Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Identification of photo-inactive phytochrome A in etiolated seedlings and photo-active phytochrome B in green leaves of the aurea mutant of tomato

The contents of spectrophotometrically measurable phytochrome A (PhyA) and phytochrome B (PhyB) and the corresponding immunochemically detectable apoproteins (PHYA and PHYB) were examined in dark- and light-grown tissues of the aurea mutant of tomato and its wild-type (WT). The amount of PHYA in etiolated aurea seedlings was found to be about 20% of that in the WT; this PHYA showed no photoreversible changes in absorbance, no downregulation of the level of PHYA in light-grown seedlings, and no differential proteolysis of Pr and Pfr species in vitro which was seen in the case of the WT. By contrast, the amount of PHYB in aurea seedlings was not significantly different from that in WT seedlings. Phytochrome isolated from green leaves of the aurea mutant and purified by ion-exchange chromatography showed a red/far-red reversible spectral change, and its elution profile during chromatography was essentially similar to that of PHYB. The results indicate that aurea is a mutant that is deficient in photoactive PhyA at the etiolated stage, when it contains a spectrally inactive PHYA. However, the mutant contains spectrally active PhyB in its green tissue as does the WT.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Studies on glutathione metabolism in ventral prostate and chemically induced prostatic carcinoma in rats

Glutathione content and the activity of glutathione reductase were examined in ventral prostate and chemically induced 11095 squamous-cell prostatic carcinoma in rats, Castration produced a significant reduction in the levels of reduced (GSH) and oxidized (GSSG) glutathione and glutathione reductase activity in the prostate. Replacement of testosterone (50 mg/kg) daily for 7 days to castrated animals elevated the reduced glutathione level and the activity of glutathione reductase almost to normal limits, Squamous-cell carcinoma was implanted in castrated and intact animals, Tumor growth in normal rats produced a decrease of almost 30% in the weight of the ventral prostate at 21 days post-implantation, although the glutathione levels remained unaffected. Much greater activity of glutathione reductase was detected in the tumor in comparison to the values noted for the normal tissue, The tumor also showed significantly higher values for the GSH/GSSG ratio, No apparent difference could be found in the rate of the growth of tumors whether implanted in normal or castrated animals, The levels of reduced and oxidized glutathione and glutathione reductase activity also seemed identical in tumors obtained from both groups of animals, Administration of testosterone (50 mg/kg) or β-estradiol (2 mg/kg) daily for 11 days to tumor-bearing castrated animals did not alter the levels of glutathione and glutathione reductase activity. A significantly higher level of blood reduced glutathione was found in tumor-bearing rats in comparison to that seen for the normal subjects. Our results demonstrate that androgen depletion and replacement therapy influence the metabolism of glutathione in rat ventral prostate. Squamous-cell carcinoma of the prostate appears to differ from the normal tissue with respect to the observed androgen effects, There is dissimilarity in the metabolism of glutathione in the two tissues since greater activity of glutathione reductase and lower values of reduced glutathione were seen in the tumor as compared to t h o s e of the ventral prostate. Treatment with β-estradiol, an antiprostatic agent, does not seem to influence the growth or glutathione metabolism of squamous-cell carcinoma of the prostate. The observed changes in blood glutathione levels might prove to be useful as an index of rapid growth of the neoplastic tissue.     

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.     

Isolation and characterization of mesenchymal stem cells in orthopaedics and the emergence of compact bone mesenchymal stem cells as a promising surgical adjunct

The potential clinical and economic impact of mesenchymal stem cell (MSC) therapy is immense. MSCs act through multiple pathways: (1) as “trophic” cells, secreting various factors that are immunomodulatory, anti-inflammatory, anti-apoptotic, proangiogenic, proliferative, and chemoattractive; (2) in conjunction with cells native to the tissue they reside in to enhance differentiation of surrounding cells to facilitate tissue regrowth. Researchers have developed methods for the extraction and expansion of MSCs from animal and human tissues. While many sources of MSCs exist, including adipose tissue and iliac crest bone graft, compact bone (CB) MSCs have shown great potential for use in orthopaedic surgery. CB MSCs exert powerful immunomodulatory effects in addition to demonstrating excellent regenerative capacity for use in filling boney defects. CB MSCs have been shown to have enhanced response to hypoxic conditions when compared with other forms of MSCs. More work is needed to continue to characterize the potential applications for CB MSCs in orthopaedic trauma.