Direct synthesis of Rev peptide-conjugated gold nanoparticles and their application in cancer therapeutics

We have developed a simple approach for generating peptide-conjugated gold nanoparticles (AuNPs) from the Rev peptide and gold aqueous solution. The peptide functions as both a reducing agent and a capping molecule. AuNPs of various sizes (20-300 nm) and shapes (spheres, triangular plates, and polygons) can be obtained upon modulating the ratio of gold ions to the Rev peptide. Transmission electron microscopy, X-ray diffraction, and UV-vis spectroscopy were utilized to characterize these nanoparticles. Fourier-transform infrared and X-ray photoelectron spectroscopy measurements were performed to investigate chemical interactions between the Rev peptide and AuNPs. Lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed that the Rev peptide-AuNP nanocomposites exhibited exceptionally high cytotoxic effects toward mouse ovarian surface epithelial cell lines, relative to the effects of equal doses of the free Rev peptide. Our study suggests a new way of utilizing biomolecule-conjugated AuNPs as potentially effective anticancer drugs.     

Multilayered microspheres for the controlled release of growth factors in tissue engineering

Tissue regeneration may be stimulated by growth factors but to be effective, this delivery must be sustained and requires delivery vehicles that overcome the short half-life of these molecules in vivo. One promising approach is to couple growth factors to the biomaterial surface so that they are readily bioavailable. Here the layer-by-layer process was used to construct a multilayered polyelectrolyte delivery system on the surface of poly(lactic-co-glycolic) acid constructs. The system was first optimized on a planar surface before translation to a 3D microsphere system. The layers incorporated heparin to facilitate the loading of basic fibroblast growth factor and increase growth factor stability. Cross-linked capping layers also reduced any burst release. The model growth factor was released in a sustained manner and stimulated significantly higher cell proliferation in vitro on release compared with the addition of the growth factor heparin complex free in solution, demonstrating the promise of this approach.     

Mixed Magnetism for Refrigeration and Energy Conversion

The efficient coupling between lattice degrees of freedom and spin degrees of freedom in magnetic materials can be used for refrigeration and energy conversion. This coupling is enhanced in materials exhibiting the giant magnetocaloric effect. First principle electronic structure calculations on hexagonal MnFe(P, Si) reveal a new form of magnetism: the coexistence of strong and weak magnetism in alternate atomic layers. The weak magnetism of Fe layers (disappearance of local magnetic moments at the Curie temperature) is responsible for a strong coupling with the crystal lattice while the strong magnetism in adjacent Mn‐layers ensures Curie temperatures high enough to enable operation at and above room temperature. Varying the composition on these magnetic sublattices gives a handle to tune the working temperature and to achieve a strong reduction of the undesired thermal hysteresis. In this way we design novel materials based on abundantly available elements with properties matched to the requirements of an efficient refrigeration or energy‐conversion cycle.     

RET mutational spectrum in Hirschsprung disease: evaluation of 601 Chinese patients

Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial).Here we present the screening for RVs in the RET CDS and intron/exon boundaries of 601 Chinese HSCR patients, the largest number of patients ever reported. We identified 61 different heterozygous RVs (50 novel) distributed among 100 patients (16.64%). Those include 14 silent, 29 missense, 5 nonsense, 4 frame-shifts, and one in-frame amino-acid deletion in the CDS, two splice-site deletions, 4 nucleotide substitutions and a 22-bp deletion in the intron/exon boundaries and 1 single-nucleotide substitution in the 5' untranslated region. Exonic variants were mainly clustered in RET the extracellular domain. RET RVs were more frequent among patients with the most severe phenotype (24% vs. 15% in short-HSCR). Phasing RVs with the RET HSCR-associated haplotype suggests that RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants were found in 250 Chinese controls.     

Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors

The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated. Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50–400 M); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration. The highest ginsenoside yield was obtained at 200 M MJ. In the second experiment, 200 M MJ was added on day 15 during the cultivation. The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment. Rb group gisnsenosides accumulated more than Rg group ginsenosides. Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly. Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides. These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.     

Effect of individual amino acids and glucose on activation and germination of Rhizopus oligosporus sporangiospores in tempe starter

AIM: To understand the conditions promoting activation and germination of spores, and to contribute to the control of tempe starters. METHODS AND RESULTS: Using microscopic counts of fluorescent labelled spores, the following results were obtained: (1) L-alanine plays an important role (of the same order as that of peptone) in stimulation of germination of dormant spores. Alanine can satisfy the requirements of carbon as well as nitrogen for spore germination; (2) L-proline, on the other hand, inhibits alanine uptake presumably by blocking/congesting transporters of spore cells, resulting in apparent low viability on agar media; (3) L-leucine and L-isoleucine slightly favour spore germination while L-arginine and L-lysine do not have any stimulating effect; (4) The stimulatory role of glucose was only evident in the presence of phosphate (in minimal medium); when glucose is used in the absence of phosphate, either alone or in combination with single amino acids its role is hardly distinguishable; (5) Phosphate plays a facilitating role in spore germination. CONCLUSIONS: Glucose and amino acids play important roles in activation and germination of sporangiospores of Rhizopus oligosporus in tempe starter (stored for 12 months). The ability and rate of germination of dormant/old sporangiospores of R. oligosporus, depend on their ability for uptake of individual amino acids and/or glucose. SIGNIFICANCE AND IMPACT OF STUDY: New light was shed on the counteractive role of proline and the stimulating effect of phosphate. Soybeans subjected to traditional preparation for tempe making are heavily leached; germination of starter spores on such beans is sub-optimal, and bean processing could be optimized.     

A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with beta-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.     

Optimized production of active alpha-glucosidase by recombinant Escherichia coli. evaluation of processes using in vivo reactivation from inclusion bodies

During fast production in recombinant Escherichia coli, the enzyme alpha-glucosidase from Saccharomyces cerevisiae accumulates partially as inclusion bodies. The inclusion bodies are reactivated inside the cell upon temperature downshift. This in vivo reactivation was most efficient on complex medium with inclusion body production at 42 degrees C and reactivation at 30 degrees C, yielding volumetric activities 85% higher than those of extended isothermal production at low temperature. On defined medium, however, in vivo reactivation was slow. In fed-batch cultivations, feeding controls the specific growth rate independent of the temperature. Here, high growth rates fostered inclusion body formation even at low temperature. Intermediate growth rates permitted accumulation of active alpha-glucosidase without affecting the total amount of alpha-glucosidase. Low growth rates yielded similar activities and additionally prevented inclusion body formation. Moreover, high growth rates during production forestalled subsequent in vivo reactivation. Accumulation of activity after temperature reduction was possible with intermediate growth rates. The best time for temperature shift was concomitant to induction. Thus, in fed-batch culture, isothermal production at 30 degrees C and with a set growth rate of 0.12 h(-)(1) controlled by feeding was most efficient for production of active alpha-glucosidase. Compared to production under optimal conditions on complex medium, the specific and volumetric activities obtained were 3 and 45 times higher, respectively.     

Candida easanensis sp. nov., Candida pattaniensis sp. nov. and Candida nakhonratchasimensis sp. nov., three new species of yeasts isolated from insect frass in Thailand

Six strains of anamorphic yeasts isolated from insect frass collected in several regions of Thailand were assigned to the genus Candida based on the conventional taxonomic criteria used for yeast classification. These strains have Q-7 as the major ubiquinone and are suggested to have close relationships to the genus Pichia. Three strains, ST-225, ST-228 and ST-229, have identical nucleotide sequences in the D1/D2 domain of 26S rDNA and are closely related to Pichia japonica, but differ by six nucleotides (1.1% ) from this species. These three strains are considered to represent a single new species, which is described as Candida easanensis sp. nov. Two strains, ST-311 and ST-320, have identical sequences in the D1/D2 domain and resemble Pichia veronae and Pichia fabianii but differ from them by nine nucleotides (1.6%) in D1/D2 sequences. The two strains are described as Candida pattaniensis sp. nov. The remaining strain, ST-37, is related to Pichia americana and Pichia bimundalis but differs by six(1.1%) and seven (1.2%) nucleotides from these species, respectively. This strain is described as Candida nakhonratchasimensis sp. nov.     

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.