Photorespiratory serine hydroxymethyltransferase 1 activity impacts abiotic stress tolerance and stomatal closure

The photorespiratory cycle is a crucial pathway in photosynthetic organisms because it removes toxic 2‐phosphoglycolate made by the oxygenase activity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase and retrieves its carbon as 3‐phosphoglycerate. Mitochondrial serine hydroxymethyltransferase 1 (SHMT1) is an essential photorespiratory enzyme converting glycine to serine. SHMT1 regulation remains poorly understood although it could involve the phosphorylation of serine 31. Here, we report the complementation of Arabidopsis thaliana shm1‐1 by SHMT1 wild‐type, phosphorylation‐mimetic (S31D) or nonphophorylatable (S31A) forms. All SHMT1 forms could almost fully complement the photorespiratory growth phenotype of shm1‐1; however, each transgenic line had only 50% of normal SHMT activity. In response to either a salt or drought stress, Compl‐S31D lines showed a more severe growth deficiency compared with the other transgenic lines. This sensitivity to salt appeared to reflect reduced SHMT1‐S31D protein amounts and a lower activity that impacted leaf metabolism leading to proline underaccumulation and overaccumulation of polyamines. The S31D mutation in SHMT1 also led to a reduction in salt‐induced and ABA‐induced stomatal closure. Taken together, our results highlight the importance of maintaining photorespiratory SHMT1 activity in salt and drought stress conditions and indicate that SHMT1 S31 phosphorylation could be involved in modulating SHMT1 protein stability.     

Hormonal and behavioural detection of oestrus in blackbuck, Antilope cervicapra, and successful artificial insemination with fresh and frozen semen

Hormonal detection of urinary pregnanediol-3 alpha-glucuronide proved an effective method of monitoring the progress of oestrous cycles in the blackbuck; observation of sexual behaviour in a vasectomized male was, however, a more practical procedure. Good correlation was observed between the occurrence of minimal pregnanediol concentrations in females and the maximal behavioural response by the male. On the basis of intervals between periods of behaviourally detected oestrus, a mean cycle length of 16.9 days (+/- 0.62, s.e.m.) was derived from 12 cycles (4 animals). Eleven females were inseminated in this study, 8 with freshly collected semen and 3 with frozen semen; 6 calves were obtained, 1 after the use of frozen semen. Pregnancy was monitored by measurements of pregnanediol-3 alpha-glucuronide excretion and by ultrasound scanning. The mean interval between insemination and parturition was 183.3 days inclusive, ranging from 182 to 186 days.     

Cobra cardiotoxins. Purification, effects on skeletal muscle and structure/activity relationships [published errtum appears in Eur J Biochem 1988 Feb 1;171(3):727

A new preparative method for isolating homogeneous cardiotoxins from cobra venoms is described. The technique, based on reverse-phase high-pressure liquid chromatography, was used to isolate eight cardiotoxins of known sequence from four different venoms. In each case the method was found to be particularly efficient at removing trace quantities of contaminating phospholipase. Cardiotoxins isolated in this manner were found to retain their full biological activity. Without exception the purified cardiotoxins lacked powerful haemolytic activity at concentrations up to 0.01 mM (about 100 micrograms ml-1), although some lysis of human erythrocytes was induced at higher concentrations. The cardiotoxins displayed a wide range of depolarizing activity on cultured skeletal muscle, the lowest activity being associated with the highest LD50 value. Correlating variations in amino acid sequence and variations in depolarization potency revealed the importance of residues in the second and third loops, especially lysine-46, serine-48 and lysine-52, together with a number of hydrophobic residues. Further modifications of pharmacological activity were associated with the presence of additional basic residues in the first and second loops and to minor differences in secondary structure.     

Artificial insemination in the Giant panda (Ailuropoda melanoleaca)

Artificial insemination was carried out on three adult captive Giant pandas (Ailuropoda melanoleuca). Oestrus in these animals was detected by behavioural observations and by the rise in urinary oestrogen metabolites. Semen was collected from a mature male by electroejaculation. Changes in the quality of semen and the size of the testes indicated a seasonal fluctuation in sperm production. Spermatozoa were capable of penetrating zona-free hamster oocytes and remained viable for up to five days at 15–20°C in a modified Tyrode's medium. After freezing and thawing in a cryopreserving diluent, approximately 50% of spermatozoa recovered progressive motility and displayed a similar velocity to spermatozoa prior to freezing. Unusual protrusions of the acrosome of the panda spermatozoon were noted.
Following insemination, one female conceived and gave birth, after a gestation of 159 days, to two cubs, one of which subsequently died. Another female exhibited a rise in circulating progesterone but pregnancy was not confirmed.     

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.     

Inhibition of matrix metalloproteinase-9 secretion by dimethyl sulfoxide and cyclic adenosine monophosphate in human monocytes

BACKGROUND Matrix metalloproteinases(MMPs),including MMP-9,are an integral part of the immune response and are upregulated in response to a variety of stimuli.New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion.There is significant evidence for regulation of inflammation by dimethyl sulfoxide(DMSO)and 3',5'-cyclic adenosine monophosphate(cAMP),thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factorα(TNFα)secretion by human monocytes was of high interest.The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFαsecretion by distinct mechanisms.AIM To investigate the regulation of lipopolysaccharide(LPS)-stimulated MMP-9 and tumor necrosis factorαsecretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP.METHODS The paper describes a basic research study using THP-1 human monocyte cells.All experiments were conducted at the University of Missouri-St.Louis in the Department of Chemistry and Biochemistry.Human monocyte cells were grown,cultured,and prepared for experiments in the University of Missouri-St.Louis Cell Culture Facility as per accepted guidelines.Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFαproduction.Inhibitors including DMSO,cAMP regulators,and anti-TNFαantibody were added to the cells prior to LPS treatment.MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software.TNFαsecretion was analyzed by enzyme-linked immuno sorbent assay.All data is presented as the average and standard error for at least 3 trials.Statistical analysis was done using a two-tailed paired Student t-test.P values less than 0.05 were considered significant and designated as such in the Figures.LPS and cAMP regulators were from Sigma-Aldrich,MMP-9 standard and antibody and TNFαantibodies were from R&D Systems,and amyloid-βpeptide was from rPeptide.RESULTS In our investigation of MMP-9 secretion from THP-1 human monocytes,we made the following findings.Inclusion of DMSO in the cell treatment inhibited LPSinduced MMP-9,but not TNFα,secretion.Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dosedependent fashion.A cell-permeable cAMP analog,dibutyryl cAMP,inhibited both LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFαin a dose-dependent fashion.Pre-treatment of monocytes with an anti-TNFαantibody blocked LPSinduced MMP-9 and TNFαsecretion.Amyloid-βpeptide induced MMP-9 secretion,which occurred much later than TNFαsecretion.The latter two findings strongly suggested an upstream role for TNFαin mediating LPS-stimulate MMP-9 secretion.CONCLUSION The cumulative data indicated that MMP-9 secretion was a distinct process from TNFαsecretion and occurred downstream.First,DMSO inhibited MMP-9,but not TNFα,suggesting that the MMP-9 secretion process was selectively altered.Second,cAMP inhibited both MMP-9 and TNFαwith a similar potency,but at different monocyte cell exposure time points.The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFαand that TNFαmay a key component of the pathway leading to MMP-9 secretion.This temporal relationship fit a model whereby early TNFαsecretion directly led to later MMP-9 secretion.Lastly,antibody-blocking of TNFαdiminished MMP-9 secretion,suggesting a direct link between TNFαsecretion and MMP-9 secretion.     

Scaling and non-scaling of muscle activity, kinematics, and dynamics in sit-ups with different degrees of difficulty.

The purpose of this study was to investigate how the CNS adjusts motor patterns for variants of a complex axial movement-the sit-up. Adjustments were induced by changing the support surface contact and mass distribution of the body. Healthy adults performed straight-legged sit-ups, 3 s in duration, with support added to or removed from the lumbar trunk, or with mass added to the head or to the legs. Each of these interventions either increased or decreased the difficulty of the task. The study addressed the extent to which changes in sit-up difficulty are compensated by scaling of muscle activity, kinematics, and dynamics versus the extent to which they are compensated by changing discretely the motor pattern. The analysis of muscle activity, kinematics, and dynamics focused on the first 30-40% of the sit-up-the trunk flexion phase-since this is the most critical part of the movement. Our results demonstrate that, in some respects, sit-up kinematics and dynamics scaled with difficulty, but in other respects, they did not. Muscle activity also scaled, in many respects, but in more difficult sit-ups, abdominal flexor activity decreased instead of increased. Non-scaling changes in these parameters suggest that complex movements, such as the sit-up, may require discrete changes in motor pattern in order to deal with large loads, which challenge the available leverage.     

Metabolism of oestradiol-17 beta and progesterone in the white rhinoceros (Ceratotherium simum simum)

14C-Labelled oestradiol-17 beta and progesterone (50 mu Ci each) were injected i.v. into an adult female white rhinoceros and all urine and faeces collected separately over the next 4 days. The total recovery of injected label was 61%, 25% being present in the urine and 36% in the faeces. Of the radioactivity recovered, 69% was excreted on Day 2 of the collection period. Repeated extraction of samples obtained on Day 2 showed that, of the radioactivity in faeces, 92.4% was associated with unconjugated steroids whereas in the urine the proportion of conjugated and unconjugated steroids were similar (41.2% and 51.4% respectively). After phenolic separation of urinary steroids, HPLC followed by derivatization and recrystallization techniques identified progesterone as the major component of the unconjugated portion with 4-pregnen-20 alpha-ol-3-one as the principal metabolite in the conjugated fraction. Pregnanediol was not present. Oestrone appeared to be the most abundant oestrogen metabolite with smaller but significant amounts of oestradiol-17 beta and oestradiol-17 alpha in the unconjugated and conjugated fractions respectively. Small amounts of progesterone were found in the faecal extract in which the radioactivity consisted mainly of oestradiol-17 alpha and oestradiol-17 beta. The results have established the major excreted metabolites of oestradiol-17 beta and progesterone in the white rhinoceros and the development of more appropriate assay methods for monitoring ovarian function in African rhinoceroses should now be possible.