Further novel amido sugars within the glycopeptidolipid antigens of Mycobacterium avium

The individual serovars of the Mycobacterium avium complex, a source of serious and persistent infections in individuals with underlying immune deficiencies, also present an extraordinary set of novel sugar epitopes as part of their type-specific glycopeptidolipid surface antigens. Californium desorption-mass spectrometry has been successfully applied to the holistic glycopeptidolipid antigen of M. avium serovar 12 and its per-O-acetyl derivative, to arrive at the following structure, of molecular mass 1876: (Sequence: see text). The pentasaccharide hapten, released as the tetraglycosyl alditol, was subjected to methylation analysis, absolute configurational analysis, 1H NMR and fast atom bombardment-mass spectrometry to arrive at the structure: 4-(2'-Hydroxy) propionamido-4,6-dideoxy-3-O-Me-Glcp (beta 1----3)-4-O-Me-L-Rhap (alpha 1----3)-L-Rhap (alpha 1----3)-L-Rhap (alpha 1----2)-6-deoxytalitol. Two-dimensional proton correlation spectroscopy was also applied to determine the configuration of the unique distal segment of the oligosaccharide unit. The significance of this structure in the context of the fully elucidated structures of the antigens from 12 of the 31-member M. avium complex is discussed.     

Diglycosyl diacylglycerol of Mycobacterium tuberculosis.

A diglycosyl diacylglycerol was isolated from Mycobacterium tuberculosis, and its structure was established by a combination of methylation analysis, 1H nuclear magnetic resonance, and fast atom bombardment-mass spectrometry. It is a 1,2-diacyl-[beta-D-glucopyranosyl(1"----6')-beta-D-glucopyranosyl(1'---- 3)]- sn-glycerol and exists in at least five molecular species differing in fatty acyl substituents. The major constituent fatty acids were identified as iso- and anteisopentadecanoate, iso- and n-hexadecanoate, and iso- and anteisoheptadecanoate. Although glycosyl diacylglycerols are common membrane components of gram-positive bacteria, this report represents the first substantial evidence for the presence of a glycosyl diacylglycerol within a member of the Mycobacterium genus. Although the glycolipid is not a major component of M. tuberculosis, it reacts readily in enzyme-linked immunosorbent assay against rabbit antibodies raised against whole bacteria and thus may be useful for the serodiagnosis of tuberculosis.     

Antibodies to the N-terminal portion of cartilage-inducing factor A and transforming growth factor beta. Immunohistochemical localization and association with differentiating cells

Two apparently homologous proteins, designated CIF-A and CIF-B, were previously isolated from bovine bone on the basis of their cartilage-inducing activity in culture. CIF-A has been shown to probably be identical to transforming growth factor beta (TGF-beta). To address the question of tissue localization, antibodies to CIF-A were produced using a synthetic polypeptide identical to N-terminal residues 1-30. The antibodies were immunoreactive with bovine CIF-A and human TGF-beta, did not recognize CIF-B, and did not recognize other molecular weight species in crude bovine bone extracts. The antibodies were used to immunohistochemically localize CIF-A/TGF-beta in fetal bovine bone and other tissues. There was abundant staining of osteocytes throughout cancellous and cortical bone as well as chondrocytes within the articular cartilage, although growth plate-associated chondrocytes were not labeled. In addition, immunoreactive cells were detected in bone marrow (megakaryocytes and some mononuclear cells), fetal liver (hematopoietic stems cells), and the thymus (Hassall's corpuscle and some medullary thymocytes). In the kidney, the antibodies labeled a population of epithelial cells lining the calyces. Tissues which did not have detectable amounts of CIF-A/TGF-beta included the thyroid, adrenal, salivary gland, and aorta. Results presented here suggest that the factor may function in vivo as a general development and repair factor and may play a significant role in the differentiation of many cell types including chondrocytes, osteocytes, T-lymphocytes, and red blood cells.     

Chondroitin/dermatan sulfate proteoglycan in human fetal membranes. Demonstration of an antigenically similar proteoglycan in fibroblasts

A proteoglycan was isolated from fetal membranes which had been separated from human postpartum placenta. The glycosaminoglycan side chains (Mr = 55,000) were found to be composed of 75% chondroitin sulfate and 23% dermatan sulfate as determined by chondroitinase ABC or AC II digestion. NH2-terminal microsequencing of the intact proteoglycan revealed a single amino acid sequence of (sequence; see text) A rabbit antiserum raised against the intact proteoglycan reacted in sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with Mr = 45,000 and 43,000 core polypeptides from chondroitinase-treated proteoglycan. Affinity-purified antibodies from this antiserum precipitated from human embryonic fibroblast culture fluid a proteoglycan which has an approximate Mr = 120,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This proteoglycan has on the average two polysaccharide side chains. As defined by chondroitinase digestion, these chains consist of 66% dermatan sulfate and 20% chondroitin sulfate. Digestion of the glycosaminoglycan with chondroitinase ABC converted the proteoglycan to a Mr = 45,000 major and a Mr = 43,000 minor core polypeptide. Tissue immunofluorescence localized the proteoglycan to interstitial matrices, suggesting that it is a product of mesenchymal cells. The methods we have devised for the purification of the fetal membrane proteoglycan in chemical amounts and the antibodies we have prepared against it will allow studies on the structural and functional properties of the proteoglycan and on the expression of immunologically cross-reactive proteoglycans by various cells and tissues.     

Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls

We designed a rapid assay that assesses the polychlorinated biphenyl (PCB)-degradative competence and congener specificity of aerobic microorganisms, identifies strains capable of degrading highly chlorinated biphenyls, and distinguishes among those that degrade PCBs by alternative pathways. Prior attempts to assay PCB-degradative competence by measuring disappearance of Aroclors (commercial PCB mixtures) have frequently produced false-positive findings because of volatilization, adsorption, or absorption losses. Furthermore, these assays have generally left the chemical nature of the competence obscure because of incomplete gas chromatographic resolution and uncertain identification of Aroclor peaks. We avoided these problems by using defined mixtures of PCB congeners and by adopting incubation and extraction methods that prevent physical loss of PCBs. Our assay mixtures include PCB congeners ranging from dichloro- to hexachlorobiphenyls and representing various structural classes, e.g., congeners chlorinated on a single ring (2,3-dichlorobiphenyl), blocked at 2,3 sites (2,5,2'5'-tetrachlorobiphenyl), blocked at 3,4 sites (4,4'-dichlorobiphenyl), and lacking adjacent unchlorinated sites (2,4,5,2',4',5'-hexachlorobiphenyl). The PCB-degrative ability of microorganisms is assessed by packed-column gas chromatographic analysis of these defined congener mixtures following 24-h incubation with resting cells. When tested with 25 environmental isolates, this assay revealed a broad range of PCB-degradative competence, highlighted differences in congener specificity and in the extent of degradation of individual congeners, predicted degradative competence on commercial PCBs, and (iv) identified strains with superior PCB-degradative ability.     

An Artificial “gum-tree” for marmosets (Callithrix j. jacchus)

Marmosets (Callithrix, Cebuella) in the wild gouge wells in trees and eat the exudates that accumulate there. An artificial gum-tree was made of wooden dowel and filled with Acacia Senegal exudate (gum arabic) dissolved in water. Three families of marmosets avidly gouged and consumed gum from this device, showing all of the behavioral patterns described in nature. The gum-tree cost little and was easy to make.     

Hemidesmosome formation in vitro

Intact, viable sheets of adult rabbit corneal epithelium, 9 mm in diameter, were prepared by the Dispase II method (Gipson, I. K., and S. M. Grill, 1982, Invest. Ophthalmol. Vis. Sci. 23:269-273). The sheets, freed of the basal lamina, retained their desmosomes and stratified epithelial characteristics, but lacked hemidesmosomes (HD). Epithelial sheets were placed on fresh segments of corneal stroma with denuded basal laminae and incubated in serum-free media for 1, 3, 6, 18, or 24 h. Tissue was processed for electron microscopy, and the number of HD/micron membrane, the number of HDs with anchoring fibrils directly across the lamina densa from them, and the number of anchoring fibrils not associated with HDs were counted. After 6 h in culture, the number of newly formed HD was 82% of controls (normal rabbit corneas), and by 24 h the number had reached 95% of controls. At all time periods studied, 80-86% of HDs had anchoring fibrils directly across the lamina densa from them. Anchoring fibrils not associated with HDs decreased with culture time. These data indicate that the sites where anchoring fibrils insert into the lamina densa may be nucleation sites for new HD formation. Corneal epithelial sheets placed on two other ocular basal laminae, Descemet's membrane and lens capsule, had not formed HDs after 24 h in culture. These two laminae do not have anchoring fibrils associated with them. Rabbit epithelial sheets placed on the denuded epithelial basal lamina of rat and human corneas formed new HDs. Thus, at least in these mammalian species, HD formation may involve some of the same molecular components.     

In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

Structural alterations in fibronectin correlated with morphological changes in smooth muscle cells

Nontransformed cultures of vascular smooth muscle cells proliferate until they form a confluent sheet of cells. Subsequently, the cells become reorganized to form multicellular nodules that are loosely attached to the substrate. The formation of nodules is facilitated by the addition of medium conditioned by nodular cultures. Nodulation is inhibited by the addition of fibronectin. Fibronectins derived from monolayer culture conditioned medium or from plasma are maximally effective while fibronectin isolated from nodular cell conditioned medium is inactive. Analysis by NaDodSO4-polyacrylamide gel electrophoresis reveals that the nodular cell fibronectin has a molecular weight that is about 20-30 kd less than that of monolayer cell fibronectin. Further, nodular cell conditioned medium contains an activity that can convert both plasma fibronectin and monolayer cell fibronectin to the lower molecular weight correlated with the loss of biological activity.