Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei

Cre-responsive dual-fluorescent alleles allow in situ marking of cell lineages or genetically modified cells. Here we report a dual-fluorescent allele, ROSA ^nT-nG , which directs nuclear accumulation of tdTomato in Cre-naïve lineages. Cre converts the allele to ROSA ^nG , which drives nuclear EGFP accumulation. Conditions were established for analyzing marked nuclei by flow cytometry on the basis of red–green fluorescence and ploidy, with a particular focus on liver nuclei. Hydrodynamic delivery of a Cre-expression plasmid was used to time-stamp arbitrary hepatocytes for lineage tracing. The distinct green fluorescence of nuclei from Cre-exposed lineages facilitated analyses of ploidy transitions within clones. To assess developmental transitions in liver nuclei, ROSA ^nT-nG was combined with the hepatocyte-specific AlbCre transgene, facilitating discrimination between hepatocyte and nonhepatocyte nuclei. Nuclei extracted from postnatal day 2 (P2) livers were 41 % green and 59 % red and reached a stable level of 84 % green by P22. Until P20, green nuclei were >98 % diploid (2N); at P40 they were ~56 % 2N, 43 % 4N, and <1 % 8N; and by P70 they reached a stable distribution of ~46 % 2N, 45 % 4N, and 9 % 8N. In conclusion, ROSA ^nT-nG will facilitate in vivo and ex vivo studies on liver and will likely be valuable for studies on tissues like muscle, kidney, or brain in which cells are refractory to whole-cell flow cytometry, or like trophectoderm derivatives or cancers in which cells undergo ploidy transitions.     

Diffusional mobility of parvalbumin in spiny dendrites of cerebellar Purkinje neurons quantified by fluorescence recovery after photobleaching

Ca(2+)-binding proteins (CaBPs) represent key factors for the modulation of cellular Ca(2+) dynamics. Especially in thin extensions of nerve cells, Ca(2+) binding and buffered diffusion of Ca(2+) by CaBPs is assumed to effectively control the spatio-temporal extend of Ca(2+) signals. However, no quantitative data about the mobility of specific CaBPs in the neuronal cytosol are available. We quantified the diffusion of the endogenous CaPB parvalbumin (PV) in spiny dendrites of cerebellar Purkinje neurons with two-photon fluorescence recovery after photobleaching. Fluorescently labeled PV diffused readily between spines and dendrites with a median time constant of 49 ms (37-61 ms, interquartile range). Based on published data on spine geometry, this value corresponds to an apparent diffusion coefficient of 43 microm(2) s(-1) (34-56 microm(2) s(-1)). The absence of large or immobile binding partners for PV was confirmed in PV null-mutant mice. Our data validate the common but so far unproven assumption that PV is highly mobile in neurons and will facilitate simulations of neuronal Ca(2+) buffering. Our experimental approach represents a versatile tool for quantifying the mobility of proteins in neuronal dendrites.     

1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

Beobachtungen zur Lebensweise von Pycnogonum litorale (Ström) (Pantopoda)

Zusammenfassung Am Leitdamm des Jadebusens lebt Pycnogonum litorale im Lückensystem des Miesmuschelbesatzes. Dieser bietet mit hartem Untergrund, hoher Feuchtigkeit bei Niedrigwasser, genügend Actinien als Nahrung und guter Durchströmung bei gleichzeitigem Schutz vor Vertragung—offensichtlich günstige Lebensbedingungen für Pycnogonum litorale.Der Eiablage im Februar geht eine Reiterstellung des Männchens auf dem Weibchen von durchschnittlich 24 Tagen voraus. Unter künstlichen Kurztagbedingungen kann diese Reiterstellung auch außerhalb der Fortpflanzungsperiode eingenommen werden. Die Eier werden durch Rumpfbewegungen beider Partner zu den Ovigeren des Männchens bewegt. Bei 12°C schlüpfen die Larven etwa 41 bis 46 Tage nach der Eiablage aus, bei 19°C, im Sommer, schlüpften keine Larven.Im Jadebusen leben die Larven etwa 1/2 Jahn endoparasitisch in Hydrozoen. Die an die Metamorphose anschließende juvenile Phase, in der die Tiere frei leben, dauert ein knappes Jahr, die Reifehäutung erfolgt normalerweise im Sommer des zweiten Jahres, die Fortpflanzungsperiode etwa 6 Monate später, im Winter.

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Observations on the life biology of Pycnogonum litorale (Ström) (Pantopoda)

Summary Pycnogonum litorale lives in an interstitial system, of the mussel zone on the embankment of the Jadebusen. Hard substrate, high humidity at low tide, sufficient Metridium senile as food, and active currents together with protection from drifting, constitute favourable conditions for this pycnogonid.Prior to laying egg in February, the male remains in a riding position upon the female for approximately 24 days. Under artificial short-day conditions the riding position may also be assumed outside of the reproductive period. The eggs are transported to the ovigers of the male by trunk movements of both partners. At 12°C the larvae hatch about 41–46 days after egg-laying. No larvae hatched from eggs laid during summer at 19°C.The larvae live endoparasitically in Hydrozoa for about 1/2 year. Following metamorphosis, the freeliving juvenile phase lasts barely a year. The maturation moult normally takes place in the summer of the second year, the reproductive period beginning about 6 months later, in winter.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

O(3)-(2-Acetylamino-2-deoxy-β-d-glucopyranosyl)-oleanolic acid,a novel triterpenoid glycoside from two Pithecellobium species

A new triterpenoid glycoside containing an amino sugar moiety has been isolated from Pithecellobium cubense and P. arboreum and identified as O(3)- (2-acetylamino-2-deoxy-β-d-glucopyranosyl)oleanolic acid. β-d-Glucopyranosyl-α-spinasterol was also obtained from P. cubense.     

Structure-function relationship studies of PTH(1-11) analogues containing sterically hindered dipeptide mimetics.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and reproduces all biological responses characteristic of the native intact PTH. In order to develop safer and non-parenteral PTH-like bone anabolic agents, we have studied the effect of introducing conformationally constrained dipeptide mimetics into the N-terminal portion of PTH in an effort to generate miniaturized PTH-mimetics. To this end, we have synthesized and conformationally and biologically characterized PTH(1-11) analogues containing 3R-carboxy-6S-amino-7,5-bicyclic thiazolidinlactam (7,5-bTL), a rigidified dipeptide mimetic unit. The wild type sequence of PTH(1-11) is H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-NH(2). The following pseudo-undecapeptides were prepared: [Ala(1), 7,5-bTL(3, 4), Nle(8), Arg(11)]hPTH(1-11)NH(2) (I); [Ala(1), 7,5-bTL(6, 7), Nle(8), Arg(11)]hPTH(1-11)NH(2) (II); [Ala(1), Nle(8), 7,5-bTL(9, 10), Arg(11)]hPTH(1-11)NH(2) (III). In aqueous solution containing 20% TFE, only analogue I exhibited the typical CD pattern of the alpha-helical conformation. NMR experiments and molecular dynamics calculations located the alpha-helical stretch in the sequence Ile(5)-His(9). The dipeptide mimetic unit 7,5-bTL induces a type III beta-turn, occupying the positions i - 1 and i of the turn. Analogue II exhibited an equilibrium between a type I beta-turn and an alpha-helix, and analogue III did not show any ordered structure. Biological tests revealed poor activity for all analogues (EC(50) > 0.1 mM). Apparently, the relative side-chain orientation of Val(2), Ile(5) and Met(8) can be critical for effective analogue-receptor interaction. Considering helicity as an essential property to obtain active PTH agonists, one must decorate the correctly positioned dipeptide mimetic azabicycloalkane scaffold with substitutions corresponding to the displaced amino acids.     

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na⁺/H⁺ ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via α_v integrin/CD46 in HeLa cells.Human rhinoviruses (HRVs), members of the family Picornaviridae that represent a major cause of the common cold, essentially utilize two different receptor types for host cell attachment. The 12 minor group HRVs, exemplified by HRV2, bind low-density lipoprotein receptor (LDLR), LDLR-related protein (LRP) (20), and very-LDLR (VLDLR) (29) and are internalized via the well-characterized clathrin-dependent endocytic pathway (44); however, these ligands, like others, can switch to different entry portals when the clathrin-dependent pathway is blocked (4). Once the virus arrives in endosomal carrier vesicles or late endosomes, uncoating (i.e., the release of the viral RNA genome) is triggered by the acidic pH (35, 39).The 87 major group HRVs, exemplified by HRV14, bind intercellular adhesion molecule-1 (ICAM-1). Following entry, uncoating is triggered by ICAM-1 itself (3), but the low endosomal pH facilitates this process (37). Based on inhibition of infection by the dominant negative (DN) dynamin-2 mutant dyn^K44A, it was proposed that HRV14 also follows a clathrin-dependent pathway in HeLa-H1 cells (9). However, ICAM-1 lacks a clathrin localization signal and even functions as a viral receptor when its cytoplasmic tail is replaced with a glycosylphosphatidylinositol (GPI) anchor (45). Furthermore, dynamin has also been shown to be involved in pathways other than clathrin-mediated endocytosis (CME), such as caveolae- and lipid raft-dependent entry, as a function of ligand and cell type (reviewed in references 30 and 34). Additionally, dynamin might play a role in formation and closure of circular pinocytic ruffles (31). More recently, a specific entry pathway for ICAM-1 ligands into human umbilical vein endothelial cells was identified and termed “cam-mediated endocytosis”; uptake was found to be triggered upon binding of multivalent ligands, such as immunoconjugates and immunobeads, and to occur independently from clathrin and caveolin. Inhibition by amiloride, actin depolymerization, and protein kinase C inhibitors pointed to macropinocytosis (33). So far, it is not known whether these findings are relevant to the entry pathway of HRVs via ICAM-1 as the uptake kinetics was significantly dependent on particle size. For all these reasons, involvement of clathrin in HRV14 uptake is questionable. Accordingly, we explored entry of HRV14 via ICAM-1 and compared the results with the well-characterized clathrin-dependent entry pathway of HRV2 (44). Employing pharmacological compounds, specific DN inhibitors, immunofluorescence, and electron microscopy, we demonstrate that HRV14 enters rhabdomyosarcoma ICAM-1-expressing (RD-ICAM) cells via a pathway independent of clathrin, caveolin, and flotillin.     

Response properties of thick myelinated group II afferents in the medial articular nerve of normal and inflamed knee joints of the cat.

This study investigated the responses to innocuous and noxious mechanical stimuli of joint mechanoreceptors with thick myelinated articular afferents (conduction velocities 21-65 m/sec) in the medial articular nerve of the cat's knee. In nine experiments, we examined whether acute arthritis would modify the discharge properties. The vast majority of the group II afferents were excited by gentle local stimuli and by movements in the working range of the knee. Although they encoded pressure and particular movement stimuli up to the noxious range, their responses were more closely related to the particular type of stimulus (e.g., a movement in a specific direction) than to its intensity (innocuous vs. noxious). In inflamed joints, the response patterns of group II units were similar with regard to local mechanical thresholds, thresholds for passive movements, and patterns of responses to passive movements. In both situations, most units had no resting activity. These results suggest that articular group II afferents do not play a significant role in nociception. Rather, they subserve proprioceptive functions such as deep pressure sensation and kinesthesia in normal as well as inflamed joints.