Insights into Exo- and Endoglucanase Activities of Family 6 Glycoside Hydrolases from Podospora anserina

The ascomycete Podospora anserina is a coprophilous fungus that grows at late stages on droppings of herbivores. Its genome encodes a large diversity of carbohydrate-active enzymes. Among them, four genes encode glycoside hydrolases from family 6 (GH6), the members of which comprise putative endoglucanases and exoglucanases, some of them exerting important functions for biomass degradation in fungi. Therefore, this family was selected for functional analysis. Three of the enzymes, P. anserina Cel6A (PaCel6A), PaCel6B, and PaCel6C, were functionally expressed in the yeast Pichia pastoris. All three GH6 enzymes hydrolyzed crystalline and amorphous cellulose but were inactive on hydroxyethyl cellulose, mannan, galactomannan, xyloglucan, arabinoxylan, arabinan, xylan, and pectin. PaCel6A had a catalytic efficiency on cellotetraose comparable to that of Trichoderma reesei Cel6A (TrCel6A), but PaCel6B and PaCel6C were clearly less efficient. PaCel6A was the enzyme with the highest stability at 45°C, while PaCel6C was the least stable enzyme, losing more than 50% of its activity after incubation at temperatures above 30°C for 24 h. In contrast to TrCel6A, all three studied P. anserina GH6 cellulases were stable over a wide range of pHs and conserved high activity at pH values of up to 9. Each enzyme displayed a distinct substrate and product profile, highlighting different modes of action, with PaCel6A being the enzyme most similar to TrCel6A. PaCel6B was the only enzyme with higher specific activity on carboxymethylcellulose (CMC) than on Avicel and showed lower processivity than the others. Structural modeling predicts an open catalytic cleft, suggesting that PaCel6B is an endoglucanase.     

The thoracic muscular system and its innervation in third instar Calliphora vicina Larvae. II. Projection patterns of the nerves associated with the pro‐ and mesothorax and the pharyngeal complex

We describe the anatomy of the nerves that project from the central nervous system (CNS) to the pro‐ and mesothoracic segments and the cephalopharyngeal skeleton (CPS) for third instar Calliphora larvae. Due to the complex branching pattern we introduce a nomenclature that labels side branches of first and second order. Two fine nerves that were not yet described are briefly introduced. One paired nerve projects to the ventral arms (VAs) of the CPS. The second, an unpaired nerve, projects to the ventral surface of the cibarial part of the esophagus (ES). Both nerves were tentatively labeled after the structures they innervate. The antennal nerve (AN) innervates the olfactory dorsal organ (DO). It contains motor pathways that project through the frontal connectives (FC) to the frontal nerve (FN) and innervate the cibarial dilator muscles (CDM) which mediate food ingestion. The maxillary nerve (MN) innervates the sensory terminal organ (TO), ventral organ (VO), and labial organ (LO) and comprises the motor pathways to the mouth hook (MH) elevator, MH depressor, and the labial retractor (LR) which opens the mouth cavity. An anastomosis of unknown function exists between the AN and MN. The prothoracic accessory nerve (PaN) innervates a dorsal protractor muscle of the CPS and sends side branches to the aorta and the bolwig organ (BO) (stemmata). In its further course, this nerve merges with the prothoracic nerve (PN). The architecture of the PN is extremely complex. It innervates a set of accessory pharyngeal muscles attached to the CPS and the body wall musculature of the prothorax. Several anastomoses exist between side branches of this nerve which were shown to contain motor pathways. The mesothoracic nerve (MeN) innervates a MH accessor and the longitudinal and transversal body wall muscles of the second segment. J. Morphol. 271:969–979, 2010. © 2010 Wiley‐Liss, Inc.     

Tarsal movements in flies during leg attachment and detachment on a smooth substrate

In order to understand the attachment mechanism of flies, it is important to clarify the question of how the adhesive pad (pulvillus) builds and breaks the contact with the substrate. By using normal and high-speed video recordings, the present study revealed that pulvilli are positioned on the surface in a particular way. The pulvilli are apparently loaded or pressed upon the substrate after leg contact, as evidenced by splaying of the claws. Detachment of pulvilli from the substrate may be achieved in four different modes depending on the leg (fore-, mid- or hindleg): pulling, shifting, twisting, and lifting. Lifting is the only detachment mode depending on the claws' action. Kinematics of the tarsal chain is studied in leg preparations, in which the tendon of the claw flexor muscle was pulled by tweezers and video recorded. The morphological background of tarsal movements during attachment and detachment is studied by scanning electron microscopy, fluorescent microscopy, and bright field light microscopy followed by serial semithin sectioning of pretarsal structures. Several resilin-bearing springs are involved in the recoil of the tarsal segments to their initial position, when the tendon is released after pull.     

Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury

Background

Bone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration.

Methods

Ex vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (10⁶ cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals.

Results

We demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats.

Conclusion

Transplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflamatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.     

Pancreatic β cell identity is maintained by DNA methylation-mediated repression of Arx

Adult pancreatic β cells can replicate during growth and after injury to maintain glucose homeostasis. Here, we report that β cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to α cells. We identified the lineage determination gene aristaless-related homeobox (Arx), as methylated and repressed in β cells, and hypomethylated and expressed in α cells and Dnmt1-deficient β cells. We show that the methylated region of the Arx locus in β cells was bound by methyl-binding protein MeCP2, which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of α cell lineage determination genes to maintain pancreatic β cell identity.     

The cephalic and pharyngeal sense organs of Calliphora vicina 3rd instar larvae are mechanosensitive but have no profound effect on ongoing feeding related motor patterns

The anterior segments of cyclorraphous Diptera larvae bear various sense organs: the dorsal- and terminal organ located on the cephalic lobes, the ventral- and labial organs associated with the mouthplate and the internal labral organ which lies on the dorsal surface of the esophagus. The sense organs are connected to the brain via the antennal nerve (dorsal- and labral organ) or the maxillary nerve (terminal-, ventral-, labial organ). Although their ultrastructure suggests also a mechanosensory function only their response to olfactory and gustatory stimuli has been investigated electrophysiologically. Here we stimulated the individual organs with step-, ramp-, and sinusoidal stimuli of different amplitude while extracellulary recording their afferents from the respective nerves. The external organs show a threshold of approximately 2 μm. All organs responded phasically and did not habituate to repetitive stimuli. The low threshold of the external organs combined with their rhythmically exposure to the substrate suggested a putative role in the temporal coordination of feeding. We therefore repetitively stimulated individual organs while simultaneously monitoring the centrally generated motor pattern for food ingestion. Neither the dorsal-, terminal- or ventral organ afferents had an obvious effect on the ongoing motor rhythm. Various reasons explaining these results are discussed.     

Valdoltra and osteoarticular tuberculosis among Slovenians--the 100th anniversary of the Valdoltra Hospital

The 100th anniversary of the hospital in Valdoltra, Slovenia, on the northeastern Adriatic coast near the Italian frontier--where borders have frequently changed (the town has belonged to Austria-Hungary, Italy, Yugoslavia, and Slovenia) and which experienced military occupation in the interwar period--offers an opportunity to review the professional path of this institution. The hospital was established in 1909 as an act of charity by the Trieste Friends of Children Society due to the high incidence of scrofula as well as bone and extrapulmonary tuberculosis among Trieste children. With 270 beds, it provided medical assistance to sick children and also later to adults. After the First World War, its management was assumed by the Italian Red Cross, which built an additional wing in 1934 and increased the hospital's capacity to 340 beds. After Italy's capitulation, German soldiers occupied the hospital and left it in shambles at the end of the war. In September 1945, the hospital was renovated and taken over by the Slovenian healthcare system; 400 beds were again available for treating bone tuberculosis patients. This did not last for long. By 1947, after the Treaty of Peace with Italy was signed and Valdoltra became the central Yugoslav institution for treating bone tuberculosis, the hospital had to be relocated to Rovinj, Croatia due to the political division of the Trieste region into Zones A and B. Only in 1952 did the hospital return to Valdoltra and continue its mission. In the twentieth century, tuberculosis was treated similarly everywhere until antitubercular agents were discovered. At first, conservative climatic and hygiene-dietary methods, orthopedic aids, plaster corsets, and physiotherapy were used to treat bone tuberculosis. This was followed by surgical treatment, which came into vogue after 1945, when it was supported by antibiotic treatment, and (postoperative) physiotherapy and rehabilitation. Chemotherapeutic agents and preventive outpatient BCG-vaccination proved successful in curing bone tuberculosis and other forms of tuberculosis, and the number of consumptive patients continued to decrease. The Valdoltra hospital has preserved its tradition of treating osteoarticular pathologies and has been the main Slovenian orthopedic hospital since 1961.     

Arsenic in the water,sediment and fish in the Neretva River Delta,Croatia

The Neretva River Delta in Croatia is under constant threat of pollution from various sources along the river watercourse, such as the aluminium industry and bauxite mining, intensive agriculture and untreated sewage from towns. The area is also an important fishing ground and food source for the local residents, whereby the suitability of fish for human consumption is always in question. In this paper the presence of arsenic from six sources was analysed: in water, sediment and fish organs (kidneys, liver, muscles and gonads) of 11 fish species: Lepomis gibbosus, Carassius auratus gibelio, Cyprinus carpio, Anguilla anguilla, Ameiurus nebulosus, Mugil cephalus, Leuciscus svallize, Oncorhynchus mykiss, Salmo trutta, Tinca tinca and Scardinius plotiza. The research showed that arsenic concentrations varied significantly from one source to another in the water, sediment and organs of different fish species. Average concentrations in water and sediment were 18.1 μg L^?1 and 32.7 μg kg^?1, respectively. Average arsenic levels in the fish organs were 115.9, 105.8, 76.1 and 61.9 μg kg^?1 in muscles, kidneys, gonads and livers, respectively. These values are below legally permitted concentrations, although individuals with higher than average concentrations were recorded.     

Beetles and flies collected on pig carrion in an experimental setting in Thuringia and their forensic implications

Decomposition processes and insect succession were analysed on a total of eight pig (Sus scrofa) carcasses. The survey was carried out in four different periods between November 2007 and August 2008 and on two different substrates (meadow, concrete floor close to a building). The experiments were placed in a rural site in the surroundings of Jena, Thuringia. The duration and specificity of the different decomposition stages were dependent on season, weather and quality and quantity of insect colonization. Whereas the carrion maintained a bloated appearance even after 133 days in winter, it reached the dry stage within 8 days in the summer months. The type of substrate had few effects on the decaying process, but the insects were generally more abundant on the meadow. In total, more than 57 species of Diptera belonging to 17 families and 48 species of Coleoptera belonging to 14 families were identified. Dominant species belonged to the families of Calliphoridae (n = 11 spp.), Sarcophagidae (n = 8), Muscidae (n = 9), Piophilidae (n = 3), Silphidae (n = 6), Dermestidae (n = 3), Nitidulidae (n = 4), Cleridae (n = 3) and Histeridae (n = 2). Remarkably, the rather common Nicrophorus species were completely absent, whereas the usually rare Necrodes littoralis (L.) was present in larger numbers. No distinct coincidence between the occurrence of a single species and a certain decomposition stage could be confirmed. A main objective of the study is the establishment of a forensic entomological database for Central Europe, especially Thuringia.