Generation and Characterization of Ins1-cre-driver C57BL/6N for Exclusive Pancreatic Beta Cell-specific Cre-loxP Recombination

Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic β cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F₁ mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F₁ mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F₁ fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F₁ mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic β cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.     

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules. The module approach generates uniform, scalable and vascularized tissues. The modules can be made of collagen as well as other gelable or crosslinkable materials. They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells. The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable. These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues. There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.Download video file.^{(58M, mov)}     

Adult mouse hematopoietic stem cells: purification and single-cell assays

Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34-KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.     

A Potential Yeast Actin Allosteric Conduit Dependent on Hydrophobic Core Residues Val-76 and Trp-79

Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76–79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation.     

Influence of the feature space on the estimation of hand grasping force from intramuscular EMG

The study compares the performance of different combinations of nine features extracted from intramuscular electromyogram (EMG) recordings for the estimation of grasping force within the range 0–100% maximum voluntary contraction (MVC). Single-channel intramuscular EMG was recorded from the flexor digitorum profundus (FDP) muscle from 11 subjects who exerted three force profiles during power grasping. The ability of the features to estimate force with a 1st order polynomial (poly1) and an artificial neural network (ANN) model was assessed using the adjusted coefficient of determination (R²). Willison amplitude (WAMP) and root mean square (RMS) showed the highest R² (～0.88) values for poly1. The performance of all the features to predict force significantly increased (P < 0.01) when an ANN was applied. In this case, the Modified Mean Absolute Value (MMAV) demonstrated the best performance (～0.91). The results showed that a single channel intramuscular EMG recording represents the entire grasping force range (0–100% MVC) measured from the FDP muscle. The association between EMG and force depends on the features extracted and on the model.     

Effect of green tea powder (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L. cv. Benifuuki) particle size on <Emphasis Type="Italic">O</Emphasis>-methylated EGCG absorption in rats; The Kakegawa Study

Tea polyphenols, e.g., (-)-epigallocatechin-3-O-(3-O-methyl gallate (EGCG3”Me), (-)-epigallocatechin-3-O-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-O-gallate (ECG), and (-)-epicatechin (EC), are believed to be responsible for the beneficial effects of tea. ‘Benifuuki’, a tea (Camellia sinensis L.) cultivar grown in Japan, is rich in the anti-allergic molecule epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3”Me). Pulverized Benifuuki green tea powder (BGP) is more widely distributed than leaf tea in Japan. Japanese people mix their pulverized tea with water directly, whereas it is common to drink leaf tea after extraction. However, few studies of the effects of BGP particle size on polyphenol bioavailability have been performed. This study was conducted to investigate the absorption of catechins in rats after the intragastric administration of Benifuuki green tea. Therefore, we assessed the plasma concentrations of catechins following the ingestion of BGP with different mean particle sizes (2.86, 18.6, and 76.1 μm) or Benifuuki green tea infusion (BGI) as a control in rats. The bioavailabilities of EGCG3”Me, EGCG, ECG, EGC, and EC were analyzed after the oral administration of a single dose of Benifuuki green tea (125 mg/rat) to rats. The plasma concentrations of tea catechins were determined by HPLC analysis combined with of electrochemical detection (ECD) using a coulometric array. The AUC (area under the drug concentration versus time curve; min μg/mL) of ester-type catechins (EGCG3”Me, EGCG, and ECG) for the BGP 2.86 μm were significantly higher than those in the infusion and 18.6 and 76.1 μm BGP groups, but the AUC of free-type catechins (EGC and EC) showed no differences between these groups. Regarding the peak plasma level of EGCG3”Me adjusted for intake, BGP 2.86 μm and BGI showed higher values than the BGP 18.6 and 76.1 μm groups, and the peak plasma levels of the other catechins displayed the same tendency. The present study demonstrates that the bioavailability of ester-type catechins (EGCG and ECG) can be improved by reducing the particle size of green tea, but the plasma level of EGCG3”Me in the BGI group was similar to that in the BGP 2.86 μm group. This result suggests that drinking Benifuuki green tea with a particle size of around 2 μm would deliver the anti-allergic EGCG3”Me and the anti-oxidant EGCG efficiently.     

The naturally trans-acting ribozyme RNase P RNA has leadzyme properties

Divalent metal ions promote hydrolysis of RNA backbones generating 5′OH and 2′;3′P as cleavage products. In these reactions, the neighboring 2′OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb²⁺ results in 5′ phosphate and 3′OH as cleavage products. Based on our findings, we infer that Pb²⁺ activates H₂O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb²⁺ at the cleavage site. Our data suggest that Pb²⁺ can promote cleavage of RNA by activating either an inner sphere H₂O or a neighboring 2′OH to act as nucleophile.     

Phylogeny and classification of phylum Cercozoa (Protozoa)

The protozoan phylum Cercozoa embraces numerous ancestrally biciliate zooflagellates, euglyphid and other filose testate amoebae, chlorarachnean algae, phytomyxean plant parasites (e.g. Plasmodiophora, Phagomyxa), the animal-parasitic Ascetosporea, and Gromia. We report 18S rRNA sequences of 27 culturable zooflagellates, many previously of unknown taxonomic position. Phylogenetic analysis shows that all belong to Cercozoa. We revise cercozoan classification in the light of our analysis and ultrastructure, adopting two subphyla: Filosa subphyl. nov. a clade comprising Monadofilosa and Reticulofilosa, ranked as superclasses, ancestrally having the same very rare base-pair substitution as all opisthokonts; and subphylum Endomyxa emend. comprising classes Phytomyxea (Plasmodiophorida, Phagomyxida), Ascetosporea (Haplosporidia, Paramyxida, Claustrosporida ord. nov.) and Gromiidea cl. nov., which did not. Monadofilosa comprise Sarcomonadea, zooflagellates with a propensity to glide on their posterior cilium and/or generate filopodia (e.g. Metopion;Cercomonas; Heteromitidae – Heteromita, Bodomorpha, Proleptomonas and Allantion) and two new classes: Imbricatea (with silica scales: Euglyphida; Thaumatomonadida, including Allas, Thaumatomastix) and Thecofilosea (Cryomonadida; Tectofilosida ord. nov. – non-scaly filose amoebae, e.g. Pseudodifflugia). Reticulofilosa comprise classes Chlorarachnea, Spongomonadea and Proteomyxidea (e.g. Massisteria, Gymnophrys, a Dimorpha-like protozoan). Cercozoa, now with nine classes and 17 orders (four new), will probably include many, possibly most, other filose and reticulose amoebae and zooflagellates not yet assigned to phyla.     

The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)

Mantamonasis a novel genus of marine gliding zooflagellates probably related to apusomonad and planomonad Apusozoa. Using phase and differential interference contrast microscopy we describe the type species Mantamonas plasticasp. n. from coastal sediment in Cumbria, England. Cells are ～5μm long, ～5μm wide, asymmetric, flattened, biciliate, and somewhat plastic. The posterior cilium, on which they glide smoothly over the substratum, is long and highly acronematic. The much thinner, shorter, and almost immobile anterior cilium points forward to the cell's left. These morphological and behavioural traits suggest thatMantamonasis a member of the protozoan phylum Apusozoa. Analyses of 18S and 28S rRNA gene sequences of Mantamonas plasticaand a second genetically very different marine species from coastal sediment in Tanzania show Mantamonasas a robustly monophyletic clade, that is very divergent from all other eukaryotes. 18S rRNA trees mostly placeMantamonaswithin unikonts (opisthokonts, Apusozoa, and Amoebozoa) but its precise position varies with phylogenetic algorithm and/or taxon and nucleotide position sampling; it may group equally weakly as sister to Planomonadida, Apusomonadida or Breviata. On 28S rRNA and joint 18/28S rRNA phylogenies (including 11 other newly obtained apusozoan/amoebozoan 28S rRNA sequences) it consistently strongly groups with Apusomonadida (Apusozoa).     

New genera, species, and improved phylogeny of Glissomonadida (Cercozoa)

Glissomonadida is an important cercozoan order of predominantly biflagellate gliding bacterivores found largely in soil and freshwater. Their vast diversity is largely undescribed. We studied 23 mostly newly isolated strains by light microscopy and sequenced their 18S rDNA genes; nine represent new species. For two misidentified ATCC 'Heteromita triangularis' strains, we establish novel gliding genera and species: the sandonid Mollimonas lacrima, the only glissomonad forming anterior and posterior pseudopodia, and Dujardina stenomorpha, a strongly flattened member of the new family Dujardinidae. A new strain from Oxfordshire grassland soil is the first reliably identified isolate of the virtually uniflagellate, smooth-gliding glissomonad genus, AllantionSandon, 1924. Phylogenetic analysis and cytological features reveal Allantion to be a member of Allapsidae. Sandona limna and Bodomorpha prolixa from Lake Baikal and Sandona hexamutans from volcanic Costa Rican soil are described as new species. Fifteen glissomonad strains were from grassland beside Lake Baikal. We describe two as new species of Sandona (S. heptamutans and S. octamutans); the others included strains of Sandona and Allapsa species that have already been described; and three were new species of Sandona and Allapsa but these died before being described. We discuss the ecological and evolutionary significance of these new strains.