A novel synthetic peptide polymer with cyclic RGD motifs supports serum-free attachment of anchorage-dependent cells

Cell adhesivity is a basic biological principle, which provides mechanisms for construction of multicellular organisms, tissue genesis, migration and individual cell survival. In vivo, the cell adhesive environment is provided by extracellular matrix molecules, neighboring cell surfaces and soluble factors delivered either by tissue cells or by blood circulation. The exact molecular composition of the microenvironment of a cell is not properly understood. The nondefined molecular composition of "native" adhesive components hinders their application when defined culture conditions are necessary, as, for an example, growing human cells for further clinical application. Applying large, substrate-coating molecules as backbones for carrying specific adhesive peptide motifs provides a relatively cheap, reproducible, and chemically defined group of synthetic adhesion molecules. Here, we report on the design, synthesis, and testing of a novel cyclic RGD-containing coating material, which promotes initial attachment, spreading, survival, and proliferation of a number of different cell types. The potent adhesive polypeptide-brush, composed of poly[Lys(DL-Ala(m))] branched chain polypeptide (AK) and multiple copies of cyclic(arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine) pentapeptide prevents anoikis and supports cell attachment in the absence of serum or other biological additives. The defined conditions for cell maintenance make this material a promising candidate for coating artificial cell substrates even for therapeutic applications.     

The isolation of immunogenic molecular entities from immunogenic and nonimmunogenic tumor homogenates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Summary YAC, a Moloney-virus-induced tumor of A-strain mice, is a nonimmunogenic tumor. Mice injected with the inactivated neoplastic cells and challenged with viable tumor cells did not survive longer than mice that received the challenge dose alone. The homogenate of this nonimmunogenic tumor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel slices containing isolated molecular entities were injected into various groups of mice. The mice were challenged with low doses of viable tumor cells (10–30 cells) and their survival time was recorded. Small but significant numbers of mice injected with apparent 80–90 K SDS-PAGE-isolated molecular entity rejected the tumor or survived longer than the control groups of mice. Spleen cells from mice injected with 80–90 K molecular entity inhibited the YAC tumor cotransferred with them to naive recipients (Winn assay). Spleen cells from mice injected with monoclonal antibody against nonspecific T-cell helper factor and immunized with 80–90 K SDS-PAGE-isolated molecular entity failed to inhibit the tumor growth in naive recipients, indicating that helper T cells are involved in induction of the antitumor resistance. Nylon-wool-passed splenocytes from mice injected with 80–90 K inhibited tumor growth in some of the recipient mice. Spleen cells from these mice treated with anti-Thy-1 and complement also inhibited the tumor growth in some of the recipients, suggesting that the effector cells were both T and non-T cells. C57BL/6 mice immunized with apparent 20 K SDS-PAGE-isolated molecular entity of RBL5 tumor also induced in vivo resistance to the syngeneic viable RBL5 cells, but not to the syngeneic B16 melanoma cells, indicating the specificity of the protective effect. The practical and theoretical implications of these findings are discussed.     

Treatment of deep vein thrombosis with streptokinase.

From September 1962 to May 1972 145 patients with acute or subacute deep vein thrombosis confirmed by phlebography were treated with streptokinase. During the same period 42 patients considered unfit for thrombolytic therapy were treated with herapin and oral anticoagulants. The results, assessed by repeat phlebography, in 93 of the patients treated with streptokinase were compared with those in 42 patients treated with heparin. The age, sex, and severity of occlusion were roughly similar in both groups. Streptokinase treatment was successful in 42 per cent, partially successful in 25 per cent, and unsuccessful in 32 per cent of the 93 patients compared with none, 10 per cent, and 88 percent respectively in the 42 patients treated with heparin. Streptokinase was more effective when the thrombus was in proximal rather than calf veins. Thrombi of more than six days old were readily lysed. Plasma fibrinogen levels were below 0-8 g/1 (80 mg/100 ml) in nearly all patients successfully treated. The incidence of pulmonary embolism was no greater with streptokinase than with heparin treatment. Only prolonged follow-up would show whether thrombolytic treatment would be effective in preventing late complications of deep vein thrombosis such as chronic venous insufficiency.     

Use of thermal and visible imagery for estimating crop water status of irrigated grapevine

Achieving high quality wine grapes depends on the ability to maintain mild to moderate levels of water stress in the crop during the growing season. This study investigates the use of thermal imaging for monitoring water stress. Experiments were conducted on a wine-grape (Vitis vinifera cv. Merlot) vineyard in northern Israel. Irrigation treatments included mild, moderate, and severe stress. Thermal and visible (RGB) images of the crop were taken on four days at midday with a FLIR thermal imaging system and a digital camera, respectively, both mounted on a truck-crane 15 m above the canopy. Aluminium crosses were used to match visible and thermal images in post-processing and an artificial wet surface was used to estimate the reference wet temperature (T(wet)). Monitored crop parameters included stem water potential (Psi(stem)), leaf conductance (g(L)), and leaf area index (LAI). Meteorological parameters were measured at 2 m height. CWSI was highly correlated with g(L) and moderately correlated with Psi(stem). The CWSI-g(L) relationship was very stable throughout the season, but for that of CWSI-Psi(stem) both intercept and slope varied considerably. The latter presumably reflects the non-direct nature of the physiological relationship between CWSI and Psi(stem). The highest R(2) for the CWSI to g(L) relationship, 0.91 (n=12), was obtained when CWSI was computed using temperatures from the centre of the canopy, T(wet) from the artificial wet surface, and reference dry temperature from air temperature plus 5 degrees C. Using T(wet) calculated from the inverted Penman-Monteith equation and estimated from an artificially wetted part of the canopy also yielded crop water-stress estimates highly correlated with g(L) (R(2)=0.89 and 0.82, respectively), while a crop water-stress index using 'theoretical' reference temperatures computed from climate data showed significant deviations in the late season. Parameter variability and robustness of the different CWSI estimates are discussed. Future research should aim at developing thermal imaging into an irrigation scheduling tool applicable to different crops.     

Pyrrolidine-constrained phenethylamines: The design of potent, selective, and pharmacologically efficacious dipeptidyl peptidase IV (DPP4) inhibitors from a lead-like screening hit

A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable parallel chemistry, and protein co-crystal structural data guided the optimization of N-substituents. Employing this strategy, a >400x improvement in potency over the initial hit was realized in rapid fashion. Optimized compounds are potent and selective inhibitors with excellent pharmacokinetic profiles. Compound 30 was efficacious in vivo, lowering blood glucose in ZDF rats that were allowed to feed freely on a mixed meal.     

Membrane defects of the tolerant B cell. I. Failure of antigen-induced capping.

In order to study the membrane function of tolerant B antigen-binding cells, tolerance to the trinitrophenyl (TNP) determinant was induced in mice by injecting the reactive form of the hapten, trinitrobenzene sulfonic acid (TNBS). By appropriate transfer experiments, Fidler and Golub (J. Immunol.112, 1891, 1974) had previously shown that this form of tolerance is a B-cell property, induced and expressed in the absence of T cells. Hapten inhibition demonstrated the TNP-specificity of receptors on TNP-donkey erythrocyte(TNP-D)-binding cells in tolerant and nontolerant mice. About 88% of these cells were B cells by immunofluorescence, and the remainder were T cells. In the tolerant mice, challenge with TNP-sheep erythrocytes failed to expand the TNP-binding population, but sheep erythrocyte binders and anti-sheep plaque-forming cells expanded normally. Despite little or no change in TNP-binding cell numbers after tolerance induction, the TNP-binding cells of tolerant animals could not cap their receptors, in contrast to the sheep erythrocyte-binding cells from the same animals which capped normally. Although there is no anti-TNP plaque-forming cell response when tolerogen and immunogen are given simultaneously, capping failure is not evident until 2–4 days after tolerogen exposure. By Day 7, substantial recovery of immune responsiveness had occurred, yet even 12 months after a single dose of tolerogen there was no restoration of capping. Thus despite the association of both capping failure and unresponsiveness with tolerogen exposure, these lymphocyte functional defects appeared not to be causally related.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland

The possibility that prostaglandin E₂ (PGE₂) may play a role in luteinizing hormone (LH) release was examined using an model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE₂ or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE₂ content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood.It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting β cells renders them susceptible to the autoimmune attack, and is associated with a diminution in β-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on β cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in β cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing β cells is implicated in β-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated.