Production of pharmaceutical proteins in milk

There is every reason to expect that it will be possible within the next few years to begin to use farm animals to produce large quantities of some of the human proteins that are needed for the treatment of disease. Revolutionary new opportunities for the production of novel proteins in milk have been created by the development of methods for gene transfer. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue specific hormonally induced expression in the mammary gland. Studies with three genes, ovine -lactoglobulin, rat -casein and whey acidic protein of rat and mouse, suggest that they may all meet this requirement. Fragments of the ovine -lactoglobulin, murine whey acidic protein and rabbit -casein genes have directed production of novel proteins in the milk of transgenic mice, sheep, rabbits and pigs. The proteins were biologically active and usually co-migrated with authentic proteins. In early experiments, protein concentration was low, but our recent observations suggest that fusion genes containing genomic clones direct production of concentrations of protein that are suitable for commercial exploitation. In the longer term, two approaches may offer the potential of more reliable expression. Control elements capable of directing expression that is independent of site of insertion of the gene, but dependent on the number of copies of the gene, have been identified for a small number of genes. The availability of such elements for the milk protein genes would increase the reliability of gene expression considerably. Alternatively, targeted mutation of genes may allow the insertion of coding sequences within an existing gene so avoiding position effects.     

Anti-bovine beta-lactoglobulin antibodies react with a human lactoferrin fragment and bovine beta-lactoglobulin present in human milk

Human milk samples react against anti-bovine beta-lactoglobulin rabbit antibodies, as measured by a competitive radioimmunoassay. Immunoreactivity was positive even in milk from mothers consuming a diet free of cow's milk. An increase with a diet rich in cow's milk proteins was detected by immunoelectrophoresis. The human milk fraction cross-reacting with anti-bovine beta-lactoglobulin antibodies corresponds to the 20 kDa fragment from the N-terminal end of human lactoferrin. Three regions of this fragment exhibit sequence homology with a sequence contained in cow's beta-lactoglobulin (between residues 124 and 141).     

Association of different mobile elements to generate novel integrative elements

Among the more important problems in modern hospitals is the prevalence of bacterial pathogens expressing resistance to multiple antimicrobial agents. The frequency of multiresistance suggests mechanisms by which bacterial species can concentrate and efficiently exchange a variety of resistance determinants. Mechanisms by which this occurs include insertion of transposons within transposons, coalescence through the activity of insertion sequences and the employment of integrons. In some instances, more than one of these mechanisms is involved in creating large multiresistance genetic elements. The association of the elements with transferable elements or transposons may promote rapid dissemination among clinical strains, and create further opportunities for inclusion of additional resistance determinants.     

Casein, a prohormone with an immunomodulating role for the newborn?

Maternal colostrum and milk, the earliest food of the newborn, should not only be considered as supplying nutrients, but also as agents providing protection against aggressions from the new environment. Indeed by enzymatic digestion of the main milk proteins, the caseins, biologically active peptides are released; they may be implicated in the stimulation of the newborn's immune system. From this point of view a 'strategic active zone' has been characterized in beta-casein. A possible role of casein as a 'prohormone' for the newborn is suggested.     

Molecular mechanisms of spider silk

Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins has revealed an association between the protein sequences and structure-property relationships. However, before spider silks can be subject to genetic engineering for commercial applications, the complete protein sequences and their functions, as well as the details of the spinning mechanism, will require additional progress and collaborative efforts in the areas of biochemistry, molecular biology and material science. Novel approaches to reveal additional molecular constituents embedded in the spider fibers, as well as cloning strategies to manipulate the genes for expression, will continue to be important aspects of spider biology research. Here we summarize the molecular characteristics of the different spider fibroins, the mechanical properties and assembly process of spidroins and the advances in protein expression systems used for recombinant silk production. We also highlight different technical approaches being used to elucidate the molecular constituents of silk fibers. Received 28 February 2006; received after revision 14 April 2006; accepted 22 May 2006 X. Hu and K. Vasanthavada contributed equally to this work.     

Helix insertion into bilayers and the evolution of membrane proteins

Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic α-helical membrane protein insertion are discussed.     

Heat shock protein gene expression during Xenopus development

Stress-induced heat shock protein gene expression is developmentally regulated during early embryogen esis of the frog, Xenopus laevis. For example, a number of heat shock protein genes, such as hsp70, hsp90, and ubiquitin are not heat-inducible until after the midblastula stage of embryogenesis. Furthermore, the family of small heat shock protein genes, hsp30, are differentially expressed after the midblastula stage as well as being regulated at the level of mRNA stability. Many of these stress proteins are also synthesized constitutively during oogenesis and embryogenesis during which they may act as molecular chaperones as well as being involved in sequestering proteins in an inactive state until required by the developing embryo. Furthermore the induction of these stress protein genes has been correlated with enhanced thermoresistance. During stressful conditions heat shock proteins probably prevent aggregation or misfolding of damaged protei ns within the embryo.     

RNA processing and human disease

Gene expression involves multiple regulated steps leading from gene to active protein. Many of these steps involve some aspect of RNA processing. Diseases caused by mutations that directly affect RNA processing are relatively rare compared with mutations that disrupt protein function. The vast majority of diseases of RNA processing result from loss of function of a single gene due to mutations in cis-acting elements required for pre-messenger RNA (mRNA) splicing. However, a few diseases are caused by alterations in the trans-acting factors required for RNA processing and in the vast majority of cases it is the pre-mRNA splicing machinery that is affected. Clearly, alterations that disrupt splicing of pre-mRNAs from large numbers of genes would be lethal at the cellular level. A common theme among these diseases is that only subsets of genes are affected. This is consistent with an emerging view that different subsets of exons require different sets of cis-acting elements and trans-acting factors.     

Glycine-rich proteins as structural components of plant cell walls

Glycine-rich proteins (GRPs) have been found in the cell walls of many higher plants and form a third group of structural protein components of the wall in addition to extensins and proline-rich proteins. The primary sequences of GRPs contain more than 60% glycine. GRPs are localized mainly in the vascular tissue of the plant, and their coding genes provide an excellent system to analyze the molecular basis of vascular-specific gene expression. In French bean, the major cell wall GRP has been localized at the ultrastructural level in the modified primary cell wall of protoxylem. Immunological studies showed that it forms a major part of these highly extensible and specialized cell walls. Specific digestion of GRP1.8 from bean by collagenase suggests that it shares structural similarities with collagen. The protein is synthesized by living protoxylem cells as well as xylem parenchyma cells. After cell death, GRPs are exported from neighboring xylem parenchyma cells to the protoxylem wall, a rare example of protein transport between cells in plants. We propose that GRPs are part of a repair system of the plant during the stretching phase of protoxylem.     

Chromosomal organization at the level of gene complexes

Metazoan genomes primarily consist of non-coding DNA in comparison to coding regions. Non-coding fraction of the genome contains cis-regulatory elements, which ensure that the genetic code is read properly at the right time and space during development. Regulatory elements and their target genes define functional landscapes within the genome, and some developmentally important genes evolve by keeping the genes involved in specification of common organs/tissues in clusters and are termed gene complex. The clustering of genes involved in a common function may help in robust spatio-temporal gene expression. Gene complexes are often found to be evolutionarily conserved, and the classic example is the hox complex. The evolutionary constraints seen among gene complexes provide an ideal model system to understand cis and trans-regulation of gene function. This review will discuss the various characteristics of gene regulatory modules found within gene complexes and how they can be characterized.     

Characterization of a thermosensitive protein from human milk whey

Summary A human milk whey protein, which aggregates at room temperature and resolubilizes when cooled, was purified by chromatography on hydroxyapatite. The present study demonstrated that the thermosensitive protein is a nonphosphorylated form of -casein.We thank Dr J.J. Pahud for preparation of anti Na-caseinate antiserum and Miss D. Fumeaux for technical assistance.     

Absence of a robust innate immune response in rat neurons facilitates persistent infection of Borna disease virus in neuronal tissue

Borna disease virus (BDV) persistently infects neurons of the central nervous system of various hosts, including rats. Since type I IFN-mediated antiviral response efficiently blocks BDV replication in primary rat embryo fibroblasts, it has been speculated that BDV is not effectively sensed by the host innate immune system in the nervous system. To test this assumption, organotypical rat hippocampal slice cultures were infected with BDV for up to 4 weeks. This resulted in the secretion of IFN and the up-regulation of IFN-stimulated genes. Using the rat Mx protein as a specific marker for IFN-induced gene expression, astrocytes and microglial cells were found to be Mx positive, whereas neurons, the major cell type in which BDV is replicating, lacked detectable levels of Mx protein. In uninfected cultures, neurons also remained Mx negative even after treatment with high concentrations of IFN-α. This non-responsiveness correlated with a lack of detectable nuclear translocation of both pSTAT1 and pSTAT2 in these cells. Consistently, neuronal dissemination of BDV was not prevented by treatment with IFN-α. These data suggest that the poor innate immune response in rat neurons renders this cell type highly susceptible to BDV infection even in the presence of exogenous IFN-α. Intriguingly, in contrast to rat neurons, IFN-α treatment of mouse neurons resulted in the up-regulation of Mx proteins and block of BDV replication, indicating species-specific differences in the type I IFN response of neurons between mice and rats.     

Genetic studies of diseases

Genomic alterations lead to cancer complexity and form a major hurdle for comprehensive understanding of the molecular mechanisms underlying oncogenesis. In this review, we describe recent advances in studying cancer-associated genes from a systems biology point of view. The integration of known cancer genes onto protein and signaling networks reveals the characteristics of cancer genes within networks. This approach shows that cancer genes often function as network hub proteins which are involved in many cellular processes and form focal nodes in information exchange between many signaling pathways. Literature mining allows constructing gene-gene networks, in which new cancer genes can be identified. The gene expression profiles of cancer cells are used for reconstructing gene regulatory networks. By doing so, genes which are involved in the regulation of cancer progression can be picked up from these networks, after which their functions can be further confirmed in the laboratory.