Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.     

Origin of metazoan stem cell system in sponges: first approach to establish the model (Suberites domuncula)

It is established that Porifera (sponges) represent the earliest phylum which branched off from the common ancestor of all multicellular animals, the Urmetazoa. In the present study, the hypothesis is tested if, during this transition, pluripotent stem cells were formed which are provided-similar to the totipotent cells (archaeocytes/germ cells)-with a self-renewal capacity. As a model system, primmorphs from the sponge Suberites domuncula were used. These 3D-cell aggregates were cultivated in medium (RPMI 1640/seawater) either lacking silicate and ferric iron or in medium which was supplemented with these 'morphogenetic' factors. As molecular markers for the potential existence of stem cells in primmorphs, two genes which encode proteins found in stem cells of higher metazoan species, were cloned from S. domuncula. First, the noggin gene, which is present in the Spemann organizer of amphibians and whose translation product acts during the formation of dorsal mesoderm derivatives. The second gene encodes the mesenchymal stem cell-like protein. Both cDNAs were used to study their expression in primmorphs in dependence on the incubation conditions. It was found that noggin expression is strongly upregulated in primmorphs kept in the presence of silicate and ferric iron, while the expression of the mesenchymal stem cell-like protein was downregulated. These data are discussed with respect to the existence of stem cells in sponges.     

Acinar primary cell culture from the digestive gland of Pecten maximus (L.): an original model for ecotoxicological purposes

The purpose of this study was to establish an original primary acinar cell culture model for the mollusc bivalve Pecten maximus (L.), and to define its values and limits for subsequent ecotoxicological applications. To prevent microbial contaminations occurring frequently in invertebrate cell cultures, a perfusion of the stomach-digestive gland complex was performed in situ using a sterile salt solution containing broad-range antibiotics. Digestive acini were isolated using a pronase enzyme that was removed by several washings of the acinar suspension, after which their viability and functionality were determined by three different assays: fluorescein diacetate (FDA) de-esterification, 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium (MTT) reduction and neutral red (NR) incorporation describing de-esterification, mitochondrial dehydrogenase and lysosome activity, respectively. The kinetic conditions for these assays were defined beforehand. The results showed that digestive acini could be maintained in vitro both cytologically and functionally for at least 96 h, which is sufficient for many ecotoxicological applications. Preliminary contamination assays, according to the function studied (cell esterases, mitochondrial respiration, lysosomal incorporation), indicated that polycyclic aromatic hydrocarbons had a negative effect on the survival of acini in vitro.     

Cell culture of bivalves: tool for the study of the effects of environmental stressors.

Spontaneous beating cells can be isolated from the heart of the oyster (Crassostrea gigas) and cultured for more than two months. They form adherent contractile networks in culture conditions. They show muscarinic and beta-adrenergic reactivity thus showing that they are functional cardio-myocytes: Acetylcholine induced a dose dependent decrease in spontaneous beating rate via an increase in potassium conductance, this effect being blocked by atropine. Epinephrine induced a dramatic increase in calcium conductance which was blocked by high concentrations of propranolol but not by sotalol and reversed by verapamil. Tributyltin and cadmium induced a dose and time dependent decrease mainly in inward ionic conductances, leading to a decrease or even a total suppression of the beating rate. Present study indicates that this model could be used as a sensitive test to study the effects of some marine pollutants at the cellular level in molluscs.     

Human galectin-8 isoforms and cancer

Galectins are animal lectins that can specifically bind beta-galactosides. Thirteen galectins have already been described. This review focuses on a specific member of this family: galectin-8. This galectin was discovered in prostate cancer cells eight years ago and has been studied extensively in the last few years. The galectin-8 gene ( LGALS8) encodes numerous mRNAs by alternate splicing and the presence of three unusual polyadenylation signals. These mRNAs encode six different isoforms of galectin-8: three belong to the tandem-repeat galectin group (with two CRDs linked by a hinge peptide) and three to the prototype group (with one CRD). Various studies showed that galectin-8 is widely expressed in tumor tissues as well as in normal tissues. The level of galectin-8 expression may correlate with the malignancy of human colon cancers and the degree of differentiation of lung squamous cell carcinomas and neuro-endocrine tumors. Recently, the differences in galectin-8 expression levels between normal and tumor tissues have been used as a guide for the selection of strategies for the prevention and treatment of lung squamous cell carcinoma. These experiments are still under investigation, but demonstrate the potential of galectin-8 research to enhance our understanding of, and possibly prevent, the process of neoplastic transformation.     

Sustainable Production of Bioactive Compounds by Sponges—Cell Culture and Gene Cluster Approach: A Review

Sponges (phylum Porifera) are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms. Protected by a highly complex immune system, as well as by the capacity to produce efficient antiviral compounds (e.g., nucleoside analogues), antimicrobial compounds (e.g., polyketides), and cytostatic compounds (e.g., avarol), they have not become extinct during the last 600 million years. It can be assumed that during this long period of time, bacteria and microorganisms coevolved with sponges, and thus acquired a complex common metabolism. It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms. As a consequence, both the host cells and the microorganisms lost the ability to grow independently from each other. Therefore, it was—until recently—impossible to culture sponge cells in vitro. Also the predominant number of symbiotic bacteria proved to be nonculturable. In order to exploit the bioactive potential of both the sponge and the symbionts, a 3D-aggregate primmorph culture system was established; also it was proved that one bioactive compound, avarol/avarone, is produced by the sponge Dysidea avara. Another promising way to utilize the bioactive potential of the microorganisms is the cloning and heterologous expression of enzymes involved in secondary metabolism, such as the polyketide synthases. From the consortium German Center of Excellence [BiotecMarin]. Dedicated to Dr. Paul J. Scheuer (University of Hawaii) who created the basis for the progress in the biomedical application of the bioactive potential of the marine environment.     

Plasmid-encoded determinants for bacteriocin production and immunity in a Lactococcus lactis strain and purification of the inhibitory peptide.

Lactococcin, a bacteriocin produced by Lactococcus lactis subsp. lactis ADRIA 85LO30, was purified as a 2.3-2.4 kDa peptide. Six non-bacteriocin-producing (Bac-) and non-immune (Imm-) strains were isolated after curing experiments. These strains had in common the loss or modification of two plasmids: pOS4 (32 kb) and pOS5 (70 kb). By comparing pOS5 and several modified plasmids, a DNA region from pOS5 of about 10 kb, which was necessary for wild-type bacteriocin production and immunity, was identified.     

Abnormal distribution of CD45 isoforms expressed by CD4+ and CD8+ T cells in rheumatoid arthritis

CD45RO+ T cells are referred to as memory or helper-inducer while CD45RA+ T cells are regarded as naive or suppressor-inducer T cells. The former population predominates in the peripheral blood and even more in the synovial fluid of patients with rheumatoid arthritis, to the expense of the latter population. Within the CD45RB+ compartment, there appears to be more of the fully-differentiated than of the early-differentiated CD4+ T cells. In spite of the fact that these lymphocytes are close to undergoing apoptosis, this programmed cell death is inhibited in the rheumatoid synovium.