Partial-pKD1 plasmids provide enhanced structural stability for heterologous protein production in Kluyveromyces lactis

We describe a patient with leukocytosis with all the stages of neutrophilic series, peripheral dominant myeloblast proliferation, marked dysplasia of myeloid and erythroid series, and extramedullary hematopoiesis of the lymph nodes. A cytogenetic study of the bone marrow cells showed normal karyotype, and molecular analysis of the leukemic cells showed negative for BCR-ABL by RT-PCR. After chemotherapy, the patient went into complete remission with a normal blood and bone marrow profile with no dysplasia. On relapse, the hematological findings showed a typical bone marrow dominant acute myeloid leukemia, with the leukemic cells having a chromosomal abnormality. The patient exhibited the combined features of myeloproliferative disorder, myelodysplastic syndrome, peripheral dominant myeloblast proliferation (so-called peripheral leukemia) and typical acute myeloid leukemia throughout the clinical course. This is thought to be a rare overlapping disease involving these distinct hematological conditions that do not usually occur in the same patient.     

KAR5 encodes a novel pheromone-inducible protein required for homotypic nuclear fusion

KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.     

A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger

The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10-20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase (glaA), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any glaA coding sequence. The portion of glaA used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.     

The use of heterologous promoters for adeno-associated virus (AAV) protein expression in AAV vector production

OBJECTIVE: There are few nonproprietary papers addressing the mechanical strength of intramedullary nails; none address the characteristics of the proximal and distal ends of these devices. Our objective was to provide such data. DESIGN: Independent testing of eight femoral intramedullary nail systems at the proximal, middle, and distal regions was undertaken to evaluate strength and flexural rigidity (stiffness). METHODS: Each device, usually a reconstruction nail, was forty-two to forty-six centimeters in length. Four or five nails of each available size (range 9 to 13 millimeters in diameter) were tested for each system. The nails were cut into proximal, middle, and distal thirds. Each nail section was loaded to failure using a four-point bend test on a custom fixture (modification of the American Society of Testing Materials standard test). RESULTS: Significant variations (p < 0.05) were found in strength and stiffness between the middle and the proximal or distal aspects of some rods. A significant difference (p < 0.05) was observed when comparing the properties of earlier designs with the properties of more recent designs. Newer rod designs all performed in a similar manner with regard to strength. Strength and rigidity increased with increasing rod diameter in some but not all systems. CONCLUSIONS: Although none of the newer designs appeared to have superior static strength, the individual systems had significant variations in their mechanical properties (bending rigidity), particularly in the proximal and distal sections. It is important that the surgeon become familiar with the individual characteristics of strength and rigidity for the particular devices available and how these might impact fracture healing. Consideration of this information could alter the decision to select one system over another in a complex fracture situation.     

A novel technique for containerless protein crystallization

Heterogeneous nucleation, which is often detrimental to the production of suitable crystals for X-ray diffraction, can be induced by the contact of a crystallization sample with the walls of its supporting vessel. A novel method for creating a 'containerless' environment for the growth of protein crystals is described. Contact between the container walls and a crystallization drop is eliminated by suspending the drop between two oils of different density: one of higher and the other of lower density than that of water and the common precipitating agents. A number of proteins were crystallized in 2-10 microliters drops using this procedure. It was found that the number of crystals obtained in such suspended drops was reduced significantly compared with the number of crystals obtained in trials where the crystallization drop was situated at the bottom of a vial under a single layer of oil. This method has potential in controlling heterogeneous nucleation.     

EspE, a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine-phosphorylated 90 kDa protein

We have previously shown that there were differential and dramatic decreases of cyclin and cyclin-dependent kinase (CDK) activities in cardiomyocytes during the neonatal period. The activity of CDKs control cell cycle progression, and this activity is regulated positively and negatively by association of CDKs with cyclins and cyclin-dependent kinase inhibitors (CKIs), respectively. While the INK family (p15(INK4B)/p16(INK4A)/p18(INK4C)/p19(INK4D)) of CKIs is not detectable in hearts, the KIP/CIP family (p21(CIP1), p27(KIP1) and p57(KIP2)) of CKIs is detectable in most organs including the heart. Differential and dramatic changes of the KIP/CIP family (p21(CIP1), p27(KIP1) and p57(KIP2)) of CKIs were detected in rat hearts during development. The mRNA and protein levels of p21(CIP1) and p57(KIP2) were readily detectable in hearts at gestational and early postnatal periods and decreased thereafter. The mRNA levels of p27(KIP1) in ventricles were high during the gestational period, and did not change until day 30 postnatal, then were decreased slightly in 90-day-old rats. The protein levels of p27(KIP1) increased significantly in the early postnatal period, then were expressed persistently, although levels decreased slightly in the adult period. However, protein levels of p27(KIP1) in atria did not change during development. Variable immuno-staining patterns of p27(KIP1) were observed at different periods of development and in various locations in myocardium. During the gestational period, approximately 35-50% of myocardial cells in the cardiac wall were p27(KIP1) immuno-positive and were distributed diffusely. These p27(KIP1) immunopositive cells increased predominantly in endocardial and mid-portion areas of ventricular myocardium at the early postnatal period. This heterogenous pattern of p27(KIP1) protein expression persisted to adult hearts though the percentage of p27(KIP1) immuno-positive cells decreased slightly. High magnification revealed that more than 50% of adult cardiomyocytes were p27(KIP1) immuno-positive and that p27(KIP1) was located solely in nuclei. These results indicate that p27(KIP1) may be an important inhibitor of CDK activities in cardiomyocytes during early postnatal development and may block the re-entrance of adult cardiomyocytes into the cell cycle after injury.     

Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis

Elongation factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes. It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi. We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes. In vitro reconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2. Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2 that encode EF2. Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid. The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target. These findings are of particular significance due to the need for new antifungal agents.     

Arabidopsis as a model host for studying plant-pathogen interactions

Because the molecular biology and genetics of Arabidopsis thaliana are so well defined, it is potentially a superb subject for research on plant-pathogen interactions. Viruses, bacteria and fungi that infect Arabidopsis and are representative pathogens of economically important plants have recently been described. The search now is for a pathogenic fungus with tractable genetics to combine with a direct analysis of plant resistance genes.     

A new resource-rich carbonate luminescent host KNa_5Ca_5(CO_3)_8:A case study activated by Eu~(3+) for red-phosphor purpose

Exploring and designing new luminescent host with rich resource is beneficial to reducing the cost and the large-scale application of luminescent materials.Herein,a series of novel Eu~(3+)-activated KNa_(5-)Ca_5(CO_3)_8 red-emitting phosphors with different Eu~(3+) concentrations were successfully synthesized under mild hydrothermal condition.XRD and SEM characterizations show that all as-prepared KNa_5 Ca_5(CO_3)_8:Eu~(3+) samples crystallize in a hexagonal structure,P63 mc(No.186) space group,and show monodispersed hexagonal sheet mo rphologies,within a side length interval about of 2-3μm and a thickness of about 0.6 μm,Eu~(3+) doping within 6 mol% concentration has little influence on the crystal structure of the host,as indicated by XRD,and FT-IR results,but thermal stability changes as shown by the TG-DTA.KNa_5 Ca_5(CO_3)_8 phosphors with different Eu~(3+) concentrations can emit red-light under effectively excitation by near-UV light.CIE chromaticity coo rdinates of all KNa_5 Ca_5(CO_3)_8:Eu~(3+) phosphors were calculated to locate in red region,and CCT of each sample is the range from 2815 to 3650 K,very close to the tungsten lamp light.Besides,concentration quenching phenomenon appearing at 5 mol% is found to be the result of electric multipolar interaction of Eu3+activators,and the decay lifetime is about1.63 ms,which almost unchanges with Eu~(3+) doping concentration.Importantly,thermal quenching analysis reveals good thermal stability with thermal activation energy of about 0.216 eV.The results reported here demonstrate that KNa_5 Ca_5(CO_3)_8 is a promising fluorescent host for LEDs applications.     

Identification of a novel Rac3-interacting protein C1D

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.     

Identification of a novel MCM3-associated protein that facilitates MCM3 nuclear localization

MCM3 is essential for the initiation of DNA replication and also participates in controls that ensure DNA replication is initiated once per cell cycle. In a two-hybrid screen for proteins that interact with human MCM3, we identified and cloned a novel protein of which the calculated molecular weight is 80,291. A specific antibody against the protein identified a 80-kDa protein in HeLa cell extract, indicating the protein actually expressed in cells. The interaction of these proteins was confirmed by immunoprecipitation assay. Moreover, we clarified a nuclear localization signal of human MCM3, and we find that mutagenesis on the nuclear localization signal of MCM3 affected the binding of newly isolated MCM3-assosiated protein, Map80. Map80 was expressed in Escherichia coli as a fusion with His6 tag and purified with sequential column chromatographies. The addition of recombinant Map80 stimulated the amount of nuclear localized MCM3. These results suggest that Map80 is involved in the nuclear localization pathway of MCM3.     

Increased production of amyloid precursor protein provides a substrate for caspase-3 in dying motoneurons

Biochemical and molecular mechanisms of neuronal cell death are currently an area of intense research. It is well documented that the lumbar spinal motoneurons of the chick embryo undergo a period of naturally occurring programmed cell death (PCD) requiring new gene expression and activation of caspases. To identify genes that exhibit changed expression levels in dying motoneurons, we used a PCR-based subtractive hybridization protocol to identify messages uniquely expressed in motoneurons deprived of trophic support as compared with their healthy counterparts. We report that one upregulated message in developing motoneurons undergoing cell death is the mRNA for amyloid precursor protein (APP). Increased levels of APP and beta-amyloid protein are also detected within dying motoneurons. The predicted peptide sequence of APP indicates two potential cleavage sites for caspase-3 (CPP-32), a caspase activated in dying motoneurons. When peptide inhibitors of caspase-3 are administered to motoneurons destined to undergo PCD, decreased levels of APP protein and greatly reduced beta-amyloid production are observed. Furthermore, we show that APP is cleaved by caspase-3. Our results suggest that differential gene expression results in increased levels of APP, providing a potential substrate for one of the cell death-activated caspases that may ultimately cause the demise of the cell. These results, combined with information on the toxic role of APP and its proteolytic by-product beta-amyloid, in the neurodegenerative disease Alzheimer's, suggest that events of developmental PCD may be reactivated in early stages of pathological neurodegeneration.     

Cloning of follistatin-related protein as a novel autoantigen in systemic rheumatic diseases

In an attempt to identify autoantigens of synovium in rheumatoid arthritis (RA), we constructed lambda phage expression cDNA libraries from synovium and screened them by IgG purified from synovial fluids, both of which were derived from RA patients. As a result of this unique combination of the libraries and probes, we cloned follistatin-related protein (FRP) as a novel autoantigen in systemic rheumatic diseases. FRP is a secreted protein containing a similar amino acid sequence to follistatin, an inhibitor of activin. FRP was first cloned as a transforming growth factor-beta1-inducible protein (called TSC-36) from a mouse osteoblastic cell line and was suggested to have some roles in the negative regulation of cellular growth. Immunoblotting analyses detected synovial fluid and serum anti-FRP antibodies of IgG class more frequently in RA than any other systemic rheumatic diseases and controls. Synovial fluid anti-FRP antibodies appeared in 44% of RA (n = 18) and none of osteoarthritis (OA) (n = 15) patients. Serum antibodies were detected in 30% of RA (n = 67), 17% of systemic sclerosis (n = 18), 10% of systemic lupus erythematosus (n = 51) and Sj?gren's syndrome (n = 10), and none of polymyositis/dermatomyositis (n = 13) patients and healthy subjects (n = 30). These antibodies recognized an EC domain, an extracellular Ca2+ binding module. In anti-FRP antibody-positive RA patients, serum C-reactive protein level and erythrocyte sedimentation rate were more elevated than negative patients (P < 0.05 and P < 0.01, respectively). FRP gene expression was higher in RA than OA synovium (P < 0.05). However, there was no difference between these groups in the amount of synovial FRP, suggesting its elevated turnover in RA. As follistatin inhibits activin, FRP might inhibit some growth factor-like molecule. Detection of anti-FRP antibodies, possibly having disease-promoting effects as the blocking antibodies, could be one of the markers for clinical evaluation of systemic rheumatic diseases.     

A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice

A genetic locus of Pseudomonas aeruginosa was identified that is highly and specifically inducible during infection of neutropenic mice. This locus, ppkA, encodes a protein that is highly homologous to eukaryote-type serine/threonine protein kinases. A ppkA null mutant strain shows reduced virulence in neutropenic mice compared to the wild type. Overexpression of the PpkA protein greatly inhibited the growth of Escherichia coli or P. aeruginosa. However, a single amino acid change at the catalytic site of the kinase domain eliminated the toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial cells.     

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.