The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of SR proteins and influence pre-mRNA splicing.

The three members of the Clk family of kinases (Clk1, 2, and 3) have been shown to undergo conserved alternative splicing to generate catalytically active (Clk) and inactive (ClkT) isoforms. The prototype, murine Clk1 (mClk1), is a nuclear dual-specificity kinase that can interact with, and cause the nuclear redistribution of, SR proteins. In this study, we demonstrate that the human Clk2 and Clk3 (hClk2 and 3) are also found within the nucleus and display dual-specificity kinase activity. The truncated isoforms, hClk2(T) and hClk3(T), colocalize with SR proteins in nuclear speckles. We also show catalytically active hClk2 and hClk3 cause the redistribution of SR proteins and can regulate the alternative splicing of a model precursor mRNA substrate in vivo.

Antibody response to keyhole limpet hemocyanin (KLH) treatment in patients with superficial bladder carcinoma.

The dynamics of specific KLH-antibody production after intracutaneous and intravesical instillation was analysed. Nine patients (male, n = 7; female, n = 2, mean age 68.6 years, range 47-75) with primary superficial carcinomas of the bladder were intracutaneously immunized with 1 mg Keyhole limpet hemocyanin (KLH) after the complete resection of the tumors. Treatment was continued for 6 consecutive weeks, monthly for one year and thereafter bimonthly for 2 subsequent years, consisting of 20 mg KLH in 20 ml saline introduced intravesically. The antibodies against KLH in patient sera were determined by means of a specially developed direct enzyme-linked immunosorbent assay (ELISA; according to H. von der Kammer, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany). Blood was taken for antibody-titer examination before treatment and 8 weeks after treatment. The KLH-antibody titer increased significantly (Mann-Whitney-Test P = 0.02) after KLH therapy in bladder cancer patients, however the level varied considerably from patient to patient. 6 of 9 patients (67%) presented increased serum antibody titers to KLH after immunotherapy. 4 patients (44.4%) remained free of tumor during the established follow-up period of 10-45 months (median 30.7 months). One patient without increased antibody titer to KLH was free of tumor, 2 patients however, suffered from tumor recurrence after the KLH course. 2 patients presented with tumor recurrence in spite of increased antibody titers. No evidence of tumor progression occurred in patients with recurrence after KLH therapy. 4 of 5 patients (80%) without tumor recurrence presented with a positive skin test. Of patients with tumor recurrence, 50% had a negative skin test. 44.4% KLH-treated patients had tumor recurrence The recurrence rate was 1.6. The time to recurrence was 8.75 months. KLH instillation did not induce major side effects. Positive skin test reactivity and KLH antibody response were more commonly seen in responding patients (i.e. those who remained tumor free after therapy) than in non-responders. The production of KLH antibodies, apparently is the biological response to the antigen stimulus of KLH.

The gene for the major protein (SVSP109) of bovine semen: structure and promoter analysis.

We have characterized the gene of SVSP109, specifying the bovine secretory protein SVSP109, which is synthesized in a tissue- as well as species-specific manner. Approximately 1.3 kb upstream of the SVSP109 gene, the 3'-end of another gene designated HG5 was identified. The HG5 gene fragment comprises exon (n-1) and exon (n), separated by an intron. Exon (n) contains the complete 3'-untranslated region, whereas exon (n-1) encodes the C-terminal part of a hitherto unknown protein with high homology to SVSP109. The sequenced region of 6956 bp of a bovine genomic clone comprised the complete SVSP109 gene, which is made up of five exons and four introns. Primer extension analysis and RTPCR of poly(A+)RNA from seminal vesicle revealed that the first exon 1 extends to a position 34 bp downstream of the TATA sequence. Employing a CAT assay, a definitive but weak promoter activity was detected in pCATeSVSP15 (base pairs -639 to +574) and pCATeSVSP10 (base pairs -639 to +198); pCATeSVSP6 (base pairs -262 to +65) displayed promoter activity similar to the positive control. We conclude from these results that the TATA sequence located at position -34 is part of the functional promoter of the SVSP109 gene.

Promoter analysis of the bovine gene for seminalplasmin.

In this study we mapped the transcriptional initiation site of the gene for seminalplasmin (SAP) by primer extension analysis, situated 125 nucleotides upstream of the translational initiation site of the SAP-specific mRNA. We showed that the TATA-box in position -30 of the SAP gene is part of a functional promoter. A 280 bp region of the 5'-flanking region exerted a strong positive effect on promoter activity. In this region we identified consensus sequences for the transcriptional control elements AP1, AP2, PEA3 and GATA.

A monoclonal antibody generated against a recombinant peptide fragment of the B3 domain of carcinoembryonic antigen reacts with intact carcinoembryonic antigen.

The chemical synthesis of a gene coding for a polypeptide of 77 amino acid residues (designated ceaB3) representing a fragment of the CEA-B3 domain of carcinoembryonic antigen (CEA) was achieved. The ceaB3 fragment was cloned into the plasmid pLZPWB1 at the C-terminus of a derivative lacZMF of the lacZ gene, devoid of methionine and cysteine amino acid residues. The fusion protein lacZMF-ceaB3 represented approx. 30% of total proteins expressed after induction. The fusion protein was formed as inclusion bodies. Simple washing steps led to an insoluble fusion protein which was of approx. 80% purity. Another fusion gene was generated by inserting ceaB3 between the malE gene encoding maltose binding protein (mbp) and lacZ alpha of the pmal-c2 vector. Expression of the resulting pmal-c2-ceaB3-lacZ alpha yielded the fusion protein mbp-ceaB3-lacZ alpha with a molecular mass of 57.94 kDa, which was obtained as a soluble protein in almost homogeneous form after affinity chromatography employing amylopectin. Polyclonal sheep anti-CEA antiserum specifically reacted with fusion proteins lacZMF-ceaB3 and mbp-ceaB3-lacZ alpha. A monoclonal antibody CEA/HK2 was generated employing lacZMF-ceaB3 for immunization and CEA for screening purposes. The mAB CEA/HK2 specifically recognized CEA in immunoblots. The described experimental strategy should be generally applicable for generation of fusion proteins. These fusion proteins are suitable for epitope characterization of existing antibodies, production of regiospecific polyclonal or monoclonal antibodies.

Characterization by cDNA cloning of two new human protein kinases. Evidence by sequence comparison of a new family of mammalian protein kinases.

Two new human cDNAs, designated phclk2 and phclk3, which have a high identity to the cDNA of the human protein kinase clk, were characterized. Typical features of hclk2 and hclk3 proteins are non-homologous N-terminal regions and the presence of the C-terminal protein kinase domain, which is characteristic for serine/threonine-type kinases. We also identified the differentially spliced forms phclk2(139) and phclk3(152) with deletions of 88 and 97 nt, respectively, which lead to changes in the open reading frames. hclk2(139) and hclk3(152) proteins do not possess a protein kinase domain and are nearly identical to the N-terminal regions of the above-mentioned protein kinases. We verified that differentially spliced variants also exist for hclk1 as well as for a mouse clk protein kinase. It was shown that shorter and longer alternatively spliced mRNAs co-exist in different human tissues. According to Southern analysis, hclk2 and hclk3 appear to be specified by single copy genes. The genes for hclk2 as well as for hclk3 were localized to human chromosomes 1 and 15, respectively.

A human amyloid precursor-like protein is highly homologous to a mouse sequence-specific DNA-binding protein.

From a cDNA sequence, we have deduced the amino acid sequence for a human amyloid precursor-like protein (APPH) with > 92% identity to the CDEI binding protein (CDEBP) of the mouse and the fragmentary rat protein YWK-II of unknown function. Expression of APPH was found in all tissues examined. A striking homology of APPH to human amyloid precursor protein (APP) was observed. Overall identity accounts for 52.7%. However, there are three domains of APPH with remarkably higher similarities, corresponding to amino acid sequence positions 47-204 (76.6%), 308-567 (67.7%), and 694-763 (69.9%). Using an APPH antiserum, we localized APPH in nuclei of human interphase cells and found an increased synthesis of APPH in mitotic cells. Our results indicate that the highly conserved proteins human APPH, mouse CDEBP, and rat YWK-II are apparently homologues of a CDEI binding protein with indispensible function in mammalian genome segregation.

Porcine luteal cells express monocyte chemoattractant protein-2 (MCP-2): analysis by cDNA cloning and northern analysis.

From an expression library in lambda UniZAP, derived from porcine corpus luteum (CL), a clone lambda MCP9 was detected by hybridization with a porcine MCP-1 specific probe. A pBluescriptSK-derivative pMCP9 was generated from lambda MCP9 by in vivo excision and was shown to contain an open reading frame (ORF) encoding a protein highly homologous to bovine monocyte chemoattractant protein-2 (MCP-2). Comparison of amino acid sequences of known MCPs identified the protein encoded by pMCP9 as porcine MPC-2. The 3' untranslated region of pMCP9 was completed by 3' RACE. Northern analysis using RNA from porcine luteal cells and probes specific for porcine MCP-1 and MCP-2 revealed that porcine luteal cells express both MCPs. According to Southern analysis MCP-2, like MCP-1, is specified by a single copy gene.

Characterization of the bovine monocyte chemoattractant protein-1 gene.

Bovine monocyte chemoattractant protein-1 (bovine MCP-1) cDNA has recently been characterized and shown to be highly expressed in bovine seminal vesicles. In an attempt to isolate the MCP-1 gene, we screened a bovine genomic lambda EMBL-3 library with an MCP-1 specific probe pH42. A positive clone lambda MCP1 was subjected to restriction analysis and a pH42-positive EcoRI-fragment of 3.8 kb was subcloned and sequenced. The bovine MCP-1 gene consists of three exons separated by two introns. We undertook to identify homologous sequences 5'-upstream of the transcriptional start site within the genes of rat, mouse, human and bovine MCP-1. Six conserved sequence stretches located within 400 bp upstream of the transcriptional start site in MCP-1 genes were detected. Interestingly an approximately 45 bp long region, displaying identities in the range of 68-72% relative to the human sequence, covers in the rat gene the -141/88 region responsible for TPA induction of gene expression.

Localization and structural characterization of an oligosaccharide O-linked to bovine PDC-109. Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa.

PDC-109 (13 kDa) is the most abundant component, and the major heparin-binding protein, of bovine (Bos taurus) seminal plasma. Here, we show that PDC-109 contains a single O-linked oligosaccharide (NeuNAc alpha(2-6)-Gal beta(1-3)-GalNAc-) attached to Thr11. Immunoquantitation of PDC-109 indicates that its concentration in seminal plasma is 15-20 mg/ml. Though PDC-109 is not present on epididymal sperm, ejaculated spermatozoa on average are coated with (9.5 +/- 0.3) x 10(6) molecules of PDC-109/cell. This value remained constant in swim-up sperm and decreased to (7.7 +/- 0.4) x 10(6)/spermatozoon after incubation for 24 h in capacitation medium at 39 degrees C. These data substantiate the hypothesis that PDC-109 may be one of the seminal plasma components that enhance the fertilizing capacity of bull spermatozoa upon interaction with heparin-like glycosaminoglycans present in the female genital tract.

Establishment of a partially informative porcine somatic cell hybrid panel and assignment of the loci for transition protein 2 (TNP2) and protamine 1 (PRM1) to chromosome 3 and polyubiquitin (UBC) to chromosome 14.

Porcine lymphocytes and fibroblasts were fused with 3 different permanent rodent cell lines, and 21 stable somatic cell hybrid lines were established. These hybrid cell lines were characterized cytogenetically by sequential QFQ banding and chromosome painting using fluorescence in situ hybridization with porcine DNA. The lines were further characterized by PCR analysis with primer pairs derived from genes with confirmed mapping information. Using this panel, we assigned the locus encoding polyubiquitin (UBC) to chromosome 14, and the transition protein 2 locus (TNP2) and protamine loci (PRM1 and PRM2) to chromosome 3. Two chromosomal localizations have been further refined by radioactive in situ hybridization. UBC maps to chromosome 14q12-q15 and TNP2 to 3p11-p12.

cDNA cloning identified a calmodulin-binding protein in bovine seminal plasma as bovine C-type natriuretic peptide.

A basic protein of apparent molecular weight 15 kD, designated bSVSP15, was purified from bovine seminal vesicle secretion to homogeneity, employing affinity absorption to calmodulin-Sepharose and reverse-phase HPLC. Immunoblotting identified bSVSP15 in bovine seminal plasma and seminal vesicle secretion, but it was not present either in extracts of bovine ampulla, epididymis, and testis or in serum or follicular fluid. When added to cAMP phosphodiesterase, bSVSP15 inhibited the activation of enzymatic activity by calmodulin in a reversible manner. Immunoscreening of a lambda gt11 expression cDNA library from bovine seminal vesicle tissue yielded two positive clones, pSVS4 and pSVS5, which were characterized by sequencing. Both sequences are identical, except for the 3' region. Because the derived amino acid sequence comprises, with an identity of 81%, the amino-terminal 21 residues of bSVSP15, cDNA clones pSVS4 and pSVS5 represent bSVSP15-specific mRNAs. The mature protein bSVSP15 contains 101 residues and is preceded by 25 residues of a signal sequence, characteristic for secretory proteins. Northern analysis identified two bSVSP15-specific mRNAs of 900 bp and 1200 bp, respectively. Sequence comparison yielded high homologies to human C-type natriuretic peptide. We conclude from this result that bSVSP15 is identical with the hitherto unknown bovine C-type natriuretic peptide.

The gene for the amyloid precursor-like protein APLP2 is assigned to human chromosome 11q23-q25.

The human amyloid precursor-like protein APLP2 is a highly conserved homologue of a sequence-specific DNA-binding mouse protein with a predicted function in the cell cycle. Somatic cell hybrids segregating human chromosomes were used to assign the APLP2 gene to chromosome 11. Fluorescence in situ hybridization confirmed this assignment and further localized the gene to q23-q25.

Porcine luteal cells express monocyte chemoattractant protein-1 (MCP-1): analysis by polymerase chain reaction and cDNA cloning.

RT PCR employing poly(A+)RNA from porcine luteal cells and a combination of primers designed from the known bovine MCP-1 cDNA identified the luteal cells as a source of MCP-1. This finding is corroborated by results from Northern analysis using total RNA from luteal cells. To characterize the complete porcine MCP-1 cDNA, poly (A+)RNA was isolated from porcine corpus luteum, transcribed into cDNA and the latter cloned into the expression vector lambda Uni-ZapXR. A digoxigenin-labeled DNA probe of 375 bp was obtained by PCR and employed to screen the library. From the positive clones pMCP5, pMCP7 and pMCP10, the clone pMCP5 was selected and both strands of the cDNA insert were sequenced. The cDNA insert was 742 bp long, with an open reading frame (ORF) encoding a protein of 99 amino acid residues which by comparison with known amino acid sequences of MCPs yielded highest identities with MCP-1 sequences. We therefore assume that pMCP5 encodes the amino acid sequence for porcine MCP-1.

[Adjuvant immunotherapy with keyhole limpet hemocyanin in category PT 2 N+ and PT 3-4, No-N+, Mo renal cell carcinoma]. / Adjuvante Immuntherapie mit Keyhole Limpet Hemocyanin (KLH) beim Nierenzellkarzinom der Kategorie PT 2 N+ und PT 3-4, No-N+, Mo.

This study was a prospective randomized trial to compare adjuvant immunotherapy with Keyhole Limpet Hemocyanin (KLH) after radical nephrectomy. From January 1983 to December 1988, 50 patients underwent radical nephrectomy for category PT 2 N+ and PT 3-4, No-N+, Mo renal cell carcinoma. Postoperatively 25 patients were given adjuvant treatment with the biological response modifier, Keyhole Limpet Hemocyanin (KLH), and 25 patients were in the control group. In each group 2 patients were lost to follow-up. The mean follow-up time was 55 months. Adjuvant treatment with KLH did not appear to improve the prognosis in renal cell carcinoma patients. The 5-year survival rate was 60% in the KLH group and 56.5% in the control group. Progress was seen in 9/23 in the KLH group, 10/23 in the controls. The median survival in patients showing progress was 27 and 28 months in the two groups, respectively. Studies with a combination of low dose cyclophosphamide and KLH revealed a positive immunostimulating effect, which may provide the rationale for further research concerning different KLH doses, schedules or therapeutic combinations.

Cloning of the gene for bovine monocyte chemoattractant protein-2.

Bovine monocyte chemoattractant protein-1 (bovine MCP-1) cDNA has recently been characterized and shown to be highly expressed in bovine seminal vesicles secretory epithelium as well as in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear leukocytes (PMNLs). In an attempt to isolate the MCP-1 gene, we screened a bovine genomic cosmid library with a MCP-1-specific probe pH42. A positive clone, c11/1, was subjected to restriction analysis and fragments probed with pH42 by southern blotting. pH42-positive fragments were subcloned and sequenced. The sequence revealed three exon-like regions that coded for a protein displaying an identity of 51% with bovine MCP-1. Employing this sequence information from c11/1, the c11/1-specific cDNA was generated from poly(A)+RNA of bovine PMNLs by reverse transcription and a combination of polymerase chain reaction (PCR) methods. The assembled c11/1 cDNA comprised a 5' UTR coding region as well as 3' UTR for the gene product c11/1. Amino acid sequence comparison of the bovine c11/1 gene product with human monocyte chemotactic proteins yielded the highest sequence identity with human MCP-2, and it is assumed that the c11/1 gene product represents the bovine MCP-2. The exon/intron structure of the bovine MCP-2 gene was found to be similar to the human MCP-1 gene. The bovine MCP-2 gene consists of three exons separated by two introns. In the 5'-flanking region of the 3.3-kb gene, a TATA box as well as an AP-1 sequence motif were identified. The bovine MCP-2 is specified by a single-copy gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization by cDNA cloning of the mRNA of human ribosomal protein L8.

From a lambda UNI-ZAP XR cDNA library derived from poly(A)+RNA of human ovarian granulosa cells a cDNA clone pHG51 was isolated. Sequence analysis showed significant homology to the C-terminal region of rat ribosomal protein L8 cDNA. The 5'-end of the cDNA was completed by PCR with cloned total cDNA. Aligning of DNA sequences from PCR clones with the sequence of the pHG51 insert yielded the full-length cDNA. From the open reading frame of the cDNA an amino acid sequence for a polypeptide of 257 residues was derived, which was identical with rat ribosomal protein L8 and also possessed a high degree of identity to ribosomal proteins L8 of other species. It is therefore assumed that the characterized cDNA represents the mRNA of the human ribosomal protein L8. Southern analysis revealed that human ribosomal protein L8 is specified by multi copy genes.

The complete cDNA coding sequence for the mouse CDEI binding protein.

This work describes the cDNA sequence of the mouse CDEI binding protein (CDEBP), comprising the complete coding sequence. The cDNA encodes a protein of 695 amino acid residues. The derived amino acid sequence displays a sequence identity to human amyloid precursor-like protein (APLP) of > 92%.

The potential use of prostatic secretory protein of 94 amino acid residues (PSP94) as a serum marker for prostatic tumor.

The serum concentrations of prostatic secretory protein of 94 amino acid residues (PSP94) as well as those of prostate-specific antigen (PSA) were determined in 40 patients with established prostatic carcinoma, prior to transurethral resection of the prostate. In a comparison with a control group of healthy men (n = 40) and a group of patients with histologically established benign prostatic hyperplasia (n = 40) no significant differences in PSP94 serum concentrations between the groups were observed. Similarly, correlations of PSP94 serum concentrations with prostatic carcinoma stages or grades were not detected. In contrast, and as expected, PSA behaved as a prostate tumor marker of known sensitivity and specificity. A correlation of PSP94 and PSA concentrations in sera of patients with benign prostatic hyperplasia and/or prostatic carcinoma could not be verified. PSP94 apparently does not fulfill the criteria of a serum marker for monitoring adenomas and/or carcinomas of the prostate.

Characterization of the gene for seminalplasmin, a secretory protein of the bovine seminal vesicle.

As part of an attempt to understand the androgen-regulated expression of seminalplasmin, a major basic protein of bovine seminal vesicle secretion, we have characterized the bovine seminalplasmin gene. The compact gene of approximate size of 2.1 kb is organized in four exons and three introns. Regulatory sequences involved in regulation of transcription could not be identified by simple sequence homologies. A putative promoter element TATAA is located 29 bp upstream of exon 1.