Measurement of lens protein aggregation in vivo using dynamic light scattering in a guinea pig/UVA model for nuclear cataract

The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA-irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA-exposed lens nuclei increased five-fold in intensity compared to controls (P < 0.05). The UVA-induced increase in apparent size of lens nuclear small diameter proteins was three-fold (P < 0.01), and the size of large diameter aggregates was more than four-fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA-irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA-irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA-induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA-irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.     

Studies on relationship between Na,K-ATPase activity and sperm capacitation in guinea pig

In order to elucidate the biochemical mechanism of sperm capacitation, the relationship between plasmalemma Na,K-ATPase, capacitation and acrosome reaction was investigated. Plasmalemma of guinea pig spermatozoa was isolated by sucrose gradient centrifugation for the determination of Na,K-ATPase activity. As far as the authors are aware, the Na,K-ATPase activity in plasmalemma of the guinea pig has never been detected. By treating sperm plasmalemma with 0.05% DOC (deoxycholate), enzyme activity could be quantitatively measured. After spermatozoa were incubated in capacitation medium for 8 h, Na,K-ATPase activity was found to be decreased to 35.6% as compared with that before incubation. The spermatozoa incubated for 10.5 h in capacitation medium containing 1 and 5 mumol/L ouabain showed 46.5% and 64.4% acrosome reactions respectively, while the acrosome reaction of the control group was only 27.4%. The above results suggest that the decrease in the Na,K-ATPase activity of guinea pig spermatozoa may be a prerequisite for capacitation. Experimental results demonstrated for the first time that Na,K-ATPase activity exists in the sperm plasmalemma of the guinea pig. It was further found that the decrease of Na,K-ATPase activity of plasmalemma enhances sperm capacitation. It is suggested that sperm capacitation in the guinea pig is possibly induced by the decrease in plasmalemma Na,K-ATPase and, as a consequence, the intracellular Na+ is increased, which would benefit the exchange of Na+out/Ca++in and the onset of acrosome reaction.     

Determination of prostaglandin profiles in lipopolysaccharide‐challenged guinea pig spleen

We previously reported that splenic extract from lipopolysaccharide (LPS)‐challenged guinea pigs inhibits the exaggerated febrile response of splenectomized guinea pigs, suggesting that the spleen generates an inhibitory factor. Earlier results indicate that the factor is a lipid. In an effort to identify this factor, lipid fractions, isolated from splenic extracts of control and LPS‐challenged guinea pigs, were analyzed with emphasis on identifying and quantifying prostanoids, which according to current knowledge are the likely bioactive factors. Prostaglandins have been extensively implicated in central and peripheral thermoregulation, and thus these lipids were targeted for characterization in the spleen. Analysis was done on the splenic extracts using solid‐phase extraction, analytical and preparative thin‐layer chromatography (TLC) and high‐performance liquid chromatography–mass spectrometry (HPLC‐MS/MS). Four prostaglandins (PGs, 6‐keto‐PGF_1α, PGF_2α, PGE₂ and PGD₂) were identified and quantified. Our data shows that these PG levels are doubled in LPS‐treated guinea pig spleen compared with the control group. The methods used in this investigation to characterize PG in the spleen offer significant advantages over immunoassays previously used to identify and quantify PG in the spleen and other biological tissues. These methods will be utilized in further research needed to definitively characterize the role of splenic‐derived PG in modulation of the febrile response induced by LPS. Copyright © 2012 John Wiley & Sons, Ltd.     

Transfer of diclofenac sodium across excised guinea pig skin on high-frequency pulse iontophoresis. I. Equivalent circuit model

High frequency pulse iontophoresis of diclofenac sodium across excised guinea-pig skin was carried out in vitro. An equivalent circuit model was constructed to simulate the time courses of voltage drop across the donor solution and the skin. Parameter values obtained by the least-squares adaptation of the model to observed data were consistent with expectation and validated the proposed model.     

Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes

The electron spin resonance (ESR) spectra of alkyl spin labels were observed in the excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. The spectrum of 5-doxylstearic acid (5-NS) in the stratum corneum and order parameter obtained from the spectrum, indicated that the spin label was present in highly ordered lipid lamella. On the other hand, the spectrum of methyl ester of 5-NS (5-NMS) and its apparent rotational correlation time calculated from the spectrum, showed only a weakly immobilized component in the stratum corneum as well as in the whole excised skin. The ester spin label seemed to be scarcely present in the rigid lipid lamella, but mainly in the relatively fluid environment. On the other hand, cationic alkyl spin labels showed quite different spectra depending on their alkyl chain lengths. Long-chain 4-(N,N-dimethyl-N,-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15) seemed to be present in the protein region of the stratum corneum as we recently reported, whereas hydrophilic quaternary ammonium spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-1) seemed to be present in the bulk water of the skin, even in delipidized skin. These findings indicated that the different interaction and different localization of the alkyl spin labels depended on their electronic charge as well as their alkyl chain lengths.     

Five subtypes of muscarinic receptors are expressed in gastric smooth muscles of guinea pig

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (I(CCh)) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited I(CCh) dose-dependently with IC(50) of 1.1 0.1 nM (n = 6). GTPgS-induced current, however, was not inhibited by 10 nM 4-DAMP. I(CCh) was not recorded in pertussis-toxin (PTX)-pretreated smooth muscle cells of gastric antrum. I(CCh) values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 3 pA (n = 6) in the control and PTX-treated cells, respectively (P 0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that G(o) protein is important for activation of ACh-activated NSC channels, our results suggest that I(CCh) might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.     

Involvement of β adrenergic receptors in spasmolytic effect of caulerpine on guinea pig ileum

Previously, we demonstrated that caulerpine has spasmolytic effect on guinea pig ileum. The aim of this study was to investigate pathways of its spasmolytic action. We test caulerpine against phasic contractions induced by carbachol in the circular layer of guinea pig ileum and this alkaloid did not inhibit these contractions, indicating that caulerpine did not interfering with the mobilisation of Ca²⁺ from intracellular stores. Additionally, the spasmolytic effect of caulerpine did not involve K⁺ channels. Furthermore, we observed that α₂-adrenergic receptors were not involved in the spasmolytic effect of caulerpine, since the relaxation curve induced by caulerpine was not shifted in the presence of yohimbine (α₂-adrenergic antagonist). However, in the presence of propranolol (β-adrenergic antagonist), the relaxation curve induced by caulerpine was right-shifted, resulting in a fivefold increase in EC₅₀. Thus, a possible mechanism for the spasmolytic action of caulerpine is the activation of β-adrenergic receptors.     

Identification of a botulinum neurotoxin A protease inhibitor displaying efficacy in a cellular model

Herein we describe a small-molecule, non-peptidic, inhibitor for botulinum neurotoxin A protease, that displays for the first time efficacy in a cell-based assay.