Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

Human T cell responses to the Epstein-Barr nuclear antigen-1 (EBNA-1) as evaluated by synthetic peptides

A panel of synthetic peptides derived from Epstein-Barr virus (EBV) nuclear antigen 1 (EB-NA-1) was used to examine human T cell responses to this antigen. In six of seven normal persons with past EBV infection, T cell precursors specific for five peptides (P27, amino acid residues 83-101;P62, 148-166;E31, 353-367;E41, 368-381; and E11, 461-474) were detectable. The precursor frequencies were in the range of 1:20,000 to less than 1:100,000 peripheral blood mononuclear cells as determined by limiting dilution analyses. Only two of these peptides were predicted as alpha-helices; all peptides were glycine-rich. Four other peptides were not reactive in the seven individuals tested. T cell responses were not detectable in donors without prior EBV infection. Infectious mononucleosis patients investigated 4-6 weeks after diagnosis had likewise no detectable peptide-specific T cell precursors. Thus, it appears that T cells recognizing peptides from EBNA-1 arise and persist in people with past EBV infection.     

Ethnobotany of purple coneflower (Echinacea angustifolia,Asteraceae) and OtherEchinacea Species

The purple coneflower, Echinacea angustifolia, was the most widely used medicinal plant of the Plains Indians. It was used for a variety of ailments, including toothache, coughs, colds, sore throats, snakebite, and as a painkiller. H. C. F. Meyer used it as a patent medicine in the 1870s and introduced it to the medical profession. Recent scientific research (mostly German) onEchinacea species has shown that they possess immunostimulatory activity. Increased cultivation ofE. purpurea andE. angustifolia may be needed to meet the increased demand for its roots and to alleviate the effects of overharvesting of wild stands.     

Biological degradation of 2,4-dichlorophenoxyacetic acid: chloride mass balance in stirred tank reactors.

A mass balance was developed for the degradation of 2,4-dichlorophenoxyacetic acid by a mixed culture. Batch culture experiments showed the degradation to be an acid-producing step. Inorganic chloride concentration consistently correlated with the expected value and with base consumption to maintain a constant pH.     

The receptors for prolactin and growth hormone are localized in the same region of human chromosome 5

Prolactin receptor (PRLR) and growth hormone receptor (GHR) are encoded by members of a gene family containing regions of identical sequences. To determine their chromosomal locations, cDNA probes for these genes were used. Analysis of hybridization to several somatic cell hybrids, together with hybridization in situ to metaphase chromosomes, resulted in the assignment of the loci for both receptors to human chromosome 5 in the region p13----p14. Thus, these proteins may be encoded by a cluster of related genes that evolved from a common ancestral gene, in a manner consonant with that of their ligands.     

Deficient glycosylation of arylsulfatase A in pseudo arylsulfatase-A deficiency

Summary Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.Abbreviations PD Pseudo arylsulfatase-A Deficiency - ARA Arylsulfatase-A     

The human PDGF receptor α-subunit gene maps to chromosome 4 in close proximity to c-kit

Summary The gene encoding the -subunit of the human platelet-derived growth factor receptor (PDGFRA) maps to band q11–q12 of chromosome 4 by in situ hybridization, which was confirmed by Southern analysis of a Chinese hamster × human cell hybrid that retains only human chromosome 4.