Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids

Transient-receptor-potential channels (TRPs) underlie the sensing of chemicals, heat, and mechanical force. We expressed the rat TRPV1 and TRPV4 subtypes in yeast and monitored their activities in vivo as Ca²⁺ rise using transgenic aequorin. Heat and capsaicin activate TRPV1 but not TRPV4 in yeast. Hypotonic shocks activate TRPV4 but not TRPV1. Osmotic swelling is modeled to activate enzyme(s), producing polyunsaturated fatty acids (PUFAs) to open TRPV4 in mammalian cells. This model relegates mechanosensitivity to the enzyme and not the channel. Yeast has only a single Δ9 fatty-acid monodesaturase and cannot make PUFAs suggesting an alternative mechanism for TRPV4 activation. We discuss possible explanations of this difference.     

Synthesis and characterization of thermosensitive graft copolymer of N-isopropylacrylamide with biodegradable carboxymethylchitosan

A novel thermosensitive and hydrogel was designed and synthesized by graft copolymerization of N-isopropylacrylamide (NIPAAm) with biodegradable carboxymethylchitosan (CMCS). The influence of the content of CMCS grafted on the properties of the resulted hydrogels was examined. The morphology of the hydrogels was observed by scanning electron microscopy (SEM), their thermal property was characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and deswelling/swelling kinetics upon external temperature changes. In comparison with the conventional PNIPAAm hydrogels, the resulted hydrogels have improved thermosensitive properties, including enlarged water content at room temperature and faster deswelling/swelling rate upon heating. The strategy described here presents a potential alternative to the traditional synthesis techniques for thermosensitive hydrogels.     

Small heat shock proteins prevent aggregation of citrate synthase and bind to the N-terminal region which is absent in thermostable forms of citrate synthase

Citrate synthase (CS) is often used in chaperone assays since this thermosensitive enzyme aggregates at moderately increased temperatures. Small heat shock proteins (sHsps) are molecular chaperones specialized in preventing the aggregation of other proteins, termed substrate proteins, under conditions of transient heat stress. To investigate the mechanism whereby sHsps bind to and stabilize a substrate protein, we here used peptide array screening covering the sequence of porcine CS (P00889). Strong binding of sHsps was detected to a peptide corresponding to the most N-terminal α-helix in CS (amino acids Leu₁₃ to Gln₂₇). The N-terminal α-helices in the CS dimer intertwine with the C-terminus in the other subunit and together form a stem-like structure which is protruding from the CS dimer. This stem-like structure is absent in thermostable forms of CS from thermophilic archaebacteria like Pyrococcus furiosus and Sulfolobus solfatacarium. These data therefore suggest that thermostabilization of thermosensitive CS by sHsps is achieved by stabilization of the C- and N-terminae in the protruding thermosensitive softspot, which is absent in thermostable forms of the CS dimer.     

S-Adenosylhomocysteine hydrolase (AdoHcyase) deficiency: enzymatic capabilities of human AdoHcyase are highly effected by changes to codon 89 and its surrounding residues

Recently, S-adenosylhomocysteine hydrolase deficiency was confirmed for the first time in an adult. Two missense mutations in codons 89 (A>V) and 143 (Y>C) in the AdoHcyase gene were identified [N.R.M. Buist, B. Glenn, O. Vugrek, C. Wagner, S. Stabler, R.H. Allen, I. Pogribny, A. Schulze, S.H. Zeisel, I. Bari?, S.H. Mudd, S-Adenosylhomocysteine hydrolase deficiency in a 26-year-old man, J. Inh. Metab. Dis. 29 (2006) 538-545]. Accordingly, we have proven the Y143C mutation to be highly inactivating [R. Beluzi?, M. Cuk, T. Pavkov, K. Fumi?, I. Bari?, S.H. Mudd, I. Jurak, O. Vugrek, A single mutation at tyrosine 143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound co-factor NAD, Biochem. J. 400 (2006) 245-253]. Now we report that the A89V exchange leads to a 70% loss of enzymatic activity, respectively. Circular dichroism analysis of recombinant p.A89V protein shows a significantly reduced unfolding temperature by 5.5 degrees C compared to wild-type. Gel filtration of mutant protein is almost identical to wild-type indicating assembly of subunits into the tetrameric complex. However, electrophoretic mobility of p.A89V is notably faster as shown by native polyacrylamide gel electrophoresis implicating changes to the overall charge of the mutant complex. 'Bioinformatics' analysis indicates that Val(89) collides with Thr(84) causing sterical incompatibility. Performing site-directed mutagenesis changing Thr(84) to 'smaller' Ser(84) but preserving similar physico-chemical properties restores most of the catalytic capabilities of the mutant p.A89V enzyme. On the other hand, substitution of Thr(84) with Lys(84) or Gln(84), thereby introducing residues with higher volume in proximity to Ala(89) results in inactivation of wild-type protein. In view of our mutational analysis, we consider changes in charge and the sterical incompatibility in mutant p.A89V protein as main reason for enzyme malfunction with AdoHcyase deficiency as consequence.     

Effects of water temperature on growth and sex ratio of juvenile Nile tilapia Oreochromis niloticus (Linnaeus) reared in geothermal waters in southern Tunisia

Growth of juvenile Nile tilapia, Oreochromis niloticus (Maryut strain) was studied under laboratory conditions. Four thermal regimes (22, 26, 30, and 34 °C) were tested on 480 20-day-old fry.     

Sleep propensity is modulated by circadian and behavior-induced changes in cutaneous temperature

A close relation between sleep and body temperature has been noted already for a long time. Although a correlation is indisputable, there is at present hardly evidence for a causal involvement of sleep in changes in body temperature. Concerning the reverse, a causal involvement of body temperature in sleep has been demonstrated: if core or skin temperature changes activate thermoregulatory processes aimed at heat loss or heat preservation, sleep is usually disrupted. We have recently proposed that sleep propensity is also affected by more subtle changes in skin temperature, within the thermoneutral range (Van Someren (2000). Chronobiol. Int. 17, 313–354). These changes are likely to modulate the firing properties of thermosensitive neurons in brain areas involved in sleep regulation. Subtle changes in skin temperature occur daily under control of the circadian timing system. They could provide this system with an additional signal pathway to support its neuronal and neurohormonal signals to enforce circadian modulation of sleep propensity. Subtle changes in skin temperature also result from behavior, and could contribute to the changes in sleep propensity resulting from these behaviors. The present review summarizes the neurobiological background and correlational physiological and behavioral data in support of the involvement of skin temperature in the modulation of sleep propensity. It moreover points out the type of experimental investigations needed to support or refute the hypothesis.     

Indirect hand and forearm vasomotion: Regional variations in cutaneous thermosensitivity during normothermia and mild hyperthermia

In this experiment, hand and forearm vasomotor activity was investigated during localised, but stable heating and cooling of the face, hand and thigh, under open-loop (clamped) conditions. It was hypothesised that facial stimulation would provoke the most potent vascular changes. Nine individuals participated in two normothermic trials (mean body temperature clamp: 36.6 °C; water-perfused suit and climate chamber) and two mildly hyperthermic trials (37.9 °C). Localised heating (+5 °C) and cooling (−5 °C) stimuli were applied to equal surface areas of the face, hand and thigh (perfusion patches: 15 min), while contralateral forearm or hand blood flows (venous-occlusion plethysmography) were measured (separate trials). Thermal sensation and discomfort votes were recorded before and during each thermal stimulation. When hyperthermic, local heating induced more sensitive vascular responses, with the combined thermosensitivity of both limb segments averaging 0.011 mL·100 mL⁻¹·min⁻¹·mmHg⁻¹·°C⁻¹, and 0.005 mL·100 mL⁻¹·min⁻¹·mmHg⁻¹·°C⁻¹ during localised cooling (P<0.05). Inter-site comparisons among the stimulated sites yielded minimal evidence of variations in local thermal sensation, and no differences were observed for vascular conductance (P>0.05). Therefore, regional differences in vasomotor and sensory sensitivity appeared not to exist. When combined with previous observations of sudomotor sensitivity, it seems that, during mild heating and cooling, regional representations within the somatosensory cortex may not translate into meaningful differences in thermal sensation or the central integration of thermoafferent signals. It was concluded that inter-site variations in the cutaneous thermosensitivity of these thermolytic effectors have minimal physiological significance over the ranges investigated thus far.     

Modulate the phase transition temperature of hydrogels with both thermosensitivity and biodegradability

The synthesis and characterization of thermoresponsive hydrogels on the basis of N-isoproplyarylamide (NIPAAm) and acrylamide (AAm) copolymers crosslinked with a novel biodegradable crosslinker (PEG-co-PLA) were carried out in this study. Swelling measurement results demonstrated that four gels of PNAM5, PNAM10, PNAM12 and PNAM15 are thermoresponsive. The equilibrium swelling ratio and degradation of the hydrogels strongly depend on hydrogels composition. The morphology of the hydrogels was observed by scanning electron microscopy (SEM), and their thermal property was characterized by differential scanning calorimetry (DSC). The results show that the proportion of AAm in the copolymer has notable effect on the low critical solution temperature (LCST) of the hydrogel. When the molar ratio of AAm to NIPAAm was increased from 1:10 to 3:10 the LCST of the copolymer increased from 39.7 to 64.2 °C. The compression modulus of PNAM15 is of the highest among other hydrogels, because PNAM15 hydrogel has a more compact structure.     

Suppression of the thermosensitive replication phenotype of the derivative plasmid of pI9789::Tn552 in Staphylococcus aureus may involve integration of the plasmid into the host chromosome

Abstract Plasmid-chromosome co-integration was found to be the mechanism of choice to overcome thermosensitivity of replication of the plasmid pS1 in PS80d and RN4220 strains of Staphylococcus aureus . The integration of the plasmid was sometimes accompanied by deletion of a specific section of the plasmid pS1 in PS80d. Growth of bacteriophage on strains containing the integrated plasmid and the subsequent use of the phage in transduction gave transductants containing plasmids that had regained their replication thermosensitivity. These plasmids had not acquired any detectable chromosomal DNA. The 16-kb EcoRI fragment of the PS80d chromosome that hybridizes to pS1 is the target for recombination in many cases, but apparently other sites are also used. This fragment contains sequence homologous to parts of the transposon Tn552 and it is probable that site-specific recombination is involved in the integration. The possible mechanisms for the integrations and the deletions are discussed.