Storage globulins pass through the Golgi apparatus and multivesicular bodies in the absence of dense vesicle formation during early stages of cotyledon development in mung bean

During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane.     

Autocrine and paracrine actions of ATP in rat carotid body

Carotid bodies are peripheral chemoreceptors that detect lowering of arterial blood O(2) level. The carotid body comprises clusters of glomus (type I) cells surrounded by glial-like sustentacular (type II) cells. Hypoxia triggers depolarization and cytosolic [Ca(2+)] ([Ca(2+)](i)) elevation in glomus cells, resulting in the release of multiple transmitters, including ATP. While ATP has been shown to be an important excitatory transmitter in the stimulation of carotid sinus nerve, there is considerable evidence that ATP exerts autocrine and paracrine actions in carotid body. ATP acting via P2Y(1) receptors, causes hyperpolarization in glomus cells and inhibits the hypoxia-mediated [Ca(2+)](i) rise. In contrast, adenosine (an ATP metabolite) triggers depolarization and [Ca(2+)](i) rise in glomus cells via A(2A) receptors. We suggest that during prolonged hypoxia, the negative and positive feedback actions of ATP and adenosine may result in an oscillatory Ca(2+) signal in glomus cells. Such mechanisms may allow cyclic release of transmitters from glomus cells during prolonged hypoxia without causing cellular damage from a persistent [Ca(2+)](i) rise. ATP also stimulates intracellular Ca(2+) release in sustentacular cells via P2Y(2) receptors. The autocine and paracrine actions of ATP suggest that ATP has important roles in coordinating chemosensory transmission in the carotid body.     

Direct fluorimetric sensing of UV-excited analytes in biological and environmental samples using molecularly imprinted polymer nanoparticles and fluorescence polarization

A rapid, robust, sensitive and economic sensing method, based on a molecularly imprinted polymer (MIP) synthetic antibody mimic, and fluorescence polarization analysis, for the direct detection of UV-excited fluorescent analytes in food and environmental samples was developed. Fluoroquinolone (FQ) antibiotics were used as fluorescent model analytes. Water-compatible MIP nanoparticles were synthesized with enrofloxacin (ENRO) as the imprinting template. Fluorescence polarization measurements then allow the direct determination of the amount of ENRO and other structurally related piperazine-based fluoroquinolones that bind to the MIP. No separation step was required since this technique distinguishes in situ analyte molecules bound to the MIP from the free analyte in solution. This assay was successfully applied for the first time to determine FQs in real samples, i.e. tap water and milk, without any prior concentration step, by simply adding a known amount of MIP. No interference by the sample components was observed even though the excitation was in the UV region. In tap water, a low limit of detection of 0.1 nM for ENRO was achieved with 5 μg mL(-1) of MIP. In milk, ENRO and danofloxacin, whose MRLs have been fixed at 0.28 μM and 0.08 μM, respectively, could be selectively measured and distinguished from other families of antibiotics. The procedure is very easy and practical as it consists of simply precipitating the milk proteins with acetonitrile and adding buffer and MIP to the supernatant before reading the polarization values with a spectrofluorimeter.     

The testicular and epididymal expression profile of PLCζ in mouse and human does not support its role as a sperm-borne oocyte activating factor

Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides.     

Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 μm) to low (7 μm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.     

Tensor-based morphometry and stereology reveal brain pathology in the complexin1 knockout mouse

Complexins (Cplxs) are small, soluble, regulatory proteins that bind reversibly to the SNARE complex and modulate synaptic vesicle release. Cplx1 knockout mice (Cplx1(-/-)) have the earliest known onset of ataxia seen in a mouse model, although hitherto no histopathology has been described in these mice. Nevertheless, the profound neurological phenotype displayed by Cplx1(-/-) mutants suggests that significant functional abnormalities must be present in these animals. In this study, MRI was used to automatically detect regions where structural differences were not obvious when using a traditional histological approach. Tensor-based morphometry of Cplx1(-/-) mouse brains showed selective volume loss from the thalamus and cerebellum. Stereological analysis of Cplx1(-/-) and Cplx1(+/+) mice brain slices confirmed the volume loss in the thalamus as well as loss in some lobules of the cerebellum. Finally, stereology was used to show that there was loss of cerebellar granule cells in Cplx1(-/-) mice when compared to Cplx1(+/+) animals. Our study is the first to describe pathological changes in Cplx1(-/-) mouse brain. We suggest that the ataxia in Cplx1(-/-) mice is likely to be due to pathological changes in both cerebellum and thalamus. Reduced levels of Cplx proteins have been reported in brains of patients with neurodegenerative diseases. Therefore, understanding the effects of Cplx depletion in brains from Cplx1(-/-) mice may also shed light on the mechanisms underlying pathophysiology in disorders in which loss of Cplx1 occurs.     

The CDK Subunit CKS2 Counteracts CKS1 to Control Cyclin A/CDK2 Activity in Maintaining Replicative Fidelity and Neurodevelopment

CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCF(SKP2) to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2(-/-) knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2(-/-) cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In?vivo, Cks2(-/-) cortical progenitor cells are limited in their capacity to differentiate into mature neurons,?a phenotype akin to animals lacking p27. We propose that?the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation.     

Discovery of novel 5-(ethyl or hydroxymethyl) analogs of 2'-'up' fluoro (or hydroxyl) pyrimidine nucleosides as a new class of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium inhibitors

Discovery of novel antimycobacterial compounds that work on distinctive targets and by diverse mechanisms of action is urgently required for the treatment of mycobacterial infections due to the emerging global health threat of tuberculosis. We have identified a new class of 5-ethyl or hydroxy (or methoxy) methyl-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium bovis, Mycobacterium tuberculosis (H37Ra, H37Rv) and Mycobacterium avium. A series of 2'-'up' fluoro (or hydroxy) nucleosides (1, 2, 4-6, 9, 10, 13, 16, 18, 21, 24) was synthesized and evaluated for antimycobacterial activity. Among 2'-fluorinated compounds, 1-(3-bromo-2,3-dideoxy-2-fluoro-β-d-arabinofuranosyl)-5-ethyluracil (13) exhibited promising activity against M. bovis and Mtb alone, and showed synergism when combined with isoniazid. The most active compound emerging from these studies, 1-(β-d-arabinofuranosyl)-4-thio-5-hydroxymethyluracil (21) inhibited Mtb (H37Ra) (MIC(50)=0.5 μg/mL) and M. bovis (MIC(50)=0.5 μg/mL) at low concentrations, and was ten times more potent against Mtb (H37Ra) than cycloserine (MIC(50)=5.0 μg/mL), a second line drug. It also showed an additive effect when combined with isoniazid. Compound 21 retained sensitivity against a rifampicin-resistant (H37Rv) strain of Mtb (MIC(50)=1 μg/mL) at concentrations similar to that for a rifampicin-sensitive (H37Rv) strain, suggesting that it has no cross-resistance to a first-line anti-TB drug. In addition, the replication of M. avium was also inhibited by 21 (MIC(50)=10 μg/mL). No cellular toxicity of 13 or 21 was observed up to the highest concentration tested (CC(50)>100 μg/mL). These observations offer promise for a new drug treatment regimen to augment and complement the current chemotherapy of TB.     

A purine-selective nucleobase/nucleoside transporter in PK15NTD cells

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with K(m) values of 2.5 +/- 0.7 microM for [(3)H]adenine, 6.4 +/- 0.5 microM for [(3)H]guanine, 1.1 +/- 0.1 mM for [(3)H]guanosine, and 4.2 +/- 0.5 mM [(3)H]adenosine. The uptake of [(3)H]guanine (0.05 microM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5-1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine > thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2' and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [(3)H]guanine: 2-chloro-2'-deoxyadenosine > 2-chloroadenosine > 2'3'-dideoxyadenosine = 2'-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2'-deoxyadenosine and 2-chloroadenosine inhibited [(3)H]guanine uptake with IC(50) values of 68 +/- 5 and 99 +/- 10 microM, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-beta-D-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 (IC(50) of approximately 1 microM), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells.