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PubMed2005

Morphometric and immunohistochemical study of the omasum of red deer during prenatal development.

The red deer is an important study species because of its value in the national economy and because it provides a wealth of genetic material. To date, there has been little research into the prenatal development of the stomach of ruminants, and none of the red deer. We therefore performed a histological evaluation of the ontogenesis of the omasum in the red deer. Histomorphometric and immunohistochemical analyses were carried out on 50 embryos and fetuses of deer from the initial stages of prenatal life until birth. For test purposes, the animals were divided into five experimental groups: Group I (1.4-3.6 cm crown-rump length, CRL; 30-60 days, 1-25% of gestation); Group II (4.5-7.2 cm CRL; 67-90 days, 25-35% of gestation); Group III (8-19 cm CRL; 97-135 days, 35-50% of gestation); Group IV (21-33 cm CRL; 142-191 days, 50-70% of gestation); and Group V (36-40 cm CRL; 205-235 days, 75-100% of gestation). At 67 embryonic days, the omasum wall was differentiated, and comprised three layers: the epithelial layer, pluripotential blastemic tissue and serosa. The stratification of the epithelial layer was accompanied by changes in its structure, with the appearance of four laminae of different sizes; in order of appearance these were: primary at 67 days, secondary at 90 days, tertiary at 97 days and quaternary at 135 days. At around mid-gestation, lateral evaginations were formed from the stratum basale of the primary and secondary smaller laminae. These were the primitive corneum papillae. From 205 days, the corneum papillae were present in all four sizes of laminae. The histodifferentiation of the lamina propia-submucosa, tunica muscularis and serosa showed patterns of development similar to those reported for the rumen and reticulum of red deer. The omasum of red deer during prenatal life, especially from 67 days of gestation, was shown to be an active structure with full secretory capacity. Its histological development, its secretory capacity (detected by the presence of neutral mucopolysaccharides) and its neuroendocrine nature (detected by the presence of positive non-neuronal enolase cells and the neuropeptides vasoactive intestinal peptide and neuropeptide Y) were parallel to the development of the rumen and the reticulum. However, its prenatal development was later than that of the omasum in sheep, goat and cow.

Animals↗
PubMed2005

The mitochondrial complex I inhibitor rotenone triggers a cerebral tauopathy.

Reduced activity of the mitochondrial respiratory chain--particularly complex I--may be implicated in the etiology of both Parkinson's disease and progressive supranuclear palsy, although these neurodegenerative diseases differ substantially as to their distinctive pattern of neuronal cell loss and the predominance of cerebral alpha-synuclein or tau protein pathology. To determine experimentally whether chronic generalized complex I inhibition has an effect on the distribution of alpha-synuclein or tau, we infused rats systemically with the plant-derived isoflavonoid rotenone. Rotenone-treated rats with a pronounced metabolic impairment had reduced locomotor activity, dystonic limb posture and postural instability. They lost neurons in the substantia nigra and in the striatum. Spherical deposits of alpha-synuclein were observed in a few cells, but cells with abnormal cytoplasmic accumulations of tau immunoreactivity were significantly more numerous in the striatum of severely lesioned rats. Abnormally high levels of tau immunoreactivity were found in the cytoplasm of neurons, oligodendrocytes and astrocytes. Ultrastructurally, tau-immunoreactive material consisted of straight 15-nm filaments decorated by antibodies against phosphorylated tau. Many tau+ cell bodies also stained positive for thioflavin S, nitrotyrosine and ubiquitin. Some cells with abnormal tau immunoreactivity contained activated caspase 3. Our data suggest that chronic respiratory chain dysfunction might trigger a form of neurodegeneration in which accumulation of hyperphosphorylated tau protein predominates over deposits of alpha-synuclein.

Amyloid beta-Peptides↗
PubMed2006

Regulation of taurine transport in rat hippocampal neurons by hypo-osmotic swelling.

Taurine, an important mediator of cellular volume regulation in the central nervous system, is accumulated into neurons and glia by means of a highly specific sodium-dependent membrane transporter. During hyperosmotic cell shrinkage, net cellular taurine content increases as taurine transporter activity is enhanced via elevated gene expression of the transporter protein. In hypo-osmotic conditions, taurine is rapidly lost from cells by means of taurine-conducting membrane channels. We reasoned that changes in taurine transporter activity also might accompany cell swelling to minimize re-accumulation of taurine from the extracellular space. Thus, we determined the kinetic and pharmacological characteristics of neuronal taurine transport and the response to osmotic swelling. Accumulation of radioactive taurine is strongly temperature dependent and occurs via saturable and non-saturable pathways. At concentrations of taurine expected in extracellular fluid in vivo, 98% of taurine accumulation would occur via the saturable pathway. This pathway obeys Michaelis-Menten kinetics with a Km of 30.0 +/- 8.8 microm (mean +/- SE) and Jmax of 2.1 +/- 0.2 nmol/mg protein min. The saturable pathway is dependent on extracellular sodium with an effective binding constant of 80.0 +/- 3.1 mm and a Hill coefficient of 2.1 +/- 0.1. This pathway is inhibited by structural analogues of taurine and by the anion channel inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and 5-nitro-2-(3 phenylpropylamino) benzoic acid (NPPB). NPPB, but not DIDS, also reduces the ATP content of the cell cultures. Osmotic swelling at constant extracellular sodium concentration reduces the Jmax of the saturable transport pathway by approximately 48%, increases Kdiff for the non-saturable pathway by 77%, but has no effect on cellular ATP content. These changes in taurine transport occurring in swollen neurons in vivo would contribute to net reduction of taurine content and resulting volume regulation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1978

Enolase from spores and cells of Bacillus megaterium: two-step purification of the enzyme and some of its properties.

A simple two-step procedure for purification of enolase from germinated spores or vegetative cells of Bacillus megaterium is described. The procedure resulted in a 1,200-fold purification with production of homogeneous enzyme in approximately 75% yield; the enzymes from spores and cells seemed identical. The molecular weight of the native enzyme was 335,000, with a subunit molecular weight of 42,000. The enzyme required Mg2+ and was inhibited by ethylenediaminetetraacetic acid and fluoride ions. The Michaelis constants for 2-phosphoglyceric acid and Mg2+ were 7.1 X 10(-4) and 4.7 X 10(-4) M, respectively.

Bacillus megaterium↗
PubMed1993

A complex regulatory element from the yeast gene ENO2 modulates GCR1-dependent transcriptional activation.

The GCR1 gene product is required for maximal transcription of many yeast genes including genes encoding glycolytic enzymes. Transcription of the yeast enolase gene ENO2 is reduced 50-fold in strains carrying a gcr1 null mutation. cis-acting sequences that are sufficient for GCR1-dependent regulation of ENO2 expression were identified by using an enhancerless CYC1 promoter which is not normally dependent on GCR1 for expression. A 60-bp ENO2 sequence that was sufficient to provide high-level, GCR1-dependent transcriptional activation of the CYC1 promoter was identified. This 60-bp element could be subdivided into a 30-bp sequence containing a novel RAP1-binding site and a GCR1-binding site which did not activate CYC1 transcription and a 30-bp sequence containing a novel enhancer element that conferred moderate levels of GCR1-independent transcriptional activation. The 60-bp CGCR1-dependent upstream activator sequence is located immediately downstream from previously mapped overlapping binding sites for the regulatory proteins ABFI and RAP1. Evidence is presented that the overlapping ABFI- and RAP1-binding sites function together with sequences that bind GCR1 and RAP1 to stage transcriptional activation of ENO2 expression.

Base Sequence↗
PubMed1995

Transcription of the human beta enolase gene (ENO-3) is regulated by an intronic muscle-specific enhancer that binds myocyte-specific enhancer factor 2 proteins and ubiquitous G-rich-box binding factors.

To provide evidence for the cis-regulatory DNA sequences and trans-acting factors involved in the complex pattern of tissue- and stage-specific expression of the beta enolase gene, constructs containing fragments of the gene fused to the chloramphenicol acetyltransferase gene were used in transient-transfection assays of C2C12 myogenic cells. Deletion analysis revealed the presence of four major regions: two negative regions in the 5'-flanking sequence, a basal promoter region which directs expression at low levels in proliferating and differentiated muscle cells, and a positive region within the first intron that confers cell-type-specific and differentiation-induced expression. This positive regulatory element is located in the 3'-proximal portion of the first intron (nucleotides +504 to +637) and acts as an enhancer irrespective of orientation and position from the homologous beta enolase promoter or the heterologous thymidine kinase promoter, conferring in both cases muscle-specific expression to the linked reporter gene. Deletion of a putative myocyte-specific enhancer factor 1 (MEF-1) binding site, containing a canonical E-box motif, had no effects on muscle-specific transcription, indicating that this site is not required for the activity of the enhancer. Gel mobility shift assays, competition analysis, DNase I footprinting, and mutagenesis studies indicated that this element interacts through an A/T-rich box with a MEF-2 protein(s) and through a G-rich box with a novel ubiquitous factor(s). Mutation of either the G-rich box or the A/T-rich box resulted in a significantly reduced activity of the enhancer in transient-transfection assays. These data indicate that MEF-2 and G-rich-box binding factors are each necessary for tissue-specific expression of the beta enolase gene in skeletal muscle cells.

Animals↗
PubMed1993

A primary cell culture system of luteinizing hormone releasing hormone neurons derived from embryonic olfactory placode in the rhesus monkey.

The purpose of this study is to establish a primary LHRH cell culture system using embryonic olfactory placode and to examine whether LHRH cells derived from olfactory placode and the migratory pathway of LHRH neurons mature in vitro. Six monkey fetuses at the ages of E34-E36 were delivered surgically and the area including the olfactory placode (PL) and the areas that encompass the migratory pathway (MP) were dissected out. The tissues were cut into small pieces and plated on collagen- or poly-L-lysine-coated glass coverslips in medium M199. Cultures were maintained for up to 33 days and immunostained for LHRH, GnRH-associated peptide, neurofilament protein, neuron-specific enolase, and glial fibrillary acidic protein. LHRH positive cells were also positive for neurofilament proteins neuron-specific enolase, and GnRH-associated peptides, but negative for glial fibrillary acidic protein. In the first week of culture, LHRH cells remained within the explants of PL, were rounded (average dimensions: 13.0 x 11.3 microns) and stained lightly. By the second week a number of LHRH cells (15.7 x 13.6 microns) with neurites started to migrate out from PL explants, whereas some still remained in the PL. By the third week a large number of LHRH cells (19.3 x 9.4 microns) had migrated out from the PL. They were fusiform in shape with clear nuclei and extended long varicose neurites up to 500 microns in length. A few "pioneer" LHRH cells appeared to lead the migration of 100-400 LHRH cells forming 1-3 major migratory paths. In contrast, LHRH cells from MP explants migrated out sooner than those from PL explants. LHRH cells from the ventral part of the MP, which is close to the PL, migrated out by 1-2 weeks and formed several migratory paths, whereas LHRH cells from the dorsal part of the MP, which is farther from the PL, were scattered widely around explants and their neurites were extended tortuously. Cultured LHRH cells released LHRH into the media and responded to challenge with high K+. The results indicate that 1) primary LHRH neurons can be obtained from the embryonic PL and their migratory pathway, 2) these neurons migrate and mature in culture and 3) they are accessible for cellular and molecular studies.

Animals↗
PubMed2002

Desmoplastic astrocytoma of infancy: a case report with histopathologic and immunohistochemistry profile.

The clinicopathological features and immunohistochemistry profile of desmoplastic cerebral astrocytoma of infancy are discussed in a 4 month old male infant who presented with an increasing head circumference more pronounced in the last two weeks prior to admission to the University Pediatric Hospital. This is a rare tumor that occurs in infants within the first two years of life and it is characterized by a massive, often cystic, supratentorial lesion usually in the frontoparietal region. It has a biphasic histologic pattern with an astrocytic and desmoplastic component and a good prognosis after total or near total surgical resection. This patient represents the first case of desmoplastic cerebral astrocytoma of infancy diagnosed in the Puerto Rico Medical Center.

Astrocytoma↗
PubMed2004

In vitro culture and induced differentiation of adult rat neural stem cells from the corpus striatum.

OBJECTIVE: To investigate the in vitro multipotential differentiation of neural stem cells from adult rat corpus striatum. METHODS: The neural stem cells isolated from adult rat corpus striatum were cultured in serum-free medium to obtain cell suspension before monoclonal subculturing and differential induction. Immunocytochemical staining and reverse transcriptional PCR (RT-PCR) were performed to identify the properties of the differentiated cells. RESULTS: Numerous cell clusters were formed in the phase of monoclonal culture, and different types of cells were observed 3 d after induction with fetal bovine serum. The differentiated cells contained cells positive for nestin, neuron-specific enolase (NSE) positive cells, and glial fibrillary acidic protein (GFAP) positive cells. RT-PCR identified expressions of the transcripts for neural cell-associated genes including brain factor-1, gamma-aminobutyric acid alpha-receptor gamma-subunit, tyrosine hydroxylase and tryptophan hydroxylase. CONCLUSION: The cells separated from adult rat corpus striatum possess the ability of self-proliferation and multipotential differentiation, and are identified as the stem cells of the central nervous system.

Animals↗
PubMed2005

[Study on effect of danshensu in directional differentiation of mesenchymal stem cells into neuron-like cells].

OBJECTIVE: To study the effect of Danshensu (DSS) on directional differentiation of mesenchymal stem cells (MSC) into neuron-like cells. METHODS: MSC were separated from bone marrow with density gradient centrifugation, wall sticking screening and amplified in vitro. Flow cytometry was used to monitor the expression of surface antigens. DSS contained in non-serum L-DMEM was used to induce differentiation of MSC to neuronlike cells, and the effect of DSS when different concentration and acting time used was explored. And levels of neuron-specific enolase (NSE), neurofilament protein (NF-M), nestin, and expression of glial fibrillary acidic protein (GFAP) were measured by immunohistochemical method. RESULTS: After being propagated and amplified in vitro, MSC were positively expressed for CD29, CD44, CD166, and negatively expressed for CD14, CD34, CD45, HLA-DR. After induction of DSS, MSC exhibited the typical form of perikaryon with pyknotic cell body and prominence projected like that of neuron. These cells were positively expressed in NSE, NF-M and nestin, and negatively expressed in GFAP. CONCLUSION: DSS could induce differentiation of MSC to neuron-like cells in vitro, the action is concentration- and time-dependent.

Bone Marrow Cells↗
PubMed2005

[Tumor markers of childhood cancer].

Tumor markers are the substances which are produced from malignant cells and are detectable from peripheral blood or body fluid. These markers are used for the evaluation of treatment effectiveness or the detection of relapse. In most cases of pediatric cancer, specific molecular abnormalities of tumor cells have been able to be identified. Evaluation of these molecular markers is critical for the diagnosis and the choice of treatment. Recently, these molecular markers have also come to be used for the detection of minimal residual disease. Such a system can be regarded as a kind of tumor marker.

Biomarkers, Tumor↗
PubMed1990

Immunohistochemical studies of retinoblastoma.

Various immunohistochemical markers were detected in 27 cases of human retinoblastoma by immunohistochemical avidin-biotin-peroxidase complex and peroxidase-antiperoxidase techniques using mono- and polyclonal antibodies. The glial markers, glial fibrillary acidic protein and Leu-7, were detected in the natural retinal component and perivascular glial cells within the tumor mass. In 4 among 27 cases Leu-7 was positive in the glial cells which were interspersed randomly among tumor cells and not associated with blood vessels. The neural marker, neuron-specific enolase, stained strongly positive in undifferentiated tumor cells and weakly positive in rosettes and fleurettes in most of the cases (21/27). Opsin, the photoreceptor marker, which is present only in the outer segment of photoreceptor cells, was detected in tumor cells of fleurettes in one case and also in rosettes in 6 cases. These results support the view that retinoblastoma has a predominantly neuronal nature with significant photoreceptor-like differentiation and rarely glial differentiation.

Adult↗
PubMed1989

[Composite carcinoma of the prostate combining a small cell carcinoma and an adenocarcinoma. Apropos of a case].

A case of combined adenocarcinoma and small cell carcinoma of the prostate is described in a 58-year-old-man. Prostatic acid phosphatases and neuron specific enolase were found elevated in the serum. At autopsy the lung was free of tumor. The liver was replaced by numerous metastatic nodules and a voluminous mesenteric metastasis extended into the wall of the vessels (aorta and vena cava). Microscopic examination showed a small cell carcinoma component of the oat cell type and an adenocarcinoma component constituting 10% of the total tumor volume. By immunostaining, the small cell carcinoma component is neuron specific enolase+ and prostatic specific antigen-. The adenocarcinoma component is neuron specific enolase- and prostatic specific antigen+.

Acid Phosphatase↗
PubMed1986

[Clinicopathological study of supratentorial tumors with multipotential differentiation in childhood. Primitive neuroectodermal tumor].

Three cases of supratentorial tumor in childhood were studied clinico-pathologically in an attempt to clarify its histological character. Case 1: A 3-year-old boy. Carotid angiogram revealed avascular lesion in the left parietal lobe. Twice operations and radiotherapy were performed. Ten months after the second operation, he died. Surgical specimen at the first operation was composed mainly of round tumor cells. The tumor tissue contained many collagen fibers. At the periphery of this tissue, medulloblastomatous areas consisting of closely aggregated hyperchromatic small round cells were found. There were perivascular rosettes and Homer Wright rosettes. In part, tubular and papillary arrangement of cells was also present. Astrocytomatous and oligodendrogliomatous structures were also present. Surgical specimens at the second operation showed the predominance of sarcomatous areas consisting of spindle-shaped cells with abundant argyrophilic fibers. Case 2: A 10-year-old girl. CT scan revealed a heterogeneous enhanced mass with a cyst and calcification in the right parietal lobe. Operation and radiotherapy were performed. Twelve months after operation, she is still alive. Most of the surgical specimens showed sarcomatous structure with abundant argyrophilic fibers. In these tissues, there were medulloblastomatous and ependymomatous features. Papillary arrangement of cells was also present. In part, there was oligodendrogliomatous structure. Case 3: A 2-year-old girl. CT scan revealed a heterogeneous enhanced mass in the right frontal lobe. The tumor tissue was composed of an aggregation of undifferentiated small round cells with Homer Wright rosettes.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Neoplasms↗
PubMed1975

Manual determination of intra-erythrocytic 2,3-diphosphoglycerate in whole blood by enzymatic analysis, and comparison with ion-exchange chromatography.

A manual modification of an automated method [Atkinson, K. F., Clin. Chem. 18, 1001 (1972)] for enzymatic assay of 2,3-diphosphoglycerate is described, which is suitable for small laboratories. Samples are easily prepared for analysis, and preparations are stable for several months. Virtually all of the color generated in the colorimetric assay is produced by the diphosphoglycerate-catalyzed reaction. The coefficient of variation for multiple samples from a single subject was 2.4%. Delayed preparation of samples, particularly samples from some acidotic subjects, is shown to result in altered results. The enzyme method is compared with analysis by ion-exchange chromatography.

Chromatography, Ion Exchange↗
PubMed1993

Expression of oligodendrocyte-associated genes in cell lines derived from human gliomas and neuroblastomas.

Two putative human oligodendroglioma cell lines were examined for the expression of the oligodendrocyte-associated genes, 2',3'-cyclic nucleotide-3'-phosphodiesterase, myelin basic protein, myelin proteolipid proteins, and myelin-associated glycoprotein. The expression of these genes also was examined in control astrocytoma and neuroblastoma cell lines. In addition, the expression of the non-oligodendrocyte-specific genes, glial fibrillary acidic protein (GFAP), neuron-specific enolase and neurofilaments (NF) NF-L and NF-M also were examined. All the cell lines expressed 2',3'-cyclic nucleotide 3'-phosphodiesterase, neuron-specific enolase, and vimentin, and none expressed myelin-associated glycoprotein. The "oligodendrocyte-specific" myelin proteolipid protein mRNAs and the "neuron-specific" NF-L mRNA were expressed in the two astrocytoma cell lines, which also expressed GFAP. Expression of intermediate filament protein genes was more restricted. The astrocytoma, neuroblastoma, and oligodendroglioma cell lines expressed only GFAP, NF-M, and cytokeratin K7, respectively. These results: (a) provide molecular data confirming the classification of the two cell lines as oligodendrogliomal and suggest that their molecular profiles are indicative of immature oligodendrocytes; (b) demonstrate the expression of cytokeratins in oligodendrogliomal cell lines and suggest that apparent GFAP expression in oligodendrogliomas detected by immunocytochemical methods may be due to cross-reactivity with cytokeratins, with which they share common polypeptide sequence; and (c) indicate that astrocytoma cell lines can exhibit a "mixed" phenotype, expressing genes associated with fully differentiated oligodendrocytes and neurons.

Astrocytoma↗