Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.     

A new species of the genus <Emphasis Type="Italic">Careproctus</Emphasis> (Liparidae) from the Sea of Japan

A new snailfish, Careproctus notosaikaiensis, is described on the basis of 6 specimens collected from the central part of the Sea of Japan, off Saikai, western coast of the Noto Peninsula, Ishikawa, Japan. The new species is distinguished from other congeners by the following combination of the characters: vertebrae 57–58, 10–12 abdominal and 46–48 caudal; dorsal-fin rays 52; anal-fin rays 46–47; principal caudal-fin rays 10; pectoral-fin rays 35–37; pectoral fin with a deep notch; proximal pectoral radials 4 (3 + 1), round; gill slit extending to pectoral-fin ray 4–7; teeth strongly trilobed; pleural ribs 2 pairs; 2 suprabranchial pores; chin pores paired in the same pit; pyloric caeca 20–29; dorsal and anal fins with dark margins and stomach black in preserved specimens.     

<Emphasis Type="Italic">Oncorhynchus kawamurae</Emphasis> “Kunimasu,” a deepwater trout,discovered in Lake Saiko, 70 years after extinction in the original habitat,Lake Tazawa,Japan

Oncorhynchus kawamurae (Osteichthyes: Salmonidae) (common name “Kunimasu”), a species endemic to Lake Tazawa, Akita Prefecture, Japan, was believed to have been extinct since 1940. However, nine specimens were discovered in March and April 2010 in Lake Saiko, Yamanashi Prefecture, one of the lakes to which eyed eggs of the species were introduced in 1935. These were identified as O. kawamurae because of having 47–62 pyloric caeca, 37–43 gill-rakers, a black-colored body, and spawning at 30–40 m depth in early spring, which are unique characteristics within Oncorhynchus. Furthermore, the distinctiveness of Kunimasu from sympatric kokanee (O. nerka) was supported by microsatellite DNA data.     

Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin–biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions:     

Taxonomic review of East Asian <Emphasis Type="Italic">Pleuronichthys</Emphasis> (Pleuronectiformes: Pleuronectidae), with description of a new species

A review of East Asian frog flounders, genus Pleuronichthys (family Pleuronectidae), recognized Pleuronichthys japonicus sp. nov. and P. cornutus (Temminck and Schlegel 1846). Pleuronichthys japonicus sp. nov. is characterized by small, dark, rounded spots or marbled markings on the ocular side of the body, rounded cycloid scales somewhat irregularly arranged, usually 12 abdominal vertebrae, 67–80 (modally 75) dorsal-fin rays, 48–59 (modally 55) anal-fin rays, and a short branch of the supratemporal lateral line usually present on both sides. Pleuronichthys cornutus is characterized by densely distributed small, dark, irregular spots on the ocular side of the body, elongate cycloid scales somewhat regularly arranged, usually 13 abdominal vertebrae, 72–88 (modally 77) dorsal-fin rays, 52–65 (modally 58) anal-fin rays, and a branch of the supratemporal lateral line usually absent on both sides. Whereas P. cornutus is distributed from Miyagi Prefecture (Tohoku District) southward along the Pacific coast of Japan to the Bungo Channel, from Akita Prefecture (Tohoku District) southward along the Sea of Japan coast through the Tsushima Strait to the East China Sea, Yellow and Bohai Seas, the Taiwan Strait, and northern Chinese coast of the South China Sea, P. japonicus is distributed from southern Hokkaido southward along the Sea of Japan and Pacific coasts of Japan to the southern East China Sea. Geographic variations were found in caudal vertebrae and anal-fin ray counts, and caudal-peduncle depth in P. cornutus, and in ocular side body coloration, body depth, and head length in P. japonicus. Pleuronichthys lighti Wu 1929 was regarded as a junior synonym of P. cornutus.     

Amperometric biosensor based on enzymes immobilized in hybrid mesoporous membranes for the determination of acetylcholine

Acetylcholine sensor is successfully prepared by using immobilized enzymes, i.e., acetylcholinesterase and choline oxidase within separate hybrid mesoporous silica membranes with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide produced by two sequential enzyme reactions. The determination range and the response time are 6.0–800 μM and within approximately 3 min, respectively. The sensor is very stable compared to free enzymes and 80% of the initial response was maintained even after storage for 80 days. These results show that two enzymes are successfully immobilized and well stabilized, and at the same time, two sequential enzyme reactions efficiently proceed within the separate hybrid mesoporous membranes. Further, we studied the possible detection of organophosphorus pesticides in terms of the inhibition of acetylcholinesterase activity, i.e., the decrease of current response, and demonstrated that the nanomolar concentrations of pesticide (DZN-oxon) can be detected with our sensor.     

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.