Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70(S6K)

Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.     

Amlodipine decreases fibrosis and cardiac hypertrophy in spontaneously hypertensive rats: persistent effects after withdrawal

Our objective was to examine the effect of chronic treatment with amlodipine on blood pressure, left ventricular hypertrophy, and fibrosis in spontaneously hypertensive rats and the persistence of such an effect after drug withdrawal. We investigated the effects of treatment with 2, 8 and 20 mg/kg/day of amlodipine given orally for six months and at three months after drug withdrawal. Systolic blood pressure was measured using the tail-cuff method. At the end of the study period, the heart was excised, the left ventricle was isolated, and the left ventricle weight/body weight ratio was calculated as a left ventricular hypertrophy index. Fibrosis, expressed as collagen volume fraction, was evaluated using an automated image-analysis system on sections stained with Sirius red. Age-matched untreated Wistar-Kyoto and SHR were used as normotensive and hypertensive controls, respectively. Systolic blood pressure was reduced in the treated SHR in a dose-dependent way and after amlodipine withdrawal it increased progressively, without reaching the values of the hypertensive controls. Cardiac hypertrophy was reduced by 8 and 20 mg/kg/day amlodipine, but when treatment was withdrawn only the group treated with 8 mg/kg/day maintained significant differences versus the hypertensive controls. All three doses of amlodipine reduced cardiac fibrosis and this regression persisted with the two highest doses after three months without treatment. We concluded that antihypertensive treatment with amlodipine is accompanied by a reduction in left ventricular hypertrophy and regression in collagen deposition. Treatment was more effective in preventing fibrosis than in preventing ventricular hypertrophy after drug withdrawal.     

Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae)

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the "stacked" gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of (125)I-labeled Cry1Ab protein ((125)I-Cry1Ab) and (125)I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either (125)I-Cry1Ab or (125)I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with (125)I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. (125)I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, (125)I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.     

A Critical View of “On TB Vaccines,Patients’ Demands,and Modern Printed Media in Times of Biomedical Uncertainties: Buenos Aires, 1920–1950”

The putative Pueyo’s vaccine was a commercial venture that obtained marketing authorization in 1946, a turbulent period of Argentine history. After a few months, health authorities withdrew financial support from the state to buy the vaccine and required patients to sign a written consent to receive that product. An independent investigation did not find any evidence of benefit in non-clinical and clinical evaluation of the putative vaccine.     

Arginine does not rescue p.Q188R mutation deleterious effect in classic galactosemia

Background

Classic galactosemia is a rare genetic metabolic disease with an unmet treatment need. Current standard of care fails to prevent chronically-debilitating brain and gonadal complications.Many mutations in the GALT gene responsible for classic galactosemia have been described to give rise to variants with conformational abnormalities. This pathogenic mechanism is highly amenable to a therapeutic strategy based on chemical/pharmacological chaperones. Arginine, a chemical chaperone, has shown beneficial effect in other inherited metabolic disorders, as well as in a prokaryotic model of classic galactosemia.The p.Q188R mutation presents a high prevalence in the Caucasian population, making it a very clinically relevant mutation. This mutation gives rise to a protein with lower conformational stability and lower catalytic activity. The aim of this study is to assess the potential therapeutic role of arginine for this mutation.

Methods

Arginine aspartate administration to four patients with the p.Q188R/p.Q188R mutation, in vitro studies with three fibroblast cell lines derived from classic galactosemia patients as well as recombinant protein experiments were used to evaluate the effect of arginine in galactose metabolism. This study has been registered at https://clinicaltrials.gov (NCT03580122) on 09 July 2018. Retrospectively registered.

Results

Following a month of arginine administration, patients did not show a significant improvement of whole-body galactose oxidative capacity (p =?0.22), erythrocyte GALT activity (p =?0.87), urinary galactose (p =?0.52) and urinary galactitol levels (p =?0.41). Patients’ fibroblasts exposed to arginine did not show changes in GALT activity. Thermal shift analysis of recombinant p.Q188R GALT protein in the presence of arginine did not exhibit a positive effect.

Conclusions

This short pilot study in four patients homozygous for the p.Q188R/p.Q188R mutation reveals that arginine has no potential therapeutic role for galactosemia patients homozygous for the p.Q188R mutation.

     

Active transport and enzymes of the erythrocyte membrane under protein deprivation

1. Starvation for 3 days causes membrane damage of the rat erythrocyte manifested by several alterations. The adenosine-triphosphatase activity is decreased but that of acetylcholinesterase is not affected. 2. The ouabain-sensitive adenosine-triphosphatase activity increases at the expense of the non-sensitive enzyme moiety. 3. The Rb(+) uptake is not altered but the galactose transport is accelerated by the stated experimental conditions. 4. The modifications induced by starvation do not recover on re-feeding.     

Studies on the oxidation–reduction systems of the erythrocyte

1. Starvation for 3 days produces a decrease in methaemoglobin-reductase and glutathione-reductase activities, but it does not alter the glucose 6-phosphate-dehydrogenase activity of the rat erythrocyte. 2. The feeding of a protein-free diet for 11 days causes greater changes in the first two enzymes and also a diminution of the third. Under this experimental condition slight decreases in protein and haemoglobin contents were noted. 3. The experimental animals did not show methaemoglobinaemia, probably because the activity of methaemoglobin diaphorase is preserved. 4. The GSH content was not affected but the stability of the tripeptide in the presence of an oxidizing agent was diminished.     

Detailed Comparative Map of Human Chromosome 19q and Related Regions of the Mouse Genome

One of the larger contiguous blocks of mouse–human genomic homology includes the proximal portion of mouse chromosome 7 and the long arm of human chromosome 19. Previous studies have demonstrated the close relationship between the two regions, but have also indicated significant rearrangements in the relative orders of homologous mouse and human genes. Here we present the genetic locations of the homologs of 42 human chromosome 19q markers in the mouse, with an emphasis on genes also included in the human chromosome 19 physical map. Our results demonstrate that despite an overall inversion of sequences relative to the centromere, apparent “transpositions” of three gene-rich segments, and a local inversion of markers mapping near the 19q telomere, gene content, order, and spacing are remarkably well conserved throughout the lengths of these related mouse and human regions. Although most human 19q markers have remained genetically linked in mouse, one small human segment forms a separate region of homology between human chromosome 19q and mouse chromosome 17. Three of the four rearrangements of mouse versus human 19q sequences involve segments that are located directly adjacent to each other in 19q13.3–q13.4, suggesting either the coincident occurrence of these events or their common association with unstable DNA sequences. These data permit an unusually in-depth examination of this large region of mouse–human genomic homology and provide an important new tool to aid in the mapping of genes and associated phenotypes in both species.