In vitro flower formation from thin epidermal cell layers of a partial somatic hybrid between Petunia hybrida (Hort.) and Nicotiana plumbaginifolia (Viv.)

In vitro flowers have been obtained by culturing thin epidermal cell layers of a partial somatic intergeneric hybrid. The phenotype of these flowers differs from that of flowers formed on seed-grown plants (in situ flowers) and from that of flowers of either parental line. In addition, modifications in the phenotype were observed when cultures were sustained for more than four months. Dimorphic leaves present in juvenile and adult stages of mother plants of Nicotiana plumbaginifolia and the somatic hybrid were formed on different ends of the thin epidermal cell layers. No anomalies were observed during microsporogenesis and in the meiotic and mitotic figures of the somatic hybrid, which resembled those of Nicotiana plumbaginifolia.     

A 19-kDa human erythrocyte molecule H19 is involved in rosettes, present on nucleated cells, and required for T cell activation. Comparison of the roles of H19 and LFA-3 molecules in T cell activation

We have previously described a molecule on the SRBC surface which, in addition to the sheep equivalent of LFA-3, is involved in the process of rosette formation. It is made of a single, 14- to 19-kDa, polypeptide chain, and we termed this molecule S14. We have now identified, on the human E a molecule with a similar Mr albeit somewhat higher (19 kDa). The mAb against H19 efficiently block autologous or homologous rosettes by binding to human E. In addition, purified H19 molecules block rosettes made with human E and SRBC in a dose-dependent manner. The H19 molecule, like LFA-3, is not limited to the E surface, but is also present on many nucleated cells, including T cells and monocytes. Moreover H19, like LFA-3, is required for T cell activation: when we stimulated whole PBMC anti-H19 blocked [H3]TdR incorporation triggered via CD3, but not via CD2, in contrast to anti-LFA-3 that inhibited activation via both pathways. When a mixture of highly purified T-PBL and autologous paraformaldehyde fixed accessory cells (AC) was cultivated, anti-H19 or anti-LFA-3 mAb bound to AC blocked T cell proliferation. When high amounts of rIL-1 (100 U/ml) were added to purified T-PBL, no AC were required to sustain their proliferation upon stimulation via CD2, contrary to stimulation via CD3. When lower amounts of rIL-1 (10 U/ml) were used, fixed AC were still necessary to sustain proliferation via CD2. In this latter situation, anti-H19 mAb bound to AC could no longer inhibit T cell proliferation, whereas the anti-LFA-3 mAb was still inhibitory. When T-PBL were stimulated via CD2 in the presence of 100 U/ml of rIL-1, anti-LFA-3 did not induce any inhibition. Thus the inhibitory effect of anti-H19 and anti-LFA-3 mAb can both be accounted for by an effect on the AC molecules only, and not on the T cell molecules. F(ab')2 fragments of anti-H19 mAb produced the same pattern of inhibition as the whole Ig molecule, excluding an effect via the FcR. Moreover, purified preparations of the H19 molecules also produced inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diastolic Dysfunction of Aging Is Independent of Myocardial Structure but Associated with Plasma Advanced Glycation End-Product Levels

Background

Heart failure is associated with abnormalities of myocardial structure, and plasma levels of the advanced glycation end-product (AGE) N^ε-(carboxymethyl)lysine (CML) correlate with the severity and prognosis of heart failure. Aging is associated with diastolic dysfunction and increased risk of heart failure, and we investigated the hypothesis that diastolic dysfunction of aging humans is associated with altered myocardial structure and plasma AGE levels.

Methods

We performed histological analysis of non-ischemic left ventricular myocardial biopsies and measured plasma levels of the AGEs CML and low molecular weight fluorophores (LMWFs) in 26 men undergoing coronary artery bypass graft surgery who had transthoracic echocardiography before surgery. None had previous cardiac surgery, myocardial infarction, atrial fibrillation, or heart failure.

Results

The patients were aged 43–78 years and increasing age was associated with echocardiographic indices of diastolic dysfunction, with higher mitral Doppler flow velocity A wave (r = 0.50, P = 0.02), lower mitral E/A wave ratio (r = 0.64, P = 0.001), longer mitral valve deceleration time (r = 0.42, P = 0.03) and lower early diastolic peak velocity of the mitral septal annulus, e’ (r = 0.55, P = 0.008). However, neither mitral E/A ratio nor mitral septal e’ was correlated with myocardial total, interstitial or perivascular fibrosis (picrosirius red), immunostaining for collagens I and III, CML, and receptor for AGEs (RAGE), cardiomyocyte width, capillary length density, diffusion radius or arteriolar dimensions. Plasma AGE levels were not associated with age. However, plasma CML levels were associated with E/A ratio (r = 0.44, P = 0.04) and e’ (r = 0.51, P = 0.02) and LMWF levels were associated with E/A ratio (r = 0.49, P = 0.02). Moreover, the mitral E/A ratio remained correlated with plasma LMWF levels in all patients (P = 0.04) and the mitral septal e’ remained correlated with plasma CML levels in non-diabetic patients (P = 0.007) when age was a covariate.

Conclusions

Diastolic dysfunction of aging was independent of myocardial structure but was associated with plasma AGE levels.     

Tolvaptan inhibits ERK-dependent cell proliferation, Cl⁻ secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin

In autosomal dominant polycystic kidney disease (ADPKD), arginine vasopressin (AVP) accelerates cyst growth by stimulating cAMP-dependent ERK activity and epithelial cell proliferation and by promoting Cl(-)-dependent fluid secretion. Tolvaptan, a V2 receptor antagonist, inhibits the renal effects of AVP and slows cyst growth in PKD animals. Here, we determined the effect of graded concentrations of tolvaptan on intracellular cAMP, ERK activity, cell proliferation, and transcellular Cl(-) secretion using human ADPKD cyst epithelial cells. Incubation of ADPKD cells with 10(-9) M AVP increased intracellular cAMP and stimulated ERK and cell proliferation. Tolvaptan caused a concentration-dependent inhibition of AVP-induced cAMP production with an apparent IC(50) of ～10(-10) M. Correspondingly, tolvaptan inhibited AVP-induced ERK signaling and cell proliferation. Basolateral application of AVP to ADPKD cell monolayers grown on permeable supports caused a sustained increase in short-circuit current that was completely blocked by the Cl(-) channel blocker CFTR(inh-172), consistent with AVP-induced transepithelial Cl(-) secretion. Tolvaptan inhibited AVP-induced Cl(-) secretion and decreased in vitro cyst growth of ADPKD cells cultured within a three-dimensional collagen matrix. These data demonstrate that relatively low concentrations of tolvaptan inhibit AVP-stimulated cell proliferation and Cl(-)-dependent fluid secretion by human ADPKD cystic cells.     

Alleviation of Cadmium Toxicity in Brassica juncea L. (Czern. & Coss.) by Calcium Application Involves Various Physiological and Biochemical Strategies

Calcium (Ca) plays important role in plant development and response to various environmental stresses. However, its involvement in mitigation of heavy metal stress in plants remains elusive. In this study, we examined the effect of Ca (50 mM) in controlling cadmium (Cd) uptake in mustard (Brassica juncea L.) plants exposed to toxic levels of Cd (200 mg L⁻¹ and 300 mg L⁻¹). The Cd treatment showed substantial decrease in plant height, root length, dry weight, pigments and protein content. Application of Ca improved the growth and biomass yield of the Cd-stressed mustard seedlings. More importantly, the oil content of mustard seeds of Cd-stressed plants was also enhanced with Ca treatment. Proline was significantly increased in mustard plants under Cd stress, and exogenously sprayed Ca was found to have a positive impact on proline content in Cd-stressed plants. Different concentrations of Cd increased lipid peroxidation but the application of Ca minimized it to appreciable level in Cd-treated plants. Excessive Cd treatment enhanced the activities of antioxidant enzymes superoxide dismutase, ascorbate peroxidase and glutathione reductase, which were further enhanced by the addition of Ca. Additionally, Cd stress caused reduced uptake of essential elements and increased Cd accumulation in roots and shoots. However, application of Ca enhanced the concentration of essential elements and decreased Cd accumulation in Cd-stressed plants. Our results indicated that application of Ca enables mustard plant to withstand the deleterious effect of Cd, resulting in improved growth and seed quality of mustard plants.     

Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor

Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal-regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.     

Structural Mechanism of Trimeric HIV-1 Envelope Glycoprotein Activation

HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.     

Analysis of creatine, creatinine, creatine-d3 and creatinine-d3 in urine, plasma, and red blood cells by HPLC and GC-MS to follow the fate of ingested creatine-d3

Creatine, which is increasingly being used as an oral supplement, is naturally present in the body. Studies on the fate of a particular dose of creatine require that the creatine be labeled, and for studies in humans the use of a stable isotopic label is desirable. The concentrations of total creatine and total creatinine were determined using HPLC. Creatine and creatinine were then separated using cation exchange chromatography and each fraction was derivatized with trifluoroacetic anhydride and the ratio of the deuterated:undeuterated species determined using GC-MS. Ratios of creatine:creatine-d(3), and creatinine:creatinine-d(3), and the concentrations of each of these species, were able to be determined in urine, plasma and red blood cells. Thus, the uptake of labeled creatine into plasma and red blood cells and its excretion in urine could be followed for a subject who ingested creatine-d(3). Creatine-d(3) was found in the plasma and red blood cells 10 min after ingestion, while creatine-d(3) and creatinine-d(3) were found in the urine collected after the first hour.