Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca²⁺ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca²⁺_o. Thirty minutes preincubation with bumetanide, a potent Na⁺,K⁺,Cl^– cotransport (NKCC) inhibitor, decreased Cl^–_i content, nifedipine-sensitive ⁴⁵Ca uptake and contractions triggered by modest depolarization ([K⁺]_o=36 mM). Elevation of [K⁺]_o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl^–_i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.     

Characterisation of bioactive and resorbable polylactide/Bioglass composites by FTIR spectroscopic imaging

Formation, size and distribution of hydroxyapatite domains in resorbable composites made of poly(DL-lactide) foams and Bioglass particles after exposure to a solution of phosphate-buffer saline (PBS) for different periods of time have been analysed with FTIR imaging using the micro-ATR-IR approach. The spectral information of 4096 spectra measured simultaneously with the IR microscope equipped with a focal plane infrared array detector allowed us to obtain chemical images showing the distribution of Bioglass particles in the composites. FTIR imaging in micro-ATR mode allowed to obtain images with enhanced spatial resolution. A random distribution of hydroxyapatite domains with average size of ca. 10 microm on the surface of the composites was found after exposure to PBS for 14 and 28 days. The further growth of the hydroxyapatite domains after exposure to PBS for 63 days was detected. The spectroscopic imaging method introduced here promises to become a powerful method for characterisation of resorbable polymer composites containing bioactive inorganic phases developed for bone tissue engineering scaffolds. The accurate detection of hydroxyapatite domains and the imaging of their location in the scaffold structure is required to provide an assessment of the composites bioactivity.     

Two Frodo/Dapper homologs are expressed in the developing brain and mesoderm of zebrafish.

Members of the Wnt family of extracellular proteins play essential roles during many phases of vertebrate embryonic development. The molecular mechanism of their action involves a complex cascade of intracellular signaling events, which remains to be understood completely. Recently, two novel cytoplasmic modulators of Wnt signaling, Frodo and Dapper, were identified in Xenopus. We report isolation of their homologs in zebrafish, and show that these genes, frd1 and frd2, are expressed in restricted domains during embryogenesis. Both genes are expressed during early gastrulation in the future mesendoderm, and continue to be expressed in distinct patterns in the forming neurectoderm and mesoderm. Comparative sequence analysis and similar expression patterns argue that frd1 is the zebrafish ortholog of Frodo and Dapper, whereas frd2 is a more divergent member of the same family. Our data suggest important roles for zebrafish frd1 and frd2 in patterning the neural plate and several mesodermal derivatives.     

Presentation of alpha-galactosylceramide by murine CD1d to natural killer T cells is facilitated by plasma membrane glycolipid rafts

CD1 molecules are non-polymorphic major histocompatibility complex class I-related proteins that bind and present glycolipid antigens to T-cell antigen receptors (TCR) expressed by alphabeta T cells or natural killer-like T cells (NKT). Anti-metastatic properties of NKT cells reactive to the CD1d-binding antigen alpha-galactosylceramide (alpha-GalCer) are now being explored as a contributor to tumour cell killing. In this study, we tested the hypothesis that presentation of alpha-GalCer by murine CD1d (mCD1d) to mCD1d-restricted NKT cells was facilitated by plasma membrane glycolipid rafts. Confocal microscopy of mCD1d-transfected A20 B cells (A20mCD1d) demonstrated that mCD1d was raft-localized. This observation was confirmed by immunoblotting of raft fractions isolated on sucrose density gradients. Raft disruption by the cholesterol-binding agent nystatin, or short-chain ceramides, inhibited presentation of low concentrations of alpha-GalCer to NKT cells. Inhibition of antigen presentation was reversed by treatment of A20mCD1d cells with higher alpha-GalCer concentrations, or removal of raft-disrupting agents. These data indicate that partitioning of mCD1d into membrane rafts increases the capacity of antigen-presenting cells to present limiting quantities of glycolipid antigens, perhaps by stabilizing mCD1d/antigen structures on the plasma membrane and optimizing TCR engagement on NKT cells.     

The molecular structure of the DNA fragments eliminated during chromatin diminution in Cyclops kolensis

Presumptive somatic cells of the copepod Cyclops kolensis specifically eliminate a large fraction of their genome by the process of chromatin diminution. The eliminated DNA (eDNA) remains only in the germline cells. Very little is known about the nature of the sequences eliminated from somatic cells. We cloned a fraction of the eDNA and sequenced 90 clones that total 32 kb. The following organizational patterns were demonstrated for the eDNA sequences. All do not contain open reading frames. Each fragment contains 1-3 families of short repeats (10-30 bp) highly homologous within families (87%-100%). Most repeats are separated by spacers up to 50 bp long. Homologous regions were found between fragments, motifs from 15-300 bp in length. Among fragments there occur groups in which the same motifs are ordered in the same fashion. However, spacers between the motifs differ in length and nucleotide composition. Ubiquitous motifs (those occurring in all fragments) were identified. Analysis of motifs revealed submotifs, each occurring within several motifs. Thus, motifs may be regarded as mosaic structures composed of submotifs (short repeats). Taken together, the results provide evidence of a high organizational ordering of the DNA sequences restricted to the germline. With this in mind, it appears incorrect to refer to this part of the genome as junk. Moreover, eDNA is redundant for only the somatic cells-its function is to be sought in germline cells.     

Effects of Working Memory Load on Performance and Cardiovascular Activity in Younger and Older Workers

Background

Working memory (WM) declines with ageing, and this may cause problems in older workers who have to do complex work requiring WM.

Purpose

We tested the assumption that an increase in WM load negatively affects performance and results in impaired cardiovascular adaptation to changing task demands in older workers relative to younger ones.

Method

Thirty-three younger (29?±?3?years) and 32 older (55?±?3?years) workers had to perform a visual 0-back (low WM load) and 2-back (high WM load) task. Heart rate (HR), heart rate variability (HRV), beat-to-beat blood pressure (BP) and baroreflex were registered.

Results

In the high WM load condition, older adults responded more slowly and less accurately than younger adults, while no age effects in the low WM load condition were found. Older workers showed a higher systolic blood pressure (SBP) reactivity to high WM load as well as a diminished post-task recovery of SBP and HRV than younger workers. Factor analysis demonstrated a close relationship between HR, baroreflex and HRV and their modulation by a common factor (??vagal tone??) in the younger group. By contrast, HR was more related to the ??sympathetic?? factor in the older group.

Conclusion

The data suggest that older workers as compared with younger ones are impaired in tasks requiring WM, which is accompanied by enhanced cardiovascular ??costs?? in terms of increased SBP and reduced vagal control over HR.     

A sensitive high throughput ELISA for human eosinophil peroxidase: A specific assay to quantify eosinophil degranulation from patient-derived sources

Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.     

Retinal ganglion cell topography and spatial resolution in the Japanese smelt Hypomesus nipponensis (McAllister, 1963)

Vision plays a crucial role in the life of the vast majority of vertebrate species. The spatial arrangement of retinal ganglion cells has been reported to be related to a species’ visual behavior. There are many studies focusing on the ganglion cell topography in bony fish species. However, there are still large gaps in our knowledge on the subject. We studied the topography of retinal ganglion cells (GCs) in the Japanese smelt Hypomesus nipponensis, a highly visual teleostean fish with a complex life cycle. DAPI labeling was used to visualize cell nuclei in the ganglion cell and inner plexiform layers. The ganglion cell layer was relatively thin (about 6-8 μm), even in areas of increased cell density (area retinae temporalis), and was normally composed of a single layer of cells. In all retinal regions, rare cells occurred in the inner plexiform layer. Nissl-stained retinae were used to estimate the proportion of displaced amacrine cells and glia in different retinal regions. In all retinal regions, about 84.5% of cells in the GC layer were found to be ganglion cells. The density of GCs varied across the retina in a regular way. It was minimum (3990 and 2380 cells/mm² in the smaller and larger fish, respectively) in the dorsal and ventral periphery. It gradually increased centripetally and reached a maximum of 14,275 and 10,960 cells/mm² (in the smaller and larger fish, respectively) in the temporal retina, where a pronounced area retinae temporalis was detected. The total number of GCs varied from 177 × 10³ (smaller fish) to 212 × 10³ cells (larger fish). The theoretical anatomical spatial resolution (the anatomical estimate of the upper limit of visual acuity calculated from the density of GCs and eye geometry and expressed in cycles per degree) was minimum in the ventral periphery (smaller fish, 1.46 cpd; larger fish, 1.26 cpd) and maximum in area retinae temporalis (smaller fish, 2.83 cpd; larger fish, 2.75 cpd). The relatively high density of GCs and the presence of area retinae temporalis in the Japanese smelt are consistent with its highly visual behavior. The present findings contribute to our understanding of the factors affecting the topography of retinal ganglion cells and visual acuity in fish.     

Localization of HTLV-1 and HIV-1 Proviral Sequences in Chromosomes of Persistently Infected Cells

Integration sites for HTLV-1 and HIV-1proviruses were detected by FISH on the chromosomes of HTHIV27 cells persistently infected by HIV-1 (strain IIIB). HTLV-1 signals were found on 9 loci of chromosomes 4, 6, 9, 15 and 16. Integration sites of GC-rich HTLV-1 provirus are located in GC-rich isochores, confirming an ‘isopycnic’ integration, namely an integration in which the GC level of the host sequences around the integration site match the GC level of the provirus. This conclusion is not only derived from the compositional map of human chromosomes, but also from HTLV-1 hybridization on compositional fractions of human DNA. Integration of GC-poor HIV- 1 provirus was found on 4 loci of chromosomes 2, 7, 17 and 19. One copy of a complete HIV-1 provirus, which is active, was integrated in H1 isochores, whereas other defective copies were located in GC-poor L isochores. These results are discussed in terms of regional integration of retroviral sequences. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dynamics of quadrupedal locomotion of monkeys: implications for central control

We characterized the three-dimensional kinematics and dynamics of quadrupedal gait of young adult rhesus and cynomolgus monkeys while they walked with diagonal and lateral gaits at 0.4–1.0 m/s on a treadmill. Rigid bodies on the wrist, ankle, and back were monitored by an optical motion detection system (Optotrak). Kinematic data could be normalized using characteristic stride length, reducing variance due to different gait styles, to emphasize common characteristics of swing and stance parameters among animals. Mean swing phase durations fell as walking speed increased, but the swing phase durations increased at each walking velocity as a linear function of increases in amplitude, thereby following a main sequence relationship. The phase plane trajectories of the swing phases, i.e., plots of the relation of the rising and falling limb velocity to limb position in the sagittal (X–Z) plane, had unique dynamic characteristics. Trajectories were separable at each walking velocity and increases in swing amplitude were linearly related to peak swing velocities, thus comprising main sequences. We infer that the swing phase dynamics are set by central neural mechanisms at the onset of the swing phases according to the intended amplitude, which in turn is based on the walking velocity in the stance phases. From the many dynamic similarities between swing phases and rapid eye movements, we further suggest that the swing phases may be generated by neural mechanisms similar to those that produce saccades and quick phases of nystagmus from a signal related to sensed or desired walking velocity. Grants: This work was supported by National Institute of Health Grants EY11812, EY04148, DC05204, and EY01867.