Influence of thyroidal status on pituitary content of thyrotropin beta- and alpha-subunit, growth hormone, and prolactin messenger ribonucleic acids

Thyroidectomized rats were used to study the effects of a single injection of T3 on pituitary mRNA synthesis and hormone secretion. T3 was injected ip at doses of 0, 0.2, 1, or 5 micrograms/100 g body weight, and and animals were killed 24 h later. T3 caused a significant decrease in serum TSH, but caused no significant change in either serum GH or PRL. Pituitary mRNA was quantified by slot blot hybridization with cDNA probes specific for alpha-TSH, beta-TSH, PRL, and GH. We found that both the alpha and beta mRNA subunits decreased, that PRL mRNA remained relatively unchanged, and that GH mRNA increased with increasing T3 dose. The data show that a single dose of T3 can profoundly influence mRNA levels in the anterior pituitary; the lowest dose of T3 caused maximum inhibition of alpha-TSH mRNA while beta-TSH mRNA declined further in a dose-dependent manner.     

Effect of capsulectomy on the hemodynamics and viability of random-pattern skin flaps raised on expanded skin in the pig

Skin flaps constructed on expanded skin usually include the underlying capsular tissue. It has been hypothesized that capsulectomy may jeopardize the viability of the expanded skin flap. The experiments reported herein were designed to test this hypothesis. Specifically, we studied the hemodynamics and viability of random-pattern skin flaps (8 X 20 cm) raised on delayed bipedicle flaps (group A) and on expanded skin pockets with capsulectomy at the time of flap elevation (group B) or with intact underlying capsular tissue (group C). Each group was randomly assigned to each flank in 16 pigs. Skin pockets were expanded by inflation of subcutaneous silicone tissue expanders with sterile saline (299 +/- 7 ml; X +/- SEM) over a period of 3 weeks. At the end of this period, the bipedicle flaps were constructed. Eight days later, random-pattern skin flaps were raised on bipedicle flaps and skin pockets. The length and area of skin flap viability, judged by the fluorescein dye test performed 1 day postoperatively, were not significantly different (p greater than 0.05) among groups A, B, and C (n = 31 to 32). There also were no significant differences (p greater than 0.05) in total skin capillary blood flow measured 1 day postoperatively (A = 2.6 +/- 0.4, B = 2.4 +/- 0.4, and C = 2.7 +/- 0.6 ml/min per flap; n = 15 to 16) and in skin viability assessed 7 days postoperatively (A = 74 +/- 2, B = 75 +/- 2, and C = 76 +/- 2 percent; n = 16) among delayed skin flaps and skin flaps raised on expanded skin pockets with or without capsulectomy. The results of this flap viability study were confirmed in 5 minipigs in a separate experiment. We conclude that capsulectomy did not have a detrimental effect on the hemodynamics and viability of random-pattern skin flaps raised on expanded skin. Furthermore, we hypothesize that skin flaps raised on expanded skin are similar to delayed skin flaps in that the skin blood flow is optimally augmented; therefore, the capsular tissue does not add significant blood supply to the overlying skin.     

Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH.

We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.     

Expression of a human multidrug resistance cDNA (MDR1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia.

The human multidrug resistance gene (MDR1) encodes a drug efflux pump glycoprotein (P-glycoprotein) responsible for resistance to multiple cytotoxic drugs. A plasmid carrying a human MDR1 cDNA under the control of a chicken beta-actin promoter was used to generate transgenic mice in which the transgene was mainly expressed in bone marrow and spleen. Immunofluorescence localization studies showed that P-glycoprotein was present on bone marrow cells. Furthermore, leukocyte counts of the transgenic mice treated with daunomycin did not fall, indicating that their bone marrow was resistant to the cytotoxic effect of the drug. Since bone marrow suppression is a major limitation to chemotherapy, these transgenic mice should serve as a model to determine whether higher doses of drugs can cure previously unresponsive cancers.     

Propranolol antagonizes hypotension induced by alpha-blockers but not by sodium nitroprusside or methacholine

A pressor response has been observed with propranolol, a nonselective beta-adrenoceptor antagonist, in animals given a nonselective alpha-adrenoceptor antagonist. This study investigates whether a pressor response to propranolol occurs in conscious unrestrained rats following a hypotensive response induced by phentolamine (nonselective alpha-antagonist), prazosin (selective alpha 1-antagonist) and (or) rauwolscine (selective alpha 2-antagonist), sodium nitroprusside (smooth muscle relaxant), or methacholine (muscarinic agonist). The rats were subjected to a continuous infusion of a hypotensive agent or normal saline followed by i.v. injection of propranolol. The infusion of phentolamine significantly decreased mean arterial pressure (MAP). Subsequent injection of propranolol restored MAP to the control level. Prazosin and rauwolscine each caused a small but not significant decrease in MAP which was reversed by propranolol. Concurrent infusions of prazosin and rauwolscine caused a significant decrease in MAP. Subsequent injection of propranolol caused a large pressor response which increased MAP to 20% above control MAP prior to the administration of drugs. Nitroprusside or methacholine each caused a significant decrease in MAP, but the hypotension was not antagonized by propranolol. The concurrent infusions of a low dose of nitroprusside and prazosin caused a significant decrease in MAP which was reversed by propranolol. The infusion of saline did not alter MAP, and propranolol did not cause a pressor response. It is concluded that propranolol antagonizes the hypotensive effect of an alpha-blocker but not that of sodium nitroprusside or methacholine. Our results suggest the presence of a specific interaction between alpha- and beta-antagonists.     

Linkage analysis of chromosome 17 markers in British and South African families with neurofibromatosis type 1

Nine markers from the pericentromeric region of chromosome 17 were typed in 16 British and five South African families with neurofibromatosis type 1 (NF1). The markers--p17H8, pHHH202, and EW204--were linked to NF1 at recombination fractions less than 1%. No evidence of locus heterogeneity was detected. Inspection of recombinant events in families informative for several markers suggests that the NF1 gene is located between the markers EW301 (cen-p11.2) and EW206 (cen-q12) and possibly distal to pHHH202 (q11.2-q12).     

Detection of surface charge-related properties in model membrane systems by aqueous two-phase partition

Unilamellar vesicles composed of phosphatidylcholine (PC) and either phosphatidic acid (PA) or phosphatidylglycerol (PG) partition to the upper poly(ethylene glycol) (PEG)-rich phase of a charge-sensitive 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 7, consistent with the vesicles bearing a net negative charge. When prepared in the presence of a pH gradient (interior acidic), PC/PA vesicles exhibit an increased partition to the top PEG-rich phase, consistent with a redistribution of the PA from the inner to the outer monolayer of the vesicle bilayer. Conversely, when prepared in the presence of a pH gradient (interior basic), PC/PG vesicles exhibit a decreased top-phase partition, consistent with a redistribution of the PG from the outer to the inner monolayer of the vesicle bilayer. Unilamellar vesicles composed of PC and stearylamine partition to the lower dextran-rich phase of a 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 8.5, consistent with the vesicles bearing a net positive charge. When prepared in the presence of a pH gradient (interior acidic), conditions under which the stearylamine is trapped on the inner monolayer of the bilayer, the vesicles now partition predominantly to the interface in a manner similar to vesicles composed of PC alone. These results demonstrate that partitioning in aqueous two-phase polymer systems is a sensitive method for monitoring the asymmetry of charged lipids in model membrane systems and also suggests that partitioning in charge-sensitive systems depends only on the physical nature of the exterior surface of the membrane.     

Establishment and characterization of a human chorionic gonadotropin (hCG) producing cell line (RTSG) from an ovarian epithelial cancer

A new cell line designated RTSG established in vitro from the pleural effusion of a patient with metastatic ovarian epithelial cancer has been subcultured 46 times for more than 2 years. The cells grew in a monolayered sheet, showing a tendency to pile up, with the population doubling in 48 hrs. Electron-microscopically, desmosomes were characteristically observed, suggesting the cells were of epithelial origin. Chromosomal analysis revealed aneuploidy with a tetraploid mode. The heterotransplanted tumors in nude mice were histopathologically classified as a poorly differentiated adenocarcinoma, whereas the original tumor consisted mainly of mucinous and serous cystadenocarcinoma and only partly of poorly differentiated adenocarcinoma. The cells secreted hCG (38.8 mIU/day/10(6) cells) and beta-hCG (6.1 ng/day/10(6) cells) in spent medium. Immunocytologic +-and-histochemical staining for tumor markers of the original tumor, the cultured cells and the transplanted tumors also revealed the localization of not only hCG and beta-hCG but also CA19-9 and CA-125 whose values had been elevated in the preoperative serum (hCG: 10 mIU/ml, CA19-9: 6,400 U/ml, CA-125: 225 U/ml). Results of PAS, Alcian-blue and Mucicarmine strains indicated that most of the PAS-positive substances in the cultured cells and the transplanted tumors were consistent with glycogen while the original tumor mainly contained mucin except for the lesion of poorly differentiated adenocarcinoma with glycogen. These results suggested that the cultured cells might originate from poorly differentiated adenocarcinoma cells in the original tumor.