Optimal Detection of Subtle Gadolinium Leakage in CSF with Heavily T2-Weighted Fluid-Attenuated Inversion Recovery Imaging.

Pericortical enhancement on postcontrast FLAIR images is a marker for subtle leptomeningeal blood-brain barrier leakage. We explored the optimal FLAIR sequence parameters for the detection of low gadolinium concentrations within the CSF. On the basis of phantom experiments and human in vivo data, we showed that detection of subtle pericortical enhancement can be facilitated by using a relatively long TE. Future studies should choose their FLAIR sequence parameters carefully when assessing pericortical enhancement due to subtle blood-brain barrier leakage.

Pericortical Enhancement on Delayed Postgadolinium Fluid-Attenuated Inversion Recovery Images in Normal Aging, Mild Cognitive Impairment, and Alzheimer Disease.

BACKGROUND AND PURPOSE: Breakdown of BBB integrity occurs in dementia and may lead to neurodegeneration and cognitive decline. We assessed whether extravasation of gadolinium chelate could be visualized on delayed postcontrast FLAIR images in older individuals with and without cognitive impairment. MATERIALS AND METHODS: Seventy-four individuals participated in this study (15 with Alzheimer disease, 33 with mild cognitive impairment, and 26 with normal cognition). We assessed the appearance of pericortical enhancement after contrast administration, MR imaging markers of cerebrovascular damage, and medial temporal lobe atrophy. Three participants who were positive for pericortical enhancement (1 with normal cognition and 2 with mild cognitive impairment) were followed up for approximately 2 years. In vitro experiments with a range of gadolinium concentrations served to elucidate the mechanisms underlying the postcontrast FLAIR signals. RESULTS: Postcontrast pericortical enhancement was observed in 21 participants (28%), including 6 individuals with Alzheimer disease (40%), 10 with mild cognitive impairment (30%), and 5 with normal cognition (19%). Pericortical enhancement was positively associated with age (P < .02) and ischemic stroke (P < .05), but not with cognitive status (P = .3). Foci with enhanced signal remained stable across time in all follow-up cases. The in vitro measurements confirmed that FLAIR imaging is highly sensitive for the detection of low gadolinium concentrations in CSF, but not in cerebral tissue. CONCLUSIONS: Postcontrast pericortical enhancement on FLAIR images occurs in older individuals with normal cognition, mild cognitive impairment, and dementia. It may represent chronic focal superficial BBB leakage. Future longitudinal studies are needed to determine its clinical significance.

[E-learning in orthopedics and traumatology. A comparative pilot study on acceptance and knowledge acquisition among users and non-users]. / E-Learning in der Orthopädie und Unfallchirurgie : Eine vergleichende Pilotstudie zur Akzeptanz und zum Wissenszuwachs unter Nutzern und Nichtnutzern.

BACKGROUND: Additional internet-based learning tools (e-learning) are successfully used in the curricula of many disciplines and are highly accepted among students. However, in orthopedics and traumatology e-learning is underrepresented and scientific papers are rare. The aim of the present pilot study was to evaluate the acceptance of the e-learning module network for students in traumatology and orthopedics (NESTOR) among users and non-users and to analyze the effect of this additional learning tool on knowledge acquisition. MATERIAL AND METHODS: A total of 544 students were asked to complete evaluation questionnaires at the end of two semesters using different ones for NESTOR users and non-users. The gain of knowledge was analyzed by two written knowledge tests (pre-post test, 20 multiple choice questions) at the beginning and end of the semester comparing these two groups. RESULTS: A total of 191 students took part in the evaluation and 152 completed both written tests. The NESTOR users showed a high acceptance of the e-learning system and non-users considered e-learning beneficial as well. Reasons given for not using NESTOR were lack of time, lack of information about the existence of NESTOR and a lack of interest in this discipline and e-learning in general. Both groups significantly increased their level of knowledge during the course of the semester (p < 0.01), whereas users scored significantly higher in the post-test (p < 0.05). CONCLUSION: The presented data support the high acceptance among users and the benefit of the e-learning project NESTOR in teaching students in orthopedics and traumatology. Based on experience and these results the permanent implementation of an additional e-learning module in the curriculum can be recommended for other faculties. In this process the critical comments of the non-users determined in the present study should be addressed.

Rage signalling promotes intestinal tumourigenesis.

Development of colon cancer is a multistep process that is regulated by intrinsic and extrinsic cellular signals. Extrinsic factors include molecular patterns that are derived from either pathogens (PAMPs) or cellular damage (DAMPs). These molecules can promote tumourigenesis by activation of the innate immune system, but the individual contribution of ligands and their receptors remains elusive. The receptor for advanced glycation end products (Rage) is a pattern recognition receptor that binds multiple ligands derived from a damaged cell environment such as Hmgb1 and S100 protein. Here we show that Rage signalling has a critical role in sporadic development of intestinal adenomas, as Apc(Min/+) Rage(-/-) mice are protected against tumourigenesis.

[Implementation of the eLearning project NESTOR. A network for students in traumatology and orthopedics]. / Implementierung des eLearning-Projekts NESTOR. Ein Netzwerk für Studierende der Traumatologie und Orthopädie.

BACKGROUND: Modern internet-based information technologies offer great possibilities to create and improve teaching methods for students. The eLearning tool NESTOR (Network for Students in Traumatology and Orthopedics) presented here was designed to complement the existing clinical teaching in orthopedics and traumatology at the Charité, University Medicine Berlin. MATERIALS AND METHODS: Using a learning management system, videos, podcasts, X-ray diagnosis, virtual patients, tests and further tools for learning and study information were combined. After implementation the eLearning project was evaluated by students. RESULTS: The NESTOR project offers various possibilities for knowledge acquisition. Students using the program voluntarily showed a high acceptance whereby 82.4% were very satisfied with the contents offered and 95.3% supported the idea of a future use of NESTOR in teaching. The blended learning approach was positively evaluated by 93.5% of the students. The project received the eLearning seal of quality of the Charité University Medicine Berlin. CONCLUSION: Using complex eLearning tools, such as the NESTOR project represents a contemporary teaching approach in the teaching of traumatology and orthopedics and should be offered in a blended learning context as they are well accepted by students.

[Monosegmental anterior lumbar interbody fusion with the SynFix-LR™ device. A prospective 2-year follow-up study]. / Die monosegmentale ventrale Spondylodese mit dem SynFix-LR™. Eine prospektive 2-Jahres-Studie.

BACKGROUND: With anterior lumbar interbody fusion (ALIF) alone, the morbidity associated with a posterior approach can be avoided. In this study we evaluated the use of a PEEK cage with an integrated angle-stable locking plate (SynFix-LR™). MATERIAL AND METHODS: Thirty-two patients with osteochondrosis at L4/5 or L5/S1 were treated with the SynFix-LR™. Follow-up at 0, 3, 6, 9, 12, and 24 months included the Oswestry Disability Index (ODI), visual analog scale (VAS), and questions regarding satisfaction and use of pain medication. The fusion rate was assessed by X-ray and computed tomography (CT) examination. RESULTS: A significant reduction of the ODI and VAS was achieved (p<0.05) with a high rate of patient satisfaction. After 2 years, 79% of the patients were able to dispense with long-term use of analgesics. We observed a fusion rate of 93% (X-ray) and 70% (CT) at final follow-up. CONCLUSION: The SynFix-LR™ device is a suitable option for the treatment of monosegmental osteochondrosis at L4/5 and L5/S1 with comparable or superior results in comparison to posterior or combined fusion techniques.

Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10.

Hoff, E. F., Cook, S. H., Sherman, G. D., Harper, J. M., Ferguson, D. J. P., Dubremetz, J. F., and Carruthers, V. B. 2001. Toxoplasma gondii: Molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Experimental Parasitology, 97, 77-88. During host cell invasion, Toxoplasma gondii secretes proteins from specialized organelles (micronemes and rhoptries) located at the apical end of the parasite. The contents of the micronemes appear to be crucial to T. gondii invasion, as inhibition of microneme secretion prevents parasite entry into host cells. Here we describe a new T. gondii microneme protein, TgMIC10. Molecular characterization of a full-length TgMIC10 cDNA revealed that TgMIC10 lacks homology to any previously characterized proteins, although a homologue, NcMIC10, was identified in a closely related parasite, Neospora caninum. TgMIC10 has an unusually long secretory leader sequence of 58 amino acids; the mature TgMIC10 is 18 kDa, possesses nine diglutamic acid repeats and an imperfect repeat sequence (RK(R/Y)HEEL), and is entirely devoid of cysteines. Antibodies raised against recombinant TgMIC10 recognized the native TgMIC10 and localized the protein to the micronemes in indirect immunofluorescence and immunoEM experiments. Comparison of immunofluorescence images indicates that TgMIC10 expression is higher in T. gondii tachyzoites, which are responsible for active infection, than in bradyzoites, which are responsible for latent infection.

Mycobacterium triplex infection in a liver transplant patient.

Mycobacterium triplex was first named in 1996 as an acid-fast bacillus with features that most resemble Mycobacterium simiae and Mycobacterium avium-intracellulare complex but which possesses a distinct mycolic acid pattern as well as a distinctive 16S rRNA hypervariable region. It has been isolated from lymph node, sputum, and cerebrospinal fluid specimens, but to date only rare clinical cases of this organism have been reported in the literature. The following is a case report of M. triplex that was isolated from the pericardial and peritoneal fluid of a 13-year-old female liver transplant patient.

Overexpression of endonuclease III protects Escherichia coli mutants defective in alkylation repair against lethal effects of methylmethanesulphonate.

Endonuclease III of Escherichia coli is normally involved in the repair of oxidative DNA damage. Here, we have investigated a possible role of EndoIII in the repair of alkylation damage because of its structural similarity to the alkylation repair enzyme 3-methyladenine DNA glycosylase II. It was found that overproduction of EndoIII partially relieved the alkylation sensitivity of alkA mutant cells. Site-directed mutagenesis to make the active site of EndoIII more similar to AlkA (K120W) had an adverse effect on the complementation and the mutant protein apparently inhibited repair by competing for the substrate without base release. These results suggest that EndoIII might replace AlkA in some aspect of alkylation repair, although high expression levels are needed to produce this effect.

Reversal-rate dependent differences in the EEG gamma-band during multistable visual perception.

It is an often reported observation in the literature on multistable perception that the reversal rate within a given observation time is subject to a high interindividual variability. Recently, we reported frontal gamma-band enhancement during multistable visual perception. The aim of the present study was to investigate whether changes in the gamma-band correspond to the variability of the reversal rate. Therefore, a total of 25 observers were divided into two subgroups according to their reversal rate during a 400-s observation period of a reversible pattern based on apparent motion. Subjects with more than 40 reversals within the 400-s were defined as high-rate switchers (HRS). Subjects with a reversal rate below 40 switches were defined as low-rate switchers (LRS). EEG was recorded from frontal, central, parietal, and occipital locations of both hemispheres. The results showed significantly higher gamma activity for the HRS in both phase-locked and non-phase-locked oscillations. Both subgroups showed the highest gamma amplitudes at frontal locations. The results support the involvement of attentional top-down processing in figure reversal. It is concluded that the higher gamma activity for the HRS reflects states of higher arousal, alertness and/or attention according to their fast reversal rate.

Cell-cycle regulation, intracellular sorting and induced overexpression of the human NTH1 DNA glycosylase involved in removal of formamidopyrimidine residues from DNA.

Endonuclease III (Nth) of Escherichia coli is a DNA glycosylase essential for the removal of oxidised pyrimidine base residues from DNA. Several eukaryotic homologues have recently been identified and shown to have biochemical properties similar to those of Nth. However, some of the eukaryotic counterparts also appear to remove imidazole ring-opened purine residues (faPy), a property not shared by the enzymes of bacterial origin. Here, we show that the human enzyme also possesses efficient faPy DNA glycosylase activity as indicated both from studies of the purified protein and induced overexpression of the human NTH1 cDNA in HeLa cells. We constructed green fluorescent protein-tagged hNTH1 fusion proteins to study the cellular localisation of hNTH1 and found strong and exclusive sorting to the nucleus. Studies with synchronised cells showed that the expression of hNTH1 is regulated during the cell cycle with increased transcription during early and mid S-phase.

Prevention of mother-to-child HIV transmission in resource-poor countries: translating research into policy and practice.

Each year, an estimated 590,000 infants acquire human immunodeficiency virus type 1 (HIV) infection from their mothers, mostly in developing countries that are unable to implement interventions now standard in the industrialized world. In resource-poor settings, the HIV pandemic has eroded hard-won gains in infant and child survival. Recent clinical trial results from international settings suggest that short-course antiretroviral regimens could significantly reduce perinatal HIV transmission worldwide if research findings could be translated into practice. This article reviews current knowledge of mother-to-child HIV transmission in developing countries, summarizes key findings from the trials, outlines future research requirements, and describes public health challenges of implementing perinatal HIV prevention interventions in resource-poor settings. Public health efforts must also emphasize primary prevention strategies to reduce incident HIV infections among adolescents and women of childbearing age. Successful implementation of available perinatal HIV interventions could substantially improve global child survival.

Schizosaccharomyces pombe retrotransposon Tf2 mobilizes primarily through homologous cDNA recombination.

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5' untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons.

Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites.

The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.

Novel assays based on human growth hormone receptor as alternatives to the rat weight gain bioassay for recombinant human growth hormone.

Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as 'receptor' in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyzed the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the Kd (approximately 0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC-P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16-20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of < or = 5% vs. an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4-16%. Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitro alternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.

Defective pH regulation of acidic compartments in human breast cancer cells (MCF-7) is normalized in adriamycin-resistant cells (MCF-7adr).

Alkalinization of normally acidic intracellular compartments or acidification of a mildly alkaline cytoplasm by biochemical or genetic manipulation has been demonstrated to inhibit both endocytosis and secretion (Tartakoff, 1983a; Cosson et al., 1989; Mellman et al., 1986; Davoust et al., 1987; Cosson et al., 1989; van Deurs et al., 1989; Maxfield & Yamashiro, 1991; Hansen et al., 1993). These results provide the basis for the conclusion that the maintenance of pH gradients between acidic vesicular compartments and a mildly alkaline cytoplasm is an essential biochemical requirement for the correct functioning of the endocytotic and secretory machinery. Tumor cells have been shown to have an abnormally acidic cytoplasmic pH (Warburg, 1956; Simon & Schindler, 1994). Here we report that the intracellular vesicular compartments in tumor cells (MCF-7) derived from a human breast cancer fail to acidify. This failure results in a significant decrease in the pH gradient (0.9 pH unit) between the vesicular luminal compartments and the cytoplasm. These defects are correlated with a disruption in the organization and function of the trans-Golgi network (TGN) and the pericentriolar recycling compartment (PRC). In marked distinction, drug-resistant tumor cells (MCF-7adr) derived from the MCF-7 line that are resistant to the most widely employed chemotherapeutic drug, adriamycin, appear normal in both acidification and organization of the PRC and TGN. Treatment of drug-resistant MCF-7adr cells with nigericin and monensin, ionophores demonstrated to disrupt vesicular acidification (Tartakoff, 1983b), leads to a resensitization of these cells to adriamycin. Drug sensitivity is proposed to result from an acidification defect within vesicles of the recycling and secretory pathways. A functional consequence of this defect is the diminished capacity of cells to remove cytotoxic drugs from the cytoplasm by sequestration of protonated drugs within the vesicles, followed by drug secretion through the activity of the secretory and recycling pathways.

Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts.

This work describes the isolation and characterization of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants in normal and Escherichia coli tag gene expressing Chinese hamster fibroblast, RJKO, cells. It was previously shown that increased removal of 3-alkylated adenine, effected by 3-methyladenine DNA glycosylase I (Tag), reduces the frequencies of hprt mutations induced by alkylating agents which produce mostly N-alkylation (MMS and EMS) to half the normal rate. In order to identify which type of mutation is suppressed by increased 3-alkyladenine repair we have determined the DNA base sequence changes of the hprt cDNA in 61 independent MMS- and EMS-induced mutant clones. For both cell types and irrespective of the agent used, the majority of mutations were GC to AT transitions originating in the non-transcribed strand. Only 6/55 base substitutions occurred at AT base pairs: five AT to GC transitions and one AT to CG transversion. Six mutations were found to be deletions. These results indicate that 3-alkylated adenines in DNA are not directly premutagenic. The fact that the mutation frequency is reduced by increased 3-alkyladenine removal might be explained by postulating the existence in mammalian cells of an SOS-like response turned on by cytotoxic lesions like 3-alkyladenine, or, alternatively, that increased removal of 3-alkyladenine increases the number of single-strand breaks in DNA, which stalls DNA replication and allows a prolonged time for DNA repair by the alkyltransferase.

Increased removal of 3-alkyladenine reduces the frequencies of hprt mutations induced by methyl- and ethylmethanesulfonate in Chinese hamster fibroblast cells.

We have previously reported the isolation of mammalian cell lines expressing the 3-methyladenine DNA glycosylase I (tag) gene from E. coli. These cells are 2-5 fold more resistant to the toxic effects of methylating agents than normal cells (15). Kinetic measurements of 3-methyladenine removal from the genome in situ show a moderate (3-fold) increase in Tag expressing cells relative to normal as compared to a high (50-fold) increase in exogenous alkylated DNA in vitro by cell extracts. Excision of 7-methylguanine is as expected, unaffected by the tag+ gene expression. The frequency of mutations formed in the hypoxanthine phosphoribosyl transferase (hprt) locus was investigated after methylmethanesulfonate (MMS), ethylmethanesulfonate (EMS), N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) exposure. Tag expression reduced the frequency of MMS and EMS induced mutations to about half the normal rate, whereas the mutation frequency in cells exposed to NMU or NEU is not affected by the tag+ gene expression. These results indicate that after exposure to compounds which produce predominantly N-alkylations in DNA, a substantial proportion of the mutations induced is formed at 3-alkyladenine residues in DNA.

Trypanosome metabolism of myristate, the fatty acid required for the variant surface glycoprotein membrane anchor.

The trypanosome variant surface glycoprotein (VSG) is anchored to the outer leaflet of the parasite plasma membrane by a glycosyl phosphatidylinositol (GPI). The VSG anchor is unique among GPIs in containing exclusively dimyristoylglycerol as its lipid moiety. Myristate is incorporated into the anchor precursor by sequential deacylation and specific reacylation with myristate. Although myristate is required for the VSG anchor, trypanosomes cannot synthesize this fatty acid and must import their entire supply from the host bloodstream, where it exists in low abundance. Chemical analysis of these parasites reveals that most of their myristate is in VSG protein, with no major lipid storage form. Unexpectedly, when these cells are radiolabeled with [3H]myristate in culture, most of the label is incorporated into phospholipids, with little into VSG. This apparent contradiction is explained by the fact that trypanosomes in culture medium elongate much of the [3H]myristate into palmitate and stearate, probably because the medium (with only 5% serum) contains limiting amounts of these fatty acids. In contrast, trypanosomes radiolabeled in whole blood (with higher concentrations of palmitate and stearate) do not modify most of the [3H]myristate, and instead utilize the major portion of it for GPI synthesis. Our studies suggest that bloodstream trypanosomes have evolved highly efficient means of directing myristate into the GPI biosynthetic pathway.