Pivotal roles of alpha-melanocyte-stimulating hormone and the melanocortin 4 receptor in leptin stimulation of prolactin secretion in rats

Leptin, the obese gene product, was reported to stimulate prolactin (PRL) secretion, but the neuroendocrine mechanism underlying this hormonal response is largely unknown. Thus, in this study we examined the involvement of several important PRL regulators in the leptin-induced PRL secretion in male rats. Compared with the values in normally fed rats, food deprivation for 3 days significantly decreased both PRL and leptin levels in the plasma. These changes were reverted to normal by a 3-day constant infusion of 75 microg/kg/day of leptin to the fasted rats, while 225 microg/kg/day of leptin further elevated both PRL and leptin levels. These four groups of animals were used for the following experiments. Results of dopamine and serotonin turnover studies in the brain and the pituitary indicated that neither of these biogenic amines plays a primary role in mediating leptin's effects on PRL. Repeated intracerebroventricular injections over 72 h of neutralizing antibodies against vasoactive intestinal peptide, PRL-releasing peptide, or beta-endorphin, did not significantly suppress the leptin actions. However, both the blockade of the melanocortin (MC) 4 receptor (R) and the immunoquenching of brain alpha-melanocyte-stimulating hormone (alpha-MSH) completely abolished the leptin-induced PRL release, and the stimulation of the MC4-R, but not the MC3-R, significantly elevated PRL levels in the fasted rats. These results suggest that alpha-MSH, a cleaved peptide from pro-opiomelanocortin of which synthesis is stimulated by leptin, may be the pivotal neuropeptide in the brain mediating the leptin's stimulatory influence on PRL secretion. It was also suggested that the MC4-R may be the primary subtype of the MC-Rs mediating this action of alpha-MSH.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Involvement of Norepinephrine in the Production of a Flower-Inducing Substance in the Water Extract of Lemna

A flower-inducing substance (FIS) is produced by incubationof the pellet of centrifuged ho-mogenates of Lemna paucicostata441 (P441) with commercial enzyme preparations such as catalase,cellulase, lipase and proteinase K. The active component inthese preparations was identified as tyrosine. The tyrosinemetabolites, dopa, dopamine, norepinephrine (NE) and epinephrine,were also effective for the production of FIS, and NE the mosteffective. Addition of only 1 µg NE to the pellet obtainedfrom 100 mg fresh weight of P441 produced FIS without incubation.NE added to the pellet heated after resuspension also producedFIS but that added to the pellet resuspended in boiling waterimmediately after separation did not. A heat-stable substance(s)produced by a heat-unstable reaction in the pellet may reactwith NE to produce FIS or interact with NE to induce flowering.About 400 ng per g fresh weight of NE was detected in the waterextract of P441 plants, but only about 0.5 ng in the hot-acidicwater extract of the plants, which suggests that most of theNE was produced after homogenization. (Received October 17, 1990; Accepted December 28, 1990)     

Evaluation of the bacterial endotoxin test for quantification of endotoxin contamination of porcine vaccines.

We investigated the application of the bacterial endotoxin test for the quantification of the endotoxin contamination of various commercial porcine vaccines. In endotoxin-spiked samples, Freund's complete adjuvant and aluminum hydroxide gel adjuvant failed to interfere with the results of the endotoxin test, and both recovery ratios were within the permissible range mentioned in the Japanese Pharmacopoeia. At the various dilutions tested, none of the adjuvants in commercial porcine vaccines caused noteworthy interference in the test. In addition, none of the 39 samples of porcine vaccines approved in Japan induced an interfering effect in the endotoxin test. Our findings suggest that the bacterial endotoxin test using endotoxin-specific Limulus amoebocyte lysate (LAL) can detect endotoxin contamination in commercial porcine vaccines containing either oil or aluminum adjuvants.     

Preparation of New Derivatives from α-Dihydro Grayanotoxin-II

Rhodojaponin-III and 10,14-epoxy-10-deoxy grayanotoxin-III were derived from grayanotoxin-I and -III, respectively. Preparation of 13 new derivatives from α-dihydro grayanotoxin-II was discussed. Some of them showed higher physiological activity than that of natural grayanotoxins.     

Primary Tumor-Secreted Lymphangiogenic Factors Induce Pre-Metastatic Lymphvascular Niche Formation at Sentinel Lymph Nodes in Oral Squamous Cell Carcinoma

Objectives

The objectives of this study were to evaluate the formation of lymphvascular niches in lymph nodes of patients with oral squamous cell carcinoma (OSCC), and investigate the roles of lymphangiogenic and angiogenic factors, such as vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D, expressed in the primary tumors.

Materials and Methods

Forty-four patients with previously untreated clinically late T2 or T3 OSCC of cN0 were evaluated for primary tumors and 166 sentinel lymph nodes (SLNs). Primary tumors were immunohistochemically analyzed for expressions of VEGFs. Densities of lymphatic vessels (LVD_podoplanin) and high endothelial venules (HEVD) in the SLNs were also calculated using antibodies for each marker, podoplanin and MECA-79, respectively.

Results

In 25 patients, all lymph nodes were metastasis-negative, whereas, in 19 patients, metastasis was positive for at least one lymph node (either at SLN, non-SLN, or nodal recurrence). From the analyses of 140 SLNs without metastasis, LVD_podoplanin in 50 SLNs of metastasis-positive cases was significantly higher than that in 90 SLNs of metastasis-negative cases (p = 0.0025). HEVD was not associated with lymph node metastasis. The patients with VEGF-A-High or VEGF-D-High tumors had significantly higher LVD_podoplanin than patients with their Low counterparts (p = 0.0233 and p = 0.0209, respectively). In cases with lymph node metastasis, the VEGF-D-expression score was significantly higher than in those without lymph node metastasis (p = 0.0006).

Conclusions

These results suggest that lymph node lymphangiogenesis occurs before metastasis in OSCC. VEGF-A and VEGF-D play critical roles in this process. VEGF-D is a potential predictive marker of positive lymph node metastasis in cN0 patients.     

Biosynthesis of Grayanotoxins in Leucothoe grayana Max.Incorporation of Mevalonic Acid and (—)-Kaurene into Grayanotoxin-III

The main subunits of glutenin were separated by preparative SDS-PAGE with a Laemmli system (U. K. Laemmli, Nature, 227, 680 (1970)) and their cysteine (Cys) contents were determined by amino acid analysis. Amino acid compositions of glutenin subunits, determined in the present study, were different from those determined by Danno et al. [G. Danno, K. Kanazawa and M. Natake, Agric. Biol. Chem., 40, 739 (1976)]. We found that these differences were due to the different methods of hydrolysis of subunit polypeptides. That is, hydrolysis of subunit polypeptides extracted from gel and hydrolysis of polypeptides in gel without extraction. Cys contents of glutenin subunits were determined as S-pyridylethyl cysteine (PE-Cys). Although no PE-Cys was detected in B-4 or B-4′, all other subunits were shown to have 4mol Cys per mol protein, respectively.