The Mice Drawer System (MDS) experiment and the space endurance record-breaking mice

The Italian Space Agency, in line with its scientific strategies and the National Utilization Plan for the International Space Station (ISS), contracted Thales Alenia Space Italia to design and build a spaceflight payload for rodent research on ISS: the Mice Drawer System (MDS). The payload, to be integrated inside the Space Shuttle middeck during transportation and inside the Express Rack in the ISS during experiment execution, was designed to function autonomously for more than 3 months and to involve crew only for maintenance activities. In its first mission, three wild type (Wt) and three transgenic male mice over-expressing pleiotrophin under the control of a bone-specific promoter (PTN-Tg) were housed in the MDS. At the time of launch, animals were 2-months old. MDS reached the ISS on board of Shuttle Discovery Flight 17A/STS-128 on August 28(th), 2009. MDS returned to Earth on November 27(th), 2009 with Shuttle Atlantis Flight ULF3/STS-129 after 91 days, performing the longest permanence of mice in space. Unfortunately, during the MDS mission, one PTN-Tg and two Wt mice died due to health status or payload-related reasons. The remaining mice showed a normal behavior throughout the experiment and appeared in excellent health conditions at landing. During the experiment, the mice health conditions and their water and food consumption were daily checked. Upon landing mice were sacrificed, blood parameters measured and tissues dissected for subsequent analysis. To obtain as much information as possible on microgravity-induced tissue modifications, we organized a Tissue Sharing Program: 20 research groups from 6 countries participated. In order to distinguish between possible effects of the MDS housing conditions and effects due to the near-zero gravity environment, a ground replica of the flight experiment was performed at the University of Genova. Control tissues were collected also from mice maintained on Earth in standard vivarium cages.     

Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days

Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occurred only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity.     

Dissociation between rate of hepatic lipoprotein secretion and hepatocyte microtubule content

The fact that colchicines inhibits hepatic secretion of very low density lipoprotein (VLDL) particles has been interpreted to mean that microtubules are involved in hepatic VLDL secretion. To further define this relationship, we have attempted to see if changes in hepatic VLDL secretion are associated with changes in hepatocyte microtubule or tubulin content. Accordingly, hepatic secretion of VLDL was increased in rats, and the hepatocyte content of both microtubules (using quantitative morphometric methods) and tubulin (using a time-decay colchicine binding assay) was determined. In acute experiments, VLDL secretion was increased by perfusion of isolated rat livers for 2 h with varying concentrations of free fatty acids (FFA). Results indicate that hepatic VLDL triglyceride (TG) secretion at perfusate FFA levels of 0.7 μEq/ml is threefold greater (P < 0.01) than when livers are perfused without added FFA. However, no differences are observed in the content of microtubules in these livers: specifically, microtubules occupy 0.029 percent of hepatocyte cytoplasm in livers perfused without FFA and 0.030 percent of cytoplasm in livers perfused with FFA. In chronic experiments, rats were fed for 1 wk with either standard rat chow or a hyperlipidemic (sucrose/lard) diet. With the experimental diet, plasma triglyceride levels increase threefold over controls, and liver VLDL-TG production, as determined by [(3)H]glycerol turnover studies, is 55 percent greater (P < 0.01) than controls. However, microtubules occupy 0.027 percent of the cytoplasm of hepatocyte cytoplasm whether rats are on standard or hyperlipidemic diets. Furthermore, the tubulin content of isolated hepatocytes does change, and represents 1 percent of hepatocyte soluble protein, irrespective of diet. These results suggest that increases in hepatic VLDL secretion can occur without any demonstrable change in hepatocyte assembled microtubule or tubulin content, and raise questions as to the role played by microtubules in hepatic VLDL secretion.     

cDNA cloning, characterization and chromosome mapping of the gene encoding human cartilage associated protein (CRTAP)

We have recently isolated and characterized cDNA clones coding for a novel developmentally regulated avian and mouse embryo protein, CASP for Cartilage Associated Protein. Here we describe the isolation and characterization of the gene coding for the human CASP. The comparison of the putative human and mouse protein sequences with the chick sequence revealed an overall high identity (89% and 51%, respectively). Homology search with known DNA and protein sequences showed that CASPs are related to two mammalian nuclear proteins. Here we demonstrate definitively that CASPs are distinct from these nuclear proteins. However, sequence comparison analyses suggest that all of these proteins belong to a new family. In all human tissues examined two CASP mRNA species were detected, whereas a single mRNA and three mRNAs were found in chick and mouse, respectively. The human CASP gene (CRTAP) was assigned to chromosome 3p22 by fluorescence in situ hybridization.     

Nisin inducible production of listeriolysin O in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000

Background  

Listeria monocytogenesis a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactisNZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.     

Social and behavioral organization of horses on the Giara (Sardinia): distribution and aggregation

In this paper some considerations on the environment of the 42 Kmq of the volcanic-basaltic Giara tableland are discussed. Conditioning by the environment and its effect on the distribution of a population of 712 horses is illustrated in view of their social and behavioural organization.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. Transformation by rous sarcoma virus induces a switch in the collagen type synthesis and enhances fibronectin expression

Epithelial-like chondrocytes obtained from chick embryo were transformed with Rous sarcoma virus. Cellular transformation was monitored looking at the morphology change, the cell growth, and the expression of plasminogen activator. Analysis on polyacrylamide gel of intracellular and secreted proteins showed: 1) a disappearance of the specific products of differentiated chondrocytes; 2) a switch in the collagen synthesis from the type II, the chondrocyte-specific type, to the type I, characteristic of fibroblasts and other cells of mesenchymal origin; 3) an enhancement of fibronectin synthesis. Analysis of the proteins from chondrocytes infected with Rous-associated virus 1, a virus unable to induce cell transformation in vitro, indicated that the altered expression of the differentiated proteins in Rous sarcoma virus-infected chondrocytes depended upon the action of src gene product.     

Tissue-specific expression of type XIV collagen--a member of the FACIT class of collagens.

The collagens represent a highly diverse superfamily of extracellular matrix proteins that can be divided into several distinct families. One of the families, called FACIT (fibril-associated collagens with interrupted triple-helices) family, contains molecules that appear to be associated with cross-striated fibrils composed of members of the fibrillar collagen family. We have determined a portion of the primary structure of a recently discovered member of the FACIT family, chicken alpha 1(XIV) collagen, based on cloning and sequencing cDNAs. A synthetic oligopeptide from within the carboxy-terminal non-triple-helical domain of the alpha 1(XIV) chain has been used for generating specific polyclonal antibodies. The antiserum, PS1, recognizes a 220 kDa polypeptide in immunoblots of extracts of chicken skin, tendons, and cartilage. Sequencing of a tryptic peptide generated from purified, immunoreactive material, gives a sequence identical to that derived from cDNA sequencing, providing strong support for the type XIV-specificity of PS1. We have examined the expression of type XIV collagen in developing chick embryos by immunostaining of sections from 12-day-old embryos with PS1 and by Northern blot analysis of RNA from several tissues from both 12- and 17-day-old embryos. The results show that type XIV collagen is prevalent within relatively dense connective tissues such as dermis, tendons, perichondrium, perimysium, the stroma of lungs and liver, and blood vessels.