Cold-adapted enzymes from marine Antarctic microorganisms.

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the viscosity of the medium, which is also significantly enhanced by low temperatures, contributes to slow down reaction rates. This review analyses the different challenges and focuses on a key element of life at low temperatures: cold-adapted enzymes. The molecular characteristics of these enzymes are discussed as well as the adaptation strategies which can be inferred from the comparison of their properties and three-dimensional structures with those of their mesophilic counterparts. As these enzymes display a high specific activity at low and moderate temperatures associated with a relatively high thermosensitivity, the interest in these properties is discussed with regard to their current and possible applications in biotechnology.

A perspective on cold enzymes: current knowledge and frequently asked questions.

Studies on psychrophilic enzymes to determine the structural features important for cold-activity have attracted increased attention in the last few years. This enhanced interest is due to the attractive properties of such proteins, i.e. a high specific activity and a low thermal stability, and thus, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications. This review examines the impact of low temperatures on life, the diversity of adaptation to counteract these effects and gives an overview of the features proposed to account for low thermal stability and cold-activity, following the chronological order of the catalytic cycle phases. Moreover, we present an overview of recent techniques used in the analysis of the flexibility of a protein structure which is an important concept in cold-adaptation; an overview of biotechnological potential of psychrophilic enzymes and finally, a few frequently asked questions about cold-adaptation and their possible answers.

Some like it cold: biocatalysis at low temperatures.

In the last few years, increased attention has been focused on a class of organisms called psychrophiles. These organisms, hosts of permanently cold habitats, often display metabolic fluxes more or less comparable to those exhibited by mesophilic organisms at moderate temperatures. Psychrophiles have evolved by producing, among other peculiarities, "cold-adapted" enzymes which have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency associated, however, with a low thermal stability. Thanks to recent advances provided by X-ray crystallography, structure modelling, protein engineering and biophysical studies, the adaptation strategies are beginning to be understood. The emerging picture suggests that psychrophilic enzymes are characterized by an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. Due to their attractive properties, i.e., a high specific activity and a low thermal stability, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications.

High-efficiency transduction and long-term gene expression with a murine stem cell retroviral vector encoding the green fluorescent protein in human marrow stromal cells.

Bone marrow stromal cells (MSCs) are unique mesenchymal cells that have been utilized as vehicles for the delivery of therapeutic proteins in gene therapy protocols. However, there are several unresolved issues regarding their potential therapeutic applications. These include low transduction efficiency, attenuation of transgene expression, and the technical problems associated with drug-based selection markers. To address these issues, we have developed a transduction protocol that yields high-level gene transfer into human MSCs, employing a murine stem cell virus-based bicistronic vector containing the green fluorescent protein (GFP) gene as a selectable marker. Transduction of MSCs plated at low density for 6 hr per day for 3 days with high-titer viral supernatant resulted in a gene transfer efficiency of 80+/-6% (n = 10) as measured by GFP fluorescence. Neither centrifugation nor phosphate depletion increased transduction efficiency. Assessment of amphotropic receptor (Pit-2) expression by RT-PCR demonstrated that all MSCs expressing the receptor were successfully transduced. Cell cycle distribution profiles measured by propidium iodide staining showed no correlation with the susceptibility of MSCs to transduction by the retroviral vector. Human MSCs sequentially transduced with an adenoviral vector encoding the ecotropic receptor and ecotropic retroviral vector encoding GFP demonstrated that all MSCs are susceptible to retroviral transduction. We further showed that both genes of bicistronic vector are expressed for at least 6 months in vitro and that transgene expression did not affect the growth or osteogenic differentiation potential of MSCs. Future studies will be directed toward the development of gene therapy protocols employing this strategy.

Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta.

In principle, transplantation of mesenchymal progenitor cells would attenuate or possibly correct genetic disorders of bone, cartilage and muscle, but clinical support for this concept is lacking. Here we describe the initial results of allogeneic bone marrow transplantation in three children with osteogenesis imperfecta, a genetic disorder in which osteoblasts produce defective type I collagen, leading to osteopenia, multiple fractures, severe bony deformities and considerably shortened stature. Three months after osteoblast engraftment (1.5-2.0% donor cells), representative specimens of trabecular bone showed histologic changes indicative of new dense bone formation. All patients had increases in total body bone mineral content ranging from 21 to 29 grams (median, 28), compared with predicted values of 0 to 4 grams (median, 0) for healthy children with similar changes in weight. These improvements were associated with increases in growth velocity and reduced frequencies of bone fracture. Thus, allogeneic bone marrow transplantation can lead to engraftment of functional mesenchymal progenitor cells, indicating the feasibility of this strategy in the treatment of osteogenesis imperfecta and perhaps other mesenchymal stem cell disorders as well.

Isolation and molecular characterization of the human CD34 gene.

The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5' termini of mRNAs by 5'-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5' and 3' untranslated regions, amino acid coding sequences, and 5' flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.

Absent or rare human immunodeficiency virus infection of bone marrow stem/progenitor cells in vivo.

An important question in human immunodeficiency virus (HIV) pathogenesis is whether HIV-infected bone marrow CD34+ stem/progenitor cells serve as a significant reservoir of virus in HIV-infected individuals. Our data indicate that infection of bone marrow stem/progenitor cells with HIV occurs rarely, if ever, in vivo. In the present study, CD34+ cells were immunomagnetically purified from the bone marrow of HIV-seropositive individuals, and purified cells or colony-forming cells of the granulocyte/macrophage lineage were analyzed for HIV proviral DNA by the polymerase chain reaction. No HIV DNA was detected in colony-forming cells of the granulocyte/macrophage lineage from HIV-positive patients. Furthermore, no virus was found in CD34(+)-enriched cells from six of seven samples from asymptomatic HIV-infected individuals and four of four samples from patients with AIDS-related complex or AIDS. Thus, infected stem cells are not a major source of persistent HIV and do not account for hematopoietic suppression. These findings have positive implications for the concept of marrow reconstitution with autologous stem cells, genetically engineered for HIV resistance, following marrow-ablative antiviral therapy.