Enhanced extractability of articular cartilage proteoglycans in osteoarthrosis (Short Communication)

Tissue contents of small, easily extracted, proteoglycans, relatively poor in keratan sulphate, were compared in normal and osteoarthrotic cartilage. Although the amounts of small proteoglycans were similar in each tissue, as were the collagen contents, some proteoglycans in the diseased cartilage were much more readily extracted than those in the normal tissue.     

Characterization of protein–polysaccharides of articular cartilage from mature and immature pigs

Protein-polysaccharides of femoral articular cartilage from pigs of ages 9 months and 5 weeks were compared after extraction at pH6.8 with iso-osmotic sodium acetate followed by 0.63m-calcium acetate. The cartilage from the younger animals had a higher moisture content and contained considerably larger amounts of protein-polysaccharide, but less than half as much collagen/g. dry weight, than cartilage from the older pigs. There was notably less keratan sulphate in the fractions from the less mature animals. After gel filtration on 6% agarose, elution profiles of the calcium acetate extracts were similar to those of the sodium acetate extracts of the same tissue. Chemical analyses, however, showed that in both age-groups the extraction procedure had achieved a sequential solubilization of protein-polysaccharides in that the initial extracts contained a higher proportion of keratan sulphate than those that were extracted subsequently. Both extracts from the older animals contained up to 25% of a relatively small protein-polysaccharide that was retarded on 6% agarose and that had a lower protein content and less keratan sulphate than the larger protein-polysaccharides. In contrast, in extracts from the less mature cartilage only about 5% of the protein-polysaccharides were small enough to be retarded by 6% agarose, suggesting that the small components may not be precursors of the larger. The average length of chondroitin sulphate chains, as calculated from the analytical data, was the same in the smaller protein-polysaccharides as in the larger.     

AN ELECTRON MICROSCOPIC STUDY OF PINOCYTOSIS IN AMEBA : II. The Cytoplasmic Uptake Phase

This report is devoted principally to a consideration of the fate of the pinocytotic vacuole and its content in the ameba Pelomyxa carolinensis (Chaos chaos). High resolution micrographs of the plasmalemma have shown it to consist of three layers, i.e., an outermost filamentiferous zone, a middle homogeneous zone, and an inner zone which appears to be a unit membrane. The three zones can be identified in the membranes lining the pinoyctotic tunnels and vacuoles of amebas fixed shortly after pinocytosis occurred. The first apparent change in the pinocytotic vacuole is an increase in the surface-to-volume ratio which occurs during the 1st hour of its existence. Within 24 hours the marker substance commonly collects in defecation vacuoles which can be identified by the profiles of bacteria usually found in the lumen. Occasionally, however, thorotrast can be seen in the lumen of the contractile vacuole. The thorotrast appears to enter the two excretory organelles by the coalescence of vesicular fragments of the pinocytotic vacuoles with the limiting membranes of the excretory organelles.     

Histoplasmosis in Montreal During the Fall of 1963, with Observations on Erythema Multiforme

Although Montreal is in an endemic area, significant clinical histoplasmosis with systemic manifestations has been, until recently, infrequently diagnosed. However, since the autumn of 1963, 31 cases of clinically significant histoplasmosis have been seen by the authors. These were divided into two groups: (1) patients in whom the diagnosis was established on the basis of histological and/or cultural demonstration of the fungus; (2) patients in whom the diagnosis was based on a positive histoplasmin skin test, a complement fixation antibody titre of 1:32 or greater and compatible clinical and radiological findings. An additional group of 11 patients who presented with erythema multiforme was investigated and a heretofore unrecognized relationship between histoplasmosis and erythema multiforme was established.     

Identification of a new subpopulation of triad junctions isolated from skeletal muscle; Morphological correlations with intact muscle

Summary It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca²⁺-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, weak, and breakage resistant, strong, triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.     

Human serum amyloid A protein. The assignment of the six major isoforms to three published gene sequences and evidence for two genetic loci

Serum amyloid A protein (apo-SAA) is an acute-phase reactant and an apolipoprotein of high density lipoproteins (HDL). Six major isoforms of apo-SAA occur in humans (pI 6.0, 6.4, 7.0, 7.4, 7.5, 8.0). In this report we have rationalized the phenotypic expression of apo-SAA isoforms with published apo-SAA structures predicted from apo-SAA cDNA's pA1 and pSAA82 and the genomic DNA SAAg9. The six apo-SAA isoforms fall into three pairs, pI 6.0/6.4, 7.0/7.5, and 7.4/8.0, which are products of cDNA pA1, cDNA pSAA82, and genomic DNA SAAg9, respectively. The second of each isoform pair (i.e. pI 6.4, 7.5, and 8.0) is the "primary" product: a 104-residue peptide with the NH2-terminal sequence Arg-Ser-Phe-Phe. Each primary product is processed either to a major 103-residue peptide with the NH2-terminal sequence Ser-Phe-Phe or processed to a minor 102-residue product which results from the loss of both an Arg and a Ser residue from the NH2 termini. These "secondary" products have the lower pI values of 6.0, 7.0, and 7.4, respectively. The isoelectric points of the SAAg9 products were confirmed by expression of SAAg9 in transfected mouse L-cells. Both the pI 8.0 and 7.4 isoforms were present in cellular extracts, suggesting that post-translational modification of apo-SAA may occur intracellularly. However, the greater relative abundance of the pI 7.4 isoform extracellularly suggests that the major conversion may occur after secretion. Whereas the gene corresponding to the pA1 cDNA sequence does not show allelic variation, the segregation characteristics of the pI 7.0/7.5 and 7.4/8.0 isoform pairs amongst individuals suggests that these isoforms are the products of genes (with sequences corresponding to pSAA82 and SAAg9, respectively) which are allelic variants at a single locus distinct from that for the pI 6.0/6.4 isoform pair.     

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.