The Effect of Old Age on the Free-Running Period of Circadian Rhythms in Rat

The free-running period is regarded to be an exclusive feature of the endogenous circadian clock. Changes during aging in the free-running period may therefore reflect age-related changes in the internal organization of this clock. However, the literature on alterations in the free-running period in aging is not unequivocal. In the present study, with various confounding factors kept to a minimum, it was found that the free-running periods for active wakefulness, body temperature, and drinking behavior were significantly shorter (by 12-17 min) in old than in young rats. In addition, it was found that the day-to-day stability of the different sleep states was reduced in old rats, whereas that of the drinking rhythm was enhanced. Transient cycles were not observed, nor were there any age-related differences in daily totals of the various sleep-wake states. The amplitudes of the circadian rhythms of active wakefulness, quiet sleep, and temperature were reduced, whereas those of paradoxical sleep and quiet wakefulness remained unchanged.     

Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triploidy—Observations in 154 Diandric Cases

Hydatidiform moles (HMs) are abnormal human pregnancies with vesicular chorionic villi, imposing two clinical challenges; miscarriage and a risk of gestational trophoblastic neoplasia (GTN). The parental type of most HMs are either diandric diploid (PP) or diandric triploid (PPM). We consecutively collected 154 triploid or near-triploid samples from conceptuses with vesicular chorionic villi. We used analysis of DNA markers and/or methylation sensitive-MLPA and collected data from registries and patients records. We performed whole genome SNP analysis of one case of twinning (PP+PM).In all 154 triploids or near-triploids we found two different paternal contributions to the genome (P1P2M). The ratios between the sex chromosomal constitutions XXX, XXY, and XYY were 5.7: 6.9: 1.0. No cases of GTN were observed. Our results corroborate that all triploid human conceptuses with vesicular chorionic villi have the parental type P1P2M. The sex chromosomal ratios suggest approximately equal frequencies of meiosis I and meiosis II errors with selection against the XYY conceptuses or a combination of dispermy, non-disjunction in meiosis I and meiosis II and selection against XYY conceptuses. Although single cases of GTN after a triploid HM have been reported, the results of this study combined with data from previous prospective studies estimate the risk of GTN after a triploid mole to 0% (95% CI: 0–1,4%).     

Cytosolic phospholipase A2α sustains pAKT,pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and EC₅₀ for glutamate. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [³H]glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but may also affect neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotrophic lateral sclerosis and cerebral hypoxia, which are associated with neuronal GLT1b up-regulation.     

Aerobic Training in Patients with Congenital Myopathy

Introduction

Congenital myopathies (CM) often affect contractile proteins of the sarcomere, which could render patients susceptible to exercise-induced muscle damage. We investigated if exercise is safe and beneficial in patients with CM.

Methods

Patients exercised on a stationary bike for 30 minutes, three times weekly, for 10 weeks at 70% of their maximal oxygen uptake (VO_2max). Creatine kinase (CK) was monitored as a marker of muscle damage. VO_2max, functional tests, and questionnaires evaluated efficacy.

Results

Sixteen patients with CM were included in a controlled study. VO_2max increased by 14% (range, 6–25%; 95% CI 7–20; p < 0.001) in the seven patients who completed training, and tended to decrease in a non-intervention group (n = 7; change -3.5%; range, -11–3%, p = 0.083). CK levels were normal and remained stable during training. Baseline Fatigue Severity Scale scores were high, 4.9 (SE 1.9), and tended to decrease (to 4.4 (SE 1.7); p = 0.08) with training. Nine patients dropped out of the training program. Fatigue was the major single reason.

Conclusions

Ten weeks of endurance training is safe and improves fitness in patients with congenital myopathies. The training did not cause sarcomeric injury, even though sarcomeric function is affected by the genetic abnormalities in most patients with CM. Severe fatigue, which characterizes patients with CM, is a limiting factor for initiating training in CM, but tends to improve in those who train.

Trial Registration

The Regional Committee on Health Research Ethics of the Capital Region of Denmark H-2-2013-066 and ClinicalTrials.gov H2-2013-066     

P2X1 receptor blockers reduce the number of circulating thrombocytes and the overall survival of urosepsis with haemolysin-producing Escherichia coli

Purinergic Signalling - Urosepsis is a severe condition often caused by Escherichia coli that spontaneously have ascended the urinary tract to the kidneys causing pyelonephritis and potentially...     

A Protein Disulfide Isomerase Controls Neuronal Migration through Regulation of Wnt Secretion

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