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Factors specifying cell lineages in the leech.

As in arthropods, several major organ systems in leeches, including body musculature, nervous system and nephridia, are organized into a fixed number of longitudinally iterated units called segments. Many cells, especially neurons, can be uniquely identified from segment to segment. Leech embryos comprise identified cells, which facilitates developmental analysis. So far as it is known, cell lineages in leech are largely determinate. Prior to first cleavage, cytoplasmic reorganization generates domains of yolk-deficient cytoplasm called teloplasm. In situ hybridization experiments suggest that teloplasm is enriched for polyadenylated RNAs. During the first three, unequal cell divisions, teloplasm is segregated to macromere D'; normally, this cell alone cleaves further to generate five bilateral pairs of embryonic stem cells, M, N, O/P and Q teloblasts. Centrifugation experiments have shown a causal link between inheritance of teloplasm and the cleavage pattern that generates teloblasts. Teloblasts undergo highly unequal divisions, generating a longitudinal array of segmental founder cells called m, n, o, p and q blast cells, from which the definitive segmental tissues arise via further stereotyped cell divisions. Microinjecting new-born teloblasts or their precursors with polyadenylic acid induces the formation of supernumerary teloblasts. This discovery permits further analyses of factors specifying the five cell lines generating segmental tissues of the leech.

Animals

Quantitative electron microscopy of carcinogen-induced alterations in hepatocyte rough endoplasmic reticulum. I. Chronic effect of 3'MeDAB and short-term effects of azo dyes of different carcinogenic potentials.

A new method has been developed for the quantitative analysis of an ultrastructural change in hepatocyte rough endoplasmic reticulum (ER) that is induced by liver carcinogens. Hitherto only subjective observations of this alteration had been made. Male inbred Leeds rats were fed a diet containing 0.06% of the carcinogenic azo dye 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB). Groups of treated rats, together with untreated controls, were sacrificed after 10 days, 4 weeks, 10 weeks and 17 weeks. Samples of liver tissue from each animal and from 10 3'MeDAB-induced hepatocellular carcinomas (HCC), were examined by electron microscopy. A quantitative study was carried out to investigate the effect of chronic exposure to 3'MeDAB upon the state of aggregation of the hepatocyte rough ER into parallel arrays. As early as 10 days after the start of treatment, the rough ER showed a highly significant degree of disaggregation: the mean number of ER cisternae per array fell from the control value of 6.20 to 3.73. This change was sustained throughout the experiment. At 17 weeks, comparison of the mean array size in the HCC cells with that in the surrounding hepatocytes revealed a further significant decline, from 3.46 to 2.12. Further groups of rats were fed other azo dyes for 4 weeks and subjected to the same assay. The carcinogens 4'-methyl-4-dimethylaminoazobenzene (4'MeDAB) and N,N-dimethyl-4-amino-N-acetyl-N-monomethyl-4-aminoazobenzene (DAAMAB) resembled 3'MeDAB with respect to the degree of rough ER disaggregation they induced. In contrast, the non-carcinogen 3'-trifluoromethyl-4-dimethylaminoazobenzene (3'TFMeDAB) had no such effect, while the weak initiator 2-methyl-4-dimethylaminoazobenzene (2MeDAB) induced disaggregation to a lesser degree than the strong carcinogens. At least with the azo dyes used in this study, the extent of rough ER disaggregation appears to be related to hepatocarcinogenesis.

Animals

Cytokeratins and cytokeratin filaments in subpopulations of cultured human and rodent cells of nonepithelial origin: modes and patterns of formation.

Using immunofluorescence microscopy, we observed that in several established cell culture lines derived from different nonepithelial tissues and species, cells spontaneously emerge, usually at low frequencies, which contain cytoplasmic structures decorated by antibodies specific for cytokeratins 8 and 18. This phenomenon was further examined at both the protein (gel electrophoreses of cytoskeletal proteins, followed by immunoblotting) and the RNA (Northern blots, "nuclear run-on" analysis, in situ hybridization) level. Positive cell lines included simian virus (SV40)-transformed human fibroblasts (HF-SV80, WI-38 VA13), human astrocytic glioma cells (U333 CG/343MG), rat (RVF-SMC) and hamster (BHK-21/13) cells derived from vascular smooth muscle and murine sarcoma MS-180 cells. In two cell lines (HF-SV80 and BHK-21/13), the frequency of the cytokeratin-containing cells and of the cytokeratin fibril arrays per cell was drastically increased upon treatment with 5-azacytidine. The structural appearance of the cytokeratins was variable in the different cell lines but could also differ among cells of the same culture: While small granular or comma-shaped structures or bizarrely shaped filament arrays prevailed in WI-38, RVF and normally grown BHK-21 cells, most of the other lines revealed extended normal-looking, fibrillar arrays. In one line (MS-180), the appearance of cytokeratins was associated with a morphological change, as it was only found in a subpopulation of cells that had lost their typical elongated and spindle-shaped phenotype and assumed a rounded ("coccoid") shape. Our results show that the expression of the genes encoding cytokeratins 8 and 18 is not necessarily restricted to programs of epithelial differentiation and that factors stochastically effective appear in cultured cell lines that allow the synthesis of these cytoskeletal components. Mechanisms possibly involved in this spontaneous and selective advent of cytokeratins 8 and 18 and implications for tumor diagnosis are discussed.

Animals

Culture of human neoplastic gastrointestinal smooth muscle cells.

Due to limited growth potential of primary cultures and the absence of continuous lines of healthy enteric smooth muscle, we have studied the culture behavior of neoplastic gastrointestinal smooth muscle cells. Forty-six human enteric smooth muscle neoplasms (leiomyomas and leiomyosarcomas) were studied while fresh and/or after culture in vitro and growth in vivo in athymic nude mice, with assessments made of morphology, growth characteristics, and biochemical markers of differentiation. The state of differentiation of the tumors varied, with well-differentiated tumors tending to express binding sites for the gastrointestinal hormone cholecystokinin, whereas less well-differentiated tumors did not. Poorly differentiated tumors were the easiest to establish in culture in vitro and to grow in vivo in nude mice. When the cells placed directly into culture proliferated to confluent density, they underwent morphologic differentiation from a spread, fibroblastlike shape to a slender spindle morphology, with these cells possessing fewer biosynthetic organelles and arranging themselves in characteristic "hill and valley" arrays. However, the highly differentiated characteristics of expression of desmin or cholecystokinin-binding sites were not observed in cultured cells. In contrast, cells that had been passaged in nude mice before culture displayed a proliferative phenotype and failed to undergo morphologic differentiation on reaching confluent density. Four human enteric smooth muscle cell lines (documented by chromosomal analysis) originating in stomach, jejunum, ileum, and rectum were established using this strategy.

Cholecystokinin

[Response of bone metastases to medical treatment: definition of evaluation criteria and classification trials].

Bone metastases are very frequent. Some are sensitive to the action of anticancer drugs. However, there is as yet an unsolved methodological problem in the evaluation of response to these drugs. The uniquely radiological UICC criteria are quite insufficient, in as much as they appear with a long delay and sometimes give erroneous results. In this work we give a brief review of biological and clinical knowledge about bone metastases, and we attempt to give an array of the possible evaluation criteria and their respective value. We propose as a working hypothesis a classification of responses taking into account the criteria: the urinary hydroxyproline to urinary creatinine ratio, the serum dosage of bone isoenzyme of alkaline phosphatase and propeptide of type III procollagen (P III NP), and as an essential element, an analysis of all available imaging techniques. A visual study of bone scintillation scans must precede that of radiographs and, when possible, it must be associated to computerized scintillation scanning. When metastasis are located to the pelvis, the vertebral column, or the sternum, a CT scan or better, a nuclear magnetic resonance study (IRM), is indispensable in order to have a direct measure of the tumor extension to soft tissues. Furthermore, in the case of isolated metastases, one of these imaging techniques allows a diagnostic biopsy. Finally an analysis of response at the bone level will always be associated with a measure of their duration and an evaluation of metastases to other sites.

Acid Phosphatase

Analysis of cell-differentiation lineage in human teratomas using new monoclonal antibodies to cytostructural antigens of embryonal carcinoma cells.

Human embryonal carcinoma cells sometimes display the developmental potential of early embryonic stem cells. While available data do not clearly identify a counterpart of these tumor cells in normal development, previous comparisons of human embryonal carcinoma and yolk sac carcinomas indicated that these cell types are closely related, and suggested that embryonal carcinoma cells might resemble the progenitors of extraembryonic endoderm. To analyse further cell-differentiation lineage in these tumors, we produced monoclonal antibodies to cytostructurally associated antigens of human embryonal carcinoma cells. Spleen cells from mice immunized with a detergent-insoluble extract of cultured human embryonal carcinoma cells were fused to NS-1 myeloma cells, and hybridoma supernatants were screened by indirect immunofluorescence on the immunizing cell line, then on a panel of cell lines derived from human embryonal carcinomas, yolk sac carcinomas, and a range of neoplastic and normal tissues. Monoclonal antibody GCTM-1 stained the nuclei of all human cells tested and served as a positive control; this antibody immunoprecipitated proteins of 85 and 66 k Da from human embryonal carcinoma cells. GCTM-2 recognized an epitope on a 200-k Da extracellular protein present on the surface of embryonal carcinoma cells, and stained the surface of visceral yolk sac-type carcinoma and colorectal carcinoma cells as well. Enzymatic analysis of carbohydrate residues on the GCTM-2 antigen revealed that it was a keratan sulphate proteoglycan, and suggested that the epitope recognized by the antibody lies on the core protein. In immunoblots, antibody GCTM-3 bound to a 57-k Da cytoskeletal protein expressed in human embryonal carcinoma. This antibody decorated filamentous arrays in cell lines from human embryonal carcinoma, visceral yolk sac carcinoma, parietal yolk sac carcinoma (endodermal sinus tumour), and adenocarcinoma and large cell carcinoma of the lung. Antibody GCTM-4 recognized a determinant present on a 69-k Da polypeptide, associated with a component of the lysosomal compartment, which was expressed in embryonal carcinoma cells, but no other cell type tested. The results with this antibody panel thus allow distinction between human embryonal carcinoma and yolk sac carcinoma, but provide further evidence of a close relationship between these cell types.

Animals

Electron microscopic detection of human papillomavirus particles in oral proliferative lesions.

Human papilloma virus (HPV) has been demonstrated in a series of benign proliferative lesions of skin and mucosae. To prove the distribution of HPV in the oral proliferative lesions at the ultrastructural level, we performed electron microscopic analysis of 10 specimens taken from 5 patients through large excisional biopsy. All of them were diagnosed pathologically as fibropapilloma. In each patient, specimens were taken from both clinically evident proliferative lesions and clinically normal surrounding mucosa. Obtained specimens were fixed in a glutaraldehyde solution and processed for routine ultrathin sectioning. Before electron microscopic observation, the tissue sections on copper grids were subjected to amylase digestion of glycogen granules. Spherical viral particles of 40-55 nm in diameter were detected the non-keratinized epithelial cells in all specimens examined. Of particular interest were the large amounts of viral particles found in the cytoplasmic matrix and nuclei (especially on their chromatin masses) of the cells in intermediate and surface layers, which did not form a crystal array. All the membranous cell organelles of epithelial cells were, however, devoid of viral particles. Some viral particles were distributed in the extracellular spaces of an intermediate layer. Viral particles were hardly observed in the cells of a basal/suprabasal and prickle cell layers. There were no significant differences in the HPV distribution between the cells derived from the proliferative lesion and those derived from the surrounding normal mucosa.

Adult

Pulmonary disposition of inhaled NO2-nitrogen in isolated rat lungs.

Nitrogen dioxide (NO2) is a relatively insoluble, reactive gas that, on inhalation, generates a diverse array of pulmonary toxic effects. Its uptake and transformation in isolated lungs have been shown to be proportional to inspired dose and associated with significant accumulations of the nitrite ion. However, not all absorbed NO2 is directly detectable as soluble nitrite. To further characterize its uptake and chemical disposition, we determined the chemical fate of 15NO2-nitrogen in isolated perfused (red cell-free) rat lungs that were exposed to 20 ppm 15NO2 for 60 min. Total excess 15N (relative to unexposed controls) was determined by isotope ratio mass spectrometry and total nitrogen analysis. Excess 15N was detected in whole lungs and in soluble and insoluble fractions but not in the total lipid pool. Perfusate excess 15N and nitrate correlated and accounted for all absorbed NO2 not detectable in tissue fractions. Exogenously instilled [15N]nitrite distributed within lung tissue, bound to insoluble elements, and diffused to the vascular space similar to NO2-nitrogen. Instilled [15N]nitrate did not distribute or bind like NO2-nitrogen or nitrite. Dialysis (1000 molecular weight cutoff) of cytosol, membranes, and perfusate removed excess 15N and nitrite derived from NO2, nitrite, or nitrate sources. We conclude that in isolated lungs, inhaled NO2 (1) undergoes rapid uptake and transformation in sites accessible to the pulmonary circulation; (2) does not form stable addition products with lipids; and (3) forms small-molecular-weight soluble reaction product(s) that behave similarly to nitrite, most likely indicating predominant univalent reduction of NO2 via initial hydrogen abstraction and subsequent HNO2 dissociation.

Administration, Inhalation

Characterization of transferrin binding proteins 1 and 2 in invasive type b and nontypeable strains of Haemophilus influenzae.

Haemophilus influenzae has the ability to obtain iron from human transferrin via two bacterial cell surface transferrin binding proteins, Tbp1 and Tbp2. Although a wide array of strains have been shown to express these receptor proteins, two studies have recently identified a series of isolates which appeared to lack the ability to bind transferrin. Included in this group were the members of a cryptic genospecies of nontypeable biotype IV strains which appear to possess a tropism for female urogenital tissues and are major etiologic agents of neonatal and postpartum bacteremia due to H. influenzae. The present study employed oligonucleotide primers specific for genes encoding the Tbp proteins of a type b biotype I strain of H. influenzae to probe the genomic DNAs of isolates from the previous studies. The tbpA and tbpB genes which encode Tbp1 and Tbp2, respectively, were detected in all of the strains tested either by PCR amplification directly or by Southern hybridization analysis. All of the strains displayed a transferrin binding phenotype, and affinity isolation of receptor proteins with transferrin-conjugated Sepharose recovered Tbp1 and/or Tbp2 from 11 of 14 strains, including 2 of the nontypeable biotype IV strains. In addition, all of the strains were capable of growing on human transferrin specifically, indicating that the mechanism of iron assimilation from transferrin is functional and is not siderophore mediated. These results confirm the presence of tbp genes in all of the invasive H. influenzae isolates characterized to date, suggesting that Tbp-mediated iron acquisition is important in disease which initiates from either the respiratory or urogenital mucosa.

Bacterial Proteins

Quail myoD is regulated by a complex array of cis-acting control sequences.

Quail myoD (QmyoD) is the earliest myoD family member expressed in quail somites and its transcription is initiated in response to early developmental signals. We have investigated the transcriptional regulation of QmyoD to define the cis-acting sequences required for tissue-specific and correct developmental expression. The QmyoD gene locus was isolated and sequenced and its regulatory properties were characterized. We identified three distinct regions of cis-acting regulatory sequences that control the expression of reporter gene constructs following DNA transfection into cell lines and cultured primary quail cells. The first, a complex distal control region (DCR), 11.5 kb upstream of the gene, contains three separable enhancer activities. Two of these DCR enhancer activities are tissue specific and can be autoactivated. In addition, these same two enhancers and the entire DCR direct somite- and muscle-specific expression of a reporter gene in transgenic mice. Sequence analysis of the DCR enhancers reveals clusters of E-boxes, MEF2 binding motifs, and the stretches of sequence identity with the human myoD enhancer. Second, the promoter region has sequences which act positively to direct expression in both muscle and nonmuscle cells as well as sequences that repress expression specifically in nonmuscle cells. The third control region, the PR, is located -3.3 to -5 kb from the transcription start site and directs muscle-specific expression in cultured cells. This analysis demonstrates that QmyoD has multiple control regions and that some features of myoD regulation are conserved between mammals and birds.

Animals

[Prosthetic possibilities with the Denar "Steri-Oss" implant].

Implantology, very frequently presented under only the surgical angle, must in fact be approached from a biological and functional point of view. The analysis of the future prosthetic situation will allow an overall treatment scheme to be established, in which periodontics, surgery, prosthodontics and occlusal equilibration will follow each other in order to achieve the functional rehabilitation. In the first part of this article, the various steps which make up the treatment scheme are briefly and quickly described. It is imperative to approach them according to this chronological order when there is any treatment involving implants. While implantology involved only one surgical session a few years ago, it now involves two: the first, consisting of placing the implant in the living tissues, and the second, at some months interval from the first, allowing the endo-osseous portion to be attached to the future prosthesis. These two surgical acts are inter-dependent. Given the wide array of systems proposed and the quantity of "Implant-Prosthesis" connections produced in each system, the practitioner does not always know which kind of connection to choose or how to use it. The second part of the article is devoted to the study of the various connection possibilities between the Steri-oss implants and the prosthesis. The methods for lining up the cores in relation to the others are studied in detail, as are the different possibilities for stabilising removable prosthesis. Some of the methods described can be used with other implant systems.

Dental Implantation, Endosseous

Evolution of the cryptic FMR1 CGG repeat.

We have sequenced the 5' untranslated region of the orthologous FMR1 gene from 44 species of mammals. The CGG repeat is present in each species, suggesting conservation of the repeat over 150 million years of mammalian radiation. Most mammals possess small contiguous repeats (mean number of repeats = 8.0 +/- 0.8), but in primates, the repeats are larger (mean = 20.0 +/- 2.3) and more highly interrupted. Parsimony analysis predicts that enlargement of the FMR1 CGG repeat beyond 20 triplets has occurred in three different primate lineages. In man and gorilla, AGG interruptions occur with higher-order periodicity, suggesting that historical enlargement has involved incremental and vectorial addition of larger arrays demarcated by an interruption. Our data suggest that replication slippage and unequal crossing over have been operative during the evolution of this repeat.

Animals

Comparison of linear array B-mode scanning and computed synthetic aperture ultrasonographic imaging.

The current trend in ultrasonography is toward digital systems that allow more freedom in beam formation and produce improved images. In this paper, conventional linear array B-mode sonograms of a tissue-mimicking phantom are compared to sonograms obtained with a synthetic aperture technique employing numerical image focusing. Sonograms in both cases were obtained using the same equipment to ensure identical imaging conditions. The computed synthetic aperture approach produced uniform spatial resolution of about 1 mm, which could not be achieved with the B-scan technique. In addition, the contrast resolution of the computed synthetic aperture method was found to be superior. We conclude that the computed synthetic aperture method is feasible for real-time clinical applications.

Algorithms

A Practical Workflow for Spatial Transcriptomics Data Analysis: From Data Acquisition to Advanced Analyses.

Spatial transcriptomics (ST) profiles genome-wide gene expression while preserving the two-dimensional spatial context of mRNA molecules within tissue sections, enabling studies of tissue architecture and microenvironment-associated biology. However, ST analysis remains challenging because data import, quality control, integration, deconvolution, spatial statistics, and visualization often require multiple software environments and reproducible parameter choices. This protocol presents a practical computational workflow for public ST datasets in R, beginning with data acquisition and software setup and proceeding through Seurat-based data loading, quality control, normalization, multi-sample integration, clustering, and spatially variable gene analysis. The workflow then applies complementary deconvolution strategies, including reference-guided SPOTlight analysis and unsupervised STdeconvolve topic modeling, followed by Giotto-based spatial cell-cell communication analysis and interactive region-of-interest (ROI) selection using a custom Python Dash application. By emphasizing script-based execution, explicit parameter rationales, expected outputs, and troubleshooting checkpoints, the protocol provides an adaptable framework for standard array-based ST datasets and related platforms after dataset- and platform-specific parameter evaluation.

Spatial Transcriptomics

Human in situ dosimetry: differential insertion loss during passage through abdominal wall and myometrium.

We constructed a specialized in vivo exposimetry system and determined selected ultrasonic field quantities. We examined two groups of non-pregnant women (nulliparas = 14, multiparas = 9) under conditions of full and empty bladder. A calibrated 7-element linear array hydrophone was placed in the anterior fornix of the vagina in each subject. In the full bladder condition, the sound beam traversed the anterior abdominal wall and full bladder, whereas after voiding, the sound beam traversed the abdominal wall and anteverted uterine fundus. Each study was conducted using a 3.5 MHz mechanical sector transducer. Calibration data were recorded after completion of each in vivo experiment. Data from both groups were pooled for analysis. Assuming (1) the sound path through the full bladder is loss less, the insertion loss (ILFULL) should represent the insertion loss for the abdominal wall (ILABD WALL) 8.2 +/- 5.6 dB; whereas (2) for the empty bladder condition, (ILEMPTY) represents (ILABD WALL+ILUTERUS). Subtracting ILFULL from ILEMPTY yields ILUTERUS = 5.8 +/- 6.8 dB. Therefore, knowing the respective path lengths and normalizing for frequency, the mean tissue attenuation coefficients (A) are estimated to be AABD WALL = 1.39 dB/cm-MHz and AUTERUS = 0.14 dB/cm-MHz. These attenuation data suggest that the abdominal wall is the principal source of ultrasonic energy loss.

Abdominal Muscles

Altered DNA Methylation Pattern Contributes to Differential Epigenetic Immune Signaling in the Upper Respiratory Airway of Unvaccinated COVID-19 Patients.

SARS-CoV-2 infection remains a global health concern, with its impact on host immune responses not fully understood. In a case-control study, we examined how COVID-19 affects DNA methylation patterns in the upper respiratory airway of hospitalized individuals. DNA methylation arrays were performed on nasopharyngeal samples at inclusion/hospitalization and 6 weeks post-inclusion. We found a distinct DNA methylation pattern in COVID-19 patients compared to healthy controls, identifying 510,099 differentially methylated CpGs. Within the transcription start sites (TSSs) and gene body, COVID-19 patients displayed a higher number of genes/CpGs with elevated methylation levels. Enrichment analysis of TSS-methylated genes revealed effects of SARS-CoV-2 on genes associated with type I interferons, anti-viral and inflammatory responses, and immune functions. Some CpG methylations were transient, and normalized at group level by 6 weeks post-inclusion. Several IFN-regulated genes, including OAS1, OAS3, IFIT3, and MX1, were identified. Among the top regulators were IL17A and ERK1/2, both involved in inflammatory processes. Networks nodes included IGF1 and EGF, associated with processes including tissue repair and activation of immune responses. Overall, our data suggests that COVID-19 can impact the upper airway by modifying gene methylation patterns. This could have implications for conditioning of the airways, how individuals respond to future airway infections, and therapeutic interventions.

Humans

Design and fabrication of a capillary cell culture chamber for the study of convective flow.

The use of capillary culture chambers as artificial pancreas and artificial liver devices would be aided by an improved ability to control the movement of molecules through the capillary walls. The modeling and analysis of the flow and mass transfer in capillary cell culture chambers in which cultured mammalian cells are grown to form masses with tissue density in the extra-capillary spaces is desirable as a basis for scaleup and optimization of the particular microenvironment. The relative roles of diffusion and ultrafiltration with convection in enhancing mass transfer across the capillary membranes are poorly understood in real cell cultures and the effects on culture viability of flow conditions chosen to promote convective flow across the capillary membranes and through the cultured cell masses are also of interest. In this report, experience with materials and techniques for fabricating capillary culture chambers with a more readily analyzed fully defined regular geometric relationship between 90 to 100 capillaries in a parallel bundle is described. High and low pressure capillaries were interspersed in a regular array. Evidence is presented for retention of cell viability during flow conditions which were chosen to induce convective flow through the cultured cells in such a chamber.

Artificial Organs

Cardiac tissue geometry as a determinant of unidirectional conduction block: assessment of microscopic excitation spread by optical mapping in patterned cell cultures and in a computer model.

OBJECTIVE: Unidirectional conduction block (UCB) and reentry may occur as a consequence of an abrupt tissue expansion and a related change in the electrical load. The aim of this study was to evaluate critical dimensions of the tissue necessary for establishing UCB in heart cell culture. METHODS: Neonatal rat heart cell cultures with cell strands of variable width emerging into a large cell area were grown using a technique of patterned cell growth. Action potential upstrokes were measured using a voltage sensitive dye (RH-237) and a linear array of 10 photodiodes with a 15 microns resolution. A mathematical model was used to relate action potential wave shapes to underlying ionic currents. RESULTS: UCB (block of a single impulse in anterograde direction - from a strand to a large area - and conduction in the retrograde direction) occurred in narrow cell strands with a width of 15(SD 4) microns (1-2 cells in width, n = 7) and there was no conduction block in strands with a width of 31(8) microns (n = 9, P < 0.001) or larger. The analysis of action potential waveshapes indicated that conduction block was either due to geometrical expansion alone (n = 5) or to additional local depression of conduction (n = 2). In wide strands, action potential upstrokes during anterograde conduction were characterised by multiple rising phases. Mathematical modelling showed that two rising phases were caused by electronic current flow, whereas local ionic current did not coincide with the rising portions of the upstrokes. CONCLUSIONS: (1) High resolution optical mapping shows multiphasic action potential upstrokes at the region of abrupt expansion. At the site of the maximum decrement in conduction, these peaks were largely determined by the electrotonus and not by the local ionic current. (2) Unidirectional conduction block occurred in strands with a width of 15(4) microns (1-2 cells).

Action Potentials