Impaired pressure sensation in mice lacking TRPV4

The sensation of pressure, mechanosensation, in vertebrates remains poorly understood on the molecular level. The ion channel TRPV4 is in the TRP family and is a candidate for a mechanosensitive calcium-permeable channel. It is located in dorsal root ganglia. In the present study, we show that disrupting the Trpv4 gene in mice markedly reduced the sensitivity of the tail to pressure and acidic nociception. The threshold to noxious stimuli and the conduction velocity of myelinated nerve responding to stimuli were also impaired. Activation of unmyelinated nerve was undetected. However, the mouse still retained olfaction, taste sensation, and heat avoidance. The TRPV4 channel expressed in vitro in Chinese hamster ovary cells was opened by low pH, citrate, and inflation but not by heat or capsaicin. These data identify the TRPV4 channel as essential for the normal detection of pressure and as a receptor of the high-threshold mechanosensory complex.     

Polyhydroxyalkanoate (PHA) synthesis by class IV PHA synthases employing Ralstonia eutropha PHB(-)4 as host strain

Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).     

Induction of cold stability of microtubules in cultured tobacco cells

In suspension-cultured tobacco (Nicotiana tabacum) cells, we have often encountered cold-stable microtubules (MTs). The cold-stable MTs were found in the pelleted fraction of tobacco cell homogenates. These cold-stable MTs were shown to be accompanied by unidentified filamentous structures that extended along part of their length. However, during the early hours in culture such cold-stable MTs were never observed. They were detectable from 120 h after the beginning of subculture and then their numbers increased gradually. The number of cells with cold-stable MTs eventually accounted for more than 95% of the total population of cells at the stationary phase of culture. The rapid loss of cold stability of MTs occurred when such cells were transferred to fresh medium for subculture. However, if the fresh medium was supplemented with once-used medium, the cold stability of MTs was retained. The active agent in the medium appeared to be of low molecular weight and to be heat resistant. A similar activity was detected in a pectin hydrolyzate. When an inhibitor of protein kinase, either 6-dimethylaminopurine or staurosporin, was added to the cells at an early stage of culture, when cold-stable MTs were normally completely absent, most cells acquired cold-stable MTs. It appears that acquisition or loss of cold stability of MTs in tobacco cells is regulated by the action of a kinase/phosphatase or a phosphorylation/dephosphorylation system on some MT protein(s), such as a cold stabilizer of MTs, some unidentified MT-associated filamentous structure, or even tubulin itself.     

Bacillus stearothermophilus Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin

The specificity of Bacillus stearothermophilus TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10⁸ to 10⁷ Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic Bacillus sp. strain A2-5a and maltogenic amylase from Bacillus licheniformis ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying α-amylase, bacterial liquefying α-amylase, β-amylase, and neopullulanase from Bacillus megaterium, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin.     

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.     

ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.     

Localization of neutrophil adherence-inhibiting factor in guinea pig platelets

The effect of constituents of guinea pig platelets on neutrophil adherence was examined. The platelet sonicate supernatant contained adherence-inhibiting activity which strongly inhibited neutrophil adherence to glass. When the platelet sonicate supernatant was treated with neuraminidase or trypsin, the adherence-inhibiting activity was significantly inhibited, suggesting that the adherence-inhibiting factor (AIF) is a glycoprotein. The subcellular fractionation experiments indicated that the AIF activity was present at about 40% in both the cytosol and granule fractions. From the Sephadex G-200 gel filtration analysis, AIF of cytosol fraction and granule fraction proved to be different molecules, with molecular masses of about 230 and 12 kDa, respectively. When platelets were stimulated with thrombin, about 20% of total AIF was released extracellularly without the release of the cytoplasmic enzyme lactate dehydrogenase. These results suggest the possibility that a biologically active substance, AIF, is released from platelets in response to stimuli and regulates neutrophil functions through interference with neutrophil adherence.     

Motor unit activation patterns during concentric wrist flexion in humans with different muscle fibre composition

Muscle activity was recorded from the flexor carpi radialis muscle during static and dynamic-concentric wrist flexion in six subjects, who had exhibited large differences in histochemically identified muscle fibre composition. Motor unit recruitment patterns were identified by sampling 310 motor units and counting firing rates in pulses per second (pps). During concentric wrist flexion at 30% of maximal exercise intensity the mean firing rate was 27 (SD 13) pps. This was around twice the value of 12 (SD 5) pps recorded during sustained static contraction at 30% of maximal voluntary contraction, despite a larger absolute force level during the static contraction. A similar pattern of higher firing rates during dynamic exercise was seen when concentric wrist flexion at 60% of maximal exercise intensity [30 (SD 14) pps] was compared with sustained static contraction at 60% of maximal voluntary contraction [19 (SD 8) pps]. The increase in dynamic exercise intensity was accomplished by recruitment of additional motor units rather than by increasing the firing rate as during static contractions. No difference in mean firing rates was found among subjects with different muscle fibre composition, who had previously exhibited marked differences in metabolic response during corresponding dynamic contractions. It was concluded that during submaximal dynamic contractions motor unit firing rate cannot be deduced from observations during static contractions and that muscle fibre composition may play a minor role. Accepted: 5 May 1998