid helping effect. Studies in the efficiency of transformation in the presence of non-transforming DNA support the notion that bacterial cells are indiscriminate in their uptake of donor DNA and that the helping effect occurs at a time when both transforming (T) and helping (H) DNAs have jointly entered the recipients. Furthermore, the ability of H DNA to promote transformation by T DNA is not directly altered by exposure of the former DNA to either UV-irradiation or nitrous acid. Nor does 5-bromouracil incorporation affect the capacity of H DNA to assist T DNA to transform a Challis cell.Increasing the concentration of denatured H DNA to a level that saturates the Challis bacteria in reaction mixtures produces a significant increase in the efficiency of genetic transformation. This increase in transformation frequency is greater with single DNA strands than with the corresponding amount of double strands.The extent of the helping effect with the Challis H DNA remains constant within an average molecular weight range of 3.5–7.0x10⁶ daltons. In the case of heterologous E. coli DNA, however, the helping function in this range is more pronounced as a result of decreasing molecular weight, even though the net incorporation of T DNA remains unaffected. When the average M. W. is reduced below 2×10⁶ a significant decline in the helping effect occurs in both cases.The effect of H DNA on the genetic transfer of two nonallelic antibiotic markers demonstrates that a saturating amount of non-transforming H DNA present in cells does not enhance the likelihood of co-integration of nonallelic factors. Evidence concerning the physiology of the competent cells and their ability to be helped reveals that the physiological basis of transformabilities and the helping capabilities of a culture are not identical.     

Further studies on the enhancing factor and its possible mechanism of action

In this study the nature of binding of enhancing factor (EF) and its mode of action are examined. EF binds to A431 cells through its own receptor, which is distinct from the receptor for epidermal growth factor (EGF). EF binds to the cell membrane and in turn provides a binding site for EGF. Data analyzed from Scatchard plots show that prior treatment of formalin-fixed A431 cells with EF for 30 minutes results in an increase in the number of binding sites for 125I-EGF. 3H-Thymidine incorporation studies, using the EGF-receptorless cell line NR-6, indicate that neither EF nor EGF alone stimulates the cells to synthesise DNA, but when both are added together the cells show 3H-thymidine incorporation. The role of EF may be to trap EGF and make it available to the cells through its own receptors even in the absence of EGF receptors. EF also induces anchorage-independent growth of normal fibroblasts in soft agar only in the presence of EGF.