Giant lysosomes in the renal proximal tubules--a morphological characteristic of DBA/2 and DBA/1 mouse kidneys.

The DBA/2Cr mouse is characterized by the presence of giant lysosomes located in the proximal convoluted tubules of males and proximal straight tubules of females. However, it remains unclear whether these giant lysosomes in the proximal tubules are characteristic of DBA/2Cr specifically, or are common to other DBA/2 substrains and DBA/1. The present study investigated the morphology of kidneys from DBA/2CrSlc, DBA/2JJcl, DBA/2NCrj and DBA/1JNCrj mice of both sexes. Giant lysosomes in the renal proximal tubules were found to represent common morphological characteristic of both DBA/2 and DBA/1JN.     

Reproductive lifespan and reproductive performance in SPF C3H mice: the onset of reproductive life and production efficiency (author's transl)]

To improve the production system, the onset and the termination of reproductive life of C3Hf/HeMsNrs mice mated immediately after weaning and reared for 400 days of life, were studied. From weaning females mated with a full grown male (group A), the first litter was obtained at a mean age of 47 days, suggesting the first copulation at 26 days of age. The age of males at the first copulation was estimated to be at 44 days of age from the age giving the first litters in weanling males mated with weanling (group B) and full grown (group C) females. The sex ratio of litters delivered by young dams tended to be excess in males. The reproductive performance of dams in later life was not affected by the parturition in earlier age. The production efficiency with weaned youngs per pair during the first 200 days after mating was the highest in group A. It was found from these results that the C3H females attained their sexual maturity at 5 to 6 days after weaning, being available for breeding without any deletion in reproductive performance.     

Residency and abundance of sperm whales (Physeter macrocephalus) in Nemuro Strait,Hokkaido, Japan

We examined the trend in residence patterns and abundance of male sperm whales in Nemuro Strait, Japan, based on long-term photo-identification (1,513 survey days, total 2,969 photos) between 2006 and 2017. A total of 225 unique individuals were identified during this study, with an average of 36 (SE = 2.55) new individuals identified in each season. The model chosen by maximum likelihood suggests that residence time around Nemuro Strait is 769 (SE = 372.4) days, with individuals staying in the strait about 48 days (SE = 8.36) per year. While the migration patterns of male sperm whales visiting this area are still unclear, these findings along with previous studies suggest that males move from one breeding area to another neighboring area every several weeks, shifting their home ranges gradually over a period of a few years. The abundance of sperm whales in Nemuro Strait varied greatly from year to year; from 28 (95%CI: 24–44) in 2015 and (95%CI: 22–48) in 2016 to 66 (95%CI: 57–84) in 2011. This study provides important knowledge of abundance and residency for Nemuro Strait, information which will contribute to further research on the social structure and movement pattern of male sperm whales.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.     

Lectin-histochemical and -cytochemical study of periodic acid Schiff-positive lysosome granules as a histological feature of the female mouse kidney

Renal proximal straight tubules (PST) of the female mouse contain periodic acid Schiff-positive lysosome granules. An excellent example of this is found in the kidneys of female DBA/2Cr mice. In the present study, lectin-histochemistry showed that lectin-positive granules occur in the PST of DBA/2Cr mice. Out of twenty-one lectins studied, the granules bound WGA, s-WGA, LEL, STL, DSL, GSL-II, VVL, RCA-I, ECL, PSA, LCA and PHA-E. Such granules were also observed in the proximal convoluted tubules (PCT). In addition, heterogeneous binding to the SBA or DBA was observed in the PST. Lectin-cytochemistry for s-WGA, STL, VVL, RCA-I, ECL and PSA, showed that: 1) lysosomes bind a higher level of s-WGA or STL than VVL, RCA-I, ECL or PSA; 2) PSA binding is similar in PST and PCT; 3) there are many PCT lysosomes that are negative for s-WGA, STL, VVL, RCA-I, and ECL lectin binding; and 4) s-WGA binding is highly specific to the lysosomes of the PST. Based on the binding specificities of each lectin, it was suggested that the mannose content of PST and PCT lysosomes is similar, and that PST lysosomes have a high level of N-acetylglucosamine, N-acetylgalactosamine, galactose or galactosyl (beta 1, 4) N-acetylglucosamine.     

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

Lactobacillus casei L ‐lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6‐bisphosphate (FBP), respectively, and exhibits unusually high pH‐dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 Å resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q‐axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra‐helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L ‐lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P‐axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q‐axis related dimer through the rigid NAD‐binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH‐dependent allosteric equilibrium. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Convergent signaling in the regulation of connective tissue growth factor in malignant mesothelioma: TGFβ signaling and defects in the Hippo signaling cascade

Malignant mesothelioma (MM) is a neoplasm that arises from serosal surfaces of the pleural, peritoneal and pericardial cavities with worldwide incidence, much of which is caused by asbestos exposure. Patients suffer from pain and dyspnea due to direct invasion of the chest wall, lungs and vertebral or intercostal nerves by masses of thick fibrotic tumors. Although there has been recent progress in the clinical treatment, current therapeutic approaches do not provide satisfactory results. Therefore, development of a molecularly targeted therapy for MM is urgently required. Our recent studies suggest that normal mesothelial and MM cell growth is promoted by TGFβ, and that TGFβ signaling together with intrinsic disturbances in neurofibromatosis type 2 (NF2) and Hippo signaling cascades in MM cells converges upon further expression of connective tissue growth factor (CTGF). The formation of a YAP-TEAD4–Smad3-p300 complex on the specific CTGF promoter site with an adjacent TEAD and Smad binding motif is a critical and synergistic event caused by the dysregulation of these two distinct cascades. Furthermore, we demonstrated the functional importance of CTGF through the mouse studies and human histological analyses, which may elucidate the clinical features of MM with severe fibrosis in the thoracic cavity.     

Fine structure of the mandibular gland of the Djungarian hamster (Phodopus sungarus)

The mandibular gland of the Djungarian hamster was examined by light microscopy, and transmission and scanning electron microscopies. Its acinar cells reacted with periodic acid-Schiff (PAS) and were weakly stained with alcian blue (AB). There were intercellular canaliculi between the acinar cells. These cells therefore appeared to be seromucous. The acinar epithelium was composed of light cells containing various spherical secretory granules. The granular cells of the mandibular gland possessed many acidophilic granules exhibiting a positive reaction to PAS stain. They were frequently observed at the junction of the acini and intercalated ducts in all mandibular glands examined. All of these cells were light and contained secretory granules of varying size and density. The intercalated ducts consisted exclusively of light cells possessing a few round granules of high density in the apical region. The striated ducts were comprised of two portions--a secretory portion and a typical striated portion without secretory granules. The secretory portion consisted of light, dark and specifically light epithelial cells containing acidophilic granules, which exhibited a strongly positive PAS reaction. The epithelium of typically striated portions was composed of light and dark cells containing fine vacuoles in the apical region. The mandibular gland of the Djungarian hamster revealed no histological differences between sexes.     

Reversal of Salt Preference Is Directed by the Insulin/PI3K and Gq/PKC Signaling in Caenorhabditis elegans

Animals search for foods and decide their behaviors according to previous experience. Caenorhabditis elegans detects chemicals with a limited number of sensory neurons, allowing us to dissect roles of each neuron for innate and learned behaviors. C. elegans is attracted to salt after exposure to the salt (NaCl) with food. In contrast, it learns to avoid the salt after exposure to the salt without food. In salt-attraction behavior, it is known that the ASE taste sensory neurons (ASEL and ASER) play a major role. However, little is known about mechanisms for learned salt avoidance. Here, through dissecting contributions of ASE neurons for salt chemotaxis, we show that both ASEL and ASER generate salt chemotaxis plasticity. In ASER, we have previously shown that the insulin/PI 3-kinase signaling acts for starvation-induced salt chemotaxis plasticity. This study shows that the PI 3-kinase signaling promotes aversive drive of ASER but not of ASEL. Furthermore, the G_q signaling pathway composed of G_qα EGL-30, diacylglycerol, and nPKC (novel protein kinase C) TTX-4 promotes attractive drive of ASER but not of ASEL. A putative salt receptor GCY-22 guanylyl cyclase is required in ASER for both salt attraction and avoidance. Our results suggest that ASEL and ASER use distinct molecular mechanisms to regulate salt chemotaxis plasticity.ANIMALS show various behaviors in response to environmental cues and modulate behaviors according to previous experience. To understand neuronal plasticity underlying learning, it is important to dissect neurons and molecules for sensing environmental stimuli, storing memory, and executing learned behaviors.The nematode Caenorhabditis elegans has only 302 neurons and functions of sensory neurons are well characterized (White et al. 1986; Bargmann 2006). C. elegans is attracted to odorants sensed by the AWC olfactory neurons or to salts sensed by the ASE gustatory neurons (Bargmann and Horvitz 1991; Bargmann et al. 1993). The ASE neuron class consists of a bilaterally symmetrical pair, ASE-left (ASEL) and ASE-right (ASER), which sense different sets of ions including Na⁺ and Cl⁻, respectively (Pierce-Shimomura et al. 2001; Suzuki et al. 2008; Ortiz et al. 2009). ASEL is activated by an increase in salt concentration, whereas ASER is activated by a decrease in salt concentration (Suzuki et al. 2008). In the ASE gustatory neurons, a cyclic GMP (cGMP) signaling pathway mediates sensory transduction (Komatsu et al. 1996; Suzuki et al. 2008; Ortiz et al. 2009). ASEL and ASER express different sets of receptor-type guanylyl cyclases (gcys) (Ortiz et al. 2006). Of these, gcy-22, which is specifically expressed in ASER, is important for attraction to ASER-sensed ions such as Cl⁻ (Ortiz et al. 2009).Preference for salts changes according to previous experience (known as gustatory plasticity or salt chemotaxis learning) (Saeki et al. 2001; Jansen et al. 2002; Tomioka et al. 2006). When worms are grown on a medium that contains sodium chloride (NaCl) and food (Escherichia coli), they show attraction to NaCl by using ASE neurons (Bargmann and Horvitz 1991; Suzuki et al. 2008). In contrast, after exposure to the salt under starvation conditions, they show reduced attraction to or even avoid the salt (Saeki et al. 2001; Jansen et al. 2002; Tomioka et al. 2006). In C. elegans, it was proposed that preference for a sensory cue is defined by the sensory neuron that detects the cue (Troemel et al. 1997). ASE neurons play a major role for salt attraction (Bargmann and Horvitz 1991; Suzuki et al. 2008; Ortiz et al. 2009). However, little is known about sensory neurons that drive the learned salt avoidance; it remains unclear whether ASE neurons act as salt receptors for the learned avoidance.We have previously shown that an insulin/PI 3-kinase signaling pathway is essential for salt chemotaxis learning (Tomioka et al. 2006). In C. elegans, the insulin-like signaling is composed of daf-2, age-1, and akt-1, which encode homologs of insulin receptor, PI 3-kinase, and protein kinase B, respectively (Morris et al. 1996; Kimura et al. 1997; Paradis and Ruvkun 1998). Mutants of daf-2, age-1, and akt-1 show attraction to salt even after starvation/NaCl conditioning (Tomioka et al. 2006).daf-18 encodes a homolog of phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome ten), which dephosphorylates phosphatidylinositol (3,4,5)-triphosphate and counteracts the insulin/PI 3-kinase signaling (Ogg and Ruvkun 1998; Gil et al. 1999; Mihaylova et al. 1999; Rouault et al. 1999; Solari et al. 2005). Mutants of daf-18, in which the PI 3-kinase signaling is activated, show reduced attraction to NaCl even without conditioning. Since the insulin/PI 3-kinase signaling acts in ASER, we proposed that the insulin/PI 3-kinase signaling attenuates the attractive drive of ASER (Tomioka et al. 2006).In C. elegans, diacylglycerol (DAG) regulates functions of motor neurons and sensory neurons. egl-30, which encodes the α-subunit of heterotrimeric G-protein G_q, facilitates production of DAG and enhances locomotory movements (Brundage et al. 1996; Lackner et al. 1999). In the AWC olfactory neurons, a novel protein kinase C-ɛ/η (nPKC-ɛ/η) ortholog TTX-4 (also known as PKC-1), which is one of DAG targets, plays an essential role in attraction behavior to AWC-sensed odors (Okochi et al. 2005; Tsunozaki et al. 2008). GOA-1 G_oα regulates olfactory adaptation by antagonizing G_qα–DAG signaling (Matsuki et al. 2006).This study investigated the involvement of the ASE taste receptor neurons in the starvation-induced salt avoidance. We show that both ASEL and ASER contribute to salt chemotaxis learning. Activation of the PI 3-kinase signaling and the G_q/DAG/PKC signaling acted antagonistically in reversal of ASER function, whereas these signaling pathways did not have prominent effects on ASEL function. In ASER, GCY-22 was required for both salt attraction and avoidance. These results suggest that ASE neurons are important for bidirectional chemotaxis and also suggest that distinct molecular mechanisms regulate functions of ASEL and ASER in salt chemotaxis learning.     

Recognition site for the side chain of 2-ketoacid substrate in d-lactate dehydrogenase

Replacement of Tyr52 with Val or Ala in Lactobacillus pentosus d-lactate dehydrogenase induced high activity and preference for large aliphatic 2-ketoacids and phenylpyruvate. On the other hand, replacements with Arg, Thr or Asp severely reduced the enzyme activity, and the Tyr52Arg enzyme, the only one that exhibited significant enzyme activity, showed a similar substrate preference to the Tyr52Val and Tyr52Ala enzymes. Replacement of Phe299 with Gly or Ser greatly reduced the enzyme activity with less marked change in the substrate preference. Except for the Phe299Ser enzyme, these mutant enzymes with low catalytic activity consistently stimulated NADH oxidation in the absence of 2-ketoacid substrates. However, the double mutant enzymes, Tyr52Arg/Phe299Gly and Tyr52Thr/Phe299Ser, did not exhibit synergically decreased enzyme activity or the substrate-independent NADH oxidation, but rather increased activities toward certain 2-ketoacid substrates. These results indicate that the coordinative combination of amino acid residues at two positions is pivotal in both the functional recognition of the 2-ketoacid side chain and the protection of the bound NADH molecule from the solvent. Multiplicity in such combinations appears to provide d-LDH-related 2-hydroxyacid dehydrogenases with a great variety of catalytic and physiological functions.