Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells.

The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission-fusion dynamics in pluripotent ESCs.

Quantum dot-based theranostics.

Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), have advanced the fields of molecular diagnostics and nanotherapeutics. Much of the initial progress for QDs in biology and medicine has focused on developing new biosensing formats to push the limit of detection sensitivity. Nevertheless, QDs can be more than passive bio-probes or labels for biological imaging and cellular studies. The high surface-to-volume ratio of QDs enables the construction of a "smart" multifunctional nanoplatform, where the QDs serve not only as an imaging agent but also a nanoscaffold catering for therapeutic and diagnostic (theranostic) modalities. This mini review highlights the emerging applications of functionalized QDs as fluorescence contrast agents for imaging or as nanoscale vehicles for delivery of therapeutics, with special attention paid to the promise and challenges towards QD-based theranostics.

Dynamics of Delta/Notch signaling on endomesoderm segregation in the sea urchin embryo.

Endomesoderm is the common progenitor of endoderm and mesoderm early in the development of many animals. In the sea urchin embryo, the Delta/Notch pathway is necessary for the diversification of this tissue, as are two early transcription factors, Gcm and FoxA, which are expressed in mesoderm and endoderm, respectively. Here, we provide a detailed lineage analysis of the cleavages leading to endomesoderm segregation, and examine the expression patterns and the regulatory relationships of three known regulators of this cell fate dichotomy in the context of the lineages. We observed that endomesoderm segregation first occurs at hatched blastula stage. Prior to this stage, Gcm and FoxA are co-expressed in the same cells, whereas at hatching these genes are detected in two distinct cell populations. Gcm remains expressed in the most vegetal endomesoderm descendant cells, while FoxA is downregulated in those cells and activated in the above neighboring cells. Initially, Delta is expressed exclusively in the micromeres, where it is necessary for the most vegetal endomesoderm cell descendants to express Gcm and become mesoderm. Our experiments show a requirement for a continuous Delta input for more than two cleavages (or about 2.5 hours) before Gcm expression continues in those cells independently of further Delta input. Thus, this study provides new insights into the timing mechanisms and the molecular dynamics of endomesoderm segregation during sea urchin embryogenesis and into the mode of action of the Delta/Notch pathway in mediating mesoderm fate.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

Interaction of Cryptococcus neoformans Rim101 and protein kinase A regulates capsule.

Cryptococcus neoformans is a prevalent human fungal pathogen that must survive within various tissues in order to establish a human infection. We have identified the C. neoformans Rim101 transcription factor, a highly conserved pH-response regulator in many fungal species. The rim101 multiply sign in circle mutant strain displays growth defects similar to other fungal species in the presence of alkaline pH, increased salt concentrations, and iron limitation. However, the rim101 multiply sign in circle strain is also characterized by a striking defect in capsule, an important virulence-associated phenotype. This capsular defect is likely due to alterations in polysaccharide attachment to the cell surface, not in polysaccharide biosynthesis. In contrast to many other C. neoformans capsule-defective strains, the rim101 multiply sign in circle mutant is hypervirulent in animal models of cryptococcosis. Whereas Rim101 activation in other fungal species occurs through the conserved Rim pathway, we demonstrate that C. neoformans Rim101 is also activated by the cAMP/PKA pathway. We report here that C. neoformans uses PKA and the Rim pathway to regulate the localization, activation, and processing of the Rim101 transcription factor. We also demonstrate specific host-relevant activating conditions for Rim101 cleavage, showing that C. neoformans has co-opted conserved signaling pathways to respond to the specific niche within the infected host. These results establish a novel mechanism for Rim101 activation and the integration of two conserved signaling cascades in response to host environmental conditions.

High number of antigen binding cells in unimmunized mice and possible occurrence of multispecific lymphocytes

A high frequency of Tobacco Mosaic virus (TMV) binding cells was found in spleen cells from unimmunized mice (about 3 to 4%). TMV binding is strongly inhibited by previous incubation with anti immunoglobulin antisera. After stripping of membrane receptors, a full recovery for antigen binding capacity can be observed after 24 hr culture. Experiments are presented to exclude artefactual fluorescent cells: interaction of TMV with some non immunoglobulin membrane components; interaction of fluorescent anti TMV antibody with the Fc receptor of B cells; the binding of TMV to cytophilic immunoglobulins. The occurrence of lymphocytes able to bind several non crossreactive antigens is suggested by three lines of evidence: the high number of antigen binding cells in unimmunized mice, presence of surface immunoglobulins on some TMV binding cells after complete capping of TMV receptors and the direct demonstration of lymphocytes binding TMV and hemocyanin at different membrane sites.

Advanced glycation endproduct modified basement membrane attenuates endothelin-1 induced [Ca2+]i signalling and contraction in retinal microvascular pericytes

PURPOSE: To assess the effects of advanced glycation endproduct (AGE) modification of vascular basement membrane (BM) on endothelin-1 (Et-1) induced intracellular [Ca2+] ([Ca2+]i) homeostasis and contraction in retinal microvascular pericytes (RMP). METHODS: RMPs were isolated from bovine retinal capillaries and propagated on AGE modified BM extract (AGE-BM) or non-modified native BM. Cytosolic Ca2+ was estimated using fura-2 microfluorimetry and cellular contraction determined by measurement of planimetric cell surface area. ETA receptor mRNA and protein expression was assessed by real time RT-PCR and western blotting, respectively. RESULTS: Exogenous endothelin-1 (Et-1) evoked rises in [Ca2+]i and contraction in RMPs were found to be mediated entirely through ETA receptor (ETAR) activation. Both peak and plateau phases of the Et-1 induced [Ca2+]i response and contraction were impaired in RMPs propagated on AGE modified BM. ETAR mRNA expression remained unchanged in RMPs exposed to native or AGE-BM, but protein expression for ETAR (66 kDa) was lower in the AGE exposed cells. CONCLUSIONS: These results suggest that substrate derived AGE crosslinks can influence RMP physiology by mechanisms which include disruption of ETA receptor signalling. AGE modification of vascular BMs may contribute to the retinal hemodynamic abnormalities observed during diabetes.

Intracellular antigenic changes associated with meiosis in the orthopteran Stauroderus scalaris

Monoclonal antibodies have been prepared against purified pachytene cells from grasshopper testes. Immunoblotting and immunofluorescence analyses identified those monoclonal antibodies which showed specificity for antigens in pachytene cells. Several antigenic changes were found to be associated with meiotic cells. Five monoclonal antibodies detected antigens which were located in the cytoplasm of premeiotic cells but were nuclear during meiosis. One monoclonal antibody showed a discrete cytoplasmic fluorescent pattern in meiotic, but not in premeiotic, cells. Another bound specifically to the nuclei of some epithelial cells at the base of follicles in mature testes.

Localisation of actin in the liver fluke, Fasciola hepatica

Adult and 3-week-old juvenile Fasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1% (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.

Fasciola hepatica:disruption of spermatogenesis by the microfilament inhibitor cytochalasin B

The distribution of actin filaments in the spermatogenic cells of Fasciola hepatica was determined using a fluorescent derivative of phalloidin. Actin was localised primarily in the region of separation of a secondary spermatogonium from a primary spermatogonium, in the inner faces at the centre of four-cell clusters of tertiary spermatogonia and in the cytophore region of spermatocyte and spermatid rosettes. The effect of the microfilament inhibitor cytochalasin B (100 micrograms/ml) on the ultrastructure of the spermatogenic cells was determined in vitro by transmission electron microscopy using tissue-slice material. Cytochalasin B treatment led to the formation of bi- and multinucleate cells, whose frequency increased with progressively longer incubation periods. Few typical rosettes of spermatocyte and spermatid cells were evident from 6 h onwards, being replaced by syncytial masses of cells. Spermatozoon formation became abnormal in the longer treatment periods, the spermatozoa containing variable numbers of axonemes and an altered distribution of cortical microtubules. Multiple axonemes were observed in the cytoplasm of spermatid cells. The results are discussed in relation to the established role of actin in the cytokinesis phase of cell division and to the effects of cytochalasin B on other tissues and organ systems within the fluke.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead β-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

IB1/JIP-1 controls JNK activation and increased during prostatic LNCaP cells neuroendocrine differentiation.

The scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1) is a modulator of the c-Jun N-terminal kinase (JNK) activity, which has been implicated in pleiotrophic cellular functions including cell differentiation, division, and death. In this study, we described the presence of IB1/JIP-1 in epithelium of the rat prostate as well as in the human prostatic LNCaP cells. We investigated the functional role of IB1/JIP-1 in LNCaP cells exposed to the proapoptotic agent N-(4-hydroxyphenyl)retinamide (4-HPR) which induced a reduction of IB1/JIP-1 content and a concomittant increase in JNK activity. Conversely, IB1/JIP-1 overexpression using a viral gene transfer prevented the JNK activation and the 4-HPR-induced apoptosis was blunted. In prostatic adenocarcinoma cells, the neuroendocrine (NE) phenotype acquisition is associated with tumor progression and androgen independence. During NE transdifferentiation of LNCaP cells, IB1/JIP-1 levels were increased. This regulated expression of IB1/JIP-1 is secondary to a loss of the neuronal transcriptional repressor neuron restrictive silencing factor (NRSF/REST) function which is known to repress IB1/JIP-1. Together, these results indicated that IB1/JIP-1 participates to the neuronal phenotype of the human LNCaP cells and is a regulator of JNK signaling pathway.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.