Oligomerization is an intrinsic property of calsequestrin in normal and transformed skeletal muscle

In skeletal muscle fibers, the high-capacity medium-affinity Ca(2+)-binding protein calsequestrin functions as the major Ca(2+)-reservoir of the sarcoplasmic reticulum. To determine the oligomeric status of calsequestrin, immunoblotting of microsomal proteins following chemical crosslinking was performed. Diagonal non-reducing/reducing two-dimensional gel electrophoresis was employed to unequivocally differentiate between cross-linked species of 63 kDa calsequestrin and calsequestrin-like proteins of higher relative molecular mass. Since chronic low-frequency stimulation has a profound effect on the expression of many muscle-specific protein isoforms, we investigated normal and conditioned muscle fibers. Calsequestrin was found to exist in a wide range of high-molecular-mass clusters in normal and chronically stimulated skeletal muscle fibers. Hence, oligomerization is an intrinsic property of this important Ca(2+)-binding protein and does not appear to be influenced by the fast-to-slow transformation process. Although fiber-type specific differences exist in the physiology of the skeletal muscle Ca(2+)-regulatory system, oligomerization of calsequestrin seems to be essential for proper functioning.     

Questionability of the benefits of routine laparotomy as the surgical approach for pheochromocytomas and abdominal paragangliomas

BACKGROUND: Improvement of preoperative imaging of pheochromocytomas and abdominal paragangliomas may render routine laparotomy questionable as the surgical approach of choice for these lesions. METHODS: We studied the records of 100 patients with chromaffin tumors who underwent abdominal exploration. The disease was familial in 28 patients and was malignant in 19. Seventy-five patients had intraadrenal disease (bilateral in 13). Computed tomography (CT), metaiodobenzylguanidine (MIBG) scintigraphy, and magnetic resonance imaging (MRI) were performed since 1979, 1984, and 1987 in 97, 73, and 43 patients, respectively. False-positive and false-negative results were defined as any discrepancy between imaging results and surgical findings. RESULTS: Overall accuracy of preoperative localization was 85% with CT scan, 77% with MIBG scintigraphy, and 86% with MRI. In unilateral pheochromocytoma, accuracy was 94% with CT scan, 80% with MIBG scintigraphy, and 96% with MRI. When all three studies were performed (n = 38), overall accuracy was 97% and only one extraadrenal tumor in a patient with familial pheochromocytoma was overlooked. CONCLUSIONS: The outstanding accuracy of available imaging techniques questions the strategy of routine laparotomy for sporadic and seemingly benign pheochromocytomas, favoring more elective approaches such as the posterior approach or laparoscopy.     

Prevalence of serological markers of autoimmunity in hemodialysis patients with hepatitis C virus

Pulmonary arteriovenous malformation can occur in up to 25% of patients after a classic Glenn shunt. Although unproven, exclusion of hepatic venous blood from the lungs has been proposed as a possible cause. We present a patient born with anomalous hepatic venous drainage into the left atrium with an intact atrial septum in whom pulmonary arteriovenous malformation developed in childhood. This was reversed after diversion of the hepatic venous drainage to the right atrium, supporting exclusion of hepatic venous flow as the cause of pulmonary arteriovenous malformation. The association with the hepatopulmonary syndrome is discussed.     

ARS Presidential Address: Estrogen therapy: a causal role in endometrial cancer?

Publicity associated with recent reports of a greater frequency of adenocarcinoma of the endometrium among women who take exogenous estrogens has created confusion among physicians and fear among patients. An objective review of available data on estrogen effects and current epidemiologic evidence suggests that estrogen may play an indirect role in the development of endometrial carcinoma. Until definitive data are available, physicians should exercise caution in prescribing estrogens, monitoring the status of the endometrium at regular intervals by sampling and pathologic analysis.     

Cyclic changes of cervical mucus enzymes related to the time of ovulation. I. Alkaline phosphatase

The concentration of alkaline phosphatase in cervical mucus was serially determined during a menstrual cycle in five normal ovulatory women and correlated with the time of ovulation as monitored by the basal body temperature and radioimmunoassay of serum lutenizing hormone (LH), progesterone, and estradiol. The activity of alkaline phosphatase decreased significantly at midcycle just prior to the LH surge and began to rise after ovulation. Self-detection of cervical mucus alkaline phosphatase may provide a practical method of ovulation prediction.     

Technical note: Validation of an in-house bovine serum enzyme immunoassay for progesterone measurement

Measuring circulating progesterone (P4) of dairy cows is a key component of many research studies dealing with basic and applied reproduction physiology. The gold standard in dairy cows for the measurement of P4 in serum is radioimmunoassay (RIA), but it generates radioactive waste and requires licensed facilities. The purpose of this study was to develop and validate an in-house competitive enzyme immunoassay (EIA) to measure the P4 concentration in serum of dairy cattle. The secondary objective was to validate a commercial EIA. In the present study, a competitive EIA was developed using commercially available antibodies and conjugates. Ninety-six well microtiter plates were coated with the secondary antibody and incubated overnight. Following a washing step, the wells were blocked using the primary antibody. Serum samples were prepared by first extracting P4 using petroleum ether, then diluted in working conjugate solution. Samples were pipetted into the coated and blocked plates, then the matching HRP conjugate label (P4-3-HRP, East Coast Bio, North Berwick, ME) was added. The plates were incubated for 2 h, then washed. The substrate solution was added, and the plate was incubated up to 1 h at room temperature in the dark until a blue color had developed. A stop solution was added, and the optical density measured on a microplate reader was set at 450 nm. The binding proportion was calculated by a visible spectrum absorbance reader, and the amount of P4 was calculated using a log-logit regression line. The commercial EIA was executed as suggested by the manufacturer. The validation of the in-house EIA was done by calculating the inter- and intraassay coefficients of variation (CV) and evaluating the parallelism of diluted samples. The results from the in-house and commercial EIA were also compared with the ones from the RIA graphically (scatterplots and Bland-Altman plots) and statistically, using the Spearman correlation coefficient (r) and the Cohen's kappa statistics using a threshold of 1.0 ng/mL (κ). For the in-house EIA, the intraassay CV were all <10%, but the interassay for samples with small and large P4 concentration had CV of 12.5 and 11.0%, respectively. The correlations between the results from the EIA and the RIA were strong (in-house: r = 0.90; commercial: r = 0.83). At small concentrations (<1.0 ng/mL), however, the correlation with the gold standard was weak (in-house: r = 0.27; commercial: r = 0.14). This was likely due to the lack of accuracy at small concentrations, also shown by the absence of parallelism in samples ≤0.4 ng/mL. In conclusion, results from both the in-house and commercial EIA strongly correlated with the gold standard, but less so at smaller concentrations. The in-house EIA offers good accuracy to measure P4 in samples with a concentration >0.4 ng/mL, and a perfect agreement with RIA using a threshold of 1.0 ng/mL.