The Genome of the Lepidopteran Mamestra brassicae has a Vertebrate-like Content of Methyl-cytosine

Mamestra brassicae genomic DNAs, isolated from larvae and adult tissues and from in vitro cultured CRL-8003 cells, were enzymatically hydrolysed to nucleosides that were separated by HPLC. HPLC analysis showed that 5mC content in cabbage moth larvae, adults and cultured cells was 8.9±0.5, 9.3%±0.2 and 10.2%±0.4 respectively. Cabbage moth 5mC content results the highest reported till now in insects and it is similar to the typical vertebrate one. Analysis of MspI and HpaII restriction pattern on M. brassicae DNA showed that a portion of its genome was methylated at CpG sites. Moreover, the absence of small digestion products after MspI digestion suggested that CpG are not clustered in the cabbage moth genome. Finally, methylation of repeated DNAs has been studied. Comparison of the restriction pattern of MspI and HpaII after hybridisation with the hobo, mariner, 28S and 5S rDNA probes did not evidence any difference indicating the absence of CpG methylation in all the studied repeated DNAs.     

PCDH11 is X/Y homologous in Homo sapiens but not in Gorilla gorilla and Pan troglodytes

Protocadherin X (PCDHX) and Protocadherin Y (PCDHY) are cell-surface adhesion molecules expressed predominantly in brain. The human PCDH11X/Y gene pair is located in the non-pseudoautosomal X-Y homologous region (Xq21.3/Yp11.2). The possible existence of PCDH11 gene dosage differences between human and non-human primates is of evolutionary significance with respect to species differences and escape from X inactivation, and has been repeatedly debated. Previous investigations on the X/Y homologous status of PCDH11 and adjacent sequences in non-human primates have highlighted the complexity of the molecular pattern and evolutionary history of this genomic region. This paper provides for the first time direct evidence for the absence of the PCDH11 genefrom the Y chromosome of chimpanzee (Pan troglodytes) as well as gorilla (Gorilla gorilla). By confirmingthe suspected lack of X-Y homologous status for PCDH11 in non-human primates, our results reinforce the hypothesis of a hominid-specific role for this gene in brain development.     

Increased arginine-vasopressin response to hypertonic stimulation and upright posture in idiopathic hyperprolactinemia

To evaluate the possible influence of idiopathic hyperprolactinemia on the arginine-vasopressin (AVP) response to osmotic and pressure-volumetric stimuli, 14 idiopathic hyperprolactinemic women and 13 normoprolactinemic women were studied during a hypertonic saline infusion test (0.51M NaCl infusion for 2h) and an orthostatic test (standing upright and maintaining an orthostatic position for 20min). In both experimental conditions, the AVP response was significantly higher in women with idiopathic hyperprolactinemia than in normal normoprolactinemic women. These results indicate that in women hyperprolactinemia influences the AVP response to hyperosmotic and hypovolemic stimuli.     

High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid chromatography

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ∼10 min and reverse-phase (RP)-HPLC in fluorescence in ∼21 min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.     

Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin

The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.     

Microsatellite instability in IVS3 of murine c-fes gene: Tumor-associated rearrangement and mammalian divergence

The murine lymphomacrophage hybrids ESb, EbF₁, EbF_2-c4, which express c-fes constitutively, were found by Southern analysis to bear a c-fes deletion of almost 100 bp. The deleted allele was transmitted to the metastatic hybrids by their nonexpressing, poorly metastatic T-lymphoma progenitor Eb, which also has a structurally normal c-fes allele. PCR amplification and sequencing of fes cDNA spanning exons 3–5, where the deletion mapped, ruled out any involvement of coding sequences in the rearrangement. PCR amplification of the as yet unsequenced murine c-fes IVS3 and IVS4 showed they are about 50% longer than their human and feline homologs. Sequencing of IVS4 showed no difference between tumor and control DNA. Sequencing of part of the ∼2600-bp IVS3 was guided by the restriction analysis of PCR products from control and hybrid DNAs. This showed that differences from the control appeared to be mainly located in the 900-bp HindIII-EcoRI fragment, localized in the middle of IVS3. As all three hybrids had the same restriction map, this fragment was sequenced in one of them (ESb). A run of >200 CA repeats was found in control DNA, and a reduction in the CA_n microsatellite accounted for most of the c-fes deletion in the ESb hybrid. Interestingly, the 50% reduction in the size of human and feline c-fes IVS3 as compared with the murine homolog is mostly due to contraction of the same microsatellite. Received: 6 November 1995 / Accepted: 2 May 1996     

Systematic Review of the Literature and Evidence-Based Recommendations for Antibiotic Prophylaxis in Trauma: Results from an Italian Consensus of Experts

Background

Antibiotic prophylaxis is frequently administered in severe trauma. However, the risk of selecting resistant bacteria, a major issue especially in critical care environments, has not been sufficiently investigated. The aim of the present study was to provide guidelines for antibiotic prophylaxis for four different trauma-related clinical conditions, taking into account the risks of antibiotic-resistant bacteria selection, thus innovating previous guidelines in the field.

Methods

The MEDLINE database was searched for studies comparing antibiotic prophylaxis to controls (placebo or no antibiotic administration) in four clinical traumatic conditions that were selected on the basis of the traumatic event frequency and/or infection severity. The selected studies focused on the prevention of early ventilator associated pneumonia (VAP) in comatose patients with traumatic brain injury, of meningitis in severe basilar skull fractures, of wound infections in long-bone open fractures. Since no placebo-controlled study was available for deep surgical site-infections prevention in abdominal trauma with enteric contamination, we compared 24-hour and 5-day antibiotic prophylaxis policies. A separate specific research focused on the question of antibiotic-resistant bacteria selection caused by antibiotic prophylaxis, an issue not adequately investigated by the selected studies. Randomised trials, reviews, meta-analyses, observational studies were included. Data extraction was carried out by one author according to a predefined protocol, using an electronic form. The strength of evidence was stratified and recommendations were given according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria.

Results

Uncertain evidence deserving further studies was found for two-dose antibiotic prophylaxis for early VAP prevention in comatose patients. In the other cases the risk of resistant-bacteria selection caused by antibiotic administration for 48 hours or more, outweighed potential benefits.

Conclusions

When accounting for antibiotic-resistant bacteria selection we found no evidence in favour of antibiotic prophylaxis lasting two or more days in the studied clinical conditions.     

Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration – but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research.     

Cutting edge: Autocrine TGF-beta sustains default tolerogenesis by IDO-competent dendritic cells

CD8(-) and CD8(+) dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8(+) DCs relies on autocrine TGF-beta, which sustains the activation of IDO in response to environmental stimuli. CD8(-) DCs do not produce TGF-beta, yet externally added TGF-beta induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8(-) DCs correlates with activation of the PI3K/Akt and noncanonical NF-kappaB pathways. These data are the first to link TGF-beta signaling with IDO in controlling spontaneous tolerogenesis by DCs.     

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.