苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26Aa和cry28Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26Aa和cry28Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26Aa在仅消除cry26Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26Aa所在质粒有关。     

Signals from the Brainstem Sleep/Wake Centers Regulate Behavioral Timing via the Circadian Clock

Sleep-wake cycling is controlled by the complex interplay between two brain systems, one which controls vigilance state, regulating the transition between sleep and wake, and the other circadian, which communicates time-of-day. Together, they align sleep appropriately with energetic need and the day-night cycle. Neural circuits connect brain stem sites that regulate vigilance state with the suprachiasmatic nucleus (SCN), the master circadian clock, but the function of these connections has been unknown. Coupling discrete stimulation of pontine nuclei controlling vigilance state with analytical chemical measurements of intra-SCN microdialysates in mouse, we found significant neurotransmitter release at the SCN and, concomitantly, resetting of behavioral circadian rhythms. Depending upon stimulus conditions and time-of-day, SCN acetylcholine and/or glutamate levels were augmented and generated shifts of behavioral rhythms. These results establish modes of neurochemical communication from brain regions controlling vigilance state to the central circadian clock, with behavioral consequences. They suggest a basis for dynamic integration across brain systems that regulate vigilance states, and a potential vulnerability to altered communication in sleep disorders.     

Synthesis, deprotection, analysis and purification of RNA and ribozymes.

Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.     

Endogenous Peptide Discovery of the Rat Circadian Clock: A FOCUSED STUDY OF THE SUPRACHIASMATIC NUCLEUS BY ULTRAHIGH PERFORMANCE TANDEM MASS SPECTROMETRY*

Understanding how a small brain region, the suprachiasmatic nucleus (SCN), can synchronize the body''s circadian rhythms is an ongoing research area. This important time-keeping system requires a complex suite of peptide hormones and transmitters that remain incompletely characterized. Here, capillary liquid chromatography and FTMS have been coupled with tailored software for the analysis of endogenous peptides present in the SCN of the rat brain. After ex vivo processing of brain slices, peptide extraction, identification, and characterization from tandem FTMS data with <5-ppm mass accuracy produced a hyperconfident list of 102 endogenous peptides, including 33 previously unidentified peptides, and 12 peptides that were post-translationally modified with amidation, phosphorylation, pyroglutamylation, or acetylation. This characterization of endogenous peptides from the SCN will aid in understanding the molecular mechanisms that mediate rhythmic behaviors in mammals.Central nervous system neuropeptides function in cell-to-cell signaling and are involved in many physiological processes such as circadian rhythms, pain, hunger, feeding, and body weight regulation (1–4). Neuropeptides are produced from larger protein precursors by the selective action of endopeptidases, which cleave at mono- or dibasic sites and then remove the C-terminal basic residues (1, 2). Some neuropeptides undergo functionally important post-translational modifications (PTMs),1 including amidation, phosphorylation, pyroglutamylation, or acetylation. These aspects of peptide synthesis impact the properties of neuropeptides, further expanding their diverse physiological implications. Therefore, unveiling new peptides and unreported peptide properties is critical to advancing our understanding of nervous system function.Historically, the analysis of neuropeptides was performed by Edman degradation in which the N-terminal amino acid is sequentially removed. However, analysis by this method is slow and does not allow for sequencing of the peptides containing N-terminal PTMs (5). Immunological techniques, such as radioimmunoassay and immunohistochemistry, are used for measuring relative peptide levels and spatial localization, but these methods only detect peptide sequences with known structure (6). More direct, high throughput methods of analyzing brain regions can be used.Mass spectrometry, a rapid and sensitive method that has been used for the analysis of complex biological samples, can detect and identify the precise forms of neuropeptides without prior knowledge of peptide identity, with these approaches making up the field of peptidomics (7–12). The direct tissue and single neuron analysis by MALDI MS has enabled the discovery of hundreds of neuropeptides in the last decade, and the neuronal homogenate analysis by fractionation and subsequent ESI or MALDI MS has yielded an equivalent number of new brain peptides (5). Several recent peptidome studies, including the work by Dowell et al. (10), have used the specificity of FTMS for peptide discovery (10, 13–15). Here, we combine the ability to fragment ions at ultrahigh mass accuracy (16) with a software pipeline designed for neuropeptide discovery. We use nanocapillary reversed-phase LC coupled to 12 Tesla FTMS for the analysis of peptides present in the suprachiasmatic nucleus (SCN) of rat brain.A relatively small, paired brain nucleus located at the base of the hypothalamus directly above the optic chiasm, the SCN contains a biological clock that generates circadian rhythms in behaviors and homeostatic functions (17, 18). The SCN comprises ∼10,000 cellular clocks that are integrated as a tissue level clock which, in turn, orchestrates circadian rhythms throughout the brain and body. It is sensitive to incoming signals from the light-sensing retina and other brain regions, which cause temporal adjustments that align the SCN appropriately with changes in environmental or behavioral state. Previous physiological studies have implicated peptides as critical synchronizers of normal SCN function as well as mediators of SCN inputs, internal signal processing, and outputs; however, only a small number of peptides have been identified and explored in the SCN, leaving unresolved many circadian mechanisms that may involve peptide function.Most peptide expression in the SCN has only been studied through indirect antibody-based techniques (19–29), although we recently used MS approaches to characterize several peptides detected in SCN releasates (30). Previous studies indicate that the SCN expresses a rich diversity of peptides relative to other brain regions studied with the same techniques. Previously used immunohistochemical approaches are not only inadequate for comprehensively evaluating PTMs and alternate isoforms of known peptides but are also incapable of exhaustively examining the full peptide complement of this complex biological network of peptidergic inputs and intrinsic components. A comprehensive study of SCN peptidomics is required that utilizes high resolution strategies for directly analyzing the peptide content of the neuronal networks comprising the SCN.In our study, the SCN was obtained from ex vivo coronal brain slices via tissue punch and subjected to multistage peptide extraction. The SCN tissue extract was analyzed by FTMS/MS, and the high resolution MS and MS/MS data were processed using ProSightPC 2.0 (16), which allows the identification and characterization of peptides or proteins from high mass accuracy MS/MS data. In addition, the Sequence Gazer included in ProSightPC was used for manually determining PTMs (31, 32). As a result, a total of 102 endogenous peptides were identified, including 33 that were previously unidentified, and 12 PTMs (including amidation, phosphorylation, pyroglutamylation, and acetylation) were found. The present study is the first comprehensive peptidomics study for identifying peptides present within the mammalian SCN. In fact, this is one of the first peptidome studies to work with discrete brain nuclei as opposed to larger brain structures and follows up on our recent report using LC-ion trap for analysis of the peptides in the supraoptic nucleus (33); here, the use of FTMS allows a greater range of PTMs to be confirmed and allows higher confidence in the peptide assignments. This information on the peptides in the SCN will serve as a basis to more exhaustively explore the extent that previously unreported SCN neuropeptides may function in SCN regulation of mammalian circadian physiology.     

Mice deficient in endothelin-converting enzyme-2 exhibit abnormal responses to morphine and altered peptide levels in the spinal cord

An increasing body of evidence suggests that endothelin-converting enzyme-2 (ECE-2) is a non-classical neuropeptide processing enzyme. Similar to other neuropeptide processing enzymes, ECE-2 exhibits restricted neuroendocrine distribution, intracellular localization, and an acidic pH optimum. However, unlike classical neuropeptide processing enzymes, ECE-2 exhibits a non-classical cleavage site preference for aliphatic and aromatic residues. We previously reported that ECE-2 cleaves a number of neuropeptides at non-classical sites in vitro; however its role in peptide processing in vivo is poorly understood. Given the recognized roles of neuropeptides in pain and opiate responses, we hypothesized that ECE-2 knockout (KO) mice might show altered pain and morphine responses compared with wild-type mice. We find that ECE-2 KO mice show decreased response to a single injection of morphine in hot-plate and tail-flick tests. ECE-2 KO mice also show more rapid development of tolerance with prolonged morphine treatment and fewer signs of naloxone-precipitated withdrawal. Peptidomic analyses revealed changes in the levels of a number of spinal cord peptides in ECE-2 KO as compared to wild-type mice. Taken together, our findings suggest a role for ECE-2 in the non-classical processing of spinal cord peptides and morphine responses; however, the precise mechanisms through which ECE-2 influences morphine tolerance and withdrawal remain unclear.     

Urotensin II in Invertebrates: From Structure to Function in Aplysia californica

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.     

Analysis of endogenous d-amino acid-containing peptides in Metazoa

Peptides are chiral molecules with their structure determined by the composition and configuration of their amino acid building blocks. The naturally occurring amino acids, except glycine, possess two chiral forms. This allows the formation of multiple peptide diastereomers that have the same sequence. Although living organisms use l-amino acids to make proteins, a group of d-amino acid-containing peptides (DAACPs) has been discovered in animals that have at least one of their residues isomerized to the d-form via an enzyme-catalyzed process. In many cases, the biological functions of these peptides are enhanced due to this structural conversion. These DAACPs are different from those known to occur in bacterial cell wall and antibiotic peptides, the latter of which are synthesized in a ribosome-independent manner. DAACPs have now also been identified in a number of distinct groups throughout the Metazoa. Their serendipitous discovery has often resulted from discrepancies observed in bioassays or in chromatographic behavior between natural peptide fractions and peptides synthesized according to a presumed all-l sequence. Because this l to d post-translational modification is subtle and not detectable by most sequence determination approaches, it is reasonable to suspect that many studies have overlooked this change; accordingly, DAACPs may be more prevalent than currently thought. Although diastereomer separation techniques developed with synthetic peptides in recent years have greatly aided in the discovery of natural DAACPs, there is a need for new, more robust methods for naturally complex samples. In this review, a brief history of DAACPs in animals is presented, followed by discussion of a variety of analytical methods that have been used for diastereomeric separation and detection of peptides.     

The amphioxus (Branchiostoma floridae) genome contains a highly diversified set of G protein-coupled receptors

Background

When natural hybridization occurs at sites where the hybridizing species differ in abundance, the pollen load delivered to the rare species should be predominantly from the common species. Previous authors have therefore proposed a hypothesis on the direction of hybridization: interspecific hybrids are more likely to have the female parent from the rare species and the male parent from the common species. We wish to test this hypothesis using data of plant hybridizations both from our own experimentation and from the literature.

Results

By examining the maternally inherited chloroplast DNA of 6 cases of F1 hybridization from four genera of plants, we infer unidirectional hybridization in most cases. In all 5 cases where the relative abundance of the parental species deviates from parity, however, the direction is predominantly in the direction opposite of the prediction based strictly on numerical abundance.

Conclusion

Our results show that the observed direction of hybridization is almost always opposite of the predicted direction based on the relative abundance of the hybridizing species. Several alternative hypotheses, including unidirectional postmating isolation and reinforcement of premating isolation, were discussed.     

Comparative analysis of neuropeptide cleavage sites in human, mouse, rat, and cattle

Neuropeptides are an important class of signaling molecules that result from complex and variable posttranslational processing of precursor proteins and thus are difficult to identify based solely on genomic information. Bioinformatics prediction of precursor cleavage sites can support effective biochemical characterization of neuropeptides. Neuropeptide cleavage models were developed using comprehensive human, mouse, rat, and cattle precursor data sets and used to compare predicted neuropeptide processing across these species. Logistic regression and artificial neural network models were used to predict cleavages based on amino acid and physiochemical properties of amino acids at precursor sequence locations proximal to cleavage. Correct cleavage classification rates across species and models ranged from 85% to 100%, suggesting that amino acid and amino acid properties have major impact on the probability of cleavage and that these factors have comparable effects in human, mouse, rat, and cattle. The variable accuracy of each species-specific model to predict cleavage sites indicated that there are species- and precursor-specific processing patterns. Prediction of mouse cleavages using rat models was highly accurate, yet the reverse was not observed. Sensitivity and specificity revealed that logistic models are well suited to maximize the rate of true noncleavage predictions with moderate rates of true cleavage predictions; meanwhile, artificial neural networks maximize the rate of true cleavage predictions with moderate to low true noncleavage predictions. Logistic models also provided insights into the strength of the amino acid associations with cleavage. Prediction of neuropeptide cleavage sites using human, mouse, rat, and cattle models are available at . Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Allison Tegge and Bruce Southey contributed equally to this work.     

Characterization of a novel gastropod toxin (6-bromo-2-mercaptotryptamine) that inhibits shaker K channel activity

A novel potassium channel antagonist has been purified from the defensive mucus secreted by Calliostoma canaliculatum, a marine snail found in the temperate coastal waters of the western Pacific. The toxin is expelled from the hypobranchial gland as part of a defensive response and is contained within a viscous matrix that minimizes dilution and degradation. The active compound was isolated by multistage microbore HPLC separations followed by bioactivity assays. Nuclear magnetic resonance, combined with electrospray ionization Fourier-transform ion cyclotron resonance and electrospray ionization ion trap mass spectrometry indicate that the active component is a heretofore unknown indole-derivative, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT). Exudates from the hypobranchial glands of various marine mollusks have been sources for dye compounds such as 6-6 dibromoindigo, the ancient dye Tyrian purple. BrMT represents the first correlation of a hypobranchial gland exudate with a molecular response. Voltage clamp experiments with a number of K channel subtypes indicate that BrMT inhibits certain voltage-gated K channels of the Kv1 subfamily.