Humoral primary immune response in vitro in a homologous mouse system: replacement of fetal calf serum by a 2-mercaptoethanol or macrophage-activated fraction of mouse serum.

The primary immune response in mouse spleen cell cultures against heterologous red cell antigens is dependent on the medium being supplemented with selected batches of fetal calf serum. Mouse serum itself is not able to support this response. The active immune response-supporting component in fetal calf serum seems to be a distinct factor (s), which has been partially purified by Sephadex G-100 filtration and termed MaSF-2-mercaptoethanol-activated serum factor. In this report it is demonstrated that MaSF is also present in mouse serum. For functional detection, mouse MaSF has to be separated from higher m.w. inhibitors, and has to be activated by 2-ME. After separation and activation mouse MaSF can support the primary immune response in a completely homologous in vitro culture system. Evidence is presented that MaSF can also be activated by macrophages. It is concluded that macrophages and 2-ME have the same mode of action in the primary immune response in vitro, i.e., induction of lymphocyte competence by activation of a serum factor.     

Heterologous expression of bacteriocin E-760 in Chlamydomonas reinhardtii and functional analysis

The use of antimicrobial peptides (AMPs) synthesized by bacteria (bacteriocins) is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production. The bacteriocin E-760 isolated from the genus Enterococcus sp. has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria. In this study, the expression of a chimeric protein coding for E-760 in the nucleus of C. reinhardtii was evaluated, as well as, its antibacterial activity. The synthetic gene E-760S was inserted into the genome of C. reinhardtii using Agrobacterium tumefaciens. A transgenic line was identified in TAP medium with hygromycin and also by PCR. The increment in the culture medium temperature of the transgenic strain at 35 °C for 10 minutes, increased the production level of the recombinant protein from 0.14 (Noninduced culture, NIC) to 0.36% (Induced culture, IC) of total soluble proteins (TSP); this was quantified by an ELISA assay. Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log, Streptococcus agalactiae in 0.48 U log, Enterococcus faecium in 0.36 U log, Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae, the activity was 0.07 U log. These results demonstrate that the nucleus transformation of C. reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.     

Two newly identified SipA domains (F1, F2) steer effector protein localization and contribute to Salmonella host cell manipulation

Salmonella Typhimurium causes bacterial enterocolitis. The type III secretion system (TTSS)-1 is a key virulence determinant of S. Typhimurium mediating host cell invasion and acute enterocolitis. The TTSS-1 effector protein SipA is transported into host cells, accumulates in characteristic foci at the bacteria-host cell interface, manipulates signalling and affects virulence. Two functional domains of SipA have previously been characterized: The N-terminal SipA region (amino acids 1-105) mediates TTSS-1 transport and the C-terminal SipA 'actin-binding' domain (ABD; amino acids 446-685) manipulates F-actin assembly. Little is known about the central region of SipA. In a deletion analysis we found that the central SipA region harbours two distinct functional domains, F1 and F2. They are involved in SipA focus formation and host manipulation. The F1 domain (amino acids 170-271) drives SipA focus formation and domain F2 (amino acids 280-394) enhances this process by mediating SipA-SipA interactions. SipA variants lacking the F1-, the F2- or the actin binding domain were attenuated in virulence assays, namely host cell invasion and/or virulence in a mouse model for enterocolitis. Our results show that the newly identified SipA domains have distinct functions. Nevertheless, cooperation between the SipA domains F1, F2 and ABD is required to promote Salmonella virulence.     

The Tyrosine Kinase Inhibitor Sunitinib Affects Ovulation but Not Ovarian Reserve in Mouse: A Preclinical Study

The aim of the study was to evaluate ovarian toxicity of tyrosine kinase inhibitor (TKI) sunitinib, since only scarce data are available on gonadal function after this treatment. Six-week-old female mice received orally, once daily, vehicle or sunitinib (50 mg/kg/d) during 5 weeks. Fertility parameters were analyzed from ovulation to litter assessment. Sunitinib exposure significantly reduced (i) corpora lutea number per ovary (1.1 ± 0.38 in sunitinib group versus 4 ± 0.79 in control group, p<0.01) and (ii) serum Anti Müllerian hormone (AMH) levels in sunitinib treated mice (12.01 ± 1.16) compared to control mice (14.33 ± 0.87 ng/ml, p< 0.05). However, primordial and growing follicles numbers per ovary were not different in both groups. After treatment withdrawal, female mice in both groups were able to obtain litters. These data could be helpful to counsel clinicians and patients, when fertility preservation methods are discussed, before TKI treatment in girls and young women.     

Safe biotechnology. 8. Transport of infectious and biological materials

The transport of infectious and biological material is regulated by a number of international organizations. This mini-review has been compiled to increase awareness within the scientific community of problems caused by differences in terminology (such as infectious materials/substances, biological products, diagnostic specimens, genetically modified microorganisms) and certain technical aspects of the main international guidelines, and to assist policy makers in the creation of harmonized guidelines. A list of relevant Internet resources has been compiled. Received: 3 March 1997 / Accepted: 8 March 1997     

Permeability characteristics of cell-membrane pores induced by ostreolysin A/pleurotolysin B,binary pore-forming proteins from the oyster mushroom

Proteins from the oyster mushroom, 15 kDa ostreolysin A (OlyA), and 59 kDa pleurotolysin B (PlyB) with a membrane attack complex/perforin (MACPF) domain, damage cell membranes as a binary cytolytic pore-forming complex. Measurements of single-channel conductance and transmembrane macroscopic current reveal that OlyA/PlyB form non-selective ion-conducting pores with broad, skewed conductance distributions in N18 neuroblastoma and CHO-K1 cell membranes. Polyethylene-glycol 8000 (hydrodynamic radius of 3.78 nm) provides almost complete osmotic protection against haemolysis, which strongly suggests a colloid-osmotic type of erythrocyte lysis. Our data indicate that OlyA/PlyB form transmembrane pores of varied sizes, as other pore-forming proteins with a MACPF domain.     

A new ant genus from the Greater Antilles and Central America,Zatania (Hymenoptera: Formicidae), exemplifies the utility of male and molecular character systems

The ant genus Prenolepis (Hymenoptera: Formicidae) is the nominal member of the recently established Prenolepis genus‐group within the subfamily Formicinae. Our molecular phylogenetic analyses using fragments from five nuclear genes (arginine kinase, carbomoylphosphate synthase, elongation factor 1‐alpha F1, elongation factor 1‐alpha F2, wingless) and one mitochondrial gene (cytochrome oxidase I) indicate that this genus is polyphyletic. Although the majority of Prenolepis species was found to belong to the same monophyletic group (Prenolepis sensu stricto), a smaller subset of Prenolepis species, all found in either Central America or the Greater Antilles, was robustly inferred to comprise a distinct lineage that is sister to the Old World genus Paraparatrechina. Here we describe this newly discovered lineage within the larger Prenolepis genus‐group clade. The genus Zatania, gen.n. is composed of five extant species (Zatania albimaculata, Zatania cisipa, Zatania gibberosa, Zatania gloriosa, sp.n. and Zatania karstica) and one Dominican amber fossil species (Zatania electra^?, sp.n. ). These are medium‐sized ants (generally between 2.5 and 3 mm in total length) that are characterized by having long scapes and legs, and elongated mesosomata. A reliance on worker‐based taxonomy has previously prevented the discovery of this new lineage because of worker convergence consisting of various combinations of elongated mesosomata, long scapes and legs, and a constriction immediately behind the pronotum, observed in several distinct lineages within the Prenolepis genus‐group. However, we did find that male morphology complements our molecular results in revealing important diagnostic and potentially phylogenetically informative characters. Our study highlights the value for ant systematics to expand beyond its traditional foundation of worker‐based morphology and embrace character systems from other castes and molecular data.