The association of HLA with young-onset keratoconus in Japan

PURPOSE: To report the association of HLA antigens with keratoconus in Japanese patients. DESIGN: Observational consecutive case series. METHODS: In 90 consecutive Japanese keratoconus patients, HLA class I (HLA-A, -B, -C) and class II (HLA-DR, -DQ) were analyzed. RESULTS: Compared with control frequencies, based on mean gene frequencies for the Japanese population, higher frequencies of HLA-A26, B40, and DR9 antigens were found in patients whose conditions were diagnosed before 20 years of age (chi(2) = 6.45, P =.01; chi(2) = 6.78, P =.01; chi(2) =3.99, P =.05, respectively), but were not found in patients whose conditions were diagnosed later in life. Men were significantly younger at diagnosis than were women. No obvious relation was found between HLA antigens and other clinical data. CONCLUSION: HLA-A26, B40, and DR9, which were found relatively frequently in the ancient Japanese population, seem to be associated with keratoconus in younger individuals.     

Validation of scanning laser Doppler flowmetry for retinal blood flow measurements in animal models

PURPOSE: To evaluate the validity of scanning laser Doppler flowmetry in retinal blood flow measurement in vivo using rabbit and monkey eyes and the microsphere technique. METHODS: A commercially available scanning laser Doppler flowmeter (SLDF), pigmented rabbits and cynomolgus monkeys were used. In rabbits, SLDF measurements at a retinal field approximately one papillary diameter away from the optic nerve head where discrete retinal vessels were not visible (vascularized medullary field) were compared with those at a field approximately one papillary diameter below the ONH (nonvascularized extramedullary field). The effect of intraocular pressure (IOP) on SLDF measurements was studied by elevating the IOP manometrically in both rabbit and monkey eyes. Retinal and choroidal blood flow measurements using the microsphere technique and an SLDF measurements were performed simultaneously in the same rabbit eye before and 30 min after 0.3 mg/kg intravenous injection of lomerizine, a calcium antagonist, or intravitreal injection of 20 microl of 10(-6) M endothelin-1. RESULTS: The blood flow measurement with an SLDF (SLDF flow) obtained from the medullary field in rabbits were 304 +/- 63 in an arbitrary unit (n = 24), while 392 +/- 39 in the extramedullary field. SLDF flow did not significantly changed by changes in IOP when it was below 50 mmHg, significantly decreasing with IOP elevation above that level in rabbits and a similar tendency was also seen in monkeys. It showed a significant correlation with retinal blood flow measured by the microsphere technique (r = 0.596, P < 0.0001) in rabbits; no correlation was found with the choroidal blood flow rate (r = 0.021, P > 0.1). CONCLUSIONS: SLDF measurements is thought to mainly reflect retinal circulation in rabbits and monkeys.     

Subconjunctival administration of bucillamine suppresses choroidal neovascularization in rat

PURPOSE: Bucillamine is an antirheumatic drug with antiangiogenic properties that is currently used in clinical practice. Because bucillamine inhibits the production of VEGF, it is possible that this drug may inhibit choroidal neovascularization (CNV). Thus, the effect of bucillamine on the eyes of rats with experimental CNV was investigated in vivo by subconjunctival injection or oral intake. METHODS: CNV was induced in rat eyes by diode laser photocoagulation. The intensity of fluorescein leakage from the photocoagulated lesions was studied 7 and 14 days after photocoagulation. The areas of CNV lesions were measured histologically and studied immunohistochemically at days 4, 7, and 14. In addition, the concentration of the drug in ocular tissue and blood was measured by high-performance liquid chromatography-tandem mass spectrometry after the drug was delivered orally or subconjunctivally. RESULTS: After subconjunctival injection, fluorescein leakage from the CNV lesions decreased significantly compared with the control eyes throughout the study period. Histologic and immunohistochemical analyses 4, 7, and 14 days after photocoagulation demonstrated that the average size of the CNV lesions was reduced in the bucillamine-treated eyes compared with the control eyes. Subconjunctival injection maximized the ocular drug concentration while minimizing the blood concentration of the drug compared with oral intake. CONCLUSIONS: Subconjunctival injection of bucillamine significantly reduced the leakage and size of experimental CNV. These results suggest that bucillamine may be beneficial in treating CNV and that further studies can be considered to evaluate this possibility.     

Expression of protein gene product 9.5 in the anterior lens epithelial cells of atopic cataracts

PURPOSE: To investigate the mechanisms of atopic cataract by immunohistochemically observing protein gene product 9.5 in lens epithelial cells (LECs) of atopic cataracts. SETTING: Departments of Ophthalmology and Anatomy, Shiga University of Medical Science, Shiga, Japan. METHODS: Specimens of anterior capsule obtained from 6 patients with atopic cataract and 8 patients with senile cataract were stained immunohistochemically and examined by light, electron, and confocal microscopy. RESULTS: In the LECs of the atopic cataracts, the arrangement and cell shape were varied. Immunohistochemistry for protein gene product 9.5 demonstrated selective localization, unlike with senile cataracts. This was especially prevalent in the spindle-shaped cells around the central opaque lesion. Protein gene product 9.5 positive cells overlapped with major basic protein positive cells in the atopic cataracts. CONCLUSIONS: The results indicate that the properties of LECs of atopic cataracts are different from those of senile cataracts. Protein gene product 9.5 and major basic protein seem to be concentrated in atopic cataracts.     

In vivo nitric oxide transfer of a physiological NO carrier, dinitrosyl dithiolato iron complex, to target complex

Dinitrosyl dithiolato iron complex (DNIC) has been identified as an endogenous NO carrier, yet in vivo mechanisms of NO donation remain undefined. Transnitrosylation, in which a coordinated NO group is transferred to another metal complex, has been observed in transition-metal-nitrosyl chemistry. In this study, we used three kinds of iron dithiocarbamate complexes (Fe-DTCs) as NO acceptors to elucidate in vivo transnitrosylation of diglutathionyl dinitrosyl iron complex [DNIC-(GS)(2)]. Fe-DTCs were administered to mice after the injection of DNIC-(GS)(2) and electron paramagnetic resonance (EPR) spectra were measured both in the resected organs and in the upper abdomen of living mice. The spectral feature gradually changed from an initial DNIC-(GS)(2) signal to mononitrosyl iron dithiocarbamate one, suggesting that NO-Fe-DTC was formed through in vivo reaction of DNIC-(GS)(2) with Fe-DTC. The spectral results in in vitro and in vivo systems indicate that NO-Fe-DTCs can be formed not only by the transfer of coordinated NO-group(s) in DNIC-(GS)(2) but also by the abstraction of Fe-NO group in DNIC-(GS)(2) by free DTC ligands. Transnitrosylation proceeded more rapidly in blood than in liver and kidney; and more efficiently in kidney than in liver. Further, the ability to accept NO from DNIC was dependent on water-solubility of Fe-DTCs. Thus, in vivo transnitrosylation from DNIC to exogenous iron complex could be observed and this reaction was influenced by biological constituents and properties of iron complex. These results demonstrate that the transnitrosylation from DNIC to intrinsic NO acceptors like metalloproteins has a probable significance in in vivo NO transfer process.     

Four highly oxygenated isopimarane-type diterpenes of Orthosiphon stamineus

Four highly oxygenated isopimarane-type diterpenes, named orthosiphols O, P and Q and nororthosiphonolide A, have been isolated from the aerial parts of Orthosiphon stamineus from Myanmar, together with three known diterpenes, orthosiphols D and E and orthosiphonone A. Their structures were determined on the basis of extensive spectral analysis. All the isolated compounds displayed mild antiproliferative activities against highly liver metastatic colon 26-L5 carcinoma and human HT-1080 fibrosarcoma cell lines.     

Quinapril treatment restores the vasodilator action of insulin in fructose-hypertensive rats

1. Angiotensin-converting enzyme (ACE) inhibitors have been shown to improve insulin-resistance both experimentally and clinically. We therefore investigated the effects of quinapril, which has high tissue specificity for ACE, regarding the contribution of insulin to vascular contractions, as well as insulin sensitivity in a dietary rat model of insulin resistance. 2. Male Sprague-Dawley rats were divided into three groups: (i) rats fed normal chow (normal diet group); (ii) rats fed fructose-rich chow containing 40% fructose and 7% lard (fructose diet group); and (iii) rats fed fructose-rich chow plus quinapril (10 mg/kg per day; quinapril-treated group). 3. After 2 weeks, we evaluated systolic blood pressure, insulin sensitivity as assessed by steady state plasma glucose (SSPG) levels, response of aortic rings to phenylephrine (10-9 to 10-6 mol/L) in the presence or absence of insulin and the response of aortic rings to acetylcholine. 4. Feeding rats fructose-rich chow resulted in an elevation of blood pressure (P < 0.01) and SSPG levels (P < 0.01). Quinapril treatment significantly prevented increases in both blood pressure and SSPG, with a return to the levels seen in the normal diet group. 5. In the absence of insulin, the maximal contractile response to phenylephrine did not differ between the three groups. However, in the presence of insulin (100 mU/mL), the contractile response to phenylephrine (10-6 mol/L) was reduced by 22.8 +/- 1.2% in the normal diet group, although no insulin effects were observed in the fructose diet group (P < 0.01). Quinapril restored the inhibitory effect of insulin on phenylephrine-induced contractions. 6. In addition, the reduction in relaxation induced by acetylcholine in the fructose diet group was significantly reversed by quinapril treatment. 7. It is concluded that the fructose diet impairs the vasodilator effects of insulin as well as acetylcholine-induced relaxation in rat thoracic aortas. Quinapril prevented deterioration in the responses of the aortic rings, suggesting that ACE inhibitors may be useful for treating vascular insulin resistance.     

Estimation of intradermal disposition kinetics of drugs: II. Factors determining penetration of drugs from viable skin to muscular layer

To develop a more efficient transdermal delivery system, it is very important to regulate the intradermal disposition of drugs after topical application. We tried to elucidate the factors determining the intradermal disposition kinetics, especially drug penetration from the viable skin to the muscular layer, mainly based on the six-compartment model, including the contralateral skin and muscle for ten model drugs with different physicochemical characteristics. In vivo transdermal absorption study was performed for six model drugs using the stripped-skin rats. The fitting analyses by the six-compartment model gave the theoretical curves describing the observed data very well and the reasonable pharmacokinetic parameters, showing the pharmacokinetic model should be useful for the estimation of the intradermal disposition kinetics of drugs applied topically again. The simulation study using the pharmacokinetic parameters obtained above could show the relative contribution of the direct penetration and the distribution from the systemic circulation to the muscular distribution of drugs. The largest contribution of direct penetration was observed for antipyrine (90.8%) and the smallest was for felbinac (43.3%). Among the pharmacokinetic parameters obtained above, the clearance from the viable skin to the muscle (CL(vs-m)) was found to be significantly correlated with the unbound fraction of drugs in the viable skin (fu(vs)). Although the clearance from the viable skin to the plasma (CL(vs-p)) also tended to increase as fu(vs) increased, the ratio of CL(vs-m) to CL(vs-p) was significantly correlated with fu(vs), meaning that the larger amount of unbound drug in the viable skin significantly contributes to the direct penetration into the muscle more than to the systemic absorption. On the other hand, k(direct) values obtained in in vitro penetration study-the penetration rate constant of drugs from the viable skin to the muscular layer-were found to be correlated with CL(vs-m) values for seven model drugs. Therefore, adding the in vitro experiments for the other three model drugs, the multiple linear regression analysis of k(direct) was performed for ten model drugs in terms of fu(vs), logarithm of the partition coefficient (Log P) and molecular weight. The results clearly showed the largest and significant contribution of fu(vs) to the direct penetration of drugs from the viable skin to the muscular layer, indicating that a drug with the higher value of fu(vs) in the viable skin can penetrate more into the muscular layer.     

Computational consideration of cisplatin hydrolysis and acid dissociation in aqueous media: effect of total drug concentrations

Cisplatin (cis-DDP) is subject to nucleophilic displacement of chloride in water, forming aquated species, subsequently liberating hydrogen ion(s) with increasing pH. This study intends to theoretically analyze the hydrolysis and polyprotic dissociation behavior of cis-DDP in various aqueous media. A mathematical model was expressed by nonlinear simultaneous equations in terms of the total drug concentration, pH and pCl based on the hydrolysis and acid dissociation constants already published. Some of the interesting simulation results include that (1) in water, cis-DDP behaves in a very complicated manner, highly depending on the total drug concentration, pH and pCl, (2) in normal saline, about 3% of the total concentration is a positively charged chloro-aqua that may be very reactive, (3) in assumed blood (pH 7.4, [Cl(-)]=0.11 mol/l, mu=0.15), the drug is stabilized at the level of 85% and the remnants are the chloro-hydroxo (11%) and the chloro-aqua (4%), (4) in assumed intracellular conditions (pH 7.1, [Cl(-)]=0.01 mol/l, mu=0.15), the drug is converted to a large extent to various species including the parent species (44%), the chloro-hydroxo (30%), hydroxo-aqua (2%), chloro-aqua (24%) diaqua (less than 1%) and dihydroxo (null). The results of this analysis may provide a useful preliminary knowledge of existing species in a system concerned and a rationale for re-evaluating the reactions between cis-DDP and various nucleophilic substances already reported while there are somewhat conflicting interpretations of some cis-DDP reactions.     

Stimulation of beta(3)-adrenoceptors causes phosphorylation of p38 mitogen-activated protein kinase via a stimulatory G protein-dependent pathway in 3T3-L1 adipocytes

1. This study deals with phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via beta(3)-adrenoceptor (AR) and the signal transduction pathway in 3T3-L1 adipocytes. 2. beta(3)-AR agonist BRL37344A (10 nM) caused phosphorylation and activation of p38 MAPK in 3T3-L1 adipocytes but not in fibroblasts. BRL37344A and also the other beta(3)-AR agonists, CGP12177A and SR58611A, caused p38 MAPK phosphorylation in dose-dependent manners. 3. The p38 MAPK phosphorylations by BRL37344A (10 nM), CGP12177A (100 nM), and SR58611A (10 nM) were not antagonized by beta(1)- and beta(2)-ARs antagonist 1-propranolol (100 nM) but blocked by beta(3)-AR antagonist SR59230A (10 microM), suggesting the phosphorylation was caused via beta(3)-AR. 4. The phosphorylations of p38 MAPK were completely abolished by treatment with cholera toxin (CTX) but not pertussis toxin (100 ng ml(-1), 24 h). Activation of Gs by CTX (100 ng ml(-1)) and adenylyl cyclase by forskolin mimicked p38 MAPK phosphorylation. 5. p38 MAPK phosphorylation by BRL37344A was reduced to almost 50% by cyclic AMP-dependent protein kinase (PKA) inhibitors such as H89 (10 microM) and PKI (10 microM). A src-family tyrosine kinases inhibitor PP2 (1 microM) also halved the p38 MAPK phosphorylation. Combined use of H89 (10 microM) and PP2 (10 microM) did not bring about further inhibition. 6. These results suggest that beta(3)-AR caused phosphorylation of p38 MAPK via Gs protein and partly through a pathway involving PKA and src-family kinase(s), although the contribution of the unidentified pathway remains to be clarified.