Non-physiological Condition Research Articles

Effect of Hydroxypropyl-β-cyclodextrin on the Ocular Bioavailability of Dexamethasone from a pH-Induced Mucoadhesive Hydrogel

Purpose: Dexamethasone (DXN) is an effective anti-inflammatory drug in the treatment of acute and chronic eye disease such as uveitis. It is relatively lipophilic and permeates biological membranes quite easily. However, its low aqueous solubility limits its clinical usefulness. To circumvent this problem Hydroxypropyl-β-cyclodextrin (HP-β-CD) was used as solubilizer and penetration enhancer for DXN. The purpose of this study was to develop HP-β-CD based pH-induced mucoadhesive hydrogel for ophthalmic delivery of DXN to treat uveitis.Materials and methods: The formation of inclusion complex of DXN with HP-β-CD was characterized in solution and solid states by phase solubility, X-ray diffractometry and IR spectrum analyses. To improve ocular retention and sustained action Carbopol 980 NF and sodium carboxymethylcellulose (NaCMC) were added to the formulations as phase transition and mucoadhesive agents, respectively.Results: The HP-β-CD-based hydrogel system enhanced the solubility of DXN and the apparent stability constant (k′) of the DXN-HP-β-CD inclusion complex was found to be 258.62 M−1. The optimum concentrations of Carbopol 980NF and NaCMC for the mucoadhesive hydrogel were 0.2% (w/v) and 0.4% (w/v), respectively. This mucoadhesive hydrogel could flow freely under non-physiological condition and showed the character of pseudoplastic fluid under both physiological and non-physiological conditions. In vitro release of DXN from the HP-β-CD complex in simulated tear fluid (STF, pH− 7.4), was influenced significantly by the properties and concentration of Carbopol and NaCMC. In vivo studies in rabbit eye showed a marked improvement in anti-inflammatory activity of mucoadhesive hydrogel-treated eye compared with a marketed solution formulation in a uveitis-induced rabbit eye model.Conclusion: The developed HP-β-CD-based mucoadhesive system is a viable alternative to conventional eye drops of DXN due to its ability to enhance bioavailability through its longer precorneal residence time and ability to sustain the release of the drug.

Transgenic Mice Expressing Cyclooxygenase-2 in Hepatocytes Reveal a Minor Contribution of This Enzyme to Chemical Hepatocarcinogenesis

Cyclooxygenase-2 (COX-2) has been associated with cell growth regulation, tissue remodeling, and carcinogenesis. Ectopic expression of COX-2 in hepatocytes constitutes a nonphysiological condition ideal for evaluating the role of prostaglandins (PGs) in liver pathogenesis. The effect of COX-2-dependent PGs in chronic liver disease, hepatitis, fibrosis, and chemical hepatocarcinogenesis, has been investigated in transgenic (Tg) mice that express human COX-2 in hepatocytes and in Tg hepatic human cell lines. We have used three different complementary approaches: i) diethylnitrosamine (DEN)-induced chemical hepatocarcinogenesis in COX-2 Tg mice, ii) DEN/phenobarbital treatment of human COX-2 Tg hepatocyte-like cells, and iii) COX-2 Tg hepatocyte-like cells implants in nude mice. The data suggest that PGs produced by COX-2 in hepatocytes promoted mild hepatitis in 60-week-old mice, as assessed by histological examination, but failed to contribute to the development of liver fibrogenesis after methionine- and choline-deficient diet treatment. Moreover, liver injury, collagen content, and hepatic stellate cell activation were equally severe in wild-type and COX-2 Tg mice. The contribution of COX-2-dependent PGs to the development of DEN-induced hepatocarcinogenesis was evaluated in Tg mice, Tg hepatocyte-like cells, and nude mice and the analysis revealed that COX-2 expression favors the development of preneoplastic foci without affecting malignant transformation. Endogenous COX-2 expression in wild-type mice is a late event in the development of hepatocellular carcinoma.

Urinary C‐Peptide Excretion: A Novel Alternate Measure of Insulin Sensitivity in Physiological Conditions

Insulin sensitivity (IS) is measured by the euglycemic-hyperinsulinemic clamp under a nonphysiological condition. Daily C-peptide urinary excretion may be a physiological index of IS, because C-peptide is co-secreted with insulin as a function of nutrient intake and IS. The amount of (2)H(2)O released from glycolytic glucose metabolism after [6,6-(2)H(2)]-glucose ingestion was recently proposed as a physiological measure of IS. We compared these IS surrogates to the gold standard (euglycemic-hyperinsulinemic clamp). Thirty (15 male/15 female) sedentary, nondiabetic participants (27.2 +/- 4.0 (s.d.) kg/m(2), 35 +/- 12 years) were admitted for 3 days to our in-patient unit. After a 10-h fast, they received 60 g of glucose and 15 g of [6,6-(2)H(2)]-glucose. Before glucose ingestion and hourly thereafter for 4 h, plasma glucose and insulin concentrations, and plasma deuterium enrichment were determined. Plasma (2)H(2)O production divided by insulin response was used as the glycolytic index. On day 2, subjects spent 23 h in a metabolic chamber (eucaloric diet, 50% carbohydrate, 30% fat). Urinary C-peptide excretion was divided by energy intake yielding the C-peptide to energy intake ratio (CPEP/EI). After leaving the chamber (day 3, 10-h fast), IS was measured by a 2-h clamp (120 mU/m(2)/min). Average IS (clamp) was 7.3 +/- 2.6 mg glucose/kg estimated metabolic body size/min (range: 3.6-13.2). These values were inversely correlated with CPEP/EI (r = -0.62; P < 0.01) and positively with the glycolytic rate (r = 0.60; P < 0.01). In nondiabetic subjects, two novel estimates of IS--daily urinary C-peptide urinary excretion and glycolytic rate during an oral glucose tolerance test --were related to IS by a clamp.

Abstract 1102: Regulation of protein citrullination through p53/PADI4 network in DNA damage response

Abstract Upon wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However its involvement in post-translational modifications of proteins has not been well characterized so far. We here report the novel role of p53 in the regulation of protein citrullination. Citrullination (also referred as deimination) is one form of PTMs converting an arginine residue in protein to a citrulline residue. This reaction is mediated by a Ca2+-dependent enzyme called as peptidylarginine deiminase. Citrullination of proteins results in a loss of positive charge and causes a significant biochemical change. On the other hand, citrullinated peptides/proteins are recognized as a non-self protein and subsequently activate immune systems. Hence, protein citrullination is now considered to play a pivotal role in various human diseases, but any roles of citrullination in carcinogenesis were not reported so far. We found p53 transactivated peptidylarginine deiminase type 4 (PADI4) through intronic p53-binding site and ectopic expression of p53 or PADI4 induced protein citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their citrullination, indicating the regulation of protein citrullination in a p53/PADI4 dependent manner. We identified that PADI4 citrullinated histone chaperon protein, nucleophosmin (NPM1) at arginine 197 residue in vivo under physiological condition. NPM1 was shown to continuously shuttle between the nucleus and cytoplasm and translocate from nucleoli to nucleoplasm as a response to DNA damage. Therefore, we investigated the effect of protein citrullination on subcellular localization of NPM1. Immunocytochemical analysis revealed that citrullination of NPM1 by PADI4 resulted in its translocation from nucleoli to nucleoplasm, while PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly knockdown of PADI4 attenuated p53-mediated growth inhibitory activity, demonstrating the significance of PADI4-mediated protein citrullination in the p53 signaling pathway. Most of previous studies assessing enzymatic activities of PADIs were conducted under non-physiological condition using Ca ionophore or at higher Ca concentration. To our knowledge, this is the first report demonstrating the physiological regulatory mechanism of protein citrullination. Thus, p53 that can directly or indirectly modulate biological property of various proteins through the protein citrullination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1102.

Sodium alginate based mucoadhesive system for gatifloxacin and its in vitro antibacterial activity.

The objective of this study was to formulate sodium alginate based ophthalmic mucoadhesive system of gatifloxacin and its in vitro antibacterial potential on pathogenic microorganisms, Staphylococcus aureus and Escherichia coli. Sodium carboxymethylcellulose (NaCMC) was added to the formulations to enhance the gel bioadhesion properties. The prepared formulations were evaluated for their in vitro drug release, gelation behaviour, rheological behavior, and mucoadhesion force. All formulations in non-physiological and physiological condition showed pseudo plastic behavior. Increase in the concentration of sodium alginate and sodium CMC enhanced the mucoadhesive force significantly. In vitro release of gatifloxacin from the system in simulated tear fluid (STF, pH – 7.4), was influenced significantly by the properties and concentration of sodium alginate, NaCMC. Significant reduction in total bacterial count was observed between control and treatment groups with both the test organisms.

Variation of the oxidation state of verdoheme in the heme oxygenase reaction

Heme oxygenase (HO) converts hemin to biliverdin, CO, and iron applying molecular oxygen and electrons. During successive HO reactions, two intermediates, α-hydroxyhemin and verdoheme, have been generated. Here, oxidation state of the verdoheme-HO complexes is controversial. To clarify this, the heme conversion by soybean and rat HO isoform-1 (GmHO-1 and rHO-1, respectively) was compared both under physiological conditions, with oxygen and NADPH coupled with ferredoxin reductase/ferredoxin for GmHO-1 or with cytochrome P450 reductase for rHO-1, and under a non-physiological condition with hydrogen peroxide. EPR measurements on the hemin-GmHO-1 reaction with oxygen detected a low-spin ferric intermediate, which was undetectable in the rHO-1 reaction, suggesting the verdoheme in the six-coordinate ferric state in GmHO-1. Optical absorption measurements on this reaction indicated that the heme degradation was extremely retarded at verdoheme though this reaction was not inhibited under high-CO concentrations, unlike the rHO-1 reaction. On the contrary, the Gm and rHO-1 reactions with hydrogen peroxide both provided ferric low-spin intermediates though their yields were different. The optical absorption spectra suggested that the ferric and ferrous verdoheme coexisted in reaction mixtures and were slowly converted to the ferric biliverdin complex. Consequently, in the physiological oxygen reactions, the verdoheme is found to be stabilized in the ferric state in GmHO-1 probably guided by protein distal residues and in the ferrous state in rHO-1, whereas in the hydrogen peroxide reactions, hydrogen peroxide or hydroxide coordination stabilizes the ferric state of verdoheme in both HOs.

Effects of Sperm Pre-Treatment on Chromosomal Integrity Following Intracytoplasmic Sperm Injection in Mice.

In intracytoplasmic sperm injection (ICSI), spermatozoa are selected by operator using a vague indicator such as progressive motility and morphological normality. Generally, non-capacitated and/or acrosome intact spermatozoon is injected into an oocyte for initiation of fertilizing process. In many species, it is believed that injection of capacitated or acrosome reacted spermatozoa is not essential for initiation of fertilization following ICSI. However, it is unknown whether the non-physiological condition like non-capacitated and acrosome intact sperm injection may induce any harmful effects on ICSI embryos or not. Therefore, we evaluated the zygotic chromosomal integrity after micro-injection of in vitro capacitated or acrosome reacted spermatozoa using a mouse model. The cytogenetic effects of another widely used sperm pre-treatment such as dithiothreitol (DTT), disulfide bond reducing agent, were also examined. For oocytes collection, B6D2F1 female mice (7-12 weeks old) were superovulated by injection of PMSG and hCG 48 h apart. The oocytes were recovered from oviducts between 14 and 16 h after hCG injection followed by denuding of their cumulus cells. Spermatozoa were collected from the cauda epididymis of male B6D2F1 mice (7-12 weeks old). Before ICSI, collected spermatozoa were underwent four treatments: i) 1 mg/ml methyl-β-cyclodextrin (MBCD) for 90 min for capacitation, ii) 0.02% lysolecithin (LL) and iii) 0.02% Triton X-100 (TX) for 1 min for disruption of acrosome, and iv) 5 mM DTT for 10, 30 and 60 min for reduction of disulfide bonds. The treated spermatozoa were injected within 30 min after treatment. After 19 to 21 h of ICSI, the presumptive zygotes were fixed by gradual-fixation/air drying methods on glass slides. The chromosomes on slides were stained with 2% Giemsa in buffered saline (pH 6.8) for 10 min. The fertilization rates of MBCD, LL and TX groups were 97, 94 and 97%, respectively, and 59, 69 and 60% of zygotes for MBCD, LL and TX groups, respectively, had normal chromosome sets. In non-treated control group, 87% of zygotes had normal chromosome sets. Although there was no significant difference among treatment groups, the proportions of zygotes having normal chromosome sets were significantly lower in sperm treatment groups than that of non-treated control (P<0.05). The fertilization rates (92 to 98%) did not vary among different periods of DTT treatments. However, chromosomal integrity decreased in a time depending manner (10 and 30 min vs. 60 min: 63 and 56% vs. 17%; P<0.001). Furthermore, severe chromosomal aberration (10 or more aberrations per one karyoplate) was observed in higher proportion (28%) of zygotes treated with 60 min DTT than those of 10 min (0%) and 30 min (8%) of counterparts (P<0.05). When compared with non-treated control group, DTT treatment for any duration had harmful effects on chromosomal integrity of zygotes (non-treatment vs. 10 and 30 min: 13% vs. 37 and 36%; P<0.05, non-treatment vs. 60 min: 13% vs. 55%; P<0.001). These results clearly show that sperm pre-treatment prior ICSI is a detriment to their genetic safety, even if the duration of treatment is extremely short, 1 min exposure to LL or TX. Moreover, prolonged sperm treatment such as DTT for 60 min increases the genetic risk as indicated by severe aberrant in their chromosomes. In conclusion, chromosomal integrity of spermatozoa treated with any chemicals should be confirmed prior ICSI.

Runx2 Is a Target of Mechanical Unloading to Alter Osteoblastic Activity and Bone Formation in Vivo

Molecular mechanisms underlying unloading-induced reduction of bone formation have not yet been fully understood. In vitro, Runx2 has been suggested to be involved in mechanical signaling in osteoblasts. However, the roles of Runx2 in vivo during the bone response to mechanical stimuli have not yet been known. The purpose of this paper was to examine the roles of Runx2 in unloading-induced bone loss in vivo. Tail suspension was conducted for 2 wk using 9- to 11-wk-old Runx2 heterozygous knockout mice (Runx2(+/-)) and wild-type (Wt) littermates. Bones were subjected to two-dimensional micro-x-ray computed tomography, bone histomorphometry and RT-PCR analyses. Loss of half Runx2 gene dosage-exacerbated unloading-induced bone loss in trabecular and cortical envelopes. Unloading-induced reduction in mineral apposition rate and bone formation rate in cortical bone as well as trabecular bone was exacerbated in Runx2(+/-) mice, compared with Wt mice. Bone resorption parameters were not significantly affected by unloading or Runx2(+/-) genotype. Basal Runx2 and osterix mRNA levels in bone were reduced by 50% in Wt, whereas unloading in Runx2(+/-) mice did not further alter Runx2 and osterix mRNA levels. In contrast, osteocalcin mRNA levels were reduced by unloading, regardless of Runx2 gene dosage. These data demonstrated that full Runx2 gene dosage is required for maintaining normal function of osteoblasts in mechanical unloading or nonphysiological condition. Finally, we propose Runx2 as a critical target gene in unloading to alter osteoblastic activity and bone formation in vivo.

Effects of Restricted Feeding on Daily Fluctuations of Hepatic Functions Including P450 Monooxygenase Activities in Rats

Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly.

Effects of Mild Versus Deep Hypothermia on a Cloned Human Brain Glutamate Transporter (GLT-1) Expressed in Chinese Hamster Ovary Cells

Glutamate transporters, widely distributed in the brain and spinal cord, maintain extracellular glutamate concentrations below neurotoxic levels. In cerebral ischemia/anoxia, the glutamate transporter runs in reverse and releases glutamate into the extracellular space, causing irreversible neuronal damage. Although hypothermia reduces the elevation of extracellular glutamate concentration during cerebral ischemia/anoxia, little is known about the effect of hypothermia on the glutamate transporter. A human glial glutamate transporter (hGLT-1) cDNA was isolated by screening a human cerebral cortical library, and cloned cDNA was stably transfected in Chinese hamster ovary (CHO) cells. Effects of deep hypothermia (22 to 23 degrees C) on uptake and release of L-glutamate via hGLT-1 were investigated by whole-cell patch-clamp. The control study was performed at 34 to 35 degrees C. The hGLT-1 transporter had the capacity to take up extracellular L-glutamate under essentially physiological ionic conditions, whereas this transporter promoted release of L-glutamate under a nonphysiological condition mimicking complete ischemia. Deep hypothermia decreased a) uptake and b) release of L-glutamate via hGLT-1 to a) 4.8+/-4.8% (P < .01, n = 7) and b) 19.0+/-4.5% (P < .01, n = 15) of control values, respectively. The results suggest that deep hypothermia is a potent inhibitor of glutamate uptake by intact glial cells as well as glutamate release from glial cells under certain pathophysiological circumstances. The balance between these antagonistic effects of hypothermia may attenuate the elevation of the extracellular glutamate concentration during ischemia/anoxia.

Development of a microcontroller-based automatic control system for the electrohydraulic total artificial heart.

An automatic physiological control system for the actively filled, alternately pumped ventricles of the volumetrically coupled, electrohydraulic total artificial heart (EHTAH) was developed for long-term use. The automatic control system must ensure that the device: 1) maintains a physiological response of cardiac output, 2) compensates for an nonphysiological condition, and 3) is stable, reliable, and operates at a high power efficiency. The developed automatic control system met these requirements both in vitro, in week-long continuous mock circulation tests, and in vivo, in acute open-chested animals (calves). Satisfactory results were also obtained in a series of chronic animal experiments, including 21 days of continuous operation of the fully automatic control mode, and 138 days of operation in a manual mode, in a 159-day calf implant.

Direct estimation of base-pair exchange kinetics in oligo-DNA by a combination of NOESY and ROESY experiments.

A new method for the determination of the kinetics of exchange of the imino protons of DNA duplex is reported using a combination NOESY and ROESY experiments at short mixing times (< or = 20 ms). These results have been compared with the commonly used longitudinal relaxation approach through the T1 measurement. To calculate kex and pi ex by ROESY-NOESY experiment, the volume of the cross-peaks between imino protons and water in the NOESY and ROESY spectra have been measured separately from the magnetization term. This work shows that the present approach for the measurement of the kinetics of slow exchanging imino protons of DNA duplex is comparable to the saturation recovery experiment in which the exchange rate can be accelerated by the addition of a base catalyst. The present ROESY-NOESY approach has been found to be particularly useful and reasonably accurate for the measurement of exchange kinetics of both the fast- and slow-exchanging imino protons in DNA duplex both under non-physiological and physiological condition where the saturation recovery method can not be used.

Effect of alkaline cations on cyclic 3′ 5′-AMP stimulated lipolysis in rat adipocytes

In Krebs-Ringer phosphate medium, cyclic AMP had little effect on production of free fatty acids by fat cells in vitro, whereas dibutyryl cyclic AMP or epinephrine stimulated the production of free fatty acids. On the other hand, although under non-physiological condition, cyclic AMP was found to stimulate the lipolysis in simple KC1-Tris medium. The stimulation level was similar to that elicited by dibutyryl cyclic AMP. Cyclic AMP also stimulated lipolysis in LiCl-Tris or RbCl-Tris medium, but not in NaCl-Tris medium. In KC1-Tris medium, addition of divalent alkaline cations (Mg 2+, Ca 2+ and Sr 2+, at 40 mM) completely inhibited the stimulation by cyclic AMP, but not those by dibutyryl cyclic AMP and epinephrine. No cyclic AMP-induced lipolysis was observed in homogenized or freeze-thawed cells.

Correlation between thermal stability maps and genetic maps of double-stranded DNAs

The positional correlation of boundaries of cooperatively melting regions with boundaries of protein coding regions and mere open reading frames, and with cleavage sites of restriction enzymes and S1 nuclease are statistically examined for ΦX174, G4, fd, SV40, BKV, and polyoma DNAs. A statistically significant correlation does exist in the case of boundaries of protein coding regions, but none is detected for boundaries of mere open reading frames or cleavage sites of restriction enzymes and S1 nuclease. The significant correlation disappears when the cooperativity of melting of the DNA double strand decreases to a nonphysiological condition. The result presents the first evidence showing that a physical property of DNAs correlates with their biological function.

Cardiac Lymph in Electrical Ventricular Fibrillation: An Experimental Study

The flow velocity of cardiac lymph during electrical ventricular fibrillation under normothermic cardiopulmonary bypass was studied experimentally in dogs. The time needed for the cardiac lymph node to become stained after injection of dye into the apex myocardium of the left ventricle was measured as an indicator in determining flow velocity of cardiac lymph. The flow velocity was markedly decelerated immediately after the commencement of electrical ventricular fibrillation. It was accelerated, however, after 2 hours of continuous electrical ventricular fibrillation. The difference between the two values was significant ( p < 0.01). Absent contractility of the heart influenced the deceleration of flow velocity of cardiac lymph immediately after the commencement of electrical ventricular fibrillation. Acceleration after 2 hours involved stasis of cardiac lymph as a result of absent contractility and increment of lymph production due to the nonphysiological condition of the myocardium.

STUDIES ON THE NATURE AND FORMATION OF THE OTOLITH

Otolith and Maculae were removed in the condition as fresh as possible by means of the particular method from frog, guinea-pig and hen.The nature and formation of otolith were studied and the results were as follows:(1) It was shown by pressure method, X-ray diffraction technique and electron microscope that the otolith was in a gel-like condition in vivo.But when otolithic membrane became to non-physiological condition by the chemical and physical stimuli, the extremely samll crystals got to gather and developed to form the otolith which has been observed as large crystals.(2) Ca45 was used as a tracer to study the formation of otolith.The rise and fall of Ca45 as tracer were investigated.Ca45 moved rapidly to the otolith and mostly disappeared in 72 hours aftet the injection.It seemed that there were calcium metabolism was present in the otolith.(3) Carbonic anhydrase was demonstrated in the otolithic organs of hen and frog, especially in high concentration in the otolithic membrane and so called Smith's extramacular portion.(4) As the results of the administration of various enzyme inhibitors, it was demonstrated that carbonic anhydrase was related to the formation of calcium carbonate in the shell.(5) From these studies, it seems that carbonic anhydrase plays an important part in the formation of otolith.