Genetic knockouts and knockins in human somatic cells

Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.     

Metabolic syndrome in Yup'ik Eskimos: the Center for Alaska Native Health Research (CANHR) Study

Objective: This study investigated the prevalence of metabolic syndrome and its defining components among Yup'ik Eskimos. Research Methods and Procedures: A cross‐sectional study design that included 710 adult Yup'ik Eskimos ≥18 years of age residing in 8 communities in Southwest Alaska. The prevalence of metabolic syndrome was determined using the recently updated Adult Treatment Panel III criteria. Results: The prevalence of metabolic syndrome in this study cohort was 14.7%, and varied by sex with 8.6% of the men and 19.8% of the women having metabolic syndrome. This is lower than the prevalence of 23.9% in the general U.S. adult population. The most common metabolic syndrome components/risk factors were increased waist circumference and elevated blood glucose. High‐density lipoprotein (HDL) cholesterol levels in Yup'ik Eskimos were significantly higher, and triglycerides lower than levels reported in National Health and Nutritional Examination III. Discussion: Compared with other populations, metabolic syndrome is relatively uncommon in Yup'ik Eskimos. The higher prevalence among Yup'ik women is primarily explained by their large waist circumference, suggesting central body fat accumulation. Further increases in metabolic syndrome risk factors among Yup'ik Eskimos could lead to increases in the prevalence of type 2 diabetes and cardiovascular disease, once rare in this population.     

Continuous fetal heart rate monitoring during late gestation in cattle by means of Doppler ultrasonography: reference values obtained by computer-assisted analysis

Continuous fetal heart rate (FHR) monitoring using transabdominal Doppler ultrasonography can be assumed to provide information about the viability of the bovine fetus during late gestation, as has been found in humans. To be able to recognize unfavourable fetal conditions, first the normal ranges of FHR parameters in cattle should be established. Therefore, in this study we aimed to determine the normal ranges of computerized FHR parameters, like basal fetal heart rate (BHR), number of accelerations and decelerations per hour and short and long term variation (STV and LTV) during the last 3 weeks before calving (n = 21 cows). Each cow had one recording in each of three episodes of 7 days before parturition. As recording time in the cow is limited, we also studied whether these FHR parameters differ between recordings of 30 and 60 min duration (n = 31 pairs of recordings). The outcomes of FHR recordings with a duration of 30 or 60 min did not differ significantly, except for a higher percentage of signal loss in the 60 min recordings. Therefore, determination of normal ranges was performed in 30 min recordings. BHR decreased from 3 to 2 weeks (114 to 109 bpm; P < 0.0001) before parturition and then remained constant until 2 days before calving. The mean number of accelerations per hour ranged between 4.4 and 5.0 h(-1) and did not change significantly with time. Compared to 3 weeks before parturition, STV was significantly higher at 2 weeks (P < 0.05), but not at 1 week before parturition (8.1, 10.0, and 9.2 ms, respectively). Changes in LTV showed a time course comparable to that of STV, but significance was not reached (51.4, 58.6, and 58.4 ms for respectively 3, 2 and 1 weeks before parturition). No decelerations were found during the period understudy. In conclusion, this study has provided normal ranges of bovine computerized FHR parameters during the last 3 weeks of gestation, allowing a comparison with data from cows with compromised gestations in future.     

Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-γ receptor-deficient (IFN-γR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-γR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4⁺ cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-γR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-κB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts.     

Importance of a hydrophobic pocket for peptide binding in lactococcal OppA

Lactococcal oligopeptide-binding protein A (OppA) binds peptides with widely varied lengths and sequences. We previously hypothesized that a hydrophobic pocket in OppA preferentially binds a hydrophobic peptide side chain and thus determines its binding register. Two crystal structures of OppA with different nonapeptides now indeed show binding in different registers.     

A comparison of the predicted and X-ray structures of angiogenin. Implications for further studies of model building of homologous proteins

The three-dimensional structure of human angiogenin has been determined by X-ray crystallography and is compared here with an earlier model which predicted its structure, based on the homology of angiogenin with bovine pancreatic ribonuclease A. Comparison of the predicted model and crystal structure shows that the active-site histidine residues and the core of the angiogenin molecule, including most of the-strands and-helices, were predicted reasonably well. However, the structure of the surface loop regions and residues near the truncated C-terminus differs significantly. The C-terminal segment includes the active-site residues Asp-116, Gln-117, and Ser-118; Gln-117 in particular has been shown to be important in affecting the ribonucleolytic activity of angiogenin. Also, the orientation of one helix in the model differed from the orientation observed experimentally by about 20°, resulting in a large displacement of this chain segment. The difficulty encountered in predicting the surface loop regions has led to a new algorithm [Palmer and Scheraga (1991),J. Comput. Chem.,12, 505–526; (1992),J. Comput. Chem.,13, 329–350] for predicting the conformations of surface loops.     

Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints

We have developed a new technique for the physical mapping of barley chromosomes using microdissected translocation chromosomes for PCR with sequence-tagged site primers derived from >300 genetically mapped RFLP probes. The positions of 240 translocation breakpoints were integrated as physical landmarks into linkage maps of the seven barley chromosomes. This strategy proved to be highly efficient in relating physical to genetic distances. A very heterogeneous distribution of recombination rates was found along individual chromosomes. Recombination is mainly confined to a few relatively small areas spaced by large segments in which recombination is severely suppressed. The regions of highest recombination frequency (相似文献