Overnight (1 mg) dexamethasone suppression testing reliably distinguishes non-cushingoid obesity from Cushing's syndrome.

To determine the sensitivity of the overnight 1-mg dexamethasone suppression test in diagnosing Cushing's syndrome, we evaluated the cortisol responses of 55 subjects (25 non-obese individuals with body mass index less than 25 kg/m2, 20 obese individuals with body mass index greater than 30 kg/m2, and 10 patients with surgically proven Cushing's syndrome) following ingestion of 1 mg dexamethasone at midnight. The basal 8 AM plasma cortisol levels among non-obese and obese individuals and patients with Cushing's syndrome were 310 +/- 85, 377 +/- 91, and 813 +/- 270 nmol/L, respectively. Following 1 mg of dexamethasone, Cushing's syndrome patients showed minimal suppression of cortisol to 609 +/- 180 nmol/L (P = 0.79). Non-obese and obese individuals suppressed to 18.7 +/- 6.0 nmol/L (P less than 0.001) and 22 +/- 7.1 nmol/L (P = 0.003), respectively. The results demonstrated similar cortisol responses to overnight dexamethasone suppression in obese and non-obese groups, and clearly distinguished these subjects from those with Cushing's syndrome. Obesity is not a confounding factor in the 1-mg dexamethasone suppression test.     

Antibodies to the N-terminal portion of cartilage-inducing factor A and transforming growth factor beta. Immunohistochemical localization and association with differentiating cells

Two apparently homologous proteins, designated CIF-A and CIF-B, were previously isolated from bovine bone on the basis of their cartilage-inducing activity in culture. CIF-A has been shown to probably be identical to transforming growth factor beta (TGF-beta). To address the question of tissue localization, antibodies to CIF-A were produced using a synthetic polypeptide identical to N-terminal residues 1-30. The antibodies were immunoreactive with bovine CIF-A and human TGF-beta, did not recognize CIF-B, and did not recognize other molecular weight species in crude bovine bone extracts. The antibodies were used to immunohistochemically localize CIF-A/TGF-beta in fetal bovine bone and other tissues. There was abundant staining of osteocytes throughout cancellous and cortical bone as well as chondrocytes within the articular cartilage, although growth plate-associated chondrocytes were not labeled. In addition, immunoreactive cells were detected in bone marrow (megakaryocytes and some mononuclear cells), fetal liver (hematopoietic stems cells), and the thymus (Hassall's corpuscle and some medullary thymocytes). In the kidney, the antibodies labeled a population of epithelial cells lining the calyces. Tissues which did not have detectable amounts of CIF-A/TGF-beta included the thyroid, adrenal, salivary gland, and aorta. Results presented here suggest that the factor may function in vivo as a general development and repair factor and may play a significant role in the differentiation of many cell types including chondrocytes, osteocytes, T-lymphocytes, and red blood cells.     

Processing of digestive vacuoles in Tetrahymena and the effects of dichloroisoproterenol

The digestive-lysosomal system in Tetrahymena has been extensively studied; however, the various vacuole stages and the existence of a required processing period prior to defecation have not been clearly defined. In this study the presence of such a required processing period and the rate of DV defecation in Tetrahymena thermophila were determined. Like the cycle in Paramecium, a digestive cycle in Tetrahymena consisted of two periods: the processing period was 45 min and the defecation period was approximately 2 h, making the complete cycle approximately 3 h. During the defecation period vacuole egestion followed the kinetics of a first-order rate reaction and had a rate constant of 0.0187/min and a t1/2 of 37 min (82 min into the cycle). Using the naphthol AS-TR phosphate-hexazotized rosanilin method to visualize acid phosphatase activity at the light microscopic level, DVs became positive beginning at 10 min. The number of positive DVs increased to a maximum of 13% when DVs were 20-min old and declined to 5-7% beyond 30 min. Although dichloroisoproterenol (DCI) has been reported by others to stimulate vacuole defecation, we found it inhibited the defecation rate. The extent of inhibition depended on the age of the DVs when exposed to DCI. Vacuole formation was completely blocked in cells preexposed to 40 microM DCI for only 10 min; however, upon further exposure, cells could recover from this inhibition. The time required for complete recovery increased with increasing DCI concentrations. If DCI was given to cells simultaneously with latex beads, it was found to exert a dose-dependent inhibitory effect on DV formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and sensitive assays for phagosomal acidification in Paramecium and Tetrahymena

Biochemical and cytochemical procedures were developed to measure the rate of phagosomal acidification for phagosomal pH ranging from 5 to 2.5. These assays were based on the pH-dependent inactivation with time of horseradish peroxidase (HRP) activity, a result attributable to the dissociation of this enzyme to a colorless protein and ferriprotoporphyrin in acidic solutions. When preincubated in buffers of varying pH, the rate of HRP inactivation followed a sigmoid curve, with the highest rate of inactivation between 4.3 and 3.5 when using citrate-phosphate buffer and between pH 3.4 and 2.8 when using the universal ABC buffer. This inactivation was temperature but not concentration dependent. When Paramecium caudatum, members of the P. aurelia complex or Tetrahymena thermophila was pulsed briefly with HRP and small fluorescent beads, the loss of HRP activity, measured biochemically in cell homogenates and/or cytochemically in phagosomes, was rapid and followed the kinetics of a first-order rate reaction. Both assays gave similar values for the rate constant for acidification and similar rates of inhibition when P. caudatum was exposed to a proton ionophore, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. These assays can readily be adapted to other phagocytic cells as long as a rapid procedure is available for removing all unphagocytosed HRP and latex beads. These procedures are sensitive and rapid thus allowing many samples to be quickly prepared and analyzed.     

Retrieval of lysosomal membrane and acid phosphatase from phagolysosomes of Paramecium caudatum

Little is known about the fate of lysosomal membrane in phagocytic cells. Because the age of the digestive vacuoles in Paramecium caudatum can be easily determined, we have been able to study the dynamic membrane events in the older vacuoles. Late in the phagolysosomal stage (DV-III) the vacuole membrane undergoes a burst of tubule formation. The tubules expand into vesicles which have characteristics resembling lysosomes in both thin sections and freeze-fracture replicas. The tubules also contain acid phosphatase activity when they arise from acid phosphatase-reactive vacuoles. We conclude that after active digestion lysosomal membrane is retrieved in whole or in part along with some membrane-associated hydrolases. A logical extension of these results is that the lysosome-like vesicles, after being recharged with hydrolases by fusing with primary lysosomes, are recycled back to DV-II for reuse.     

Elucidation of a minimal immunoreactive site of vertebrate calmodulin

The heptapeptide AsnTyrGluGluPheValGlnNH₂ corresponding to residues 137–143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem.256, 4205–4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127–144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only sevenresidue segment in the 135–145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.     

Cardiac and vascular actions of sarafotoxin S6b and endothelin-1

Snake venom-derived sarafotoxin S6B (SRT) and porcine endothelium-derived endothelin-1 (ET) have striking structural similarities. In conscious, freely-moving rats, ET (0.67 nmol/kg) produced a transient tachycardia and fall in arterial blood pressure which was followed by a long-lasting increase in arterial pressure, bradycardia, decrease in cardiac output (CO) and marked increase in total peripheral resistance. In contrast, SRT (0.67 nmol/kg) produced only the sustained cardiovascular responses. The sustained cardiovascular effects of SRT or ET were similarly attenuated by nifedipine. SRT and ET (30 nM) produced vasoconstriction in the isolated perfused mesenteric vascular bed without initial vasodilation. SRT and ET had potent positive inotropic and negative chronotropic effects on isolated perfused hearts and induced toxic reactions including coronary vasospasm, arrhythmias, A-V block and ventricular fibrillation. In addition to SRT lacking the initial depressor response in vivo, several differences in the activities of the peptides were also observed. ET produced greater and longer-lasting actions than SRT in producing pressor and vasoconstrictor responses in all 3 preparations, and in its ability to induce toxic effects on the heart.     

Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-α has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling. © 1994 Wiley-Liss, Inc.     

Effects of dimethylsulfoxide (DMSO) on the digestive-lysosomal system in Paramecium caudatum

The effects of DMSO (dimethylsulfoxide) on cell growth and on the digestive-lysosomal system of axenically grown Paramecium caudatum were studied. A general protocol of exposing cells to different concentrations of DMSO at the beginning of each of the four processes in the digestive cycle enabled us to analyze the effect of DMSO at each step. Vacuole formation and the beginning of a digestive cycle were initiated by adding latex beads to the cells. Maximum cell densities at stationary phase of growth were found to be inversely proportional to DMSO between 0.5 and 1.75%, and the duration of the generation time was exponentially proportional. At 2% DMSO cellular division was completely blocked, and above 2% it was cytotoxic. P. caudatum survived for 8 h in 4% DMSO and died instantaneously in 10%. This inhibitory effect on growth was reversible, though this reversibility might depend on the duration and level of DMSO exposure. DMSO exerted a dose- and time-dependent inhibitory effect on the rate of DV formation but had little effect on the acidification-condensation and the lysosome fusion-digestion processes. The size of the DV formed was also reduced, and this effect was dose-but not time-dependent; vacuole size reduction occurred immediately with DMSO exposure, and no further reduction was observed during exposures of up to 24 h. DMSO at 3 and 4% inhibited vacuole defecation, but the cells could overcome this inhibition when exposed to DMSO for longer periods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen-specific stimulation of T cell extracellular acidification by MHC class II-peptide complexes.

A specific T cell response to a preformed complex of detergent-solubilized MHC class II molecule and cognate antigenic peptide was observed by monitoring the extracellular acidification. An increase in this rate was observed when the resting 4R3.9 T cell clone specific for the peptide fragment MBP(1-14) of myelin basic protein was exposed to preformed detergent-solubilized IAk-MBP(1-14)A4 complexes. MBP peptide alone, IAk alone, or complexes of IAs-proteolipid protein(139-151) and IAd-OVA(323-339), did not cause significant increases in the acidification rates of the MBP(1-14)-restricted 4R3.9 T cell clone. In addition, BW 5147 T lymphoma cells, which lack TCR, did not show any increase in rate when exposed to IAk-MBP(1-14)A4 complexes. Similar increases in acidification rate were observed in the presence of IL-2, anti-CD3 and anti-TCR antibodies. The enhanced acidification responses were blocked by genistein, a tyrosine kinase inhibitor.