NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Functional domains for assembly of histones H3 and H4 into the chromatin of Xenopus embryos

Histones H3 and H4 have a well defined structural role in the nucleosome and an established role in the regulation of transcription. We have made use of a microinjection strategy using Xenopus embryos to define the minimal structural components of H3 and H4 necessary for nucleosome assembly into metazoan chromosomes in vivo. We find that both the N-terminal tail of H4, including all sites of acetylation, and the C-terminal α-helix of the H4 histone fold domain are dispensable for chromatin assembly. The N-terminal tail and an N-terminal α-helix of H3 are also dispensable for chromatin assembly. However, the remainder of the H3 and H4 histone folds are essential for incorporation of these proteins into chromatin. We suggest that elements of the histone fold domain maintain both nucleosomal integrity and have distinct functions essential for cell viability.

Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): Evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry

Homologues of Drosophilia transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca2+ stores. In agreement with this hypothesis, cells expressing TRPs display novel Ca2+-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca2+ entry and to test for a role of their respective binding partners in Ca2+ entry. C7 and an F2q-containing fragment of IP3R decreased both forms of Ca2+ entry. In contrast, F2g enhanced the two forms of Ca2+ entry. We conclude that store depletion-activated Ca2+ entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing Ca2+ from stores and activating Ca2+ influx in response to either increasing IP3 or decreasing luminal Ca2+.

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

An Intronic Enhancer Is Required for Deflagellation-induced Transcriptional Regulation of a Chlamydomonas reinhardtii Dynein Gene

Chlamydomonas reinhardtii flagellar regeneration is accompanied by rapid induction of genes encoding a large set of flagellar structural components and provides a model system to study coordinate gene regulation and organelle assembly. After deflagellation, the abundance of a 70-kDa flagellar dynein intermediate chain (IC70, encoded by ODA6) mRNA increases approximately fourfold within 40 min and returns to predeflagellation levels by ∼90 min. We show by nuclear run-on that this increase results, in part, from increased rates of transcription. To localize cis induction elements, we created an IC70 minigene and measured accumulation, in C. reinhardtii, of transcripts from the endogenous gene and from introduced promoter deletion constructs. Clones containing 416 base pairs (bp) of 5′- and 2 kilobases (kb) of 3′-flanking region retained all sequences necessary for a normal pattern of mRNA abundance change after deflagellation. Extensive 5′- and 3′- flanking region deletions, which removed multiple copies of a proposed deflagellation-response element (the tub box), did not eliminate induction, and the IC70 5′-flanking region alone did not confer deflagellation responsiveness to a promoterless arylsulfatase (ARS) gene. Instead, an intron in the IC70 gene 5′-untranslated region was found to contain the deflagellation response element. These results suggest that the tub box does not play an essential role in deflagellation-induced transcriptional regulation of this dynein gene.

Adaptor Complex-independent Clathrin Function in Yeast

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and α-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or α-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the β subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

The roles of specific xanthophylls in photoprotection

Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the α- or β-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (α-carotene branch), zeaxanthin, and antheraxanthin (β-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), α-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.

Subdivisions of auditory cortex and processing streams in primates

The auditory system of monkeys includes a large number of interconnected subcortical nuclei and cortical areas. At subcortical levels, the structural components of the auditory system of monkeys resemble those of nonprimates, but the organization at cortical levels is different. In monkeys, the ventral nucleus of the medial geniculate complex projects in parallel to a core of three primary-like auditory areas, AI, R, and RT, constituting the first stage of cortical processing. These areas interconnect and project to the homotopic and other locations in the opposite cerebral hemisphere and to a surrounding array of eight proposed belt areas as a second stage of cortical processing. The belt areas in turn project in overlapping patterns to a lateral parabelt region with at least rostral and caudal subdivisions as a third stage of cortical processing. The divisions of the parabelt distribute to adjoining auditory and multimodal regions of the temporal lobe and to four functionally distinct regions of the frontal lobe. Histochemically, chimpanzees and humans have an auditory core that closely resembles that of monkeys. The challenge for future researchers is to understand how this complex system in monkeys analyzes and utilizes auditory information.

Sex and the single cell: meiosis in yeast.

Recent studies of Saccharomyces cerevisiae have significantly advanced our understanding of the molecular mechanisms of meiotic chromosome behavior. Structural components of the synaptonemal complex have been identified and studies of mutants defective in synapsis have provided insight into the role of the synaptonemal complex in homolog pairing, genetic recombination, crossover interference, and meiotic chromosome segregation. There is compelling evidence that most or all meiotic recombination events initiate with double-strand breaks. Several intermediates in the double-strand break repair pathway have been characterized and mutants blocked at different steps in the pathway have been identified. With the application of genetic, molecular, cytological, and biochemical methods in a single organism, we can expect an increasingly comprehensive and unified view of the meiotic process.

Abnormal junctions between surface membrane and sarcoplasmic reticulum in skeletal muscle with a mutation targeted to the ryanodine receptor.

Junctions that mediate excitation-contraction (e-c) coupling are formed between the sarcoplasmic reticulum (SR) and either the surface membrane or the transverse (T) tubules in normal skeletal muscle. Two structural components of the junctions, the feet of the SR and the tetrads of T tubules, have been identified respectively as ryanodine receptors (RyRs, or SR calcium-release channels), and as groups of four dihydropyridine receptors (DHPRs, or voltage sensors of e-c coupling). A targeted mutation (skrrm1) of the gene for skeletal muscle RyRs in mice results in the absence of e-c coupling in homozygous offspring of transgenic parents. The mutant gene is expected to produce no functional RyRs, and we have named the mutant mice "dyspedic" because they lack feet--the cytoplasmic domain of RyRs anchored in the SR membrane. We have examined the development of junctions in skeletal muscle fibers from normal and dyspedic embryos. Surprisingly, despite the absence of RyRs, junctions are formed in dyspedic myotubes, but the junctional gap between the SR and T tubule is narrow, presumably because the feet are missing. Tetrads are also absent from these junctions. The results confirm the identity of RyRs and feet and a major role for RyRs and tetrads in e-c coupling. Since junctions form in the absence of feet and tetrads, coupling of SR to surface membrane and T tubules appears to be mediated by additional proteins, distinct from either RyRs or DHPRs.

Acesso ao tratamento oncológico no município de São Paulo: o câncer de mama como condição traçadora

Acompanhando a tendência mundial, o Brasil apresenta um processo de envelhecimento de sua população, caracterizado pelo aumento das condições crônicas, inclusive do câncer. O quadro convoca mudanças profundas nos sistemas de saúde, demandando a implantação de Redes de Atenção, a fim de garantir acesso a todos os níveis de atenção, superando a fragmentação do cuidado. Com o intuito de conhecer os avanços no que se refere à atenção oncológica em rede, analisou-se o acesso ao tratamento do câncer em São Paulo, especialmente a partir do surgimento da Lei dos sessenta dias. Foram considerados os sistemas de monitoramento da atenção oncológica no município, além de analisados os itinerários assistenciais de usuárias, utilizando o câncer de mama como condição traçadora. Não foi possível identificar uma redução do tempo de espera para iniciar o tratamento, a partir do banco do Registro Hospitalar de Câncer de são Paulo, considerando que não há completude na base a partir de 2013, sendo observado que o tempo indicado na lei foi ultrapassado nos dois anos anteriores. Da mesma forma, notou-se um aumento da proporção de estádios avançados nesse período. Ainda com relação à variável tempo, as informações no SIGA demonstraram que, em 2013, o tempo médio para uma consulta em Onco-mastologia nos serviços de gestão municipal que estão sob regulação foi de apenas 4 dias. Por meio dos Sistemas de Informação Ambulatorial e Hospitalar, observou-se um aumento estatisticamente significativo na produção de radioterapia e de cirurgias oncológicas entre os anos 2011 e 2014, e uma tendência de redução dos procedimentos quimioterápicos. O Sistema de Informação sobre Câncer de Mama demonstrou aumento no percentual de mamografias alteradas, aspecto que, ao ser analisado em conjunto com o aumento da proporção de estadiamentos avançados, pode ser indicativo de maior dificuldade no acesso ao diagnóstico precoce do câncer de mama. Observou-se que a judicialização esteve muito relacionada a acesso a medicamentos quimioterápicos, de prescrição após a entrada nos serviços especializados, o que confirma que o acesso ao tratamento de câncer de mama no município não apresenta grandes barreiras. Um importante efeito visualizado com o surgimento da Lei foi a padronização dos protocolos de acesso aos serviços de gestão municipal e estadual. Entretanto, a rede de oncologia em São Paulo continua fragmentada dentre seus componentes estruturais, as ações permanecem no plano da construção de fluxos de encaminhamento, ficando restrita à atenção especializada. A atenção oncológica na cidade é atravessada pelo setor privado, o que deixa na dependência dos prestadores a disponibilização de vagas para acesso e o fluxo interno de cada serviço. O poder ainda continua com os grandes prestadores, não sendo bem conhecidos os caminhos para o acesso a algumas instituições, nem publicizadas as informações sobre fila e tempo de espera. A legislação sozinha não é indutora de melhoria de acesso, nem muito menos de garantia de integralidade. Um importante desafio para o SUS é a integração dos serviços e a construção de redes de atenção com centralidade na APS, garantindo, acima de tudo, o diagnóstico em tempo oportuno e a efetiva gestão sobre os serviços privados contratados de média e alta complexidade.

Método de Rorscharch em adultos: evidências psicométricas da Escola Francesa

O Método de Rorschach é internacionalmente utilizado e reconhecido como instrumento de avaliação psicológica em processos de investigação da personalidade. Para embasar seu adequado uso no contexto do Brasil, contínuos estudos sobre suas evidências psicométricas fazem-se necessários, sobretudo sobre seus indicadores de validade, precisão e referenciais normativos. Nesse contexto, este trabalho objetivou: a) verificar indicadores de precisão e validade do Rorschach (Escola Francesa ou de Paris), por meio da reavaliação de adultos não pacientes do estudo normativo de Pasian (1998), para checagem da estabilidade temporal (estrutural e funcional) das características de personalidade examinadas pelo método (ESTUDO 1); b) revisar e atualizar padrões normativos e atlas da Escola Francesa do Rorschach em adultos, avaliando-se eventuais especificidades de produção associadas ao sexo, à idade e à escolaridade (ESTUDO 2). No primeiro estudo foram reavaliados 88 adultos do estudo de Pasian (1998), com idade entre 34 a 69 anos, de ambos os sexos e de diferentes níveis de escolaridade (baixa, média e alta). No segundo estudo compôs-se nova amostra com 102 adultos, entre 18 e 65 anos, com sinais de desenvolvimento típico, distribuídos equitativamente em relação ao sexo e com diferentes graus de escolaridade (baixa, média e alta). Para elaboração dos dados normativos foram incluídos 66 adultos do Estudo 1, totalizando 168 casos nessa nova amostra (86 mulheres e 82 homens). Os participantes residiam no interior do Estado de São Paulo e foram avaliados, individualmente, por: a) instrumento de rastreamento de saúde mental (SRQ-20) - no estudo 1, utilizado para caracterização da amostra e no estudo 2 como critério de seleção dos participantes; b) instrumento de avaliação intelectual (Teste de Inteligência Não Verbal - INV, forma C - apenas no Estudo 2) para controle cognitivo da amostra; c) critério de classificação econômica Brasil (ABEP) e d) Método de Rorschach (Escola de Paris). Cada instrumento de avaliação psicológica foi aplicado, codificado e sistematizado conforme seus respectivos manuais técnicos. Especificamente o Método de Rorschach foi avaliado pelas diretrizes da Escola Francesa, sendo cada protocolo (de cada um dos dois estudos) examinado independentemente por dois avaliadores, chegando-se a uma classificação final dos casos. Calculou-se o índice de concordância entre examinadores pelo coeficiente Kappa para as quatro categorias de classificação das respostas do Rorschach (localização, determinante/qualidade formal, conteúdo e banalidades). Os resultados foram sistematizados inicialmente em termos descritivos (média, desvio-padrão, mediana, valor mínimo e máximo), realizando-se análises inferenciais específicas para as amostras de cada estudo em função de seus objetivos centrais. No Estudo 1, os achados relativos aos índices de correlação entre as duas avaliações dos 88 voluntários variaram entre 0,72 a -0,005, evidenciando estabilidade em um conjunto das características de personalidade dos adultos examinados após 15 anos, configurando evidências empíricas de precisão e de validade de método projetivo. Houve variáveis do Rorschach que não apresentaram resultados estáveis entre os dois momentos avaliativos (fórmulas vivenciais), sugerindo se tratarem de indicadores técnicos relativos ao funcionamento da personalidade, mais do que componentes estruturais. As análises do Estudo 2 apontaram reduzida influência dos fatores relacionados ao sexo, à escolaridade e à idade sobre as variáveis do Rorschach, não indicando a necessidade de normas específicas para grupos em termos desses fatores. Por fim, foi elaborado novo atlas de referência do Método de Rorschach (Escola de Paris) no contexto brasileiro, seguindo-se as diretrizes técnicocientíficas nacionais e internacionais da área. Os dados fortalecem a relevância dos estudos de natureza psicométrica para embasar adequadas análises interpretativas desse instrumento de avaliação psicológica (FAPESP e CAPES/PDSE).

Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics

Background: Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Results: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Conclusions: Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS’s mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.

Short peptide alpha helices induced by multiple metal clips

Alpha helices are key structural components of proteins and important recognition motifs in biology. New techniques for stabilizing short peptide helices could be valuable for studying protein folding, modeling proteins, creating artificial proteins, and may aid the design of inhibitors or mimics of protein function. We previously reported* that 5-15 residue peptides, corresponding to the Zn-binding domain of thermolysin, react with [Pd(en)(ONO,),]in DMF-d’ and 90% H,O 10% DzO to form a 22-membered [Pd(en)(H*ELTH*)]2+ macrocycle that is helical in solution and acts as a template in nucleating helicity in both Cand N- terminal directions within the longer sequences in DMF. ~f~~&g7$$& d&qx~m ~. y AC&q& In water, however, there was less a-helicity observed, testifying to #..q,& &$--Lb &l-- &.$;,J~p?:~~q&~+~~ ’ w w the difficulty of fixing intramolecular amide NH...OC H-bonds in 6,“;;” ( k.$ U”C.a , p d$. competition with the H-bond donor solvent water. To expand the utility of [Pd(en)(H*XXXH*)]*+ as a helix- @r4”8 & oJ#:& &G& @-qd ,‘d@-gyp promoting module in solution, we now report the result that Ac- ‘$4: %$yyy + H*ELTH*H*VTDH*-NH,(l), AC-H*ELTH*AVTDYH*ELTH*- NH, (2) and AC-H*AAAH*H*ELTH*H*VTDH*-NH* (3) react with multiple equivalents of [Pd(en)(ONO,),] to produce exclusively 4-6 respectively in both DMF-d7 and water (90% Hz0 10% D,O). Mass spectrometry, 15N- and 2D ‘H- NMR spectroscopy, and CD spectra were used to characterise the structures 4-6, and their three dimensional structures were calculated from NOE restraints using simulated annealing protocols. Results demonstrate (a) selective coordination of metal ions at (i, i+4) histidine positions in water and DMF, (b) incorporation of 2 and 3 a turn-mimicking modules [Pd(en)(HELTH)]2+ in lo-15 residue peptides, and (c) facile conversion of unstructured peptides into 3- and 4- turn helices of macrocycles, with well defined a-helicity throughout and more structure in DMF than in water.

Semi-analytical solution for nonlinear vibration of laminated FGM plates with geometric imperfections

This paper investigates the nonlinear vibration of imperfect shear deformable laminated rectangular plates comprising a homogeneous substrate and two layers of functionally graded materials (FGMs). A theoretical formulation based on Reddy's higher-order shear deformation plate theory is presented in terms of deflection, mid-plane rotations, and the stress function. A semi-analytical method, which makes use of the one-dimensional differential quadrature method, the Galerkin technique, and an iteration process, is used to obtain the vibration frequencies for plates with various boundary conditions. Material properties are assumed to be temperature-dependent. Special attention is given to the effects of sine type imperfection, localized imperfection, and global imperfection on linear and nonlinear vibration behavior. Numerical results are presented in both dimensionless tabular and graphical forms for laminated plates with graded silicon nitride/stainless steel layers. It is shown that the vibration frequencies are very much dependent on the vibration amplitude and the imperfection mode and its magnitude. While most of the imperfect laminated plates show the well-known hard-spring vibration, those with free edges can display soft-spring vibration behavior at certain imperfection levels. The influences of material composition, temperature-dependence of material properties and side-to-thickness ratio are also discussed. (C) 2004 Elsevier Ltd. All rights reserved.