RESUMO
A direct reverse transcription-polymerase chain reaction (RT-PCR) method for detecting the chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd) to screen for a viroid-free chrysanthemum plant at a small plant size was established and named microtissue direct RT-PCR. A razor or syringe needle was used for RNA template preparations. Under a stereoscopic microscope, a razor or syringe needle was used to pierce, a tissue sample to a depth of 0.1-0.2mm, and the sample was directly transferred to the RT mixtures. Methods using razors or needles for the preparation of templates could detect CSVd and CChMVd with a high sensitivity. The most sensitive method used a razor or syringe needle to acquire template from the shoot tips. Using the microtissue direct RT-PCR method, both viroids could be detected from the high- and low-viroid-concentration plants. The microtissue direct RT-PCR method was more sensitive than a conventional template preparation method. Using the microtissue direct RT-PCR method established in this study, the laborious subculture step could be omitted because detecting viroids and screening for viroid-free plants even at a small plant size before the subculture could be possible. In addition, the microtissue direct RT-PCR method could also be a powerful tool for clarifing the viroid distribution among microtissues, such as shoot apical meristems.
Assuntos
Chrysanthemum/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Viroides/isolamento & purificação , Doenças das Plantas/virologia , Brotos de Planta/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Sensibilidade e Especificidade , Seringas , Moldes Genéticos , Viroides/genéticaRESUMO
It has been observed that the frequency of individuals with Lewis-negative erythrocytes is significantly higher in cancer patients than in healthy controls. In this study, 20 of the 66 (30.3%) patients with various cancers were typed as Lewis negative from their erythrocytes, while the same frequency in healthy controls was 11.1%. These 20 patients were divided into three groups based on the presence of Lewis blood group antigens and alpha 1-->4-fucosyltransferase in their salivas: group I, 6 patients who had both Lewis antigens and alpha 1-->4-fucosyltransferase activity; group II, 8 patients who had no Lewis antigens but possessed alpha 1-->4-fucosyltransferase activity; group III, 6 patients who had neither Lewis antigens nor alpha 1-->4-fucosyltransferase activity. The genotyping of Le genes by the PCR-RFLP methods, which have been developed and established by us recently, demonstrated that all 14 patients from groups I and II possess Le gene homozygously (Le/Le) or heterozygously (Le/le), whereas all 6 patients from group III were le/le homozygotes. Only the 6 patients from group III were identified as the genuine Lewis-negative individuals. The immunohistochemical staining of the colorectal tumors also showed that the Lewis antigens could be detected on the tumors from groups I and II but not from group III.
Assuntos
Eritrócitos/imunologia , Neoplasias Esofágicas/sangue , Fucosiltransferases/análise , Neoplasias Gastrointestinais/sangue , Antígenos do Grupo Sanguíneo de Lewis/análise , Saliva/imunologia , Sequência de Bases , Genótipo , Humanos , Dados de Sequência Molecular , Fenótipo , Saliva/enzimologiaRESUMO
A novel lectin (RSL) which recognizes blood group H type 1 and type 2 (Fuc alpha 1-->2Gal beta 1-->3/4GlcNAc beta-R), and N-acetyllactosamine (Gal beta 1-->4GlcNAc beta-R) was purified from rabbit serum using affinity chromatography on Synsorb H type 2 beads, gel filtration and preparative polyacrylamide gel electrophoresis. The lectin agglutinated human O type red cells, and the hemagglutination reaction was inhibited by H type 1 and type 2 haptens, N-acetyllactosamine and human salivas from secretor individuals. The molecular weight of the lectin was estimated to be approximate 650,000 and 65,000 on Sephacryl S-400 gel filtration and SDS-polyacrylamide gel electrophoresis, respectively.
Assuntos
Amino Açúcares/metabolismo , Haptenos/metabolismo , Lectinas/sangue , Animais , Sequência de Carboidratos , Células Cultivadas , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Haptenos/classificação , Testes de Inibição da Hemaglutinação , Testes de Hemaglutinação , Humanos , Lectinas/isolamento & purificação , Lectinas/metabolismo , Dados de Sequência Molecular , Peso Molecular , CoelhosRESUMO
An N-acetyl-D-galactosamine-binding lectin from Falcata japonica seeds was purified by affinity column chromatography of N-acetyl-D-galactosamine coupled to epoxy-activated Sepharose 6B. A 1000-fold purification of lectin was obtained from the crude extracts. The purified lectin agglutinated blood group A red cells, but neither blood group B nor O red cells. Polyacrylamide gel electrophoresis of the lectin showed one diffuse band. Molecular weights of 125,000 and 117,000 were estimated by gel filtration and ultracentrifugal analysis, respectively. SDS-polyacrylamide gel electrophoresis of the lectin also showed a single band which has a molecular weight of 34,000. Therefore, the lectin molecule was estimated to be a tetramer composed of four identical non-covalently bound subunits. F. japonica lectin was a glycoprotein containing 5% total carbohydrate, and the amino acid composition was characterized by a high content of aspartic acid, serine and glycine, a low content of methionine and the absence of cysteine.
Assuntos
Acetilgalactosamina , Galactosamina , Lectinas/isolamento & purificação , Lectinas de Plantas , Sistema ABO de Grupos Sanguíneos , Aminoácidos/análise , Carboidratos/análise , Cromatografia de Afinidade , Galactosamina/análogos & derivados , Testes de Hemaglutinação , Humanos , Peso MolecularRESUMO
We have isolated a novel phospholipid/ganglioside-binding protein from rabbit sera or platelet-free plasma. Using an affinity chromatography of a commercial gel (Sephacryl S-series gel, Pharmacia) column and a preparative polyacrylamide gel electrophoresis, the protein can be easily purified. The protein agglutinates human red cells irrespective of the ABO blood types, and its hemagglutination reaction is specifically inhibited by some phospholipids (phosphatidylserine and phosphatidylglycerol) and ganglioside (N-acetylneuraminyl-galactosylglucosyl ceramide, GM3). The hemagglutination and its inhibition reactions are independent on any divalent cations (Ca2+, Mg2+, Mn2+, Ni2+). The protein seems to be assembled as multimers of disulfide-bonded molecular of 86 kDa and 59 kDa subunits.
Assuntos
Proteínas de Transporte/sangue , Hemaglutininas/sangue , Animais , Sequência de Carboidratos , Carboidratos/farmacologia , Proteínas de Transporte/isolamento & purificação , Ceramidas , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Eritrócitos/fisiologia , Gangliosídeos/metabolismo , Glicoproteínas/farmacologia , Hemaglutinação/efeitos dos fármacos , Testes de Hemaglutinação , Hemaglutininas/isolamento & purificação , Humanos , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Fosfolipídeos/farmacologia , CoelhosRESUMO
We have previously reported that the elevated activities of serum alpha 1,3fucosyltransferase reverted to normal levels after curative removal of the tumors. To determine the origin of elevated serum alpha 1,3fucosyltransferase, blood samples were obtained from both the drainage vein and the artery in patients with different stages of colorectal cancer at surgery. The enzyme levels in all samples from the drainage vein were found to be higher than the levels in the artery that fed the tumor. Hence, the origin of elevated alpha1,3fucosyltransferase in serum was thought to be the tumor rather than the liver that is the normal source of serum alpha1,3fucosyltransferase. When serum samples not only from colorectal cancer patients but also from patients with gastric, liver, lung, pancreas, bladder and esophagus cancer were treated with anti-FUTVI antibody, the measured activities of alpha1,3fucosyltransferase were markedly reduced. Further, secretion of alpha1,3fucosyltransferase from human colorectal carcinoma cells was also detected in the culture medium by Western immuno-blot analysis with anti-FUTVI antibody.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Fucosiltransferases/sangue , Idoso , Anticorpos/imunologia , Biomarcadores Tumorais/imunologia , Western Blotting , Divisão Celular , Neoplasias Colorretais/patologia , Feminino , Fucosiltransferases/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [3H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Gal beta1,3[3OMeGal beta1,4GlcNAc beta1,6]alphaBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.
Assuntos
Adesão Celular/efeitos dos fármacos , Oligossacarídeos/farmacologia , Omento/patologia , Animais , Sequência de Carboidratos , Separação Celular , Neoplasias do Colo/patologia , Neoplasias do Colo/ultraestrutura , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Metástase Neoplásica , Oligossacarídeos/química , Cavidade Peritoneal/patologia , Células Tumorais CultivadasRESUMO
A simple method for the determination of ABH blood group antigens in secretions has been developed. Blood group ABH specific monoclonal antibodies were covalently bound to blue dyed microspheres of acryl polymer with a diameter of 2.2 microns. The dyed microspheres coated with anti-A, -B and -H antibodies were found to be agglutinated specifically on a plastic microtiter plate by the corresponding blood group antigens secreted in saliva. The agglutination reactions with saliva samples were also observed rapidly and conveniently in a glass capillary tube which contained the same antibody immobilized dyed microspheres. The procedure provides a simple and sensitive method for the determination of blood group antigens through the visible agglutination reaction of dyed microspheres despite the invisibility of the antigens.
Assuntos
Sistema ABO de Grupos Sanguíneos/análise , Técnicas Imunológicas , Saliva/imunologia , Aglutinação , Anticorpos Monoclonais/imunologia , HumanosRESUMO
Three IgM class anti-H monoclonal antibodies (1E3, 1E5 and 3H1) were obtained from a BALB/c mouse immunized with human O type saliva. These antibodies were found to agglutinate red cells from O group and A and B subgroups but not from Bombay and para-Bombay individuals whose H antigen was barely detected by anti-H reagents. The agglutination reactions of these antibodies were inhibited by H antigens from human tissues. It was also demonstrated that both 1E3 and 3H1 reacted with H disaccharide (Fuc alpha 1-->2Gal beta), H type 1 (Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta), H type 2 (Fuc alpha 1-->2Gal beta 1-->4GlcNAc beta), H type 3 (Fuc alpha 1-->2Gal beta 1-->3GalNAc alpha) and H type 4 (Fuc alpha 1-->2Gal beta 1-->3GalNAc beta) but not with Lea (Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc beta), Leb (Fuc alpha 1-->2Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc beta), X (Gal beta 1-->4[Fuc alpha-->3]GlcNAc beta) or Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta). On the other hand, 1E5 was found to react with H type 1, H type 2, Leb and Y. Because of the unique reactivities against various fucosyl linkages these monoclonal antibodies could be useful not only as anti-H reagents but also as reagents for the structural analysis of fucosylated glycoconjugates.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Anticorpos Monoclonais/imunologia , Saliva/imunologia , Adsorção , Animais , Sequência de Carboidratos , Carboidratos/imunologia , Hemaglutinação , Humanos , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
Capsiate is a capsaicin-like ingredient of a non-pungent cultivar of red pepper, CH-19 sweet. To elucidate the mechanisms underlying the non-pungency of capsiate, we investigated whether capsiate activates the cloned capsaicin receptor, TRPV1 (VR1). In patch-clamp experiments, capsiate was found to activate TRPV1 expressed transiently in HEK293 cells with a similar potency as capsaicin. Capsiate induced nociceptive responses in mice when injected subcutaneously into their hindpaws with a similar dose dependency as capsaicin. These data indicate that the non-pungent capsiate is an agonist for TRPV1 and could excite peripheral nociceptors. In contrast to this, capsiate did not induce any significant responses when applied to the skin surface, eye or oral cavity of mice, suggesting that capsiate requires direct access to nerve endings to exhibit its effects. Capsiate was proved to have high lipophilicity and to be easily broken down in normal aqueous conditions, leading to less accessibility to nociceptors. Another highly lipophilic capsaicin analogue, olvanil, was similar to capsiate in that it did not produce irritant responses when applied to the skin surface, although it could activate TRPV1. Taken together, high lipophilicity and instability might be critical determinants for pungency and so help in understanding the effects of capsaicin-related compounds.
Assuntos
Capsaicina/farmacologia , Nociceptores/efeitos dos fármacos , Dor/induzido quimicamente , Receptores de Droga/agonistas , Animais , Comportamento Animal/efeitos dos fármacos , Capsaicina/análogos & derivados , Capsaicina/química , Células Cultivadas , Fenômenos Químicos , Físico-Química , Eletrofisiologia , Olho/efeitos dos fármacos , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Irritantes/farmacologia , Masculino , Camundongos , Boca/efeitos dos fármacos , Terminações Nervosas/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Medição da Dor/efeitos dos fármacos , Ratos , Pele/efeitos dos fármacos , Paladar/efeitos dos fármacosRESUMO
Carcinoembryonic antigen (CEA) levels were determined in the peritoneal washings from 44 patients with gastric cancer to evaluate the usefulness for a predictor of postsurgical prognosis. Seventeen of the 21 patients (80.9%) with serosal invasion showed elevated levels of CEA, whereas most of the patients with no serosal invasion (22/23) showed low levels of CEA and did not develop peritoneal metastasis. All patients with positive cytology showed elevated levels of CEA in the peritoneal washings. Therefore, CEA levels in the peritoneal washings could be an adjunctive tool for predicting the postsurgical prognosis in gastric cancer.
Assuntos
Antígeno Carcinoembrionário/análise , Cavidade Peritoneal/patologia , Neoplasias Gástricas/imunologia , Humanos , Metástase Neoplásica , Prognóstico , Neoplasias Gástricas/patologiaRESUMO
A novel ex vivo method to determine the cell adhesion of cancer cells to the peritoneum was described. The wells of a microtiter plate were filled with cell suspension and sealed using mouse peritoneum. The peritoneum was fixed using a plastic cover and the plate was turned upside down and incubated for cell adhesion. After incubation for 80 min, the plate was centrifuged and non-adherent cells were assayed by MTT assay. Human cancer cells (MKN28, MKN45, MKN74, KM12C and KM12SM) adhered to the mouse peritoneum as well as cells from mouse (Colon26) and the ratio of cells attached to the peritoneum was estimated to be between 12.8 and 66.4%. This simple method could be useful to investigate the adhesion molecule associated with peritoneal dissemination.
Assuntos
Adesão Celular , Neoplasias/patologia , Cavidade Peritoneal/citologia , Animais , Bioensaio , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Células Tumorais CultivadasRESUMO
Serum alpha(1----3)-L-fucosyltransferase activity was measured in 29 ovarian cancer patients with active disease, 26 ovarian cancer patients with no clinical evidence of disease and 23 healthy females. N-Acetyl-2'-O-methyl-lactosamine was used as the acceptor for the enzyme. The level of the enzyme activity was significantly (P less than 0.05) higher in the serum of patients with known tumor when compared to healthy controls and patients with no clinical evidence of disease.
Assuntos
Amino Açúcares/sangue , Ensaios Enzimáticos Clínicos/métodos , Fucosiltransferases/sangue , Hexosiltransferases/sangue , Neoplasias Ovarianas/diagnóstico , Feminino , Humanos , Peso MolecularRESUMO
Levels of alpha(1----3)-L-fucosyltransferase activities were measured in salivas of patients with ovarian cancer. GDP-L-Fuc:GlcNAc alpha(1----3)-fucosyltransferase was found elevated in patients known to have epithelial ovarian cancer, irrespective of their ABH and Lewis blood group phenotypes. GDP-L-Fuc: Glc alpha(1----3)-fucosyltransferase was also elevated in both Lewis positive and negative patients, although the enzyme activity was very low or absent in Lewis negative healthy controls.
Assuntos
Fucosiltransferases/análise , Hexosiltransferases/análise , Antígenos do Grupo Sanguíneo de Lewis/genética , Neoplasias Ovarianas/enzimologia , Saliva/enzimologia , Feminino , Humanos , Neoplasias Ovarianas/sangueRESUMO
Cells of uterine endometrial cancer cell line SNG-II were classified into two groups according to their reactivity with anti-uterine endometrial cancer monoclonal antibody (MSN-1), whose recognition antigen is mainly the Lewis(b) antigens; those that strongly reacted with MSN-1 (SNG-S group) and those that weakly reacted with it (SNG-W group). The SNG-S showed a higher activity of a 1-->4-fucosyltransferase activity than that of the SNG-W. The expression of Lewis(b) antigen was stronger in the SNG-S than that in the SNG-W. Therefore, the expression of uterine endometrial cancer-specific fucosylated carbohydrate could be mainly controlled by alpha-fucosyltransferase activities.
RESUMO
(alpha 1----3)-L-Fucosyltransferase activity was measured in serum samples from 90 gastric cancer patients, 10 patients with benign diseases and 100 healthy controls. The enzyme activity was significantly elevated in the serum samples of patients with cancer compared to those from patients with benign diseases (P less than 0.01) and healthy controls (P less than 0.001). The elevation of the enzyme activity was found to correlate strongly with the clinical stage of disease. The sensitivity of (alpha 1----3)-L-fucosyltransferase was also demonstrated to be high in comparison with the tumor-associated antigens, such as carcinoembryonic antigen and sialylated Lewis X-i. Follow-up studies of (alpha 1----3)-L-fucosyltransferase in 11 cancer patients with disease at different stages showed that the enzyme activity could be useful for monitoring the post-surgical course of the disease. These results suggest that (alpha 1----3)-L-fucosyltransferase activity has a clinically important potential as a tumor marker in gastric cancer.
Assuntos
Fucosiltransferases/sangue , Hexosiltransferases/sangue , Neoplasias Gástricas/enzimologia , Biomarcadores Tumorais/análise , Antígeno Carcinoembrionário/análise , Seguimentos , Glicolipídeos/análise , Humanos , Antígenos CD15 , Estadiamento de Neoplasias , Neoplasias Gástricas/imunologiaRESUMO
Fucosyl glycoproteins were fractionated from a sialoglycoprotein preparation of human erythrocyte membrane by using Aleuria aurantia lectin (AAL) coupled to Sepharose 4B. The affinity eluates were characterized as having high fucose content and significant H activity as measured in terms of N-acetylgalactosamine (GalNAc) incorporation with A1-enzyme and hemagglutination inhibition assay with anti-H sera, and the unadsorbed fractions contained low levels of fucose and were devoid of apparent H activity. Neuraminidase treatment of the material improved the recovery of the affinity eluate. Thus, 66% of the applied asialoglycoprotein was recovered in the eluate, though only 10% of the untreated material was bound and eluted. Moreover, a fucose-rich and H-active fraction was obtained through the affinity chromatography of the previously unbound fraction after neuraminidase treatment. In sodium dodecyl sulfate-gel electrophoresis, the main component of both the unadsorbed and eluted fraction was revealed to be PAS-1 glycoprotein. These results indicate that AAL-Sepharose was effective for isolating fucose-containing compounds from the membrane glycoprotein especially after neuraminidase treatment. The reasons for the appearance of H activity in the affinity eluates are discussed.
Assuntos
Membrana Eritrocítica/análise , Fucose , Glicoproteínas/sangue , Assialoglicoproteínas/sangue , Cromatografia de Afinidade/métodos , Fucose/análise , Glicoproteínas/isolamento & purificação , Humanos , Hidrólise , Lectinas , NeuraminidaseRESUMO
Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Gal beta 1-->4Glc, Gal beta 1-->4GlcNAc, asialo-alpha 1-acid glycoprotein, and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-ceramide, but not to Gal beta 1-->3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by beta-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-alpha 1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5 mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAc alpha 2-->3Gal beta-1,4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the beta-galactosyl residue. Although the GalNAc beta 1-->3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Gal alpha 1-->4Gal beta-1,3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc-ceramide. Hence, the GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure is synthesized by the novel Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase.
Assuntos
N-Acetilgalactosaminiltransferases/sangue , Oligossacarídeos/biossíntese , Sistema do Grupo Sanguíneo P/biossíntese , Trissacarídeos , Acetilgalactosamina/metabolismo , Sequência de Carboidratos , Glicolipídeos/química , Glicolipídeos/metabolismo , Humanos , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/imunologia , Sistema do Grupo Sanguíneo P/química , Sistema do Grupo Sanguíneo P/imunologia , Especificidade por SubstratoRESUMO
To clarify the somesthetic functions of the supplementary motor area (SMA), we recorded the cortical potentials following the median nerve electric stimulation directly from the SMA and investigated the modulation caused by voluntary movements in two patients with intractable SMA seizures. The evoked potentials over the SMA consisted of positive (61.5ms) and negative (100.0 ms) peaks, which were enlarged by voluntary movements of the stimulated hand. The present finding is in strong contrast with the attenuation (gating) of the response at the primary sensorimotor area (SM1) and suggests that the voluntary movements differently modulate the somatosensory functions of SMA and SM1.
Assuntos
Potenciais Somatossensoriais Evocados/fisiologia , Córtex Motor/fisiologia , Movimento/fisiologia , Volição/fisiologia , Adulto , Estimulação Elétrica , Eletromiografia , Epilepsia , Feminino , Humanos , Masculino , Nervo Mediano/fisiologia , Pessoa de Meia-Idade , Córtex Somatossensorial/fisiologiaRESUMO
To elucidate the functional localization and somatotopic organization of pain perception in the human cerebral cortex, we studied the regional cerebral blood flow using positron emission tomography during selective painful stimulation in six normal subjects. Response to a painful stimulus was elicited using a special CO2 laser, which selectively activates nociceptive receptors, to the hand and foot. Multiple brain areas, including bilateral secondary somatosensory cortices (SII) and insula, and the frontal lobe and thalamus contralateral to the stimulus side, were found to be involved in the response to painful stimulation. While our data indicate that the bilateral SII play an important role in pain perception, they also indicate that there is no pain-related somatotopic organization in the human SII or insula.