Cathepsins B, K, and L are regulated by a defined collagen type II peptide via activation of classical protein kinase C and p38 MAP kinase in articular chondrocytes

Degradation of the extracellular matrix (ECM) is a prominent feature in osteoarthritis (OA), which is mainly because of the imbalance between anabolic and catabolic processes in chondrocytes resulting in cartilage and bone destruction. Various proteases act in concert to degrade matrix components, e.g. type II collagen, MMPs, ADAMTS, and cathepsins. Protease-generated collagen fragments may foster the destructive process. However, the signaling pathways associated with the action of collagen fragments on chondrocytes have not been clearly defined. The present data demonstrate that the N-terminal telopeptide of collagen type II enhances expression of cathepsins B, K, and L in articular chondrocytes at mRNA, protein, and activity levels, mediated at least in part through extracellular calcium. We also demonstrate that the induction is associated with the activation of protein kinase C and p38 MAP kinase.     

Brain synaptic junctional proteins at the acrosome of rat testicular germ cells.

Proteins of the presynaptic exocytic machinery have been found associated with the acrosome of male germ cells, suggesting that the sperm acrosome reaction and neurotransmission at chemical synapses may share some common mechanisms. To substantiate this hypothesis, we studied the expression and ultrastructural localization of prominent pre- and postsynaptic protein components in rat testis. The presynaptic membrane trafficking proteins SV2 and complexin, the vesicular amino acid transporters VGLUT and VIAAT, the postsynaptic scaffolding protein ProSAP/Shank, and the postsynaptic calcium-sensor protein caldendrin, could be identified in germ line cells. Immunogold electron microscopy revealed an association of these proteins with the acrosome. In addition, evidence was obtained for the expression of the plasmalemmal glutamate transporters GLT1 and GLAST in rat sperm. The novel finding that not only presynaptic proteins, which are believed to be involved in membrane fusion processes, but also postsynaptic elements are present at the acrosome sheds new light on its structural organization. Moreover, our data point to a possible role for neuroactive amino acids in reproductive physiology.     

Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.     

The halophilic alkalithermophile Natranaerobius thermophilus adapts to multiple environmental extremes using a large repertoire of Na+(K+)/H+ antiporters

Natranaerobius thermophilus is an unusual extremophile because it is halophilic, alkaliphilic and thermophilic, growing optimally at 3.5 M Na⁺, pH^55°C 9.5 and 53°C. Mechanisms enabling this tripartite lifestyle are essential for understanding how microorganisms grow under inhospitable conditions, but remain unknown, particularly in extremophiles growing under multiple extremes. We report on the response of N. thermophilus to external pH at high salt and elevated temperature and identify mechanisms responsible for this adaptation. N. thermophilus exhibited cytoplasm acidification, maintaining an unanticipated transmembrane pH gradient of 1 unit over the entire extracellular pH range for growth. N. thermophilus uses two distinct mechanisms for cytoplasm acidification. At extracellular pH values at and below the optimum, N. thermophilus utilizes at least eight electrogenic Na⁺(K⁺)/H⁺ antiporters for cytoplasm acidification. Characterization of these antiporters in antiporter-deficient Escherichia coli KNabc showed overlapping pH profiles (pH 7.8–10.0) and Na⁺ concentrations for activity ( K _0.5 values 1.0–4.4 mM), properties that correlate with intracellular conditions of N. thermophilus . As the extracellular pH increases beyond the optimum, electrogenic antiport activity ceases, and cytoplasm acidification is achieved by energy-independent physiochemical effects (cytoplasmic buffering) potentially mediated by an acidic proteome. The combination of these strategies allows N. thermophilus to grow over a range of extracellular pH and Na⁺ concentrations and protect biomolecules under multiple extreme conditions.     

Changes in heart rate variability of athletes during a training camp.

Heart rate variability (HRV) reflects regulatory processes of the cardiovascular system and reveals fractal characteristics. In this paper we investigated standard HRV parameters and scaling characteristics in ten athletes before, during, and after a 2-week training camp to assess the effects of short-term overtraining on cardiovascular control. High-resolution ECGs were recorded over 30 min under resting conditions 1 week before the training camp, after 1 week of training in the camp, and after 3-4 days of recovery. Standard HRV analysis was performed according to Task Force recommendations. Scaling characteristics were assessed, applying detrended fluctuation analysis (DFA). Standard HRV analysis showed significant changes in meanNN and rmssd during the training camp. DFA revealed three distinct regions of scale-invariance and significant alterations during the training camp. In conclusion, HRV might be used to monitor the training state in athletes.     

Isolation and characterization of a xanthophyll-rich fraction from the thylakoid membrane of Dunaliella salina(green algae).

Long-term acclimation to irradiance stress (HL) of the green alga Dunaliella salina Teod. (UTEX 1644) entails substantial accumulation of zeaxanthin along with a lowering in the relative amount of other pigments, including chlorophylls and several carotenoids. This phenomenon was investigated with wild type and the zea1 mutant of D. salina, grown under conditions of low irradiance (LL), or upon acclimation to irradiance stress (HL). In the wild type, the zeaxanthin to chlorophyll (Zea/Chl)(mol : mol) ratio was as low as 0.009 : 1 under LL and as high as 0.8 : 1 under HL conditions. In the zea1 mutant, which constitutively accumulates zeaxanthin and lacks antheraxanthin, violaxanthin and neoxanthin, the Zea/Chl ratio was 0.15 : 1 in LL and 0.57 : 1 in HL. The divergent Zea/Chl ratios were reflected in the coloration of the cells, which were green under LL and yellow under HL. In LL-grown cells, all carotenoids occurred in structural association with the Chl-protein complexes. This was clearly not the case in the HL-acclimated cells. A beta-carotene-rich fraction occurred as loosely bound to the thylakoid membrane and was readily isolated by flotation following mechanical disruption of D. salina. A zeaxanthin-rich fraction was specifically isolated, upon mild surfactant treatment and differential centrifugation, from the thylakoid membrane of either HL wild type or HL-zea1 mutant. Such differential extraction of beta-carotene and Zea, and their separation from the Chl-proteins, could not be obtained from the LL-grown wild type, although small amounts of Zea could still be differentially extracted from the LL-grown zea1 strain. It is concluded that, in LL-grown D. salina, xanthophylls (including most of Zea in the zea1 strain) are structurally associated with and stabilized by the Chl-proteins in the thylakoid membrane. Under HL-growth conditions, however, zeaxanthin appears to be embedded in the lipid bilayer, or in a domain of the chloroplast thylakoids that can easily be separated from the Chl-proteins upon mild surfactant treatment. In conclusion, this work provides biochemical evidence for the domain localization of accumulated zeaxanthin under irradiance-stress conditions in green algae, and establishes protocols for the differential extraction of this high-value pigment from the green alga D. salina.