Substantial oxygen consumption by aerobic nitrite oxidation in oceanic oxygen minimum zones.

Oceanic oxygen minimum zones (OMZs) are globally significant sites of biogeochemical cycling where microorganisms deplete dissolved oxygen (DO) to concentrations <20 µM. Amid intense competition for DO in these metabolically challenging environments, aerobic nitrite oxidation may consume significant amounts of DO and help maintain low DO concentrations, but this remains unquantified. Using parallel measurements of oxygen consumption rates and 15N-nitrite oxidation rates applied to both water column profiles and oxygen manipulation experiments, we show that the contribution of nitrite oxidation to overall DO consumption systematically increases as DO declines below 2 µM. Nitrite oxidation can account for all DO consumption only under DO concentrations <393 nM found in and below the secondary chlorophyll maximum. These patterns are consistent across sampling stations and experiments, reflecting coupling between nitrate reduction and nitrite-oxidizing Nitrospina with high oxygen affinity (based on isotopic and omic data). Collectively our results demonstrate that nitrite oxidation plays a pivotal role in the maintenance and biogeochemical dynamics of OMZs.

Immunochip analysis identifies novel susceptibility loci in the human leukocyte antigen region for acquired thrombotic thrombocytopenic purpura.

Essentials Genetic predisposition to acquired thrombotic thrombocytopenic purpura (aTTP) is mainly unknown. Genetic risk factors for aTTP were studied by Immunochip analysis and replication study. Human leukocyte antigen (HLA) variant rs6903608 conferred a 2.5-fold higher risk of developing aTTP. rs6903608 and HLA-DQB1*05:03 may explain most of the HLA association signal in aTTP. Click to hear Dr Cataland's presentation on acquired thrombotic thrombocytopenic purpura SUMMARY: Background Acquired thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy associated with the development of autoantibodies against the von Willebrand factor-cleaving protease ADAMTS-13. Similarly to what has been found for other autoimmune disorders, there is evidence of a genetic contribution, including the association of the human leukocyte antigen (HLA) class II complex with disease risk. Objective To identify novel genetic risk factors in acquired TTP. Patients/Methods We undertook a case-control genetic association study in 190 European-origin TTP patients and 1255 Italian healthy controls by using the Illumina Immunochip. Replication analysis in 88 Italian cases and 456 controls was performed with single-nucleotide polymorphism (SNP) TaqMan assays. Results and conclusion We identified one common variant (rs6903608) located within the HLA class II locus that was independently associated with acquired TTP at genome-wide significance and conferred a 2.6-fold increased risk of developing a TTP episode (95% confidence interval [CI] 2.02-3.27, P = 1.64 × 10-14 ). We also found five non-HLA variants mapping to chromosomes 2, 6, 8 and X that were suggestively associated with the disease: rs9490550, rs115265285, rs5927472, rs7823314, and rs1334768 (nominal P-values ranging from 1.59 × 10-5 to 7.60 × 10-5 ). Replication analysis confirmed the association of HLA variant rs6903608 with acquired TTP (pooled P = 3.95 × 10-19 ). Imputation of classic HLA genes followed by stepwise conditional analysis revealed that the combination of rs6903608 and HLA-DQB1*05:03 may explain most of the HLA association signal in acquired TTP. Our results refined the association of the HLA class II locus with acquired TTP, confirming its importance in the etiology of this autoimmune disease.

Potential application value of Doppler ultrasonography in neoadjuvant chemotherapy for breast cancer.

BACKGROUND: The aim of this study was to evaluate the efficacy of the ultrasonography with Power Doppler, by quantifying the vascular density by Vascularity Index (VI), to predict the pathologic response to neoadjuvant chemotherapy (NAC) for breast cancer (BC). METHODS: For this prospective observational study, 20 patients were recruited with a histological diagnosis of infiltrating breast carcinoma and indication for NAC. Patients were submitted to ultrasonography with Power Doppler. Tumor vascular density was evaluated by computer software SysArea©1, before treatment, after 2 or 3 cycles of chemotherapy and at the end of treatment. The pathologic response was analyzed. The sensitivity, specificity and positive and negative predictive values for the histological response were also calculated and P-values <0.05. RESULTS: VI showed a sensitivity of 88.88%, specificity of 100%, positive predictive value of 100% and negative predictive value of 91.66% for the pathologic response, as well as being strongly associated with it. The variation of VI, in terms of either an increase or decrease after 2 or 3 cycles of chemotherapy, significantly predicted the final histological response. CONCLUSIONS: The analysis of VI was predictive and showed a strong correlation with histological response to neoadjuvant chemotherapy for BC.

Congenital factor XIII deficiency in Pakistan: characterization of seven families and identification of four novel mutations.

Deficiency of coagulation factor XIII (FXIII) belongs to the rare bleeding disorders and its incidence is higher in populations with consanguineous marriages. The aims of this study were to characterize patients and relatives from seven families with suspected FXIII deficiency from Pakistan and to identify the underlying mutations. As a first indicator of FXIII deficiency, a 5M urea clot solubility test was used. Plasma FXIII A- and B-subunit antigen levels were determined by ELISA. FXIII activity was measured with an incorporation assay. Sequencing of all exons and intron/exon boundaries of F13A was performed, and a novel splice site defect was confirmed by RT-PCR analysis. Genetic analysis revealed six different mutations in the F13A gene. Two splice site mutations were detected, a novel c.1460+1G>A mutation in the first nucleotide of intron 11 and a previously reported c.2045G>A mutation in the last nucleotide of exon 14. Neither of them was expressed at protein level. A novel nonsense mutation in exon 4, c.567T>A, p.Cys188X, was identified, leading in homozygous form to severe FXIII deficiency. Two novel missense mutations were found in exons 8 and 9, c.1040C>A, p.Ala346Asp and c.1126T>C, p.Trp375Arg, and a previously reported missense mutation in exon 10, c.1241C>T, p.Ser413Leu. All patients homozygous for these missense mutations presented with severe FXIII deficiency. We have analysed a cohort of 27 individuals and reported four novel mutations leading to congenital FXIII deficiency.

Estudio comparativo de la biodistribución de 99mTc-HYNIC-Lys3-bombesina obtenido con los coligandos EDDA/tricina y AN/tricina / Comparative study of the biodistribution of 99mTc-HYNIC-Lys3-Bombesin obtained with the EDDA/tricine and NA/tricine as coligands

El propósito de la presente investigación fue evaluar la biodistribución en animales sanos y en modelos de tumores de los radiofármacos 99mTc-EDDA/tricina-HYNIC-Lys3-bombesina (HYNIC-Lys3-BN) y 99mTc-AN/tricina-HYNIC-Lys3-BN. La biodistribución y la farmacocinética fueron realizados durante 24 horas para lo cual se utilizaron 24 ratas wistar sanas a las cuales se les administró 37,0±0,8 MBq/rata de cada radiofármaco y para el estudio de modelo de tumor fueron utilizados 20 ratones desnudos CD-1 a los que se les injertaron tumores de próstata (PC3). Diez días después fueron calculados los volúmenes tumorales y aplicados 40,00±0,04 MBq/ratón de cada radiofármaco. Ambos mostraron alta pureza radioquímica con valores de 98,08±0,25% para el compuesto con EDDA/tricina y de 95,1±0,3% para el conjugado con AN/tricina. La captación del radiofármaco con AN/tricina fue significativamente mayor en órganos del sistema retículo endotelial de ratas Wistar sanas durante 24h, específicamente en hígado y bazo. No se encontraron diferencias significativas entre los tiempos medios de eliminación de la sangre para ambos compuestos. El volumen promedio de crecimiento tumoral fue 0,93±0,02cm3 y la afinidad por tumores mostró una unión creciente y específica de ambos radiofármacos, siendo significativamente mayor para el conjugado con EDDA/tricina. Este resultado permitió corroborar la relación directa que existe entre la densidad de receptores del péptido liberador de la gastrina (PLGr) y la variación de la acumulación de los radiofármacos en el tumor. El uso de EDDA/tricina como coligando para marcar HYNIC-Lys3-BN con 99mTc, es más apropiado que AN/tricina(AU)

The aim of present investigation was to evaluate biodistribution in healthy animals and in tumor models of the radiopharmaceuticals 99mTc-EDDA/tricine-HYNIC-Lys3-Bombesin (HYNIC-Lys3-BN) and 99mTc-NA/tricine-HYNIC-Lys3-BN. Biodistribution and pharmacokinetics were carried out over 24hours. To do so, 24 healthy Wistar rats were used and were administered 37.0±0.8 MBq/rat of each radiopharmaceutical. For the tumor model study, 20 CD-1 nude mice were used and prostate tumors (PC3) were implanted in all the mice. Ten days later, tumor volumes were calculated and 40.00±0.04 MBq/mice of each radiopharmaceutical were injected. Both showed high radiochemical purity: 98.08±0.25% for EDDA/tricine product and 95.1±0.3% for the conjugate with NA/tricine. Uptake of the radiopharmaceutical with NA/tricine was significantly higher in organs of the reticulo-endothelial system of healthy Wistar rats during 24h, specifically in the liver and spleen. Both labeled compounds showed no significant differences between their blood elimination half lives. Average of tumor growth was 0.93± 0.02cm3 and affinity for tumors showed a growing and specific binding of both radiopharmaceuticals, although it was significantly higher for the EDDA/tricine conjugate. This outcome made it possible to corroborate the direct relationship between the density of gastrin releasing peptide and its receptors (GRPr) and the variation of the accumulation of the radiopharmaceuticals in the tumor. Use of EDDA/tricine as coligand is more appropriate than NA/tricine for labeling of HYNIC-Lys3-BN with 99mTc(AU)

[Comparative study of the biodistribution of (99m)Tc-HYNIC-Lys3-Bombesin obtained with the EDDA/tricine and NA/tricine as coligands]. / Estudio comparativo de la biodistribución de (99m)Tc-HYNIC-Lys3-bombesina obtenido con los coligandos EDDA/tricina y AN/tricina.

The aim of present investigation was to evaluate biodistribution in healthy animals and in tumor models of the radiopharmaceuticals (99m)Tc-EDDA/tricine-HYNIC-Lys3-Bombesin (HYNIC-Lys3-BN) and (99m)Tc-NA/tricine-HYNIC-Lys3-BN. Biodistribution and pharmacokinetics were carried out over 24 hours. To do so, 24 healthy Wistar rats were used and were administered 37.0 ± 0.8 MBq/rat of each radiopharmaceutical. For the tumor model study, 20 CD-1 nude mice were used and prostate tumors (PC3) were implanted in all the mice. Ten days later, tumor volumes were calculated and 40.00 ± 0.04 MBq/mice of each radiopharmaceutical were injected. Both showed high radiochemical purity: 98.08 ± 0.25% for EDDA/tricine product and 95.1 ± 0.3% for the conjugate with NA/tricine. Uptake of the radiopharmaceutical with NA/tricine was significantly higher in organs of the reticulo-endothelial system of healthy Wistar rats during 24h, specifically in the liver and spleen. Both labeled compounds showed no significant differences between their blood elimination half lives. Average of tumor growth was 0.93 ± 0.02 cm(3) and affinity for tumors showed a growing and specific binding of both radiopharmaceuticals, although it was significantly higher for the EDDA/tricine conjugate. This outcome made it possible to corroborate the direct relationship between the density of gastrin releasing peptide and its receptors (GRPr) and the variation of the accumulation of the radiopharmaceuticals in the tumor. Use of EDDA/tricine as coligand is more appropriate than NA/tricine for labeling of HYNIC-Lys3-BN with (99m)Tc.

Nutritional status of patients with gynecologic and breast cancer / Estado nutritivo de pacientes con cáncer ginecológico o de mama

Objective: To identify the preoperative nutritional status of women with gynecologic or breast cancer, in correlation with disease site and staging as well as previous treatments. Subjects and methods: A cross-sectional study of 250 women evaluated by Body Mass Index (BMI) and Subjective Global Assessment (SGA). For data analysis, the chisquare test was applied. Results: Breast cancer was the most frequent cancer, predominating in 56.2%. The median age of the patients was 52 years. In about 57% of these women, the tumor was restricted to clinical stages 0, I and II and 77% of the women had not undergone any other oncologic treatment prior to surgery. Subjective Global Assessment detected 76% of nourished women and 24% undernourished women, while Body Mass Index identified 34% of nourished women, 3.6% undernourished women and 62.4% overweight/obese women. A low level of diagnostic agreement between normal nutrition and malnutrition by both methods was observed (63.8%; kappa (95% CI) = 0.0884 (-0.07-0.24). No correlation between nutritional evaluation and previous treatment and disease staging was observed. Concerning anatomic site, it was subjectively observed that women with cancer of the uterine corpus were more malnourished than the rest (p = 0.02). Conclusions: The findings suggest that a more careful evaluation should be employed to identify preoperative nutritional status in women with gynecologic or breast cancer (AU)

Objetivo: Identificar el estado nutritivo preoperatorio de mujeres con cáncer ginecológico o de mama, en correlación con la localización de la enfermedad y su estadificación, así como de tratamientos previos. Sujetos y métodos: Estudio transversal de 250 mujeres evaluadas mediante Índice de masa corporal (IMC) y Valoración global subjetiva (VGS). Se aplicó la prueba de Chi cuadrado para el análisis de los datos. Resultados: el cáncer de mama fue el tipo más frecuente, predominando en el 56,2%. La edad media de las pacientes fue de 52 años. En cerca del 57% de ellas, el tumor estaba limitado a los estadios clínicos 0, I y II, y el 77% de ellas no habían sido sometidas a ningún otro tratamiento oncológico antes de la cirugía. La valoración global subjetiva detectó un 76% de las mujeres bien nutridas y 24% malnutridas, mientras que el Índice de masa corporal identificó un 34% de las mujeres bien nutridas, un 3,6% de ellas malnutridas, y un 62,4% de mujeres con sobrepeso/obesidad. Se observó un nivel bajó de concordancia para nutrición normal y malnutrición entre ambos métodos (63,8%; kappa (IC 95%) = 0,0884 (-0,07-0,24). No se observó ninguna correlación entre la evaluación nutricional y tratamiento previo o estadificación de la enfermedad. Con respecto a la localización anatómica, se observó subjetivamente que las mujeres con cáncer del cuerpo del útero presentaban mayor desnutrición que el resto (p = 0,02). Conclusiones: Los hallazgos sugieren que se debería realizar una evaluación más minuciosa para identificar el estado nutritivo de las mujeres con cáncer ginecológico o de mama (AU)

Imagen de sepsis asociada a una histiocitosis X / No disponible

No disponible

Nutritional status of patients with gynecologic and breast cancer.

OBJECTIVE: To identify the preoperative nutritional status of women with gynecologic or breast cancer, in correlation with disease site and staging as well as previous treatments. SUBJECTS AND METHODS: A cross-sectional study of 250 women evaluated by Body Mass Index (BMI) and Subjective Global Assessment (SGA). For data analysis, the chi-square test was applied. RESULTS: Breast cancer was the most frequent cancer, predominating in 56.2%. The median age of the patients was 52 years. In about 57% of these women, the tumor was restricted to clinical stages 0, I and II and 77% of the women had not undergone any other oncologic treatment prior to surgery. Subjective Global Assessment detected 76% of nourished women and 24% undernourished women, while Body Mass Index identified 34% of nourished women, 3.6% undernourished women and 62.4% overweight/obese women. A low level of diagnostic agreement between normal nutrition and malnutrition by both methods was observed (63.8%; kappa (95% CI) = 0.0884 (-0.07-0.24). No correlation between nutritional evaluation and previous treatment and disease staging was observed. Concerning anatomic site, it was subjectively observed that women with cancer of the uterine corpus were more malnourished than the rest (p = 0.02). CONCLUSIONS: The findings suggest that a more careful evaluation should be employed to identify preoperative nutritional status in women with gynecologic or breast cancer.

Nueva formulación para marcar ciprofloxacina con 99mTc / New formulation to label ciprofloxacin with 99mTc

No disponible

Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast.

We constructed representative large-insert bacterial artificial chromosome (BAC) libraries of two human pathogens (Trypanosoma brucei and Giardia lamblia) using a new hybrid vector, pTARBAC1, containing a yeast artificial chromosome (YAC) cassette (a yeast selectable marker and a centromere). The cassette allows transferring of BACs into yeast for their further modification. Furthermore, the new hybrid vector provides the opportunity to re-isolate each DNA insert without construction of a new library of random clones. Digestion of a BAC DNA by an endonuclease that has no recognition site in the vector, but which deletes most of the internal insert sequence and leaves the unique flanking sequences, converts a BAC into a TAR vector, thus allowing direct gene isolation. Cotransformation of a TAR vector and genomic DNA into yeast spheroplasts, and subsequent recombination between the TAR vector's flanking ends and a specific genomic fragment, allows rescue of the fragment as a circular YAC/BAC molecule. Here we prove a new cloning strategy by re-isolation of randomly chosen genomic fragments of different size from T. brucei cloned in BACs. We conclude that genomic regions of unicellular eukaryotes can be easily re-isolated using this technique, which provides an opportunity to study evolution of these genomes and the role of genome instability in pathogenicity.

Avaliaçäo intra-operatória do estádio do carcinoma do endométrio / Intraoperative evaluation of endometrial carcinoma staging

A nova classificaçäo por estádios apra o câncer do corpo uterino adotado pela Federaçäo Internacional de Ginecologia e Obstetrícia (FIGO), em 1988, é baseada no exame anatomopatológico intra-operatório da peça de histerectomia, näo irradiada. Objetivo. Através do estudo intra-operatório com a biópsia de congelaçäo, reconhecer os fatores de risco para metástases ganglionares intra-abdominais e selecionar as pacientes que realmente devem ser submetidas à linfadenectomia da ilíaca comum e periaórtica. Métodos. Säo considerados fatores de maior risco para metástase: neoplasias pouco diferenciadas (grau histológico 3), invasäo miometrial profunda (mais da metade do miométrio), tipos histológicos especiais mais agressivos e extensäo da neoplasia ao colo uterino. O exame intra-operatório dura cerca de 30 minutos, quando o útero é aberto lateralmente e säo feitos vários cortes transversais completos para selecionar as áreas suspeitas de maior invasäo miometrial e cervical. Posteriormente, säo comparados os dados do exame de congelaçäo com as lâminas permanentes, em cortes de parafina. REsultados. A comparaçäo dos dados preliminares intra-operatórios com os preparados permanentes revelou alta taxa de concordância, ao redor de 90 por cento, para as diferentes variáveis estudadas. Conclusäo. O procedimento proposto pela FIGO para a nova classificaçäo de estádios do câncer do corpo uterino está sendo avaliado. A alta taxa de concordância encontrada fala a favor da confiabilidade do exame intra-operatório e da sua execuçäo. Tratamento radioterápico complementar é indicado conforme o estádio da doença. Como é um procedimento recente, somente mediante avaliaçäo uniforme dos estádios e protocolos de tratamento poderemos melhor avaliar estas modificaçöes

[Intraoperative evaluation of endometrial carcinoma staging]. / Avaliação intra-operatória do estádio do carcinoma do endométrio.

The new International Federation of Gynecology and Obstetrics (FIGO) staging system for endometrial cancer is based on intraoperative examination of hysterectomy specimens not submitted to radiation. PURPOSE--This system aims to recognize the risk factors for intrabdominal lymph node metastases not detected by the earlier staging system, permitting to select the patients who must be submitted to lymphadenectomy. The major risks for metastases are: poorly differentiated neoplasms (grade 3), deep myometrial invasion (more than half of the thickness of the myometrium) and certain more aggressive histological types. METHODS--Intraoperative examination takes about 30 minutes. The uterus is laterally opened and several complete transversal cuts are made in order to choose the suspected deeper myometrial or cervical invasion areas for histological frozen examination. The findings were compared with the permanent slides. RESULTS--Good correlations between frozen and permanent sections were obtained, with an accuracy of about 90%. CONCLUSION--This new FIGO staging is under evaluation. The comparative results show a higher degree of confidence on the method performance. The complementary radiotherapic treatment is indicated according to the disease stage.

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water.

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

Carcinoma in situ da mama e sua defecçäot através da termografia de placa / In situ breast carcinoma defection through of plate thermography

Os autores avaliam a termografia de placa realizada em controles periódicos e empregada através de uma metodologia de interpretaçäo diagnóstica adequada na detecçäo do carcinoma "in situ" de mama, quando entäo, praticamente, näo há manifestaçöes clínicas da sua presença. Baseando-se no diagnóstico angiotermográfico, foram realizadas 127 biópsias da mama em 82 mulheres, sendo 45 bilaterais. O estudo histológico revelou no total de 16 carcinomas, sendo 15 "in situ" e um microinvasor, sendo, em três casos, bilaterais. Destes seis eram ductais e nove lobulares. Foram diagnosticadas, ainda, cinco hiperplasias lobulares atípicas. Desta forma, podemos concluir que a termografia de placa é um método propedêutico eficiente no diagnóstico do carcinoma "in situ" da mama

Cocoa: a new mouse model for platelet storage pool deficiency.

We describe genetic, haematological and biochemical properties of a new mouse pigment mutant, cocoa (coa). Cocoa is a recessive mutation located on the centromeric end of chromosome 3 near the Car-2 locus. The mutation causes increased bleeding time accompanied by symptoms of platelet storage pool deficiency (SPD), including decreased platelet serotonin and decreased visibility of dense granules as analysed by electron microscopy of unfixed platelets. Dense granules were visible in normal numbers when platelets were incubated with the fluorescent dye, mepacrine. The intragranular environment, however, was abnormal as indicated by decreased flashing of mepacrine-loaded dense granules after exposure to ultraviolet light. Unlike the previously described seven mouse pigment mutations with SPD in which pigment granules, platelet dense granules and lysosomes are affected, the cocoa mutant had normal secretion of lysosomal enzymes from kidney proximal tubule cells and platelets. The cocoa mutation thus represents an example of a single gene which simultaneously affects melanosomes and platelet dense granules but probably does not affect lysosomes. The results indicate that melanosomes and platelet dense granules share steps in synthesis and/or processing. Cocoa may be a model for cases of human Hermansky-Pudlak syndrome in which functions of melanosomes and platelet dense granules, but not lysosomes, are involved.

Establishment of plasma membrane domains in hepatocytes. I. Characterization and localization to the bile canaliculus of three antigens externally oriented in the plasma membrane.

A membrane fraction denoted N2 upper was isolated from homogenates of rat liver by sucrose gradient centrifugation. This fraction, which was enriched 65-fold over the homogenate in 5'-nucleotidase activity, was used as an immunogen in goats. The antisera obtained contained antibodies to three predominant polypeptides in the N2 upper membrane fraction, as shown by crossed immunoelectrophoresis. These polypeptides had molecular weights of 105,000, 110,000, and 160,000 after recovery from the crossed immunoelectrophoretic gels and are denoted PM105, PM110, and PM160. Each was a distinct polypeptide, as shown by the distinct peptide patterns resulting from limited proteolysis in the presence of detergents. The three polypeptides were synthesized by primary cultures of hepatocytes and were externally oriented at the surface of these cells, as shown by their accessibility in situ to iodination catalyzed by lactoperoxidase. They were not detectable in the serum by crossed immunoelectrophoresis. The three antigens were present at very low (PM110) or nondetectable (PM105, PM160) concentrations in intracellular membrane fractions derived from the Golgi and smooth and rough endoplasmic reticulum of liver. The antigens also were reduced in concentration in a plasma membrane fraction most likely derived from the sinusoidal surface of the hepatocyte. The three membrane antigens bind to concanavalin A; hence, they are probably glycoprotein constituents of a discrete domain of the hepatocyte plasma membrane. Immune complexes were isolated after crossed immunoelectrophoresis and injected into rabbits. Each of the antisera obtained was reactive to one of the membrane polypeptides. Sections of fixed rat livers were reacted with each of the antibodies and then the primary antibody was localized by indirect immunocytochemical methods using horseradish peroxidase or colloidal gold as labels. Each of the three antigens was localized by this method to the bile canalicular domain of the hepatocyte plasma membrane.