Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1-1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.     

Elevated serum silicon levels in women with silicone gel breast implants

The metatolic fate of silicone gel leaked from an intact or ruptured prosthesis is unknown. In this study, serum was blindly assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) for elemental silicon in 72 women with silicone gel breast implants and 55 control women (mean age 48 yr, both groups). Blood was drawn and processed using silicon-free materials. The mean silicon level in controls was 0.13±0.07 mg/L (range 0.06–0.35 mg/L), whereas in implant patients, the mean was significantly higher at 0.28±0.22 mg/L (range 0.06–0.87 mg/L) (P<0.01, Student'st-test with correction for unequal variances). Using the mean of the control group +2 SD as a cutoff for normal range (0.27 mg/L), 25/72 (34.7%) implant patients exceeded this value, compared with 2/55 (3.6%) controls. There was no significant correlation between past rupture of one or both implants, current rupture at the time of the blood draw, or the number of years with implants and silicon levels. The results suggest that serum silicon levels are elevated in many women with silicone gel breast implants. The chemical species involved and kinetics of this elevation remain to be determined.     

A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification

Tissue specific genes that contain high GC segments are difficult to amplify by standard PCR. We report an improved method for successful amplification of gene segment that has >70% GC base pairs. This new method of touch down PCR differed by having an initial annealing temperature (Ta) 1.5°C below the primers melting temperature that descended 0.2°C per cycle for 20 cycles and continued thereafter at fixed Ta for next 15 cycles. Different co-solvents were tested with this method to improve the result and betaine proved better than the other co-solvents. This new method is economical, fast and specific in amplifying GC rich region of other genes also.     

Heparan sulfate dissociates serum amyloid A (SAA) from acute-phase high-density lipoprotein, promoting SAA aggregation

Inflammation-related (AA) amyloidosis is a severe clinical disorder characterized by the systemic deposition of the acute-phase reactant serum amyloid A (SAA). SAA is normally associated with the high-density lipoprotein (HDL) fraction in plasma, but under yet unclear circumstances, the apolipoprotein is converted into amyloid fibrils. AA amyloid and heparan sulfate (HS) display an intimate relationship in situ, suggesting a role for HS in the pathogenic process. This study reports that HS dissociates SAA from HDLs isolated from inflamed mouse plasma. Application of surface plasmon resonance spectroscopy and molecular modeling suggests that HS simultaneously binds to two apolipoproteins of HDL, SAA and ApoA-I, and thereby induce SAA dissociation. The activity requires a minimum chain length of 12-14 sugar units, proposing an explanation to previous findings that short HS fragments preclude AA amyloidosis. The results address the initial events in the pathogenesis of AA amyloidosis.     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.     

New culture system for human embryonic stem cells: autologous mesenchymal stem cell feeder without exogenous fibroblast growth factor 2

Human embryonic stem (hES) cells have been successfully maintained using human-cell feeder systems or feeder-free systems. However, despite advances in culture techniques, hES cells require supplementation with fibroblast growth factor 2 (FGF-2), an exogenous stemness factor, which is needed to sustain the authentic undifferentiated status. We developed a new culture system for hES cells; this system does not require supplementation with FGF-2 to obtain hES cells that are suitable for tissue engineering and regenerative medicine. This culture system employed mesenchymal stem cells derived from hES cells (hESC-MSCs) as autologous human feeder cells in the absence of FGF-2. The hES cell line SNUhES3 cultured in this new autologous feeder culture system maintained the typical morphology of hES cells and expression of pluripotency-related proteins, SSEA-4, TRA-1-60, OCT4, and alkaline phosphatase, without development of abnormal karyotypes after more than 30 passages. RNA expression of the pluripotency-related genes OCT4 and NANOG was similar to the expression in SNUhES3 cells maintained on xenofeeder STO cells. To identify the mechanism that enables the cells to be maintained without exogenous FGF-2, we checked the secretion of FGF-2 from the mitomycin-C treated autofeeder hESC-MSCs versus xenofeeder STO cells, and confirmed that hESC-MSCs secreted FGF-2 whereas STO cells did not. The level of FGF-2 in the media from the autofeeder system without exogenous FGF-2 was comparable to that from the xenofeeder system with addition of FGF-2. In conclusion, our new culture system for hES cells, which employs a feeder layer of autologous hESC-MSCs, supplies sufficient amounts of secreted FGF-2 to eliminate the requirement for exogenous FGF-2.     

Critical dependence of blood-borne biomarker concentrations on the half-lives of their carrier proteins

Until recently, the low-abundance (LA) range of the serum proteome was an unexplored reservoir of diagnostic information. Today it is increasingly appreciated that a diagnostic goldmine of LA biomarkers resides in the blood stream in complexed association with more abundant higher molecular weight carrier proteins such as albumin and immunoglobulins. As we now look to the possibility of harvesting these LA biomarkers more efficiently through engineered nano-scale particles, mathematical approaches are needed in order to reveal the mechanisms by which blood carrier proteins act as molecular ‘mops’ for LA diagnostic cargo, and the functional relationships between bound LA biomarker concentrations and other variables of interest such as biomarker intravasation and clearance rates and protein half-lives in the bloodstream. Here we show, by simple mathematical modeling, how the relative abundance of large carrier proteins and their longer half-lives in the bloodstream work together to amplify the total blood concentration of these tiny biomarkers. The analysis further suggests that alterations in the production of biomarkers lead to gradual rather than immediate changes in biomarker levels in the blood circulation. The model analysis also points to the characteristics of artificial nano-particles that would render them more efficient harvesters of tumor biomarkers in the circulation, opening up possibilities for the early detection of curable disease, rather than simply better detection of advanced disease.     

An enzyme cycling method for measurement of allantoin in human serum

Current conventional measurement of allantoin levels in human serum uses an HPLC method. However, performing this assay is time-consuming and sample-intensive, and it requires expensive equipment. We have developed a novel enzyme cycling method for measuring allantoin concentrations in human serum. In the first step, serum allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5), and endogenous ammonia is simultaneously removed by the action of glutamine synthetase II (EC 6.3.1.2). In the second step, l-methionine sulfoximine is used to inhibit glutamine synthetase II, and ammonia is liberated from allantoate by the activity of allantoate amidohydrolase (EC 3.5.3.9). In the final step, the ammonia is then converted to NAD by NAD synthetase (EC 6.3.1.5). Subsequent action of glucose dehydrogenase (EC 1.1.1.47) and diaphorase (EC 1.6.99.2) in the presence of glucose and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) acts to cycle the formed NAD between its oxidized and reduced forms, resulting in the production of WST-1 formazan, which is monitored at 450 nm. The assay standard curve is linear from 0 to 70 μM allantoin. The level of allantoin in healthy subjects was measured to be 8.2 ± 3.1 μM (n = 30).     

Stereoselective routes towards the synthesis of unusual bis(amino acids) and cyclic amino acids

Summary Stereoselective syntheses are described of bridged bis(glycines) as conformationally constrained substitutes for cystine, and of cyclic α-amino acids where the α-carbon of the amino acid is part of a five-, six- or seven-membered ring which may hold a hydroxy group as a threonine analogue.     

Ability of human sera to neutralise the activity of Vero cytotoxins VT1, VT2 and variant forms of VT2

Abstract Sera, from 17 patients with diarrhoea or haemolytic uraemic syndrome, and six healthy adults, were tested for neutralisation of Vero cytotoxins (VT). For all 17 patients there was evidence of infection with Escherichia coli O157. Sera from two controls but from none of the patients neutralised VT1, although two patients were infected by strains producing VT1 and VT2. Sera from all six controls and 14 patients neutralised VT2 derived from strains 933 and E32511, but not variant forms of VT2 derived from strains E32511, E57, B2F1 and H.1.8. This neutralising activity warrants further investigation, especially as many 0157 VTEC carry both VT2 and VT2 variant genes.