Thioredoxin reductase-1 knock down does not result in thioredoxin-1 oxidation

The active site of thioredoxin-1 (Trx1) is oxidized in cells with increased reactive oxygen species (ROS) and is reduced by thioredoxin reductase-1 (TrxR1). The purpose of the present study was to determine the extent to which the redox state of Trx1 is sensitive to changes in these opposing reactions. Trx1 redox state and ROS generation were measured in cells exposed to the TrxR1 inhibitors aurothioglucose (ATG) and monomethylarsonous acid (MMA(III)) and in cells depleted of TrxR1 activity by siRNA knock down. The results showed that all three treatments inhibited TrxR1 activity to similar extents (90% inhibition), but that only MMA(III) exposure resulted in oxidation of Trx1. Similarly, ROS levels were elevated in response to MMA(III), but not in response to ATG or TrxR1 siRNA. Therefore, TrxR1 inhibition alone was not sufficient to oxidize Trx1, suggesting that Trx1-independent pathways should be considered when evaluating pharmacological and toxicological mechanisms involving TrxR1 inhibition.     

Smcy transgene does not rescue spermatogenesis in sex-reversed mice

In mouse, the Sxr^b deletion interval (delta Sxr^b) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia. This deletion interval is approximately 1–2 Mb and contains eight known genes. In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxr^b interval including Zfy1, Ube1y, Smcy, and Eif2s3. Two male and one female founder mice, transgenic for all four genes, were sterile. However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified. Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y. The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers. Introduction of the transgene into an X Sxr^b/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males. These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy. Received: 16 June 2000 / Accepted: 25 September 2000     

Tyrosine phosphorylation of phospholipase D1 by v-Src does not per se result in activation

The relationship between tyrosine phosphorylation and activation of phospholipase D1 (PLD1) by v-Src was examined. Co-expression of v-Src and PLD1 in COS-7 cells resulted in increased activity and marked tyrosine phosphorylation of PLD1. PLD activity was increased in membranes or immunoprecipitates prepared from these cells. Dephosphorylation of the immunoprecipitated enzyme by tyrosine phosphatase or phosphorylation by c-Src produced no changes in its activity. Tyrosine phosphorylation induced by v-Src caused a shift of the enzyme from the Triton-soluble to the Triton-insoluble fraction. v-Src and PLD1 could be co-immunoprecipitated from cells co-expressing these and were co-localized in the perinuclear region as assessed by immunofluorescence. Mutation of the palmitoylation sites of PLD1 significantly reduced tyrosine phosphorylation by v-Src. It is concluded that tyrosine phosphorylation of PLD1 by v-Src does not per se alter its activity. It is proposed that activation of PLD1 by v-Src in vivo may involve association/colocalization of the two proteins.     

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B₁₂ metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.     

High-fat feeding does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle

Apoptosis and autophagy are critical in normal skeletal muscle homeostasis; however, dysregulation can lead to muscle atrophy and dysfunction. Lipotoxicity and/or lipid accumulation may promote apoptosis, as well as directly or indirectly influence autophagic signaling. Therefore, the purpose of this study was to examine the effect of a 16-week high-fat diet on morphological, apoptotic, and autophagic indices in oxidative and glycolytic skeletal muscle of female rats. High-fat feeding resulted in increased fat pad mass, altered glucose tolerance, and lower muscle pAKT levels, as well as lipid accumulation and reactive oxygen species generation in soleus muscle; however, muscle weights, fiber type-specific cross-sectional area, and fiber type distribution were not affected. Moreover, DNA fragmentation and LC3 lipidation as well as several apoptotic (ARC, Bax, Bid, tBid, Hsp70, pBcl-2) and autophagic (ATG7, ATG4B, Beclin 1, BNIP3, p70 s6k, cathepsin activity) indices were not altered in soleus or plantaris following high-fat diet. Interestingly, soleus muscle displayed small increases in caspase-3, caspase-8, and caspase-9 activity, as well as higher ATG12-5 and p62 protein, while both soleus and plantaris muscle showed dramatically reduced Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) levels. In conclusion, this work demonstrates that 16 weeks of high-fat feeding does not affect tissue morphology or induce a global autophagic or apoptotic phenotype in skeletal muscle of female rats. However, high-fat feeding selectively influenced a number of apoptotic and autophagic indices which could have implications during periods of enhanced muscle stress.     

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.     

Promiscuous thymic expression of an autoantigen gene does not result in negative selection of pathogenic T cells

"Promiscuous" thymic expression of peripheral autoantigens can contribute to immunological tolerance in some cases. However, in this study we show that thymic mRNA expression alone cannot predict a contribution to thymic tolerance. Autoimmune gastritis is caused by CD4+ T cells directed to the alpha (H/Kalpha) and beta (H/Kbeta) subunits of the gastric membrane protein the H+/K+ ATPase. H/Kalpha mRNA is expressed in the thymus, but H/Kbeta expression is barely detectable. In this study, we demonstrate that thymic H/Kalpha in wild-type mice or mice that overexpressed H/Kalpha did not result in negative selection of pathogenic anti-H/Kalpha T cells. However, negative selection of anti-H/Kalpha T cells did occur if H/Kbeta was artificially overexpressed in the thymus. Given that H/Kalpha cannot be exported from the endoplasmic reticulum and is rapidly degraded in the absence of H/Kbeta, we conclude that H/Kalpha epitopes are unable to access MHC class II loading compartments in cells of the normal thymus. This work, taken together with our previous studies, highlights that thymic autoantigen expression does not necessarily result in the induction of tolerance.     

Inactivation of monoamine oxidase by allylamine does not result in flavin attachment

[1-3H]Allylamine was synthesized by sodium boro[3H]hydride reduction of acrolein followed by direct conversion of the [1-3H]allyl alcohol to N-allylphthalimide with triphenylphosphine, diethylazodicarboxylate, and phthalimide. The protecting group was removed with hydrazine. Inactivation of beef liver mitochondrial monoamine oxidase with [1-3H]allylamine led to incorporation of 1-6 eq of inactivator/active site depending upon the length of incubation time. Inactivation and radioactivity incorporation coincided; however, after 1 eq of tritium was incorporated and 5% enzyme activity remained, additional radioactivity continued to become incorporated into the enzyme. The optical spectrum of the FAD coenzyme changed during inactivation from that of oxidized to reduced flavin. Following dialysis of the inactivated enzyme, the spectrum remained reduced, but denaturation in urea rapidly resulted in reoxidation of the flavin. Under these same denaturing conditions, 96% of the radioactivity associated with the enzyme remained bound, therefore indicating that allylamine attachment is not to the flavin coenzyme but rather to an active site amino acid residue. The adduct also was stable to base and, to a lesser degree, acid treatment. Although allylamine and N-cyclopropylbenzylamine appear to be oxidized by monoamine oxidase to give 3-(amino acid residue) propanal adducts, two different amino acids seem to be involved because of a difference in stability of the adducts. The mechanisms for inactivation of monoamine oxidase by allylamine and reactivation by benzylamine are discussed in relation to previously reported results.     

Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration

Prion protein (PrP) plays a crucial role in prion disease, but its physiological function remains unclear. Mice with gene deletions restricted to the coding region of PrP have only minor phenotypic deficits, but are resistant to prion disease. We generated double transgenic mice using the Cre-loxP system to examine the effects of PrP depletion on neuronal survival and function in adult brain. Cre-mediated ablation of PrP in neurons occurred after 9 weeks. We found that the mice remained healthy without evidence of neurodegeneration or other histopathological changes for up to 15 months post-knockout. However, on neurophysiological evaluation, they showed significant reduction of afterhyperpolarization potentials (AHPs) in hippocampal CA1 cells, suggesting a direct role for PrP in the modulation of neuronal excitability. These data provide new insights into PrP function. Furthermore, they show that acute depletion of PrP does not affect neuronal survival in this model, ruling out loss of PrP function as a pathogenic mechanism in prion disease and validating therapeutic approaches targeting PrP.     

Airway closure in humans does not result in overestimation of plethysmographic lung volume

Exercise Physiol. 52: 638-641, 1982) have shown in dogs that airway closure may induce rib cage deformation and nonhomogeneous alveolar pressure swings, and they have suggested that this could lead to thoracic gas volume (TGV) overestimation by body plethysmography. However, in humans the rib cage is less easy to distort than in dogs. In four healthy volunteers we measured TGV by plethysmography before (B) and during (D) the occlusion of the middle and lower right lobes by a balloon (attached to a double-lumen catheter) positioned in the intermediate right bronchus. Subjects were trained to perform panting maneuvers preferentially with intercostals and accessory muscles or the diaphragm. Five to eleven TGV measurements were made in each subject with each panting pattern B and D occlusion. Balloon inflation resulted in no change in TGV whether low [13.3 +/- 3.4 (SD) cmH2O] or high (46.8 +/- 8.4 cmH2O) transdiaphragmatic pressures (Pdi) were used: TGV 4.0 +/- 0.4 (B) vs. 4.0 +/- 0.4 liters (D) and 4.3 +/- 0.4 (B) vs. 4.3 +/- 0.4 liters (D) for low and high Pdi, respectively. Thus, in trained subjects performing maneuvers aimed to distort the rib cage, no pressure difference was observed between the occluded and the nonoccluded lung during panting against the closed shutter. We conclude that it is unlikely that the mechanism proposed by Brown et al. might explain errors in lung volume measurements by body plethysmography in humans.     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.     

Overexpression of cyclin E does not influence homologous recombination in Chinese hamster cells

Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination.     

Evaluation of DNA damage in different stages of mouse spermatogenesis after testicular X irradiation

To evaluate whether DNA alterations in mature spermatozoa could stem from DNA damage induced in immature germ cells, testis cells and spermatozoa were analyzed by the comet assay and by the sperm chromatin structure assay 14, 45 and 100 days after in vivo X irradiation of the testes. These times were selected, according to the mouse seminiferous epithelium cycle, to follow the DNA damage induced in different germ cell compartments. The cytotoxic action was assessed by DNA flow cytometric analysis of testicular cells. A dose-dependent increase of DNA damage in testis cells was observed 14 days after irradiation, whereas mature sperm cells were not affected. On the other hand, an increase in DNA strand breaks was seen in spermatozoa 45 days after treatment. DNA damage returned to the control levels 100 days after irradiation. The methods used to evaluate DNA damage gave comparable results, emphasizing the correlation between DNA fragmentation and susceptibility of sperm chromatin to denaturation. Both techniques showed the high radiosensitivity of differentiating spermatogonia. The overall results showed that DNA damage induced in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa.