Neuronal apoptosis following human brain injury

Neuronal apoptosis has been investigated in paraffin-embedded brain tissue from 103 individuals who had sustained blunt head injury by use of the in situ nick translation (ISNT) technique. In order to provide reliable data for a forensic wound age estimation, a quantitative morphometric analysis was performed. Apoptotic neuronal cells could be detected in a cortical contusion with a wound age of 45 min at the earliest and in the majority of the cases with postinfliction intervals up to 2 weeks, numerous ISNT-positive cells were found adjacent to the traumatically injured area. The presented data indicate that neuronal apoptosis peaks at about 1 day and persists for at least 22 weeks after blunt head injury. The time-dependent occurrence of apoptotic cells can contribute to a forensic timing of cortical contusions and complements other immunohistochemical parameters, especially in the early postinfliction interval.     

Recurrent intussusceptions in an infant that were terminated by laparoscopic ileocolonic pexie

For children with ileocolic intussusceptions, laparoscopy has been proposed as an emergency intervention, but it has not been elaborated for elective prevention of recurrencies. We report about an infant who developed his first ileocolic intussusception at the age of 12 months. Radiologic devagination was successful, but had to be repeated for two consecutive recurrences within several days. Six months later, he presented with another episode of intussusception, which again was managed conservatively. At this time, preventive surgery seemed indicated. Diagnostic laparoscopy using three trocars and 5-mm instruments showed an insufficient closure of the ileocecal valve, allowing the surgeon easily to provoke an intussusception. Consequently, the distal ileum was attached to the ascending colon with several interrupted 3-0 sutures. The infants postoperative course was uneventful. Oral feeding was started immediately, and he could be discharged after 3 days. Within a follow-up period of 1 year, no evidence of intussusception was noted. We conclude that for children with recurrent episodes of intussusception, laparoscopic ileocolonic pexie presents a beneficial strategy for protective surgery.     

<Emphasis Type="Italic">DAT1</Emphasis> and <Emphasis Type="Italic">DRD4</Emphasis> genes involved in key dimensions of adult ADHD

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder often persisting in adulthood. Genetic studies of ADHD mainly focused on the Dopamine Transporter (DAT1) and the Dopamine Receptor 4 (DRD4) genes. Nevertheless, polymorphisms of these genes explain only a small fraction of the assigned risk, suggesting that intermediate dimensions and environmental factors should also be considered. We investigated in 77 adult ADHD subjects compared to 474 controls, how polymorphisms within the genes coding for DAT1 (40-bp VNTR in 3′UTR), the Dopamine Receptor 2 (DRD2) (rs1799732) and DRD4 (48-bp VNTR in exon 3), may modulate the expression of the disorder. By genotyping DAT1, we detected a new 9.5R allele showing a deletion of 40 bp and also an insertion of 19 bp compared to the 10R allele. This novel allele was found to be significantly protective for ADHD (p < 0.0001). Another significant difference was found in the distribution of DRD4 48-bp VNTR 6R allele when comparing patients and controls (p = 0.0007). In addition significant results were also found for DAT1 9.5R allele, which was associated with impulsiveness (p = 1.98 × 10^?4) and trait anger scores (p = 7.66 × 10^?4). Moreover, impulsiveness scores were partly modulated by an interaction between the DRD4 48-bp VNTR 6R allele and childhood maltreatment (p = 0.01), however, this result did not resist correction for multiple comparisons. Altogether, our results show the putative involvement of DAT1 and DRD4 genes in the aetiology of ADHD with a main role in modulation of key dimensions of the disorder.     

Influence of Mast Cells on the Expression of Adhesion Molecules on Circulating and Migrating Leukocytes in Acute Pancreatitis-Associated Lung Injury

Pancreatitis-associated lung injury is an early-occurring and severe complication, still associated with substantial mortality. A number of inflammatory cells and their products are involved in the initiation and progress of the condition. In the present study, acute pancreatitis (AP) was induced by the intraductal infusion of 5% sodium taurodeoxycholate in the rat. Pulmonary endothelial barrier dysfunction was measured by plasma exudation of radiolabeled albumin. Expression of PECAM-1, ICAM-1, and L-selectin on neutrophils (CD11b⁺) and monocytes/macrophages (CD11b/c⁺), obtained from circulation and lung tissue, was measured 1 and 6 hours after AP induction (n = 10 rats/time point/group). Plasma levels of histamine and serotonin were determined. The role of mast cells was evaluated by pretreatment with the mast cell stabilizer cromolyn. Intraperitoneal administration of cromolyn downregulated pancreatitis-induced systemic increase of histamine at 1 hour (513 ± 82 vs. 309 ± 50, p < 0.05). Cromolyn prevented a decreased expression of PECAM-1 on circulatory neutrophils and monocytes/macrophages and against an increased expression of ICAM-1 and PECAM-1 on pulmonary neutrophils and monocytes/macrophages 6 hours after AP induction (about 40% vs. 10%, p < 0.01). The mast cell stabilizer also prevented pancreatitis-induced pulmonary endothelial barrier dysfunction at 6 hours. Thus, our data indicate that mast cells may play a critical role in the activation of leukocytes during the initiation of pancreatitis-associated lung injury by altering phenotypes of adhesion molecules.     

Multi-metal tolerance of DHHC palmitoyl transferase-like protein isolated from metal contaminated soil

Ecotoxicology - The microbiota inhabiting in metal polluted environment develops strong defense mechanisms to combat pollution and sustain life. Investigating the functional genes of the eukaryotic...     

The in-planta induced ecp2 gene of the tomato pathogen Cladosporium fulvum is not essential for pathogenicity

During the colonization of tomato leaves, the fungal pathogen Cladosporium fulvum excretes low-molecular-weight proteins in the intercellular spaces of the host tissue. These proteins are encoded by the ecp genes which are highly expressed in C. fulvum while growing in planta but are not, or are only weakly, expressed in C. fulvum grown in vitro. To investigate the function of the putative pathogenicity gene ecp2, encoding the 17-kDa protein ECP2, we performed two successive disruptions of the gene. In the first of these, the ecp2 gene was interrupted by a hygromycin B resistance gene cassette. In the second gene disruption, the ecp2 gene was completely deleted from the genome, and replaced by a phleomycin resistance gene cassette. Both disruption mutants were still pathogenic on tomato seedlings, indicating that the C. fulvum ecp2 gene is not essential for pathogenicity in tomato.     

The dominant <Emphasis Type="Italic">Hc.Sdh</Emphasis><Superscript>R</Superscript> carboxin-resistance gene of the ectomycorrhizal fungus <Emphasis Type="Italic">Hebeloma cylindrosporum</Emphasis> as a selectable marker for transformation

In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.Sdh ^R that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe–S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 μg l⁻¹ carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 μg l⁻¹ of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.Sdh ^R cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0–30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60–95%. The authors Chrisse Ngari and Jean-Philippe Combier contributed equally to this work.     

Alterations of Adhesion Molecule Expression and Inflammatory Mediators in Acute Lung Injury Induced by Septic and Non-septic Challenges

The lung is frequently the first failing organ during the sequential development of multiple organ dysfunction under both septic or non-septic conditions. The present study compared polymorphisms of tumor necrosis factor (TNFα), monocyte chemoattractant protein-1 (MCP-1), and adhesion molecule (AM) expression on circulating, recruited, and migrating leukocytes in the development of lung injury after induction of acute pancreatitis (AP) or abdominal sepsis by cecal ligation and puncture (CLP). Pulmonary alveolar barrier and endothelial barrier permeability dysfunction were measured. The expression of AMs (CD11b, CD11b/c, CD31, CD54 and CD62L) on leukocytes isolated from blood, lung tissue, and bronchoalveolar space were measured by flowcytometry. Plasma exudation to the interstitial tissue and the bronchoalveolar space significantly increased 1 and 3 hours after induction of pancreatitis and to the bronchoalveolar space from 6 hours after sepsis. Bronchoalveolar levels of MCP-1 significantly increased earlier than plasma exudation to the alveoli in both pancreatitis and sepsis. Alterations in expression of adhesion molecules on bronchoalveolar lavage (BAL) leukocytes can represent a marker reflecting leukocyte activation in the lung tissue, since both BAL and lung tissue leukocytes showed similar patterns of changes. Expression of adhesion molecules on circulating leukocytes increased 1 hour after induction of pancreatitis. Activating phenotypes of circulating, lung tissue and bronchoalveolar leukocytes may thus be responsible for the-development and severity of secondary lung injury.Supported by grants from the Swedish Research Council (grant no 11236), the Crafoord Foundation, Ake Wiberg Foundation, Maj Bergvall Foundation, Golje Foundation and Clas Groschinsky Foundation.     

Proteinase-activated receptor 1- and 4-promoted migration of Hep3B hepatocellular carcinoma cells depends on ROS formation and RTK transactivation

Purpose

There is growing evidence for a role of proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, in cancer. We have previously shown that PAR₁ and PAR₄ are able to promote the migration of hepatocellular carcinoma (HCC) cells suggesting a function in HCC progression. In this study, we assessed the underlying signalling mechanisms.

Methods

Using Hep3B liver carcinoma cells, RTK activation was assessed by Western blot employing phospho-RTK specific antibodies, ROS level were estimated by H₂DCF-DA using confocal laser scanning microscopy, and measurement of PTP activity was performed in cell lysates using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as a substrate.

Results

Thrombin, the PAR₁ selective agonist peptide TFLLRN-NH₂ (PAR₁-AP), and the PAR₄ selective agonist peptide, AYPGKF-NH₂ (PAR₄-AP), induced a significant increase in Hep3B cell migration that could be blocked by inhibitors targeting formation of reactive oxygen species (ROS), or activation of hepatocyte-growth factor receptor (Met), or platelet-derived growth factor receptor (PDGFR), respectively. The involvement of these intracellular effectors in PAR_1/4-initiated migratory signalling was further supported by the findings that individual stimulation of Hep3B cells with the PAR₁-AP and the PAR₄-AP induced an increase in ROS production and the transactivation of Met and PDGFR. In addition, PAR₁- and PAR₄-mediated inhibition of total PTP activity and specifically PTP1B. ROS inhibition by N-acetyl-l-cysteine prevented the inhibition of PTP1B phosphatase activity induced by PAR₁-AP and the PAR₄-AP, but had no effect on PAR_1/4-mediated activation of Met and PDGFR in Hep3B cells.

Conclusions

Collectively, our data indicate that PAR₁ and PAR₄ activate common promigratory signalling pathways in Hep3B liver carcinoma cells including activation of the receptor tyrosine kinases Met and PDGFR, the formation of ROS and the inactivation of PTP1B. However, PAR_1/4-triggered Met and PDGFR transactivation seem to be mediated independently from the ROS-PTP1B signalling module.