Adenomyoma associated with high level of CA 125 and CA 19-9: case report.

PURPOSE OF INVESTIGATION: A rare case of increasing CA 125 and CA 19-9 levels increasing in a woman with adenomyoma is described. METHODS: A 39-year-old nullipara woman with CA 125 = 1,796 U/ml and CA 19-9 = 177 U/ml was submitted to abdominal and pelvic MRI, gastric endoscopy, colonoscopy, hysteroscopy, pelvic Doppler and PET scan. None of the exams revealed any apparent malignant disease. RESULTS: Six months of gonadotropin releasing hormone agonist treatment reduced CA 125 and CA 19-9 levels. However, after contraceptive pill use the markers were again elevated, and a laparoscopic hysterectomy was performed, and normal CA 125 and CA 19-9 levels were achieved. CONCLUSIONS: Adenomyoma may be associated with high levels of CA 125 and CA 19-9.

Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants.

To understand the mechanisms of mRNA transport in eukaryotes, we have isolated Saccharomyces cerevisiae temperature-sensitive (ts) mutants which accumulate poly(A)+ RNA in the nucleus at the restrictive temperature. A total of 21 recessive mutants were isolated and classified into 16 complementation groups. Backcrossed mRNA transport-defective strains from each complementation group have been analyzed. A strain which is ts for heat shock transcription factor was also analyzed since it also shows nuclear accumulation of poly(A)+ RNA at 37 degrees C. At 37 degrees C the mRNA of each mutant is characterized by atypically long polyA tails. Unlike ts pre-mRNA splicing mutants, these strains do not interrupt splicing of pre-mRNA at 37 degrees C; however four strains accumulate oversized RNA polymerase II transcripts. Some show inhibition of rRNA processing and a further subset of these strains is also characterized by inhibition of tRNA maturation. Several strains accumulate nuclear proteins in the cytoplasm when incubated at semipermissive temperature. Remarkably, many strains exhibit nucleolar fragmentation or enlargement at the restrictive temperature. Most strains show dramatic ultrastructural alterations of the nucleoplasm or nuclear membrane. Distinct mutants accumulate poly(A)+ RNA in characteristic patterns in the nucleus.

Differentiation-dependent expression of laminin-8 (alpha 4 beta 1 gamma 1) mRNAs in mouse 3T3-L1 adipocytes.

We report that laminin-8 (alpha 4 beta 1 gamma 1) is the specific isoform of laminin synthesized in adipocytes. Reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from mouse 3T3-L1 cells with paired primers for alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, beta 1, beta 2, beta 3, gamma 1 and gamma 2 laminins yielded amplified fragments only for alpha 4, beta 1 and gamma 1. A polyclonal antibody against mouse laminin-1 (alpha 1 beta 1 gamma 1) precipitated alpha 4 in addition to beta 1 and gamma 1, while the antibody against a deduced peptide sequence of mouse alpha 4 in addition to beta 1 and gamma 1 in addition to alpha 4. Thus, laminin-8 (alpha 4 beta 1 gamma 1) is the only isoform expressed in 3T3-L1 cells. Northern blots showed that the levels of alpha 4, beta 1 and gamma 1 mRNAs increased 2.5-fold during adipose conversion of 3T3-L1 cells. A 1062 bp cDNA fragment cloned by RT-PCR demonstrated a polymorphism in the mouse alpha 4 gene which would lead to five amino acid changes in the domain G.

Cloning and molecular characterization of the acetamidase-encoding gene (amdS) from Aspergillus oryzae.

We have isolated an acetamidase-encoding gene (amdS) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus nidulans gene as a probe. The gene is located on a 3.5-kb SacI fragment and its nucleotide (nt) sequence was determined. Compared with the A. nidulans amdS gene, the coding region of A. oryzae gene consists of seven exons interrupted by six introns and encodes 545 amino acid (aa) residues. The deduced aa sequence has a high degree of homology with that of the A. nidulans acetamidase protein. Three introns (IVS-1, IVS-2, and IVS-4) exist at the same positions as those of A. nidulans amdS, whilst three additional introns (IVS-3, IVS-5, and IVS-6) are also present. There is no preference in its codon usage (G + C content in the third position of codons is 51%). Gene disruption experiments demonstrate that the resulting mutants show significantly reduced growth on acetamide-containing medium, indicating that the A. oryzae amdS gene encodes a functional acetamidase that is required for acetamide utilization. Transcriptional analysis by Northern blot reveals a 1.8-kb transcript in RNA extracted from mycelium grown in medium containing acetamide or acetate plus beta-alanine as the sole carbon and nitrogen sources.

Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae.

The glucoamylase-encoding gene (glaA) from Aspergillus oryzae was cloned using its cDNA as a probe, which had been isolated previously. From comparison of nucleotide (nt) sequences of genomic clones with its cDNA, the glaA gene was found to contain four short putative introns, 45-56 nt in length. The A. oryzae glaA gene shared 62% homology at the nt level with the A. niger glaA gene with the four introns located at the same position. The 5'-flanking region contained a TATA box at nt-72 from the start codon, and two putative CAAT sequences at nt-87 and -331. Genomic Southern analysis and physical mapping showed that the glaA gene is located on the smallest chromosome (3.4 Mb) of six separated bands of chromosomes. Clones containing the glaA gene, when re-introduced intro A. oryzae, resulted in a three- to eightfold increase in glucoamylase activity.

Disulfide-bonding between Drosophila laminin beta and gamma chains is essential for alpha chain to form alpha betagamma trimer.

Assembly of Drosophila laminin alpha, beta and gamma chains was analyzed by immunoprecipitation of the lysate from metabolically radiolabeled Kc 167 cells with chain-specific antibodies followed by two dimensional electrophoresis in which non-reducing and reducing SDS gel electrophoresis are combined. Precipitation of monomeric beta (or gamma) with anti-gamma (or -beta) antibody revealed that beta and gamma form stable dimer before they are disulfide-bonded to each other. In contrast, alpha associates with neither monomeric beta, monomeric gamma nor betagamma dimer without disulfide-bonding but only with disulfide-bonded betagamma dimer to form alpha betagamma trimers. These results thus demonstrated that the interchain disulfide-boding between beta and gamma is essential for alpha to form alpha betagamma trimer. We also found that the alpha betagamma trimer can be secreted with alpha chain either disulfide-bonded or not bonded to the disulfide-bonded betagamma dimer.

Isolation of a new lytic enzyme for hiochi bacteria and other lactic acid bacteria.

A microorganism producing a lytic enzyme preparation that could rapidly lyse bacterial cells such as hiochi bacteria and other lactic acid bacteria was screened. The microorganism was identified as Streptomyces fulvissimus. The enzyme produced by this organism lysed boil-denatured cells quicker than intact cells of hiochi bacteria. A mutant strain of S. fulvissimus producing the enzyme exhibiting high activity against intact cells of hiochi bacteria was screened on plates, containing intact cells inactivated with UV irradiation. The optimal pH for lytic activity against intact cells of the hiochi bacterium Lactobacillus casei S-4 was from 3.5 to 4.0, and the optimum temperature was close to 50 degrees C. This enzyme activity was stable between pH 3.5 and pH 8.0 and up to 60 degrees C. The enzyme exhibits N-acetyl glucosaminidase and muramidase activities. The effects of adjusting the pH and using different inducers for enzyme production were investigated. Chitin was the most effective inducer of enzyme production. Intact DNA was easily isolated from the cells of many lactic acid bacteria following lysis with the enzyme. It is thought that this enzyme will be a good biotechnological tool.

Potential molecular chaperones involved in laminin chain assembly.

To explore potential molecular chaperones involved in the intracellular assembly of laminin chains, bovine aortic endothelial cells were treated with a thiol cleavable divalent cross-linking reagent, dithio-bis-(succinimidylpropionate), and cellular proteins cross-linked to laminin chains were co-immunoprecipitated with anti-laminin antiserum. Sodium dodecylsulfate (SDS) gel electrophoresis of the precipitate under reducing condition showed polypeptides with estimated sizes of 80, 60 and 50 kDa together with laminin chains. Two dimensional electrophoresis, in which non-reducing and reducing SDS electrophoresis were combined, suggested that many molecules of these polypeptides were cross-linked to each laminin chain. Sepharose CL-4B beads conjugated with E8 fragment of mouse laminin-1 was prepared. Affinity chromatography with the beads of microsomal proteins from rat liver showed that Bip and HSP70 associated to laminin chains and dissociated upon ATP hydrolysis. Protein-disulfide isomerase also showed affinity to the column. GRP94 and calnexin showed strong affinity and were washed out only with a detergent solution. Thus, many molecular chaperones are suggested to be involved in the intracellular assembly of laminin chains.

Three heterotrimeric laminins produced by human keratinocytes.

Laminins are a family of glycoproteins composed of alpha,beta and gamma chains. Five alpha(alpha1-alpha5), three beta (beta1-beta3) and twogamma (gamma1 and gamma2) chains have been cloned fromhuman and their replaceable assembly into heterotrimers producesthe variety of laminins. Reverse transcription-polymerase chainreaction of mRNAs showed that human keratinocytes express thealpha3, alpha5, beta1, beta3, gamma1 andgamma2 genes at high level among the ten cloned lamininchains. Western blot and immunoprecipitation of the cell lysatewith antiserum directed against mouse laminin-1(alpha1beta1gamma1) detected two trimers with thecomposition of alphaxbeta1gamma1 (probablylaminin-10 with the composition of alpha5beta1gamma1and alphaybeta1gamma1. Meanwhile, antiserum directedagainst a synthetic peptide of human alpha3 detected onlyalpha3beta3gamma2 trimer (laminin-5). We thus show thatkeratinocytes produce three heterotrimeric laminins. We couldnot detect the assembly of alpha3 with beta1 and gamma1chains to form alpha3beta1gamma1 (laminin-6) in keratinocytes.

Electrophoretic karyotype and gene assignment to chromosomes of Aspergillus oryzae.

An electrophoretic karyotype of Aspergillus oryzae was obtained by transverse altering-field electrophoresis. Seven chromosomal bands were found. With Schizosaccharomyces pombe chromosomes as size standards, we estimated the sizes of the chromosomes to be 7.0, 5.2, 5.0, 4.5, 4.0, 3.7, and 2.8 megabase pairs (Mbp). The chromosomal DNA bands were identified with use of 13 cloned genes including ribosomal DNA. The intensity of ethidium staining and the results of Southern blotting with 100 random clones isolated from A. oryzae suggested that the smallest band migrated as doublet and that the total genome size was approximately 35 Mbp.

Involvement of growth-associated protein-43 with irreversible neurite outgrowth by dibutyryl cyclic AMP and phorbol ester in NG108-15 cells.

Simultaneous treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) and dibutyryl cyclic AMP (diBu-cAMP) for 72 h induced neurites in NG108-15 cells significantly longer than treatment with each alone. Treatment for 72 h with both drugs induced irreversible neurite extension and a decline in protein kinase C activity, although neurites extended by diBu-cAMP alone disappeared after the withdrawal of the drug. The expression of growth-associated protein-43 (GAP-43) mRNA was also observed by a combined application of TPA and diBu-cAMP. The increased level of GAP-43 mRNA induced by treatment with both drugs for 72 h was maintained at least 24 h after withdrawal of the drugs. In cells transfected with GAP-43 cDNA, neurites induced by treatment with diBu-cAMP alone for 72 h were maintained at least 48 h after removal of the drugs. These results suggest that GAP-43 could be involved in the maintenance of elongated neurites and that a decline in protein kinase C activity may be involved in the accumulation of GAP-43.

Cloning, characterization and overproduction of nuclease S1 gene (nucS) from Aspergillus oryzae.

The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced. It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in length. The nucS gene had an open-reading frame of 963 base pairs and coded for a protein of 287 amino acid residues, comprising the signal peptide of 20 amino acids and a mature protein of 267 amino acids. The deduced amino acid sequence agreed well with the published amino acid sequence except for one substitution. Southern hybridization analysis showed that the nucS gene existed as a single copy in the A. oryzae chromosome. When the structural gene of nucS was fused with the promoter of the glaA gene and introduced into A. oryzae, the yield of secreted nuclease S1 increased about 100-fold compared with the recipient strain.

High level secretion of calf chymosin using a glucoamylase-prochymosin fusion gene in Aspergillus oryzae.

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1-603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1-511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.

A novel culture method for high level production of heterologous protein in Saccharomyces cerevisiae.

A high level production system for heterologous protein by cold culture of yeast transformants at 15 degrees C was developed. The yeast transformants, carrying a plasmid containing cDNA for Aspergillus oryzae alpha-amylase (Taka-amylase A) or human lysozyme synthetic DNA, were cultivated in a selective medium for 1 or 2 days until full growth at 30 degrees C. The yeast cells were harvested by centrifugation from the culture fluid and then were transferred to YPD medium. These inoculated broths were incubated for 2 days at 15 degrees C and then for another 2 days at 30 degrees C. By the cold culture method described above, higher amounts of Taka-amylase A (28.6 mg/liter) and human lysozyme (6.1 mg/liter) were produced by the yeast transformants compared to those by conventional methods. Heterologous protein productions using YEp, YCp, and YIp types of yeast expression vectors with ADH1 or GAPDH promoter by the cold culture method showed effective productivity of about 2-fold compared to those by the conventional method of culture at 30 degrees C. The high level production of heterologous protein by this method was not specific to the S. cerevisiae strains examined.

Characteristic expression of three amylase-encoding genes, agdA, amyB, and glaA in Aspergillus oryzae transformants containing multiple copies of the agdA gene.

In Aspergillus oryzae wild-type strains, the expression of the agdA gene encoding alpha-glucosidase (AGL) is induced by maltose at the transcriptional level in a similar manner to the amyB gene encoding Takaamylase A (TAA) and the glaA gene encoding glucoamylase (GLA). In A. oryzae transformants containing multiple copies of the agdA gene, a high-level of AGL activity was observed. This was accompanied by a significant reduction in TAA and GLA activities. Moreover, transformants with the highest AGL activity showed the lowest degree of TAA and GLA activities. Northern blot analyses showed that the transcriptional levels of amyB and glaA in the AGL-overproducing transformant were drastically reduced when large amounts of agdA mRNA were detected in maltose-grown mycelia. In addition, the glucose concentration of the maltose-containing medium that was used to grow the AGL-overproducing transformant for RNA extraction was higher than that of the control transformant. These results suggest that the reduced expression of the amyB and glaA genes in the AGL-overproducing transformant was due to either titration of a common regulatory protein(s) involved in maltose induction or carbon catabolite repression.

Cloning and nucleotide sequence of the ribonuclease T1 gene (rntA) from Aspergillus oryzae and its expression in Saccharomyces cerevisiae and Aspergillus oryzae.

A genomic DNA encoding ribonuclease (RNase) T1 from Aspergillus oryzae was cloned using a synthetic oligonucleotide probe. The cloned gene (designated rntA) encoded functional RNase T1, since an A. oryzae transformant with multiple copies of the rntA gene showed higher RNase T1 activity (over 200 times) than a transformant with a vector. A cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) with primers corresponding to the 5' terminus and 3' terminus of the reading frame of the rntA gene. Nucleotide sequencing analysis of both DNAs found that RNase T1 had a prepro-sequence consisting of 26 amino acids and the rntA gene had only one intron (114 bp) in the region encoding the signal sequence. The A. oryzae transformant with cDNA controlled by the amyB promoter also showed higher activity (over 300 times), indicating that the cloned cDNA encoded functional RNase T1. On the other hand, the Saccharomyces cerevisiae transformant with cDNA controlled by the GAL1 promoter could not grow on a medium containing galactose. These results suggests that A. oryzae may have a protection mechanism from RNase T1.

Cloning and nucleotide sequence of the acid protease-encoding gene (pepA) from Aspergillus oryzae.

We have cloned a genomic DNA sequence encoding the acid protease (PEPA) from Aspergillus oryzae using a 0.6-kb fragment as a probe. This fragment was amplified by the polymerase chain reaction (PCR) using oligonucleotide primers designed from the partial amino acid sequences of peptide fragments of the purified protein. Nucleotide sequencing analysis has shown that the cloned gene (designated pepA) encodes 404 amino acid residues and contains 3 putative introns ranging in length from 50 to 61 nucleotides. The deduced amino acid sequence of the A. oryzae PEPA has a high degree of homology (67%) to the A. awamori PEPA. Comparison with the amino acid sequence of A. awamori PEPA suggests that the A. oryzae PEPA may consist of a 78 amino acid prepro-peptide and 326 amino acid mature protein. The amino acid composition of the mature protein was almost consistent with that of the acid protease purified from A. oryzae reported previously. Southern hybridization analyses showed that the pepA gene exists as a single copy in the A. oryzae chromosome. The cloned gene was found to be functional, since transformants of A. oryzae containing multiple copies of the pepA gene showed a 2-6 fold increase in acid protease activity compared with the recipient strain.

Secretion of calf chymosin from the filamentous fungus Aspergillus oryzae.

Active calf chymosin was secreted from Aspergillus oryzae transformants when the chymosin cDNA was expressed under the control of glucoamylase gene (glaA) promoter. Secreted prochymosin was autocatalytically activated to the chymosin (0.07-0.16 mg/l). Western blot analysis showed that a secreted protein immunoreactive with an anti-chymosin antibody was of similar size to authentic chymosin. Northern blot analysis revealed that mRNA of the chymosin cDNA was expressed at as high level as that of the glaA gene. The size and the level of the transcript were different among transformants, due to the integration position of the plasmid on the chromosome.

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae.

Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T2 gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T2 revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T2 activity than wild-type strains.