Half-maximal Dose Research Articles

Inhibition of osteogenesis in vitro by a cigarette smoke-associated hydrocarbon combined with Porphyromonas gingivalis lipopolysaccharide: reversal by resveratrol.

Smoking and infection with Gram-negative bacterial pathogens are risk factors for alveolar bone loss. The aims of this study were: 1) to examine the combined effects of an aryl hydrocarbon, benzo[a]pyrene (BaP), that is concentrated in cigarette smoke, and lipopolysaccharide (LPS) extracted from Porphyromonas gingivalis on osteogenesis in a rat bone marrow cell (RBMC) model of osteogenesis; and 2) to determine whether resveratrol (Res), an aryl hydrocarbon receptor antagonist, could reverse the putative inhibitory effects of BaP + LPS on osteogenesis. LPS of P. gingivalis strain 2561 was introduced in various concentrations to the RBMC in 96-well plates and kept in culture for 8 to 12 days. The same protocol was used for studying BaP and LPS + BaP combinations. Following the incubation periods, parameters of osteogenesis were measured, including formation of mineralized bone nodules, alkaline phosphatase activity, and total cell protein. Transcription of the pro-inflammatory cytokine interleukin (IL)-1beta in the cultures was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Bone nodule formation generally decreased significantly with increasing LPS concentrations (P<0.05), whereas total cell protein decreased only slightly (P>0.05). BaP in previously high concentrations alone also caused a significant dose-dependent decrease in bone nodule formation (P<0.05) but when half maximal doses were used, significant decreases were most often seen when LPS was added. Hence, in combination, the inhibitory effects of LPS + BaP on osteogenesis were additive, inhibiting bone nodule formation up to 9-fold. Resveratrol partially reversed the inhibitory effects of low concentrations of LPS alone, and completely reversed the inhibition of nodule formation when low concentrations of LPS were combined with BaP. IL-1beta expression generally fluctuated inversely to the inhibitory activity of LPS, LPS + BaP, and LPS + BaP + Res combinations. Smoke-derived aryl hydrocarbons and bacterial LPS may act additively to inhibit bone formation. The findings may explain, in part, why net periodontal bone loss is greater and bone healing is less successful in smokers than non-smokers with periodontal infections. Reversal of the inhibitory effects in vitro by resveratrol suggests that this phytoalexin should be studied further for its potential therapeutic value, given its aryl hydrocarbon receptor antagonism and apparent anti-inflammatory activity.

The Fast Tumble Signal in Bacterial Chemotaxis

We have analyzed repellent signal processing in Escherichia coli by flash photorelease of leucine from photolabile precursors. We found that 1), response amplitudes of free-swimming cell populations increased with leucine jump concentration, with an apparent Hill coefficient of 1.3 and a half-maximal dose of 14.4 μM; 2), at a 0–0.5 mM leucine concentration jump sufficient to obtain a saturation motile response, the swimming cell response time of ∼0.05 s was several-fold more rapid than the motor response time of 0.39 ± 0.18 s measured by following the rotation of cells tethered by a single flagellum to quartz coverslips; and 3), the motor response time of individual cells was correlated with rotation bias but not cell size. These results provide information on amplification, rate-limiting step, and flagellar bundle mechanics during repellent signal processing. The difference between the half-maximal dose for the excitation response and the corresponding value reported for adaptation provides an estimate of the increase in the rate of formation of CheYP, the phosphorylated form of the signal protein CheY. The estimated increase gives a lower limit receptor kinase coupling ratio of 6.0. The magnitude and form of the motor response time distribution argue for it being determined by the poststimulus switching probability rather than CheYP turnover, diffusion, or binding. The temporal difference between the tethered and swimming cell response times to repellents can be quantitatively accounted for and suggests that one flagellum is sufficient to cause a measurable change of direction in which a bacterium swims.

Functional analysis of the SGNP I in the pupal diapause of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae)

Helicoverpa assulta suboesophageal ganglion neuropeptide I (Has-SGNP I) is a 24-amino acids peptide amide, which shows 62.5% similarity with the diapause hormone of Bombyx mori (Bom-DH). It has been demonstrated that embryonic diapause is induced by DH in B. mori. Injection of synthetic amidated Has-SGNP I terminated pupal diapause in a dose-dependent manner. Therefore, Has-SGNP I might be referred to a “diapause termination hormone” in H. assulta (Has-DTH). The maximal dose of Has-DTH for diapause termination was 1.0 μg and the half-maximal dose 0.4 μg. The time required for diapause termination of Has-DTH was 2–3 days longer than that of 20-hydroxyecdysone. During the pupal stage, DTH mRNA content in the SGs of nondiapausing pupae was always higher than in diapausing pupae using the combined method of quantitative RT-PCR and Southern blot. DTH gene also expressed at a low level while diapausing pupae were chilled at 4 °C, but increased rapidly and largely after being transferred to 25 °C. Using a competitive ELISA, Has-DTH-like immunoreactivity in the haemolymph showed the same pattern as that of Has-DTH gene expression. Those results indicated that Has-DTH gene expression was related to diapause development and could be activated by low temperature. Has-DTH might be useful to elucidate the mechanism of diapause termination in pupal diapause species.

The rostral ventrolateral medulla mediates sympathetic baroreflex responses to intraventricular angiotensin II in rabbits

The present study examined the role of the rostral ventrolateral medulla (RVLM) in mediating the pressor and renal sympathetic baroreflex effects of intraventricularly administered angiotensin II (Ang II) in urethane anaesthetised rabbits. Microinjection of Ang II over a wide range of medullary sites showed that pressor responses were observed only in the RVLM. Ang II was particularly potent in producing a transient pressor response at this site with a half maximal dose of 9 fmol. The administration of the Ang II antagonist Sar 1-Ile 8-Ang II (10 pmol) bilaterally into the RVLM inhibited the pressor response to local and fourth ventricular Ang II, but not the pressor response to RVLM applied glutamate. To determine the contribution of the RVLM to the renal sympathetic baroreflex effects of Ang II, blood pressure–renal sympathetic nerve activity (RSNA) curves were constructed with intravenous infusion of phenylephrine or nitroprusside before and after Ang II, vehicle or glutamate infusions into the RVLM. Ang II infusion of 4 pmol/min into the RVLM increased blood pressure by 8±3 mm Hg and shifted the renal sympathetic baroreflex curve to the right. The maximum RSNA evoked by lowering blood pressure increased by 36±6%, similar to the effect seen with fourth ventricular Ang II and RVLM glutamate. These studies suggest that the major medullary pressor site of action of Ang II when injected into the hindbrain cerebro-spinal fluid of anaesthetized rabbits is the RVLM where it facilitates baroreflex control of RSNA.

Targeting and blocking B7 costimulatory molecules on antigen-presenting cells using CTLA4Ig-conjugated liposomes: in vitro characterization and in vivo factors affecting biodistribution.

CTLA4Ig, a fusion protein of CTLA-4 and Fc of immunoglobulin (Ig) heavy chain, inhibits the essential costimulatory signal for full T cell activation via blocking the interaction between CD28 and B7 molecules and renders T cell nonresponsiveness. CTLA4Ig has been used to control deleterious T cell activation in many experimental systems. We hypothesized that by conjugating CTLA4Ig to liposomes the efficacy of CTLA4Ig could be enhanced through multivalent ligand effect, superior targetability, and modification of the fate of ligated costimulatory molecules. Consistent with this hypothesis, liposome-conjugated CTLA4Ig bound to B7 and blocked their binding sites more efficiently than free CTLA4Ig, lowering the half maximal dose for B7 blocking by an order of the magnitude. These results were similar both in B7-1 expressing p815 cells and in activated macrophages. Moreover, CTLA4Ig-liposomes underwent rapid internalization upon cell surface binding through B7 molecules. In allogenic mixed lymphocyte reaction assays, the CTLA4Ig-liposomes were tested to show effective inhibition of T cell proliferation. In vivo, however, when CTLA4Ig-liposomes were injected into mice, a significant fraction was localized to the reticuloendothelial system (RES), presumably because of its binding to Fc receptors expressed on tissue macrophages. The Fc receptor-mediated uptake could be alleviated by coinjection of anti-FcR monoclonal antibody. In the mouse engrafted with pancreatic islets of Langerhans underneath the capsule of one kidney, despite the increased localization in RES, enhanced accumulation of CTLA4Ig-conjugated liposome was observed in the engrafted kidney compared to the contralateral kidney. We show that the conjugation of CTLA4Ig to liposome could increase the efficiency of the targeting by increasing the binding avidity at cellular level and by increasing the concentration at the target site in in vivo system. The biodistribution and circulation time data suggested that the CTLA4Ig-liposomes could be improved upon minimizing the FcR-mediated uptake by Fc receptor-bearing cells. Thus, the strategy of conjugating CTLA4Ig to liposomes could be exploited for immune intervention in transplantation and autoimmune diseases for the efficient blocking of costimulation.

Polyphenol-enriched extract of oil palm fronds (Elaeis guineensis) promotes vascular relaxation via endothelium-dependent mechanisms.

Plant-based polyphenolic compounds have been reported to possess cardiovascular health benefits. Several dietary sources, including herbs and spices, fruits and vegetables, and tea and wine, contain an array of biologically active compounds that have been shown to be effective in retarding oxidation of low-density lipoproteins (LDL) and promoting vascular relaxation. In the present study four different plant sources, both edible and non-edible, were evaluated for potential activity. Organic extracts enriched in polyphenols were prepared from palm fronds (Elaesis guineensis); lemongrass (Cymbopogon citrates); papaya shoots (Carica papaya) and green chilli (Capsicum frutescenes) and tested for their ability to prevent in vitro oxidation of LDL, and for potential vascular relaxation actions. Rings of rat thoracic aorta and isolated perfused mesenteric vascular beds were mounted in organ baths, contracted using a half-maximal dose of noradrenaline and exposed to cumulative additions of test extracts. Palm frond extract resulted in considerable relaxation (>75%) in both preparations and was found to be endothelium-dependent as removal of endothelium or inhibition of endogenous nitric oxide (NO) led to a total loss in relaxant activity. Lemongrass extract caused a greater relaxation action in the mesenteric preparation compared to aortic rings, and appears to be mediated via NO-independent and non-prostanoid mechanisms. Of the extracts tested, palm fronds also demonstrated the highest antioxidant capacity, as determined by the ferric reducing activity/potential assay, and resulted in a significant delay (P < 0.05) in the oxidation of LDL. Collectively, these preliminary findings lend further support to the potential cardiovascular actions of plant polyphenols and also identify oil palm fronds as containing constituents that promote vascular relaxation via endothelium-dependent mechanisms.

IGF-I regulates osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand in vitro and OPG in vivo.

IGF-I, a ubiquitous polypeptide, plays a key role in longitudinal bone growth and acquisition. The most predominant effect of skeletal IGF-I is acceleration of the differentiation program for osteoblasts. However, in vivo studies using recombinant human (rh) IGF-I and/or rhGH have demonstrated stimulation of both bone formation and resorption, thereby potentially limiting the usefulness of these peptides in the treatment of osteoporosis. In this study, we hypothesized that IGF-I modulates bone resorption by regulating expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) in bone cells. Using Northern analysis in ST2 cells, we found that human IGF-I suppressed OPG mRNA in a time- and dose-dependent manner: 100 micro g/LIGF-I (13 nM) decreased OPG expression by 37.0 +/- 1.8% (P < 0.002). The half maximal inhibitory dose of IGF-I was reached at 50 micro g/liter ( approximately 6.5 nM) with no effect of IGF-I on OPG message stability. Conditioned media from ST2 cells confirmed that IGF-I decreased secreted OPG, reducing levels by 42%, from 12.1-7 ng/ml at 48 h (P < 0.05). Similarly, IGF-I at 100 micro g/liter (13 nM) increased RANKL mRNA expression to 353 +/- 74% above untreated cells as assessed by real-time PCR. In vivo, low doses of rhGH when administered to elderly postmenopausal women only modestly raised serum IGF-I (to concentrations of 18-26 nM) and did not affect circulating OPG concentrations; however, administration of rhIGF-I (30 micro g/kg.d) for 1 yr to older women resulted in a significant increase in serum IGF-I (to concentrations of 39-45 nM) and a 20% reduction in serum OPG (P < 0.05). In summary, we conclude that IGF-I in a dose- and time-dependent manner regulates OPG and RANKL in vitro and in vivo. These data suggest IGF-I may act as a coupling factor in bone remodeling by activating both bone formation and bone resorption; the latter effect appears to be mediated through the OPG/RANKL system in bone.

Economics of factor adjusted herbicide doses: a simulation analysis of best efficacy targeting strategies ( BETS)

This paper explores short and long run economic outcomes of fixed label herbicide doses versus a flexible “best efficacy targeting strategy” ( BETS), which is factor adjusted to current weather and density of weeds. A herbicide efficacy model is combined with water balance, wheat yield, yield loss and weed seedbank dynamics models to construct a bioeconomic simulation model. Results with long run weather records from two contrasting rain fed wheat districts and a range of weed densities showed BETS was superior to static maximum label or half maximum dose rates at both locations, in terms of Hamiltonians representing mean net present values of current plus future benefits and costs of weed management. BETS also resulted in lower overall herbicide use, except in the case of the highest weed density where the half max dose was lower. These positive results raise the question whether such benefits from factor adjusting dose can be realised more generally, at other locations and in the cases of other weeds, crops and herbicides.

Ligand-specific regulation of proteasome-mediated proteolysis of estrogen receptor-alpha.

Proteasome-mediated proteolysis modulates the cellular concentration of estrogen receptor-alpha (ERalpha) and is induced by treatment of cells with 17beta-estradiol. Herein, we show that multiple receptor agonists, including 17alpha-estradiol and estriol as well as the antagonist ICI-182780, stimulate proteasome-dependent proteolysis of ERalpha in a process that requires ligand binding to the receptor. Proteolysis of receptor depends on ligand concentration, and there exists a direct correlation between ligand-binding affinity and the half-maximal dose of ligand required to stimulate receptor degradation. Furthermore, introduction of a point mutation into the receptor ligand-binding pocket yields a stable receptor resistant to proteolysis. Interestingly, although all ligands stimulate receptor degradation, the extent to which overall ER levels are affected varies with each ligand and is not related to ligand-binding affinity or activation of transcription. These results demonstrate ligand-specific regulation of ERalpha proteolysis, and they introduce the concept that cellular receptor concentration is governed not only at the level of induction of proteolysis but also by the efficiency with which the receptor is degraded.

9-Carboxymethyl-5H,10H-imidazo[1,2-a]indeno[1,2-e]pyrazin-4-one-2-carbocylic Acid (RPR117824): Selective Anticonvulsive and Neuroprotective AMPA Antagonist

Excessive release of glutamate, a potent excitatory neurotransmitter, is thought to play an important role in a variety of acute and chronic neurological disorders, suggesting that excitatory amino acid antagonists may have broad therapeutic potential in neurology. Here, we describe the synthesis, pharmacological properties and neuroprotective activity of 9-carboxymethyl-imidazo-[1-2a]indeno[1-2e]pyrazin-4-one-2-carboxylic acid (RPR117824), an original selective AMPA antagonist. RPR117824 can be obtained through a six-step synthesis starting from (1-oxo-indan-4-yl) acetic acid, which has been validated on a gram-scale with an overall yield of 25%. Monosodium or disodium salts of the compound exhibit excellent solubility in saline (≥10 g/L), enabling intravenous administration. RPR117824 displays nanomolar affinity (IC50=18 nM) for AMPA receptors and competitive inhibition of electrophysiological responses mediated by AMPA receptors heterologously expressed in Xenopus oocytes (KB=5 nM) and native receptors in rat brain slices (IC50=0.36 μM). In in vivo testing, RPR117824 behaves as a powerful blocker of convulsions induced in mice or rats by supramaximal electroshock or chemoconvulsive agents such as pentylenetetrazole, bicuculline, isoniazide, strychnine, 4-aminopyridine and harmaline with half maximal effective doses ranging from 1.5 to 10 mg/kg following subcutaneous or intraperitoneal administration. In disease models in rats and gerbils, RPR117824 possesses significant neuroprotective activity in global and focal cerebral ischemia, and brain and spinal cord trauma.

Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity.

Obesity in humans and in rodents is usually associated with high circulating leptin levels and leptin resistance. To examine the molecular basis for leptin resistance, we determined the ability of leptin to induce hypothalamic STAT3 (signal transducer and activator of transcription) signaling in C57BL/6J mice fed either low-fat or high-fat diets. In mice fed the low-fat diet, leptin activated STAT3 signaling when administered via the intraperitoneal (ip) or the intracerebroventricular (icv) route, with the half-maximal dose being 30-fold less when given by the icv route. The high-fat diet increased body-weight gain and plasma leptin levels. After 4 weeks on the diet, hypothalamic STAT3 signaling after ip leptin administration was equivalent in both diet groups. In contrast, peripherally administered leptin was completely unable to activate hypothalamic STAT3 signaling, as measured by gel shift assay after 15 weeks of high-fat diet. Despite the absence of detectable signaling after peripheral leptin at 15 weeks, the mice fed the high-fat diet retained the capacity to respond to icv leptin, although the magnitude of STAT3 activation was substantially reduced. These results suggest that leptin resistance induced by a high-fat diet evolves during the course of the diet and has at least two independent causes: an apparent defect in access to sites of action in the hypothalamus that markedly limits the ability of peripheral leptin to activate hypothalamic STAT signaling, and an intracellular signaling defect in leptin-responsive hypothalamic neurons that lies upstream of STAT3 activation.

Antimicrobial peptides derived from heme-containing proteins: hemocidins.

Deprived of heme and partially unfolded hemoglobin, myoglobin and cytochrome c display microbicidal activity against a broad spectrum of microorganisms with half maximal lethal dose estimated at micromolar concentrations. The intact proteins were ineffective. Antibacterial activity of these apohemoproteins was also sustained after digestion to approximately 50 amino acids long peptides but further fragmentation abolished microbicidal properties. The most active fragment of apomyoglobin (corresponding to 56-131 region) showed a pronounced effect on the E. coli membrane permeabilization and its action was sensitive to salt as well as to divalent cations concentrations. The membrane-directed effect was specific toward bacteria but no lipopolysaccharide binding properties were observed. No hemolytic properties, even at high peptide concentrations were found; however, a slight but dose-independent cytotoxic effect was observed on fibroblasts and hepatoma cells. The presented data suggest a 'carpet-like' mechanism of the membrane-directed activity and may result from exceptional abilities of hemoprotein-derived peptides to form alpha-helical structures. We postulate that the antimicrobial peptides obtained from the heme-containing proteins should be named hemocidins, in contrast to, e.g., hemorphins displaying opioid-like activity.

Role of cholecystokinin in the anorexia produced by duodenal delivery of peptone in rats.

We used the cholecystokinin receptor antagonist devazepide to assess the importance of CCK in mediating the anorexia produced by 2-h duodenal infusions of peptone, a protein digest, at dark onset in nonfasted rats. Peptone alone (0.14-2.24 g/h) suppressed food intake dose dependently by 18-96%, with an approximate half-maximal dose of 1 g/h. Peptone-induced reductions in caloric ingestion were comparable to the caloric loads infused. Devazepide alone (30-1,000 microgram/kg) stimulated food intake dose dependently by 30-73%, with a minimal effective dose of 100 micrograms/kg. Devazepide appeared to reverse the anorexic response to peptone (1.1 g/h) dose dependently by 29-65%, with a minimal effective dose of 30 micrograms/kg. The magnitudes of these devazepide-induced effects were similar to, and in some cases were larger than, those produced when the same doses of devazepide were administered alone. Coadministration of devazepide (1,000 micrograms/kg) and a lower peptone dose (0.8 g/h) produced similar results. These results suggest that an essential CCK mechanism plays a significant role in mediating the satiety response to duodenal delivery of protein.

Pituitary adenylate cyclase-activating polypeptide is an auto/paracrine stimulator of acute progesterone accumulation and subsequent luteinization in cultured periovulatory granulosa/lutein cells.

Recently, we have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) is transiently expressed in steroidogenic ovarian cells during the periovulatory period. This prompted us to establish an in vitro system in which the potential local regulatory role of PACAP during periovulatory progesterone production could be examined. Granulosa/lutein cells from PMSG- and human CG (hCG)-stimulated immature rats were used. The cells were isolated from preovulatory follicles 4-6 h after the hCG injection, at which time the transient ovarian PACAP expression begins in vivo. By immunocytochemistry on intact cells and RIA on cell extracts and culture medium, granulosa/lutein cells were found to accumulate and secrete PACAP during incubation. Furthermore, the cells responded to exogenous PACAP 38 with a rapid (10(-7) M induced a peak value 20-fold higher than controls at 2 h) and dose-dependent accumulation of progesterone. PACAP 38 (5 x 10(-9) M), in combination with an approximately half-maximal dose of hCG (1 ng/ml), showed an additive effect on progesterone accumulation. Immunoneutralization of endogenously released PACAP was performed using the IgG fraction from a specific PACAP antiserum that dose-dependently inhibits the progesterone accumulating effect of exogenous PACAP 38. The acute effects of endogenously released PACAP were studied during 8 h of incubation of granulosa/lutein cells with anti-PACAP IgG (100 microg/ml). A significant reduction in progesterone accumulation was observed after 4, 6, and 8 h [38.7% (P < 0.05), 41.2% (P < 0.02), and 50% (P < 0.002), respectively], compared with nonimmune IgG (100 microg/ml) treated cultures. The long-term effects on luteinization induced by endogenously released PACAP were studied after incubation of the cells with anti-PACAP IgG or nonimmune IgG for 24 h, followed by incubation for 9 days in serum-containing medium. Under these conditions, nonimmune IgG-treated cells assumed a luteal phenotype, accumulating large and stable amounts of progesterone and acquiring hypertrophic cell bodies with numerous lipid droplets and distinct nucleoli in the large nuclei. Anti-PACAP IgG-treated cells displayed morphological and functional signs of impaired luteinization being smaller and more irregular and with progesterone accumulation being significantly lower throughout the incubation period [56.4% (P < 0.02), 69.2% (P < 0.05), 43.8% (P < 0.02), and 52.2% (P < 0.02) at 1, 4, 7, and 10 days, respectively]. Together, these findings support an auto- or paracrine role for PACAP during gonadotropin-induced acute periovulatory progesterone production and subsequent luteinization in granulosa/lutein cells.

Thyroid hormone effects on mouse oocyte maturation and granulosa cell aromatase activity.

In the present study we evaluated the role of T3 on the in vitro processes of mouse cumulus cell-oocyte complex expansion, oocyte meiotic maturation, and granulosa cell aromatase activity. Results obtained from cumuli oophori isolated from immature and adult mice ovaries demonstrated that T3 at all concentrations tested (0.1-100 nM) did not affect basal or FSH-induced cumulus expansion or interfere with oocyte meiotic maturation up to metaphase II stage. On the contrary, T3 inhibited in a time- and dose-dependent manner FSH-induced aromatase activity in cultured granulosa cells obtained from either adult or immature female mice. The half-maximal dose (ED50) of T3 inhibition was 0.87 +/- 0.21 nM, which is in agreement with the reported dissociation constant of T3 nuclear receptor (Kd = 0.4-5 nM) in mammalian granulosa cells. Time-course experiments demonstrated higher sensitivity to T3 of adult granulosa cells with respect to immature granulosa cells in culture. Indeed, in immature granulosa cells T3 inhibition became significantly evident only after 6 days of hormonal treatment, whereas in adult granulosa cells the inhibitory effect was present after only 2 days of treatment. (Bu)2cAMP- or 3-isobutyl-1-methyl-xanthine-stimulated aromatase activity was also significantly decreased by T3, thus suggesting that the inhibition was downstream from cAMP formation. Lastly, analysis of aromatase messenger RNA (mRNA) levels by semiquantitative RT-PCR demonstrated the ability of FSH to increase aromatase mRNA level in cultured granulosa cells by 2.4 +/- 0.5-fold. In agreement with the effect on enzyme activity, the stimulatory effect of FSH on aromatase mRNA level was greatly reduced after T3 cotreatment. In conclusion, T3 inhibition of aromatase activity may be of physiological relevance in the complex multihormonal regulation of mammalian follicle development and may contribute to explaining the alteration in female reproductive functions after thyroid hormone hypo- or hypersecretion.

Identification and Characterization of Receptor for Mammalian Hepatopoietin That Is Homologous to Yeast ERV1

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-alpha, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (Kd) of 2 and 0.7 pM, and a receptor density of about 10, 000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. The Kd values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with 125I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.

Different contractile properties between intralobar and extralobar pulmonary arteries of the rabbit.

In pulmonary circulation, small muscular resistance arteries are known to have different receptor properties and sensitivity to neurotransmitters from those of large elastic conduit arteries. It is, however, not yet certain whether the different properties are primarily due to the diameter or the location of arteries. In the present study, we compared the contractile responses to various agonists among large extralobar (ELPA, diameter: 2-3 mm), large intralobar (ILPA, diameter: 2-3 mm), and small intralobar pulmonary arteries (SPA, diameter: 300-500 microm) of the rabbit. There were no differences in normalized dose-response curves to KCl among three groups. Half maximum doses (EC50 in mM) were 38.0+/-2.0 (n=8, mean+/-SEM) in ELPA, 36.9+/-2.4 (n=10) in ILPA, and 39.0+/-0.9 (n=12) in SPA. Responses to phenylephrine, epinephrine, histamine, serotonin, and PGF2alpha were normalized and expressed as a relative contraction against maximum tension to KCl. In ELPA, the contractile responses to various agents showed the following sequence: KCl>epinephrine>phenylephrine>serotonin>PGF2alpha>histamine. In ILPA and SPA, the sequence was: KCl>histamine>PGF2alpha>serotonin. There was little response to phenylephrine and epinephrine in ILPA and SPA. These results demonstrate that the difference of contractile responses between ELPA and ILPA was more prominent than that between ILPA and SPA, suggesting that the location is more important than the diameter itself in determining the characteristic contractile responses of pulmonary arteries.

Endotoxin and cytokines increase hepatic sphingolipid biosynthesis and produce lipoproteins enriched in ceramides and sphingomyelin.

Alterations in triglyceride and cholesterol metabolism often accompany inflammatory diseases and infections. We studied the effects of endotoxin (lipopolysaccharide [LPS]) and cytokines on hepatic sphingolipid synthesis, activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid synthesis, and lipoprotein sphingolipid content in Syrian hamsters. Administration of LPS induced a 2-fold increase in hepatic SPT activity. The increase in activity first occurred at 16 hours, peaked at 24 hours, and was sustained for at least 48 hours. Low doses of LPS produced maximal increases in SPT activity, with half-maximal effect seen at approximately 0.3 microg LPS/100 g body weight. LPS increased hepatic SPT mRNA levels 2-fold, suggesting that the increase in SPT activity was due to an increase in SPT mRNA. LPS treatment also produced 75% and 2.5-fold increases in hepatic sphingomyelin and ceramide synthesis, respectively. Many of the metabolic effects of LPS are mediated by cytokines. Interleukin 1 (IL-1), but not tumor necrosis factor, increased both SPT activity and mRNA levels in the liver of intact animals, whereas both IL-1 and tumor necrosis factor increased SPT mRNA levels in HepG2 cells. IL- produced a 3-fold increase in SPT mRNA in HepG2 cells, and the half-maximal dose was 2 ng/mL. IL-1 also increased the secretion of sphingolipids into the medium. Analysis of serum lipoprotein fractions demonstrated that very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein isolated from animals treated with LPS contained significantly higher amounts of ceramide, glucosylceramide, and sphingomyelin. Taken together, these results indicate that LPS and cytokines stimulate hepatic sphingolipid synthesis, which results in an altered structure of circulating lipoproteins and may promote atherogenesis.

Multivalent cations and ligand affinity of the type 1 insulin-like growth factor receptor on P2A2-LISN muscle cells.

Mouse P2A2-LISN myoblasts are transfected cells that overexpress the human type 1 insulin-like growth factor (IGF) receptor. Because the type 1 IGF receptor is the major binding site for both IGF-I and IGF-II, this cell line is an excellent model to determine the effect of multivalent cations on ligand binding specifically to this type of receptor. Competitive binding assays were performed to characterize IGF binding and Scatchard analysis to quantify affinity (Ka). 125I-IGF-I, 125I-IGF-II, and 125I-R3-IGF-I bind only to the type 1 IGF receptor on these cells. Zn2+ increased binding of the three ligands to the type 1 IGF receptor by 17 to 35%. Cd2+ significantly increased binding of 125I-IGF-I, although by only 8%. La3+ and Cr3+ did not effect binding. Au3+ decreased IGF binding by approximately 56%. Scatchard analysis produced nonlinear concave-down plots yielding binding constants for high and low affinity sites. Zn2+ increased the strength of only the high affinity sites. Au3+ decreased the affinity of both high and low affinity sites. Zn2+ increased binding with a half-maximal effect between 40 microM and 60 microM. Half-maximal dose of Au3+ was >130 microM. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) and only these cations were found to affect receptor binding indicating similar mechanisms of action. Thus, multivalent cations may alter IGF binding to cell surface receptors ultimately controlling growth. Physiologically this may be especially important for the growth promoting effects of Zn2+.

Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor.

Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.