Phytochemical Profiling of Leaf, Stem, and Tuber Parts of Solena amplexicaulis (Lam.) Gandhi Using GC-MS

Objective. To explore the possible bioactive compounds in the methanolic extracts of leaf, stem, and tuber parts of the medicinal climber, Solena amplexicaulis, using GC-MS. Methods. GC-MS analysis of the plant extracts were performed by using GC-MS-5975C [Agilent] and mass spectra of the compounds found in the extract was matched with the data in the library of National Institute of Standards and Technology (NIST). Results. Thirty-five compounds were determined to be present in the parts studied. The active principles with their retention time, molecular formula, molecular weight, peak area, structure, category of the compounds, and activities were predicted. The most prevailing compounds were phytol (38.24%) in leaf, 4-(4-ethoxyphenyl) but-3-en-2-one (56.90%) in stem, and 9,17-octadecadienal, (Z)- (21.77%) in tuber. Conclusion. This study revealed that the species S. amplexicaulis is a good source of many bioactive compounds like terpenes, triazines, esters, alkanes, alcohols, hydrocarbons, aldehydes, amides, and so forth. That justifies the traditional usage of this species.

Interaction of high molecular weight kininogen binding proteins on endothelial cells

SummaryCell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endothelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular complexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.

ACTH-like bioactivity and immunoactivity in fetal lamb pituitaries at 0.65 and 0.95 gestation

We wished to determine if the concentration of bioactive ACTH-like activity increased during development and if there was heterogeneity in ovine fetal anterior pituitary ACTH activity as measured by bioassay and radioimmunoassay (RIA). We obtained anterior pituitaries from eight sheep fetuses (four at 0.65 and four at 0.95 gestation; term 145 +/- 5 days) and extracted and homogenized them in ice-cold 5N acetic acid, 0.3% phenylmethanesulfonyl fluoride (PMSF) and 0.2% BSA. Fractionation of each pituitary extract was performed by size-exclusion chromatography using Sepadex G-50. The ACTH-like immunoactivity (ALI) profile for each pituitary showed two well-defined peaks. One eluted with human ACTH1-39 and the other eluted with the high molecular weight fraction in the void volume. Four fractions from the first peak representing the high molecular weight forms of ACTH activity and four fractions from the second peak representing the low molecular weight forms of ACTH activity were pooled separately. These two pools were subjected to reverse-phase chromatography (RPC) on a C-8 column using a linear gradient of 70% acetonitrile in 0.8% trifluoroacetic acid over a 60 min period. Based upon the RIA, the high molecular weight forms of ACTH from the G-50 column were resolved into three main fractions, one eluting similar to the standard ACTH1-39 and the remaining two eluting after that. The low molecular weight forms of ACTH from the G-50 column were resolved into three peaks, before, with, and after the standard. We used collagenase-dispersed rat adrenal cells to test the ACTH-like bioactivity (ALB) of the crude extracts and of the different fractions obtained from the RPC of the high and low molecular weight material. The concentration of ACTH-like bioactivity in the crude extracts was similar at the two stages of gestation. However, there was a trend for the low molecular weight peak to have more peptide eluting with human ACTH1-39 and higher ratios of ALB/ALI than did the high molecular weight peak. These results suggest that multiple ACTH molecular forms with different ALB/ALI ratios are present in the ovine fetal pituitary and that there is no selective increase in ACTH1-39 concentration in the fetal pituitary in late gestation.

Bradykinin stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity

In the present investigation, we evaluated the potential of bradykinin (BK), histamine, and serotonin to induce the release of neutrophil and monocyte chemotactic activity (NCA and MCA) from bronchial epithelial cells (BEC). BK significantly stimulated BEC to release NCA and MCA in a dose- and time-dependent manner. Histamine weakly but significantly induced the release of both NCA and MCA in a similar fashion. Serotonin did not stimulate BEC. Checkerboard analysis showed that the NCA and MCA released in response to BK were chemotactic. Molecular-sieve column chromatography by Sephadex G-75 revealed that BK induced a single low-molecular-weight peak (approximately 400 Da) for both NCA and MCA. The releases of NCA and MCA in response to BK and histamine were inhibited by lipoxygenase inhibitors (P < 0.01). The released NCA was inhibited by leukotriene B4 (LTB4) receptor antagonist (P < 0.01) and was slightly inhibited by platelet-activating factor receptor antagonist. LTB4 was increased in BK-stimulated BEC supernatant (P < 0.01). BK B2-receptor antagonist attenuated the release of NCA and MCA. These data suggest that BK and histamine may stimulate BEC to release NCA and MCA and may modulate neutrophil and monocyte recruitment into the airways in patients with asthma.

The presence of atrial natriuretic peptide in canine cerebrospinal fluid and its possible origin in the brain

ABSTRACT The presence of atrial natriuretic peptide (ANP) in canine cerebrospinal fluid (CSF) was clearly demonstrated and an attempt was made to determine its origin as either the brain or the atrium. The concentration of ANP in canine CSF was 0·78 ±0·37 pmol/l (n = 31) and showed no evident correlation with that in plasma (r =0·12). Physiological doses of human α-ANP (α-hANP) were continuously infused intravenously into nine dogs, and ANP concentrations in CSF and plasma were examined six to eight times within a 120-min period following this. The ANP level in CSF was not influenced by the systemic administration of α-hANP up to 180 min. Only one low molecular weight peak corresponding to α-hANP could be obtained from the CSF samples, while both low and high molecular weight peaks were observed for plasma ANP by gel permeation chromatography. In the atrial and hypothalamic tissue extracts the same kinds of peaks were also evident. These results prove the presence of ANP in canine CSF and that it does not come from blood that has seeped across the blood–CSF barriers, but suggest that it may originate from the brain. J. Endocr. (1988) 119, 127–131

Chromatographic characteristics of pro-opiomelanocortin-derived peptides from the rat transplantable tumour 7315a

ABSTRACT Adrenocorticotrophin (ACTH) and other proopiomelanocortin (POMC)-derived peptides produced by the 7315a corticomammotrophic tumour have been poorly studied although they elicit profound hypertrophy and hyperplasia in the adrenal glands of recipient Buffalo rats. Tumour extracts were chromatographed on Sephadex G-75 and fractions monitored for POMC-derived peptides by four radioimmunoassay (RIA) systems: ACTH, α-MSH, β-lipotrophin (β-LPH)/endorphin and N-terminal POMC (N-POMC). Chromatograms were compared with those of pars distalis extracts from normal Buffalo rats. All four RIA systems detected immunoreactive material in tumour extracts. ACTH, β-LPH/ endorphin and N-POMC were present in approximately equimolar amounts (ACTH content 93·40 ± 5·27 (s.e.m.) pmol/g) whereas α-MSH was present in smaller amounts (2·83± 0·13 pmol/g). Total peptide content correlated well with tumour weight. ACTH immunoreactivity (IR) in Sephadex chromatograms was located in a large 20 000 mol. wt peak, an ACTH(1–39) peak and a smaller peak coinciding with ACTH(1–24). The latter two peaks showed biological activity consistent with ACTH(1–39) and an ACTH (1–24)-like peptide respectively. The β-LPH/endorphin RIA revealed a peak eluting at approximately 20 000 mol. wt which could not be ascribed to any known POMC peptide containing the endorphin sequence. A β-LPH-like peak, a β-endorphin-like peak and a smaller-sized peak, which contained the bulk of the β-LPH/endorphin IR, were detected; the low molecular weight peak probably representing α- or γ-endorphin. The N-POMC RIA revealed a 20 000 mol. wt peak and a wide peak which could not be completely resolved into two peaks, and which probably represented N-POMC(1–95) and (N-POMC(1–74). No 30 000 mol. wt precursor could be detected with any of the RIA systems employed. Sephadex chromatography of material released from perfused dispersed tumour cells revealed identical IR peaks with all RIA systems used. Glycosylation of tumour POMC peptides was assessed by Concanavalin A–agarose (Con A) chromatography of pooled IR peaks from Sephadex chromatograms. The 20 000 mol. wt (both IR-ACTH and IR-N-POMC) peak, IR-N-POMC(1–95) and IR-N-POMC(1–74) peaks all bound to Con A and were specifically eluted with methylglucoside. Sephadex G-75 and Con A chromatography of pooled pars distalis extracts from normal Buffalo rats were performed. They showed significant differences, compared with tumour extract chromatograms, with all the RIA systems employed. It is concluded that the 7315a tumour processes POMC in a different manner when compared with normal Buffalo rat pars distalis and that it may be used as an interesting model in which to study the processing of POMC. In addition it may shed light on the role of POMC-derived peptides, other than ACTH(1–39), on adrenal growth and function. J. Endocr. (1987) 112, 417–425

Distribution of radioactive silver in the subcellular fractions of various tissues of the rat and its binding to low molecular weight proteins

In view of the electron microscopic evidence that silver does not penetrate cellular barriers, the distribution of radioactive silver in rat blood and subcellular fractions of liver, kidneys, spleen, and forebrain was studied. It was found that 24 h after a single intraperitoneal injection high levels of radioactivity were reached which decreased at different rates in the various tissues studied. In plasma, liver, and kidneys there was an initial rapid loss of radioactivity which was followed by a slower rate of loss. In the blood, forebrain, and spleen the loss of radioactivity was linear and somewhat slower than in the other three tissues. The cytosols of the liver and kidneys contained 60% while those of the forebrain and spleen contained 30% of the total radioactivity found in the tissue homogenates. Gel filtration on Sephadex G-75 showed that all cytosols contained two peaks of radioactivity; a high molecular weight peak which eluted just after the void volume and a low molecular weight peak. The amount of radioactivity in both peaks was, however, much lower in the chromatographic peaks of the forebrain and spleen than that found in those of the liver and kidneys. Furthermore, the spleen had a comparatively very small low molecular weight radioactive peak. In vitro experiments with liver cytosol showed similar results to those found in vivo in that the high molecular weight radioactive peak could be removed by heat. It is concluded that silver does enter cells and that silver thionein exists in the cytosols of forebrain, spleen, kidney, and liver.

Low molecular weight nonheme iron and a highly labeled heme pool in the reticulocyte

The ligands that bind iron and heme in reticulocyte and may regulate their distribution are not known. Guinea pig reticulocyte hemolysates were labeled with 59Fe and filtered through molecular weight sieving columns. 59Fe-containing peaks of ferritin, transferrin, and hemoglobin were identified, as well as a low molecular weight peak containing predominantly nonheme iron, and a 17,000 mol wt heme containing peak with a specific activity (59Fe/heme) 20-fold greater than hemoglobin. We note that the low molecular weight peak, the existence of which has been postulated, has been identified for the first time without the addition of a chelating agent. We speculate that the 17,000 mol wt peak is an alpha-chain pool.

Low molecular weight nonheme iron and a highly labeled heme pool in the reticulocyte

The ligands that bind iron and heme in reticulocyte and may regulate their distribution are not known. Guinea pig reticulocyte hemolysates were labeled with 59Fe and filtered through molecular weight sieving columns. 59Fe-containing peaks of ferritin, transferrin, and hemoglobin were identified, as well as a low molecular weight peak containing predominantly nonheme iron, and a 17,000 mol wt heme containing peak with a specific activity (59Fe/heme) 20-fold greater than hemoglobin. We note that the low molecular weight peak, the existence of which has been postulated, has been identified for the first time without the addition of a chelating agent. We speculate that the 17,000 mol wt peak is an alpha-chain pool.

Developments in column chromatography for the separation and characterization of casein micelles

SUMMARYSkim-milk was fractionated by permeation chromatography on CPG-10 (300 nm) at 20 °C with synthetic milk serum. The elution profile, which was highly reproducible, showed 3 partly resolved peaks. On treatment with 0.1% glutaralde-hyde before chromatography, a similar profile was obtained indicating no appreciable micellar dissociation during chromatography of the milk. The casein composition of the eluent fractions, determined by quantitative chromatography on hydroxyapatite, showed that 12.1% of total casein was associated with the low molecular weight peak. The micelle content of ²- and κ-casein increased with increasing elution volume, and the ±s/κ ratio showed a progressive diminution from 4·3 to 2·3. Large differences were found in the colloidal phosphate contents and the weight average diameters of the micelle fractions which clearly show that CPG-10 fractionates on the basis of molecular size.