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.

A novel bradykinin-like peptide from skin secretions of the frog, Rana nigrovittata

A bradykinin-like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin-N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Ranakinin-N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration-dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin-N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in the absence of a dibasic site for trypsin-like proteinases and an unusual -AEVA- insertion in the N-terminal part of ranakinin-N. The -AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first reported bradykinin-like peptide with a neutral net charge at the N-terminus. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Phylogeny and evolution of the Drosophila nasuta subgroup based on mitochondrial ND4 and ND4L gene sequences

The sequences of the mitochondrial ND4 gene (1339 bp) and the ND4L gene (290 bp) were determined for all the 14 extant taxa of the Drosophila nasuta subgroup The average A + T content of ND4 genes is 76.5% and that of ND4L genes is 83.5%. A total of 114 variable sites were scored. The ND4 gene sequence divergence ranged from 0 to 5.4% within the subgroup. The substitution rate of the ND4 gene is about 1.25% per million years. The base substitution of the genesis strongly transition biased. Neighbor-joining and parsimony were used to construct a phylogeny based on the resultant sequence data set. According to these trees, five, distinct mtDNA clades can be identified. D. niveifrons represents the most diverged lineage. D, sulfurigaster bilimbata and D. kepulauana form two independent lineages. The other two clades are the kohkoa complex and the albomicans complex. The Kohkoa complex consists of D. sulfurigaster sulfurigaster, D. pulaua, D. kohkoa, and Taxon-F. The albomicans complex can be divided into two groups: D. nasuta, D. sulfurigaster neonasuta, D. sulfurigaster albostrigata, and D.. albomicans from Chiangmai form one group; and D. pallidifrons, Taxon-I, Taxon-J, and D. albomicans from China form the other group. High genetic differentiation was found among D. albomicans populations. Based on our phylogenetic results, we hypothesize that D. niveifrons diverged first from the D, nasuta subgroup in Papua New Guinea about 3.5 Mya. The ancestral population spread to the north and when it reached Borneo, it diversified sequentially into the kohkoa complex, D. s. bilimbata, and D. kepulauana. About 1 Mya, another radiation occurred when the ancestral populations reached the Indo-China Peninsula, forming the albomicans complex. Discrepancy between morphological groupings and phylogenetic results suggests that the male morphological traits may not be orthologous. (C) 1999 Academic Press.

Stepwise loss of motilin and its specific receptor genes in rodents

Specific interactions among biomolecules drive virtually all cellular functions and underlie phenotypic complexity and diversity. Biomolecules are not isolated particles, but are elements of integrated interaction networks, and play their roles through specific interactions. Simultaneous emergence or loss of multiple interacting partners is unlikely. If one of the interacting partners is lost, then what are the evolutionary consequences for the retained partner? Taking advantages of the availability of the large number of mammalian genome sequences and knowledge of phylogenetic relationships of the species, we examined the evolutionary fate of the motilin (MLN) hormone gene, after the pseudogenization of its specific receptor, MLN receptor (MLNR), on the rodent lineage. We speculate that the MLNR gene became a pseudogene before the divergence of the squirrel and other rodents about 75 mya. The evolutionary consequences for the MLN gene were diverse. While an intact open reading frame for the MLN gene, which appears functional, was preserved in the kangaroo rat, the MLN gene became inactivated independently on the lineages leading to the guinea pig and the common ancestor of the mouse and rat. Gain and loss of specific interactions among biomolecules through the birth and death of genes for biomolecules point to a general evolutionary dynamic: gene birth and death are widespread phenomena in genome evolution, at the genetic level; thus, once mutations arise, a stepwise process of elaboration and optimization ensues, which gradually integrates and orders mutations into a coherent pattern.

新生豚鼠高胆红素血症时P50抑制变化的研究

目的:评价新生豚鼠高胆红素血症时P50抑制值[即T/C值(T为实验,C为对照)]的动态变化对神经系统的毒性作用.方法:出生5～7天的新生豚鼠30只,随机分成5组,每组6只.其中,第一组为正常对照组(C),其余4组为实验组(T).5组新生豚鼠均在硫喷妥钠麻醉下行颅骨电极包埋术,待手术麻醉清醒后,分别测各组新生豚鼠的T/C值.检测完毕,分别向其中2组实验组动物腹腔注入胆红素溶液100 μg/g,4 h、8 h后观察;另2组实验组动物腹腔注入胆红素溶液200 μg/g,4 h、8 h后观察.正常对照组动物均向腹腔注入生理盐水0.5 ml.各组动物在观察完行为学变化和T/C值检测后,再迅速处死动物,取脑组织,在光镜、电镜下观察脑组织结构的变化.结果:实验豚鼠在注入胆红素溶液后,除Tlb组变化不明显外(P＞0.05),其余各组豚鼠T/C值的变化差异均有统计学意义(P＜0.01).不同实验组与正常对照组的T/C值的变化差异也均有统计学意义(P＜0.01).结论:P50抑制(T/C)在高胆红素血症不同时段均有明显变化,可以较早期地(在胆红素聚集阶段)预测胆红素对神经系统的毒性作用,为临床预防和评价高胆红素血症对新生儿神经系统损伤提供一种新的方法.

La peche dans le fleuve Niger

The fish production of the River Niger can best be estimated from a country by country evaluation of the tonnage exported and that consumed locally. All exported and some locally consumed fish are preserved by smoking or sun drying, a process which entails a loss of weight. Coefficients to correct for this of between 2.6 to 4 have been calculated depending on the type of product. A further loss occurs due to handling and to insect attack, which may account for up to 40% of the production. Taking the above factors into account the productions estimated for the various countries of the Niger River basin are as follows: Guinea (3,600 t), Mali (90,000 t), Upper Volta and Ivory Coast (negligible), Niger (5,200 t), Dahomey (1,200 t), Nigeria (25,000 t), Cameroon (3,000 t). A total production of 128,000 t is, therefore, obtained for the basin as a whole, excluding the Kainji Reservoir. At this level of production, there have been no intimations of overfishing from any part of the basin, and there is unanimity that fishing could be intensified. On the basis of the estimates of existing production and local estimates of potential production it is possible that up to 200,000 t of fish could be produced annually from the basin as a whole.

烙铁头血小板活化素延长豚鼠到大鼠的异种移植心脏存活时间

研究烙铁头m小板活化索(TMVA)对大鼠血小板聚集的抑制作用以及对豚鼠 到大鼠的异种移植心存活时间的影响。方法(1)TMVA对血小板聚集率的影响：将Wistar大鼠随 机分为3组，每组5只，各组经阴茎背静脉注射不同剂量(20、50或100 pg／kg)的TMVA，于注射前 以及注射后0．5 h和24 h时抽血，测定血小板聚集率。(2)TMVA对豚鼠到大鼠的异种移植心存活 时间的影响：以豚鼠为供者，Wistar大鼠为受者，制作颈部异位心脏移植模型，实验组于移植心恢复 血液循环前0．5 h经静脉给予TMVA 50弘g／kg，另设不使用TMVA的对照组。观察移植心的存活 时间，停跳后的移植心组织行病理学检查，并用放射免疫法检测心肌组织内6一酮一前列腺素F，。 (6一keto-PGF。。)及血栓素B2(TXt笺)的含量。结果(1)静脉给予5()或100 pg／kg的TMVA后0．5 h即能完全抑制血小板聚集。(2)实验组移植心的存活时间为1()～135 min，中位数为42 min；对照组 为4～16 min，中位数为5 min，两组比较，差异有统计学意义(P

Characterization of a new bradykinin-potentiating peptide (TmF) from Trimeresurus mucrosquamatus

A novel bradykinin-potentiating peptide (BPP), designated as TmF, has been purified to homogeneity from the venom of Trimeresurus mucrosquamatus by 70% cold methanol extraction, Sephadex G-15 gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). The amino acid sequence of TmF was determined to be pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro (pGlu denotes pyroglutamic acid), which shared high homology with other BPPs. The molecular mass of TmF was 1.1107 kD as determinated by electrospray ionization-mass spectrometry (ESI-MS), which was in accordance with the calculated value of 1.1106 kD. The potentiating "unit" of TmF to bradykinin-induced (BK-induced) contraction on the guinea-pig ileum in vitro was (1.13 +/- 0.3) unit (mg/L), and TmF (5.0 x 10(-4) mg/kg) increased the pressure-lowering-effect of bradykinin (5.0 x 10(-5) mg/kg) with approximate descent value of (14 +/- 2) mmHg. In addition, TmF inhibited the conversion of angiotensin I to angiotensin 11, 2 x 10(-3) mg of TmF caused 50% inhibition (IC50) of angiotensin-converting enzyme (ACE) hydrolyzing activity to bradykinin.

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 mug/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 mug/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB2 and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 mug/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB2 in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.

Nonconstant quality of auditory filters in the porpoises, Phocoena phocoena and Neophocaena phocaenoides (Cetacea, Phocoenidae)

Simultaneous tone-tone masking in conjunction with the envelope-following response (EFR) recording was used to obtain tuning curves in porpoises Phocoena phocoena and Neophocaena phocaenoides asiaeorientalis. The EFR was evoked by amplitude-modulated probes with a modulation rate of 1000 Hz and carrier frequencies from 22.5 to 140 kHz. Equivalent rectangular quality Q(ERB) of the obtained tuning curves varied from 8.3-8.6 at lower (22.5-32 kHz) probe frequencies to 44.8-47.4 at high (128-140 kHz) frequencies. The QERB dependence on probe frequency could be approximated by regression lines with a slope of 0.83 to 0.86 in log-log scale., which corresponded to almost frequency-proportional quality and almost constant bandwidth of 34 kHz. Thus, the frequency representation in the porpoise auditory system is much closer to a constant-bandwidth rather that to a constant-quality manner. (c) 2006 Acoustical Society of America.

Circulation in the western tropical Pacific Ocean and its seasonal variation

An assimilation data set based on the GFDL MOM3 model and the NODC XBT data set is used to examine the circulation in the western tropical Pacific and its seasonal variations. The assimilated and observed velocities and transports of the mean circulation agree well. Transports of the North Equatorial Current (NEC), Mindanao Current (MC), North Equatorial Countercurrent (NECC) west of 140degreesE and Kuroshio origin estimated with the assimilation data display the seasonal cycles, roughly strong in boreal spring and weak in autumn, with a little phase difference. The NECC transport also has a semi-annual fluctuation resulting from the phase lag between seasonal cycles of two tropical gyres' recirculations. Strong in summer during the southeast monsoon period, the seasonal cycle of the Indonesian throughflow (ITF) is somewhat different from those of its upstreams, the MC and New Guinea Coastal Current (NGCC), implying the monsoon's impact on it.

Origins and pathways of the subsurface and intermediate water masses of the Indonesian Throughflow derived from historical and Argo data

On the basis of Argo data and historic temperature/salinity data from the World Ocean Database 2001 (WOD01 origins and spreading pathways of the subsurface and intermediate water masses in the Indonesian Throughflow (ITF) region were discussed by analyzing distributions of salinity on representative isopycnal layers. Results were shown that, Subsurface water mostly comes from the North Pacific Ocean while the intermediate water originates from both the North and South Pacific Ocean, even possibly from the Indian Ocean. Spreading through tire Sulawesi Sea, the Makassar Strait, and the Flores Sea, the North Pacific subsurface water and the North Pacific Intermediate water dominate the western part of the Indonesian Archipelago. Furthermore its the depth increases, the features of the North Pacific sourced water masses become more obvious. In the eastern part of the waters, high salinity South Pacific subsurface water is blocked by a strong salinity front between Halmahera and New Guinea. Intermediate water in the eastern interior region owns salinity higher than the North Pacific intermediate water and the antarctic intermediate water (AAIW), possibly coming from the vertical mixing between subsurface water and the AAIW from the Pacific Ocean, and possibly coming front the northward extending of the AAIW front the Indian Ocean as well.

太平洋西部边界及边界流对赤道暖水库形成和演化的影响

A model of equatorial ocean is used to study the roles of the Pacific western boundary and the Mindanao Current (MC) in the evolution of the equatorial warm pool. The model consists of the single baroclinic mode of a two-layer ocean, with the parameterization of the anomalous increment of the interface representing the SST difference from its long-term-space-mean. The ocean is driven by a wind path in the middle ocean with a real or an artificial geometry assigned at the western and eastern boundaries. In order to test the role of the MC, the western boundary current is introduced into the model by a boundary condition at a position, real and unreal, respectively. The model experiments show that the warm pool, which is insensitive to the longitudinal width of the wind band in middle ocean, results mainly from the accumulation o the eastly-drifted warm water in the equatorial western Pacific. It is the dominant factor for the formation of the warm pool that, at a very low latitude, the Papua New Guinea coast intersects the longitudinally lined Philippine Islands at an obtuse angle. In contrast, the western Atlantic boundary, which inclines poleward from the equator at some 135 degrees, could guide the warm water there moving to a higher latitude. On the other hand, the equatorial warm pool in the western equatorial Pacific is very sensitive to the assignment of th Mindanao Current at 7.5°N and displaces southward, with a stronger southern branch than the northern one. We attribute this asymmetry to the combined effect of the western boundary and the MC upon the equatorial warm away from the equator. A by-product of our solutions is the possible mechanism of the "secondary warm pool" in the eastern Pacific north of the equator. It is suggested that, mainly or partly, the "secondary warm pool" results from the cooperation of the southeast monsoon in eastern Pacific and the eastern boundary hindering the propagation of the Kelvin wave poleward alongshore.

Capitães do Fim

Tese apresentada à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Doutor em Ciências Sociais, especialidade em Sociologia

Comparative immunogenicity of HIV-1 clade C envelope proteins for prime/boost studies.

BACKGROUND: Previous clinical efficacy trials failed to support the continued development of recombinant gp120 (rgp120) as a candidate HIV vaccine. However, the recent RV144 HIV vaccine trial in Thailand showed that a prime/boost immunization strategy involving priming with canarypox vCP1521 followed by boosting with rgp120 could provide significant, although modest, protection from HIV infection. Based on these results, there is renewed interest in the development of rgp120 based antigens for follow up vaccine trials, where this immunization approach can be applied to other cohorts at high risk for HIV infection. Of particular interest are cohorts in Africa, India, and China that are infected with clade C viruses. METHODOLOGY/PRINCIPAL FINDINGS: A panel of 10 clade C rgp120 envelope proteins was expressed in 293 cells, purified by immunoaffinity chromatography, and used to immunize guinea pigs. The resulting sera were collected and analyzed in checkerboard experiments for rgp120 binding, V3 peptide binding, and CD4 blocking activity. Virus neutralization studies were carried out with two different assays and two different panels of clade C viruses. A high degree of cross reactivity against clade C and clade B viruses and viral proteins was observed. Most, but not all of the immunogens tested elicited antibodies that neutralized tier 1 clade B viruses, and some sera neutralized multiple clade C viruses. Immunization with rgp120 from the CN97001 strain of HIV appeared to elicit higher cross neutralizing antibody titers than the other antigens tested. CONCLUSIONS/SIGNIFICANCE: While all of the clade C antigens tested were immunogenic, some were more effective than others in eliciting virus neutralizing antibodies. Neutralization titers did not correlate with rgp120 binding, V3 peptide binding, or CD4 blocking activity. CN97001 rgp120 elicited the highest level of neutralizing antibodies, and should be considered for further HIV vaccine development studies.