Identification and molecular cloning of a novel neuromedin U analog from the skin secretions of toad Bombina maxima

Amphibian skin contains rich neuropeptides. In the present study, a novel neuromedin U (NmU) analog was isolated from skin secretions of Chinese red belly Load Bombina maxima. Being 17-amino acids long, its primary structure was established as DSSGIVGRPFFLFRPRN-NH2, in which the C-terminal 8-residue segment (FFLFRPRN) is the same as that of rat NmU, while the N-terminal part DSSGIVGRP shows a great sequence variation compared with those of NmU peptides from different resources. The peptide, named Bm-NmU-17, was found to elicit concentration-dependent contractile effects on smooth muscle of rat uterus horns. The cDNA Structure of the peptide, as obtained by a 3'-RACE strategy and subsequently cloning from a skin cDNA library, was found to contain a coding region of 438 nucleotides. The encoded precursor is composed of 145 amino acids with a single copy of Bm-NmU-17 located towards the C-terminus. The sequence of the peptide is preceded by a dibasic site (Lys-Arg) and followed by the sequence of Gly-Arg-Lys, providing the sites of cleavage and releasing of the mature peptide. (c) 2005 Elsevier B.V. All rights reserved.

Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions

In mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel two domain TFF protein from frog Bombina maxima skin secretions (Bm-TFF2) has been purified and cloned. It activated human platelets in a dose-dependent manner and activation of integrin a(11b)beta(3) was involved. Aspirin and apyrase did not largely reduce platelet response to Bm-TFF2 (a 30% inhibition), indicating that the aggregation is not substantially dependent on ADP and thromboxane A2 autocrine feedback. Elimination of external Ca2+ with EGTA did not influence the platelet aggregation induced by Bm-TFF2, meanwhile a strong calcium signal (cytoplasmic Ca2+ release) was detected, suggesting that activation of phospholipase C (PLC) is involved. Subsequent immunoblotting revealed that, unlike in platelets activated by stejnulxin (a glycoprotein VI agonist), PLC gamma 2 was not phosphorylated in platelets activated by Bm-TFF2. FITC-labeled Bm-TFF2 bound to platelet membranes. Bm-TFF2 is the first TFF protein reported to possess human platelet activation activity. (c) 2005 Elsevier Inc. All rights reserved.

Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification

Antimicrobial peptides secreted by the skin of many amphibians play an important role in innate immunity. From two skin cDNA libraries of two individuals of the Chinese red belly toad (Bombina maxima), we identified 56 different antimicrobial peptide cDNA sequences, each of which encodes a precursor peptide that can give rise to two kinds of antimicrobial peptides, maximin and maximin H. Among these cDNA, we found that the mean number of nucleotide substitution per non-synonymous site in both the maximin and maximin H domains significantly exceed the mean number of nucleotide substitution per synonymous site, whereas the same pattern was not observed in other structural regions, such as the signal and propiece peptide regions, suggesting that these antimicrobial peptide genes have been experiencing rapid diversification driven by Darwinian selection. We cloned and sequenced seven genes amplified from skin or liver genomic DNA. These genes have three exons and share the same gene structure, in which both maximin and maximin H are encoded by the third exon. This suggests that alternative splicing and somatic recombination are less likely to play a role in creating the diversity of maximins and maximin Hs. The gene trees based on different domain regions revealed that domain shuffling or gene conversion among these genes might have happened frequently.

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile(1)-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar raito of 1:58, it augmented the activity of bradykinin up to two-fold. (C) 2004 Elsevier B.V. All rights reserved.

Maximins S, a novel group of antimicrobial peptides from toad Bombina maxima

Amphibian skin secretions are rich in antimicrobial peptides acting as important components of innate defense system against invading microorganisms. A novel type of peptide, designated as maximin S, was deduced by random sequencing of 793 clones from a constructed Bombina maxima skin cDNA library. The putative primary structures of maximin S peptides can be grouped into five species, in which maximin S I has 14 amino acid residues and the rest of maximin S peptides (S2-S5) all have 18 amino acid residues. Unlike most of the amphibian antimicrobial peptides so far identified, the newly characterized four maximin S precursors are composed of maximin S I and different combinations of tandem repeated maximin S2-S5 linked by internal peptides. Except maximin S I, the predicted secondary structures of maximin S2-S5 show a similar amphipathic alpha-helical structure. MALDI-TOF mass spectrometry analysis of partially isolated skin secretions of the toad indicates that most of the deduced maximin S peptides are expressed. Two deduced maximin S peptides (S1, S4) were synthesized and their antimicrobial activities were tested. Maximin S4 only had an antibiotic activity against mycoplasma and had no antibacterial or antifungal activity toward tested strains. Maximin S1 had no activity under the same conditions. (C) 2004 Elsevier Inc. All rights reserved.

Apoptotic activity of frog Bombina maxima skin albumin

Albumin, the most abundant protein components of blood plasma, is synthesized and secreted by liver cells in vertebrates. Recently, it was demonstrated that frog Bombina maxima albumin is also expressed in skin. Both B. maxima albumins from skin and serum

Melanoma cell growth inhibition by beta gamma-CAT, which is a novel non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin

In vertebrates, non-lens beta gamma-crystallins are widely expressed in various tissues but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained

Acute toxicity of beta gamma-CAT, a naturally existing non-lens beta gamma-crystallin and trefoil factor complex from frog Bombina maxima skin secretions

In vertebrates, non-lens beta gamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained

RFID Tag Location Systems

We describe a methods of locating an RFID tag. One method comprises: transmitting tag location signals from a plurality of different transmit antennas, wherein said antennas are spaced apart by more than a near field limit distance at a frequency of a said signal; receiving a corresponding plurality of receiving return signals from said tag; and processing said tag return signals to determine a range to said tag; wherein said transmitting comprises transmitting at a plurality of different frequencies; wherein said processing comprises determining a phase difference at said plurality of different frequencies to determine said range, and wherein said determining of said phase difference determines a phase difference between either i) two or more of said transmit signals resulting in a maxima in the returned signal RSSI or ii) a first transmit signal and its corresponding return signal; and wherein said determining of said range to said tag uses said return signals weighted responsive to a respective received signal strength of the return signal. Further data which may be used for averaging may be generated by using the above techniques along with changes in the polarisation state of the transmit and receive antennas and/or physical reconfiguration of the antennas (e.g. switch the transmit and receive elements).

Weighted least squares with expectation-maximization algorithm for burst detection in U.K. water distribution systems

Flow measurement data at the district meter area (DMA) level has the potential for burst detection in the water distribution systems. This work investigates using a polynomial function fitted to the historic flow measurements based on a weighted least-squares method for automatic burst detection in the U.K. water distribution networks. This approach, when used in conjunction with an expectationmaximization (EM) algorithm, can automatically select useful data from the historic flow measurements, which may contain normal and abnormal operating conditions in the distribution network, e.g., water burst. Thus, the model can estimate the normal water flow (nonburst condition), and hence the burst size on the water distribution system can be calculated from the difference between the measured flow and the estimated flow. The distinguishing feature of this method is that the burst detection is fully unsupervised, and the burst events that have occurred in the historic data do not affect the procedure and bias the burst detection algorithm. Experimental validation of the method has been carried out using a series of flushing events that simulate burst conditions to confirm that the simulated burst sizes are capable of being estimated correctly. This method was also applied to eight DMAs with known real burst events, and the results of burst detections are shown to relate to the water company's records of pipeline reparation work. © 2014 American Society of Civil Engineers.

Determining of the delay time for a heating ventilating and air-conditioning plant using two weighted neural network approach

This paper presents an two weighted neural network approach to determine the delay time for a heating, ventilating and air-conditioning (HVAC) plan to respond to control actions. The two weighted neural network is a fully connected four-layer network. An acceleration technique was used to improve the General Delta Rule for the learning process. Experimental data for heating and cooling modes were used with both the two weighted neural network and a traditional mathematical method to determine the delay time. The results show that two weighted neural networks can be used effectively determining the delay time for AVAC systems.

Application of multi-weighted neuron for iris recognition

In this paper, from the cognition science point of view, we constructed a neuron of multi-weighted neural network, and proposed a new method for iris recognition based on multi-weighted neuron. In this method, irises are trained as "cognition" one class by one class, and it doesn't influence the original recognition knowledge for samples of the new added class. The results of experiments show the correct rejection rate is 98.9%, the correct cognition rate and the error recognition rate are 95.71% and 3.5% respectively. The experimental results demonstrate that the correct rejection rate of the test samples excluded in the classes of training samples is very high. It proves the proposed method for iris recognition is effective.

Research on multi-degree-of-freedom neurons with weighted graphs

In this paper, we redefine the sample points set in the feature space from the point of view of weighted graph and propose a new covering model - Multi-Degree-of-Freedorn Neurons (MDFN). Base on this model, we describe a geometric learning algorithm with 3-degree-of-freedom neurons. It identifies the sample points secs topological character in the feature space, which is different from the traditional "separation" method. Experiment results demonstrates the general superiority of this algorithm over the traditional PCA+NN algorithm in terms of efficiency and accuracy.