Linkage analysis of seven kindreds with the X-linked lymphoproliferative syndrome (XLP) confirms that the XLP locus is near DXS42 and DXS37

Summary Analysis of seven kindreds indicates that the XLP locus exhibits 1% recombination with DXS42 (lod = 17.5) and no recombination with DXS37 (lod = 13.3).     

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.     

Localization of the gene for human heart fatty acid binding protein to chromosome 1p32-1p33

Among 639 spontaneous abortions between the 8th and 14th week of gestation 342 (53.5%) revealed an abnormal karyotype. While the rate of trisomies distinctly increased with advancing maternal age, a decrease in the rate of 45,X conceptuses and polyploidies was observed among abortions from older women. The overall relation of XXXXXXYY among the tetraploidies was 1411 and that of XXXXXYXYY among the triploidies was 26 361. However, when the latter was related to maternal age, a reversal of the XXXXXY ratio of 12 in the younger to 21 in the older age groups became evident. Furthermore a decrease in the rate of paternally derived partial hydatidiform moles was found among the triploid abortion specimens from older women. From these observations we conclude that digyny plays a major role in the origin of triploidy in the increased maternal age groups, while diandry related to immaturity of oocytes and impairment of oocyte cortical function is more frequent in triploid abortions from younger women.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Time- and dose-dependent biom arker responses in flounder (Platichthys flesus L.) exposed to benzo[a]pyrene, 2,3,3′,4,4′, 5-hexachlorobiphenyl (PCB-156) and cadmium

Responses in flounder (Platichthys flesus) towards benzo [a]pyrene (BaP), 2,3,3′,4,4′,5-hexachlorobiphenyl (PCB-156), and cadmium (Cd) were investigated in time-course and dose-response studies of selected biomarkers. Measurements of biliary fluorescent BaP metabolites and hepatic concentrations of PCB-156 and cadmium showed that the injected toxicants were rapidly m obilized from the muscle to the liver, but a depot effect was indicated in the highest dose groups of BaP and PCB-156 (12 mg kg^-1 bodyweight). Clearest biomarker responses were found in the induction of hepatic cytochrome P450 1A (CYP1A) enzymes as a response towards BaP and PCB-156 exposure. Maximum induction of CYP1A dependent 7-ethoxyresorufin O-deethylase (EROD) activity was observed after 2 and 8 days in BaP and PCB-156-treated flounder, respectively. Positive dose-effect relationships were observed towards both compounds, but the CYP1A induction was more persistent with PCB exposure than with BaP exposure. In Cd-exposed fish, the hepatic level of metallothionein responded more slowly with highest levels observed after 16 days in the time-study. In the combined BaP + Cd treatment, the CYP1A induction was only slightly suppressed. Aspartate aminotransferase in serum appeared to be responsive towards BaP, but also towards the acetone vehicle in controls in the first part of the exposure period. Hematocrit as well as hepatic activities of aldrin epoxidase, glutathione S-transferase, and UDP-glucuronyl transferase were not responsive to any treatm ent in the present study. In general, the results demonstrate that selected biom arkers in flounder are responsive to PAH, PCB, and heavy metal pollutant exposure, indicating the applicability of this species in future environmental pollution monitoring programmes.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Effects of citrinin on iron-redox cycle

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.     

Pap smear screening in women under 30 in the Norwegian Coordinated Cervical Cancer Screening Program, with a comparison of immediate biopsy vs Pap smear triage of moderate dysplasia

OBJECTIVE: Top provide population based estimates of Pap smear screening results among women under 30 and to compare the outcomes of 2 triage options for women under 30 with a moderate dysplastic Pap smear: immediate biopsy vs. Pap smear follow-up. STUDY DESIGN: All Pap smears, histologically verified cervical intraepithelial lesion (CIN) 2/3 and invasive cervical cancer in 1999-2002 from women in Norway under 30 in 2000-2002 were analyzed. Due to different triage recommendations in different laboratories, 836 women under 30 with a moderately dysplastic Pap smear were recommended for immediate biopsy, while 109 were recommended for a Pap smear within 6 months. Risk estimates for triage outcome were estimated by multinominal logistic regression. RESULTS: The prevalence of squamous intraepithelial lesion was highest around 20 years. The first moderate dysplasia was diagnosed at age 16, as was CIN 2/3. Of the women with Pap smear triage, one third were diagnosed with a CIN 2+ lesion, one third had a normal outcome, while the last third were in uncertain state 12 months later. Among those recommended for immediate biopsy, nearly 70% were diagnosed with a CIN 2+, thus having odds of 4.2 (95% CI 2.5-7.1) higher than did women with Pap smear triage of being diagnosed with CIN2+ 12 months later. CONCLUSION: The choice of triage has a profound impact on the proportion of young women diagnosed with CIN 2+ after a moderately dysplastic Pap smear.     

Importance of the core structure of flavones in promoting inhibition of the mitochondrial respiratory chain

Flavonoids are a large group of polyphenolic compounds that have received considerable attention because of their biological and physiological importance. The flavone (2-phenyl-4H-1-benzopyran-4one) used in this work is found in some cereal grains and generates several biological activities, including: apoptosis induction, cell cycle arrest, caspase activation and inhibition of tumor cell proliferation. However, its effects on the hepatic mitochondrial metabolism are still unknown. We evaluated the effect of flavone on the metabolism of mitochondria isolated from rat liver. Polarographic experiments using 200 μmol L^?1 flavone and rat liver mitochondria oxidizing glutamate or succinate indicated that both substrates underwent: (i) reduction of state 3 respiration; (ii) stimulation of state 4 respiration; (iii) reduction of the respiratory control coefficient; and (iv) reduction of the ADP/O ratio. An analysis of the activity of enzymatic complexes in the respiratory chain showed that flavone acts between complexes I and III. Flavone reduced the membrane electric potential at doses of 100, 150 and 200 μmol L^?1. Flavone at certain doses (75–200 μmol L^?1) reduced mitochondrial swelling in the presence of valinomycin and KNO₃, suggesting that flavone could induce changes in mitochondrial membrane properties. These results demonstrate that the inhibition of mitochondrial enzymes in the respiratory chain coupled with the effects on membrane properties are promoted by the core structure of flavones, and these effects may be in part responsible for the cytotoxic effects of flavones.     

Importance of factors determining the effective lifetime of a mass, long-lasting, insecticidal net distribution: a sensitivity analysis

Background

Long-lasting insecticidal nets (LLINs) reduce malaria transmission by protecting individuals from infectious bites, and by reducing mosquito survival. In recent years, millions of LLINs have been distributed across sub-Saharan Africa (SSA). Over time, LLINs decay physically and chemically and are destroyed, making repeated interventions necessary to prevent a resurgence of malaria. Because its effects on transmission are important (more so than the effects of individual protection), estimates of the lifetime of mass distribution rounds should be based on the effective length of epidemiological protection.

Methods

Simulation models, parameterised using available field data, were used to analyse how the distribution's effective lifetime depends on the transmission setting and on LLIN characteristics. Factors considered were the pre-intervention transmission level, initial coverage, net attrition, and both physical and chemical decay. An ensemble of 14 stochastic individual-based model variants for malaria in humans was used, combined with a deterministic model for malaria in mosquitoes.

Results

The effective lifetime was most sensitive to the pre-intervention transmission level, with a lifetime of almost 10 years at an entomological inoculation rate of two infectious bites per adult per annum (ibpapa), but of little more than 2 years at 256 ibpapa. The LLIN attrition rate and the insecticide decay rate were the next most important parameters. The lifetime was surprisingly insensitive to physical decay parameters, but this could change as physical integrity gains importance with the emergence and spread of pyrethroid resistance.

Conclusions

The strong dependency of the effective lifetime on the pre-intervention transmission level indicated that the required distribution frequency may vary more with the local entomological situation than with LLIN quality or the characteristics of the distribution system. This highlights the need for malaria monitoring both before and during intervention programmes, particularly since there are likely to be strong variations between years and over short distances. The majority of SSA's population falls into exposure categories where the lifetime is relatively long, but because exposure estimates are highly uncertain, it is necessary to consider subsequent interventions before the end of the expected effective lifetime based on an imprecise transmission measure.