C2360, a nuclear protein expressed in human proliferative cytotrophoblasts, is a representative member of a novel protein family with a conserved coiled coil-helix-coiled coil-helix domain

In this study, we describe the identification of nine novel genes isolated from a unique human first-trimester cDNA library generated from the placental bed. One of these clones, called C2360 and located on chromosome 10q22, was selected as it showed restricted expression in placental bed tissue as well as in JEG3 choriocarcinoma cells with absent expression in adult tissues. We show that the expression is restricted to first-trimester proliferative trophoblasts of the proximal column and show that C2360 is a nuclear protein. No detectable transactivation potential was observed for different domains of the protein. Secondary structure prediction showed that C2360 is a representative member of a eukaryotic family of proteins with a low conservation at the amino acid level, but with strong conservation at the structural level, sharing the general domain (coiled coil 1)-(helix 1)-(coiled coil 2)-(helix 2), or CHCH domain. Each alpha-helix within this domain contains two cysteine amino acids, and these intrahelical cysteines are separated by nine amino acids (C-X(9)-C motif). The fixed position within each helix indicated that both helices could form a hairpin structure stabilized by two interhelical disulfide bonds. Other proteins belonging to the family include estrogen-induced gene 2 and the ethanol-induced 6 protein. The conserved motif was found in yeast, plant, Drosophila, Caenorhabditis elegans, mouse, and human proteins, indicating that the ancestor of this protein family is of eukaryotic origin. These results indicate that C2360 is a representative member of a multifamily of proteins, sharing a protein domain that is conserved in eukaryotes.     

Presenilins in memory, Alzheimer's disease, and therapy

Presenilins are considered to be the catalytic subunits of the gamma-secretase complex and are therefore drug targets for Alzheimer's disease. They are also essential for the fine tuning of the immunological system and for memory and synaptic plasticity. Genetic ablation in the forebrain results in a progressive neurodegenerative process that is independent from Abeta generation. The question arises as to what extent these observations should influence our thinking on the pathogenesis of Alzheimer's disease and on strategies to further develop gamma-secretase inhibitors.     

Factors influencing glycosylation of Trichoderma reesei cellulases. II: N-glycosylation of Cel7A core protein isolated from different strains

A systematic analysis of the N-glycosylation of the catalytic domain of cellobiohydrolase I (Cel7A or CBH I) isolated from several Trichoderma reesei strains grown in minimal media was performed. Using a combination of chromatographic, electrophoretic, and mass spectrometric methods, the presence of glucosylated and phosphorylated oligosaccharides on the three N-glycosylation sites of Cel7A core protein (from T. reesei strains Rut-C30 and RL-P37) confirms previous findings. With N-glycans isolated from other strains, no end-capping glucose could be detected. Phosphodiester linkages were however found in proteins from each strain and these probably occur on both the alpha1-3 and the alpha1-6 branch of the high-mannose oligosaccharide tree. Evidence is also presented for the occurrence of mannobiosyl units on the phosphodiester linkage. Therefore the predominant N-glycans on Cel7A can be represented as (ManP)(0-1)GlcMan(7-8)GlcNAc2 for the hyperproducing Rut-C30 and RL-P37 mutants and as (Man(1-2)P)(0-1-2)Man(5-6-7)GlcNAc2 for the wild-type strain and the other mutants. As shown by ESI-MS, random substitution of these structures on the N-glycosylation sites explains the heterogeneous glycoform population of the isolated core domains. PAG-IEF separates up to five isoforms, resulting from posttranslational modification of Cel7A with mannosyl phosphodiester residues at the three distinct sites. This study clearly shows that posttranslational phosphorylation of glycoproteins is not atypical for Trichoderma sp. and that, in the case of the Rut-C30 and RL-P37 strains, the presence of an end-capped glucose residue at the alpha1-3 branch apparently hinders a second mannophoshoryl transfer.     

Fatty acid production from amino acids and alpha-keto acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and alpha-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of alpha-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and alpha-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from alpha-keto acids only. BL2 also converted alpha-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and alpha-keto acids and that carbon metabolism is important in regulating this event.     

The Rb tumor suppressor is required for stress erythropoiesis

The retinoblastoma tumor suppressor gene plays important roles in cell cycle control, differentiation and survival during development and is functionally inactivated in most human cancers. Early studies using gene targeting in mice suggested a critical role for pRb in erythropoiesis, while more recent experiments have suggested that many of the abnormal embryonic phenotypes in the Rb null mouse result from a defective placenta. To address this controversy and determine whether Rb has cell intrinsic functions in erythropoiesis, we examined the effects of Rb loss on red cell production following acute deletion of pRb in vitro and under different stress conditions in vivo. Under stress conditions, pRb was required to regulate erythroblast expansion and promote red cell enucleation. Acute deletion of Rb in vitro induced erythroid cell cycle and differentiation defects similar to those observed in vivo. These results demonstrate a cell intrinsic role for pRb in stress erythropoiesis and hematopoietic homeostasis that has relevance for human diseases.     

Exploiting dendritic cells for cancer immunotherapy: genetic modification of dendritic cells

Dendritic cells (DCs) are pivotal regulators of immune reactivity and immune tolerance. The observation that DCs can recruit naive T cells has invigorated cancer immunology and led to the proposal of DCs as the basis for vaccines designed for the treatment of cancer. Designing effective strategies to load DCs with antigens is a challenging field of research. The successful realization of gene transfer to DCs will be highly dependent on the employed vector system. Here, we review various viral and non-viral gene transfer systems, and discuss their distinct characteristics and possible advantages and disadvantages in respect to their use in DC-based immunotherapy.     

Satisfaction (not) guaranteed: re-evaluating the use of animal models of type 1 diabetes

Without a doubt, rodent models have been instrumental in describing pathways that lead to pancreatic beta-cell destruction, evaluating potential causes of type 1 diabetes and providing proof-of-principle for the potential of immune-based interventions. However, despite more than two decades of productive research, we are still yet to define an initiating autoantigen for the human disease, to determine the precise mechanisms of beta-cell destruction in humans and to design interventions that prevent or cure type 1 diabetes. In this Perspective article, we propose that a major philosophical change would benefit this field, a proposition that is based on evaluation of situations in which rodent models have provided useful guidance and in which they have led to disappointments.     

Amylopectin molecular structure reflected in macromolecular organization of granular starch

For lintners with negligible amylose retrogradation, crystallinity related inversely to starch amylose content and, irrespective of starch source, incomplete removal of amorphous material was shown. The latter was more pronounced for B-type than for A-type starches. The two predominant lintner populations, with modal degrees of polymerization (DP) of 13-15 and 23-27, were best resolved for amylose-deficient and A-type starches. Results indicate a more specific hydrolysis of amorphous lamellae in such starches. Small-angle X-ray scattering showed a more intense 9-nm scattering peak for native amylose-deficient A-type starches than for their regular or B-type analogues. The experimental evidence indicates a lower contrasting density within the "crystalline" shells of the latter starches. A higher density in the amorphous lamellae, envisaged by the lamellar helical model, explains the relative acid resistance of linear amylopectin chains with DP > 20, observed in lintners of B-type starches. Because amylopectin chain length distributions were similar for regular and amylose-deficient starches of the same crystal type, we deduce that the more dense (and ordered) packing of double helices into lamellar structures in amylose-deficient starches is due to a different amylopectin branching pattern.     

Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity

Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes--the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules.     

Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex

Phosphatidylinositol 4-kinasebeta (PI4Kbeta) plays an essential role in maintaining the structural integrity of the Golgi complex. In a search for PI4Kbeta-interacting proteins, we found that PI4Kbeta specifically interacts with the GTP-bound form of the small GTPase rab11. The PI4Kbeta-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kbeta abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane. We propose that a novel function of PI4Kbeta is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane.