Effect of Active Oxygen Radicals on Protein and Carbohydrate Moieties of Recombinant Human Erythropoietin

Our previous study showed that active oxygen radicals generated from a Fenton system and a xanthine plus xanthine oxidase system caused serious loss of in vivo bioactivity of recombinant human erythropoietin (EPO), a highly glycosylated protein. In the present study, we characterized the oxidative modifications to the protein and carbohydrate moiety of EPO, which lead to a reduction of its bioactivity. In vitro bioactivity was reduced when EPO was treated with oxygen radi cals generated from a Fenton system in the presence of 0.016 mM H₂0₂, and the reduction was directly proportional to the loss of in vivo bioactivity. SDS-PAGE analysis showed that dimer formation and degradation was observed under more severe conditions (Fenton reaction with 0.16 mM H₂0₂). The tryptophan destruction was detected at 0.016 mM H₂O₂ and well correlated with the loss of in vitro bioactivity, whereas loss of other amino acids were occurred under more severe conditions. Treatment with the Fenton system did not result in any specific damage on the carbohydrate moiety of EPO, except a reduction of sialic acid content under severe condition. These results suggest that active oxygen radicals mainly react with the protein moiety rather than the carbohydrate moiety of EPO. Destruction of tryptophan residues is the most sensitive marker of oxidative damage to EPO, suggesting the importance of tryptophan in the active EPO structure. Deglycosylation of EPO caused an increase of susceptibility to oxygen radicals compared to intact EPO. The role of oligosaccharides in EPO may be to protect the protein structure from active oxygen radicals.     

Translating tissue culture results into animal models: the case of Salmonella typhimurium

Investigators use both in vitro and in vivo models to better understand infectious disease processes. Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relevant variability encompassing in vivo animal models. In an effort to understand how Salmonella initiates disease at the intestinal epithelium, in vitro models have served a useful purpose in allowing investigators to identify molecular mechanisms responsible for Salmonella invasion of host cells and stimulation of host inflammatory responses. Identification of these molecular mechanisms has generated hypotheses that are now being tested using in vivo models. Translating the in vitro findings into the context of an animal model and subsequently to human disease remains a difficult challenge for any disease process.     

Regulation of proliferation of rat cartilage and bone by sex steroid hormones

We have demonstrated previously that 17β-estradiol (E₂) stimulates proliferation of skeletal tissues, both in vivo and in vitro, as measured by increased DNA synthesis and creatine kinase (CK) specific activity. The effect of E₂ on bone is sex specific. E₂ is active only in females and androgens only in males. By contrast, in cartilage of both sexes, dihydrotestosterone (DHT) as well as E₂ stimulates CK specific activity and DNA synthesis. In bone, we find that sex steroids stimulate skeletal cell proliferation in gonadectomized as well as in immature rats. Ovariectomized (OVX) rats, between 1 and 4 weeks after surgery, show stimulation of CK by E₂. The basal activity and response of CK changes with the varying endogenous levels of E₂ in cycling rats, in which the highest basal activity is at proestrus and estrus and the highest response is in diestrus. In rats of all ages tested, both the basal and stimulated specific activity of CK is higher in diaphysis and epiphysis than in the uterus, or in the adipose tissue adjacent to the uterus, which has a response similar to that of the uterus itself. The effect of E₂ in vivo, and in chrondroblasts and osteoblasts in vitro, is inhibited by high levels of the antiestrogen tamoxifen which, by itself, in similar high concentrations, shows stimulatory effects. In addition to the sex steroids, skeletal cells are also stimulated by secosteroid and peptide calciotrophic hormones. The interactions of the sex steroids and the other calciotrophic hormones. These results provide the first steps towards understanding the regulation of bone cell proliferation and growth by the concerted action of a variety of hormones and growth factors.     

EGF responsiveness of hepatocytes after partial hepatectomy

We investigate the effect of EGF on IP₃ production, PLCγ phosphorylation, calcium transients in rat hepatocytes isolated in quiescent liver (G₀ phase of cell cycle) and at 4 h (G₁ phase of cell cycle) and 24 h (M phase of cell cycle) after partial hepatectomy. Our results show that EGF does not utilize IP₃ and calcium as its signal transduction molecules when the hepatocytes are in vivo stimulated to entry in the cell cycle. In particular the growth factor does not phosphorylate PLCγ and induces a decrease in IP₃ content. These data suggest that EGF utilizes different signal transduction to send information from receptor to nucleus during PH with respect to the quiescent liver.     

The Loss of In Vivo Activity of Recombinant Human Erythropoietin by Active Oxygen Species

The effects of active oxygen species on the in vivo activity of recombinant human erythropoietin (EPO) treated by Fenton system, xanthine (X) plus xanthine oxidase (XO) system and hydrogen peroxide (H₂O₂) has been studied by means of counting the increase in number of hemolyser-resistant cells (HRCs) in EPO-injected mice. The results showed that both Fenton and X plus XO systems caused a significant reduction of the activity in proportion to the concentration of generated active oxygen species. Meanwhile, the treatment of EPO with H₂O₂ alone resulted in a relatively slight reduction of the activity. Electrophoretic studies on the structure of EPO revealed that its main protein band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) disappeared in proportion with the extent of exposure to active oxygen generating systems. Both Fenton and X plus XO systems caused a significant loss of fluorescence in the pyridylamino (PA-) sugar chain in proportion to the concentration of generated active oxygen species, and no degradation products in the sugar chain part of the PA-sugar chain were detected. This showed that aromatic groups in EPO were sensitive to attack by active oxygen species. These results provide evidence that hydroxyl radical and other active oxygen species have a potential to react with EPO, leading to a reduction of its in vivo activity.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Effects of progestagens and Org OD14 in in vitro and in vivo tumor models

Sex steroids, in particular estradiol (E2) and progesterone (P4), play, together with other hormones and growth factors, a role in the development of normal breast tissue. The effect of four progestagens (norethisterone, 3-ketodesogestrel, gestodene and P4) and Org OD14, a steroid with weak estrogenic, progestagenic and androgenic properties were studied on growth of breast tumor cells in vitro using two subclones of MCF-7 (H and A) and T47D (S and A) cells. In addition, we investigated the effects of 3-ketodesogestrel, gestodene and Org OD14 on the growth of 7,12-dimethyl-benz(a)anthracene(DMBA)-induced mammary tumors in rats. In the in vitro assays with MCF-7 cells norethisterone, 3-ketodesogestrel and gestodene stimulated growth only at high doses (10⁻⁷ M), whereas P4 had no effect. Gestodene was more potent than 3-ketodesogestrel and norethisterone. Org OD14, stimulated cell growth at a dose of 10⁻⁸ M, while E2 is active at 10⁻¹⁰ M. In T47D-A cells similar effects were found, but the subclone S did not respond to the progestagens and Org OD14. The two T47D subclones also reacted differently to progestagens during growth stimulation with E2. In T47D-S the progestagens and Org OD14 inhibited, while in T47D-A these compounds did not modulate the effect of E2. In the DMBA model we found that gestodene and 3-ketodesogestrel were able to inhibit tumor growth to the same extent. Surprisingly, Org OD14 was even more effective in the DMBA model using the therapeutic approach. Using the prophylaxic approach tumor development was delayed and tumor growth was strongly suppressed. The inhibitory effects of Org OD14 on tumor growth in the DMBA model may be attributed to its mixed hormonal profile. From these studies we conclude that different cell lines and even subclones thereof respond quite differently to steroids. Both in vitro and in vivo studies are required to judge whether synthetic steroids might be involved in an increased risk for the development of breast tumors.     

The metabolism of dihydrotachysterols: Renal side chain and non-renal nuclear hydroxylations in vivo and in vitro

The metabolism of dihydrotachysterol (DHT), a hydrogenated analogue of vitamin D, has been studied in vivo using man and rat and in vitro using the perfused rat kidney, and hepatoma (3B) and osteosarcoma (UMR-106) cell lines. In vivo a large number of metabolites appeared in the plasma of rats given DHT₂ and DHT₃. Of particular interest was a compound more polar than 25-hydroxy-DHT, which has been designated compound H. Further study of this compound showed that it was composed of two components, one (Ha) being in much lower concentration than the other (Hb). The production of T2/H (peak H from DHT₂) was demonstrated in human plasma after administration of oral DHT₂. Comparison of the metabolites formed in vivo with those isolated from the rat kidney perfused with 25-hydroxy-DHT₃ in vitro showed that 25-hydroxy-DHT₃ was metabolized along two metabolic pathways previously described for vitamin D, culminating in the production of 25-hydroxy-DHT₃-23,26-lactone and 23,25-dihydroxy-24-oxo-DHT₃. The osteosarcoma cell line metabolized 25-OH-DHT₃ in vitro along the same two metabolic pathways already demonstrated in the perfused rat kidney. More polar metabolites than compound H seen in rat plasma in vivo were shown to be metabolites of compound H and similar metabolites were also produced in the osteosarcoma cell line from chemically synthesized 1,25-dihydroxy-DHT₃. The hepatoma cell line 25-hydroxylated DHT and no feed-back inhibition was observed. Use of the hepatoma cell to 25-hydroxylate a number of chemically synthesized 1-hydroxy-DHTs indicated that compound Ha was indistinguishable from 1,25-dihydroxy-DHT whereas compound Hb is possibly 1β,25-dihydroxy-DHT. Studies with the VDR in both chick gut and calf thymus indicated that 1,25-dihydroxy-DHT is very effective in displacing radiolabelled 1,25-dihydroxyvitamin-D₃ and is thus most likely to be the calcaemic metabolite of DHT.     

Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays

Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity.

This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays.

In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor , with a lower potency but comparable efficacy to that of 17 β-estradiol.

Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F_2, and bradykinin than tissues obtained from estradiol valerate treated rats.

The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F_2, and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 β-estradiol.

Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Control of aromatase in breast cancer cells and its importance for tumor growth

Three approaches have been taken to elucidate further the biological importance of intratumoural aromatase activity. (i) MCF₇ and T47D hormone-dependent breast cancer cell lines both showed detectable aromatase activity in vitro. The up-regulation of this by TGF indicates the possible existence of an autocrine growth stimulatory loop involving aromatase. (ii) Both tamoxifen and cytotoxic chemotherapy caused the suppression of aromatase activity in breast carcinomas in vivo. Aromatase activity prior to treatment did not predict for clinical response to tamoxifen. (iii) Transfection of aromatase into MCF₇ cells led to their growth being stimulated by low doses of androgens and this was inhibited by the aromatase inhibitor CGS 16949A.     

Specificity of the action of parathyroid hormone upon mitochondrial metabolism: Cyanogen bromide-derived parathyroid-hormone peptides

The mitochondrial response to cyanogen bromide-treated parathyroid hormone was studied as a means of testing further the relationship between the structure and the effects in vitro of this hormone. The treated hormone and appropriate control hormone were tested in a standard bioassay and in a mitochondrial assay system in vitro.

Reaction of more than 90 % of the methionine residues in the hormone resulted in total inactivation of the hormone both in vivo and in vitro. This result disagrees with previously published data.     

Interaction of glucocorticoid analogues with the human glucocorticoid receptor

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

A method for the design of 3D scaffolds for high-density cell attachment and determination of optimum perfusion culture conditions

The application of in vitro cultured cells in tissue engineering or drug screening, aimed at complex soft tissues such as liver, requires in vivo physiological function of the cultured cells. For this purpose, the scaffold in which cells are cultured should provide a microenvironment similar to an in vivo one with a three-dimensional extracellular matrix, a high supply capacity of O₂ and nutrients, and high cell density. In this paper, we propose a method to design (1) the geometry of the scaffold, with a surface/volume ratio optimized to allow high-density (5×10⁷ cells/mL) cell culture and (2) culture conditions that will supply optimal quantities of oxygen and nutrients. CFD modeling of mass transport was used to determine the shear stress as well as O₂ and glucose metabolism in the scaffold (20 mm width–35 mm length) for various flow rates. Validation of the model was done through comparison with flow resistance and micro-PIV experiments. CFD analysis showed the maximum metabolic rate densities for this scaffold are 6.04×10⁻³ mol/s/m³ for O₂ at 0.71 mL/min and 1.91×10⁻² mol/s/m³ for glucose at 0.35 mL/min.     

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.