RESUMO
STUDY DESIGN: Prospective experimental. OBJECTIVES: To compare the reliability and repeatability of the electrical perceptual threshold (EPT) and Semmes-Weinstein monofilament (SWM) tests for cutaneous sensibility. EPT and SWM tests have potential as quantitative and sensitive adjuncts to the American Spinal Injuries Association (ASIA) Impairment Scale (AIS) assessment of spinal cord injury (SCI). SETTING: London, UK. METHODS: EPT and SWM tests were carried out on 40 neurologically healthy individuals (20 male). One examiner carried out all the tests. Each individual was examined for EPT and SWM sensitivity at ASIA key points on four dermatomes (C4, T1, T6, L4) on both sides of the body. The tests were repeated after an interval of approximately 1 week. Intra-rater reliability was determined using intra-class correlation coefficients (ICC). Repeatability was determined using the method of Bland and Altman. RESULTS: There were no significant differences in the mean values of EPT or SWM between assessments for any dermatome. Significant difference in mean values for both EPT and SWM were observed between some dermatomes. ICC ranged from 0.67 to 0.81 for the EPT and from 0.46 to 0.61 for the SWM. Higher ICC for the EPT compared with the SWM was again revealed when male and female subjects were assessed separately. Correlation between EPT and SWM was weak or (largely) absent. CONCLUSION: EPT has better reliability than SWM in healthy subjects. However, as both tests have the potential to add sensitivity and resolution to the AIS assessment, a further comparison of their repeatability in SCI is warranted.
Assuntos
Exame Neurológico/métodos , Limiar Sensorial , Traumatismos da Medula Espinal/diagnóstico , Estimulação Elétrica/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estimulação Física/métodos , Reprodutibilidade dos Testes , Traumatismos da Medula Espinal/complicações , Tato/fisiologia , Adulto JovemRESUMO
BACKGROUND AND PURPOSE: Subtle changes in the intracellular reduction-oxidation (redox) state can modulate nuclear factor-kappaB (NF-kappaB) activity. Thioredoxin-1 (Trx) is a small, ubiquitous, redox-active thiol (-SH) protein that, with thioredoxin reductase-1 (TrxR), modifies the redox status of NF-kappaB pathway components. PMX464 is a novel thiol-reactive quinol thought to inhibit the Trx/TrxR system. The aim of this work was to investigate whether PMX464 inhibited NF-kappaB-mediated proinflammatory activation of human type II alveolar epithelial cells (A549). EXPERIMENTAL APPROACH: Intercellular adhesion molecule-1 (ICAM-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and CXCL8, NF-kappaB DNA binding, nuclear translocation of NF-kappaB p65 subunit, IkappaBalpha degradation, IkappaB phosphorylation and IkappaB kinase (IKK) activity were assessed in A549 cells stimulated with IL-1beta with or without PMX464 pretreatment. Effects of PMX464 on ICAM-1 expression in human lung microvascular endothelial cells (HLMVEC) were also investigated. For comparison, selected measurements (ICAM-1 and IkappaB-alpha phospho-IkappaB-alpha) were made on A549 cells after RNA interference-mediated silencing (siRNA) of Trx. KEY RESULTS: PMX464 reduced ICAM-1, GM-CSF and CXCL8 expression in IL-1beta-stimulated A549 cells and ICAM-1 in HLMVEC. PMX464 inhibited IL-1beta-induced NF-kappaB DNA binding, nuclear translocation of NF-kappaB p65 subunit and factors involved in NF-kappaB activation; specifically, IkappaBalpha degradation, IkappaB phosphorylation and IkappaB kinase (IKK) activity in A549. By contrast, Trx siRNA did not alter ICAM-1 expression or IkappaBalpha degradation/phosphorylation in IL-1beta-stimulated A549 cells. CONCLUSION AND IMPLICATIONS: PMX464 inhibits a proinflammatory response in A549 cells targeting the NFkappaB pathway above IKK. The lack of effect with Trx siRNA suggests that PMX464 acts on thiol proteins, in addition to Trx, to elicit anti-inflammatory responses in lung epithelial cells.
Assuntos
Benzotiazóis/farmacologia , Cicloexanonas/farmacologia , Células Epiteliais , Hidroquinonas/farmacologia , NF-kappa B/metabolismo , Alvéolos Pulmonares , Tiorredoxinas/antagonistas & inibidores , Animais , Benzotiazóis/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cicloexanonas/química , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Hidroquinonas/química , Immunoblotting , Microscopia Confocal , Neutrófilos/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , RNA Interferente Pequeno/farmacologia , Tiorredoxinas/genéticaRESUMO
OBJECTIVE: The clinical and functional assessment of back muscles in human spinal cord injury (SCI) has received little attention. The aim of this study was to develop a method to assess the level of a thoracic spinal cord lesion based on the reflex activation of back muscles. METHODS: In 11 control subjects and in 12 subjects with clinically complete thoracic SCI (T2-T12), either a spinous process or an erector spinae muscle was prodded to elicit short latency reflexes recorded electromyographically at the spinal level of stimulation. An electromagnetic servo, attached to a blunt probe, applied stimuli at a frequency of 1 Hz and amplitude of 3 mm. Two trials of 50 mechanical prods were conducted at each site. RESULTS: Reflexes were evoked in control subjects in 82% of trials when the spinous process was prodded, and in 80% of trials when the muscle was prodded. In contrast, reflexes in SCI subjects could be elicited in 90-100% of trials two segments either above or below the lesion. Reflex responses in control subjects had a mean (SEM) latency of 5.72 (0.53) ms when the spinous process was prodded, and 5.42 (0.42) ms when the muscle was prodded. In the SCI subjects, responses had slightly (but insignificantly) longer latencies both above and below the lesion to either stimulus. The amplitude of reflex responses, expressed as a percentage of the background EMG, was on average 2-3 times larger at the three vertebral levels spanning the lesion in SCI subjects than at sites above or below the lesion or at any level in control subjects. CONCLUSION: We propose that the size of these mechanically evoked reflexes may be useful in determining the level of thoracic SCI. Furthermore, the reflexes might provide a valuable tool with which to monitor recovery after an intervention to repair or improve function of a damaged spinal cord.
Assuntos
Mecanorreceptores/fisiologia , Neurônios Motores/fisiologia , Músculo Esquelético/fisiologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/patologia , Adulto , Dorso/inervação , Dorso/fisiologia , Estudos de Casos e Controles , Eletromiografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/inervação , ReflexoRESUMO
Glucose- and caffeine-containing energy drinks are said to influence the cognitive and cellular function within the brain. In this study, we have used the size of motor-evoked potentials (MEPs) produced in response to transcranial magnetic stimulation (TMS) of the motor cortex as an index of corticospinal excitability after ingestion of Lucozade and control drinks of glucose-containing or caffeine-containing carbonated water or carbonated water alone. With local ethical approval and informed consent, 10 healthy volunteers took part; surface electromyographic (EMG) recordings were taken from the thenar muscles of the dominant hand. In each assessment, 15 TMS stimuli were delivered over the motor cortex at an intensity of 1.1 T. Six subjects ingested a 380-ml bottle of carbonated Lucozade drink containing 68 g of glucose and 46 mg caffeine. Four subjects took part in three control trials drinking: (A) carbonated water with caffeine, (B) carbonated water with glucose and (C) carbonated water alone. Assessments were made before and at 30-min intervals after each drink. Mean fasting blood glucose concentrations and mean areas of MEPs rose after the Lucozade, remaining elevated for 90 min. Similar rises in MEP areas were seen in trials after drinking carbonated water with caffeine or with glucose, but not after drinking carbonated water alone. No change was seen in the M-wave evoked by electrical stimulation of the ulnar nerve. We conclude that Lucozade can affect the size of MEPs to activation of the motor cortex with fixed-intensity TMS. The underlying mechanism is likely to relate to the combined effects of caffeine and glucose on the brain.
Assuntos
Bebidas , Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Glucose/farmacologia , Medula Espinal/efeitos dos fármacos , Adulto , Glicemia/metabolismo , Córtex Cerebral/fisiologia , Estimulação Elétrica , Campos Eletromagnéticos , Eletromiografia , Potencial Evocado Motor/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Motor/efeitos dos fármacos , Córtex Motor/fisiologia , Nervo Ulnar/fisiologiaRESUMO
STUDY DESIGN: Preliminary longitudinal clinical trial. OBJECTIVES: To test the efficacy of repetitive transcranial magnetic stimulation (rTMS) in modulating corticospinal inhibition and improving recovery in stable incomplete spinal cord injury (iSCI). SETTING: National Spinal Injuries Centre, Stoke Mandeville Hospital, Bucks, UK and Division of Neuroscience, Imperial College Faculty of Medicine, Charing Cross Hospital, London, UK. METHODS: Four stable iSCI patients were treated with rTMS over the occipital cortex (sham treatment) and then over the motor cortex (real treatment). Patients were assessed using electrophysiological, clinical and functional measures before treatment, during sham treatment, during the therapeutic treatment and during a 3-week follow-up period. RESULTS: Cortical inhibition was reduced during the treatment week. Perceptual threshold to electrical stimulation of the skin, ASIA clinical measures of motor and sensory function and time to complete a peg-board improved and remained improved into the follow-up period. CONCLUSION: In this preliminary trial, rTMS has been shown to alter cortical inhibition in iSCI and improve the clinical and functional outcome. SPONSORSHIP: This work was supported by the International Spinal Research Trust.
Assuntos
Terapia por Estimulação Elétrica , Magnetismo , Inibição Neural/fisiologia , Traumatismos da Medula Espinal/reabilitação , Adulto , Eletromiografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Atividade Motora/fisiologia , Limiar da Dor/fisiologia , Resultado do TratamentoRESUMO
Clinical practice and scientific research may soon lead to treatments designed to repair spinal cord injury. Repair is likely to be partial in the first trials, extending only one or two segments below the original injury. Furthermore, treatments that are becoming available are likely to be applied to the thoracic spinal cord to minimise loss of function resulting from damage to surviving connections. These provisos have prompted research into the improvement of clinical and physiological tests designed (1) to determine the level and density of a spinal cord injury, (2) to provide reliable monitoring of recovery over one or two spinal cord segments, and (3) to provide indices of function provided by thoracic spinal root innervation, presently largely ignored in assessment of spinal cord injury. This article reviews progress of the Clinical Initiative, sponsored by the International Spinal Research Trust, to advance the clinical and physiological tests of sensory, motor and autonomic function needed to achieve these aims.
Assuntos
Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Eletrofisiologia , Potencial Evocado Motor , Potenciais Somatossensoriais Evocados , Humanos , Músculo Esquelético/inervaçãoRESUMO
Chronic fatigue syndrome (CFS) is characterised by fatigue and musculosketetal pain, the severity of which is variable. Simple reaction times (SRTs) and movement times (SMTs) are slowed in CFS. Our objective is to correlate the day-to-day changes in symptomatology with any change in SRT, SMT or corticospinal excitability. Ten CFS patients were tested on two occasions up to two years apart. Motor evoked potentials (MEPs) to transcranial magnetic stimulation (TMS) of the motor cortex were recorded from the thenar muscles. Threshold TMS strength to evoke MEPs was measured to index corticospinal excitability. SRTs and SMTs were measured. The percentage change in both SRTs and SMTs between the two test sessions correlated with the percentage change in corticospinal excitability assessed according to threshold TMS intensity required to produce MEPs. This study provides evidence that changing motor deficits in CFS have a neurophysiological basis. The slowness of SRTs supports the notion of a deficit in motor preparatory areas of the brain.
Assuntos
Potencial Evocado Motor , Síndrome de Fadiga Crônica/fisiopatologia , Atividade Motora/fisiologia , Tempo de Reação/fisiologia , Adulto , Eletromiografia/métodos , Feminino , Humanos , Modelos Lineares , Magnetismo , Masculino , Pessoa de Meia-Idade , Córtex Motor/fisiopatologia , Movimento/fisiologia , Desempenho Psicomotor , Medula Espinal/fisiopatologiaRESUMO
OBJECTIVES: The sympathetic skin response (SSR) is a technique to assess the sympathetic cholinergic pathways, and it can be used to study the central sympathetic pathways in spinal cord injury (SCI). This study investigated the capacity of the isolated spinal cord to generate an SSR, and determined the relation between SSR, levels of spinal cord lesion, and supraspinal connections. METHODS: Palmar and plantar SSR to peripheral nerve electrical stimulation (median or supraorbital nerve above the lesion, and peroneal nerve below the lesion) were recorded in 29 patients with SCI at various neurological levels and in 10 healthy control subjects. RESULTS: In complete SCI at any neurological level, SSR was absent below the lesion. Palmar SSR to median nerve stimuli was absent in complete SCI with level of lesion above T6. Plantar SSR was absent in all patients with complete SCI at the cervical and thoracic level. In incomplete SCI, the occurrence of SSR was dependent on the preservation of supraspinal connections. For all stimulated nerves, there was no difference between recording from ipsilateral and contralateral limbs. CONCLUSIONS: No evidence was found to support the hypothesis that the spinal cord isolated from the brain stem could generate an SSR. The results indicate that supraspinal connections are necessary for the SSR, together with integrity of central sympathetic pathways of the upper thoracic segments for palmar SSR, and possibly all thoracic segments for plantar SSR.
Assuntos
Resposta Galvânica da Pele/fisiologia , Pele/inervação , Traumatismos da Medula Espinal/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Adulto , Tronco Encefálico/fisiopatologia , Estimulação Elétrica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Exame Neurológico , Nervos Periféricos/fisiopatologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/diagnósticoRESUMO
OBJECTIVES: To study the effect of repeating electrical peripheral nerve stimulation on latency, duration and amplitude of the sympathetic skin response (SSR). METHODS: SSRs were elicited in all limbs by median and peroneal nerves stimuli. In 10 subjects, 20 stimuli were applied at random time intervals (15-20 s). Another test was performed in 7 subjects using the same protocol, but switching the stimulation site every 5 or 10 stimuli without warning. RESULTS: The mean amplitude of right palmar response to right peroneal nerve stimulation decreased from 5.05+/-0.76 (SEM) mV at the first stimulus to 1.23+/-0.42 mV at the 20th stimulus (P<0.001). The latency did not change significantly (1473+/-82 to 1550+/-90 ms, P>0.1), while the duration increased (1872+/-356 to 3170+/-681 ms, P<0.001). Stimulation and recording at other sites showed similar trends. Changing the stimulation site failed to alter the adaptation process in terms of amplitude, latency or duration. CONCLUSIONS: Changes in amplitude and duration of the SSRs to repeated electrical stimuli can occur in presence of constant latency and appear to be independent of the source of sensory input. Peripheral sweat gland mechanisms may be involved in the loss of amplitude and increase in duration of the SSR during habituation.
Assuntos
Potenciais Evocados/fisiologia , Habituação Psicofisiológica/fisiologia , Nervo Mediano/fisiologia , Nervo Fibular/fisiologia , Pele/inervação , Sistema Nervoso Simpático/fisiologia , Adulto , Estimulação Elétrica , Eletrocardiografia , Feminino , Humanos , Recém-Nascido , Masculino , Metacarpo/fisiologia , Metatarso/fisiologia , Pessoa de Meia-Idade , Articulação do PunhoRESUMO
We have assessed corticospinal function in 19 post-coma patients severely brain-injured by anoxia or physical trauma. Eleven patients were unresponsive (Category 1) and eight demonstrated minimal, non-verbal responses to simple commands (Category 2). Motor evoked potentials (MEPs) could be elicited in hand and leg muscles in nine Category 1 and all eight Category 2 patients in response to transcranial magnetic stimulation (TMS). In comparison with normal subjects, threshold to TMS was significantly elevated in Category 1 but not in Category 2. Central conduction times were within the normal range except for two patients (one in each category) in whom they were prolonged. The variability in MEP amplitude to constant TMS was not significantly different from normal in either category. The size of MEPs recorded simultaneously in different hand muscles were correlated in all three groups. The presence of H-reflexes in hand muscles was associated with an absence of MEPs or a high threshold to TMS. Variability of MEPs was substantially greater than that of H-reflexes. We conclude that brain injury of a severity that may preclude consciousness and voluntary movement does not invariably predicate a non-functional motor cortex and corticospinal system.
Assuntos
Lesões Encefálicas/fisiopatologia , Córtex Motor/fisiopatologia , Tratos Piramidais/fisiopatologia , Adulto , Limiar Diferencial/fisiologia , Potencial Evocado Motor/fisiologia , Feminino , Reflexo H/fisiologia , Humanos , Magnetismo , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Condução Nervosa/fisiologia , Estimulação Física , Fatores de TempoRESUMO
The IkappaB kinases (IKKs) lie downstream of the NF-kappaB-inducing kinase (NIK) and activate NF-kappaB by phosphorylation of IkappaBalpha. This leads to IkappaBalpha degradation and release of NF-kappaB. In U937 monocytic cells, interleukin (IL)-1beta (1 ng/ml) and tumor necrosis factor (TNF)-alpha; 10 ng/ml) induced kappaB-dependent transcription equally. However, IKK activity was strongly induced by TNF-alpha but not by IL-1beta. This was consistent with IkappaBalpha phosphorylation and degradation, yet TNF-alpha-induced NF-kappaB DNA binding was only 30-40% greater than for IL-1beta. This was not explained by degradation of IkappaBbeta, IkappaBepsilon, or p105 nor nuclear translocation of NF-kappaB. IkappaBalpha complexes or degradation-independent release of NF-kappaB. Dominant negative (NIK) repressed TNF-alpha and IL-1beta-induced kappaB-dependent transcription by approximately 60% and approximately 35%, respectively. These data reveal an imprecise relationship between IKK activation, IkappaBalpha degradation, and NF-kappaB DNA binding, suggesting the existence of additional mechanisms that regulate NF-kappaB activation. Finally, the lack of correlation between DNA binding and transcriptional activation plus the fact that PP1 and genistein both inhibited kappaB-dependent transcription without affecting DNA binding activity demonstrate the existence of regulatory steps downstream of NF-kappaB DNA binding. Therapeutically these data are important as inhibition of the NIK-IKK-IkappaBalpha cascade may not produce equivalent reductions in NF-kappaB-dependent gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , Interleucina-1/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ativação Enzimática , Regulação da Expressão Gênica , Genes Reporter , Genisteína/farmacologia , Humanos , Quinase I-kappa B , Leupeptinas/farmacologia , Ligases/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Fosforilação , Pirazóis/farmacologia , Pirimidinas/farmacologia , Células U937RESUMO
Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme unique to photosynthetic organisms and has a key role in regulating the photosynthetic Calvin cycle through which nearly all carbon enters the biosphere. This makes SBPase an appropriate target for intensive study. We have expressed wheat SBPase in Escherichia coli either with or without an N-terminal polyhistidine tag. The identity of the recombinant SBPases was confirmed by SDS-PAGE analysis and immunological detection with a specific antibody. Recombinant SBPase with a polyhistidine tag (His-SBPase) was obtained in soluble, active form and purified by one-step metal-chelate chromatography. Like the native enzyme, recombinant His-SBPase was specific for the substrate sedoheptulose-1,7-bisphosphate and required the presence of a reducing agent for activity. Polyclonal antibodies were raised against recombinant SBPase and were then used to determine relative levels of the enzyme in plant extracts. The availability of large amounts of active recombinant SBPase will also allow detailed structural studies by site-directed mutagenesis and X-ray crystallography.
Assuntos
Cloroplastos/enzimologia , Escherichia coli/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos , Sequência de Bases , Cromatografia de Afinidade , DNA de Plantas/genética , Expressão Gênica , Monoéster Fosfórico Hidrolases/metabolismo , Plasmídeos/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Especificidade por Substrato , Fosfatos Açúcares , Triticum/enzimologia , Triticum/genéticaRESUMO
The large raspberry aphid, Amphorophora idaei, has several biotypes described by their abilities to overcome plant resistance genes. Bioassays of field populations showed a strong shift towards A1 resistance-breaking biotypes since the 1960s. RFLP analysis of the rDNA cistron was used to study variation found within and between standard clones of three A. idaei biotypes and twenty-nine field populations collected over 3 years. Probing genomic DNA with the ribosomal DNA probe pBG 35 produced consistent differences in RFLPs between standard clones of biotypes. However, analysis of field populations gave more complex RFLP patterns that were not biotype-specific, unlike characteristic intergenic spacer (IGS) patterns reported for Schizaphis graminum biotypes. All but one sample collected from separate fields showed considerable genetic diversity within populations, attributed to alate migrations of parthenogenetic females in summer and males in autumn.
Assuntos
Afídeos/genética , DNA Ribossômico/genética , Animais , Afídeos/classificação , Sequência de Bases , Sondas de DNA/análise , Sondas de DNA/genética , Feminino , Genes de Insetos , Genes de Plantas/genética , Marcadores Genéticos , Variação Genética , Masculino , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Especificidade da EspécieRESUMO
Discharges of gamma motoneurones were recorded from cut filaments of the nerve to the gastrocnemius medialis muscle in the cat decerebrated at an intercollicular level. Gamma motoneurones exhibited a background discharge in the absence of intentional stimulation, or could be made to discharge by continuous, innocuous stimulation of the skin of the heel. The discharges were periodic and regular (low coefficient of variation of interspike intervals), and no correlation was observed between the discharges of pairs of individual gamma efferents. Electrolytic lesion of the ipsilateral pontine and medullary reticular formation in the nucleus subcoeruleus, the nucleus reticularis gigantocellularis or the nucleus reticularis magnocellularis, invariably decreased regularity of discharge and resulted in short term synchrony. Lesions of the peri-aqueductal grey, the nucleus raphe dorsalis or the midline raphe nuclei did not induce synchrony. Surgical lesions in the locus coeruleus caused irregular firing and synchrony only when the lesion extended into the adjacent nucleus subcoeruleus. We conclude that monoaminergic neurones of the nucleus subcoeruleus, or a closely associated tegmental field, with axons descending through the gigantocellularis and magnocellularis fields, are the most likely origin of the bulbospinal control of synchronizing influences on gamma motoneurone discharge.
Assuntos
Tronco Encefálico/fisiologia , Locus Cerúleo/fisiologia , Bulbo/fisiologia , Neurônios Motores/fisiologia , Ponte/fisiologia , Núcleos da Rafe/fisiologia , Formação Reticular/fisiologia , Animais , Axônios/fisiologia , Mapeamento Encefálico , Gatos , Cerebelo/fisiologia , Estado de Descerebração , Eletrofisiologia/métodos , Músculos/inervação , Condução Nervosa/fisiologia , Fatores de TempoRESUMO
The accumulation of amyloplast DNA during endosperm development was studied in two cultivars of spring wheat, Triticum aestivum L. 'Chinese Spring' (CS) and 'Spica', small and relatively larger-grained cultivars, respectively. Endosperms were isolated between 9 and 45 days post anthesis (dpa) and the amyloplast DNA content of endosperm nucleic-acid extracts was measured by quantitative hybridisation with a homologous chloroplast-DNA probe. The endosperm cells of CS and Spica accumulated amyloplast DNA during development in a similar way. In both cultivars there was a large increase in the amount of plastid DNA (ptDNA) per endosperm between 9 and about 15 dpa, after which there was no further increase. Because nuclear DNA continued to accumulate until 24 dpa, the percentage contribution of amyloplast DNA to total DNA fluctuated in both cultivars during development, reaching maxima at 12 dpa of about 1.00% and 0.85%, and dropping to apparently constant levels of 0.60% and 0.52% in CS and Spica, respectively, by 24 dpa. In both cultivars, the average number of ptDNA copies per amyloplast was calculated to increase from about 10 copies at 9 dpa to about 50 copies in the mature amyloplasts at 31 dpa. However, the heavier endosperms of Spica contain more cells than those of CS and the varieties therefore differed in the amount of ptDNA that accumulated per endosperm: Spica endosperms accumulated 110 ng of ptDNA by 15 dpa, compared with only 85 ng in CS. The apparent accumulation of ptDNA copies in wheat amyloplasts during endosperm development contrasts with the decline in chloroplast-DNA copies in wheat chloroplasts during leaf development.