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[Expression of endocrine-paracrine cells in prostatic carcinoma].

Endocrine-Paracrine cells (EP cells) in prostatic carcinomas were screened by immunohistochemical tests for neuron specific enolase, chromogranin, and serotonin and by Grimelius method. Formalin fixed, paraffin-embedded sections from 60 prostatic carcinomas were used. EP cells were detected in 16 cases (27%). The number of EP cells in hormone independent prostatic carcinomas were significantly larger than hormone dependent (p less than 0.05) and latent prostatic carcinomas (p less than 0.01). Five cases of prostatic carcinomas with abundant EP cell proliferation died of widespread metastases within 4 years, irrespective of hormone treatment. The pathologic finding was classified into the category of adenocarcinoma, partly showing carcinoid or small cell carcinoma-like features. EP cells were found in perineural invading cancer cells and also immunoreactive to both prostate specific antigen and prostate specific acid phosphatase. It is suggested that the proliferation of EP cells in prostatic carcinomas is related with the sensitivity to hormone treatment.

Acid Phosphatase↗

Potent inhibitor of N-myristoylation: a novel molecular target for cancer.

N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the NH(2) terminus of the glycine residue of a number of important proteins that have diverse biological functions and thus have been proposed as potential targets for chemotherapeutic drug design. Earlier, we demonstrated that NMT is more active in colonic epithelial neoplasms than in corresponding normal-appearing colonic tissue. Furthermore, an increased expression of NMT was also observed in gallbladder carcinoma. In the present study, we report a novel protein inhibitor of NMT. This protein caused a potent concentration-dependent inhibition of human NMT with half-maximal inhibition at 4.5 +/- 0.35 nM. This study will serve as a template for further investigations in the area of protein myristoylation.

Acyltransferases↗

[Mature cystic teratoma of the ovary with a small ganglioneuroma].

A case of ganglioneuroma arising within a cystic mature teratoma of the ovary in a 34-year-old woman is reported. Patient underwent right adnexectomy. The ovary was completely replaced by a bilocular cystic lesion, measuring 8 cm in diameter and filled with adipose tissue and pilosebaceous material. Microscopically the cyst was composed by a mature cystic teratoma containing skin with dermal appendages, fatty tissue and bronchial epithelium. Furthermore a nodule (0.5 cm in size) composed of mature ganglion cells, axons and Schwann cells, was identified. Ganglion cells were positive for NSE and synaptophysin, while Schwann cells stained positively with S100 protein and GFAP. To the best of our knowledgment this is the first reported cases of ganglioneuroma arisen within a cystic mature teratoma of the ovary.

Adult↗

Paraganglioma of the cauda equina. A case report and review of the literature.

A case of paraganglioma of the cauda equina is reported. The patient was a 55-year-old Japanese woman who complained of lower back pain and gradual weakening of the left lower extremity, she was diagnosed as having a spinal cord tumor, and the tumor was removed surgically. Histologically, the tumor was encapsulated, and consisted of solid nests of large, polyhedral epithelioid cells with abundant eosinophilic cytoplasm. The nests were separated from each other by a thin fibrovascular stroma. Grimelius staining revealed neurosecretory granules in the cytoplasm of the tumor cells. This was further confirmed by electron microscopic observation. The tumor cells were immunohistochemically positive for neuron-specific enolase (NSE), but negative for glial fibrillary acidic protein (GFAP). On the basis of the histologic, immunohistochemical and electron microscopic features of the tumor cells, the tumor was diagnosed as paraganglioma. The cauda equina is a rare location for this type of tumor, and only a limited number of cases have been reported.

Cauda Equina↗

Model peptides reveal specificity of N alpha-acetyltransferase from Saccharomyces cerevisiae.

N alpha-Acetylation is a major co-translational modification occurring at the alpha-NH2 group of eukaryotic cytosolic proteins. In order to understand better the specificity of N alpha-acetyltransferase, we used the purified enzyme from yeast (Lee, F.-J. S., Lin, L.-W., and Smith J. A. (1988) J. Biol. Chem. 263, 14948-14955) and synthetic peptides mimicking the NH2 terminus of yeast and human proteins. Alcohol dehydrogenase I-(1-24) and 8 of the 19 synthetic analogues with substitutions at the NH2-terminal residue were N alpha-acetylated with varying efficiency. Penultimate amino acid substitutions, except for proline, had little influence on N alpha-acetylation. Substitution of sequences from N alpha-acetylated proteins into the yeast sequences which cannot be N alpha-acetylated demonstrated that not only the first 3 NH2-terminal residues but also more carboxyl-terminal residues were important for determining the specificity of N alpha-acetyltransferase. Two other peptides mimicking yeast mitochondrial cytochrome c oxidase (subunit VI) and ATPase inhibitor, which are naturally non-acetylated, were efficiently acetylated. In addition, recombinant human alcohol dehydrogenase I and basic fibroblast growth factor, which are naturally N alpha-acetylated, were not acetylated post-translationally.

Acetylation↗

Purification and characterization of neuron-specific gamma gamma enolase from human neuroblastoma: comparison with the brain enzyme.

Neuron-specific gamma gamma enolase was purified from a neuroblastoma tissue obtained at surgical resection. The final preparation showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a mobility identical to that of gamma gamma enolase purified from human brain. The values of specific activity (about 80 units/mg), optimal pH (6.9), and Km for 2-phosphoglycerate (about 3 X 10(-5) M) of gamma gamma enolase purified from neuroblastoma were very similar to those of gamma gamma enolase purified from brain. The results of peptide mapping analysis after limited proteolysis, and amino acid analysis also indicate there was no difference between the enzymes purified from neuroblastoma and brain.

Amino Acids↗

Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase.

The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.

Amino Acid Sequence↗

Detection of neuron-specific gamma-enolase messenger ribonucleic acid in normal human leukocytes by polymerase chain reaction amplification with nested primers.

BACKGROUND: NNE (non-neuronal alpha-enolase) is a glycolytic enzyme detected in most tissues. NSE (neuron-specific gamma-enolase) is detected in normal neurons and tumors such as neuroblastoma. Staining with antibodies against NSE is therefore used to detect neuroblastoma cells invading bone marrow. Since staining of normal leukocytes has been reported we asked whether bona fide NSE is in fact expressed in normal blood and marrow. EXPERIMENTAL DESIGN: We designed nested coding region specific primers for NSE and NNE and, after reverse transcription of mRNA, we amplified the coding region between these primers in a semi-nested polymerase chain reaction. In order to distinguish both iso-mRNAs from each other, we amplified a long (1,047 bp) template in a first round of 30 cycles with primers specific for NNE or NSE. One percent of this product was used in a second round of 30 cycles in which both sense primers and two nested anti-sense primers of alternate specificities yielding shorter products of discernible sizes (768 bp or 619 bp) were added together in the same reaction tube. With this combination of four primers, only that shorter product was amplified to visibility, the specificity of which was homologous to the template produced in the first 30 cycles. Restriction enzyme digestion of the amplified products was used to verify this polymerase chain reaction-based approach for the distinction of isoforms of RNA. RESULTS: This semi-nested polymerase chain reaction clearly allows for the distinction of mRNA for NNE or NSE and shows the presence of transcripts for NSE in normal human leukocytes from blood and bone marrow. CONCLUSIONS: This method exploiting short stretches of nucleotide differences in the coding regions for priming can more generally be applied to the distinction of all isoforms of RNA where nested specific primers can be designed. However, the presence of NSE specific transcripts in normal human leukocytes invalidates the use of this highly sensitive method as a disease marker in neuroblastoma.

Base Sequence↗

Concentrations of nucleotides, nucleosides, purine bases, oxypurines, uric acid, and neuron-specific enolase in the cerebrospinal fluid of children with sepsis.

To determine the effects of sepsis on cerebral energy metabolism, the cerebrospinal fluid adenosine monophosphate, inosine monophosphate, inosine, adenosine, guanosine, hypoxanthine, xanthine, and urate concentrations were determined by high-performance liquid chromatography and the neuron-specific enolase levels by means of an enzyme immunoassay method in 32 children with sepsis, without meningitis, aged between 2 months and 13 years, and in 160 age-matched controls. The septic group had significantly higher cerebrospinal fluid concentrations of inosine, adenosine, xanthine, and urate than controls. These results suggest that sepsis could provoke some degree of neuronal hypoxia and significant alterations of cerebral energy metabolism homeostasis.

Adolescent↗

[Acetate metabolism in Ectothiorhodospira shaposhnikovii growing in the dark].

When the cells of Ectothiorhodospira shaposhnikovii assimilated 1- and 2-14C-acetate for a short period of time in the dark under aerobic conditions, the greatest amount of 14C was found after 5 sec in malate, succinate and aspartate. The content of 14C in these compounds decreased in due time, but increased in phosphoglyceric acid and in phosphoric esters of sugars, citrate, alanine and glutamate. The composition and kinetics of labeled products formed during the assimilation of 14C-acetate by the cells in the dark did not depend on the presence of thiosulfate. The cells of Ectothiorhodospira shaposhnikovii grown in the dark, like those grown in the light, contained all enzymes of the citric acid cycle with an exception of alpha-ketoglutarate dehydrogenase. Moreover, they produced enzymes of the glyoxylate shunt, malate synthase and isocitrate lyase, whose activity was higher than that in cells grown in the light. The activity of ribulosediphosphate carboxylase in cells grown in the dark was much lower than in cells grown under phototrophic conditions in a medium with acetate. Cells grown either in the dark or in the light displayed also the activity of phosphopyruvate carboxylases (E.C. 4.1.1.3.1 and 4.1.1.38) and pyruvate carboxylase (E.C. 6.4.1.1). The results suggest that the utilization of acetate in the dark under aerobic conditions by the cells of E. shaposhnikovii is related to the operation of the glyoxylate cycle and the citric acid cycle.

Acetates↗

Small cell carcinoma of the prostate. II. Immunohistochemical and electron microscopic studies of 18 cases.

To evaluate the histogenesis of small cell carcinoma of the prostate, 18 cases of this tumor (9 pure small cell and 9 combined adeno- and small cell carcinoma) were studied using immunohistochemical methods. Seven of the small cell components also were assessed by electron microscopic examination. Using neuron-specific enolase (NSE), prostatic acid phosphatase (PAP), and prostate-specific antigen (PSA) on tissue sections, three distinctive immunostaining patterns of small cell carcinoma components were identified: staining positive for NSE and negative for PSA and PAP (10 cases), staining positive for PSA and PAP and negative for NSE (3 cases), and negative reaction for all three antigens (5 cases). Electron microscopic study demonstrated neurosecretory granules in two cases. Based on the immunostaining and electron microscopic findings, small cell carcinomas of the prostate appear to be a heterogeneous group of tumors. Some of them are neuroendocrine carcinomas whereas others are poorly differentiated adenocarcinomas or, possibly, reserve cell carcinomas. Differences in immunostaining patterns or presence and absence of adenocarcinoma component do not reflect any differences in the uniformly poor prognosis of small cell carcinomas, in which median survivals is 7.7 months. The authors believe that, because of such heterogeneity, small cell carcinomas of the prostate arise from multipotential prostatic epithelium and that an origin from specific neuroendocrine cells need not be implicated.

Acid Phosphatase↗

Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides.

Analysis of tryptic digests of proteins using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry commonly results in superior detection of arginine-containing peptides compared with lysine-containing counterparts. The effect is attributable in part to the greater stability of the arginine-containing peptide ions associated with the sequestration of the single ionizing proton on the arginine side-chain. Reaction of peptides with O-methylisourea resulted in conversion of lysine to homoarginine residues with consequent improved detection during MALDI-MS. Analysis of the underivatized tryptic digest of the yeast protein, enolase, revealed peptides representing 20% of the protein; the corresponding figure after derivatization was 46%.

Alcohol Dehydrogenase↗

Immunohistochemistry of retinoblastomas.

Immunohistochemical stains using neuronal and glial marker proteins were applied to retinoblastomas tissues from 14 children. Among the neurofilament triplet proteins, NF68Kd positive cells were observed in 12. Few NF160Kd positive cells were noted in 2, and NF210Kd positive cells were not detected. The positive ratio of NF68Kd and gamma-enolase seems to relate to the Flexner-Wintersteiner rosettes. Gamma-enolase positive cells were observed in 13. The distribution in tumor tissues was broader than that of NF68Kd positive cells. The immunoreactivities of NF68Kd were in parallel with those of gamma-enolase. Few GFAP positive cells were present around blood vessels, while S-100 protein and MBP positive cells were never observed. Our results indicate that retinoblastoma possesses predominantly neuronal properties, albeit in an immature form.

Child↗

Clinical pathology of primitive gliomas in the cerebrum.

To clarify the biological features of primitive gliomas in the cerebrum and clearly distinguish them from malignant or anaplastic gliomas and glioblastomas, we studied eight cases clinically and pathologically. Our evaluations included immunohistochemical and electron microscopic observations. We divided the patients into two groups, children and young adults. Most tumours appeared as ring-like, enhanced masses on computed tomography and avascular or ring-like, vascular masses on angiography. Macroscopically, the tumours were well demarcated and contained cysts. Occassionally we found tumour dissemination. Microscopically, the tumours were composed of small, round cells without remarkable structural features. Ependymal, astroglial, and oligodendroglial differentiation was evident, in varying proportions; tumours in which the differentiated areas constituted more than half of the mass were classified as poorly differentiated gliomas. By these criteria, this series comprised four undifferentiated and four poorly differentiated gliomas. Cell anaplasia and polymorphism were rare in both undifferentiated and differentiated areas of the tumours. Immunohistochemical and electron microscopic examinations also revealed glial differentiation. These primitive gliomas appear to be biologically similar, but not identical, to cerebellar medulloblastomas. In this series, five patients died because of recurrence or dissemination. Whole brain and spinal irradiation should be considered after total or subtotal surgical removal.

Adolescent↗

Complex of brain D-phosphoglycerate mutase and gamma enolase and its reactivation by D-glycerate 2,3-bisphosphate.

The dissociabilities of dimeric gamma enolase, alpha enolase, and phosphoglycerate mutase of brain origin were tested using fluorescein isothiocyanate attached covalently to these enzymes. The dissociation constant of dimeric gamma enolase is lower (Kd = 0.03 microM) than that of the alpha enolase (Kd = 3 microM), while dimeric mutase seems to be nondissociable in the concentration range 0.1-10 microM, at pH 7.3 in 50 mM imidazole buffer at 20 degrees C. Interaction of neuron-specific gamma enolase with D-phosphoglycerate mutase was detected with the same fluorescence-labeling technique as well as by a kinetic analysis. The determined dissociation constant of the enolase-mutase complex was found to be in the range 5-40 microM, independent of the technique used. A mixed type of inhibition in the binding of D-glycerate-2-P and mutase to the D-glycerate-2-P binding site on enolase was observed in the absence of D-glycerate-2,3-P2. However, the inhibition of the enolase activity by brain D-phosphoglycerate mutase in the D-glycerate-2-P----phosphoenolpyruvate transformation is almost fully reverted by D-glycerate-2,3-P2, probably via the proper coordination of the active centers in the ternary complex of enolase, D-phosphoglycerate mutase, and their common intermediate, D-glycerate-2-P. The mechanism of intermediate transfer by consecutive enzyme pairs in a nondivergent metabolite flux (around the transformation of D-glycerate-2-P) is examined and conclusions of the present experiments are compared with the results of an extended analysis performed earlier with a divergent metabolite flux (around the transformation of multiusage triosephosphates, D-glyceraldehyde-3-P, and dihydroxyacetone phosphate).

2,3-Diphosphoglycerate↗

Intracerebral administration of antisense oligodeoxynucleotides to GAD65 and GAD67 mRNAs modulate reproductive behavior in the female rat.

Increased GABA activity in the medial hypothalamus (HYP) and midbrain central gray (MCG), but not the preoptic area (POA), facilitates sexual receptivity in the female rat [40]. In the current experiments, ovariectomized females were chronically treated with estrogen (via silastic capsules) to maintain a continuously high level of lordosis response. Administration of crystalline antisense oligodeoxynucleotide to the GABA synthetic enzyme, GAD67, into the HYP and MCG significantly and reversibly reduced lordosis response for 1-2 days, but did not inhibit lordosis when administered into the POA. Administration of a control oligonucleotide, consisting of the same nucleotide bases but in a scrambled sequence, did not significantly modulate behavior when infused into any brain areas. When oligodeoxynucleotide antisense to GAD67 was suspended in oil and then infused into the HYP or MCG it was more effective and resulted in less inter-animal variability. Subsequent experiments involving infusions into the MCG compared the effectiveness of antisense oligonucleotides to the two different forms of GAD, known as GAD65 and GAD67. Oligodeoxynucleotides antisense to the mRNA for either gene were effective at reducing lordosis behavior but with a different time course. Oligonucleotide antisense to GAD67 significantly reduced behavior within 24 h of infusion and there was full recovery by 4 days post-infusion. GAD65 antisense oligonucleotide did not significantly reduce behavior until 48 h post infusion and animals did not fully recover to pretest levels of lordosis until 5 days post-infusion. When antisense oligonucleotide for the two genes was administered simultaneously, the inhibition of lordosis was maximal at 24 h and stayed depressed for 4 days. There did not appear to be an additive effect of the two different antisense oligonucleotides when administered together. Tissue GABA levels in HYP and MCG of individual rats assayed by HPLC were no longer correlated with lordosis score after antisense oligonucleotide infusion but were after infusions of scrambled control oligos. Immunoblotting for the two forms of GAD revealed that GAD67 antisense oligonucleotide infusion led to significant decreases in both GAD67 and GAD65 protein levels as compared to infusions of scrambled control oligo. In addition, the levels of a neuronal marker, neuron-specific enolase, also decreased (although nonsignificantly) suggesting either a temporary shutdown of protein synthesis or a degeneration of GABAergic neurons after GAD67 antisense oligonucleotide infusion.

Animals↗