Preadipocyte differentiation in vitro: identification of a highly active adipogenic agent

A highly active adipogenic agent was identified in an in vitro adipose conversion system. This agent, ADD 4743 (or ADD), was synthesized by Takeda Chemical Industries (Osaka) as a 3-hydroxy derivative of an oral antidiabetic agent, ciglitazone, and has been presumed to be an active metabolite of the latter substance. When ST 13 mouse preadipose cells were treated with micromolar concentrations of ADD they rapidly and uniformly converted into lipid-accumulating adipocytelike cells within 8-11 days after cell seeding. The degree of adipose conversion and lipid accumulation induced by ADD far exceeded those of the previously known inducing agents such as indomethacin plus insulin. The highly potent adipogenic activity of ADD was confirmed with two other preadipose cell lines (3T3 L1 and RMT rat preadipose cells). In addition to adipogenic activity, ADD inhibited cell proliferation of preadipose cells specifically. Activity of ADD induced lipid accumulation and growth inhibition of ST 13 cells, exhibiting very similar dose-response relationships. Cell proliferation or triacylglycerol content of nonadipocytic mesenchymal cells or epithelial cells were not affected by ADD. These observations strongly suggest that ADD-induced growth inhibition is not due to the nonspecific toxicity of the drug but is tightly associated with the adipocytic character of the treated cells. The present observation provides evidence that ADD would be a powerful agent in studies that involve preadipocyte differentiation.     

Modulation of rat guanylate cyclase activity in vitro by chemical carcinogens.

The nucleotide cyclic GMP has been reported to be involved in cell proliferation and malignant transformation. Nitroso chemical carcinogens activate the enzyme guanylate cyclase (EC 4.6.1.2) which catalyzes the production of cyclic GMP. The present investigation demonstrates that compounds from other major classes of carcinogens including (1) alpha-halo ethers (chloromethyl methyl ether); (2) aromatic amines (benzidine and B-naphthylamine); (3) polycyclic hydrocarbons (1,2-benzanthracene and acridine); (4) azo dyes (p-dimethylaminoazobenzene), and (5) aflatoxins (B1, B2, G1, G2) produced a striking and significant inhibition of guanylate cyclase over a general concentration range of 0.5-13 mmol/1 in a variety of tissues. Some of the nitrosamides which increase guanylate cyclase activity, increase DNA synthesis whereas carcinogens which decrease guanylate cyclase activity inhibit DNA or RNA synthesis suggesting a relationship between cyclic GMP, DNA synthesis, and chemical carcinogenesis.     

Endothelin is a positive inotropic agent in human and rat heart in vitro

We have investigated the response to endothelin of isolated atrial and ventricular trabeculae from failing human hearts obtained at transplant. Results indicate that endothelin exerts a significant positive inotropic effect on human atrial and ventricular tissue, with increases in developed tension of 74.6 +/- 14.1% (+/- SEM) and 9.9 +/- 4.0%, respectively. Further studies on rat cardiac muscle demonstrate that the greater inotropic effect on atrial than ventricular muscle is also exhibited by the rat heart in vitro, with 39.9 +/- 10.7% and 17.1 +/- 5.9% increases in developed tension for atria and papillary muscle, respectively. Studies in rat atria also provide no evidence for an effect of endothelin on the frequency of spontaneous contractions. These results suggest that the potential exists for regulation of cardiac function in humans and rats by endothelial-derived factors such as endothelin, possibly via augmentation of atrial systole.     

Everolimus is an active agent in medullary thyroid cancer: a clinical and in vitro study

Everolimus, an mTOR inhibitor, which has been demonstrated to induce anti-tumour effects in different types of neuroendocrine tumours, has never been evaluated in patients with medullary thyroid cancer (MTC). The aim of this study was to evaluate the in vitro and in vivo effects of everolimus in combination with octreotide in MTC. Two patients with progressive metastatic MTC and high calcitonin levels were treated with everolimus 5-10 mg/day. Both patients were under treatment with octreotide LAR at the study entry. An in vitro study was also performed to assess everolimus effects on MTC cell lines (TT and MZ-CRC-1 cells). A tumour response was observed in both patients. Serum calcitonin decreased by 86% in patient 1 and by 42% in patient 2. In TT and MZ-CRC-1 cells, everolimus induced a significant dose-dependent inhibition in cell proliferation. This effect seems to be related to a cell cycle arrest in G(0) /G(1) phase in both cell lines and to the induction of cellular senescence in TT cells. Everolimus in combination with octreotide may be active as anti-tumour therapy in patients with progressive metastatic MTC, suggesting to further evaluate this agent in MTC patients in a large prospective study.     

Modulation of a neuronal network by electrical high frequency stimulation in striatal slices of the rat in vitro

The effects of the GABA(A) receptor antagonist bicuculline, the D2-like receptor antagonist sulpiride and the D1-like receptor antagonist SCH-23390 on the electrical high frequency stimulation (HFS)-evoked gamma-aminobutyric acid (GABA) and dopamine (DA) release were measured from slices of the rat striatum by means of HPLC method with electrochemical detection. HFS with 130Hz stimulated veratridine-activated GABAergic neurons resulting in an increased GABA outflow while DA outflow decreased. In the presence of the GABA(A) receptor antagonist bicuculline extracellular GABA and DA outflow were enhanced. When the competitive dopamine D2-like receptor antagonist S-(-)-sulpiride was added to incubation medium, the HFS-evoked stimulatory effect on GABA outflow declined to values found after veratridine (1microM) without HFS. After co-incubation of sulpiride and the competitive D1-like receptor antagonist R-(+)-SCH-23390, the effect of sulpiride on HFS plus veratridine-evoked GABA outflow was completely reversed. Neither sulpiride nor SCH-23390 had any influence on the effect of HFS on veratridine-induced DA outflow. No effect of HFS on glutamate outflow was observed in all experiments. These results led us to suggest that in our model HFS primarily affects GABAergic neurons. These neurons are embedded in a neuronal network with a GABA-dopamine circuit, and thus, HFS interacts with a neuronal network, not only with one neurotransmitter system or one neuron population.     

Modulation of a mitochondrial function by oat phytochrome in vitro

Previous data in the literature have indicated that phytochrome could alter the rate of reduction of exogenously added NADP by a pea mitochondrial preparation in vitro. These results could not be duplicated using a mitochondrial preparation isolated from etiolated oat seedlings. Further experimentation demonstrated that the addition of Pr to the preparation, in combination with a far red light illumination, could significantly reduce the rate of oxidation of NADH by the external dehydrogenases of oat mitochondria. This response was characterized by a 15% decrease in reaction velocity at saturating substrate concentrations and a 2-fold increase in apparent K_m as compared to values obtained after Pfr plus red light treatment. The response was photoreversible, the rate of oxidation of exogenous NADH being determined by the last light illumination given to the mitochondrial preparation. The interaction between phytochrome and the mitochondria was apparently occurring at the level of the inner mitochondrial membrane. A requirement for these results was that the mitochondria be isolated from plants that were illuminated with white or red light before extraction; mitochondria from unirradiated plants showed no dehydrogenase response to treatments with phytochrome plus actinic light.     

Inhibition of active sodium transport by cytochalasin B in rat jejunum in vitro

The effects of cytochalasin B on electrophysiological properties and sodium transport in rat jejunum in vitro are described. Stripped paired rat jejunal segments were maintained in Ussing chambers with Leibovitz's (L-15) tissue culture medium bubbled with 100% oxygen. L-15 medium contains galactose as the only sugar, and an assortment of amino acids and cofactors to nourish the tissue. Electrophysiological parameters of short-circuit current (Isc) and transepithelial potential difference could be maintained for up to 4 h in control tissues. Upon application of cytochalasin B (20 micrograms/ml), on the mucosal side, Isc and potential difference fell within 1 h from 1.93 +/- 0.12 to 1.09 +/- 0.14 (mean +/- S.E.) muequiv./cm2 per h and from 5 to 2.5 mV. Tissue resistance remained unchanged at approx. 110 omega X cm2 for up to 4 h. 22Na net flux was 4.1 +/- 0.9 muequiv./cm2 per h during the last control period and fell to zero within 1 h after cytochalasin B treatment. Transmission electron micrographs revealed no gross morphological changes at this dose. Absorptive junctional morphology was apparently not altered by cytochalasin B treatment, a finding which was consistent with the stable transepithelial electrical resistance observed during exposure to this drug. Active sodium transport processes coupled to hexose, amino acid, and chloride movements are all possible in L-15 medium. However, following exposure to 20 micrograms/ml cytochalasin B, all net sodium transport is completely inhibited. The data are consistent with the hypothesis of a common regulator for active sodium transport processes which is modulated through structural changes in cytoskeletal organization.     

Modulation of branched-chain amino acid oxidation in rat hemidiaphragms in vitro by glucose and ketone bodies

Branched-chain amino acid metabolism in hemidiaphragms from 40 h-starved rats is influenced by the provision of glucose as co-substrate. Glucose inhibits 14CO2 production from [l-14C]valine and [U-14C]valine but stimulates 14CO2 production from [l-14C]leucine, [U-14C]leucine and [U-14C]isoleucine. In the presence of glucose, ketone bodies inhibit alanine release and 14CO2 production from [l-14C]valine, [l-14C]leucine and [U-14C]isoleucine, but inhibition is not observed in the absence of glucose as cosubstrate. Glucose-dependent inhibition by ketone bodies of branched-chain amino acid oxidation via inhibition of the branched-chain 2-oxo acid dehydrogenase complex or branched-chain amino acid aminotransferase may account in part for the reported hypoalanaemic action of ketone bodies in vivo.     

Modulation of active potassium transport by aldosterone

• 1.1. The role of aldosterone on active potassium transport across lizard colon under voltage-clamped conditions has been investigated.
• 2.2. Control colons exhibited no net potassium flux (J^k_net) despite of the existence of active opposite unidi ectional fluxes.
• 3.3. An important net secretory potassium flux was found in short-circuited aldosterone-stimulated colons.
• 4.4. Mucosal amiloride did not change (J^k_net) either in control or aldosterone-stimulated colons.
• 5.5. Luminal barium alters K ⁺ transport in a manner consistent with the presence of barium-sensitive conductances at the apical membrane of both control and aldosterone-treated colons.
• 6.6. The effects of ouabain and barium on control and aldosterone-induced potassium flows were consistent with a model involving basolateral uptake by an Na ⁺-K ⁺-ATPase and conductive exit across the apical membrane.
• 7.7. The stimulatory effect of aldosterone on potassium secretion is associated with parallel increases of both basolateral K ⁺ entry and the apical conductive pathway.

     

Studies on the effects of the antitumor agent camptothecin and derivatives on DNA. Camptothecin potentiated cleavage of DNA by bleomycin in vitro

Addition of sodium camptothecin (2a, Fig. 1) in comparable low concentrations to the glycopeptide antitumor antibiotic bleomycin (BLM) leads to enhanced rates of single-strand scission of PM2-covalently closed circular DNA, whereas sodium camptothecin alone has no effect. A similar enhancement of DNA scission by sodium camptothecin is produced with the 1 : 1 bleomycin-iron complex alone or in conjunction with NADPH as an additional reductant. The interpretation that camptothecin may substitute for the reducing requirement of the antibiotic is supported by its oxidation at 37°C by the 1 : 1 bleomycin iron complex, by iron salts or more efficiently by hydrogen peroxide to the known hemiacetal (3, Fig. 1).Electrochemical studies of 2a, its analogues and selected model compounds established that the α-pyridone ring D is most susceptible to a one-electron reduction at a reversible potential of ?0.95 ± 0.01 V. The reduced camptothecin is a transient species readily capable of donating an electron. This process may by compatible with a coupled reduction of the sequestered Fe(III) in the glycopeptide antibiotic necessary for the expression of antibiotic and antitumor properties. The results may provide a mechanistic rationale for the observed potentiation of the antitumor activity of bleomycin by camptothecin in vivo.     

Modulation of granulocyte colony stimulating factor conformation and receptor binding by methionine oxidation

Biosimilars offer an avenue for potential cost savings and enhanced patient access to various emerging therapies in a budget neutral way. Biosimilars of the granulocyte colony stimulating factor (GCSF) are an excellent example in this regard with as many as 18 versions of the drug being currently approved across globe for treatment of neutropenia. Here, we identified oxidation of the various methionine residues in GCSF as a key heterogeneity that adversely impact its efficacy. In agreement with earlier studies, it was found that oxidation of Met 122 and Met 127 significantly contributes toward reduction of GCSF efficacy, measured using binding affinity to the GCSF receptor. The combination of molecular dynamics simulation along with structural characterization studies established that oxidation of Met 127 and Met 122 brings about a small local conformational change around the B‐C loop in GCSF structure due to slight displacement of Asp113 and Thr117 residues. The simulation studies were validated using fluorescence quenching experiments using acrylamide as quencher and site‐directed mutagenesis by replacing Met 122 and Met 127 residues with alanine. The results of this study lead to an enhanced mechanistic understanding of the oxidation in GCSF and should be useful in protein engineering efforts to design stable, safe, and efficacious GCSF product. In addition, the structure‐function information can provide targets for protein engineering during early drug development and setting specifications of allowable limits of product variants in biosimilar products.     

Modulation of granulocyte functions by peptide YY in the rat: Age-related differences in Y receptors expression and plasma dipeptidyl peptidase 4 activity

It has been acknowledged that aging exerts detrimental effects on cells of the innate immune system and that neuropeptides, including neuropeptide Y (NPY) and NPY-related peptides fine-tune the activity of these cells through a receptor specific mechanism. The present study investigated the age-dependent potential of peptide YY (PYY) to modulate different granulocyte functions. The PYY reduced the carrageenan-elicited granulocyte accumulation into the air-pouch of aged (24 months) rats, and markedly decreased the phagocytosis of zymosan, as well as the H₂O₂ production, when applied in vivo (20 μg/air-pouch). The anti-inflammatory effect of PYY was less prominent in adult (8 months) and young (3 months) rats. However, the proportions of granulocytes expressing Y1, Y2 and Y5 receptor subtypes were significantly lower in both aged and young rats when compared to adult rats. Furthermore, the aging was found to be associated with the diminished dipeptidyl peptidase 4 (DP4, an enzyme converting the NPY and PYY to Y2/Y5 receptor selective agonists) activity in plasma. In conclusion, the diverse age-related anti-inflammatory effect of PYY in rats originates from different expression levels of Y1, Y2, and Y5 receptor subtypes in addition to different plasma DP4 activity.