A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

Cytostatic and cytolytic activities of macrophages regulation by prostaglandins

Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis.     

Quinone induced stimulation of hexose monophosphate shunt activity in the guinea pig lens: role of zeta-crystallin.

The response of the hexose monophosphate shunt (HMS) in organ-cultured guinea pig lens to 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone (juglone) has been investigated. Both these compounds, which are substrates of guinea pig lens zeta-crystallin (NADPH:quinone oxidoreductase), were found to cause increases in the rate of 14CO2 production from 1-14C-labelled glucose. Exposure of lenses to 15 microM 1,2-naphthoquinone or 20 microM juglone yielded 5.9- and 7-fold stimulation of HMS activity, respectively. Unlike hydrogen peroxide-induced stimulation of HMS activity, these effects were not abolished by preincubation with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU). While hydrogen peroxide produced substantial decrements in lens glutathione (GSH) levels, incubation with quinones was not associated with a similar reduction in GSH concentration. Protein-bound NADPH content in quinone-exposed guinea pig lenses was decreased, with a concomitant increase in the amounts of free NADP+. This finding supported the involvement of zeta-crystallin bound NADPH in the in vivo enzymic reduction of quinones. Hydrogen peroxide, on the other hand, caused decreases in the level of free NADPH alone, serving to confirm our earlier inference that quinone stimulated increases in the guinea pig lens HMS could be mediated through zeta-crystallin NADPH:quinone oxidoreductase activity.     

Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.

zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.     

Zeta-crystallin from guinea pig lens is capable of functioning catalytically as an oxidoreductase

zeta-Crystallin, a major taxon-specific protein of the guinea pig lens, has been shown to be distantly related to the alcohol/polyol dehydrogenase family and to specifically bind NADPH. The capacity of zeta-crystallin to function catalytically was investigated in the present study. zeta-Crystallin exhibited an NADPH-dependent oxidoreductase activity with 2,6-dichlorophenolindophenol (DCIP). The NADPH:DCIP oxidoreductase activity of zeta-crystallin exhibits a linear response with increasing protein concentration, and saturation kinetics with NADPH and DCIP. This activity was abolished by heat inactivation and immunoadsorption of the protein. Dicumarol, Cibacron blue, manganese, and sulfhydryl reagents were inhibitory.     

Extremely high levels of NADPH in guinea pig lens: correlation with zeta-crystallin concentration

Zeta-crystallin, a major "taxon-specific" protein of the guinea pig lens, specifically binds NADPH. Analysis of pyridine nucleotide levels in guinea pig lens revealed values for NADPH approximately 50-fold higher than in other lenses. Indeed to our knowledge the values reported are higher than have been observed in any tissue. A clear correlation exists between NADPH and zeta-crystallin contents of the lens both in normal guinea pigs during development and in a line of guinea pigs with a mutation in the gene for zeta-crystallin. Heterozygotes for this mutation had a 50% reduction in NADPH, while homozygotes have only about 6% of the normal level. NADP+ levels were also markedly elevated suggesting that redox cycling of the NADPH is occurring.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.     

250 million years of bindin evolution

Bindin plays a central role in sperm-egg attachment and fusion in sea urchins (echinoids). Previous studies determined the DNA sequence of bindin in two orders of the class Echinoidea, representing 10% of all echinoid species. We report sequences of mature bindin from five additional genera, representing four new orders, including the distantly related sand dollars, heart urchins, and pencil urchins. The six orders in which bindin is now known include 70% of all echinoids, and indicate that bindin was present in the common ancestor of all extant sea urchins more than 250 million years ago. Over this span of evolutionary time there has been (1). remarkable conservation in the core region of bindin, particularly in a stretch of 29 amino acids that has not changed at all; (2). conservation of a motif of basic amino acids at the cleavage site between preprobindin and mature bindin; (3). more than a twofold change in length of mature bindin; and (4). emergence of high variation in the sequences outside the core, including the insertion of glycine-rich repeats in the bindins of some orders, but not others.