α‐Crystallins are members of the family of small heat‐shock proteins. The conformation and mode of action of these ‘junior chaperones’ are unknown. To investigate the structure and chaperone‐like ...activity, four mutants of bovine αA‐crystallin were generated by site‐directed mutagenesis. In comparison with wild‐type αA‐crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat‐protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat‐protective capacity were observed. Although it is assumed that small heat‐shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in α‐crystallin can also influence the efficiency of substrate binding.
Determining the pharmacokinetics and safety of solifenacin succinate, a once-daily, oral antimuscarinic agent indicated for treatment of overactive bladder, in subjects with hepatic impairment. In ...this open-label study, 16 subjects (eight with moderate hepatic impairment defined as a Child-Pugh score of 7-9, eight healthy) received a single oral 10 mg solifenacin dose. Blood and urine were collected for pharmacokinetic assessments. Pharmacokinetic parameters (primary：area under the plasma concentration-time curve from time 0 to infinity AUC0-∞ and maximum plasma concentration Cmax) and safety were evaluated for solifenacin and its metabolites. There were no clinically relevant differences in safety. Moderate hepatic impairment increased AUC0-∞, by 60%, and the mean elimination half-life of solifenacin and several of its metabolites was longer versus healthy subjects. Mean Cmax values were comparable between the groups. A single oral dose of solifenacin was well tolerated in hepatically impaired and healthy subjects；however, moderate hepatic impairment influenced solifenacin pharmacokinetics. In patients with mild hepatic impairment, solifenacin may be used without special caution；however, in patients with moderate hepatic impairment, doses greater than 5 mg are not recommended and the 5 mg dose should be used with caution.
The amine-donor substrate specificity of tissue-type transglutaminase has been studied in a series of recombinant alpha A-crystallin mutants. These mutant proteins have been provided with a potential ...substrate lysine residue, flanked by different amino acid residues, in the C-terminal extended arm of alpha A-crystallin. A biotinylated amine-acceptor hexapeptide was used as a probe for labelling the amine-donor sites. Wild-type bovine alpha A-crystallin does not function as an amine-donor substrate for tissue-type transglutaminase. Yet, upon introduction of a lysine residue at the C-terminal or penultimate position, all mutant alpha A-crystallins act as amine-donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine-donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.
1. Uncomplicated insulin-dependent diabetes mellitus is associated with generalized vasodilatation. This vasodilatation is believed to contribute to the development of microvascular complications. ...The endothelium plays an important role in the regulation of vascular tone. 2. To investigate the role of endothelial mediators, we measured plasma endothelin levels and studied the vascular effects of intravenous L-arginine (the precursor of NO) in 10 male type 1 diabetic patients and 10 non-diabetic subjects. 3. The baseline plasma endothelin level was significantly lower in the diabetic patients mean 1.7 (SD 0.5) versus 2.1 (0.4) pmol/l; P < 0.05 than in the control subjects. 4. During L-arginine infusion, plasma cyclic GMP (the second messenger for NO) increased in the control subjects from 5.1 (2.9) to 6.9 (2.9) nmol/l; P < 0.05 versus saline and in the diabetic patients from 4.6 (1.8) to 5.7 (2.2) nmol/l; P = 0.09. L-Citrulline (a by-product of NO synthesis from L-arginine) increased in both groups. The responses to L-arginine were not significantly different between the control subjects and the diabetic patients. The plasma atrial natriuretic peptide level did not change in either group during infusion of L-arginine or of an equal volume of isotonic saline. 5. Blood pressure decreased slightly during L-arginine administration in both groups. In control subjects, the extracellular fluid volume in the lower leg increased during L-arginine infusion as compared with saline; in the diabetic patients both L-arginine and saline increased the extracellular fluid volume.
alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several ...non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.
To evaluate the pharmacokinetics, safety, and tolerability of solifenacin in patients with mild, moderate, or severe renal disease, eighteen patients with renal disease and six healthy volunteers ...received a single oral dose of solifenacin (10 mg). Pharmacokinetic parameters were assessed from blood samples drawn over a 360-h period. Safety and tolerability were also evaluated. Total mean ± S. D. exposure (ng , h/mL) to solifenacin in healthy individuals (1190 ± 403) was increased in patients with renal disease (mild: 1784 ± 792, moderate: 1559 ± 555, severe: 2530 ± 700), and elimination half-life (mean ± S. D. h) was prolonged (healthy: 68.2 ± 27.2, mild: 89.1 ± 34.5, moderate: 90.6 ± 27.3, severe: 111 ± 38.3). A significant correlation was found between creatinine clearance and pharmacokinetic parameters for exposure and apparent oral clearance. No deaths or serious adverse events occurred during the study. Solifenacin 10 mg was well tolerated in patients with renal disease. Solifenacin displays a higher exposure and a prolonged half-life in patients with renal impairment, especially severe. Therefore, while no special cautions are necessary for patients with mild/moderate renal impairment, patients with severe renal impairment should receive no more than 5 mg solifenacin once daily.
The rudimentary eyes of the mole rat Spalax ehrenbergi have lost their visual function, but are still required for the control of circadian rhythms. It has previously been found that ...alphaA-crystallin, a major eye lens protein in other mammals, evolved much faster in the mole rat than in rodents with normal vision. Yet, although mole rat alphaA-crystallin seems superfluous as a lens protein, its rate of change is still much slower than that of pseudogenes, suggesting some remaining function. The authors therefore studied the structure and function of recombinant mole rat alphaA-crystallin. Circular dichroism (CD), tryptophan fluorescence and gel permeation analyses indicated that the overall structure and stability of mole rat alphaA-crystallin are comparable to that of rat alphaA-crystallin. However, the chaperone-like activity of mole rat alphaA-crystallin is considerably lower than that of its rat orthologue. Two-dimensional NMR spectroscopy of mole rat alphaA-crystallin suggests that this may be in part due to a diminished flexibility of the C-terminal extension, which is thought to be important for the chaperoning capacity. Overall, mole rat alphaA-crystallin appears to still be a viable protein, confirming that it has some as yet elusive role, despite the loss of its primary lens function.
αA-Crystallin is a member of the small heat shock protein family that is abundantly expressed as a structural component in the vertebrate eye lens. In lenses of rodents and some other mammals, there ...occurs a minor variant of αA-crystallin, which has an insertion of 23 amino acid residues. This variant, αAins-crystallin, results from differential integration of an optional exon into a small fraction of the mRNA. We have studied whether this alternative splicing is caused by a non-consensus cytosine in the 5' splice site adjacent to the optional exon. After replacement of the aberrant cytosine in the hamster αA-crystallin gene by a consensus thymine, and transient transfection of this gene in Chinese Hamster Ovary cells, the optional exon is indeed almost completely spliced into the mature mRNA. In contrast, replacement of the cytosine by adenine or guanine completely abolishes the splicing of the optional exon. Our results confirm that alternative splicing of the αA-crystallin primary transcript is mainly due to a non-consensus 5' splice site nucleotide. However, we conclude that the small size of the optional exon is probably an additional contributing factor and therefore it seems that the splicing mechanism is based on recognition of exons rather than introns.PUBLICATION ABSTRACT
aA-Crystallin is a member of the small heat shock protein family that is abundantly expressed as a structural component in the vertebrate eye lens. In lenses of rodents and some other mammals, there ...occurs a minor variant of aA-crystallin, which has an insertion of 23 amino acid residues. This variant, aAins-crystallin, results from differential integration of an optional exon into a small fraction of the mRNA. We have studied whether this alternative splicing is caused by a non-consensus cytosine in the 5' splice site adjacent to the optional exon. After replacement of the aberrant cytosine in the hamster aA-crystallin gene by a consensus thymine, and transient transfection of this gene in Chinese Hamster Ovary cells, the optional exon is indeed almost completely spliced into the mature mRNA. In contrast, replacement of the cytosine by adenine or guanine completely abolishes the splicing of the optional exon. Our results confirm that alternative splicing of the aA-crystallin primary transcript is mainly due to a non-consensus 5' splice site nucleotide. However, we conclude that the small size of the optional exon is probably an additional contributing factor and therefore it seems that the splicing mechanism is based on recognition of exons rather than introns.
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![The rodent [alpha]A-crystal...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0301-4851/small "The rodent [alpha]A-crystallin gene: mutagenesis of a non-consensus 5'-splice site to study alternative splicing in vivo")