Addressing differences in cancer: a framework for synergistic programming in cancer prevention and control.

Background: Cancer remains a leading cause of death worldwide and continues to disproportionately impact certain populations. Several frameworks have been developed that illustrate the multiple determinants of cancer. Expanding upon the work of others, we present an applied framework for cancer prevention and control designed to help clinicians, as well as public health practitioners and researchers, better address differences in cancer outcomes. Methods: The framework was developed by the Cancer Prevention and Control Research Network's Health Behaviors Workgroup. An initial framework draft was developed based on workgroup discussion, public health theory, and rapid literature review on the determinants of cancer. The framework was refined through interviews and focus groups with Federally Qualified Health Center providers (n=2) and cancer patients (n=2); participants were asked to provide feedback on the framework's causal pathways, completeness, and applicability to their work and personal life. Results: The framework provides an overview of the relationships between sociodemographic inequalities, social and structural determinants, and key risk factors associated with cancer diagnosis, survivorship, and cancer morbidity and mortality across the lifespan. The framework emphasizes how health-risk behaviors like cigarette smoking interact with psychological, psychosocial, biological, and psychosocial risk factors, as well as healthcare-related behavior and other chronic diseases. Importantly, the framework emphasizes addressing social and structural determinants that influence health behaviors to reduce the burden of cancer and improve health equity. Aligned with previous theory, our framework underscores the importance of addressing co-occurring risk factors and disease states, understanding the complex relationships between factors that influence cancer, and assessing how multiple forms of inequality or disadvantage intersect to increase cancer risk across the lifespan. Conclusions: This paper presents an applied framework for cancer prevention and control to address cancer differences. Because the framework highlights determinants and factors that influence cancer risk at multiple levels, it can be used to inform the development, implementation, and evaluation of interventions to address cancer morbidity and mortality.

Decadal forest dynamics in logged and unlogged sites at Uppangala, Western Ghats, India.

Selective logging disrupts forests, changing their structure and species composition. Long-term monitoring helps in identifying the factors influencing it and aids in designing management plans. We conducted a quantitative re-assessment of trees ≥ 30 cm girth at breast height in four 1 ha plots in logged and two 1 ha plots in adjacent unlogged compartments of Uppangala forest continuum in the Western Ghats, India to compare the structural and compositional changes after a decade (2010-2021). Altogether, four species disappeared and three species were newly recruited. Mean species richness and stem density of both the forest sites decreased. Logged plots showed a slight increase in basal area (2.5%) and biomass (5.1%), whereas unlogged plots showed a decline in basal area (3.92%) and biomass (2.9%). As compared to unlogged plots, all the demographic rates were higher for logged forest sites. Across the six individual plots, the growth rates varied significantly owing to wood density and forest strata categories. Non-metric multidimensional scaling (NMDS) identified three groups with significant difference in species composition, where logged and unlogged plots have a distinct composition except for one plot. Although species richness and stem diversity remained stable, the species composition is different 37 years after logging, and the impacts of logging are still evident in the forest.

Nucleated succession by an endemic palm Phoenix pusilla enhances diversity of woody species in the arid Coromandel Coast of India.

BACKGROUND AND AIMS: Phoenix pusilla, an endemic shrubby palm, was used as a model nurse plant in degraded tropical dry evergreen forest (TDEF) landscapes. This choice was informed by traditional ecological knowledge of the Irula tribe of south India. We tested whether the presence of P. pusilla in water-stressed arid regions improves conditions for other species to establish, resulting in nucleated succession. Success would point the way forward for establishing species-rich woodland in abandoned farm land on the south-eastern Coromandel Coast of India. METHODOLOGY: Spatial associations of woody species in the natural landscape were studied. Experimental tests of nurse plant potential examined the extent to which P. pusilla (i) promoted seed germination, (ii) seedling emergence and (iii) establishment of two TDEF species, and (iv) ameliorated soil and microclimatic conditions over 8 months. PRINCIPAL RESULTS: Phoenix pusilla cooled the soil by up to 50 % and decreased radiation by up to 9-fold, especially in summer. Soil organic matter and water-holding capacity increased, as did seedling number and seedling height of tested TDEF species. The presence of P. pusilla favoured a greater abundance (20 %) of woody plants with a bias towards primary (11) rather than secondary (2) species, indicating species specificity of the effect. CONCLUSIONS: Phoenix pusilla ameliorated abiotic stresses present in open ground to create a patchy species-rich mosaic. This nucleated succession created using P. pusilla provided an important refuge for primary TDEF species. This effect can be expected to have impact at the landscape scale and may prove useful in managing landscapes and in biodiversity conservation. The conservation value of these patchy landscapes deserves to be more widely recognized as they persist in populated areas and thus merit protection. The value of traditional tribal knowledge in identifying a highly effective nurse species is highlighted by this study.

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies.

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of naïve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.

Evaluation of the growth inhibitory activities of Triphala against common bacterial isolates from HIV infected patients.

The isolation of microbial agents less susceptible to regular antibiotics and the rising trend in the recovery rates of resistant bacteria highlights the need for newer alternative principles. Triphala has been used in traditional medicine practice against certain diseases such as jaundice, fever, cough, eye diseases etc. In the present study phytochemical (phenolic, flavonoid and carotenoid) and antibacterial activities of aqueous and ethanol extracts of Triphala and its individual components (Terminalia chebula, Terminalia belerica and Emblica officinalis) were tested against certain bacterial isolates (Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, S. flexneri, Staphylococcus aureus, Vibrio cholerae, Salmonella paratyphi-B, Escherichia coli, Enterococcus faecalis, Salmonella typhi) obtained from HIV infected patients using Kirby-Bauer's disk diffusion and minimum inhibitory concentration (MIC) methods. T. chebula was found to possess high phytochemical content followed by T. belerica and E. officinalis in both aqueous and ethanol extracts. Further, most of the bacterial isolates were inhibited by the ethanol and aqueous extracts of T. chebula followed by T. belerica and E. officinalis by both disk diffusion and MIC methods. The present study revealed that both individual and combined aqueous and ethanol extracts of Triphala have antibacterial activity against the bacterial isolates tested.

Effect of methanol intoxication on specific immune functions of albino rats.

Our previous studies revealed that methanol intoxication significantly altered the non-specific immune functions in albino rats. The present investigation focuses on the effect of methanol on certain specific immune functions of cell mediated immunity such as footpad thickness, leukocyte migration inhibition test (LMI) and antibody levels. In addition, serum interleukins (IL-2, IL-4, TNF-alpha and IFN-gamma), and splenic lymphocyte subsets were measured after an immune challenge. The specific immune function tests were carried out in three different groups of albino rats, which include control, 15 and 30 days methanol intoxication. Our study reports that animal body weight, organ weight ratio, lymphoid cell counts, footpad thickness, antibody titer, IL-2, TNF-alpha, IFN-gamma, Pan T cell, CD4, macrophages, MHC class II molecule expression, and B cell counts were significantly decreased compared to control animals nevertheless, LMI, IL-4, and DNA single strand breakage were increased significantly. Plasma corticosterone level was significantly increased in the 15 days group whereas the 30 days methanol intoxication group showed considerable decrease in corticosterone level compared with control animals. Therefore, our investigation concluded that repeated exposure of methanol profoundly suppressed the cell mediated and humoral immune functions in albino rats.

Effect of methanol intoxication on rat neutrophil functions.

Methanol (MeOH) toxicity, a potential problem from accidental, intentional, as well as occupational and daily ingestion of the agent, receives attention only after severe signs of intoxication have set in or death is imminent. While accidental and intentional exposures involve high doses, the occupational and ingestion forms more often reflect small daily intakes. Still, even at these low levels, little is known about the potential immunotoxic implications from these recurring exposures. As innate immunity confers a first-line of defense against infection, a study was designed to examine the effects of daily exposure to MeOH (at (1)/(4) LD(50) level, for up to 15 or 30 days) on neutrophil (PMN) functions using rats that were (or were not) injected with sheep red blood cells (SRBC) during the course of exposures. Blood samples were analyzed for total (TLC) and differential leucocyte counts (DLC), and isolated neutrophils (PMN) were assessed for changes in function by monitoring phagocytic (PI) and avidity indices (AI), nitroblue tetrazolium (NBT) reduction, and adherence. Body weights were monitored during exposures and weights of major immune system organs (i.e., spleen, thymus, lymph nodes) were assessed at sacrifice. Body and organ weight, TLC, blood PMN levels, PMN PI, and adherence were all significantly decreased in SRBC-untreated rats that received MeOH, although these cells did also display significant increases in AI and NBT reduction. With SRBC-treated rats, though the percentage of PMN in the blood increased with ongoing MeOH exposure, all the other parameters were markedly decreased in comparison to their controls. Thus, this study showed that repeated exposures to MeOH modulates PMN functions, thereby potentially altering the first line of defense in a normal immune response in exposed hosts.

Mutational analysis and biochemical characterization of the Burkholderia thailandensis DW503 quorum-sensing network.

Numerous gram-negative bacteria communicate and regulate gene expression through a cell density-responsive mechanism termed quorum sensing (QS), which involves the synthesis and perception of diffusible N-acyl-homoserine lactones (AHL). In this study we genetically and physiologically characterized the Burkholderia thailandensis DW503 QS network. In silico analysis of the B. thailandensis genome revealed the presence of at least three AHL synthases (AHS) and five transcriptional regulators belonging to the LuxIR family of proteins. Mass spectrometry demonstrated that wild-type B. thailandensis synthesizes N-hexanoyl-homoserine lactone (C6-HSL), N-octanoyl-homoserine lactone (C8-HSL), and N-decanoyl-homoserine lactone (C10-HSL). Mutation of the btaI1 (luxI) AHS gene prevented accumulation of C8-HSL in culture supernatants, enhanced beta-hemolysis of sheep erythrocytes, increased lipase production, and altered colony morphology on swarming and twitching motility plates. Disruption of the btaI3 (luxI) AHS prevented biosynthesis of C6-HSL and increased lipase production and beta-hemolysis, whereas mutagenesis of the btaI2 (luxI) allele eliminated C10-HSL accumulation and reduced lipase production. Complementation of the btaI1 and btaI3 mutants fully restored the synthesis of C8-HSL and C6-HSL to parental levels. In contrast, mutagenesis of the btaR1, btaR3, btaR4, and btaR5 (luxR) transcriptional regulators had no effect on AHL accumulation, enhanced lipase production, and resulted in extensive beta-hemolysis on sheep blood agar plates. Furthermore, interruption of the btaI1, btaR1, and btaR3 genes altered colony morphology on twitching and swarming motility plates and induced pigmentation. Additionally, phenotypic microarray analysis indicated that QS in B. thailandensis both positively and negatively affects the metabolism of numerous substrates, including citric acid, formic acid, glucose 6-phosphate, capric acid, gamma-hydroxybutyric acid, and d-arabinose. These results demonstrate that mutagenesis of the B. thailandensis QS system affects various cellular processes, including lipase production, swarming and twitching motility, beta-hemolysis of sheep erythrocytes, and carbon metabolism and/or transport.

Influence of glucose on production and N-sulfation of heparan sulfate in cultured adipocyte cells.

Altered lipoprotein lipase regulation associated with diabetes leading to the development of hypertriglyceridemia might be attributed to possible changes in content and the fine structure of heparan sulfate and its associated lipoprotein lipase. Adipocyte cell surface is the primary site of synthesis of lipoprotein lipase and the enzyme is bound to cell surface heparan sulfate proteoglycans via heparan sulfate side chains. In this study, the effect of diabetes on the production of adipocyte heparan sulfate and its sulfation (especially N-sulfation) were examined. Mouse 3T3-L1 adipocytes were exposed to high glucose (25 mM) and low glucose (5.55 mM) in the medium and cell-associated heparan sulfate was isolated and characterized. A significant decrease in total content of heparan sulfate was observed in adipocytes cultured under high glucose as compared to low glucose conditions. The degree of N-sulfation was-assessed through oligosaccharide mapping of heparan sulfate after chemical cleavages involving low pH (1.5) nitrous acid and hydrazinolysis/high pH (4.0) nitrous acid treatments; N-sulfation was found to be comparable between the adipocyte heparan sulfates produced under these glucose conditions. The activity and message levels for N-deacetylase/N-sulfotransferase, the enzyme responsible for N-sulfation in the biosynthesis of heparan sulfate, did not vary in adipocytes whether they were exposed to low or high glucose. While most cells or tissues in diabetic situations produce heparan sulfate with low-charge density concomitant with a decrease in N-sulfation, adipocyte cell system is an exception in this regard. Heparan sulfate from adipocytes cultured in low glucose conditions binds to lipoprotein lipase by the same order of magnitude as that derived from high glucose conditions. It is apparent that adipocytes cultured under high glucose conditions produce diminished levels of heparan sulfate (without significant changes in N-sulfation). In conclusion, it is possible that the reduction in heparan sulfate in diabetes could contribute to the decreased levels of heparan sulfate associated lipoprotein lipase, leading to diabetic hypertriglyceridemia.

Altered dermatan sulfate structure and reduced heparin cofactor II-stimulating activity of biglycan and decorin from human atherosclerotic plaque.

Biglycan and decorin are small dermatan sulfate-containing proteoglycans in the extracellular matrix of the artery wall. The dermatan sulfate chains are known to stimulate thrombin inhibition by heparin cofactor II (HCII), a plasma proteinase inhibitor that has been detected within the artery wall. The purpose of this study was to analyze the HCII-stimulatory activity of biglycan and decorin isolated from normal human aorta and atherosclerotic lesions type II through VI and to correlate activity with dermatan sulfate chain composition and structure. Biglycan and decorin from plaque exhibited a 24-75% and 38-79% loss of activity, respectively, in thrombin-HCII inhibition assays relative to proteoglycan from normal aorta. A significant negative linear relationship was observed between lesion severity and HCII stimulatory activity (r = 0.79, biglycan; r = 0.63, decorin; p < 0.05). Biglycan, but not decorin, from atherosclerotic plaque contained significantly reduced amounts of iduronic acid and disulfated disaccharides DeltaDi-2,4S and DeltaDi-4,6S relative to proteoglycan from normal artery. Affinity coelectrophoresis analysis of a subset of samples demonstrated that increased interaction of proteoglycan with HCII in agarose gels paralleled increased activity in thrombin-HCII inhibition assays. In conclusion, both biglycan and decorin from atherosclerotic plaque possessed reduced activity with HCII, but only biglycan demonstrated a correlation between activity and specific glycosaminoglycan structural features. Loss of the ability of biglycan and decorin in atherosclerotic lesions to regulate thrombin activity through HCII may be critical in the progression of the disease.

Permeation liquid membrane metal transport: studies of complex stoichiometries and reactions in Cu(II) extraction with the mixture 22DD-laurate in toluene/phenylhexane

The role of lauric acid (LAH) in the transport of copper(II) through a permeation liquid membrane (PLM) comprising 1,10-didecyldiaza-18-crown-6 (22DD) and lauric acid (ratio 1:1) in 1:1 v/v toluene/phenylhexane has been investigated by determining the stoichiometry of metal extraction and of the metal complex formed in the organic phase by performing 1H NMR and liquid/liquid and liquid/membrane extraction measurements. In the absence of copper(II), the 1H NMR data suggest that there is a strong interaction between the proton of LAH and the nitrogen of the 22DD macrocycle but no interaction between the aliphatic long chains of LAH and 22DD. Thus, in the organic solution, the two compounds are associated as (22DD-H)(+)-LA-, the laurate being away from (22DD-H)+. The signal intensity of the acidic proton was found to decrease when the metal Pb(II) was incorporated by the carrier after its extraction from the aqueous phase. Additionally, liquid/liquid as well as liquid/membrane extraction results reveal that Cu(II) extraction proceeds via the loss of two protons from the organic phase. The Cu(II) is found to be located in the 22DD cavity and the stoichiometry of the complex in the organic phase is (22DD-Cu)(2+)-2LA-. Metal extraction is governed by 22DD and laurate acts only as counteranion. An unexpected feature was observed in the liquid/liquid extraction which was that, at low 22DD and LAH concentrations, the slope for log(Kp) = f(pH) was 2 whereas it was much lower at high carrier concentration. This unexpected result seems to stem from impurities present in 22DD: only 0.1 mol% of impurity can indeed influence the exchange ratio of Cu(II) and H+. This type of anomaly, however, is not found in the normal procedure of liquid/membrane extraction possibly due to the lower carrier/metal molar ratio which is used in the classical PLM conditions.

Oligosaccharide sequence of human breast cancer cell heparan sulfate with high affinity for laminin.

Laminin-1 is a basement membrane glycoprotein implicated in tumor-host adhesion, which involves the cell-binding domain(s) of laminin-1 and tumor cell surface heparan sulfate (HS). The specific tumor cell surface HS oligosaccharide sequences that are necessary for binding to laminin-1 have not been characterized. To identify this laminin-binding oligosaccharide sequence, GlcNSO4-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) residues were isolated from human breast cancer cell (MCF-7)-derived HS through hydrazinolysis/high pH (4.0) nitrous acid treatment/[3H]NaBH4 reduction. These oligosaccharides were chromatographed on a laminin-1 affinity column. A high affinity dodecasaccharide was isolated and characterized. Disaccharide analysis yielded IdoA(2-SO4) --> AManR(6-SO4) as the only disaccharide upon treatment of this dodecasaccharide with nitrous acid at low pH (1.5). The sequence of laminin-binding high affinity oligosaccharide is therefore [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5[IdoA(2-SO4) --> AManR(6-SO4)]. Low affinity dodecasaccharides composed of [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5, [IdoA(2-SO4) --> GlcNSO4] were also isolated by laminin-1 affinity chromatography. Molecular modeling studies indicate that a heparin-binding peptide sequence corresponding to amino acid residues 3010-3031 (KQNCLSSRASFRGCVRNLRLSR) in the G domain of laminin-1, modeled as a right-handed alpha-helix, carries an array of basic residues well placed to bind to clusters of sulfate groups on the high affinity dodecasaccharide.

Endothelial cell heparanase modulation of lipoprotein lipase activity. Evidence that heparan sulfate oligosaccharide is an extracellular chaperone.

A unique feature of lipoprotein lipase (LpL), the rate-limiting enzyme in the hydrolysis of circulating triglycerides, is its movement from its cell of synthesis, adipocyte or myocyte, to its site of action, the luminal endothelial surface. This involves processes that allow LpL to be released from the adipocyte cell surface and transferred against the flow of interstitial fluid to the luminal surface of endothelial cells. LpL, an unstable enzyme, must retain its activity during this process. Whether a chaperone-like molecule is involved in LpL stabilization and transport is unclear. In the present study, we tested the hypothesis that endothelial cells secrete factors that release LpL and promote its transfer to the luminal endothelial surface. Incubation of adipocytes with endothelial cell conditioned medium (ECCM) led to release of about 2-fold more LpL activity than control medium. Medium from endothelial cells exposed to lysophosphatidylcholine (lyso-ECCM), a product of LpL lipolysis of lipoproteins, released approximately 3-fold more LpL than ECCM. Concomitant with the release of LpL, adipocyte cell surface heparan sulfate (HS) proteoglycans were degraded suggesting that lyso-ECCM contained a heparanase-like activity. More heparanase was found in media from the basolateral than the apical side of lysolecithin-stimulated polarized endothelial cells. In coculture experiments, lipolysis and lysolecithin stimulation of endothelial cells increased LpL release from adipocytes. LpL released by lyso-ECCM remained stable and did not lose enzymatic activity at 37 degrees C for 1 h. LpL activity was also stabilized by heparanase-digested fragments of HS (HS oligosaccharide) and by purified LpL binding decasaccharide. Moreover, LpL.HS oligosaccharide complexes crossed endothelial cell monolayers and bound to the apical side of the cells. Thus, an endothelial heparanase may play a critical role in releasing subendothelial HS bound proteins, and specific HS oligosaccharides produced by this enzyme may serve as extracellular chaperones.

Isolation of heparin-derived oligosaccharides containing 2-O-sulfated hexuronic acids, by lipoprotein lipase affinity chromatography.

Oligosaccharides (hexa to dodeca) terminating with [3H]2,5-anhydromannitol (AManR) were isolated from heparin by partial cleavage with nitrous acid at low pH (pH 1.5) followed by gel filtration and reduction with [3H]NaBH4. They were subsequently chromatographed on a lipoprotein lipase (LpL)-Sepharose column. High- and low-affinity oligosaccharides for LpL were isolated and characterized. Disaccharide analysis revealed the presence of (IdceA(2-SO4)-->AManR6-SO4) and (IdceA(2-SO4)-->AManR) as the major disaccharide products after low pH nitrous acid treatment. The oligosaccharides are, therefore, enriched in IdceA(2-SO4)-(GlcNSO4 +/- 6-SO4) sequences. Furthermore, they are found to be composed of 2-O-sulfated hexuronic acid-containing sequences, structural features, characteristic of heparin and heparan sulfate oligosaccharides with potential antiproliferative activities. These oligosaccharides may have the potential as lipase-releasing agents from endothelial and adipocyte surfaces.

Specific regulation of procoagulant activity on monocytes. Intrinsic pathway inhibition by chondroitin 4,6-disulfate.

Hypercoagulability of blood, monocytic infiltration, and changes in pericellular and extracellular matrix glycosaminoglycans (GAGs) are observed in atherosclerosis, inflammation, and neoplasia. In the present studies, monocyte procoagulants and different GAGs including chondroitin sulfate (CS) A, CSB, CSC, CSD, CSE, and heparan sulfate, were tested either in clotting assays with whole plasma or in chromogenic assays with purified coagulation proteases. Procoagulant activity in plasma was inhibited by three of the seven GAGs, including heparan sulfate, CSE, and CSB. In contrast, activity of purified coagulation protease was inhibited only by CSE, and the inhibition was observed with intrinsic (factor VIIIa/IXa) but not extrinsic (tissue factor/factor VII) components. Reciprocal titration experiments with enzyme and substrate and Scatchard type analyses were consistent with concentration-dependent inhibitory interactions between CSE and sites on both factor VIIIa and IXa. On purified phospholipids, CSE concentration resulting in half-maximal inhibition (Ki) was 5 ng/ml for interaction with factor IXa and > 500 ng/ml for interaction with factor VIIIa. The Ki values were lower for reactions on purified lipid than for reactions on monocyte surfaces and for reactions on resting than on endotoxin-stimulated monocytes. Experiments with CSE oligosaccharides of defined size indicated that the smallest CSE fragment capable of inhibitory activity was composed of 12-18 monosaccharide units. Collectively, these results indicate that factor X-activating reactions are inhibited by GAGs expressed on monocyte membranes. Inhibition is specific with respect to the structure of both the GAG and the activating protease. Lack of inhibition by added CSA, CSB, and CSC in contrast to CSE strongly suggests a direct role of 4,6-di-O-sulfated N-acetylgalactosamine GAG structures in the inhibition of intrinsic pathway protease. These findings also suggest potential pharmacologic use of CSE as specific anticoagulant in the management of prothrombotic states mediated by intrinsic pathway coagulation reactions.

Oligosaccharide sequences of endothelial cell surface heparan sulfate proteoglycan with affinity for lipoprotein lipase.

Lipoprotein lipase (LpL) catalyzes the hydrolysis of triglycerides in plasma lipoproteins at the luminal surface of the vascular endothelium. This enzyme is bound via electrostatic interactions to heparan sulfate (HS). The specific endothelial cell surface HS oligosaccharide sequences that are necessary for binding of LpL to HS have not been characterized. To identify this LpL-binding oligosaccharide sequence, oligosaccharides were isolated from bovine aortic endothelial cell-derived HS and assessed for LpL binding properties. Endothelial HS chains that were isolated from endothelial total cell-associated proteoglycans were deacetylated by complete hydrazinolysis, cleaved with nitrous acid (pH 4.5), and reduced with [3H]NaBH4. The resulting fragments composed of N-sulfated glucosamine-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) were chromatographed on a LpL-Sepharose column. A high affinity decasaccharide was isolated and characterized. Disaccharide analysis of this decasaccharide indicated that it yielded only the disaccharide IdceA(2-SO4)-->AManR(6-SO4) on treatment with nitrous acid at low pH. Therefore, the sequence of the LpL-binding decasaccharide is [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4)) alpha 1-4]4-IdceA(2-SO4) alpha 1-4AManR(6-SO4) and is distinct from those that bind antithrombin and basic fibroblast growth factor. Partial depolymerization of endothelial HS chains with hydrazine/high pH nitrous acid treatment gave rise to lipase-binding oligosaccharides larger than decasaccharide. However, further complete depolymerization of these oligosaccharides resulted in only a high affinity decasaccharide composed of repeating disaccharide units of [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4))]. These results indicate that the decasaccharide is the active fragment that binds to LpL with high affinity. Molecular modeling studies of the decasaccharide indicate that it presents a linear array of negatively charged sulfate groups that may adopt a favorable disposition to bind to peptide region(s) comprised of basic amino acid residues of LpL with high affinity.

The major proteoglycan of adult rabbit skeletal muscle. Relationship to small proteoglycans of other tissues.

We have been interested in examining the putative biological role(s) of the major proteoglycan of adult skeletal muscle. The small proteoglycans of adult rabbit skeletal muscle and tendon were extracted and purified by sequential density-gradient ultracentrifugation, ion-exchange chromatography and gel filtration. They appeared to be homogeneous by the criterion of gel electrophoresis in SDS and to yield one major product, the core protein, after digestion with chondroitin ABC lyase, also observed after gel electrophoresis. Two major products were obtained when the intact proteoglycans were cleaved by CNBr, and those peptides were separated by SDS/PAGE and by ion-exchange chromatography. Sequencing of the N-terminal amino acids of either the intact proteoglycans or the CNBr-cleaved products allowed for comparison of the muscle and tendon proteoglycan with derived amino acid sequences previously reported for bovine bone proteoglycan. The bone and tendon proteoglycan sequences were remarkably similar, whereas those of the muscle proteoglycan differed from the other two molecules. The major site of glycosaminoglycan substitution was on a peptide fragment distant from the N-terminus, and a presumptive serine residue at position 4 from the N-terminus also appeared to be substituted, perhaps with a small glycosaminoglycan chain. These results provide some insight into the diversity of small proteoglycans of the PG-II class and provide a basis for exploring their mode of genetic expression.

Collagen biosynthesis in rabbit intraarticular patellar tendon transplants.

Autogenous patellar tendon grafts were transplanted into the knees of 40 New Zealand White adult rabbits. Grafts were subsequently analyzed for rate of collagen synthesis, collagen content, collagen type, histologic change, and cyanogen bromide cleavage patterns of collagen to closely assess the nature of collagen in tendon grafts up to 2 years from the time of transplantation. Tendon grafts were placed in rabbit knees as free fragments or were attached to synovium. These studies show that tendon grafts, even without vascularization or stress, remain viable after intraarticular transfer. Vascularization produces a trend toward increased collagen synthesis, but statistical analysis suggests that control levels of collagen synthesis continue after tendon transfers into rabbit knees. Cyanogen bromide cleavage peptides showed appropriate collagen formed by unstressed autogenous tendon transplants removed from rabbit knees up to 2 years from transplantation. All tendon grafts degenerated initially, but began to form histologically healthy looking connective tissue by 18 to 24 weeks after transplantation. Overall, the results are encouraging with regard to the fate of intraarticular tendon grafts.

Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle.

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions. A chondroitin sulfate proteoglycan which constituted about 44% of the total hexuronic acid content of the muscle tissue was isolated. This proteoglycan was found to have an apparent molecular weight [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] of 95,000, consistent with its small hydrodynamic size (Kav = 0.8 on Sepharose CL-2B), and to consist of peptide and glycosaminoglycan in a weight ratio of 1.0/0.8. The average molecular weight of its core protein-oligosaccharide remnants is 50,000, as estimated by SDS-PAGE of the chondroitinase ABC digested proteoglycan. Alkaline NaB3H4 treatment of the intact proteoglycan released chondroitin sulfate chains with an average molecular weight of 21,000. Pronase digestion of the intact proteoglycan generated glycosaminoglycan-peptides with an average of two chondroitin sulfate chains per peptide. These two saccharide units account for the total glycosaminoglycans per molecule and appear to be closely spaced on the core protein.(ABSTRACT TRUNCATED AT 250 WORDS)