Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions

Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA-dependent protein kinase (DNA-PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA-PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double-strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA-PK defects in Ku70-/- embryonic stem cells can be counteracted by introducing epitope-tagged wild-type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site-specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA-PK, although two mutants showed that the roles of Ku70 in DNA-PK activation and IR repair are separate. Mutation of DNA-PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double-strand break repair.     

A prospective evaluation of a diffractive versus a refractive designed multifocal intraocular lens

OBJECTIVE: To evaluate prospectively a diffractive (811E, Pharmacia; power add +4.0 D) versus a refractive (PA154N, Allergan; power add +3.5 D) designed multifocal lens. PARTICIPANTS: Eighty patients planned for cataract surgery without additional ocular pathologies were randomized into the diffractive or refractive group, respectively. INTERVENTION: A standardized no-stitch phacoemulsification with implantation of one of the two multifocal lenses was performed in each patient. MAIN OUTCOME MEASURES: Distance and near-visual acuity, contrast sensitivity, low contrast visual acuity, glare visual acuity, and depth of focus were measured after surgery. RESULTS: All treated patients had best-corrected visual acuities of 20/30 or better. Near-uncorrected vision was significantly better (P < 0.0001) with the diffractive lens (mean, J1) than with the refractive lens (mean, J4). Low contrast visual acuity (61 +/- 12% versus 59 +/- 9%), glare visual acuity (39 +/- 19% versus 38 +/- 14%), and contrast sensitivity (1.48 +/- 0.08 versus 1.50 +/- 0.12) were not significantly different between the groups. CONCLUSIONS: Both lens designs showed satisfactory functional results with advantages for the diffractive lens design.     

Adipose tissue development during early postnatal life in ewe-reared lambs

This study examines the precise time course that brown adipose tissue (BAT) takes to adopt the characteristics of white adipose tissue in postnatal lambs. Perirenal adipose tissue was sampled from ewe-reared lambs within 1 h of birth and at 1, 2, 4, 7, 14, 21 and 30 days of age and analysed for the amount of mRNA for uncoupling protein (UCP), the amount and activity of UCP, and protein, mitochondrial protein and lipid content. This was combined with measurements of colonic temperature and jugular venous plasma concentrations of thyroid hormones and insulin-like growth factor-1 (IGF-1). Over the first 4-7 days of age, large quantities of UCP mRNA were associated with a peak in plasma triiodothyronine concentration at 2 days of age followed by a maximal amount and activity of UCP at 4 days and a basal colonic temperature of 39.3 degrees C. Between 7 and 30 days there was a large increase in lipid deposition as the amount and activity of UCP and the amount of UCP mRNA declined to basal values and colonic temperature was maintained at 40 degrees C. A significant positive relationship between perirenal adipose tissue lipid content and plasma IGF-1 concentration was observed throughout the study period. It is concluded that ovine adipose tissue maturation occurs in two distinct phases over the first month of life. The precise time scale of this process could be regulated in part by the lamb's body temperature which determines whether adipose tissue is required for heat production (i.e. BAT) or as an endogenous energy source (i.e. white adipose tissue).     

Artefacts in MR images of electrical current distribution

Artefacts in images of electrical current distribution measured by magnetic resonance imaging (MRI) are presented. These artefacts, caused by the effects of the connecting cables used to apply the electrical current to the object during the MRI scan, can lead to an underestimation of the magnitude of the electrical current measured in the experiment. The size of this underestimate depends upon the experimental geometry, but in the experiments and simulations described here, performed with a cylindrical phantom approximately 8 cm long and 8 cm in diameter, the difference can be 27%. These artefacts are reduced if the connecting cables are rigidly fixed to the object so that the induced magnetic field is correctly measured during the MRI experiment.     

Nonsecretory IgA1 autoantibodies targeting desmosomal component desmoglein 3 in intraepidermal neutrophilic IgA dermatosis

Intraepidermal neutrophilic IgA dermatosis, a rare skin disease entity manifested with blisters and pustules clinically and lower epidermal blister, acantholysis, and neutrophilic infiltration pathologically, was first reported in 1985. Although the disease is characterized by IgA autoantibodies targeting the epithelial cell surface component, the target antigen has not been determined. We investigated a patient with this disease by histopathology, direct and indirect immunofluorescence, immunoblotting, and immunoadsorption studies. The pustular lesion was characterized by blister at the lower epidermis, acantholysis, and neutrophilic infiltration. Nonsecretory IgA1 subclass autoantibodies targeting the lower epithelial cell surfaces were detected in the patient's skin and serum. The patient's IgA autoantibodies labeled a recombinant desmosomal protein desmoglein 3 on immunoblotting and the immunolabeling of epithelial cell surfaces was eliminated by preadsorption with desmoglein 3. Thus, desmoglein 3 is identified as a target antigen in intraepidermal neutrophilic IgA dermatosis. The ability of IgA1 autoantibodies to bind neutrophils may be responsible for the prominent neutrophilic infiltration observed histopathologically and for the pustular lesions observed clinically.     

Erythema migrans-like rash illness at a camp in North Carolina: a new tick-borne disease?

BACKGROUND: Borrelia burgdorferi, the causative agent of Lyme disease, has never been isolated from a patient thought to have acquired Lyme disease in any southeastern state. OBJECTIVE: To investigate 14 cases of an erythema migrans (EM)-like rash illness that occurred during 2 summers at an outdoor camp in central North Carolina in an effort to determine the etiologic, epidemiological, and clinical aspects of this illness. METHODS: Using active surveillance, we identified cases of clinically diagnosed EM in residents and staff of the camp. We collected clinical and demographic information; history of exposure to ticks; acute and convalescent serum antibodies to B. burgdorferi, Rickettsia rickettsii, and Ehrlichia chaffeensis; and cultures for spirochetes from biopsy specimens of skin lesions. Serum samples from a group of residents and staff who did not develop rashes were tested for the same antibodies. We speciated ticks removed from people and collected from vegetation. RESULTS: We identified 14 cases of EM-like rash illness during the 2 summers. Of the 14 case-patients, 10 had associated mild systemic symptoms and 1 had documented fever. All 14 case-patients had removed attached ticks, and 8 remembered having removed a tick from the site where the rash developed a median of 12 days earlier (range, 2-21 days). One tick removed from the site where a rash later developed was identified as Amblyomma americanum, the Lone Star tick; 97% of ticks collected from vegetation and 95% of ticks removed from people were A. americanum. No spirochetes were isolated from skin biopsy specimens. Paired serum samples from 13 case-patients did not show diagnostic antibody responses to B. burgdorferi or other tick-borne pathogens. CONCLUSIONS: This investigation suggests the existence of a new tick-associated rash illness. We suspect that the disease agent is carried by A. americanum ticks. In the southern United States, EM-like rash illness should no longer be considered definitive evidence of early Lyme disease.     

Mechanism of cardiovascular actions of the chromogranin A fragment catestatin in vivo

Catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL), reduces catecholamine secretion from chromaffin cells in vitro. We investigated the effects of this peptide on catecholamine release and blood pressure in vivo. Intravenous catestatin reduced pressor responses to activation of sympathetic outflow by electrical stimulation in rats, and the catestatin effect persisted even after adrenergic (alpha plus beta) blockade. Catestatin did not alter plasma norepinephrine levels, but increased plasma epinephrine 11-fold. Catestatin also blunted pressor responses to exogenous neuropeptide Y agonists. A control peptide (chromogranin A(141-160)) did not alter pressor or catecholamine responses to electrical stimulation. Pretreatment with a histamine H1 receptor antagonist blocked both the vasodepressor response to catestatin and the elevation in plasma epinephrine. Catestatin elevated endogenous circulating histamine 21-fold, and exogenous histamine mimicked both the epinephrine elevation and the vasodepressor actions of catestatin. We conclude that catestatin is a potent vasodilator in vivo whose actions appear to be mediated, at least in part, by histamine release and action at H1 receptors.     

Prediction of residual retroperitoneal mass histology after chemotherapy for metastatic nonseminomatous germ cell tumor: multivariate analysis of individual patient data from six study groups

PURPOSE: To develop a statistical model that predicts the histology (necrosis, mature teratoma, or cancer) after chemotherapy for metastatic nonseminomatous germ cell tumor (NSGCT). PATIENTS AND METHODS: An international data set was collected comprising individual patient data from six study groups. Logistic regression analysis was used to estimate the probability of necrosis and the ratio of cancer and mature teratoma. RESULTS: Of 556 patients, 250 (45%) had necrosis at resection, 236 (42%) had mature teratoma, and 70 (13%) had cancer. Predictors of necrosis were the absence of teratoma elements in the primary tumor, prechemotherapy normal alfa-fetoprotein (AFP), normal human chorionic gonadotropin (HCG), and elevated lactate dehydrogenase (LDH) levels, a small prechemotherapy or postchemotherapy mass, and a large shrinkage of the mass during chemotherapy. Multivariate combination of predictors yielded reliable models (goodness-of-fit tests, P > .20), which discriminated necrosis well from other histologies (area under the receiver operating characteristic (ROC) curve, .84), but which discriminated cancer only reasonably from mature teratoma (area, .66). Internal and external validation confirmed these findings. CONCLUSION: The validated models estimate with high accuracy the histology at resection, especially necrosis, based on well-known and readily available predictors. The predicted probabilities may help to choose between immediate resection of a residual mass or follow-up, taking into account the expected benefits and risks of resection, feasibility of frequent follow-up, the financial costs, and the patient's individual preferences.