Effect of metabolic inhibitors on vasopressin-stimulated transport systems in the toad bladder.

Vasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin-stimulated water flow or vasopressin-stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally. Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP-stimulated water flow but showed no effect on urea transport. We would conclude that water and urea transport, as examples of hormone-stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non-nucleotide pathway may be involved in the action of vasopressin.     

Synthesis of 2- [ (4-Hydroxylcarbomylphenyl) -methylamino ] - N- ( 2- aminophenyl ) acetamide

Early studies revealed that the hybrid polar/apolar compound N, N′-hexamethylene bisacetaamide (HMBA) was a very effective inducer of differentiation in a transformed cell lines. [1] The use of this polar/apolar compound to induce murine erythroleukemia cells (MELC) to undergo erythoid differentiation with suppression of oncogenicity has proved a useful model to study inducer-mediated differentiation of transformed cell. [2] To search more effective inducers, we utilized the computer to design a new target compound. Herein, we reported the synthesis of title compound.     

THE EFFECT OF ULTRAVIOLET LIGHT ON THE CYCLIC NUCLEOTIDE SYSTEM OF HUMAN FIBROBLASTS

Abstract— The concentrations of cyclic AMP and cyclic GMP in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m², and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, our results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may affect the extranuclear functions of irradiated cells.     

Cyclic AMP and low molecular weight effector(s) present in yeast extract are involved in pectin lyase production by Penicillium griseoroseum cultured on sucrose

Pectin lyase (PL) induction by organic and inorganic components of yeast extract (YE) was evaluated in Penicillium griseoroseum, cultured in a mineral medium containing sucrose, by determining PL activity (A235) and mycelial growth (mycelial dry weight). The lowest YE concentration that promoted significant PL induction without acting as a carbon source for the fungus corresponded to 0.0075%. Neither calcined YE nor a nutrient solution containing micronutrients induced PL production, indicating that the inducer was an organic compound. Vitamins, phospholipid components, amino acids, and nitrogenous bases were tested in place of YE and promoted no significant PL induction. A PL inducer compound was found to be soluble in the nucleotide fraction obtained during extraction of YE. The inducer was shown to be a thermostable polar substance dialyzable at 2000 Daltons, hydrolyzable by HCl, and activated by boiling for up to 60 min. Cyclic AMP (cAMP) exogenously added to the culture medium at 5 and 10 mM was capable of inducing PL in P. griseoroseum grown on sucrose, suggesting that at least one compound may be present in YE acting in a cooperative fashion for the maintenance of high levels of cAMP into the cell. PL activity and the level of cAMP inside the fungal cells increased after the addition of YE to the culture medium, suggesting the participation of this messenger in this enzyme's synthesis.     

Use of photolithography to encode cell adhesive domains into protein microarrays

Protein microarrays are rapidly emerging as valuable tools in creating combinatorial cell culture systems where inducers of cellular differentiation can be identified in a rapid and multiplexed fashion. In the present study, protein microarraying was combined with photoresist lithography to enable printing of extracellular matrix (ECM) protein arrays while precisely controlling "on-the-spot" cell-cell interactions. In this surface engineering approach, the micropatterned photoresist layer formed on a glass substrate served as a temporary stencil during the microarray printing, defining the micrometer-scale dimensions and the geometry of the cell-adhesion domains within the printed protein spots. After removal of the photoresist, the glass substrates contained micrometer-scale cell-adhesive regions that were encoded within 300 or 500 microm diameter protein domains. Fluorescence microscopy and atomic force microscopy (AFM) were employed to characterize protein micropatterns. When incubated with micropatterned surfaces, hepatic (HepG2) cells attached on 300 or 500 mum diameter protein spots; however, the extent of cell-cell contacts within each spot varied in accordance with dimensions of the photoresist stencil, from single cells attaching on 30 microm diameter features to multicell clusters residing on 100 or 200 microm diameter regions. Importantly, the photoresist removal process was shown to have no detrimental effects on the ability of several ECM proteins (collagens I, II, and IV and laminin) to support functional hepatic cultures. The micropatterning approach described here allows for a small cell population seeded onto a single cell culture substrate to be exposed to multiple scenarios of cell-cell and cell-surface interactions in parallel. This technology will be particularly useful for high-throughput screening of biological stimuli required for tissue specification of stem cells or for maintenance of differentiated phenotype in scarce primary cells.     

The correlation of plasma membrane microvilli and intracellular cyclic AMP content in a rat epitheloid kidney cell line.

Modulation of the intracellular concentration of cyclic AMP has been associated with a regulatory role in cell division, cell morphology, and physical properties of the plasma membrane. Untransformed rat kidney cells in culture exhibit epitheloid morphology, high intracellular cyclic AMP levels, and contact inhibition of growth. Untransformed rat kidney cells transformed with the Kirsten murine sarcoma virus exhibit a low cyclic AMP content, rapid growth rate, and a loss of contact inhibition. Scanning electron microscopy reveals a distinctive difference in the surface structure of the two cell types during Gl of the cell cycle. The surface of the transformed cell is covered with microvilli while its untransformed counterpart is devoid of microvilli. The presence of microvilli can be controlled as a function of temperature by two temperature-sensitive mutants of the Kirsten sarcoma virus (ts6t6 and ts371 cl 5). In the ts6t6 mutant, growth at 32 degrees C results in a low cyclic AMP content and the presence of microville, while growth at 39 degrees C results in a high cyclic AMP content and a decrease in microvilli. The opposite effect is seen with the ts371 cl 5 mutant. Correlation of cyclic AMP content with the presence of microvilli suggests that this surface phenomenon is a function of cyclic AMP concentration.     

DNA synthesis in cultured human fibroblasts: regulation by 3':5'-cyclic AMP.

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [3H] thymidine incorporation into acid-precipitable material, beginning after 8-12 hr and reaching maximum levels of 16-24 hr. Addition of dibutyryl-3':5'-cyclic AMP (DBcAMP) together with serum inhibited [3H] thymidine incorporation by 75-95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3':5'-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [3H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G1 was inhibited by high concentrations of 3':5'-cyclic AMP.     

环肽类组蛋白去乙酰化酶抑制剂

组蛋白乙酰化转移酶(HAT)和组蛋白去乙酰化酶(HDAC)调节组蛋白乙酰化程度，HDAC在基因表达和染色体形成等方面起着重要的调节作用。HDAC抑制剂能够引起肿瘤细胞生长停滞、诱导肿瘤细胞分化和调亡。通过对各种HDAC抑制剂结构及作用机制的研究有助于该类药物在临床上的应用和拓宽癌症治疗的适用范围。本文概述了近年来天然及合成的环肽类组蛋白去乙酰化酶抑制剂的研究进展。     

Effect of the Inducers Veratryl Alcohol, Xylidine, and Ligninosulphonates on Activity and Thermal Stability and Inactivation Kinetics of Laccase from Trametes versicolor

Laccase production from Trametes versicolor was improved in the presence of the inducers ligninosulphonates, veratryl alcohol, and xylidine respectively two-, four-, and eightfold. The thermal inactivation of the produced laccase, after partial purification with ammonium sulfate was kinetically investigated at various temperatures (60?C70?°C) and pH values (3.5, 4.5, and 5.5). The inactivation process followed first-order kinetics for all conditions tested, except for veratryl alcohol, for which a constant activity level was observed at the end of the inactivation, also after first-order decay. Enzyme thermostability was affected by the type of inducer used in the culture medium for the production of laccase and also by the pH of incubation mixture. Generally, laccase stability increased with pH increment, being more stable at pH?5.5, except with xylidine. At pHs?4.5 and 5.5, the three inducers significantly increased laccase thermal stability, with the higher effect being observed for pH?5.5 and ligninosulphonates, where increment of half-life times ranged from 3- to 20-fold, depending on the temperature.     

UV-DNA Damage in Mouse and Human Cells Induces the Expression of Tumor Necrosis Factor α

Ultraviolet light induces the expression of tumor necrosis factor α (TNFα) in many mammalian cells. We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V in-creased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane-bound TNFα expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNFa gene expression in mouse and human cells.     

Inhibitory effect of extracts of muscles of mackerel (Scomber japonicus Houttuyn) on hepatic glycogenolysis in rats

The effects of extracts of muscles of mackerel (Scomber japonicus; M-ext) on hepatic glycogenolysis were investigated by a rat liver perfusion method. M-ext inhibited glucagon- and cyclic adenosine monophosphate (AMP)-induced glycogenolysis but was ineffective on phenylephrine-induced glycogenolysis. The contents of hepatic glycogen and cyclic AMP, and phosphorylase and glycogen synthase activities in liver were measured after perfusion with glucagon. M-ext inhibited the increase of cyclic AMP and activation of phosphorylase. It is considered that M-ext inhibits hepatic glycogenolysis caused by glucagon through a cyclic AMP-dependent mechanism.     

Effects of sodium salicylate on the febrile response and increased levels of cyclic AMP in cerebrospinal fluid during endogenous pyrogen-induced fever in rabbits

To further evaluate the causality between endogenous pyrogen (EP)-induced fever and cyclic adenosine-3',5'-monophosphate (cyclic AMP) level, the effects of sodium salicylate (SS) on the febrile response and increased levels of cyclic AMP in both cerebrospinal fluid (c.s.f.) and plasma during EP-induced fever in rabbits were observed. The results suggest that cyclic AMP is probably involved in the central mediation of EP-induced fever and that increased concentration of cyclic AMP in c.s.f. associated with EP-induced fever is not the result of temperature elevation but appears to be caused by the increased synthesis in the central nervous system. In addition it is confirmed that blood is impossibly a contributory source of increased cyclic AMP in c.s.f. during EP fever, and that SS may act subsequent to the increase in cyclic AMP.     

Calculation of relative binding free energy differences for fructose 1,6-bisphosphatase inhibitors using the thermodynamic cycle perturbation approach.

An iterative, computer-assisted, drug design strategy that combines molecular design, molecular mechanics, molecular dynamics (MD), and free energy perturbation (FEP) calculations with compound synthesis, biochemical testing of inhibitors, and crystallographic structure determination of protein-inhibitor complexes was successfully used to predict the rank order of a series of nucleoside monophosphate analogues as fructose 1,6-bisphosphatase (FBPase) inhibitors. The X-ray structure of FBPase complexed with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) provided structural information used for subsequent analogue design and free energy calculations. The FEP protocol was validated by calculating the free energy differences for the mutation of ZMP (1) to AMP (2). The calculated results showed a net gain of 1.7 kcal/mol, which agreed with the experimental result of 1.3 kcal/mol. FEP calculations were performed for 18 other AMP analogues. Inhibition constants were determined for over half of these analogues, usually after completion of the calculation, and were consistent with the predictions. Solvation free energy differences between AMP and various AMP analogues proved to be an important factor in binding free energies, suggesting that increased desolvation costs associated with the addition of polar groups to an inhibitor must be overcome by stronger ligand-protein interactions if the structural modification is to enhance inhibitor potency. The results indicate that FEP calculations predict relative binding affinities with high accuracy and provide valuable insight into the factors that influence inhibitor binding and therefore should greatly aid efforts to optimize initial lead compounds and reduce the time required for the discovery of new drug candidates.     

The mode of action of hormones

Higher organisms having anatomically and functionally differentiated organ systems must be able to coordinate biochemical processes within and between the various organs or cells, even under rapidly changing environmental conditions. Hormones play an important part in the regulation processes: they promote numerous metabolic reactions and differentiation processes, and show considerable differences in the speed with which they act. Some hormones act almost instantaneously by stimulating adenyl cyclase and thus initiating the production of adenosine 3′,5′-monophosphate (cyclic AMP), which passes on the hormone message (“second messenger”) to the intracellular metabolism by modifying enzyme activities. Delayed-action hormones, mainly morphogenetic hormones, act primarily on the cell nucleus where they induce the synthesis of certain enzymes by activation of genes.     

EFFECTS OF SODIUM SALICYLATE ON THE FEBRILE RESPONSE AND INCREASED LEVELS OF CYCLIC AMP IN CEREBRO-SPINAL FLUID DURING ENDOGENOUS PYROGEN-INDUCED FEVER IN RABBITS

To further evaluate the causality between endogenous pyrogen (EP)-induced fever andcyclic adenosine- 3', 5'- monophosphate (cyclic AMP) level. the effects of sodium salicylate(SS) on the febrile response and increased levels of cyclic AMP in both cerebrospinal fluid(c.s.f.) and plasma during EP- induced fever in rabbits were observed. The results suggestthat cyclic AMP is probably involved in the central mediation of EP-induced fever and thatincreased concentration of cyclic AMP in c.s.f. associated with EP- induced fever is not theresult of temperature elevation but appears to be caused by the increased synthesis in the cen-tral nervous system. In addition it is confirmed that blood is impossibly a contributorysource of increased cyclic AMP in c.s.f. during EP fever, and that SS may act subsequentto the increase in cyclic AMP.     

Effect of salicin on induction and carbon catabolite repression of endoxylanase synthesis in Penicillium janthinellum MTCC 10889

Amongst various carbon sources, xylan was found to be the sole inducer of endoxylanase production by Penicillium janthinellum MTCC 10889 in submerged cultivation. Endoxylanase synthesis by a xylan induced culture was initially repressed after a simultaneous addition of xylose, probably by the inducer exclusion mechanism, but it was resumed and achieved its highest level at a much later stage of growth (at 120 h). Xylose added after 30 h of growth cannot exert its full repressive effect. Although glucose was proved to be a more potent repressor than xylose, supplementation of salicin, an alcoholic β-glycoside containing d-glucose, with pure xylan resulted in an about 3.22 fold increase in the enzyme synthesis at 72 h followed by constant high production of the enzyme at least until the 144th h of growth. Inducing capacity of salicin in a xylan induced culture was significantly reduced when it was added after 30 h of growth. Addition of salicin and xylan help to partially overcome the repressive effect of xylose and glucose. Failure of salicin in recovering the endoxylanase synthesis in actinomycin D and cyclohexamide inhibited the xylan induced culture indicating that salicin cannot initiate the de novo synthesis of the enzyme.     

Improved cyclic urea inhibitors of the HIV-1 protease: synthesis,potency, resistance profile,human pharmacokinetics and X-ray crystal structure of DMP 450

Background: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds.Results: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg⁻¹ DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC₉₀ suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response.Conclusions: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.     

Confocal restricted-height imaging of suspension cells (CRISC) in a PDMS microdevice during apoptosis

We have monitored and imaged cell death induced in human leukemic U937 cells over time using three-color confocal imaging. Three different apoptotic inducers, anti-Fas, TNF-alpha and Etoposide were used. Individual cascaded events such as loss of mitochondrial transmembrane potential, exposure of phosphatidyl-serine, membrane blebbing and permeabilization of the cell membrane have been observed in real time with different individual cells. From the results, an interesting heterogeneicity in the apoptotic phenotype has been observed. The CRISC method is easy to use and provides biologist with a powerful additional tool to study in real-time processes of several hours of duration such as apoptosis. We predict that the period of cell viability obtained after protein coating of the PDMS devices (>80 h) will also allow monitoring of other biological processes of longer duration or long onset time, such as mitosis, phagocytosis and differentiation.     

Isolation and Characterization of Oviduct-specific Glycoproteins from Ampulla and Isthmus Parts of Cyclic and Acyclic Buffalo for Studying Differential Microenvironment

The present study characterized the glycoproteins synthesized by buffalo oviduct. Scanning electron microscopy analyses of the ampullary and isthmic segments of cyclic and acyclic buffaloes showed ultrastructural variations in ciliated and nonciliated cells. Mucosal proteins were extracted by scrapping of different segments of oviduct and, after centrifugation, the remainder tissues were subjected to establish primary cell culture system of oviduct epithelial cells and conditioned media were prepared. Time- and concentration-dependent effects of trypsinization on the establishment of primary monolayer culture showed that 0.25% trypsin for 1–2 min at 37 °C were the optimal conditions. Total protein content in oviductal tissues and conditioned media was quantified and analyzed by SDS-PAGE which showed marked variation in different segments of the oviduct. Western blot analysis revealed five major oviduct-specific glycoproteins (OGPs) in cyclic oviduct (ampulla and isthmus) with Mw 180, 95, 75, 66 and 35 kDa in the oviduct extract and two glycoproteins with Mw 95 and 66 kDa in conditioned media. However, in acyclic oviduct (ampulla and isthmus), three glycoproteins were immunostained with Mw 180, 95 and 66 kDa in the oviduct extract and one glycoprotein with Mw 66 kDa in conditioned media. Indirect Enzyme-Linked Immuno Sorbent Assay (ELISA) results showed significant differences of OGPs in different segments of cyclic and acyclic buffaloes and, thus, indicative of segmental variation in the synthesis and secretion of glycoproteins. Oviductal extract secretes more amounts of OGPs as compared to the conditioned medium. The role of these OGPs may be defined and exploited for influencing the fertilization process and/or subsequent embryonic development.     

Hybrid cell adhesive material for instant dielectrophoretic cell trapping and long-term cell function assessment

Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.