Identification, localization, and function in steroidogenesis of PAP7: a peripheral-type benzodiazepine receptor- and PKA (RIalpha)-associated protein.

Peptide hormones and cAMP acutely stimulate steroid biosynthesis by accelerating the transport of cholesterol into the mitochondria. The peripheral-type benzodiazepine receptor (PBR) has been shown to be an indispensable element of the cholesterol transport machinery. Using the yeast two-hybrid system and PBR as bait, we identified a protein that interacts with PBR, the PBR-associated protein PAP7. Using the regulatory subunit RIalpha of PKA as bait, we also isolated PAP7. Glutathione-S-transferase -PAP7 interacted with both the mitochondrial PBR and cytosolic PKA-RIalpha in MA-10 Leydig cells. PAP7 is a novel 52-kDa protein present in mouse, rat, and human tissues, and it has a major 3-kb mRNA transcript in all tissues examined. Immunohistochemical and in situ hybridization studies indicated that PAP7 is highly expressed in the gonads, adrenal, hippocampus, and distinct brain neuronal and glial populations. Overexpression of the full length PAP7 increased the hCG-induced steroid production. However, overexpression of a partial PAP7, which includes the PBR- and PKA-RIalpha-binding domains, inhibited the hormone-stimulated cholesterol transport and steroid synthesis. Treatment of MA-10 cells with oligonucleotides antisense to PAP7 also inhibited the hCG-stimulated steroid formation, suggesting that PAP7 is a functional element of the hormone-induced signal transduction cascade leading to steroidogenesis. PAP7 may function by targeting the PKA isoenzyme to organelles rich in PBR, i.e. mitochondria, where phosphorylation of specific protein substrates may induce the reorganization of PBR topography and function.

Cholesterol binding at the cholesterol recognition/ interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide.

We previously defined a cholesterol recognition/interaction amino acid consensus (CRAC; ATVLNYYVWRDNS) in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), an outer mitochondrial membrane protein involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. We examined (i) the PBR-cholesterol interaction by UV crosslinking of the C17 side-chain containing progestin, promegestone, and (ii) the role of the CRAC domain of PBR in Leydig cell steroidogenesis by using a transducible peptide composed of the TAT domain of HIV and the CRAC domain of PBR. [(3)H]Promegestone photoincorporated into recombinant PBR, and this labeling was displaced by cholesterol. [(3)H]Promegestone also photoincorporated into the TAT-CRAC peptide. [(3)H]Promegestone crosslinking to TAT-CRAC could be displaced by cholesterol and promegestone, with IC50 values of 1 and 200 microM, respectively. TAT-CRAC efficiently transduced into MA-10 Leydig cells and inhibited the hCG- and cAMP-stimulated steroid production in a dose-dependent manner. TAT-CRAC did not affect the hCG-induced cAMP synthesis and the 22R-hydroxycholesterol-supported steroidogenesis. Mutated TAT-CRAC lost its ability to bind [(3)H]promegestone and to inhibit the hCG-stimulated steroidogenesis. These results show that TAT-CRAC binds cholesterol and competes for cholesterol interaction with endogenous PBR, suggesting that the cytosolic carboxyl-terminal domain of PBR is responsible for taking up and bringing steroidogenic cholesterol into the mitochondria.

Cell wall adaptations to multiple environmental stresses in maize roots.

A municipal solid-waste bottom slag was used to grow maize plants under various abiotic stresses (high pH, high salt and high heavy metal content) and to analyse the structural and chemical adaptations of the cell walls of various root tissues. When compared with roots of control plants, more intensive wall thickenings were detected in the inner tangential wall of the endodermis. In addition, phi thickenings in the rhizodermis in the oldest part of the seminal root were induced when plants were grown in the slag. The role of the phi thickenings may not be a barrier for solutes as an apoplastic dye could freely diffuse through them. The chemical composition of cell walls from endodermis and hypodermis was analysed. Slag-grown plants had higher amounts of lignin in endodermal cell walls when compared to control plants and a higher proportion of H-type lignin in the cell walls of the hypodermis. Finally, the amount of aliphatic suberin in both endo- and hypodermal cell walls was not affected by growing the plants on slag. The role of these changes in relation to the increase in mechanical strengthening of the root is discussed.

Location-dependent role of the human glioma cell peripheral-type benzodiazepine receptor in proliferation and steroid biosynthesis.

We examined the localization and function of the peripheral-type benzodiazepine receptor (PBR), a protein highly expressed in steroidogenic tissues and aggressive tumor cells, in cell lines derived from glioblastoma multiforme tumors. In MGM-1 cells, PBR is located in the nucleus, and cells proliferate in response to PBR ligands but do not synthesize steroids de novo. In MGM-3 cells, PBR is located in mitochondria and the cells synthesize steroids, but do not proliferate in response to PBR ligands. In glioblastoma biopsies, PBR is expressed in the nuclei of cells, while it is found in the cytosol of astrocytomas, and is absent from meningioma and medulloblastoma tumor biopsies. These data suggest that the subcellular localization of PBR defines its function.

Cycle-to-cycle variability attributed to the primary respiratory mechanism.

Wave forms attributed to the primary respiratory mechanism (PRM), as published by Viola Frymann, DO, in JAOA June 1971, were analyzed for an undescribed parameter, cycle-to-cycle variability. Tracings from this paper were independently measured by two physicians focusing on the duration of each cycle. Consistency of the measurements and interexaminer agreement were shown. The duration of individual cycles demonstrated significant cycle-to-cycle variability ranging from 0.6 second up to 6.3 seconds. The reason for variability as well as its clinical significance is unknown. The minute rate of each tracing ranged from 6.5 to 13.8 cycles per minute (0.108 to 0.230 Hz [corrected]), mean 10.8 +/- 2.3 (0.180 +/- 0.038 Hz [corrected]). (Different minute rates attributed to the PRM have been reported in other studies.) Although variability is an innate characteristic of biologic cycles, this phenomenon has not been previously reported for the PRM. The authors suggest that this variability has likely confounded previous interexaminer reliability studies and should be considered in any future studies of this type. Determination of causes of this variability present timely and fruitful avenues of research.

Update on osteopathic medical concepts and the lymphatic system.

The osteopathic medical profession has long recognized the importance of the lymphatic system in maintaining health. A review of scientific studies shows much information on the mechanisms and importance of lymph circulation. Many osteopathic manipulative techniques designed to treat patients with tissue congestion are based on early research recognizing that lymph flow is influenced by myofascial compression. Osteopathic manipulative treatment of the diaphragm was substantiated when pressure differentials created by the thoracic diaphragm were shown to influence lymph flow. Current research demonstrates that autonomically mediated, intrinsic lymphatic contractility plays a significant role in lymph propulsion, supporting the use of osteopathic manipulative techniques directed at influencing the autonomic nervous system to improve lymphatic circulation. Although research provides an explanation of how osteopathic manipulative techniques influence the lymphatic system, experimentation to test the direct influence of manipulation on lymph circulation is needed. Clinical outcomes studies are also necessary to substantiate the clinical efficacy of osteopathic manipulative techniques.