Search

A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core that reduces interaction with P-glycoprotein in bovine brain microvessel endothelial cells is described. Modification and derivatization of the C10 position were carried out using a substrate controlled hydride addition to a key C9 and C10 diketone intermediate. The analogs were tested for tubulin assembly and cytotoxicity, and were shown to retain potency similar to paclitaxel. P-glycoprotein interaction was examined using a rhodamine assay and it was found that simple hydrolysis or epimerization of the C10 acetate of paclitaxel and Taxol C can reduce interaction with the P-glycoprotein transporter that may allow for increased permeation of taxanes into the brain.

The unsubstituted, 3'-Cl, 4'-C1, and 3',4'-diCl C10 analogues of cryptophycin-24 were prepared via total synthesis and tested in vitro for cytotoxicity against MCF-7 and multi-drug-resistant MCF-7/ADR breast cancer cell lines and in a tubulin assembly assay. The ED(50) values ranged from 7.2 to 15.8 microM in the tubulin assay and from 0.05 to 3.4 nM in the cell assays. The presence of a 3'-C1 and/or 4'-C1 substituent on the C10 phenyl ring increased cytotoxicity in the MCF-7 cell line compared to the unsubstituted phenyl ring. The most potent compound in this series possessed a 3'-C1 substituent on the C10 phenyl ring. The 3'-C1 analogue had ED(50) values of 50 and 580 pM in the MCF-7 and MCF-7/ADR cell lines, respectively. Its activity was very similar to the parent compound cryptophycin-24. Substitution of the 4'-MeO group in cryptophycin-24 with a 4'-C1 moiety did not significantly affect cytotoxicity against MCF-7 and MCF-7/ADR cells compared to the parent compound. These results demonstrated that the 4'-MeO group in cryptophycin-24 is not essential and can be replaced with 3'-C1 or 4'-C1 substituents.

Epi-C3-cryptophycin-24, epi-C3-m-chlorobenzyl-cryptophycin-24, and the corresponding styrenes were synthesized and tested in vitro against the MCF-7 and multidrug-resistant MCF-7/ADR breast cancer cell lines and in an in vitro tubulin assembly assay. The results demonstrate that the S configuration at the C3 stereocenter is not required to induce potent cytotoxicity and the m-Cl substituent present on the C10 side chain did not induce any large change in activity.

Hydra, as a member of the phylum Cnidaria, is characterized by a body lining organized as an epithelial bilayer with an intervening extracellular matrix (ECM) termed the mesoglea. Previous studies have established that the mesoglea has components indicative of mammalian ECM such as type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan, and these components appear to play a critical role in hydra head regeneration. A remarkable feature of hydra is its ability to reorganize into its adult structure within 96 hr to 7 days from pellets formed from dissociated hydra cells. This regenerative model has been termed the hydra cell aggregate system. The present study has been designed to characterize the biogenesis of mesoglea in hydra cell aggregates and to determine its role in morphogenesis of aggregates. We find that hydra cell aggregates first form an epithelial bilayer by 12 hr of development and then subsequently develop a mesoglea. Morphogenesis of hydra structure then follows formation of the mesoglea. Immunofluorescence studies indicate that mesoglea components are first deposited between the epithelial bilayer by about 12-17 hr of pellet formation, and pulse-labeling studies indicate that the translation rate of matrix components peaks by 48-72 hr of development. Ultrastructural studies indicate that a mature mesoglea is formed by 48-96 hr of pellet formation. Drugs such as β-aminoproprionitrile and 2,2′-dipydridyl, which interfere with the cross-linking of collagens, and p -nitrophenyl-β-d-xylopyranoside, which interferes with the addition of GAG moieties to proteoglycan core molecules, were found to reversibly block development of hydra cell aggregates. Transmission electron microscopy studies indicate that these drugs affect the ultrastructure of the mesoglea. In addition, both polyclonal and monoclonal antibodies raised to isolated mesoglea were found to block development of hydra cell aggregates. These studies indicate that (1) mesoglea formation is rapid and precedes morphogenetic processes during aggregate development, and (2) formation of mesoglea is essential for normal morphogenesis of hydra cell aggregates.

Principal cells of the pancreatic ductal epithelium have been reported to secrete high molecular weight (HMW) glycoconjugates such as mucins into the ductal lumen. We used a human pancreatic carcinoma cell line of ductal origin (PANC-1) which has retained some of the morphological and biochemical characteristics of normal ductal principal cells as a source for isolation of HMW secretory products. The present study was designed to isolate these HMW secretory products, partially characterize them through biochemical and immunological approaches, and determine the effects of secretin on their synthesis and discharge from PANC-1 cells. Our results indicated that when PANC-1 cells are grown on collagen-coated beads in defined serum-free medium, at least three HMW secretory products could be isolated from the medium. These secretory products had a mass of approximately 200, 280, and 370 kDa based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The 200-kDa species made up proportionally less of the three in the gel-staining pattern. Polyclonal antibodies raised to the 370-kDa secretory product cross-reacted with the 280-kDa species. The 370-kDa secretory product was sulfated and wheat germ agglutinin (WGA) binding indicated that the 370-kDa species was a glycoconjugate. The 280-kDa and 200-kDa species were sulfated to a much lesser degree than the 370-kDa species and WGA binding could not be clearly demonstrated with the 280 kDa or 200 kDa species. Glycosidase and selective degradation studies, however, indicated that all three species contained glycosaminoglycan moieties. Antibodies raised to the 370-kDa secretory product localized to the epithelium of human pancreatic carcinomas but not to other cell types in this neoplastic tissue. The antibody also cross-reacted with the ductal epithelium of normal human pancreas and could be localized to centroacinar cells, the epithelium of intralobular ducts, and the epithelium of interlobular ducts. The antibody did not cross-react with goblet cells of the human small or large intestine, indicating no generalized reactivity to gastrointestinal mucins. ELISA and pulse-chase immunoprecipitation studies indicated an increase in the cellular content and synthesis of these HMW secretory products after stimulation of PANC-1 cells with 10(-8)- to 10(-11)-M secretin. We correlated secretin stimulation with the appearance of numerous membrane bound vesicles throughout the cytoplasm as monitored at the ultrastructural level. Optimal secretin concentration were in the range of 10(-10) M. The content of these vesicles was immunoreactive to the antibody raised against the 370-kDa secretory product as determined by ultrastructural immunocytochemical techniques.(ABSTRACT TRUNCATED AT 400 WORDS)

An increase of basement membrane thickness in renal glomeruli, blood vessels, and other tissues is a consistent pathological observation in individuals with diabetes mellitus. Although a number of pathological complications of the disease are thought to result from this structural abnormality in basement membranes, the mechanism(s) responsible for this glucose-mediated process remain unknown. The current study was designed to develop a non-mammalian in vivo epithelial/basement membrane model which would facilitate detailed analysis of the cellular and molecular processes which lead to thickening of basement membrane under hyperglycaemic conditions. The system developed utilizes the Cnidarian, Hydra vulgaris. Hydra lends itself to such studies because of (1) its simplified body structure which is composed of an epithelial bilayer with an intervening basement membrane (mesoglea) and (2) its extensive regenerative capacity which allows cell pellets (Hydra cell aggregates), formed from isolated Hydra cells, to develop into adult Hydra within 72-96 h. This process involves reformation of an epithelial bilayer and de novo biosynthesis of a basement membrane. Our studies indicate that exposure of developing Hydra cell aggregates to levels of D-glucose which mimic that observed in the human diabetic patient (15 mmol/l) induces a doubling of Hydra basement membrane thickness within 72-96 h of pellet formation. The same results were obtained using 15 mmol/l D-Ribose which is a highly efficient glycating agent. The data presented support the use of the Hydra cell aggregate system as a potentially powerful non-mammalian in vivo model to investigate the cellular and molecular mechanism(s) underlying glucose-mediated basement membrane thickening.

This study evaluated the suitability of some disinfection and sterilization methods for use with microdialysis probes. Disinfection or sterilization should minimize the tissue inflammatory reaction and improve the long-term health of rats on study and ensure the quality of data obtained by microdialysis sampling. Furthermore, the treatment should not negatively impact probe integrity or sampling performance. The techniques chosen for evaluation included two disinfection methods (70% ethanol and a commercial contact lens solution) and two sterilization methods (hydrogen peroxide plasma, and e-beam radiation). Linear microdialysis probes treated by these processes were compared to untreated probes removed from the manufacturer's packaging as if sterile (the control group). The probes were aseptically implanted in the livers of rats and monitored for 72 hours. The parameters chosen to evaluate probe performance were relative sample mass recovery and the relative in vivo extraction efficiency of the probe for caffeine. Post mortem bacterial counts and histopathology examination of liver tissue were also conducted. The probes remained intact and functional for the entire study period. The methods tested did not acutely alter the probes although hydrogen peroxide plasma and contact lens solution groups showed reduced extraction efficiencies. Minimal tissue damage was observed surrounding the probes and acute inflammatory reaction was mild to moderate. Low numbers of bacterial colonies from the implantation sites indicates that the health of animals in this study was not impaired. This was also true for the control group (untreated probe).

Methylphenidate (MPD), also called Ritalin, changes the extracellular levels of dopamine (DA) in the brain. This study coupled multiple-site microdialysis sampling with appropriate analytical methods to simultaneously profile the MPD concentration in blood and brain, while monitoring changes in the extracellular level of DA in the striatum of awake and freely moving rats. The animals' activity was also recorded. The maximum concentration of MPD in the blood and brain occurred during the first 20 min of sampling. The maximum DA concentration was reached in the first 20 min and gradually returned to the basal level after 3 h. The activity peak correlated well with the MPD and DA peaks and remained elevated for about 2.5 h. The ability to obtain and correlate data in this manner has the potential to reduce the number of animals required for a given study and to minimize interanimal variation.

BACKGROUND & AIMS: Cystic fibrosis is characterized by increased secretion of glycoconjugates with altered carbohydrate composition, but no specific gene products that show these changes have been identified. The aim of this study was to use a recently described sulfated mucin- like glycoprotein ( MUCLIN: formerly called gp300) as a model glycoconjugate to study such changes in the gastrointestinal system in the cystic fibrosis transmembrane conductance regulator (CFTR) knockout mouse (cftrm1Unc). METHODS: Western and Northern blots were used to determine the tissue levels of MUCLIN and its messenger RNA (mRNA) in normal and CFTR knockout mice. Immunocytochemistry was used to determine the localization of MUCLIN. RESULTS: MUCLIN is expressed in the normal mouse intestinal tract, pancreas, and gallbladder. In CFTR knockout mice, MUCLIN shows increased expression at both mRNA and protein levels in pancreas and duodenum, but not in the gallbladder. In the duodenum, MUCLIN was localized intracellularly in crypt enterocytes and on the luminal surface, and luminal surface labeling was dramatically increased in the CFTR knockout mouse. In the CFTR knockout mouse duodenum and gallbladder, MUCLIN showed retarded electrophoretic migration indicating altered posttranslational processing. CONCLUSIONS: MUCLIN shows increased expression and possibly altered posttranslational processing in the CFTR knockout mouse and will serve as a good model for understanding changes in the composition of mucous secretions in patients with this disease. (Gastroenterology 1997 Aug;113(2):521-32)

The Cnidarian, hydra, lends itself to studies related to the role of extracellular matrix (ECM) components in development because of its high regenerative capacity and its simple structure, which is organized as an epithelial bilayer with an intervening ECM termed the mesoglea. Previous immunocytochemical and biochemical studies have established that hydra mesoglea contains many of the major matrix components (e.g., fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan) associated with the ECM of vertebrate and more complex invertebrate species. Additional studies have also established that ECM components have a critical role in hydra development as monitored during head regeneration and morphogenesis of hydra cell aggregates. In the present study a monoclonal antibody (mAb52) raised to isolated hydra mesoglea was used as a probe in additional functional studies and to screen a cDNA expression library made from poly(A)+ RNA isolated from Hydra vulgaris. Immunofluorescent analysis indicated that mAb52 was localized along the entire longitudinal axis of adult polyps in what is termed the subepithelial zones of hydra mesoglea. Cytochemical studies found these subepithelial zones to be rich in anionic sites. Previous studies have shown that mAb52 blocks hydra cell aggregate development and experiments in the current study have shown that mAb52 also blocks in vivo interstitial cell (I-cell) migration in hydra grafts. Sequence analysis of cDNA clones isolated using mAb52 indicated that the protein encoded by these clones had structural homology with mammalian and Drosophila laminin B1 chain and hybridized to a single 6.75-kb band on Northern blots of total hydra RNA. One interesting difference in hydra laminin B1 was the presence of a FTGTQ amino acid sequence in place of the vertebrate YIGSR cell binding domain. Under nonreducing conditions, polyclonal antibodies against FTGTQ bound to the same > 200-kDa band on Western blots of mesoglea as mAb52 and also immunolocalized to the subepithelial zones. Under reducing conditions, anti-FTGTQ antibodies bound to a single band with a mass of approximately 200 kDa. In addition, FTGTQ peptide inhibited adhesion of dissociated hydra cells to mesoglea and anti-FTGTQ antibodies inhibited hydra cell binding to substrates coated with mesoglea or FTGTQ peptide. Anti-FTGTQ antibodies also inhibited in vivo I-cell migration in hydra grafts. Given the early divergence of Cnidarians during evolution, these studies indicate the highly conserved nature of laminin and provide additional information regarding the critical role of ECM components during hydra development.

Although the basic biochemical defect in cystic fibrosis (CF) is unknown, previous studies have indicated that errors in protein glycosylation may be involved in the pathogenesis of the disease. Utilizing human skin fibroblasts, the present study was designed to quantitatively analyze glycosylation of cell surface glycoconjugates in CF and normal cells. Cell surface glycoconjugates were analyzed using 125I-concanavilin A (Con A), 125I-WGA, and Con A-ferritin conjugates. Under our binding conditions, Con A was used as a probe for mannose residues and WGA was used as a probe for N-acetylglucosamine residues. Saturable binding of both probes was observed and appropriate sugar controls confirmed the specificity of each lectin. When compared on a DNA basis, iodinated lectin binding studies indicated that no consistent differences existed between CF and normal strains of human skin fibroblasts. Ultrastructural quantitative morphometric analysis of Con A-ferritin conjugate binding indicated that neither proteolysis of cell surface glycoconjugates or internalization of lectin probes was occurring at saturable binding concentrations. In summary, our results indicated that no consistent differences in cell surface mannose and N-acetylglucosamine residues could be detected between the normal and CF strains of human skin fibroblasts used in these studies.

Summary Pancreatic ductal cell secretion has not been well characterized due to the difficulty in obtaining sufficient quantities of purified ductal cells. To determine if the MIA PaCa-2 cell line would provide a useful model for in vitro studies of pancreatic ductal cell secretion, the present study was designed to characterize these cells in greater detail. In this investigation, the human pancreatic undifferentiated cell line, MIA PaCa-2, was compared with PANC-1 cells (a human ductal cell line previously characterized), isolated rat and human ducts, acinar cells, and nonpancreatic cell lines. The results indicate that while the morphology of the MIA PaCa-2 cell line is nonpolarized and generally atypical of either ductal or acinar cells, the cell line has retained certain biochemical similarities to ductal cells. Additional morphological studies indicated (a) the presence of intermediate filaments characteristic of epithelial cells, (b) the absence of zymogen granules, and (c) an apparent basolateral plasma membrane localization of Na+, K+-ATPase. Similar to ductal cells, biochemical analyses indicated (a) the presence of Na+, K+-ATPase based on [3H]-ouabain binding assays, (b) high levels of carbonic anhydrase, (c) low levels of γ-glutamyl transpeptidase, (d) nondetectable levels of amylase, and (e) protein composition and protein synthetic patterns comparable to PANC-1 cells. Finally, as with PANC-1 cells and isolated rat and human ducts, the major sulfated secretory product of MIA PaCa-2 cells was a protein with a molecular weight of approximately 660,000 to 1 million. These results suggest that while the MIA PaCa-2 cell line can be used to study pancreatic ductal cell secretion, it possesses less ductal-like characteristics than observed with the PANC- 1 cell line.

Elevated levels of serum and urinary L-homocitrulline (HC) are seen in citrullinemia and in several other genetic disorders of the urea cycle and lysine catabolism. In recent studies on the metabolism of HC in rats and mice we have found that HC is converted to a previously undescribed compound (Fed. Proc. 35:1478, 1976). We now report evidence indicating that the initial step in the formation of the new compound is a transamination. The conversion of HC to the cyclic derivative, in vitro, proceeds in mouse liver homogenates, but not in preparations of kidney, brain, spleen, lung, muscle or intestine. The most effective amino-group acceptors for the reaction are pyruvate and glyoxylate; α-ketoglutarate, oxaloacetate, α-ketomalonate, α-ketobutyrate, and α-ketoisocaproate are active but less effective. This transamination may represent a significant route for the disposal of HC and, possibly, other alpha-amino acid intermediates which accumulate in disorders of urea, lysine, and ammonia metabolism. This reaction may also contribute to the depletion of available α-ketoacids in clinical situations with elevated levels of HC, homoarginine, citrulline, and similar compounds.