HCC-1, a novel chemokine from human plasma.

A novel CC chemokine, HCC-1, was isolated from the hemofiltrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and 29-37% with the other human CC chemokines. Unlike MIP-1 alpha and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34+ myeloid progenitor cells. It was as effective as MIP-1 alpha, but about 100-fold less potent.

hDIP--a potential transcriptional regulator related to murine TSC-22 and Drosophila shortsighted (shs)--is expressed in a large number of human tissues.

We have cloned a 420 bp cDNA from a human fetal brain cDNA library in lambda encoding the human homologue of a DSIP-immunoreactive leucine zipper protein (DIP) isolated from porcine brain. The derived human protein (hDIP) shares a significant sequence identity with parts of the murine TSC-22 and Drosophila shs, both proteins which are discussed as functioning as transcriptional regulators. A similar role of hDIP is partially confirmed by the results of an RT-PCR analysis, demonstrating the widespread distribution of the protein among different human tissues.

A new human guanylate cyclase-activating peptide (GCAP-II, uroguanylin): precursor cDNA and colonic expression.

We have amplified, cloned, and sequenced 583 bp GCAP-II/uroguanylin-specific cDNA from human colon cDNA first strand. The cDNA codes for a putative 112 amino-acid precursor protein including the sequence of uroguanylin and GCAP-II. Northern blot hybridization revealed a high level expression of the GCAP-II gene in human colon, but not in the kidney. This expression of GCAP-II indicates a pivotal role in cGMP-mediated functions of the colon.

Hemofiltrate CC chemokines with unique biochemical properties: HCC-1/CCL14a and HCC-2/CCL15.

The hemofiltrate CC chemokines CCL14a (formerly HCC-1), CCL14b (formerly HCC-3), and CCL15 (formerly HCC-2) are encoded by mono- as well as bicistronic transcripts from a tandem gene arrangement on human chromosome 17q11.2. The transcription and splicing into several mono- and bicistronic transcripts of this gene complex are unique for human genes. No corresponding mechanism is known in nonprimate mammalian species such as mice and rats. The extremely high concentration of CCL14a in human plasma is exceptional for chemokines and led to the identification of this chemokine. Several molecular forms of CCL14a have been isolated and investigated. The mature propeptide CCL14a(1-74) is a low-affinity agonist of CCR1 which is converted to a high-affinity agonist of CCR1 and CCR5 on proteolytic processing by serine proteases. In contrast, CCL15 is characterized using molecular forms deduced from the mRNA/cDNA and shown to activate cells via CCR1 and CCR3, also dependent on the amino-terminal length. Hemofiltrate CC chemokines are chemoattractants for different types of leukocytes including monocytes, eosinophils, T cells, dendritic cells, and neutrophils. In this review, we emphasize the genomic organization, expression patterns, and biochemical properties of CCL14a, CCL14b, and CCL15. We report results of significance for the development of therapeutic strategies, especially concerning HIV infection and inflammatory diseases.

hBD-1: a novel beta-defensin from human plasma.

We report the isolation and characterization of a novel peptide with significant sequence homology to beta-defensins from human blood filtrate. The human beta-defensin-1 (hBD-1) is a short basic peptide of 36 amino acid residues. It contains six cysteines forming three intramolecular disulfide bonds. The molecular mass of hBD-1 is 3928.6 Da. Cloning of the specific cDNA confirmed the amino acid sequence of the native peptide. hBD-1 shares the nine conserved amino acids characteristic for beta-defensins from respiratory epithelial cells and neutrophils of cattle and chicken leukocytes. hBD-1 is present in nanomolar concentration in human plasma.

Casocidin-I: a casein-alpha s2 derived peptide exhibits antibacterial activity.

Here we report the isolation and characterization of an antibacterial peptide from bovine milk inhibiting the growth of Escherichia coli, and Staphylococcus carnosus. The primary structure of the peptide was revealed as a 39-amino-acid-containing fragment of bovine alpha s2-casein (position 165-203) by means of Edman amino acid sequencing and mass spectrometry. Since human milk does not contain any casein-alpha s2, these findings could explain the different influence of human and bovine milk on the gastrointestinal flora of the suckling.

LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity.

We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP-1 (liver-expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine-rich peptides, a 25-residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP-1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram-positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram-negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose-dependently exhibited sensitivity upon treatment with synthetic LEAP-1. The discovery of LEAP-1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.

Membrane topology of the 22 kDa integral peroxisomal membrane protein.

In order to study the membrane topology and the possible function of the rat liver 22 kDa integral peroxisomal membrane protein (PMP 22) at a molecular level, we have cloned PMP 22 from a lambda gt11 expression library and sequenced its cDNA. Hydropathy analysis of the deduced primary structure indicates 4 putative transmembrane segments. The accessibility to exogenous aminopeptidase of PMP 22 in intact peroxisomes suggests that the N-terminus faces the cytosol. A model of the topology of PMP 22 in the peroxisomal membrane is discussed. Homology studies revealed a striking similarity with the Mpv 17 gene product. Lack of this membrane protein causes nephrotic syndrome in mice.

Uroguanylin: gene structure, expression, processing as a peptide hormone, and co-storage with somatostatin in gastrointestinal D-cells.

Guanylin/GCAP-I and uroguanylin/GCAP-II are two structurally related peptides which play an important role in the regulation of water/electrolyte balance within the gut. In order to enable the investigation and comparison of both peptide hormones at the genomic level, we decided to clone the corresponding genes. The human gene for guanylin/GCAP-I and its 5'-flanking region have been described recently. Here, we report the three exon/two intron structure of the human uroguanylin/GCAP-II gene and its localization on chromosome 1 p35-34, as determined by radiation hybrid mapping. Together with data obtained for the guanylin/GCAP-I gene we show that these genes are localized in the same chromosomal area with other guanlyl cyclase-activating peptides like ANP etc. Northern hybridization revealed that the expression of the uroguanylin/GCAP-II gene is highest in the intestinal mucosa, especially in the ileum and colon. By means of polymerase chain reaction (PCR), an expression was also observed in the stomach where no guanylin/GCAP-I expression is detectable. Using immunohistochemical methods, uroguanylin/GCAP-II immunoreactive material was distinctly localized in D-type gastric and intestinal endocrine cells. Although the comparable data on the genomic organisation of both peptide hormones verify their high degree of relationship, this finding indicates a special task of uroguanylin/GCAP-II within the stomach, such as regulatory functions in gastric secretion. The redundant expression of the GCAP/GC-C system in the small and large intestine, however, is as yet unclear.

Molecular biological characterization of phosphodiesterase 3A from human corpus cavernosum.

The hydrolysis of the second messenger cyclic AMP (cAMP) by phosphodiesterase 3 (PDE3) is known to play an important regulatory role in the context of relaxation of cavernous smooth muscle of the penis. Thus, we investigated the PDE3A isoform from penile cavernous tissues of male patients with and without symptoms of erectile dysfunction at the molecular biological level. As revealed by reverse transcriptase polymerase chain reaction, of all tissues of the urogenital tract analyzed the expression of the PDE3A gene was highest in the corpus cavernosum. However, significant differences in the levels of gene expression were not found between the two subgroups of patients. Also, the determined nucleotide sequences of the cloned penile PDE3A cDNAs of all patients were absolutely identical. Surprisingly, some deviations could be detected in the cDNA sequences of PDE3A from human myocard and platelets. The data obtained indicate that neither the expression levels nor the sequence deviations of PDE3A are the main reasons for erectile dysfunction in men.

Association between different psychotic disorders and the DRD4 polymorphism, but no differences in the main ligand binding region of the DRD4 receptor protein compared to controls.

The dopamine D4 receptor gene (DRD4 gene) is one of the important candidate genes for schizophrenia and other psychoses. In humans, several alleles with variable repeat numbers of a 48-base-pair element within the third exon are known. The corresponding receptor proteins differ in their pharmacological properties. It might be possible that specific alleles or genotypes predispose for schizophrenia and other psychoses. The aim of the present study was to investigate and compare the frequency of the DRD4 alleles and genotypes in healthy controls and patients suffering from schizophrenia, schizoaffective or affective disorders. The DRD4 subtypes of 92 controls, 91 patients with schizophrenia, 90 patients with affective and 20 with schizoaffective disorders were identified by a combination of Southern blot technique and PCR. Statistical analysis revealed several significant differences between controls and patients, e.g. an increased frequency of the D4.7 allele among patients with schizophrenia, schizoaffective or unipolar affective disorder. The results indicate a possible role of the DRD4 gene polymorphism in the pathophysiology of psychotic diseases. When a part of the DRD4 gene sequence containing the codons for the most important amino acids for dopamine binding in 9 controls, 9 patients with schizophrenia and 10 with affective disorders were compared, no differences could be found.

hK5 and hK7, two serine proteinases abundant in human skin, are inhibited by LEKTI domain 6.

BACKGROUND: Several skin diseases and atopic disorders including Netherton syndrome and atopic dermatitis have been associated with mutations and deviations of expression of SPINK5, the gene encoding the human 15-domain serine proteinase inhibitor LEKTI. The biochemical mechanisms underlying this phenomenon have not yet been fully clarified. OBJECTIVES: To identify target proteinases of LEKTI important for processes of desquamation and inflammation of the skin which will enable the development of specific drugs. METHODS: The inhibitory activities of LEKTI domains 6 and 15 were tested on a number of commercially available serine proteinases and also on the purified kallikreins hK5 and hK7. In addition, recombinant hK5 was used. RESULTS: LEKTI domain 6 is a potent inhibitor of hK5 and hK7, whereas LEKTI domain 15 exhibits inhibitory activity on plasmin. hK5 and hK7 in particular are relevant to skin disorders. CONCLUSIONS: The inhibition of hK5 and hK7 by LEKTI domain 6 indicates an important regulatory role of LEKTI in processes of skin desquamation and inflammation, which may explain the severe pathological symptoms associated with abnormalities of SPINK5 and/or its expression. Thus, LEKTI represents a potential drug for the treatment of these disorders.

Identification and isolation of glucose dehydrogenase genes of Bacillus megaterium M1286 and their expression in Escherichia coli.

A gene encoding glucose dehydrogenase of Bacillus megaterium M1286 was isolated from a lambda-EMBL3 phage library. It is transcribed and translated in cells of the heterologous organism Escherichia coli by own control regions. The gene is located on a 1126-bp HindIII fragment. Its nucleotide sequence contains 220 bp in the 5' non-coding region, 783 bp in the coding region and 123 bp in the 3' non-coding region. The amino acid sequence, as deduced from the coding region, consists of 261 amino acids and is different from the known protein sequence of glucose dehydrogenase from B. megaterium M1286. [Jany, K. D., Ulmer, W., Fröschle, M. & Pfleiderer, G. (1984) FEBS Lett. 165, 6-10]. By using this gene as a hybridization probe a second glucose dehydrogenase gene was isolated, which was also directly expressed in E. coli. Additionally a DNA region with extended sequence homology to the hybridization probe was identified. This work indicates the existence of at least two independent glucose dehydrogenase genes in B. megaterium M1286. Homologies in the primary structures of the two different glucose dehydrogenases of B. megaterium M1286 and of the corresponding Bacillus subtilis enzyme are discussed.

Functional analysis of the human guanylin gene promoter.

Guanylin (GCAP-I, guanylate cyclase activating peptide I) and uroguanylin (GCAP-II, guanylate cyclase activating peptide II) are regulatory peptides involved in the regulation of the intestinal chloride / water balance. They share significant structural homology to the E. coli enterotoxin STa, which binds to the particulate guanylyl cyclase C causing diarrhea in mammals. In this study we report the functional analysis of the guanylin / GCAP-I gene promoter region. By means of the luciferase reporter gene assay, we demonstrate a strong promoter activity in T84 cells. Especially the first 160 bp of the 5'-flanking region of the gene seem to be essential for gene induction. Our findings are the basis for further identification of important regulatory elements of the corresponding gene.

The golden hamster aphrodisin gene. Structure, expression in parotid glands of female animals, and comparison with a similar murine gene.

The so-called lipocalins are a family of extracellular proteins that are known to typically fulfill tasks as transport proteins for small hydrophobic molecules. However, in the last decade, a large diversity has been described concerning their functions, for example as enzymes, immunomodulators, or proteins involved in coloration and pheromone action. Aphrodisin belongs to those lipocalins, which are of significant importance for the pheromonal stimulation of copulatory behavior in male hamsters. We recently succeeded in characterizing the corresponding cDNA and demonstrated the expression of the aphrodisin gene in the vagina, uterus, and Bartholin's glands of female hamsters. Here we report the structure of the aphrodisin gene and the functionality of its promoter region. We further compare the aphrodisin gene to the related gene for mouse odorant-binding protein 1a, indicating similar functions of their products. As a novelty, we show that the aphrodisin gene, in addition to the above-mentioned tissues, is also expressed in female hamster parotid glands. In contradiction to the results expected, we finally demonstrate that aphrodisin already occurs in vaginal discharge before the female animals reach fertility. These findings may lead to the identification of as yet unknown aphrodisin functions.

Nucleotide sequence of the rabbit gamma-preprotachykinin I cDNA.

We succeeded in amplifying tachykinin I specific cDNAs from cerebral tissue of guinea pig, rabbit, rat, hamster, trout and tupaia in the expected size range of 820-980 bp. The amplified 859 bp rabbit gamma-preprotachykinin I cDNA was sequenced and consisted of the whole 345 bp gamma-PPT I coding sequence including the substance P and neurokinin A coding regions and a 505 bp large 3'-nontranslated region. Both, molecular weight and sequence comparison emphasizes the very high phylogenetic age of the preprotachykinin I gene.

cDNA sequence and expression pattern of the putative pheromone carrier aphrodisin.

The cDNA sequence for aphrodisin, a lipocalin from hamster vaginal discharge which is involved in pheromonal activity, has been determined. Corresponding genomic clones were isolated and the promoter region was identified. Primer extension analysis revealed an adenosine residue as the main transcription initiation site, located 50 bp upstream of the translation start codon ATG, which is surrounded by a typical Kozak sequence. However, data from polymerase chain reaction analysis suggest the existence of at least one alternative transcription initiation site. The aphrodisin cDNA is 732 bp long and codes for the mature 151-aa aphrodisin and an additional N-terminal 16-aa secretory signal peptide. The 3' nontranslated region is 228 bp long. Among the known sequences, the aphrodisin cDNA shares the highest homology with the rat odorant-binding protein cDNA (45%), which verifies the protein data. Vaginal tissue and Bartholin's glands are the main aphrodisin gene-expressing tissues of the female hamster genital tract, as demonstrated by Northern blot analysis. Under less stringent hybridization conditions, RNA isolated from rat Bartholin's glands also showed a signal, indicating the occurrence of aphrodisin-related mRNA in this species.

Expression of different phosphodiesterase genes in human cavernous smooth muscle.

PURPOSE: Knowledge of intracellular signal propagation in smooth muscle tone regulation is of major importance in the understanding of the physiology of penile erection, and the development of new and selective pharmacological agents for the treatment of related disorders. Since phosphodiesterases (PDE) are key enzymes of the signaling pathway, we elucidate their presence and potential functional relevance in human cavernous tissue. MATERIALS AND METHODS: To identify PDE messenger RNA in human cavernous tissue, we constructed primers for 14 published PDE isoforms. Expression of the genes was then analyzed by reverse transcriptase polymerase chain reaction under standard conditions and by subsequent sequencing. RESULTS: Messenger RNA was detected in human corpus cavernosum for the human phosphodiesterase isoenzymes and isoforms PDE1A, PDE1B, PDE1C, PDE2A, PDE3A, PDE4A, PDE4B, PDE4C, PDE4D, PDE5A, PDE7A, PDE8A and PDE9A. CONCLUSIONS: A total of 13 PDE genes were expressed in human cavernous tissue, indicating a role of these enzymes in penile erection regulation. The intracellular mechanisms of hydrolyzing cyclic adenosine monophosphate and cyclic guanosine monophosphate by PDEs are more complex than assumed previously. These findings open up new possibilities in the development of drugs for the treatment of erectile dysfunction.

Intrinsic human glomerular mesangial cells can express receptors for IgG complexes (hFc gamma RIII-A) and the associated Fc epsilon RI gamma-chain.

Immune complexes localize to the glomerular mesangium in many forms of human glomerulonephritis. In this investigation, we determined whether primary cultured human glomerular mesangial cells (HMC) express mRNA and functional protein of FcR for IgG. Performing reverse transcription-PCR with subsequent control Southern blots, we showed that in contrast to human monocytes or granulocytes, mRNA for Fc gamma RI and Fc gamma RII is not detectable in either growth-arrested (48 h serum free) or unstimulated proliferating (medium with 10% FCS) HMC. However, IFN-gamma in combination with LPSE.coli (LPS), but not LPS alone elicited a significant transcription of hFc gamma RIII mRNA in resting HMC, whereas cytokines, such as IL-1 beta or TGF-beta, failed to induce any of the three Fc gamma Rs in resting HMC. Proliferating HMC showed a basal transcription of Fc gamma RIII mRNA, which was enhanced by IFN-gamma in combination with LPS. Slot-blot analysis indicated that only the Fc gamma RIII-A gene encoding the transmembrane isoform of Fc gamma RIII was expressed by HMC. For the first time, transcripts for the gamma-chain of Fc epsilon RI were found in HMC. Fc gamma RIII protein expression was detected after LPS/IFN-gamma stimulation both by specific staining of paraformaldehyde-fixed HMC and immunoprecipitation of Fc gamma RIII protein from HMC membranes. Fc gamma RIII-A receptors are functionally active, as HMC IL-6 mRNA synthesis was stimulated by heat-aggregated IgG or F(ab')2 fragments of CLBGran1 mAb only after induction of Fc gamma RIII-A. These in vitro data suggest that HMC that are basically negative for Fc gamma RIII can be stimulated to express low affinity Fc gamma Rs, and thus may participate actively in human glomerulonephritis involving immune complexes.

Porcine guanylin and uroguanylin: cDNA sequences, deduced amino acid sequences, and biological activity of the chemically synthesized peptides.

Guanylin and uroguanylin are structurally related intestinal peptide hormones which were purified from a limited number of mammals and are capable of activating the particulate guanylate cyclase-C. Although the biological functions of guanylin and uroguanylin are not yet clarified in detail, they are involved in the regulation of the intestinal water and electrolyte balance. In order to verify the general importance of this hormone system in mammals, we cloned the corresponding cDNAs from pig. Here, we present the nucleotide sequences and the deduced amino acid sequences representing porcine guanylin and uroguanylin. The expression patterns of the corresponding genes, as shown by Northern hybridization and RT-PCR analysis, resemble those of the human homologues. Further, we demonstrate the bioactivity of both porcine peptide hormones by inducing the intracellular cGMP production in human T84 cells and by ion transport experiments using porcine intestinal mucosa in the Ussing chamber.