Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins^1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N₂; <2 ppm O₂) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.     

Chlamydia trachomatis Infection and Anti-Hsp60 Immunity: The Two Sides of the Coin

Chlamydia trachomatis (CT) infection is one of the most common causes of reproductive tract diseases and infertility. CT-Hsp60 is synthesized during infection and is released in the bloodstream. As a consequence, immune cells will produce anti-CT-Hsp60 antibodies. Hsp60, a ubiquitous and evolutionarily conserved chaperonin, is normally sequestered inside the cell, particularly into mitochondria. However, upon cell stress, as well as during carcinogenesis, the chaperonin becomes exposed on the cell surface (sf-Hsp60) and/or is secreted from cells into the extracellular space and circulation. Reports in the literature on circulating Hsp and anti-Hsp antibodies are in many cases short on details about Hsp60 concentrations, and about the specificity spectra of the antibodies, their titers, and their true, direct, pathogenetic effects. Thus, more studies are still needed to obtain a definitive picture on these matters. Nevertheless, the information already available indicates that the concurrence of persistent CT infection and appearance of sf-Hsp60 can promote an autoimmune aggression towards stressed cells and the development of diseases such as autoimmune arthritis, multiple sclerosis, atherosclerosis, vasculitis, diabetes, and thyroiditis, among others. At the same time, immunocomplexes composed of anti-CT-Hsp60 antibodies and circulating Hsp60 (both CT and human) may form deposits in several anatomical locations, e.g., at the glomerular basal membrane. The opposite side of the coin is that pre-tumor and tumor cells with sf-Hsp60 can be destroyed with participation of the anti-Hsp60 antibody, thus stopping cancer progression before it is even noticed by the patient or physician.     

Human Hsp10 and Early Pregnancy Factor (EPF) and their relationship and involvement in cancer and immunity: Current knowledge and perspectives

This article is about Hsp10 and its intracellular and extracellular forms focusing on the relationship of the latter with Early Pregnancy Factor and on their roles in cancer and immunity. Cellular physiology and survival are finely regulated and depend on the correct functioning of the entire set of proteins. Misfolded or unfolded proteins can cause deleterious effects and even cell death. The chaperonins Hsp10 and Hsp60 act together inside the mitochondria to assist protein folding. Recent studies demonstrated that these proteins have other roles inside and outside the cell, either together or independently of each other. For example, Hsp10 was found increased in the cytosol of different tumors (although in other tumors it was found decreased). Moreover, Hsp10 localizes extracellularly during pregnancy and is often indicated as Early Pregnancy Factor (EPF), which is released during the first stages of gestation and is involved in the establishment of pregnancy. Various reports show that extracellular Hsp10 and EPF modulate certain aspects of the immune response with anti-inflammatory effects in patients with autoimmune conditions improving clinically after treatment with recombinant Hsp10. Moreover, Hsp10 and EPF are involved in embryonic development, acting as a growth factor, and in cell proliferation/differentiation mechanisms. Therefore, it becomes evident that Hsp10 is not only a co-chaperonin, but an active player in its own right in various cellular functions. In this article, we present an overview of various aspects of Hsp10 and EPF as they participate in physiological and pathological processes such as the antitumor response and autoimmune diseases.     

Chaperonopathies and chaperonotherapy

The study of molecular chaperones (genetics, structure, location, physiology, pathology, and therapeutics) has developed into a science with specific objectives, methods, and hypotheses, a discipline we called chaperonology. Subdisciplines of chaperonology include the study of pathological chaperones (chaperonopathies) and the analysis of their genes in sequenced genomes (chaperonomics). Chaperonopathies are pathological conditions in which one type of chaperone is deficient due to a genetic or acquired defect that modifies the chaperone's structure and/or makes the chaperone unavailable for functioning when needed. Experimental and clinical data show that chaperones and their genes can be used for treating various pathological conditions, thus justifying the development of chaperonotherapy. We discuss recent work showing that chaperonotherapy is on solid foundations: the data demonstrate that molecular chaperones counteract pathogenetic mechanisms in disease and during stress.     

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.     

The superficial antigenic mosaic of Methanobrevibacter smithii

A panel of six different monoclonal antibodies (6A to F) was generated using Methanobrevibacter smithii strain PS as immunogen. The antibodies were characterized and calibrated by standard techniques and with a novel application of the slide immunoenzymatic assay (SIA) for determination of the l-chain type of the monoclonal antibody molecule. Five (and possibly six) determinants were identified with the antibodies. Each antibody recognized one determinant exclusively, except for antibodies 6B and 6F which might recognize the same determinant, although some data suggest that antibody 6F recognizes a sixth determinant different from the other five. The determinant for antibody 6A involves Glu, Lys and Orn. It is most likely located in the region of the peptide moiety of pseudomurein which is typical of strain PS. The six antibodies reacted with whole bacterial cells unfixed or formalinized and/or heat-fixed, but did not react with the other M. smithii reference strain ALI, or with any other reference methanogen tested. However, the antibodies did react with a number of isolates from human feces considered M. smithii from morphologic, physiologic and immunologic information, and were instrumental for grouping the isolates.Abbreviations PBS phosphate buffered saline - SIA slide immunoenzymatic assay - IIF indirect immunofluorescence     

Silent generation of memory in genetic low-responder mice after a single contract with a bacterial antigen.

It is shown that immunologic memory can progressively develop following a single contact with a bacterial antigen (Escherichia coli β-d-galactosidase) in the absence of any sign of an antibody response. The relevance that this silent preparation for an anamnestic response might have in the pathogenesis of unexpected immune reactions is emphasized.     

Methanocorpusculaceae fam. nov., represented by Methanocorpusculum parvum,Methanocorpusculum sinense spec. nov. and Methanocorpusculum bavaricum spec. nov.

Two new methanogenic bacteria, Methanocorpusculum sinense spec. nov. strain DSM 4274 from a pilot plant for treatment of distillery wastewater in Chengdu (Province Sichuan, China), and Methanocorpusculum bavaricum spec. nov. strain DSM 4179, from a wastewater pond of the sugar factory in Regensburg (Bavaria, FRG) are described. Methanocorpusculum strains are weakly motile and form irregularly coccoid cells, about 1 μm in diameter. The cell envelope consists of a cytoplasmic membrane and a S-layer, composed of hexagonally arranged glycoprotein subunits with molecular weights of 90000 (Methanocorpusculum parvum), 92000 (M. sinense), and 94000 (M. bavaricum). The center-to-center spacings are 14.3 nm, 15.8 nm and 16.0 nm, respectively. Optimal growth of strains is obtained in the mesophilic temperature range and at a pH around 7. Methane is produced from H₂/CO₂, formate, 2-propanol/CO₂ and 2-butanol/CO₂ by M. parvum and M. bavaricum, whereas M. sinense can only utilize H₂/CO₂ and formate. Growth of M. sinense and M. bavaricum is dependent on the presence of clarified rumen fluid. The G+C content of the DNA of the three strains is ranging from 47.7–53.6 mol% as determined by different methods. A similar, but distinct polar lipid pattern indicates a close relationship between the three Methanocorpusculum species. The polyamine patterns of M. parvum, M. sinense and M. bavaricum are similar, but distinct from those of other methanogens and are characterized by a high concentration of the otherwise rare 1,3-diaminopropane. Quantitative comparison of the antigenic fingerprint of members of Methanocorpusculum revealed no antigenic relationship with any one of the reference methanogens tested. On the basis of the distant phylogenetic position of M. parvum and the data presented in this paper a new family, the Methanocorpusculaceae fam. nov., is defined.