Distribution of hippocampal cholinergic neurostimulating peptide (HCNP)-like immunoreactivity in organs and tissues of young Wistar rats

This report concerns the distribution of the hippocampal cholinergic neurostimulating peptide (HCNP) in tissues and organs of 11-day-old Wistar rats. HCNP, originally isolated and purified from the hippocampus of young rats, is an undecapeptide (acetyl-Ala-Ala-Asp-Ile-Ser-Gln-Trp-Ala-Gly-Pro-Leu). HCNP distribution was investigated by using immunohistochemical techniques, employing an affinity-purified rabbit antibody that specifically recognizes HCNP and its 21-kDa precursor protein. Positively stained cells were detected in a variety of tissues and organs, including salivary gland, small intestine, colon, pancreas, bronchiole, adrenal gland, testis, as well as several others. The nerve fibres around blood vessels of almost all organs expressed HCNP. Our results suggest that HCNP or its precursor, or both, may have a specific function not only in the central nervous system, but also in the peripheral nervous system, and possibly in certain specialized duct and gland cells as well.     

Natural revegetation potential of Japanese wild thyme (<Emphasis Type="Italic">Thymus quinquecostatus</Emphasis> Celak.) on serpentine quarries

Quarrying is an important industry that supports human infrastructure, but it has negative effects on human health, environments, and plant communities. In the case of the serpentine quarry on Sugashima Island (Sugashima), Japan, rapid remediation using native plants is necessary to restore the serpentine ecosystem. According to our previous work, we revealed high adaptability of Japanese wild thyme (Thymus quinquecostatus Celak.) to the severe environment of serpentine areas, and we desired to utilize this species for revegetation. However, basic information on Japanese thyme was extremely limited, in comparison with well-investigated European thymes that have been used for revegetation experiments. The aims of this work were to identify the plant functional traits of wild thymes collected from different areas of Japan and to test their potential for revegetation of serpentine quarry soil. We collected four types of wild thyme from Sugashima and other areas of serpentine, calcareous, and tuff breccia soil, and revealed varietal differences in leaf form, branching patterns, flowers, and also blooming season, seed formation ability, and seed germination percentages. Thymes from serpentine and calcareous areas could grow on soil of the serpentine quarry, and a small number of plants were able to cover the experimental plots. Among the Japanese thymes investigated, Sugashima thyme had the longest branches, and spread rapidly by forming many branches. These data suggest that Japanese thymes, especially Sugashima thyme, have good potential for revegetation of serpentine quarries.     

Physiological roles of free D- and L-alanine in the crayfish Procambarus clarkii with special reference to osmotic and anoxic stress responses

Under hyper-salinity stress from freshwater to 17 and 25 ppt seawater, red swamp crayfish Procambarus clarkii largely accumulated D- and L-alanine together with glycine, L-glutamine, and L-proline in both muscle and hepatopancreas. The increases of D- and L-alanine in muscle were the highest in all amino acids and reached 6.8- and 5.4-fold, respectively, from freshwater to 25 ppt seawater. These results indicate that both D- and L-alanine are the most potent osmolytes for intracellular isosmotic regulation in crayfish as well as other crustaceans thus far examined. Under anoxia stress below 0.1 mg/l dissolved oxygen for 12 h and subsequent recovery in normoxia for 12 h in freshwater, 17 and 25 ppt seawater, muscle ATP decreased dramatically in all salinity levels and almost depleted in seawater. Along with the decrease of muscle glycogen level, the significant increase of L-lactate was found in muscle, hepatopancreas, and hemolymph for each salinity level, suggesting the transport of L-lactate from muscle into hepatopancreas via hemolymph. Under anoxia, D- and L-alanine also largely increased in both muscle and hepatopancreas for each salinity level. The increase was much higher in seawater than in freshwater. Thus, both D- and L-alanine are possible to be anaerobic end products during prolonged anaerobiosis of this species.     

Feeding habits may explain the morphological uniqueness of brown bears on Etorofu Island,Southern Kuril Islands in East Asia

The brown bear (Ursus arctos) population on Etorofu Island, Southern Kuril Islands, has several unique morphological features: (1) the presence of white‐pelage bears within the population and (2) a larger body size than bears on a larger neighbour island, Hokkaido Island. Nevertheless, little ecological information is available about Etorofu brown bears. In the present study, we reveal the unique feeding habits of Etorofu brown bears and suggest that their unique morphological features and diet are related. The feeding habits of brown bears on Etorofu Island were assessed using carbon, nitrogen, and sulfur stable isotope analysis, and their feeding habits were compared with those of bears on the eastern side of Hokkaido Island. According to the stable isotope analysis, the dependence on salmon is great for bears on Etorofu but only slight for bears on Hokkaido. Our results suggest that the feeding habits of Etorofu brown bears may explain their unique morphological features because a white pelage colour confers an advantage when catching salmon, and a carnivorous diet can make their body size larger. The variation in feeding habits can be an important driver of the speciation and evolution of animals.     

Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5’-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5’-monophosphate (GMP) to inosine 5’-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine β-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K⁺ and NH₄⁺ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR’s was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.     

Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoa

Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa.     

Free oligosaccharides in the cytosol of Caenorhabditis elegans are generated through endoplasmic reticulum-golgi trafficking

Free oligosaccharides (FOSs) in the cytosol of eukaryotic cells are mainly generated during endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded glycoproteins. We analyzed FOS of the nematode Caenorhabditis elegans to elucidate its detailed degradation pathway. The major FOSs were high mannose-type ones bearing 3-9 Man residues. About 94% of the total FOSs had one GlcNAc at their reducing end (FOS-GN1), and the remaining 6% had two GlcNAc (FOS-GN2). A cytosolic endo-beta-N-acetylglucosaminidase mutant (tm1208) accumulated FOS-GN2, indicating involvement of the enzyme in conversion of FOS-GN2 into FOS-GN1. The most abundant FOS in the wild type was Man(5)GlcNAc(1), the M5A' isomer (Manalpha1-3(Manalpha1-6)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAc), which is different from the corresponding M5B' (Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAc) in mammals. Analyses of FOS in worms treated with Golgi alpha-mannosidase I inhibitors revealed decreases in Man(5)GlcNAc(1) and increases in Man(7)GlcNAc(1). These results suggested that Golgi alpha-mannosidase I-like enzyme is involved in the production of Man(5-6)-GlcNAc(1), which is unlike in mammals, in which cytosolic alpha-mannosidase is involved. Thus, we assumed that major FOSs in C. elegans were generated through Golgi trafficking. Analysis of FOSs from a Golgi alpha-mannosidase II mutant (tm1078) supported this idea, because GlcNAc(1)Man(5)GlcNAc(1), which is formed by the Golgi-resident GlcNAc-transferase I, was found as a FOS in the mutant. We concluded that significant amounts of misfolded glycoproteins in C. elegans are trafficked to the Golgi and are directly or indirectly retro-translocated into the cytosol to be degraded.