Specific probes for Trypanosoma (Trypanozoon) evansi based on kinetoplast DNA minicircles

Trypanosoma evansi is difficult to distinguish from other members of subgenus Trypanozoon, save for its inability to develop cyclically in the tsetse fly and its characteristic kinetoplast DNA (kDNA). We have used cloned kDNA minicircle fragments as specific probes to distinguish T. evansi from other trypanosomes of subgenus Trypanozoon. Two probes were required, each specific for one of the subgroups of T. evansi previously described. Probe A reacted only with the major isoenzyme group of T. evansi stocks, which have minicircle type A and occur in South America, Kenya, Sudan, Nigeria and Kuwait. The probe did not hybridise with various Trypanosoma brucei spp. stocks, Trypanosoma vivax, Trypanosoma congolense or Trypanosoma simiae, nor with trypanosomes of the minor isoenzyme group of T. evansi stocks found in Kenya with type B minicircles. Probe B was specific for the latter. The probes were sensitive down to a level of 100 trypanosomes in a dot blot. These probes thus provide a simple means of distinguishing T. evansi from T. brucei spp. using comparatively few trypanosomes and without resort to tsetse transmission experiments.     

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase epsilon (Pol epsilon), are reduced at stalled replication forks. In contrast, DNA polymerase alpha (Pol alpha) accumulates to higher than normal levels at stalled forks in asf1Delta cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.     

Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development

FOXL2 mutations cause gonadal dysgenesis or premature ovarian failure (POF) in women, as well as eyelid/forehead dysmorphology in both sexes (the 'blepharophimosis-ptosis-epicanthus inversus syndrome', BPES). Here we report that mice lacking Foxl2 recapitulate relevant features of human BPES: males and females are small and show distinctive craniofacial morphology with upper eyelids absent. Furthermore, in mice as in humans, sterility is confined to females. Features of Foxl2 null animals point toward a new mechanism of POF, with all major somatic cell lineages failing to develop around growing oocytes from the time of primordial follicle formation. Foxl2 disruption thus provides a model for histogenesis and reproductive competence of the ovary.     

Interobserver variability in determining MIB-1 labeling indices in oligodendrogliomas

Several studies have shown that MIB-1 labeling indices correlate well with tumor grade and prognosis in a variety of tumor types. Several factors are responsible for some degree of variability in the determination of labeling indices. Interobserver variability is one of the factors often cited as responsible for this variability. A slide from each of 30 oligodendrogliomas, stained with MIB-1 antibody, was distributed to six pathologists. The same set of slides was reviewed by each individual. Each pathologist was instructed to determine a MIB-1 labeling index by evaluating 1,000 tumor cell nuclei from the area of the slide with the most staining. The labeling index record reflected a percentage of positive-staining tumor cells. Interobserver agreement was compared. MIB-1 labeling indices ranged from 0 to 45.7. Overall agreement was good (> or =0.75) with a concordance coefficient of 0.832 (confidence interval, 0.700 to 0.909). Variability was greater among tumors with higher labeling indices as compared with tumors with labeling indices closer to 0. The overall agreement of MIB-1 labeling indices, while not perfect, was good. The generally minor variability among observers may be related to differences in the area of the slide evaluated and in differing lower thresholds for interpreting positivity. Further improvement of concordance may theoretically be attainable by further training and discussion among observers.     

Changing contexts? The effects of a primary prevention program on classroom levels of peer relational and physical victimization

Whereas school‐based prevention programs often target deficits in individual children's social skills in order to limit their aggression or exposure to peer victimization, there is increasing evidence that school‐wide and classroom‐level factors can also affect the success of these programs. This short‐term longitudinal study involved 432 elementary school students from 44 classrooms in 17 urban schools. We investigated whether classroom characteristics (average levels of social competence, emotional problems, and behavioral problems) and school‐wide characteristics (proportion of children on income assistance and implementation of a peer victimization prevention program—the Walk away, Ignore, Talk, and Seek help [W.I.T.S.] program) experienced in Grade 1 influences changes in children's reports of relational and physical victimization at the end of Grade 2. Findings showed that classroom levels of emotional problems predicted increases in relational victimization (beyond individual differences in emotional and behavioral problems). Classroom levels of behavioral problems predicted reports of increases in physical victimization (beyond individual differences). Classroom levels of social competence also interacted with individual levels of emotional problems such that children with higher levels of emotional problems in classes with more socially competent children reported more relational and physical victimization. Higher school levels of poverty and lack of program involvement also predicted higher levels of physical victimization, beyond individual and classroom effects. The capacity of the W.I.T.S. program to influence classroom level characteristics and the moderating effects of school poverty on victimization were also assessed. © 2003 Wiley Periodicals, Inc. J Comm Psychol 31: 397–418, 2003.     

Genetics of the low density lipoprotein receptor:

Fibroblast association (plasma membrane binding plus intracellular accumulation) and degradation of radioiodinated low density lipoprotein (125I-LDL) index plasma membrane LDL receptor activity. Cultured fibroblasts from 23 subjects affected with familial hypercholesterolemia (HC) and from 95 subjects without HC (non-HCs) were tested for 125I-LDL association and degradation. Both LDL receptor activity indices were twice as high in non-HC and HC heterozygous cell strains. This is compatible with a major gene effect on LDL receptor activity. However, a considerable overlap between non-HC and HC heterozygous values was found in the 125I-LDL association assay [median (range) 970 (330-2500), and 450 (250-490), respectively] and in the degradation assay [median (range) 810 (280-2020), and 470 (160-790), respectively]. The values are expressed as ng 125I-LDL X mg cell protein-1 X 4.5 h-1. These great overlaps in the LDL receptor activity indices support the view that the influence of LDL receptor activity on the HC phenotype may be smaller than believed previously. Furthermore, for the diagnosis of HC, these LDL receptor activity assays are far more expensive and have less sensitivity and specificity than simple serum cholesterol determination. The LDL receptor-dependent 125I-LDL association values for the HC heterozygous individuals clustered into four groups. Family data supported the hypothesis that this variation could be due to four different LDL receptor variants, each coded for by different alleles at the LDL receptor locus. If confirmed, this finding may have implications for the understanding of the variable expression of HC and also of the genetic impact on lipoprotein metabolism and susceptibility to atherosclerosis in non-HCs.     

Performance status and comorbidity predict transplant-related mortality after allogeneic hematopoietic cell transplantation.

Comorbidity measurements have recently been used to improve estimation of tolerance to allogeneic hematopoietic cell transplantation (HCT). We sought to determine the independent effect of comorbidity and performance status on HCT outcome and to devise a simple risk classification system for transplant-related mortality. We analyzed 105 consecutively enrolled patients who underwent HCT and received reduced intensity conditioning with fludarabine, melphalan, and alemtuzumab. Comorbid conditions were tabulated using 2 scales, the Charlson Comorbidity Index (CCI) and the Kaplan-Feinstein Scale (KFS). Comorbid conditions were found in 47% of patients by the KFS and in 27% by the CCI (P < .001). Using the Eastern Cooperative Oncology Group Performance Status (PS) scale, 34% had a PS score >0 (range, 0-2). A simple scale combining the KFS and PS enabled separation of high- from low-risk patients, with 6-month cumulative incidences 50% and 15%, respectively for transplant-related mortality (P = .001) and enhanced prognostic power over the CCI alone (P = .018). Prospective studies evaluating more comprehensive functional and comorbidity measurements are warranted.     

NADPH oxidase inhibition improves neurological outcomes in surgically-induced brain injury

Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91^phox subunit of NADPH oxidase (gp91^phox KO) and apocynin, a specific inhibitor of NADPH oxidase. Neurological function and brain edema were evaluated at 24 h post-SBI in gp91^phox KO and wild-type littermates grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into vehicle- and apocynin-treated (5 mg/kg, i.p. 30 min before SBI) groups. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 h post-SBI. The gp91^phox KO mice, but not the apocynin-treated mice showed significantly improved neurological scores. Brain edema was observed in both gp91^phox KO and wild-type groups after SBI; however, there was no significant difference between these two groups. Brain edema was also not affected by apocynin-pretreatment. LPO levels were significantly higher in SBI group in both gp91^phox KO and wild-type groups as compared to sham group. A trend, although without statistical significance, was noted towards attenuation of LPO in the gp91^phox KO animals as compared to wild-type group. LPO levels were significantly attenuated at 3 h post-SBI by apocynin-pretreatment but not at 24 h post-SBI. These results suggest that chronic and acute inhibition of NADPH oxidase activity does not reduce brain edema after SBI. Long-term inhibition of NADPH oxidase, however improves neurological functions after SBI.