Immunological Exploration of Primary Metabolite Extracted From Aqueous Stem Extract of Caralluma Fimbriata

Objective: The objective of our study is to determined its immunological property of primary (protein) metabolite extracted from aqueous stem extract of Caralluma fimbriata against specific protein antigens (lactoferrin and Ovalbumin, OVA) pertaining to determine B cell production in vitro by ELISA (enzyme linked immunosorbent assay) and also measured T cell production in vitro using immature bone marrow cells of mice exposed with Incomplete freunds adjuvant (IFA) and Concanavalin (Con) A. Methods: For these studies, estimation of protein from stem extract of Caralluma fimbriata were determined and also determining the total cellular (protein) content in immature bone marrow cells along with variable concentration of Caralluma fimbriata in presence or absence of IFA. In addition, lactoferrin (100 µg/well) and weak antigen i.e. ovalbumin (OVA; 100 µg/well) pertaining to antibody (IgG) production were determined in vitro through Elisa and also measured its proliferative response using Con A (2.5 µg/ml). Results: The results showed that aqueous stem extract of Caralluma fimbriata showed the presence of protein (0.698 mg/ml; 10 µl) content and also showed the enhancement in total cellular (protein) content in presence or absence of IFA which is determined through NanoDrop method. In addition, this aqueous stem extract showed enhancement in anti-lactoferrin and anti-OVA IgG titre in vitro at higher doses as compared to control but there is sudden decline in bone marrow cell proliferation containing Con A at higher doses. Conclusion: Our data suggest that aqueous stem extract of Caralluma fimbriata may help to raise antibodies in vitro against lactoferrin and OVA but sudden decline in Con A proliferative response in bone marrow cells. In other words, aqueous stem extract containing primary metabolite of Caralluma fimbriata could be a potent immune enhancer of B cells in vitro but inhibitor of T cells at higher doses.

Defining the differential sensitivity to norepinephrine and angiotensin II in the ovine uterine vasculature

The intact ovine uterine vascular bed (UVB) is sensitive to α-agonists and refractory to angiotensin II (ANG II) during pregnancy; the converse occurs in the systemic circulation. The mechanism(s) responsible for these differences in uterine sensitivity are unclear and may reflect predominance of nonconstricting AT2 receptors (AT2R) in uterine vascular smooth muscle (UVSM). The contribution of the placental vasculature also is unclear. Third generation and precaruncular/placental arteries from nonpregnant ( n = 16) and term pregnant ( n = 23) sheep were used to study contraction responses to KCl, norepinephrine (NE), and ANG II (with/without ATR specific inhibitors) and determine UVSM ATR subtype expression and contractile protein content. KCl and NE increased third generation and precaruncular/placental UVSM contractions in a dose- and pregnancy-dependent manner ( P ≤ 0.001). ANG II only elicited modest contractions in third generation pregnant UVSM ( P = 0.04) and none in precaruncular/placental UVSM. Moreover, compared with KCl and NE, ANG II contractions were diminished ≥ 5-fold. Whereas KCl and ANG II contracted third generation>>precaruncular/placental UVSM, NE-induced contractions were similar throughout the UVB. However, each agonist increased third generation contractions ≥ 2-fold at term, paralleling increased actin/myosin and cellular protein content ( P ≤ 0.01). UVSM AT1R and AT2R expression was similar throughout the UVB and unchanged during pregnancy ( P > 0.1). AT1R inhibition blocked ANG II-mediated contractions; AT2R blockade, however, did not enhance contractions. AT2R predominate throughout the UVB of nonpregnant and pregnant sheep, contributing to an inherent refractoriness to ANG II. In contrast, NE elicits enhanced contractility throughout the ovine UVB that exceeds ANG II and increases further at term pregnancy.

The Effect of Dilution Rate upon Protein Content and Cellular Amino Acid Profiles in Chemostat Cultures of Saccharomyces Cerevisiae CABI 039916

In the present study we examined the use of a chemostat system to investigate the impact of changes in the specific growth rate of Saccharomyces cerevisiae CABI 039916 on cellular amino acid profiles and total protein content. This experimental system allowed the unambiguous examination of the link between changes in dilution rate and the culture response, which would have been difficult in batch or fed-batch cultures. Alteration of the specific growth rate (via manipulation of the dilution rate) within a carbon and energy-limited chemostat has a significant impact on the physiology of Saccharomyces cerevisiae. Low dilution rates (<0.1h-1) led to predominantly respiratory metabolism and the maximisation of cellular protein content within the cell (58%), by contrast high dilution rates (>0.2h-1) led to respirofermentative metabolism, where the cellular protein content was minimal (~40%). The content of nearly all amino acids in the yeast protein pool fell significantly as dilution rate increased in parallel with the decline in cell protein content. By contrast, the concentration of two related key food/feed amino acids in the cell protein pool—glutamic acid and arginine could be increased within the cellular protein by 5% (increasing the dilution rate from 0.05h-1 to 0.25h-1) and 1.5% respectively (decreasing the dilution rate from 0.05h-1 to 0.2h-1). Despite previous studies showing that metabolic change was associated with major changes in free amino acid levels, the present study indicates that the total cellular yeast protein amino acid composition is largely invariant despite profound metabolic changes, with one or two key exceptions.

Physiological Responses to Starvation in the Marine Oligotrophic Ultramicrobacterium Sphingomonas sp. Strain RB2256

ABSTRACT Sphingomonas sp. strain RB2256 is representative of the ultramicrobacteria that proliferate in oligotrophic marine waters. While this class of bacteria is well adapted for growth with low concentrations of nutrients, their ability to respond to complete nutrient deprivation has not previously been investigated. In this study, we examined two-dimensional protein profiles for logarithmic and stationary-phase cells and found that protein spot intensity was regulated by up to 70-fold. A total of 72 and 177 spots showed increased or decreased intensity, respectively, by at least twofold during starvation. The large number of protein spots (1,500) relative to the small genome size (ca. 1.5 Mb) indicates that gene expression may involve co- and posttranslational modifications of proteins. Rates of protein and RNA synthesis were examined throughout the growth phase and up to 7 days of starvation and revealed that synthesis was highly regulated. Rates of protein synthesis and cellular protein content were compared to ribosome content, demonstrating that ribosome synthesis was not directly linked to protein synthesis and that the function of ribosomes may not be limited to translation. By comparing the genetic capacity and physiological responses to starvation of RB2256 to those of the copiotrophic marine bacteriumVibrio angustum S14 (J. Ostling, L. Holmquist, and S. Kjelleberg, J. Bacteriol. 178:4901–4908, 1996), the characteristics of a distinct starvation response were defined forSphingomonas strain RB2256. The capacity of this ultramicrobacterium to respond to starvation is discussed in terms of the ecological relevance of complete nutrient deprivation in an oligotrophic marine environment. These studies provide the first evidence that marine oligotrophic ultramicrobacteria may be expected to include a starvation response and the capacity for a high degree of gene regulation.

Protein prenylation is required for aldosterone-stimulated Na+ transport

Aldosterone stimulation of transcellular Na+ flux in polarized epithelial cells is dependent on at least one transmethylation reaction, but the substrate of this signaling step is unknown. Because it is clear that the majority of cellular protein methylation occurs in conjunction with protein prenylation, we examined the importance of prenylation to aldosterone-stimulated Na+ transport in the A6 cell line. Lovastatin, an inhibitor of the first committed step of the mevalonate pathway, inhibits the natriferic effect of aldosterone but does not inhibit insulin-stimulated Na+ flux. The addition of a farnesyl group does not appear to be involved in aldosterone's action. Neither alpha-hydroxyfarne-sylphosphonic acid, an inhibitor of farnesyl:protein transferase, nor N-acetyl-S-farnesyl-L-cysteine, an inhibitor of farnesylated protein methylation, inhibits the hormone-induced increase in Na+ transport. In contrast, N-acetyl-S-geranyl-geranyl-L-cysteine, an inhibitor of geranylgeranyl protein methylation, completely abolishes the aldosterone-induced increase in Na+ flux with no effect on insulin-mediated Na+ transport or cellular protein content. These data indicate that methylation of a geranylgeranylated protein is involved in aldosterone's natriferic action.

Control of Hep G2-cell triacylglycerol and apolipoprotein B synthesis and secretion by polyunsaturated non-esterified fatty acids and insulin

Non-esterified fatty acids (NEFAs) and insulin are important factors in the control of lipoprotein secretion, but the mechanism of action is unclear. The present study was undertaken to determine whether insulin and NEFAs modulated hepatic secretion of triacylglycerol and apolipoprotein B (apo-B) by regulation of hepatic intracellular apo-B content. The experiments were performed with the human hepatoblastoma cell line Hep G2, for periods of up to 72 h in the presence and absence of NEFAs and insulin. Higher concentrations of eicosapentanoate (EPA) sustained for 72 h decreased cellular protein content (at 250 microM) or caused cell death (at 750 microM), and this effect was not observed with the other NEFAs studied, whereas 75 microM-EPA did not affect cell viability. Compared with the absence of NEFA, 75 microM-EPA did not alter the intracellular triacylglycerol content, but decreased the intracellular content of apo-B by 47% (P < 0.01) and decreased secreted triacylglycerol and secreted apo-B by 13% (P < 0.05) and 21% (P < 0.01) respectively, after 72 h. However 250 microM-oleate increased the intracellular triacylglycerol by 36% (P < 0.01), intracellular apo-B by 22% (P < 0.05) and secreted triacylglycerol and apo-B by 20-30% (P < 0.05-0.01). Insulin decreased secreted triacylglycerol and apo-B in the presence of each NEFA studied by 20-30%. There was no correlation between the changes in intracellular triacylglycerol and the rate of secretion. However, when the secreted triacylglycerol or apo-B was plotted against intracellular apo-B content a significant correlation was observed (r = 0.89, P < 0.001 for both analyses). Apo-B mRNA levels did not change after 72 h incubation with oleate or EPA. These results demonstrate that EPA can be toxic to hepatocytes and that NEFAs and insulin control secretion of triacylglycerol and apo-B by regulation of the intracellular apo-B concentration, thus controlling assembly of apo-B with triacylglycerol to form lipoproteins.

Pregnancy-induced changes in ovine cerebral arteries

We examined the effects of pregnancy on the ovine cerebral vasculature by comparing several characteristics of isolated endothelium-intact segments of three intracranial arteries including the middle cerebral (MCA), posterior communicating (PC), and basilar (BAS) arteries taken from pregnant sheep (138-143 days gestation, term approximately 145 days) and nonpregnant controls. For comparison, segments of the extracranial common carotid (COM) artery were also studied. With pregnancy, vessel water content increased (5.4-5.8%) in all arteries except the PC. Additionally, cellular protein content increased in all arteries (4.4-50.0%). Arterial stiffness, as determined by passive stress-strain determinations, was significantly decreased during pregnancy in the MCA but not in the larger arteries. Maximum contractile responses, when normalized to vessel wall cross-sectional area, were consistently greater in arteries from pregnant than in those from nonpregnant animals (10.1-49.7%). Relaxation to the endothelium-independent guanylate cyclase stimulator S-nitroso-N-acetyl penicillamine (SNAP) increased with pregnancy only in the distal MCA (approximately 17%). Endothelium-dependent relaxation to the calcium ionophore A23187 decreased only in the larger and more proximal COM (-39%). Thus pregnancy was associated with an increase in production of contractile force, a decrease in peripheral vascular stiffness, a decrease in the relaxant response to A23187 in the COM, and an increase in the relaxant response to SNAP in the MCA. Together, these findings indicate that pregnancy has widespread and important vessel specific cerebrovascular consequences that affect not only arterial composition, but also contractility and endothelial reactivity.

ATP and Protein Synthesis Assays to Evaluate the Toxicities of Preservatives in Vitro

Cellular protein content, protein synthesis, ATP level and lactate dehydrogenase (LDH) activity, measured in a murine epidermal cell line (HEL/30), were used as the endpoints for determining the cytotoxicities of 17 antimicrobial chemicals. The relative toxicities of the test compounds were quantified by the determination of the concentrations inducing a 50% inhibition of [3H]-leucine incorporation into proteins (IC50), causing a 50% reduction of ATP level or final cell protein content or producing the maximal effect on LDH leakage (EC50) after 2 hours of treatment. The results indicate a good correlation between both the reduction of ATP level and inhibition of protein synthesis and the minimal inhibitory concentration (MIC) on different microorganisms, suggesting that ATP and protein synthesis assays could be useful as prescreening methods for testing the cytotoxicities of preservatives.

In Vitro Toxicity to Two Cellular Systems of the First Ten Chemicals on the MEIC List

The cytotoxic effects of the first 10 chemicals on the MEIC list were evaluated with two experimental cellular systems, monolayer cultures of rat hepatocytes and cell lines (Hep G2 and 3T3). Three endpoints were measured to evaluate cytotoxicity, intracellular LDH activity, cellular protein content and the MTT test. The results show that: 1. digoxin, amitriptyline and diazepam were the most cytotoxic chemicals (IC50:0.01-0.5mM); 2. alcoholic compounds (sopropanol, ethylene glycol, ethanol and methanol) produced the lowest toxic effects (IC50: 100–1500mM); 3. paracetamol, acetylsalicylic acid and ferrous sulphate showed an intermediate cytotoxic action (C50: 0.05–15mM); 4. regarding the sensitivity of the cellular systems, paracetamol, acetylsalicylic acid, diazepam and ferrous sulphate were more toxic to rat hepatocytes, while digoxin produced a different toxic effect on hepatic and non-hepatic cells; and 5. the other chemicals did not show significant differences in their toxicity in the different cellular systems studied.