Hans-Jürgen Mägert

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.

hBD-1: a novel β-defensin from human plasma

We report the isolation and characterization of a novel peptide with significant sequence homology to β‐defensins from human blood filtrate. The human β‐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 β‐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-αs2 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 αs2‐casein (position 165–203) by means of Edman amino acid sequencing and mass spectrometry. Since human milk does not contain any casein‐αs2, these findings could explain the different influence of human and bovine milk on the gastrointestinal flora of the suckling.

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.

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.

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.

LEKTI: a multidomain serine proteinase inhibitor with pathophysiological relevance

Proteinase inhibitors are important negative regulators of proteinase action in vivo and are thus involved in several pathophysiological processes. Starting with the isolation of two new peptides from human blood filtrate, we succeeded in cloning a cDNA encoding the precursor protein for a novel 15-domain Kazal-type-related serine proteinase inhibitor. Two of the 15 domains almost exactly match the Kazal-type pattern, whereas the other 13 domains exhibit only four instead of six cysteine residues. Since the corresponding gene is expressed in several lympho-epithelial tissues, we termed this inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI). For three of the 15 LEKTI domains, we demonstrated a significant trypsin-inhibiting activity. Recent results of another group show a relation between mutations within the LEKTI gene and the severe congenital disorder Netherton syndrome. In this review article, we give an overview of the already known data on the structure, processing, gene expression, and pathophysiological role of LEKTI.

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.

Gene expression of the phosphodiesterases 3A and 5A in human corpus cavernosum penis.

Objective: The following study was performed to evaluate the importance of phosphodiesterases 3A (PDE3A) and 5A (PDE5A) for the regulation of penile smooth muscle tone. Furthermore, indications of side effects of specific inhibitors in certain tissues as well as of a possible relation between the level of PDE3A and PDE5A gene expression and erectile dysfunction should have been deduced from the results obtained.Methods: Total ribonucleic acid was isolated from different human tissues (urogenital tract, gastrointestinal tract, cardiovascular system and central nervous system) and subjected to RTPCR analysis and Northern blotting using primers and probes specific for PDE3A and PDE5A.Results: As shown by RT–PCR and Northern blotting, PDE3A and PDE5A mRNAs exhibit a distinct distribution throughout the tissues examined but were 2–fold higher in cavernous tissue than in all other tissues investigated. However, there were no significant differences in the levels of gene expression between the two subgroups of patients.Conclusions: Very high expression levels of PDE3A and PDE5A in human cavernous tissue underscore the physiological importance of these enzymes for the regulation of penile erection, emphasizing their therapeutical and pharmacological relevance. The distribution pattern of the mRNA for the isoenzymes PDE3A and PDE5A may explain the pharmacological effects as well as the side effects of milrinone and sidenafil.

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 λgt11 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.