Focus: Personalized Medicine: Simultaneously Detection of 50 Mutations at 20 Sites in the BRAF and RAS Genes by Multiplexed Single-Nucleotide Primer Extension Assay Using Fine-Needle Aspirates of Thyroid Nodules

Fine-needle aspiration (FNA) is commonly used for primary evaluation of thyroid nodules. Twenty to 30 percent of thyroid nodules remain indeterminate after FNA evaluation. Studies show the BRAF p.V600E to be highly specific for papillary thyroid carcinoma (PTC), while RAS mutations carry up to 88 percent positive predictive value for malignancy. We developed a two-tube multiplexed PCR assay followed by single-nucleotide primer extension assay for simultaneous detection of 50 mutations in the BRAF (p.V600E, p.K601E/Q) and RAS genes (KRAS and NRAS codons 12, 13, 19, 61 and HRAS 61) using FNA smears of thyroid nodules. Forty-two FNAs and 27 paired formalin-fixed, paraffin-embedded (FFPE) tissues were tested. All BRAF p.V600E-positive FNA smears (five) carried a final diagnosis of PTC on resection. RAS mutations were found in benign as well as malignant lesions. Ninety-two percent concordance was observed between FNA and FFPE tissues. In conclusion, our assay is sensitive and reliable for simultaneous detection of multiple BRAF/RAS mutations in FNA smears of thyroid nodules.     

The tumor suppressor p53 can reduce stable transfection in the presence of irradiation

The tumor suppressor p53 is believed to play an essential role in maintaining genome stability. Although it is currently unknown how p53 is involved in this important biological safeguard, several previous publications indicate that p53 can help to maintain genome integrity through the recombination-mediated DNA repair process. The integration of linearized plasmid DNA into the host chromosome utilizes the same repair process, and the frequency can be measured by clonogenic assays in which cells that were stably transfected by plasmid integration can be scored by their colony-forming abilities. To gain insight into whether p53 has a direct role in plasmid integration into the host chromosome, we determined the frequency of stable transfection with CHO cells expressing either wild-type or mutant p53 in the presence and absence of irradiation. We found that low-dose irradiation (50 to 100 cGy) increased stable transfection frequencies in CHO cells regardless of their p53 status. However, the increase of transfection frequency was significantly lower in CHO cells expressing wild-type p53. Our data thus suggest that wild-type p53 can suppress plasmid DNA integration into the host genome. This p53 function may play a direct and significant role in maintaining genome stability.     

Evidence for a New Member of the Myosin I Family from Mammalian Brain

Myosin I is an actin-based motor responsible for powering a wide variety of motile activities in amebae and slime molds and has been found previously in vertebrates as the lateral bridges within intestinal epithelial cell microvilli. Although neurons exhibit extensive cellular and intracellular motility, including the production of ameboid-like growth cones during development, the proteins responsible for the motor in these processes are unknown. Here, we report the isolation of a partially purified protein fraction from bovine brain that is enriched for a 150-kDa protein; immunochemical and biochemical analyses suggest that this protein possesses a number of functional properties that have been ascribed to myosin I from various sources. These properties include an elevated K(+)-EDTA ATPase, a modest actin-activated Mg(2+)-ATPase, the ability to bind calmodulin, and a ready association with phospholipid vesicles made from phosphatidylserine, but not from phosphatidylcholine. The combination of these properties, together with a molecular mass of 150 kDa (most myosin I molecules found to date have molecular masses in the range 110-130 kDa) yet recognition by an anti-myosin I antibody, suggests the presence of a new member of the myosin I family within mammalian brain.     

Induction of holomycin production and complex metabolic changes by the argR mutation in Streptomyces clavuligerus NP1

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of argR in Streptomyces clavuligerus NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its argR disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in S. clavuligerus.     

Type XIX collagen purified from human umbilical cord is characterized by multiple sharp kinks delineating collagenous subdomains and by intermolecular aggregates via globular,disulfide-linked,and heparin-binding amino termini

Type XIX collagen was discovered from the sequence of rhabdomyosarcoma cDNA clones. The chain is composed of a 268-residue amino terminus, an 832-residue discontinuous collagenous region, and a 19-residue carboxyl peptide. Light microscopy immunohistochemistry of adult human tissues demonstrated that type XIX is localized in vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. It also appears to be involved in events linked to skeletal myogenesis. In this report, we have presented the first direct evidence for the molecular structure of type XIX collagen. Using human umbilical cord, native type XIX was purified by neutral salt extraction and by ion exchange and antibody affinity chromatography. Type XIX was found to represent only approximately 10(-6)% of the dry weight of tissue, making it by far the least abundant collagen ever isolated. Transmission electron microscopy after rotary shadowing revealed the appearance of rodlike structures with multiple sharp bends, a small nodule at one end of the molecule, and a total length of 240 nm. Domain-specific antibodies were used to identify the nodule as the noncollagenous amino terminus, whereas the location of most kinks corresponds to major interruptions separating the five collagenous subdomains. More than half of the type XIX molecules observed were present in oligomers of different size and complexity, resulting from association of the amino-terminal domains. Biochemical analysis demonstrated that these supramolecular aggregates are dependent upon and/or stabilized by intermolecular disulfide cross-links and that the globular amino terminus contains a high affinity, heparin-binding site. The polymorphic conformational states of this rare collagen, and its ability to self-assemble into a higher order structure provide focal points for future determination of biologically significant functions in cell-cell and/or cell-matrix interactions.     

The Analog of Ginkgo biloba Extract 761 is a Protective Factor of Cognitive Impairment Induced by Chronic Fluorosis

Ginkgo biloba extract EGb761 is widely used to treat patients with learning and memory impairment in Alzheimer’s disease and Parkinson’s disease in China. However, it is not yet clear whether the analog of EGb761 (EGb) has a protective effect on the learning and memory damage induced by chronic fluorosis. In this study, 30 Wistar rats were randomly divided into three groups: a control group, a sodium fluoride (NaF) + EGb group, and a NaF group. The rats were administered 0.5 ml water containing NaF (100 mg/l) and EGb (120 mg/kg) per day via gavage. After 3 months, the rats’ capacity for learning and memory was tested using a Y-maze. Damage to hippocampal neurons was evaluated by histological examination of the CA3 area. Superoxide dismutase (SOD) activity and the levels of glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured. Furthermore, the expression levels of Bcl-2 and Bax and the levels of cleaved Caspase3 in the hippocampus were evaluated by RT-PCR and Western blotting. The results showed that EGb could improve learning and memory abilities, enhance the activities of SOD and GSH-Px, attenuate the level of MDA, upregulate the ratio of Bcl-2/Bax, and downregulate the level of cleaved Caspase3.