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PubMed2005

Pairwise knockdowns of cdc2-related kinases (CRKs) in Trypanosoma brucei identified the CRKs for G1/S and G2/M transitions and demonstrated distinctive cytokinetic regulations between two developmental stages of the organism.

Expression of the cdc2-related kinase 3 (CRK3) together with expression of CRK1, -2, -4, or -6, were knocked down in pairs in the procyclic and bloodstream forms of Trypanosoma brucei, using the RNA interference technique. Double knockdowns of CRK3 and CRK2, CRK4, or CRK6 exerted significant growth inhibition and enriched the cells in G2/M phase, whereas a CRK3 plus CRK1 (CRK3 + CRK1) knockdown arrested cells in both G1/S and G2/M transitions. Thus, CRK1 and CRK3 are apparently the kinases regulating the G1/S and G2/M checkpoint passages, respectively, whereas the other CRKs are probably playing only minor roles in cell cycle regulation. A CRK1 + CRK2 knockdown in the procyclic form was found to cause aberrant posterior cytoskeletal morphogenesis (X. M. Tu and C. C. Wang, Mol. Biol. Cell 16:97-105, 2005). A CRK3 + CRK2 knockdown, however, did not lead to such a change, suggesting that CRK2 depletion can lead to the abnormal morphogenesis only when procyclic-form cells are arrested in the G1 phase. The G2/M-arrested procyclic form produces up to 20% stumpy anucleated cells (zoids) in the population, suggesting that cytokinesis and cell division are not blocked by mitotic arrest but are apparently driven to completion by the kinetoplast cycle. In the bloodstream form, however, G2/M arrest resulted in little zoid formation but, instead, enriched a population of cells each containing multiple kinetoplasts, basal bodies, and flagella and an aggregate of multiple nuclei, indicating failure in entering cytokinesis. The two different cytokinetic regulations between two distinct stage-specific forms of the same organism may provide an interesting and useful model for further understanding the evolution of cytokinetic control among eukaryotes.

Animals↗
PubMed2006

Use of quantitative real-time PCR to investigate the dynamics of the red tide dinoflagellate Lingulodinium polyedrum.

A new method based on quantitative real-time polymerase chain reaction (qPCR) was developed and applied to quantify the red tide dinoflagellate Lingulodinium polyedrum in natural seawater samples and in laboratory cultures. The method uses a Molecular Beacontrade mark approach to target a species-specific region of the small subunit ribosomal RNA gene. The accuracy of the method was verified by microscopical counts using cultures of the dinoflagellate isolated from coastal waters near Los Angeles, CA, and with natural water samples spiked with cultured L. polyedrum. The method was applied to document the pattern and timing of vertical migration by the dinoflagellate in a 2-m water column on an 11:13 h light/dark photoperiod established in the laboratory. Positive phototaxis of L. polyedrum resulted in dense aggregations of the dinoflagellate within the top few centimeters of the water column during the light period. This pattern of distribution was readily established by both methods, although abundances of L. polyedrum determined using qPCR were higher than abundances determined by microscopy in the morning and lower in the afternoon and evening. These differences may have been a consequence of variability in the DNA content per cell because of synchrony of cell division. Counts using both methods to analyze natural samples collected from coastal waters in the Long Beach-Los Angeles area and adjacent San Pedro Channel were in close agreement. However, the qPCR method exhibited greater sensitivity than the microscopical method when L. polyedrum was present at low abundances, and qPCR had a much higher rate of sample throughput than microscopy. The development of this new approach for enumerating L. polyedrum provides a useful tool for studying the ecology of this important red tide species.

Animals↗
PubMed1983

The behavior during the initial phase of in vitro aggregation of dissociated imaginal disc cells from Drosophila melanogaster.

Aspects of the initial phase of aggregation in vitro of dissociated imaginal disc cells from Drosophila melanogaster are described. Using the methods described certain interdisc differences in the percent decrease in single cell units during the first hour of aggregation can be demonstrated. In addition it is shown that prospective notum cells isolated from the dorsal mesothoracic disc show less of a decrease than do prospective wing cells. This difference shows up in a variety of different wild-type stocks and in several mutant stocks as well. Prospective notum cells from the mutation fu59 show only a limited ability to adhere to one another, while the percentage of single cell units from prospective wing blade cells from r9 larvae grown on pyrimidine-poor medium decrease less compared to cells from the same stock grown on RNA-supplemented medium. There is a significantly greater decrease in the percent single cell units in cultures of prospective eye cells than in cultures of prospective antennal cells. Furthermore, cells from the antennal disc of larvae bearing the homeotic mutation ssa show a significantly lower decrease in single cell units when grown at restrictive temperatures. In contrast, antennal disc cells from the homeotic mutation ophthalmoptera; eyeless Dominant, a mutation which affects the eye disc, are unaffected, while cells from the eye disc are slightly less able to reassociate with one another.

Animals↗
PubMed1989

Sequence and transcription mapping of Bacillus subtilis competence genes comB and comA, one of which is related to a family of bacterial regulatory determinants.

The complete nucleotide sequences of the comA and comB loci of Bacillus subtilis were determined. The products of these genes are required for the development of competence in B. subtilis and for the expression of late-expressing competence genes. The major 5' termini of both the comA and comB transcripts were determined. The inferred promoters of both comA and comB contained sequences that were similar to those found at the -10 and -35 regions of promoters that are used by sigma A-RNA polymerase, the primary form of this enzyme in vegetative cells. The comB gene was located approximately 3 kilobase pairs upstream of the comA gene and encoded a 409-amino-acid protein with a predicted molecular weight of 46,693. The comA locus contained two open reading frames (ORFs) and comB contained one ORF. The predicted amino acid sequence of the comA ORF1 gene product consisted of 214 amino acids, with an aggregate molecular weight of 24,132. The ORF1 product was required for competence, while ORF2, which was cotranscribed with ORF1 and encoded a predicted protein of 126 amino acids, was not. The predicted protein sequence of the comA ORF1 gene product was found to be similar to that of several members of the effector class of procaryotic signal transducers. The C-terminal portion of the predicted comA sequence contained a possible helix-turn-helix motif, which is characteristic of DNA-binding proteins. comA ORF1 was cloned on a multicopy plasmid and was shown to complement the competence-deficient phenotype caused by the comA124 insertion of Tn917lac. Also, the presence of comA ORF1 in multiple copies interfered with sporulation. Anti-peptide antibodies raised to the predicted product of comA ORF1 reacted strongly with a single protein band of about 24,000 daltons in immunoblots. The possible roles of multiple signal transduction systems in triggering the development of competence are discussed.

Amino Acid Sequence↗
PubMed2000

Identification and characterization of the ribosome-associated protein, HrpA, of Bacillus Calmette-Guérin.

HrpA was found as a ribosome-associated protein which appeared in heat-stressed Mycobacterium bovis Bacillus Calmette-Guérin. Here, we have studied the function of HrpA in vitro. HrpA is a heat shock protein belonging to a small heat shock protein family. The putative molecular mass was 17784.86 kDa. Recombinant HrpA formed large complexes of nonamer or dodecamer. HrpA prevented the aggregation of enzymes under heat shock conditions, and it formed stable complexes with partially denatured enzymes. HrpA was induced temporarily by oxygen repletion after anaerobic condition.

Amino Acid Sequence↗
PubMed1995

Analysis of zeins' ER retention in Xenopus oocytes.

Zeins, maize storage proteins, are retained in the endoplasmic reticulum (ER) during the intracellular protein targeting process. Hydrophobic interaction has been postulated as the driving force of zeins' aggregation and retention in the ER. Recently, a class of zein (the 27K zein) has been proposed to facilitate zeins' ER retention by anchoring to the ER membrane. This study investigated the significance of the two proposed mechanisms toward zeins' ER retention using Xenopus oocyte. Following injection of the total or 27K zein mRNA, zein's movement within the ER was analyzed based upon the extent of diffusion to the non-injected oocyte half. This study indicates that the total zeins freely move within the lumen of the ER, thus, suggesting that the intermolecular aggregation, leading to insolubility and exclusion from the ER lumenal fluid, may not be essential for zeins' ER retention. This study also suggests that the 27K zein may not facilitate zeins' ER retention by virtue of an anchor to the ER membrane based on its free movement in the ER. Free movement of the total and 27K zeins, under conditions where zein aggregates should form, necessitates a reevaluation of the mechanisms responsible for zein polypeptides' ER retention and protein body formation.

Animals↗
PubMed2004

Correlation of N-cadherin expression in high grade gliomas with tissue invasion.

Cadherins are Ca2+-dependent cell adhesion molecules that play an important role in tissue construction and morphogenesis in multicellular organisms. Over the last few years, reports have emerged in the literature describing the involvement of cadherins in tumor invasion and metastasis. Cadherins typically demonstrate up and down-regulation according to the biological needs of the tissue. Additionally, up-regulation of N-cadherin is thought to be important for tumor formation in early stages of tumor development. We studied N-cadherin in surgical specimens of patients with primary glioblastoma by microarray analysis and found that N-cadherin mRNA expression is up-regulated compared to normal brain. To study the effects of N-cadherin expression on invasion and metastasis in vitro and in vivo, we overexpressed N-cadherin in the rat C6 glioma cell line which normally has low levels of N-cadherin. We found that up-regulation of N-cadherin resulted in a slight decreased adhesion to type IV collagen, fibronectin, and laminin, but statistically significant decreased adhesion to type I collagen. Furthermore, increased expression of N-cadherin correlated with a dramatic decrease in invasive behavior in extracellular matrix invasion assays. We then proceeded to study these cell lines in vivo in a rat intracranial glioma model, and found that N-cadherin expression inversely correlated with invasion into surrounding tissues, irregular margins, and extracranial invasion. In summary, these data collectively demonstrate that N-cadherin levels are important in the malignant behavior of gliomas, and may serve as a prognostic indicator for patients with high-grade gliomas.

Adult↗
PubMed1971

Fine structural and radioautographic observations on dense perinuclear cytoplasmic material in tadpole oocytes.

Dense fibrous material is first seen in association with mitochondria in tadpole oogonia but is most prominent in oocytes during the extended first meiotic prophase when it aggregates into dense bodies in the perinuclear cytoplasm. The origin of this material has been attributed to 350-A nuclear granules which form cytoplasmic streamers of fibrous material upon passage through nuclear pores. This has commonly been interpreted as the transfer of ribonucleoprotein to the cytoplasm for storage. However, cytochemical reactions for nucleic acids have indicated an absence of detectable RNA in this dense material, and the results of radioautographic studies with labeled uridine, thymidine, or actinomycin D argue against the presence of nucleic acids. When sites of incorporation of tritiated amino acids were radioautographically localized, an appreciable number of silver grains were present over the dense bodies. Uptake of certain amino acids occurs fairly promptly but the degree of labeling levels off after about 6 hr, suggesting a rapid turnover of the material in the dense bodies. Attention is drawn to the similarity of the dense bodies to structures present in germ cells of a number of other species, and possible functions of the dense bodies in germ cell differentiation are considered.

Animals↗
PubMed2006

Thrombin and prothrombin are expressed by neurons and glial cells and accumulate in neurofibrillary tangles in Alzheimer disease brain.

Thrombin is a serine protease that is generated by proteolytic cleavage of its precursor, prothrombin. We previously showed that thrombin proteolyses the microtubule-associated protein tau and that phosphorylation of tau inhibits this process. To characterize further the role of thrombin in the brain, we investigated prothrombin and thrombin expression in cultured brain cells and in brains of control, Alzheimer disease (AD) and parkinsonism-dementia complex of Guam (PDCG). We show by reverse transcriptase-polymerase chain reaction that prothrombin mRNA is expressed in brain tissues, neuroblastoma cells, and cultured human astrocytes, oligodendrocytes, and microglial cells. We also show by immunohistochemistry that the proteins prothrombin and thrombin are present in brain using specific monoclonal and polyclonal antibodies for both proteins. All antibodies stained residual serum in blood vessels, as well as normal pyramidal neurons and their processes, and some astrocytes. Additionally, in AD and PDCG cases, all antibodies stained extra- and intracellular neurofibrillary tangles (NFTs), senile plaques, and reactive microglial cells. The ubiquitous expression of prothrombin and thrombin in brain cells suggests that thrombin plays an important physiological role in normal brain. The accumulation of thrombin and prothrombin in NFTs supports the hypothesis that thrombin may be involved in tau proteolysis and that failure to metabolize tau may lead to its aggregation in neurodegenerative diseases.

Aged↗
PubMed2006

The use of high-dimensional biology (genomics, transcriptomics, proteomics, and metabolomics) to understand the preterm parturition syndrome.

High-dimensional biology (HDB) refers to the simultaneous study of the genetic variants (DNA variation), transcription (messenger RNA [mRNA]), peptides and proteins, and metabolites of an organ, tissue, or an organism in health and disease. The fundamental premise is that the evolutionary complexity of biological systems renders them difficult to comprehensively understand using only a reductionist approach. Such complexity can become tractable with the use of "omics" research. This term refers to the study of entities in aggregate. The current nomenclature of "omics" sciences includes genomics for DNA variants, transcriptomics for mRNA, proteomics for proteins, and metabolomics for intermediate products of metabolism. Another discipline relevant to medicine is pharmacogenomics. The two major advances that have made HDB possible are technological breakthroughs that allow simultaneous examination of thousands of genes, transcripts, and proteins, etc., with high-throughput techniques and analytical tools to extract information. What is conventionally considered hypothesis-driven research and discovery-driven research (through "omic" methodologies) are complementary and synergistic. Here we review data which have been derived from: 1) genomics to examine predisposing factors for preterm birth; 2) transcriptomics to determine changes in mRNA in reproductive tissues associated with preterm labour and preterm prelabour rupture of membranes; 3) proteomics to identify differentially expressed proteins in amniotic fluid of women with preterm labour; and 4) metabolomics to identify the metabolic footprints of women with preterm labour likely to deliver preterm and those who will deliver at term. The complementary nature of discovery science and HDB is emphasised.

Biomarkers↗
PubMed1993

Tyrosinase gene expression in human tissues.

The occasional occurrence of primary extra-cutaneous malignant melanomas (MM) has led to the hypothesis that melanocytes derived from the neural crest may be arrested in their migration and may undergo an in situ malignant transformation. However, aggregates of nevus cells have only rarely been identified by histological examination in a few organs other than skin and eye. Tyrosinase is a melanin biosynthetic enzyme that is considered one of the most specific markers of melanocytic differentiation. We have attempted to detect cells committed to the melanocytic lineage, in human tissues, by means of tyrosinase gene expression. Total RNA was extracted from normal and neoplastic tissues and analyzed using a highly sensitive reverse transcription PCR assay with primers specific for the tyrosinase gene. Peripheral blood mononuclear cells (PBMC) from healthy subjects were used as negative controls. Tyrosinase transcripts were identified in a wide range of normal organs such as skin, lymph nodes, antrum, colon, kidney, lung, testis, ovary, breast, and peripheral nerve. Tyrosinase RNA was also detected in neoplastic samples including benign cutaneous nevi, lymph nodes involved by MM, breast carcinoma, liposarcoma, malignant lymphoma, and schwannoma. PBMC from patients with metastatic MM were also positive, while no positivity was detected in blood specimens from patients with other cancers. Therefore, it appears likely that cells expressing the tyrosinase gene are present in a wide range of human tissues. Although these cells still have to be accurately identified, one could propose that they might correspond to either fully differentiated melanocytes, melanocytic precursors, or Schwann cells bearing potentialities of melanocytic differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2004

Shear rate moderates community diversity in freshwater biofilms.

The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S(-1)) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates (198 to 305 S(-1)). The intermediate shear rate (122 S(-1)) selected for the highest proportion of coaggregating bacteria (47%, or 17 of a possible 36 coaggregation interactions). Under static conditions (<0.1 S(-1)), 41 (33%) of a possible 125 coaggregation interactions were positive. Few coaggregation (3.3%) or autoaggregation (25%) interactions occurred between the 16 planktonic strains. In conclusion, these data show that shear rates affect biofilm diversity as well as the relative proportions of aggregating bacteria.

Bacteria↗
PubMed2003

Stable-tau overexpression in human neuroblastoma cells: an open door for explaining neuronal death in tauopathies.

Many neurodegenerative disorders referred to as "tauopathies" are characterized by the accumulation and aggregation of Tau proteins into filaments. In these pathologies, Tau proteins are hyperphosphorylated and also abnormally phosphorylated. Moreover, they differ from each other by the preferential aggregation of isoforms exhibiting either three microtubule-binding repeats (3R) or four repeats (4R) Tau. To investigate the effects of an intracellular accumulation of Tau, we stably transfected neuroblastoma cell line SY5Y with either 3R or 4R Tau. Our data showed that an increase in intracellular Tau expression has led to their hyperphosphorylation. Conversely, an abnormal Tau phosphorylation and/or aggregation were never observed. Furthermore, SY5Y cells transfected with 4R Tau showed an increased susceptibility to cell death. Finally, in apoptotic conditions, Tau proteins were degraded at their carboxy terminus by caspase, leading to an apparent decrease in Tau phosphorylation in this region. Because truncated Tau generated during apoptosis are not commonly found in Tau aggregates, apoptotic processes may not be of interest in neurofibrillary degeneration.

Apoptosis↗
PubMed2007

Autophagy genes protect against disease caused by polyglutamine expansion proteins in Caenorhabditis elegans.

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington's disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.

Animals↗
PubMed1999

Specialized ability of thymic epithelial cells to mediate positive selection does not require expression of the steroidogenic enzyme p450scc.

Thymic epithelial cells are uniquely efficient in mediating positive selection, suggesting that in addition to providing peptide/MHC complexes for TCR ligation, they may also provide additional support for this process. Recent studies have shown that although engagement of either the TCR or glucocorticoid (GC) receptors can individually induce apoptosis in thymocytes, together these signals are mutually antagonistic. This had led to the suggestion that local GC production by thymic epithelial cells, by opposing TCR signaling for apoptosis, provides the basis of the ability of these cells to mediate thymocyte positive selection. In this paper we have examined this possibility directly and shown that highly purified cortical epithelial cells, which have the functional ability to mediate positive selection in reaggregate cultures, do not express mRNA for the key steroidogenic enzyme P405scc. Thus we conclude that the ability of thymic epithelial cells to support positive selection does not rely on their ability to produce GC. However, we find that P450scc mRNA is up-regulated in thymocytes on the initiation of positive selection, raising the possibility that any local protective effect of steroid production is mediated at the level of thymocytes themselves.

Animals↗
PubMed2002

[Calcitonin gene-related peptide induces chemokine interleukine-8 synthesis in human monocytes].

OBJECTIVE: To study the effect of calcitonin gene-related peptide (CGRP) on the function of immune cells. METHODS: Human monocytes were cultured with calcitonin gene-related peptide in vitro and activated with lipopolysaccharide (LPS). The IL-8 level in the supernatant was measured with ELISA and the IL-8 mRNA expression in monocytes was observed by reverse transcription polymerase chain reaction (RT-PCR). The chemotactic activity of monocytes to neutrophils and lymphocytes was analyzed with micro-chemotacxis chamber. Chemotactic index (CI) was calculated by the formula: number of monocytes migrating to the underside of membrane in the LPS + CGRP group/number of monocytes migrating to the underside of membrane in the control group. CGRP receptor antagonist CGRP8 - 37 was added into the culture to study the effect of CGRP. Blank control and cultures of monocytes with LPS or with CGRP only were used as controls. RESULTS: The level of IL-8 protein in the supernatant of the LPS + CGRP group was 1 120 pg/ml +/- 14.80 pg/ml, significantly higher than those in other groups (670 pg/ml +/- 15.10 pg/ml in LPS + CGRP + CGRP8 - 37 group). The expression of IL-8 mRNA in the LPS + CGRP group was the highest (IL-8/beta-actin = 1.845 +/- 0.587), IL-8/beta-actin in the LPS + CGRP + CGRP8 - 37 group was1.339 +/- 0.434. The chemotactic activities of the monocytes to neutrophils and lymphocytes were enhanced in the LPS + CGRP group (CI = 3.78 +/- 0.08 to neutrophils and CI = 3.4 +/- 0.27 to lymphocytes). The CI values were 1.15 +/- 0.31 and 1.21 +/- 0.06 respectively in the LPS + CGRP + CGRP 8 - 37 group. CONCLUSION: CGRP in the peripheral nerve ending induces monocytes to synthetize and secret chemotactic factor IL-8 and enhance the chemotactic activity of monocytes, thus promoting the directional migration and aggregation of neutrophils and lymphocytes to foci of inflammation.

Calcitonin Gene-Related Peptide↗
PubMed1999

In Situ Hybridization of RA Induced P19 Cells.

P19 EC cells can be induced to differentiate into neurons in vitro when they were treated with 1x10(-6) mol/L retinoic acid. In the process of neuronal differentiation, P19 cells gradually migrate and form aggregates. Aggregated cells weakly adhered to the surface of cell culture dishes are usually dropped of during the procedure of in situ hybridization. To keep cells on the coverslips, we modified the procedure by using 1 g/L gelatin solution to cover the cells and substituting proteinase K treatment with pepsin treatment which digested cellular proteins mildly. With the treatment of pepsin, RNA probe can easily go through cell membrane and bind to its target, without loss of cell from coverslips.

Journal Article↗