Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems. We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP. The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.     

Change in growth kinetics of hybridoma cells entrapped in collagen gel affected by alkaline supply

The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to ajust the pH level when using medium-circulating-fermenters.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli for glutamate 1-semialdehyde (GSA) aminomutase, which is involved in the C5 pathway for synthesis of -aminolevulinic acid (ALA), was cloned onto a multicopy plasmid, pUC18, by the complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of deletion fragments from the initial 3.5-kb chromosomal fragment allowed the isolation of a 1.7-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.7-kb DNA fragment revealed an open reading frame (ORF) that is located downstream from a potential promoter sequence and a ribosome-binding site. The ORF encodes a polypeptide of 429 amino acid residues, and the deduced molecular mass of this polypeptide is 45,043 Da. The amino acid sequence shows a high degree of homology to the HemL proteins from other organisms, and a putative binding site for pyridoxal 5-phosphate is conserved. Correspondence to: Y. Murooka     

Regional changes in muscle mass and strength following 20 days of bed rest, and the effects on orthostatic tolerance capacity in young subjects

To this day, many studies have suggested that prolonged bed rest (BR) affects on muscle mass and strength not only in gravity muscles but also in ungravity muscles. However, it is still unclear whether the decrease in regional muscle strength after BR is due to the alterations in the corresponding muscle mass, or not. On the other hand, if BR decreases the mass of antigravity muscles (UGM) as well as muscle strength and then increases tissue compliance of the antigravity muscles, orthostatic tolerance capacity will be decreased by the reduction in cardiac output (CO) in spite of the increase in myocardial contractility because the more decrease in venous return due to the more increase in blood pooling within the compliant tissues of the lower body. However, this is also unclear. To make these questions clear, the present study investigated the regional muscle mass and strength and orthostatic tolerance capacity before and after 20 days of bed rest in young subjects.     

Sporocarp ontogeny in Panus (Basidiomycotina): evolution and classification

Ontogenies of cultured Panus conchatus, P. rudis, and P. fulvus sporocarps were observed macroscopically and with scanning electron microscopy. Hymenophore differentiation in Panus involves periclinal growth of context hyphae below a closed surface palisade of hymenial elements, resulting in a cantharelloid appearance and radiate trama. This pattern is qualitatively different from that in Lentinus s. str., which suggests that lamellae of Panus and Lentinus are not homologous. Panus conchatus and P. rudis sporocarps have short stipes, develop directly from the mycelium, and mature in 5–10 d. Panus fulvus sporocarps have an elongate stipe, develop from a pseudosclerotium, and mature in about 3 wk, the first approximately 15 d of which involve apical elongation of a stipelike primordium that is able to dedifferentiate and regenerate cut apices. Panus conchatus and P. rudis sporocarps lacked regeneration ability. Panus conchatus sporocarps developed an ephemeral partial veil that was obliterated during sporocarp expansion. Outgroup comparison suggests that evolutionary changes in developmental programs in Panus have included: 1) delay in offset of primordium growth, with a corresponding increase in primordium size and time to maturation (hypermorphosis); 2) insertion of the pseudosclerotial stage in ontogeny; 3) gain of ability for dedifferentiation and regeneration; and 4) nonterminal gain or loss of veil tissue.     

Carbon uptake in Eurasian boreal forests dominates the high-latitude net ecosystem carbon budget

Arctic-boreal landscapes are experiencing profound warming, along with changes in ecosystem moisture status and disturbance from fire. This region is of global importance in terms of carbon feedbacks to climate, yet the sign (sink or source) and magnitude of the Arctic-boreal carbon budget within recent years remains highly uncertain. Here, we provide new estimates of recent (2003–2015) vegetation gross primary productivity (GPP), ecosystem respiration (R_eco), net ecosystem CO₂ exchange (NEE; R_eco − GPP), and terrestrial methane (CH₄) emissions for the Arctic-boreal zone using a satellite data-driven process-model for northern ecosystems (TCFM-Arctic), calibrated and evaluated using measurements from >60 tower eddy covariance (EC) sites. We used TCFM-Arctic to obtain daily 1-km² flux estimates and annual carbon budgets for the pan-Arctic-boreal region. Across the domain, the model indicated an overall average NEE sink of −850 Tg CO₂-C year⁻¹. Eurasian boreal zones, especially those in Siberia, contributed to a majority of the net sink. In contrast, the tundra biome was relatively carbon neutral (ranging from small sink to source). Regional CH₄ emissions from tundra and boreal wetlands (not accounting for aquatic CH₄) were estimated at 35 Tg CH₄-C year⁻¹. Accounting for additional emissions from open water aquatic bodies and from fire, using available estimates from the literature, reduced the total regional NEE sink by 21% and shifted many far northern tundra landscapes, and some boreal forests, to a net carbon source. This assessment, based on in situ observations and models, improves our understanding of the high-latitude carbon status and also indicates a continued need for integrated site-to-regional assessments to monitor the vulnerability of these ecosystems to climate change.     

Genomic Analysis of Borrelia japonica Sp. Nov. Isolated from Ixodes ovatus in Japan

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.     

Evolution of the therian face through complete loss of the premaxilla

The anatomical framework of the jawbones is highly conserved among most of the Osteichthyes, including the tetrapods. However, our recent study suggested that the premaxilla, the rostralmost upper jaw bone, was rearranged during the evolution of therian mammals, being replaced by the septomaxilla at least in the lateral part. In the present study, to understand more about the process of evolution from the ancestral upper jaw to the therian face, we re-examined the development of the therian premaxilla (incisive bone). By comparing mouse, bat, goat, and cattle fetuses, we confirmed that the therian premaxilla has dual developmental origins, the lateral body and the palatine process. This dual development is widely conserved among the therian mammals. Cell-lineage-tracing experiments using Dlx1-CreER^T2 mice revealed that the palatine process arises in the ventral part of the premandibular domain, where the nasopalatine nerve distributes, whereas the lateral body develops from the maxillary prominence in the domain of the maxillary nerve. Through comparative analysis using various tetrapods, we concluded that the palatine process should not be considered part of the ancestral premaxilla. It rather corresponds to the anterior region of the vomerine bone of nonmammalian tetrapods. Thus, the present findings indicate that the true premaxilla was completely lost during the evolution of the therian mammals, resulting in the establishment of the unique therian face as an evolutionary novelty. Reconsideration of the homological framework of the cranial skeleton based on the topographical relationships of the ossification center during embryonic development is warranted.