The innervation of the lymphoid tissue at the ileocolonic transition: an enzyme and immunohistochemical study.

Using enzyme and immunohistochemical methods on whole-mount preparations and cryostat sections, a morphologic and semiquantitative study was performed of the nervous tissue in the appendix and the ileum (areas with and without Peyer's patches) of the rabbit. The plexus submucous externus (Meissner) consists of a network of small ganglia, vaguely associated with the vascular submucosal plexus. From the nerve cell bodies, cell processes occasionally penetrate the lymphoid follicles at the junction between the mucosa and the submucosa while other extensions form a dense plexus in the lamina propria of the mucosa. No nerve fibers are present in the dome of the follicles. The plexus submucous internus (Henle), consisting of large cell bodies and large processes, closely follows the blood vessels. The numeration of the nerve fibers of the submucosal plexus endorses the histological finding that the appendix is a richly innervated lymphoid organ. In addition, the plexus myentericus (Auerbach) of the appendix is a network of small meshes, while in the ileum, in the area of Peyer's patches, the same plexus is composed of a network with large meshes. These differences point to a higher density of innervation in the appendix. Yet a specialized anatomic distribution of the innervation of lymphoepithelial structures cannot be demonstrated.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Binding of BiP to the processing enzyme lymphoma proprotein convertase prevents aggregation, but slows down maturation

Lymphoma proprotein convertase (LPC) is a subtilisin-like serine protease of the mammalian proprotein convertase family. It is synthesized as an inactive precursor protein, and propeptide cleavage occurs via intramolecular cleavage in the endoplasmic reticulum. In contrast to other convertases like furin and proprotein convertase-1, propeptide cleavage occurs slowly. Also, both a glycosylated and an unglycosylated precursor are detected. Here we demonstrate that the unglycosylated precursor form of LPC is localized in the cytosol due to the absence of a signal peptide. Using a reducible cross-linker, we found that glycosylated pro-LPC is associated with the molecular chaperone BiP. In addition, we show that pro-LPC is prone to aggregation and forms large complexes linked via interchain disulfide bonds. BiP is associated mainly with non-aggregated pro-LPC and pro-LPC dimers and trimers, suggesting that BiP prevents aggregation. Overexpression of wild-type BiP or a dominant-negative BiP ATPase mutant resulted in reduced processing of pro-LPC. Taken together, these results suggest that binding of BiP to pro-LPC prevents aggregation, but results in slower maturation.     

Bioactive peptides,networks and systems biology

Bioactive peptides are a group of diverse intercellular signalling molecules. Almost half a century of research on this topic has resulted in an enormous amount of data. In this essay, a general perspective to interpret all these data will be given. In classical endocrinology, neuropeptides were thought of as simple signalling molecules that each elicit one response. However, the fact that the total bioactive peptide signal is far from simple puts this view under pressure. Cells and tissues express many different bioactive peptides and they are also able to respond to many different bioactive peptides, indicating that multiple receptors and signal transduction pathways are present in a single cell. Therefore, the authors suggest that the bioactive peptide signalling system should be regarded in the context of network and systems biology. Bioactive peptides can best be viewed as an extension of the protein interaction network that allows regulating and fine‐tuning the metabolism of the different cells and tissues in the body. The cell thus responds to the ‘peptidome’ instead of to a single peptide. The intracellular part of this signalling network consists of the various signalling transduction cascades. Recently, new systems biology approaches have emerged for the modelling of cell signalling. The network and systems biology approach is also able to shed new light on the evolution of intercellular signalling.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

The autism candidate gene Neurobeachin encodes a scaffolding protein implicated in membrane trafficking and signaling

Autism is a developmental disorder of the central nervous system characterized by impairments in social interaction, communication and restricted repetitive and stereotyped behavior. It is generally assumed that in most cases autism has a polygenic cause, but the pathogenesis is still unknown. Neurobeachin (NBEA) has recently been identified as a candidate gene for autism in a patient with a de novo chromosomal translocation and three patients with a monoallelic deletion. This multidomain scaffolding protein has been suggested to be involved in neuronal post-Golgi membrane traffic. Knockout of Nbea in two independent mouse models has demonstrated a role in neurotransmitter release and synaptic functioning. Knockdown in a cell line has shown a role as negative regulator of secretion of large dense-core vesicles (LDCVs) and haploinsufficiency in blood platelets results in dense granules with an aberrant morphology. A potential role in vesicle transport is further supported by a study of SEL-2, the C.elegans homologue of NBEA. This protein was identified as a negative regulator of LIN-12/Notch activity, probably due to defects in endosomal trafficking. Members of the Notch pathway have also been shown to be modifiers of the NBEA homologue in Drosophila, rugose. These new insights in the function of NBEA may help identifying novel pathways affected in autistic patients. In particular, it suggests that impaired functionality of LDCVs, which contain neurotrophins, neuropeptides and monoamines, might contribute to the pathogenesis of autism in at least a subgroup of patients.     

Control of mRNA translation preserves endoplasmic reticulum function in beta cells and maintains glucose homeostasis

Type 2 diabetes is a disorder of hyperglycemia resulting from failure of beta cells to produce adequate insulin to accommodate an increased metabolic demand. Here we show that regulation of mRNA translation through phosphorylation of eukaryotic initiation factor 2 (eIF2alpha) is essential to preserve the integrity of the endoplasmic reticulum (ER) and to increase insulin production to meet the demand imposed by a high-fat diet. Accumulation of unfolded proteins in the ER activates phosphorylation of eIF2alpha at Ser51 and inhibits translation. To elucidate the role of this pathway in beta-cell function we studied glucose homeostasis in Eif2s1(tm1Rjk) mutant mice, which have an alanine substitution at Ser51. Heterozygous (Eif2s1(+/tm1Rjk)) mice became obese and diabetic on a high-fat diet. Profound glucose intolerance resulted from reduced insulin secretion accompanied by abnormal distension of the ER lumen, defective trafficking of proinsulin, and a reduced number of insulin granules in beta cells. We propose that translational control couples insulin synthesis with folding capacity to maintain ER integrity and that this signal is essential to prevent diet-induced type 2 diabetes.     

Internalization of proprotein convertase PC7 from plasma membrane is mediated by a novel motif

Proprotein convertase 7 (PC7) is a member of the subtilisin-like proprotein convertase family, which is involved in the endoproteolysis of a variety of precursor proteins. Under steady state conditions, PC7 is mainly localized in the trans-Golgi network, but a small fraction is found at the cell surface. So far, no sorting signals for membrane trafficking have been identified in PC7. In this study, we have examined the internalization of PC7 from the plasma membrane. Our results show that internalization of PC7 is mediated by clathrin-coated vesicles. After inhibition of clathrin-mediated endocytosis using hypertonic conditions or the small molecule inhibitor, Pitstop 2, PC7 accumulated at the plasma membrane. Furthermore, PC7 was present in isolated clathrin-coated vesicles. To determine the internalization motif, constructs were generated in which parts of the N and C terminus of the cytoplasmic tail of PC7 were deleted, and chimeric proteins were constructed consisting of the luminal and transmembrane domains of Tac (CD25) and parts of the cytoplasmic domain of PC7. Antibody uptake experiments as well as surface biotinylation experiments demonstrated that the region between Ala(713) and Cys(726) in the cytoplasmic domain of PC7 is essential and sufficient for the internalization of PC7 but not for trans-Golgi network localization. Individual amino acids in this region were substituted with alanine, which identified Pro, Leu, and Cys as the essential amino acids. In conclusion, internalization of PC7 depends on a short transferable sequence in the cytoplasmic tail, which contains the three crucial amino acids PLC.