Immunodetection of G proteins in human pituitary adenomas: evidence for a low expression of proteins of the Gi subfamily

G proteins mediate signal transduction in a variety of cell systems. As the expression of these proteins has not yet been investigated in detail in human pituitary tumors, we evaluated the presence of G proteins in a series of tumors including six non-functioning adenomas, five GH-secreting adenomas, three prolactinomas and one TSH-secreting adenoma, using immunoblotting and immunohistochemistry. By immunoblotting, Gi1/2alpha was undetectable in six and barely detectable in nine tumors. A similar pattern of expression was observed by probing with the antibody to Gi3alpha, which detected a very weak band in 11 tumors and no protein in four. In contrast, using large amounts of membrane proteins (40 microg), both Gi1/2alpha and Gi3alpha were detected, although at very low levels, in the negative tumors. The low expression of these proteins appeared to be specific to tumoral tissues, as both Gi1/2alpha and Gi3alpha were abundant in normal human and rat pituitary. In all tumors, Go alpha, the two Gs alpha forms, Gq/11 and G beta were present in significant amounts. Semiquantitative analysis indicated that Gs alpha was clearly detected when 2.5 microg loaded proteins were used, whereas Gi1/2alpha and Gi3alpha were barely detected with 5 microg. By immunofluorescence, all tumors studied were markedly positive for Gs alpha that was immunolocalized at the cell periphery, whereas they showed a weak positivity for Gi1/2alpha and Gi3alpha. The study is the first to provide evidence for a low expression of Gi proteins, which are involved in the transduction of inhibitory signals, in pituitary adenomas.     

Antibiotics: mechanisms of action

Medical practice rests on the foundation of science. Clinicians are constantly making practical decisions and dealing with immediate situations that demand solutions. Time should be taken to focus on those scientific principles that underlie our diagnostic and therapeutic maneuvers. This section of Pediatrics in Review presents selected topics that are relevant to practice from the areas of physiology, pharmacology, biochemistry, and other disciplines; clarification of these will augment the pediatrician's understanding of clinical procedures.     

Retinoic acid receptor expression abnormalities in lung cancer: important clues or major obstacles?

We have developed a cine display of postprocessed 3D MR images of the thoracic aorta. Maximum intensity projection (MIP) images of the thoracic aorta in each phase were reconstructed from consecutive 2D-cine MR data sets, and displayed in a cine loop. The postprocessed 3D MR images clearly showed the relationship between major cervical branches and aortic pathologies such as aneurysms, and cine display presented the flow pattern in the aorta. 3D-cine MR angiography seems to be useful for follow-up studies of thoracic aortic diseases especially in patients with renal dysfunction.     

Retinoic acid regulates selectively the expression of immediate early response genes in PC12 cells

Six modular acetabular components were evaluated to determine whether screw holes in the metal shell offer a route for fluid and debris into the acetabular bone stock. A 56-mm acetabular shell for each trial was mounted to a sealed chamber and loaded at a 25 degrees angle under axial loads of 270-2,700 N and +/- 2.5-N-m torsional load. Polystyrene microspheres (average diameter, 0.5 microm) were placed in double-deionized water at 300 mmH2O pressure in a sealed chamber above the component. The only channel between the fluid above and the collecting chamber below was through the cup-liner interface and 1 screw hole. Fluid and debris in the collecting chamber were harvested after 1,000,000 cycles. The collected sample was filtered through a 0.2-microm-pore filter and analyzed under electron microscopy for evidence of microspheres. Water and polystyrene microspheres were isolated in the collecting chamber for all trials except the Reflection cup (Smith & Nephew Orthopaedics, Memphis, TN) with a screw hole cover and the Micro-Seal cup (Whiteside Biomechanics, St. Louis, MO) with a peripheral seal. A screw placed in the screw hole of the Reflection cup failed to seal the interface. The peripheral seal around the rim of the Micro-Seal polyethylene prevented fluid and particle flow between the metal shell and polyethylene liner.     

Retinoic acid receptors regulate expression of retinoic acid 4-hydroxylase that specifically inactivates all-trans retinoic acid in human keratinocyte HaCaT cells

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), 7beta-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-Ibeta and/or tumor necrosis factor (TNF)-alpha secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7beta-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1beta secretion. TNF-alpha secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the alpha- or beta-hydroxyl radical position plays a key role in apoptosis and IL-1beta secretion.     

Chronic central leptin infusion enhances insulin-stimulated glucose metabolism and favors the expression of uncoupling proteins

Continuous (4 days) intracerebroventricular leptin infusion (12 microg/day) was performed in lean rats, and its hormonometabolic effects were determined. Intracerebroventricular leptin administration did not result in leakage of the hormone into the peripheral circulation. Thus, its effects were elicited by its presence within the central nervous system. Intracerebroventricular leptin infusion produced marked decreases in food intake and body weight gain relative to vehicle-infused fed ad libitum rats. Because decreases in food intake alter hormonometabolic homeostasis, additional control rats pair-fed to the amount of food consumed by leptin-infused ones were included in the study. Intracerebroventricular leptin-infused and vehicle-infused pair-fed rats were characterized, relative to vehicle-infused ad libitum-fed animals, by decreases in body weight and insulinemia and by increases in insulin-stimulated overall glucose utilization and muscle and brown adipose tissue glucose utilization index. Brown adipose tissue uncoupling protein (UCP)1, UCP2, and UCP3 mRNA levels were markedly decreased in pair-fed animals relative to those of fed ad libitum control animals, as were liver and white adipose tissue UCP2 and muscle UCP3 mRNA levels. In marked contrast, intracerebroventricular leptin administration was accompanied by the maintenance of high UCP1, UCP2, and UCP3 expression in all these tissues. Thus, despite analogies between leptin's effects and those of pair-feeding with regard to glucose handling, their respective underlying mechanisms differ. While leptin maintains or favors energy-dissipating mechanisms (UCP1, UCP2, and UCP3), the latter are markedly depressed in pair-fed rats. This effect of leptin may prevent subsequent excessive storage processes, thereby maintaining normal body homeostasis.     

Molecular mechanisms for cooperative folding of proteins

The folding of single-domain globular proteins exhibits the character of first-order or two-state thermodynamics. The origin of such high cooperativity in relatively small polymer systems is still not well understood. Recently, the statistical mechanics of protein folding has been studied extensively with simple protein models such as short cubic-lattice chains with contact-based interactions. While many valuable insights about protein folding were gained with such models, some concerns have also arisen, viz. that they lack the character of protein backbones whose interactions would limit the folding patterns of proteins. Here, a comparative study of the conventional cubic-lattice chain model and a fine-grained more realistic lattice protein model with both backbone and side-chain interactions is carried out. It is found that, even though both types of models exhibit a cooperative two-state folding transition to the native structure with optimized force fields, the character and origin of cooperativity of the two models are different. In the simple contact-based model, the free-energy barrier occurs at the low end of the energy scale, and the cooperativity arises from a concerted formation of native contacts among many residues in a compact state. In the other more complicated model, the free-energy barrier occurs in the intermediate energy region, and the folding cooperativity arises from collective orientational arrangements of locally structured units in semi-open conformational states. On the basis of these results, two limiting molecular mechanisms for protein folding emerge, which can be used for analyzing the folding process of real proteins.     

Interferon-gamma and retinoic acid down-regulate N-myc in neuroblastoma through complementary mechanisms of action

A method is described for the assay of the major malondialdehyde-deoxyguanosine adduct (M1G) based on immunoaffinity purification and gas chromatography/electron capture/negative chemical ionization/mass spectrometry. A stable isotope of M1G-deoxyribose ([2H2]M1G-dR) was used as an internal standard. Recovery of internal standard throughout the entire assay procedure was approximately 40%. The assay showed a linear response over a range of 10-1000 pg of M1G-dR and was verified by analysis of a synthetic. M1G-containing oligomer. The limit of detection in biological samples was 100 fmol/sample, corresponding to 3 adducts/10(8) bases for 1 mg of DNA. DNA was isolated from the blood of 10 healthy human donors, and M1G levels were measured. A mean value of 6.2 +/- 1.2 adducts/10(8) bases was obtained, with no obvious differences bases on age or cigarette smoking. A small, but statistically significant difference was observed between the levels in females (5.1 +/- 0.4 adducts/10(8) bases) and males 6.7 +/- 1.1 adducts/10(8) bases). The presence of M1G in leukocyte DNA was further verified by analysis using liquid chromatography/electrospray ionization mass spectrometry.