The beta -hairpin motif of UvrB is essential for DNA binding, damage processing, and UvrC-mediated incisions.

UvrB plays a major role in recognition and processing of DNA lesions during nucleotide excision repair. The crystal structure of UvrB revealed a similar fold as found in monomeric DNA helicases. Homology modeling suggested that the beta-hairpin motif of UvrB might be involved in DNA binding (Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) EMBO J. 18, 6899-6907). To determine a role of the beta-hairpin of Bacillus caldotenax UvrB, we have constructed a deletion mutant, Deltabetah UvrB, which lacks residues Gln-97-Asp-112 of the beta-hairpin. Deltabetah UvrB does not form a stable UvrB-DNA pre-incision complex and is inactive in UvrABC-mediated incision. However, Deltabetah UvrB is able to bind to UvrA and form a complex with UvrA and damaged DNA, competing with wild type UvrB. In addition, Deltabetah UvrB shows wild type-like ATPase activity in complex with UvrA that is stimulated by damaged DNA. In contrast to wild type UvrB, the ATPase activity of mutant UvrB does not lead to a destabilization of the damaged duplex. These results indicate that the conserved beta-hairpin motif is a major factor in DNA binding.     

Evaluation of a new fiber-optic pressure recording system for cardiovascular measurements in mice

We have tested a new fiber-optic pressure recording system, Samba, with a thin fiber [outer diameter (OD) = 0.25 mm] and a pressure sensor (length and OD = 0.42 mm) attached to the end. The accuracy of the system tested in vitro was good, with a coefficient of variation of 2.54% at 100 mmHg. The drift was <0.45 mmHg/h, and the temperature sensitivity was approximately 0.07 mmHg/1 degrees C between 22 and 37 degrees C. The frequency response characteristics were similar to a 1.4-Fr Millar catheter (0-200 Hz). Introduction of the Samba sensor from the right carotid artery into the left ventricle in six mice caused no drop in mean aortic pressure, whereas introduction of a 1.4-Fr Millar catheter (OD = 0.47 mm; n = 6) caused a pressure drop from 91.6 +/- 9.2 to 65.1 +/- 6.2 mmHg; P < 0.05. Thus the Samba sensor system may represent a new alternative to assess hemodynamic variables in the murine cardiovascular system.     

The rate of sister chromatid exchange in normal human bone marrow cells

Summary A ring chromosome 22 is described in a 6-year-old mentally retarded boy, who presented a dysmorphic syndrome. The ring chromosome 22 was inherited from the mother, in whom a 46,XX/46,XX,r(22)/45,XY,-15,-22,+t(15;22)(p11;q11) mosaic karyotype was found, indicating a high degree of instability of the chromosome(s) 22 in this woman.     

IFN-gamma and Fas ligand are required for graft-versus-tumor activity against renal cell carcinoma in the absence of lethal graft-versus-host disease

To determine the mechanisms of graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD) against a solid tumor, we established two allogeneic bone marrow transplantation models with a murine renal cell carcinoma (RENCA). The addition of 0.3 x 10(6) donor CD8(+) T cells to the allograft increased the survival of tumor-bearing mice without causing GVHD. The analysis of CD8(+) T cells deficient in cytotoxic molecules demonstrated that anti-RENCA activity is dependent on IFN-gamma and Fas ligand (FasL), but does not require soluble or membrane-bound TNF-alpha, perforin, or TRAIL. Recipients of IFN-gamma(-/-) CD8(+) T cells are unable to reject RENCA compared with recipients of wild-type CD8(+) T cells and, importantly, neither group develops severe GVHD. IFN-gamma(-/-) CD8(+) T cells derived from transplanted mice are less able to kill RENCA cells in vitro, while pretreatment of RENCA cells with IFN-gamma enhances class I and FasL expression and rescues the lytic capacity of IFN-gamma(-/-) CD8(+) T cells. These results demonstrate that the addition of low numbers of selected donor CD8(+) T cells to the allograft can mediate GVT activity without lethal GVHD against murine renal cell carcinoma, and this GVT activity is dependent on IFN-gamma and FasL.     

Accumulation of Major Life Events in Childhood and Adult Life and Risk of Type 2 Diabetes Mellitus

Background

The aim of the study was to estimate the effect of the accumulation of major life events (MLE) in childhood and adulthood, in both the private and working domains, on risk of type 2 diabetes mellitus (T2DM). Furthermore, we aimed to test the possible interaction between childhood and adult MLE and to investigate modification of these associations by educational attainment.

Methods

The study was based on 4,761 participants from the Copenhagen City Heart Study free of diabetes at baseline and followed for 10 years. MLE were categorized as 0, 1, 2, 3 or more events. Multivariate logistic regression models adjusted for age, sex, education and family history of diabetes were used to estimate the association between MLE and T2DM.

Results

In childhood, experiencing 3 or more MLE was associated with a 69% higher risk of developing T2DM (Odds Ratio (OR) 1.69; 95% Confidence Interval (CI) 1.60, 3.27). The accumulation of MLE in adult private (p-trend = 0.016) and work life (p-trend = 0.049) was associated with risk of T2DM in a dose response manner. There was no evidence that experiencing MLE in both childhood and adult life was more strongly associated with T2DM than experiencing events at only one time point. There was some evidence that being simultaneously exposed to childhood MLE and short education (OR 2.28; 95% C.I. 1.45, 3.59) and work MLE and short education (OR 2.86; 95% C.I. 1.62, 5.03) was associated with higher risk of T2DM, as the joint effects were greater than the sum of their individual effects.

Conclusions

Findings from this study suggest that the accumulation of MLE in childhood, private adult life and work life, respectively, are risk factors for developing T2DM.     

Expression of the myc/His-tagged human peptide transporter hPEPT1 in yeast for protein purification and functional analysis

The human intestinal peptide transporter hPEPT1 has been expressed in the yeast Pichia pastoris using the promoter of the glyceraldehyde-3-phosphate-dehydrogenase gene. A myc-epitope fused to a polyhistidine-tag was introduced at the C-terminus of hPEPT1 for ease of detection and purification. Yeast cells transformed with tagged hPEPT1 exhibited 30-fold increased dipeptide uptake compared to control cells with a substrate specificity and pH dependence similar to the native transporter. The tagged hPEPT1 protein was detected in crude membrane fractions of Pichia cells with an apparent molecular mass of 66 kDa and an expression level of approximately 64 pmol/mg membrane protein. These studies demonstrate that tagged hPEPT1 can be expressed functionally in P. pastoris with unaltered phenotypical characteristics allowing the yeast cells to be used for functional analysis such as screening for compounds utilizing the peptide transporter for absorption in the human intestine. Moreover, recombinant hPEPT1 can now easily be detected for further purification purposes using immobilized metal-affinity chromatography.     

Toward Sustainable Nanoproducts

Sustainable development of nanotechnology will inevitably require incorporation of life cycle thinking to analyze environmental impacts of nanomanufacturing. While many concerns have been raised regarding the human and ecological health effects of and benefits from using nanoproducts, relatively little attention has been given to the manufacturing phase. Unlike many conventional manufacturing techniques, nanomanufacturing techniques require unique facility and process design as well as operation and control. Accordingly, the environmental burden of most nanomanufacturing techniques may be more profound than that of many other conventional techniques. This article reports on a comprehensive review of the current state of technologies used in the manufacture of nanostructured materials in order to identify those attributes that contribute to environmental impacts. It provides a preliminary analysis of significant attributes of commonly used nanomanufacturing techniques, including strict material purity requirements, less tolerance for defects, low process yields and material utilization efficiencies, repetitive processing steps, the need for specialized environments (such as high temperatures or cryogenic processing), the use of toxic chemicals and solvents, the need for moderate to high vacuum, the use or generation of greenhouse gases, high energy and water consumption, and the potential for chemical exposure.