Tick neurobiology: recent advances and the post-genomic era

Increasing worldwide resistance to acaricides necessitates greater research on the identification of potential acaricide targets in ticks to aid in the control of these serious pests of medical and veterinary importance. Historically, and most likely in the future, acaricide targets are largely of neural origin, but our knowledge of tick neurobiology is surprisingly limited. The tick central nervous system is a fused nerve mass, termed the synganglion. Tick synganglion material is relatively easily accessible to most researchers and employing modern amplification methods of complementary-DNA construction is readily amenable for gene cloning investigations. The various tick neurotransmitter systems are described with emphasis on our current knowledge of both existing and potential acaricide targets at the molecular level. We describe the impact of mass gene sequencing (expressed sequence tag and genome projects), advances in bioinformatics and RNA-interference on target identification and validation.     

Biclustering as a method for RNA local multiple sequence alignment

Motivations: Biclustering is a clustering method that simultaneously clusters both the domain and range of a relation. A challenge in multiple sequence alignment (MSA) is that the alignment of sequences is often intended to reveal groups of conserved functional subsequences. Simultaneously, the grouping of the sequences can impact the alignment; precisely the kind of dual situation biclustering is intended to address. RESULTS: We define a representation of the MSA problem enabling the application of biclustering algorithms. We develop a computer program for local MSA, BlockMSA, that combines biclustering with divide-and-conquer. BlockMSA simultaneously finds groups of similar sequences and locally aligns subsequences within them. Further alignment is accomplished by dividing both the set of sequences and their contents. The net result is both a multiple sequence alignment and a hierarchical clustering of the sequences. BlockMSA was tested on the subsets of the BRAliBase 2.1 benchmark suite that display high variability and on an extension to that suite to larger problem sizes. Also, alignments were evaluated of two large datasets of current biological interest, T box sequences and Group IC1 Introns. The results were compared with alignments computed by ClustalW, MAFFT, MUCLE and PROBCONS alignment programs using Sum of Pairs (SPS) and Consensus Count. Results for the benchmark suite are sensitive to problem size. On problems of 15 or greater sequences, BlockMSA is consistently the best. On none of the problems in the test suite are there appreciable differences in scores among BlockMSA, MAFFT and PROBCONS. On the T box sequences, BlockMSA does the most faithful job of reproducing known annotations. MAFFT and PROBCONS do not. On the Intron sequences, BlockMSA, MAFFT and MUSCLE are comparable at identifying conserved regions. AVAILABILITY: BlockMSA is implemented in Java. Source code and supplementary datasets are available at http://aug.csres.utexas.edu/msa/     

Co-localization of Glutamic Acid Decarboxylase and Phosphate-activated Glutaminase in Neurons of Lateral Reticular Nucleus in Feline Thalamus

Immunohistochemical methods were used to label singly and/or in combination glutamic acid decarboxylase (GAD, the sole synthesizing enzyme for the inhibitory neurotransmitter γ-aminobutyric acid) and phosphate-activated glutaminase (GLN, a synthesizing enzyme for glutamate) in neurons of lateral reticular nucleus (LRN) of thalamus of adult cats. (1) GAD- and GLN-immunoreactivity (IR) exhibited matching regional patterns of organization within LRN. (2) GAD- and GLN-IR co-localized within most if not all LRN neuronal cell bodies as shown by light microscopy. (3) GAD- and GLN-IR had distinct subcellular localizations in LRN neurons as shown by correlative light/electron microscopy. LRN neurons are important conceptual models where strongly inhibitory cells receive predominant excitatory glutamatergic afferents (from neocortex). Consistent with known actions of intermediary astrocytes, LRN neurons demonstrate GLN enrichment synergistically coupled with glutamatergic innervation to supplement the glutamate pool for GABA synthesis (via GAD) and for metabolic utilization (via the GABA shunt/tricarboxylic acid cycle) but not, apparently, for excitatory neurotransmission. Special issue dedicated to John P. Blass.     

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.     

Post-hatching parental care behaviour and hormonal status in a precocial bird

In birds, the link between parental care behaviour and prolactin release during incubation persists after hatching in altricial birds, but has never been precisely studied during the whole rearing period in precocial species, such as ducks. The present study aims to understand how changes in parental care after hatching are related to circulating prolactin levels in mallard hens rearing ducklings. Blood was sampled in hens over at least 13 post-hatching weeks and the behaviour of the hens and the ducklings was recorded daily until fledging. Contacts between hens and the ducklings, leadership of the ducklings and gathering of them steadily decreased over post-hatching time. Conversely, resting, preening and agonistic behaviour of hens towards ducklings increased. Plasma prolactin concentrations remained at high levels after hatching and then fell after week 6 when body mass and structural size of the young were close to those of the hen. Parental care behaviour declined linearly with brood age, showed a disruption of the hen-brood bond at week 6 post-hatching and was related to prolactin concentration according to a sigmoid function. Our results suggest that a definite threshold in circulating prolactin is necessary to promote and/or to maintain post-hatching parental care in ducks.     

菝葜科种皮微结构特征及其分类学意义

在光学显微镜和扫描电镜下对菝葜科Smilacaceae3个属(菝葜属Smilax、肖菝葜属Heterosmilax和Ripogonum属)共53种5变种植物的种子形态及种皮微形态特征进行了研究。结果表明其种子形状为球形、半球形或钝三角形。在扫描电镜下种子表皮纹饰可分为7种类型,即脑纹型、粗脑纹型、网纹型、细网纹型、孔穴型、密孔穴型和细条纹型。根据种皮微形态的特征,对菝葜科内属间和属内组间的关系进行了探讨。种皮形态分析结果支持将Ripogonum属从菝葜科中分离、独立成科,支持将肖菝葜属与菝葜属合并的观点,这与孢粉学和分子证据的分析结果一致;推测肖菝葜属和菝葜属的土茯苓组sect.Coilanthus及草本组sect.Coprosmanthus的多数种类之间亲缘关系较近,菝葜组sect.China和圆锥组sect.Macranthae的大多数种类之间的亲缘关系较为密切,但种皮形态证据不支持Koyama将菝葜属分为6个组的观点。     

Hyperglycemia and hyperinsulinemia have additive effects on activation and proliferation of pancreatic stellate cells: possible explanation of islet-specific fibrosis in type 2 diabetes mellitus

Pancreatic islet fibrosis observed in Type 2 diabetes is one of the major factors leading to progressive beta-cell loss and dysfunction. Despite its importance, the mechanism of islet-restricted fibrogenesis associated with pancreatic stellate cell (PSC) activation and proliferation remains to be defined. Therefore, we studied whether the islet-specific environment represented by hyperglycemia and hyperinsulinemia had additive effects on the activation and proliferation of cultured rat PSCs. Cells were stimulated to activate and proliferate with glucose and insulin, either individually or concomitantly. Both stimuli promoted PSC proliferation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation independently, but an additive effect was also demonstrated. Blockade of ERK signaling by the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, suppressed both glucose- and insulin-induced ERK 1/2 phosphorylation and PSC proliferation. Glucose and insulin-induced ERK 1/2 phosphorylation also stimulated connective tissue growth factor gene expression. Thus, hyperglycemia and hyperinsulinemia are two crucial mitogenic factors that activate and proliferate PSCs, and the presence of both states will amplify this response.     

A recombinant allosteric lectin antagonist of HIV-1 envelope gp120 interactions

The first, critical stage of HIV-1 infection is fusion of viral and host cellular membranes initiated by a viral envelope glycoprotein gp120. We evaluated the potential to form a chimeric protein entry inhibitor that combines the action of two gp120-targeting molecules, an allosteric peptide inhibitor 12p1 and a higher affinity carbohydrate-binding protein cyanovirin (CVN). In initial mixing experiments, we demonstrated that the inhibitors do not interfere with each other and instead show functional synergy in inhibiting viral cell infection. Based on this, we created a chimera, termed L5, with 12p1 fused to the C-terminal domain of CVN through a linker of five penta-peptide repeats. L5 revealed the same broad specificity as CVN for gp120 from a variety of clades and tropisms. By comparison to CVN, the L5 chimera exhibited substantially increased inhibition of gp120 binding to receptor CD4, coreceptor surrogate mAb 17b and gp120 antibody F105. These binding inhibition effects by the chimera reflected both the high affinity of the CVN domain and the allosteric action of the 12p1 domain. The results open up the possibility to form high potency chimeras, as well as noncovalent mixtures, as leads for HIV-1 envelope antagonism that can overcome potency limits and potential virus mutational resistance for either 12p1 or CVN alone.