Behavior of Antimicrobial Peptide K4 in a Marine Environment.

K4 is a de novo peptide with antibacterial activity on human pathogens. It has a short sequence (14 amino acids), with a cationic N-terminal moiety and an amphipathic ɑ-helix structure. The present paper demonstrates its activity on Vibrio bacteria in a marine environment. It was found non-toxic on marine organisms including Artemia salina, Dicentrarchus labrax, and Magallana gigas at different developmental stages, but influenced the growth of unicellular organisms like microalgae, depending on the algal strain and on K4 concentration. Furthermore, an original approach coupling liquid chromatography (RP-HPLC) and mass spectrometry (MS/MS) allowed us to monitor the degradation time course of the peptide for the first time in conditions close to a hatchery environment, i.e., in the presence of oyster spat. We detected truncated forms over time, and the full K4 was gradually no longer found in these filter-feeder oysters. Finally, using an automated optical density meter, we monitored the growth of several aquatic bacteria identified as pathogenic on animals. K4 had a bactericidal effect on Aeromonas salmonicida and Vibrio splendidus LGP32 at concentrations below 45 µg mL-1. Our results show that K4 could be an environment-friendly alternative to antibiotics, non-toxic to several marine organisms. The use of K4 would be particularly useful to decrease the bacterial load associated with food intake in the early developmental stages of marine animals reared in hatcheries.

Calculation of the decision limit (CCalpha) and the detection capability (CCbeta) for banned substances: the imperfect marriage between the quantitative and the qualitative criteria.

Initially in the Decision 2002/657/EC the criteria for the calculation of the decision limit (CCalpha) and the detection capability (CCbeta) have been estimated as purely quantitative (alpha-error is 1% and beta-error is 5%). In 2004, the European Commission has issued a document to provide guidance for the interpretation of the 2002/657/EC. In this document it is mentioned that also qualitative criteria should be fulfilled. Therefore, the calculated CCalpha and CCbeta must be verified by using fortified samples. The method should be able to detect/identify the target component in 50% of the cases at CCalpha and in 95% of the cases at CCbeta. Analytical methods for the analysis of nitroimidazoles, nitrofurans and corticosteroids with LC-MS/MS have been validated by fortifying blank samples below and above the MRPL. CCalpha and CCbeta were calculated using the ISO 11843 approach. In addition, the frequency of methodical compliance for the qualitative criteria was determined at each concentration level. It was observed that at the calculated CCalpha and CCbeta levels the qualitative criteria were not fulfilled. It was concluded that the detection capability of the analytical method should be calculated by using decreasing fortification levels at and below the MRPL. A protocol validating methods for banned substances by limiting the number of samples is presented and the qualitative criteria for the assessment of CCalpha and CCbeta were verified based on the same set of data without the need of performing additional validation experiments.

Interlaboratory study based on a one-plate screening method for the detection of antibiotic residues in bovine kidney tissue.

A microbiological test based on one-plate, seeded with Bacillus subtilis BGA, is frequently used in Belgium as a screening method to monitor antibiotic residues in kidney tissue of slaughter animals. The procedure as described in the National Legislation recommends test agar pH 7.2 from Merck. Since this type of agar is no more commercially available, two kinds of agar medium are proposed on the market: niertest agar from Biotrading and Standard II nutrient agar from Merck. Niertest agar, which is actually used by a few laboratories, is a ready-to-use medium manufactured from test agar pH 7.2 from Merck. Its composition is identical to those of test agar pH 7.2. Standard II nutrient agar is the second alternative culture medium which is used by a higher number of laboratories. The equivalence of the standard II nutrient agar medium to both identical culture media (test agar pH 7.2 and niertest agar) was evaluated during a national collaborative trial. Results of this study as well as the experimental procedure are presented.

A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase.

The Xenopus laevis South African frog oocyte is a well suited and widely used system for protein biochemistry and functional studies. So far, two methods are commonly in use for the expression of exogenous proteins in this system. Investigators have the choice between cytoplasmic injections of in vitro synthesized cRNA or nuclear injections of cDNA. Here, we describe a new method for ion channel expression in oocytes, which consists of a coinjection of T7-driven cDNA and T7-RNA polymerase directly into the cytoplasm. This technique uses very limited amounts of purified enzyme and is also applicable to SP6 polymerase. Commercially available polymerases can also conveniently substitute for self-purified enzymes. The technique can be used for electrophysiological and biochemical analysis. In particular, high level expressions have been achieved for potassium (Shaker B, Kv1.2 and Kv1.3) and sodium (P mu 1.2) channels, and we also demonstrate efficient metabolic labeling of the calcium channel auxiliary beta 3 subunit. The properties of the channels expressed by this technique are indistinguishable from those of the channels expressed by classical methods. Expression of multi-subunit proteins was also achieved illustrating that the technique can be used for structure-function analyses. Moreover, this novel expression technique avoids many drawbacks of the two former techniques. It clearly bypasses the costly and time-consuming step of cRNA synthesis in vitro, prevents delicate cRNA manipulation and is easier to perform and more reliable than nuclear injection. Finally, it does not affect cell survival rate. These data indicate that the T7-RNA polymerase expression technique could be widely used in the future for the expression of exogenous proteins in the Xenopus oocyte system.

Distinct properties and differential beta subunit regulation of two C-terminal isoforms of the P/Q-type Ca(2+)-channel alpha(1A) subunit.

Two C-terminal splice variants (BI-1 and BI-2, now termed Ca(v)2.1a and Ca(v)2.1b) of the neuronal voltage-gated P/Q-type Ca(2+) channel alpha(1A) pore-forming subunit have been cloned (Mori et al., 1991, Nature, 350, 398-402). BI-1 and BI-2 code for proteins of 2273 and 2424 amino acids, respectively, and differ only by their extreme carboxyl-termini sequences. Here, we show that, in Xenopus oocytes, the two isoforms direct the expression of channels with different properties. Electrophysiological analysis showed that BI-1 and BI-2 have peak Ba(2+) currents (I(Ba)) at a potential of +30 and +20 mV, respectively. The different C-terminal sequence (amino acids 2229-2273) of BI-1 caused a shift in steady-state inactivation by +10 mV and decreased the proportion of fast component of current inactivation twofold. Likewise, the biophysical changes in I(Ba) caused by coexpression of the beta(4) auxiliary subunit were substantially different in BI-1- and BI-2-containing channels in comparison to those induced by beta(3). Several of these differences in beta regulation were abolished by deleting the carboxyl-terminal splicing region. By creating a series of GST fusion proteins, we identified two locations in the C-terminal (Leu2090-Gly2229 for BI-1 and BI-2, and Arg2230-Pro2424 for BI-2 only) that determine the differential interaction of beta(4) with the distinct alpha(1A) isoforms. These interactions appear to favour the binding of beta(4) to the AID site, and also the plasma membrane expression of BI-2. These results demonstrate that the final segment of the C-terminal affects alpha(1A) channel gating, interaction and regulation with/by the beta subunits. The data will have several implications for the understanding of the biophysical effects of many channelopathies in which the carboxyl-termini of alpha(1A) and beta(4) are affected.

Synaptotagmin I and IV define distinct populations of neuronal transport vesicles.

Mammalian synaptotagmins constitute a multigene family of at least 11 membrane proteins. We have characterized synaptotagmin IV using antibodies directed against the C2A domain of the protein. Antibodies reacted specifically with a protein band that migrated as a 41-44 kDa doublet. Synaptotagmin IV expression was regulated throughout development. A strong decrease in the amount detected by Western blotting occurred between postnatal day 5 and adulthood, in agreement with studies on the expression of synaptotagmin IV transcripts. In subcellular fractionation, synaptotagmin IV was not detected in the synaptic vesicle-enriched fraction. Immunofluorescence microscopy was concordant with this finding. In 6-day-old rat cerebellum and cultured hippocampal neurons the subcellular distribution of synaptotagmin IV was clearly different from that of synaptotagmin I. Synaptotagmin IV displayed a punctate non-polarized distribution on neuronal extensions, whereas synaptotagmin I staining was essentially synaptic. Synaptotagmin IV staining was also observed in the soma in strong perinuclear fluorescent puncta superimposed on that of Golgi/TGN markers. Furthermore, synaptotagmin IV was seen in the proximal part of the growth cone domain and not in the microfilament-rich region which includes filopodia. Co-localizations with the adhesion molecules vinculin and zyxin at the proximal part of growth cones were observed. Synaptotagmin IV may thus be involved in the regulation of specific membrane-trafficking pathways during brain development.

The I-II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit.

The auxiliary beta subunit is essential for functional expression of high voltage-activated Ca2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of alpha1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the alpha1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of alpha1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The beta subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length alpha1 subunit facilitates the cell surface expression of the channel in the absence of beta subunit. Our data suggest that the beta subunit favors Ca2+ channel plasma membrane expression by inhibiting an expression brake contained in beta-binding alpha1 sequences.

A new beta subtype-specific interaction in alpha1A subunit controls P/Q-type Ca2+ channel activation.

The cytoplasmic beta subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha1 interaction domain (AID) in the I-II cytoplasmic loop of alpha1 and the beta interaction domain (BID) of the beta subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha1A (BI-2) and the carboxyl terminus of beta4. Like the interaction identified previously () between the carboxyl termini of alpha1A and beta4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha1 is isoform-dependent, suggesting a role in the specificity of alpha1-beta pairing. Coexpression of beta4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha1A channel compared with beta3 (not exhibiting this interaction). Replacing the amino terminus of alpha1A with that of alpha1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.

Efficiency of retroviral transduction into hematopoietic cells by cocultivation procedure does not correlate with viral titer.

Relative transduction efficiency with retroviral vector-producing clones was assayed by cocultivating TF-1, a human CD34+ hematopoietic cell line and YR-2, a sheep B-lymphoid cell line, with LacZ containing vector-producing cells, and then by scoring the percentage of X-Gal+ cells. At the same time, viral titer was estimated by titration assay with murine fibroblasts. Results clearly demonstrated a lack of correlation between viral titer and efficiency of transduction into hematopoietic cells, which depends neither on the type of packaging cell line, PG-13 and GP-envAM12 in this study, nor on the type of LacZ containing retroviral vector. These results strongly favor consideration of interactions between producers and target cells of the study for the screening of producing cell lines.

In vivo delivery of human alpha-L-iduronidase in mice implanted with neo-organs.

Mucopolysaccharidose type I is a lysosomal storage disease caused by a deficiency in the enzyme alpha-L-iduronidase (IDUA). The existence of a secretory pathway for lysosomal enzymes and the capture of secreted molecules by distant cells through binding to mannose-6-phosphate receptors have provided a rationale for enzyme replacement therapy in lysosomal storage diseases. We have used genetically modified fibroblasts implanted into neo-organs as an in vivo delivery system for IDUA. The human IDUA cDNA was isolated and inserted into a retroviral vector where it was expressed from the phosphoglycerate kinase 1 gene promoter. MPS I fibroblasts transduced with this vector showed high levels of IDUA activity and secreted phosphorylated molecules that could be internalized by naive deficient cells. Neo-organs containing 2 x 10(7) IDUA-secreting cells were implanted into nude mice. Human and murine IDUA activities were measured in the liver and spleen of animals sacrificed 35-77 days after implantation. Human IDUA activity corresponded to 0.6-2.3% of the murine enzyme activity in the liver and to 0.1-0.3% in the spleen. These data indicated that human IDUA was secreted from neo-organs and internalized in distant tissues.

The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A.

Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10-15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.

Activity of the kappa B enhancer of the interleukin-2 receptor alpha chain in somatic cell hybrids is accompanied by the nuclear localization of NF-kappa B.

The two nuclear proteins NF-kappa B (consisting of subunits p50 an dp65) and the DNA-binding subunit of NF-kappa B (p50) by itself, also called KBF1, are constitutively expressed and localized in the nucleus of the human T-cell line IARC 301.5. In order to define the roles of these two factors, which bind to the same kappa B enhancers, in transcription activation we have prepared somatic cell hybrids between IARC 301.5 and a murine myeloma. Most hybrids express both KBF1 and NF-kappa B in their nuclei, but one hybrid expresses only KBF1. The kappa B enhancer of the gene encoding the interleukin-2 (IL-2) receptor alpha chain (IL-2R alpha) is functional only in the hybrids expressing nuclear NF-kappa B. These findings show that nuclear NF-kappa B is necessary to activate the kappa B enhancer, while KBF1 by itself is not sufficient. We propose that KBF1 is a competitive inhibitor of NF-kappa B and discuss how these factors may be involved in the transient expression of IL-2 and IL-2R alpha genes during the immune response.

Kappa B binding proteins are constitutively expressed in an IL-2 autocrine human T cell line.

The IL-2 and the IL-2-R alpha genes are both expressed transiently in normal T lymphocytes after Ag or mitogen activation. In contrast, the human T cell line, IARC 301, expresses these two genes constitutively and we have previously demonstrated that its growth depends on the autocrine production of this T cell growth factor and high affinity IL-2R. To dissect the molecular basis for the unusual persistent expression of the IL-2 and IL-2-R alpha genes in these IARC 301 T cells, we have analyzed the interactions of constitutively expressed nuclear proteins with the 5' flanking regions of the IL-2 and IL-2-R alpha genes using both DNase I footprinting and gel retardation techniques. We have found that a region in both genes (-276 to -250 for IL-2-R alpha and -203 to -183 for IL-2), which corresponds to a kappa B enhancer element, is specifically protected by nuclear proteins from IARC 301. In agreement with this finding, both the IL-2 and IL-2-R alpha promoters are active in transient transfection assays in IARC 301 cells. In contrast, mutation of the kappa B enhancer results in markedly attenuated activities of both promoters. Two proteins binding the kappa B sequence, NF-kappa B and KBF1, are constitutively expressed in IARC 301 nuclei and induced by PMA and PHA in Jurkat. They bind to the kappa B motifs with different relative affinities that may reflect their different contribution in the expression of various promoters.

Modulation of interleukin 2 internalization and interleukin 2-dependent cell growth by antireceptor antibodies.

The growth factor interleukin 2 (IL2) binds to and is internalized together with high-affinity surface receptors present on lymphoid cells. This endocytosis thus results in down-regulation of the receptors. However, it is not known if the internalization is relevant to the induction of cell growth. In the present study a rat monoclonal antibody to the P55 chain of the IL2 receptor was used to examine the role of receptor internalization in the IL2-dependent autocrine human tumor T cell line IARC 301. When given alone, this antibody did not inhibit IL2 binding, internalization, or IL2-dependent cell proliferation. However, crosslinking by anti-rat immunoglobulins, which did not affect binding of the growth factor, inhibited both IL2 internalization and cell proliferation. Besides offering a novel means for the specific inhibition of the uptake of IL2 bound to IL2 high-affinity receptors, the results are compatible with the association of this receptor-ligand uptake to the growth stimulation by IL2.

Down-regulation of high affinity interleukin 2 receptors in a human tumor T cell line. Interleukin 2 increases the rate of surface receptor decay.

We have described a human tumor T cell line, IARC 301, which constitutively expresses high affinity interleukin 2 (IL2) receptors, and showed that after binding to its receptors, IL2 is endocytosed and degraded. Here we present evidence that IL2 down-regulates its own high affinity receptors. Within 1 h, IL2 induces a 60% decrease in surface receptor expression. In order to maintain this down-regulation, IL2 concentration must be high enough for the receptors to be saturated throughout the incubation. The effect of IL2 on the kinetics of receptor internalization was investigated with two approaches. First, the initial rate of IL2 internalization was measured, and no difference could be detected whether the receptors were saturated with IL2 or only partially occupied. Second, the initial rate of surface receptor decay was followed and found to be significantly decreased in the presence of IL2. Although the half-life of IL2 receptors is very short in the absence of IL2, t 1/2 approximately 65 min, suggesting that these receptors are constantly endocytosed, it can still be reduced to t 1/2 approximately 25 min when the receptors are saturated with ligand. This suggests that occupied receptors are internalized faster than and independently from free receptors. The difference in internalization rates can explain the observed receptor down-regulation.

Autocrine growth of a human T-cell line is inhibited by cyclosporin A.

The effect of cyclosporin A (CsA), a potent immunosuppressive agent, on a human T-cell line, IARC 301, which constitutively secretes interleukin-2 (IL-2) and expresses high-affinity IL-2 receptors, was investigated. We show that CsA inhibits IARC 301 cell growth. CsA also prevents the constitutive secretion of IL-2 in this T-cell line by blocking transcription of the IL-2 gene. If exogenous IL-2 is added together with CsA for 3 days, the cells grow as well as untreated controls. Thus, under such conditions, CsA inhibits IARC 301 growth by preventing its endogenous constitutive IL-2 synthesis. This demonstrates that IL-2 stimulates the proliferation of this cell line by an autocrine pathway, in agreement with our previous data. We also show for the first time, that CsA not only can inhibit IL-2 production of T cells upon activation, but that it can also prevent ongoing constitutive IL-2 synthesis of a T-cell line. Autocrine growth stimulation of tumor cells by cytokines has been demonstrated in a few cases. CsA inhibits synthesis of several cytokines. Probing for the autocrine growth of tumor cells by studying the effect of CsA and its reversibility by cytokines on their proliferation may be simple and useful.