Constitutive secretion of β-trace protein by cultivated porcine choroid plexus epithelial cells: Elucidation of its complete amino acid and cDNA sequences

Primary porcine choroid plexus epithelial cells cultivated in chemically defined medium maintain their epithelial characteristics and form confluent monolayers. They produce a fluid the composition of which resembles cerebrospinal fluid. The present study demonstrates constitutive secretion of large amounts of β-trace protein. This intrathecally synthesized protein is a prominent polypeptide constituent of natural cerebrospinal fluid. According to the identity of amino acid sequences it has previously been tentatively identified as a prostaglandin-D synthase and as a member of the lipocalin protein family. β-Trace was purified from cell culture supernatants and was subjected to tryptic digestion and amino acid sequencing of the resulting peptides. The complete primary structure of the protein was obtained by additional isolation of the cDNA from cultured epithelial cells. The porcine 163-amino acid polypeptide showed 69% identity with the human β-trace and contained two N-glycosylation sites occupied by complex-type oligosaccharides as is the case for the human protein. The amino acid sequences around the N-glycosylation sites of mammalian β-trace proteins (porcine, human, murine, and rat) were highly conserved. The nucleotide sequence was found to be less conserved; the porcine cDNA had a strikingly high GC-content (67%). The constitutive secretion of β-trace protein from the in vitro cultivated porcine choroid plexus epithelial cells demonstrates that the cells have retained their major in vivo physiological properties: secretion of cerebrospinal fluid proteins. Therefore, this in vitro culture system may be used as a versatile tool for studying the regulation of the formation of cerebrospinal fluid. © 1996 Wiley-Liss, Inc.     

An ancient haplotype containing antimicrobial peptide gene variants is associated with severe fungal skin disease in Persian cats

Dermatophytosis, also known as ringworm, is a contagious fungal skin disease affecting humans and animals worldwide. Persian cats exhibit severe forms of the disease more commonly than other breeds of cat, including other long-haired breeds. Certain types of severe dermatophytosis in humans are reportedly caused by monogenic inborn errors of immunity. The goal of this study was to identify genetic variants in Persian cats contributing to the phenotype of severe dermatophytosis. Whole-genome sequencing of case and control Persian cats followed by a genome-wide association study identified a highly divergent, disease-associated haplotype on chromosome F1 containing the S100 family of genes. S100 calcium binding protein A9 (S100A9), which encodes a subunit of the antimicrobial heterodimer known as calprotectin, contained 13 nonsynonymous variants between cases and controls. Evolutionary analysis of S100A9 haplotypes comparing cases, controls, and wild felids suggested the divergent disease-associated haplotype was likely introgressed into the domestic cat lineage and maintained via balancing selection. We demonstrated marked upregulation of calprotectin expression in the feline epidermis during dermatophytosis, suggesting involvement in disease pathogenesis. Given this divergent allele has been maintained in domestic cat and wildcat populations, this haplotype may have beneficial effects against other pathogens. The pathogen specificity of this altered protein should be investigated before attempting to reduce the allele frequency in the Persian cat breed. Further work is needed to clarify if severe Persian dermatophytosis is a monogenic disease or if hidden disease-susceptibility loci remain to be discovered. Consideration should be given to engineering antimicrobial peptides such as calprotectin for topical treatment of dermatophytosis in humans and animals.     

The inducible antibacterial peptides of insects

Insects respond to bacterial challenge by the rapid and transient synthesis of a large number of potent antibacterial peptides that are active against many different bacteria. Two families of inducible antibacterial peptides are well characterized: the cecropins and the insect defensins. A rapidly increasing number of proline- and glycine-rich peptides are reported from various insect species together with cecropins and insect defensins. In this review, Stéphane Cociancich, Philippe Bulet, Charles Hetru and Jules A. Hoffmann give an update of our current information on the induced antibacterial peptides.     

Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect

Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger (heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi. Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.     

Selective and sensitive detection of pectin lyase activity using a colorimetric test: application to the screening of microorganisms possessing pectin lyase activity

Several methods have been described for the detection and quantification of polygalacturonase (PG) and pectin lyase (PL) activities. The most frequently used tests are the Nelson method using copper(II) and an arsenomolybdate reagent to detect PG activity, and the colorimetric method using thiobarbituric acid (TBA) to detect PL activity. We observed that none of these methods are suitable to differentiate between these two enzymatic activities. Therefore, we optimized the test conditions of the TBA method. As a result, the detection of the enzymatic beta-elimination (PL activity) became sensitive and selective. A basic pretreatment at 80 degrees C for 5 min of the solution which contains the pectin fragments of the PL activity furnished aldehydes which were condensed with TBA or its derivatives. After acidification of the medium, a pink fluorescent dye was detected spectrophotochemically (lambda = 550 nm). The interference of galacturonic acid or oligomers resulting from PG activity was completely eliminated. The most sensitive reagent was N-(pyridin-2-yl)-thiobarbituric acid. The application of this method with the new reagent was extended to the screening of microorganisms possessing the PL activity. The obtained results confirm that Aspergillus niger strain and a Saccharomyces cerevisiae SCPP strain possess this activity.     

Relationship between cerebro-spinal fluid pH and pulmonary ventilation of the South American lungfish, Lepidosiren paradoxa (Fitz.)

The respiratory control in land vertebrates (Tetrapoda) is mainly linked to regulation of acid-base status, which involves peripheral and central chemoreceptors. The lungfish (Dipnoi) might constitute the sister group of all land vertebrates (Tetrapoda) and possess a combination of real lungs and reduced gills. In this context, we evaluated the possible presence of central respiratory chemoreceptors in the South American Lungfish, Lepidosiren paradoxa. Pulmonary ventilation and respiratory frequency increased significantly with reductions of CSF pH by means of mock CSF solutions. This suggests that Lepidosiren possess central acid-base receptors.     

Receptors and signalling components of plant hormones

Recent advances in understanding plant hormonal signalling has resulted in the identification of a variety of signalling components including receptor kinases with homology to the bacterial two component system as well as serine/threonine kinases and protein phosphatases. In addition, the existence of MAP kinase pathways in plants indicates a similar role of these signalling cascades in the relay of exogenous signals into the nucleus as has been disclosed in animal cells. The emerging signalling pathways of the plant hormone abscisic acid and ethylene are presented.     

A melatonin preparation with a pulsatile liberation pattern: a new form of melatonin in replacement therapy

Using melatonin (MLT) as a circadian synchroniser in humans to treat rhythm disorders, it is desirable to have controlled-release dosage forms. Following in vitro liberation tests, one fast-release form containing 5 mg MLT (capsule A) and two oral pulsatile-dosage forms containing 10 mg MLT each (capsules B and C) were studied in a randomised, single-dose, threefold cross-over study in 15 healthy male volunteers after investigation of capsule B in dogs. Mean peak concentrations of MLT in serum (pmol/ml) were reached between 0.5 h and 0.75 h: Cmax1 20.7 (A), 16.4 (B), 9.7 (C). Capsules B and C released a second MLT pulse after about 3.5 h with Cmax2 of 13.0 and 17.5 pmol/ml, respectively. The time course of the renally excreted main metabolite 6-sulphatoxymelatonin (aMT6s) correlates with that of changes in MLT serum concentrations. The kinetic profile of the delivery system is adjusted to the pattern of sleep maintenance disturbances.