Fluorescence studies of the location and membrane accessibility of the palmitoylation sites of rhodopsin

Fluorescent fatty acid labels have been incorporated into the palmitoylation sites of rhodopsin and used to probe the membrane accessibility and location of these sites. The fluorescence properties of anthroyloxy and pyrenyl fatty acids bound to rhodopsin were investigated in a reconstituted vesicle system. Collisional quenching of fluorescence by stearic acid (DSA) labeled with doxyls in the 16, 12, and 5 positions was used to determine the membrane accessibility and disposition of the modifying fatty acids. To properly determine the membrane concentration of these quenchers, the dependence of the Stern-Volmer parameters on both quencher and vesicle concentration was determined. An analysis of these dependences provided a correction for partitioning of the quencher between the aqueous phase and the membrane. After this correction, the relative effectiveness of doxyl quenchers was 16-DSA > 12-DSA > 5-DSA. Parallel studies on free anthroyloxy and pyrenyl fatty acids incorporated into the reconstituted system showed the same dependence on quencher position. These results indicate that the labels at the palmitoylation sites of rhodopsin are situated in the membrane much as a free fatty acid. This anchoring of the palmitates in the membrane results in the formation of a fourth cytoplasmic loop.     

Sulfation of parabens and tyrosylpeptides by bacterial arylsulfate sulfotransferases

Arylsulfate sulfotransferase purified from Eubacterium A-44 has higher specific activity than the enzymes from Klebsiella K-36 and Haemophilus K-12. Propylparaben and butylparaben were good substrates among several parabens. The antibacterial activity of parabens was reduced by the sulfation of the phenolic hydroxy group. Tyrosine-containing peptides, kyotorphin, enkephalin and cholecystokinin non-sulfate, were effective as acceptor substrates by A-44, K-36 and K-12 sulfotransferases.     

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.     

Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues

Methylglyoxal synthase provides bacteria with an alternative to triosephosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP). In the present studies, the methylglyoxal synthase gene in Escherichia coli has been cloned and sequenced. The identified open reading frame (ORF) codes for a polypeptide of 152 amino acids, consistent with the 17 kDa purified protein. The sequence of this protein is not similar to any other protein of known function, including the functionally similar protein triosephosphate isomerase. The methylglyoxal synthase gene was amplified by PCR, subcloned into the pET16B expression vector, and expressed in the host E. coli BL21(DE3). Sequence comparison of the methylglyoxal protein and related ORFs from four different bacterial species revealed that four aspartic acid and no glutamic acid residues are absolutely conserved. The function of the four aspartic acid residues was tested by mutating them to either asparagine or glutamic acid. Thermal denaturation, CD spectroscopy, and gel filtration experiments showed that the mutant enzymes had the same secondary and quaternary structure as the wild-type enzyme. Kinetic characterization of both Asp 71 and Asp 101 mutant proteins shows reduced kcat/Km by 10(3)- and 10(4)-fold respectively, suggesting that they are both intimately involved in catalysis. A time-dependent inhibition of both Asp 20 and Asp 91 asparagine mutants by DHAP suggests that these two residues are involved with protecting the enzyme from DHAP or reactive intermediates along the catalytic pathway. In combination with the results of 2-phosphoglycolate binding studies, a catalytic mechanism is proposed.     

Liberation of phospholipids from Z-disks of chicken skeletal muscle myofibrils by 0.1 mM calcium ions: weakening mechanism for Z-disks during post-mortem aging of meat

Weakening of the Z-disks of skeletal muscle myofibrils contributes to the tenderization of meat during post-mortem aging. To elucidate the weakening mechanism, we compared Z-disks weakened by post-mortem aging of chicken breast muscle with those of myofibrils treated with a solution containing 0.1 mM CaCl2 and 1 microM calpastatin domain I. In both cases, the Z-disks were weakened with a corresponding liberation of their constituent phospholipids (PLs). The liberation of PLs specific to 0.1 mM calcium ions was minimal at pH 6.5 and maximal at 35 degrees C together with the Z-disk weakening. Binding of calcium ions to PLs in the Z-disks was determined by 45Ca-autoradiography. Acidic PLs were strongly radioactive and neutral PLs were appreciably radioactive. It is very probable that acidic PLs would bind electrostatically to alpha-actinin under physiological conditions, and that this interaction would be broken by the binding of calcium ions at 0.1 mM to PLs, resulting in the partial liberation of PLs from Z-disks. We conclude, therefore, that the liberation of PLs by the binding of 0.1 mM calcium ions was the main cause for Z-disk weakening during the post-mortem aging of chicken.     

Treatment of HCV positive individuals with normal serum ALT levels

A substantial number of cases of chronic hepatitis C infection can be shown to have normal or near normal serum ALT levels. Major questions that arise when such cases are seen are: should such patients be treated; if so for how long; and what is the end point of therapy. The reported experience of such patients treated by IFN is reviewed.     

Intrathyroidal parathyroid glands can be a cause of failed cervical exploration for hyperparathyroidism

BACKGROUND: The incidence of intrathyroidal parathyroid glands remains controversial. The purpose of this study was to determine the incidence in a series of patients with hyperparathyroidism. METHODS: Three hundred nine patients underwent parathyroidectomy. Patients were divided into two groups: uniglandular disease versus hyperplasia. RESULTS: Eighteen of 309 patients (6%) had abnormal intrathyroidal parathyroid glands. The incidence was 3% (7 of 222) in patients with uniglandular disease versus 15% (11 of 73) in those with hyperplasia. With a mean follow-up of 54 months, 12 patients are eucalcemic, 5 have persistent hypocalcemia, and 1 has recurrent hypercalcemia. There were no recurrent laryngeal nerve injuries. CONCLUSIONS: These data suggest that an intrathyroidal adenoma is an uncommon cause of failure, whereas abnormal intrathyroidal parathyroid tissue may be a more common cause of failure in patients with hyperplasia.