Isolation and structural characterization of the polypeptide subunits of membrane glycoprotein IIb-IIIa from human platelets

We have previously demonstrated the isolation of platelet membrane glycoprotein IIb-IIIa by affinity chromatography with a specific monoclonal antibody. We have now separated the polypeptide subunits IIb and IIIa of the isolated glycoprotein by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and have compared their structural features. Both IIb and IIIa contain approximately 15% carbohydrate, but IIIa contains a larger percentage of mannose residues, suggesting the presence of high mannose as well as complex N- linked oligosaccharide chains. The amino acid compositions are sufficiently similar to imply areas of sequence homology between the two subunits. To examine further the relationship between the subunits, we digested a mixture of 125I-IIb and 131I-IIIa with trypsin and then separated the radiolabeled peptides by high performance liquid chromatography. The resultant peptide maps of IIb and IIIa are completely different. This indicates that neither subunit is derived from the other and suggests that polypeptides IIb and IIIa are products of separate genes.     

Episodic dopamine discharge in paroxysmal hypertension. Page's syndrome revisited

Dopamine concentration, a marker of the sympathetic discharge additional to norepinephrine and epinephrine levels, was determined in 31 patients. These patients, mostly women, had essential hypertension and hypertensive episodes that mimicked pheochromocytoma, except that the patients were rather plethoric (instead of pale) and often had associated nausea, epigastric discomfort, and polyuria. During and after hypertensive paroxysms, plasma free norepinephrine and epinephrine levels did not increase, but we found a mean eightfold and 16-fold increase of free and sulfated plasma dopamine levels, respectively, and similar although less marked dopamine level increases in the urine collected following the paroxysm. The hypertensive paroxysms, spontaneous or precipitated by stimulation of the autonomic nervous system, were similar to those described by Page as simulating diencephalic stimulation. Dopamine level may be a marker of the sympathetic discharge, undetected by measurements of free norepinephrine level, and may explain some clinical features of Page's syndrome.     

Detecting fetomaternal hemorrhage: a comparison of five methods

Appropriate postpartum administration of Rh immune globulin relies on sensitive detection and accurate quantitation of fetomaternal hemorrhage (FMH). Recently, the microscopic Du test (micro Du) enhanced with polyethylene glycol (PEG Du) and flow cytometry (FC) have been advocated for this purpose. Three qualitative methods (micro Du, rosette test, and PEG Du) and two quantitative methods (acid elution and FC) for assessing FMH were evaluated with particular attention given to PEG Du and FC. In vitro studies comprised 10 series of dilutions of D+ cord cells in D- adult cells to yield D+ cell concentrations of 0.06, 0.12, 0.25, 0.50, 0.75, 1.0, and 2.0 percent. Additionally, 26 postpartum samples were tested. Of the qualitative techniques, the micro Du test was the least sensitive with 20 percent false-negative results occurring at 0.5 percent fetal cells. The PEG Du test was only slightly more sensitive and offered no clinical advantage. The rosette test was the most sensitive, consistently detecting fetal cells at concentrations of 0.25 percent or greater. FC and acid elution showed similar results, with good correlation obtained between measured and expected quantities of fetal cells (r = 0.99 and 0.96, respectively). One of 26 postpartum samples was positive by all screening techniques; acid elution and FC detected 0.3-percent concentrations of fetal cells and 0.17-percent concentrations of D+ cells, respectively. Although acid elution is a more commonly used method for quantitating FMH, FC offers an acceptable alternative that is capable of analyzing large numbers of cells with objectivity and reproducibility.     

Activation of the contact system in insect-sting anaphylaxis: association with the development of angioedema and shock

A postulated role of the contact system in anaphylactic reactions to insect stings was investigated. During prospective, in-hospital sting challenge, we collected serial blood samples from five normal volunteers and 16 patients with a history of insect-sting anaphylaxis. Activation of the contact system was assessed by measuring plasma levels of factor XIIa-C1-inhibitor and kallikrein-C1-inhibitor complexes as well as those of cleaved high molecular weight kininogen (HK). In addition, antigenic levels of (pre)kallikrein, factor XII, and HK were measured. No significant changes in contact system parameters were observed in any of the five volunteers or the four patients who did not develop an anaphylactic reaction after sting challenge. In contrast, significant changes in contact system parameters occurred in 7 of the 12 patients with anaphylactic symptoms after challenge. Peak levels of either C1-inhibitor complex were found 5 minutes after the onset of anaphylactic symptoms. The increase in C1-inhibitor was most pronounced in the 4 patients with angioedema, 2 of which also developed shock. However, activation of HK was observed in all four patients with angioedema, the two patients with shock but no angioedema, as well as in 1 of the remaining 6 patients with anaphylactic symptoms other than angioedema or shock. Thus, activation products of the contact system may be involved in the pathogenesis of angioedema and shock in insect- sting anaphylaxis.     

Gamma heavy chain disease: rapid, sustained response to cyclophosphamide and prednisone

A patient, CAL, with gamma heavy chain disease is presented who has had a complete remission lasting over 2 yr with combination chemotherapy consisting of pulsatile cyclophosphamide and prednisone. The patient exhibited many features of an atuoimmune process including a vasculitis, low serum complement levels, a positive antiglobulin (Coombs) test, Raynaud's phenomenon, and keratoconjunctivitis sicca. The CAL paraprotein was found to have several previously undescribed characteristics. It reacted with antisera to Fd, Fab, and Fab', suggesting that most of the Fd portion of the molecule was intace. CAL protein consists of two polypeptide chains of molecular weight 49,000 covalently linked to form a dimer of 95,000 molecular weight. The covalent linkage suggests that the hinge region of this gamma heavy chain is intact.     

Antifungal effects on metabolite profiles of medically important yeast species measured by nuclear magnetic resonance spectroscopy

Drug-induced inhibition of fungal growth is used in the diagnostic laboratory to predict therapeutic efficacy but is relatively slow, and determination of endpoints can be problematic. Nuclear magnetic resonance (NMR) spectroscopy identifies the metabolic complement of microorganisms while monitoring utilization of constituents of the incubation medium. This technique may provide a rapid and objective indicator of antifungal effects. We evaluated the effects of caspofungin, amphotericin B (AMB), and voriconazole on metabolic profiles of yeast species cultured in RPMI-2% glucose-morpholinepropanesulfonic acid buffer in microtiter plates in a proof-of-principle study. 1H NMR spectra were obtained using Bruker NMR spectrometers at 1H frequencies of 600 and 360 MHz. Metabolites were identified by two-dimensional correlation NMR spectra, and relative peak integrals were calculated from one-dimensional 1H NMR spectra. MICs were determined by a modification of the Clinical and Laboratory Standards Institute broth microdilution method M27-A. Utilization of glucose and branched-chain and aromatic amino acid substrates was accompanied by fungal production of acetate, acetaldehyde, ethanol, formate, fumarate, glycerol, lactate, pyruvate, and succinate. Clear-cut metabolic endpoints indicating a greater than 50% reduction in substrate utilization and fungal metabolite production which correlated with MICs were noted at 16 and 24 h for all three drugs. At 8 h, reductions of greater than 50% for selected metabolites were noted for caspofungin and AMB. Direct NMR-based observation of metabolic alterations in yeast cultures reveals changes in key metabolic pathways and should be evaluated formally as a rapid technique for determining susceptibility to antifungal drugs.