What is the evidence status of Appropriate Use Criteria (AUC)? Insight from a matching exercise with the guidelines for echocardiography.

There is interest in adapting the American Appropriate Use Criteria (AUC) for transthoracic echocardiography to Australian practice. We matched 90 of 98 AUC with the guidelines (53 appropriate, 12 sometimes appropriate, 25 rarely appropriate), but eight lacked any match. Among the matched criteria, 76 (82%) indications were concordant with the guidelines. A stronger evidence base would be desirable to settle these discrepancies before Australian adoption of AUC.

Comparative study of treatment efficacy and the incidence of post-inflammatory hyperpigmentation with different degrees of irradiation using two different quality-switched lasers for removing solar lentigines on Asian skin.

BACKGROUND: Quality-switched (QS) lasers are well-known effective treatment for removing solar lentigines. However, the high incidence of post-inflammatory hyperpigmentation (PIH) raises concern in darker skin types. This is the first study comparing efficacies and incidences of PIH in Asian skin with different degrees of irradiation between two QS lasers. METHOD: In total, 355 solar lentigines in 193 cases, skin types III-V, were randomly divided into four groups. All cases received single laser treatment. Clinical results were evaluated after 4 weeks. Groups 1 and 3 were treated 'aggressively' with endpoints of very obvious immediate whitening (IW) of the lesion. Groups 2 and 4 were treated 'mildly' with endpoints of slight IW of the lesion. Groups 1 and 2 were irradiated with the QS ruby, and groups 3 and 4 with the QS frequency doubled Nd:YAG laser. RESULTS: There were no statistically significant differences in degrees of clearance among the four groups. However, PIH incidences were very different: 33.33%, 7.47%, 23.18% and 8.47% in groups 1, 2, 3 and 4 respectively. The difference between aggressively and mildly irradiated groups (1 and 3 vs. 2 and 4) was statistically significant (P < 0.001). However, there was no statistical difference between the two aggressively or the two mildly irradiated groups. There were no significant differences between skin types. CONCLUSION: Aggressive irradiation using QS lasers resulted in a high PIH incidence, while having no advantage in efficacy. For darker skin types, mild irradiation reduces the PIH risk with no disadvantage in efficacy.

Oxygen dependence of retinal S-potential-producing cells.

Changes in the membrane potential of the S-potential-producing cells (S-cells) in the isolated retina of fish (Gerridae) were correlated with changes in oxygen concentration. During brief hypoxia the changes in potential consisted of initial depolarization and subsequent hyperpolarization to near 70 millivolts. Depolarization occurred when oxygen concentration was reduced to a level of from 13 to 10 percent, and hyperpolarization occurred on reduction from 10 to 2.5 percent; there was yariation from cell to cell. The recovery of S-cell function from anoxia was fast in oxygen but slow in air. The results show that the S-cell stops functioning in seconds without oxygen; hence this kind of cellular element in the nervous system is much more sensitive to oxygen deprivation than other cells studied thus far.

New dopaminergic and indoleamine-accumulating cells in the growth zone of goldfish retinas after neurotoxic destruction.

Juvenile goldfish were allowed to grow for 3 months after dopaminergic or indoleamine-accumulating cells in their retinas had been destroyed by intravitreal injection of 6-hydroxydopamine or 5,7-dihydroxytryptamine, respectively. New cells of each type were found growing in concentric rings at the margin of the retina. To compensate for the loss of dopaminergic innervation in retinas treated with 6-hydroxydopamine, cells in the growth zone appeared to proliferate at a higher rate than those in untreated retinas and long processes were extended into the retina by the first dopaminergic cells to appear.

Renal L-type fatty acid-binding protein mediates the bezafibrate reduction of cisplatin-induced acute kidney injury.

Fibrates, the PPAR alpha ligand-like compounds increase the expression of proximal tubule liver fatty acid binding protein (L-FABP) and significantly decrease cisplatin-induced acute kidney injury. To study whether the bezafibrate-mediated upregulation of renal L-FABP was involved in this cytoprotective effect we treated transgenic mice of PPAR agonists inducible human L-FABP expression with cisplatin in the presence or absence of bezafibrate. Blood urea nitrogen was unchanged in the first day but increased 3 days after cisplatin. While urinary L-FABP increased over 100-fold 1 day after cisplatin treatment in the transgenic mice it was significantly reduced when these transgenic mice were pretreated with bezafibrate. Cisplatin-induced renal necrosis and apoptosis were significantly reduced in bezafibrate pretreated transgenic mice and this correlated with decreased accumulation of lipid and lipid peroxidation products. Immunohistochemical analysis of kidney tissue of bezafibrate-cisplatin-treated transgenic mice showed preservation of cytoplasmic L-FABP in the proximal tubule, but this was reduced in transgenic mice treated only with cisplatin. L-FABP mRNA and protein levels were significantly increased in bezafibrate-cisplatin-treated transgenic mice when compared to mice not fibrate treated. Our study shows that the bezafibrate-mediated upregulation of proximal tubule L-FABP plays a pivotal role in the reduction of cisplatin-induced acute kidney injury.

Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.

DNA strand cleavage in vitro by 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]-indole, a direct-acting mutagen formed in the metabolism of carcinogenic 3-amino-1-methyl-5H-pyrido[4,3-b]indole.

3-Hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) is a direct-acting mutagenic compound derived by metabolic activation from 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a strongly mutagenic carcinogen. The action of N-OH-Trp-P-2 on DNA in vitro was investigated. N-OH-Trp-P-2 inactivated Bacillus subtilis transforming DNA and produced single-strand cuts in a supercoiled circular DNA (phi X174RFI) under neutral conditions. When mouse FM3A cells in culture were treated with a noncytotoxic dose of N-OH-Trp-P-2 and then the cellular DNA was examined by the alkaline elution technique, chain cleavages of the DNA were observed. Cysteamine inhibited the spontaneous degradation of N-OH-Trp-P-2 and enhanced the covalent binding of [3H]N-OH-Trp-P-2 to DNA. This finding offered an explanation for the previously observed enhancement of Trp-P-2 mutagenicity by cysteamine. In contrast cysteamine inhibited the N-OH-Trp-P-2-mediated inactivation of B. subtilis DNA as well as the strand cleavage in phi X174RFI DNA. The cleavage in phi X174RFI DNA was also inhibited by catalase. These observations indicate that the mutagenicity and DNA-cleaving activity of N-OH-Trp-P-2 are distinct from each other, that the inactivation of transforming DNA was caused mainly by strand cleavage, and that the DNA cleavage was probably caused by active oxygen radicals produced in the oxidative degradation of N-OH-Trp-P-2.

Studies of midaglizole (DG-5128). A new type of oral hypoglycemic drug in healthy subjects.

Midaglizole (DG-5128), 2-[2-(4,5-dihydro-1H-imidazol-2-yl)-1-phenylethyl]pyridine dihydrochloride sesquihydrate, is a novel alpha 2-adrenoceptor antagonist. Its effects on plasma glucose, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG) in healthy male volunteers were investigated. Volunteers received single oral administrations of midaglizole (150-500 mg), multiple increasing oral administration on 3 separate days (150-300 mg 3 times daily), or successive daily oral administration for 1 wk (200 mg 3 times daily). The hypoglycemic action of midaglizole was observed within 0.5-1.0 h after its administration and thereafter for 5 h. The maximum hypoglycemic effect was found 1.0-1.5 h after administration. Midaglizole decreased postprandial hyperglycemia in a dose-dependent manner. In the fasting state, midaglizole significantly increased IRI secretion and suppressed IRG secretion. Midaglizole inhibited epinephrine-induced platelet aggregation after successive administration for 1 wk (200 mg 3 times daily). The plasma half-life of midaglizole was only 3 h, and the drug was rapidly excreted into the urine and feces, with greater than 80% in its unchanged form, within 24 h. Midaglizole did not affect the results of any clinical or laboratory tests performed. Our data indicate that midaglizole is a possible hypoglycemic agent. Further clinical investigations are required to confirm its effects on diabetes mellitus.

Initial phase II clinical studies on midaglizole (DG-5128). A new hypoglycemic agent.

Midaglizole (DG-5128), 2-[2-(4,5-dihydro-1H-imidazol-2-yl)-1-phenylethyl]pyridine dihydrochloride sesquihydrate, is a new type of oral antidiabetic agent that has an alpha 2-adrenoceptor-antagonizing effect. As previously reported, midaglizole reduces plasma glucose, mainly by stimulation of insulin secretion, and inhibits epinephrine-induced platelet aggregation in normal human subjects. In this study, the clinical safety and efficacy of short-term administration of midaglizole were evaluated in 47 patients with non-insulin-dependent diabetes mellitus (NIDDM). After an observation period on diet or sulfonylurea treatment (1 patient was on insulin), patients received 150-250 mg 3 times a day of midaglizole for 2-4 wk, (some patients continued treatment for greater than 4 wk). In 20 of the patients first treated with diet and then switched to midaglizole treatment, fasting plasma glucose (FPG) decreased significantly from 187 +/- 10 mg/dl (mean +/- SE) to 147 +/- 13 mg/dl (P less than .05) and 120 +/- 6 mg/dl (P less than .01) 2 and 4 wk, respectively, after administration of midaglizole. Glycosylated hemoglobin (HbA1) also decreased from 12.0 +/- 0.7 to 11.3 +/- 1.1 and 10.7 +/- 0.6% after 2 and 4 wk, respectively. In 23 of the patients whose treatment was changed from sulfonylureas to midaglizole, FPG, and HbA1 levels were maintained at the same values obtained before administration of midaglizole. In patients treated with midaglizole for greater than 12 wk, FPG and HbA1 were kept at the lowered levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for association between the class I subset of the insulin gene minisatellite (IDDM2 locus) and IDDM in the Japanese population.

Although the shortest (class I) minisatellite (i.e., variable number of tandem repeats [VNTR]) alleles in the 5' region of the insulin gene are positively associated with IDDM in Caucasians, the majority of Japanese are homozygous for class I alleles. Here, we determined the exact length, in number of repeat units (RUs), of class I alleles in Japanese subjects. The distribution of class I alleles in Japanese was trimodal, with peaks located at 32/33, 41, and 44 RUs. The shortest component (i.e., 1S [25-38 RUs]) alleles were significantly increased in the IDDM group compared with the control group (54 vs. 46%; P = 0.040). The 1S/1S genotype was significantly increased in the IDDM patients (34 vs. 20%; P = 0.005; relative risk 2.1). Furthermore, the transmission disequilibrium test of Japanese families with 1S/1M or 1S/1L heterozygous parents confirmed the association of 1S alleles; 17 alleles of 1S and 6 alleles of 1M (39-41 RUs) or 1L (42-44 RUs) were transmitted to affected offspring (P = 0.022). In addition, we found tight linkage of 1S with allele 9 of the tyrosine hydroxylase gene microsatellite and allele (-) of the IGF-II gene Apa I polymorphism, but neither 9 nor (-) alleles were significantly associated with IDDM. The present study suggests that a class I subset may have a role in IDDM susceptibility in Japan. It was revealed that the difference between 1S alleles and 1M or 1L alleles is almost consistently characterized by a sequence variation generated by deletion of two copies of an ACAGGGGTCC CGGGG repeat element, implying that sequence variation of class I alleles may influence disease susceptibility.

Spectrum of N4-aminocytidine mutagenesis.

N4-Aminocytidine, a nucleoside analog, is a potent mutagen towards phages, bacteria, Drosophila and mammalian cells in culture. In vitro, biochemical studies indicate that this reagent acts by being incorporated into DNA. To elucidate the mechanism of N4-aminocytidine mutagenesis, it is essential to identify the nature of DNA sequence alterations taking place during the mutagenesis. We have analyzed the nucleotide sequence changes in the lac promoter-lacZ alpha region of M13mp2 phage induced by treatment of phage-infected Escherichia coli with N4-aminocytidine. The sequence alterations of DNA samples from 89 mutants of the phage were determined. These mutants had single point mutations, except one mutant, in which a double point mutation was detected. Several hot spots were found: however, there are no apparent relations to particular DNA sequences regarding the locations of these spots. All the mutations are transitions; neither transversions nor deletions/insertions were found. A feature in these transitions is that the A/T to G/C and G/C to A/T changes occur at approximately equal rates. The overall picture of the mutagenesis is consistent with a scheme in which misincorporation and misreplication caused by the modified cytosine structure are the key steps in the DNA replication leading to transitions. Similar nucleotide alterations were found for the mutagenesis induced by an alkylated derivative, N'-methyl-N4-aminocytidine. N4-Aminocytidine also induced reversions of these mutants; both A/T to G/C and G/C to A/T transitions again took place.

The eyes of deep-sea fish. II. Functional morphology of the retina.

Three different aspects of the morphological organisation of deep-sea fish retinae are reviewed: First, questions of general cell biological relevance are addressed with respect to the development and proliferation patterns of photoreceptors, and problems associated with the growth of multibank retinae, and with outer segment renewal are discussed in situations where there is no direct contact between the retinal pigment epithelium and the tips of rod outer segments. The second part deals with the neural portion of the deep-sea fish retina. Cell densities are greatly reduced, yet neurohistochemistry demonstrates that all major neurotransmitters and neuropeptides found in other vertebrate retinae are also present in deep-sea fish. Quantitatively, convergence rates in unspecialised parts of the retina are similar to those in nocturnal mammals. The differentiation of horizontal cells makes it unlikely that species with more than a single visual pigment are capable of colour vision. In the third part, the diversity of deep-sea fish retinae is highlighted. Based on the topography of ganglion cells, species are identified with areae or foveae located in various parts of the retina, giving them a greatly improved spatial resolving power in specific parts of their visual fields. The highest degree of specialisation is found in tubular eyes. This is demonstrated in a case study of the scopelarchid retina, where as many as seven regions with different degrees of differentiation can be distinguished, ranging from an area giganto cellularis, regions with grouped rods to retinal diverticulum.

Supplement nephropathy due to long-term, high-dose ingestion of ascorbic acid, calcium lactate, vitamin D and laxatives.

A 48-year-old Japanese woman previously in good health was found to have severe proximal tubular dysfunction with a high serum level of ascorbic acid (57.3 microg/ml, reference range: 1.9 - 15.0 microg/ml). Renal biopsy specimen showed marked tubulointerstitial damage, i.e. tubular atrophy, dilatation of tubular lumen with flattened tubular epithelial cells, vacuolization of proximal and distal tubular epithelial cells, and severe interstitial fibrosis with mild infiltration of mononuclear cells. Calcified lesions, which caused tubular obstruction or stenosis, were also seen in interstitial area adjacent to degenerated proximal tubuli. Hypokalemic nephropathy, probably due to long-term use of laxatives, was clearly shown. However, calcified lesions seemed to be caused by inappropriate excessive daily ingestion of ascorbic acid (6 000 mg/day), calcium lactate, and vitamin D because of the patient's misunderstanding that these supplements could keep her in a good health. This condition may be clinically called "supplement nephropathy".

A new reaction useful for chemical cross-linking between nucleic acids and proteins.

Cytosine in nucleic acids can be converted into N4-aminocytosine by treatment with a mixture of hydrazine and bisulfite. The hydrazino group thus formed at position 4 of the pyrimidine ring can be linked to a sulhydryl group in proteins by the use of bromopyruvate as a linker. Successful use of this scheme of chemical cross-linking between nucleic acid and protein was demonstrated in the linking of poly(C) with glutathione, and of RNA with protein in the E. coli 30 S ribosomal subunit.

Postnatal development of dopaminergic cells in the rat retina.

By means of a histofluorescence technique (FGS method), a post-natal ontogeny of dopaminergic neurons (DA-cells) in the retina was studied in rats which had been born and reared under diurnal lighting (LD), reversed lighting (DL), constant lighting (LL), or continuous dark conditions (DD). The time of first appearance of DA-cells was not altered by any of the above conditions; weakly fluorescent cell bodies were visible at about the 10th postnatal day. After the eyes were open the DA fluorescence of the cell body was gradually increased in intensity in all LD, DL, and LL rats, but not for DD rats. At the 16-17th day the processes of the cell first became visible, extending laterally to the inner plexiform layer. The somata and their processes appeared to be well developed at the postnatal 4-6th week, and they resembled those of the adult rat. On the other hand, the DA-cells in the DD rats showed a weak fluorescence in both the somata and their processes. When the DD rats were transferred and reared under LD conditions for 1-3 days, the DA fluorescence was increased. These results strongly suggest that the initiation of retinal DA synthesis is independent of environmental lighting condition, but an adequate light stimulus is required for continued normal development of DA-cells. Unlike in the rat, fluorescent cells in the guinea pig and chick retinas were observed to be fairly mature at birth or hatching. The developmental pattern of DA-cells in the three species examined coincides with the maturation of visual function.

Embryonic development of monoaminergic neurons in the chick retina.

By means of a histofluorescence technique, embryonic and postnatal development of monoaminergic neurons was followed in the chicken retina with or without intravitreal injection of monoamines 30-60 minutes before eye removal. Fluorescent cells were tentatively classified into five subsets with respect to the soma shape, localization, migration of somata during retinal development, uptake capacity (color and intensity in fluorescence), and sensitivity to neurotoxins. The five subsets of cells were endogenous dopaminergic (DA), catecholamine-accumulating (CA), indoleamine-accumulating (IA), CA-bipolarlike, and IA-bipolarlike cells. Greenish endogenous DA-cells first appeared at the 14-15th embryonic day. The cell body of DA-cells was initially fusiform and located slightly distal to the innermost level of the inner nuclear layer (INL). They became round or oval and migrated to the innermost level of the INL by day 20. Both large and small bottle-shaped CA-cells were visualized at an intermediate portion of INL by intravitreal injection of exogenously applied dopamine or noradrenaline (1-2 micrograms/eye) at day 10. Large bottle-shaped cells, like the DA cells, changed to round or oval and migrated to the innermost level of the INL by day 20. On the other hand, small bottle-shaped CA-cells retained their cell shape and location in the INL as retinal development progressed. Therefore, the large bottle-shaped CA-cells seen in an early developmental stage correspond to the DA-cells. IA-cells were visualized one or two cell rows outward in the INL first at day 13-14 by intravitreal injection of 5,6-dihydroxytryptamine or 5-hydroxytryptamine (1-5 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin levels during fasting and the glucose tolerance test and Homa's index predict subsequent development of hypertension.

OBJECTIVE: To determine whether there is a longitudinal relationship between hypertension and hyperinsulinemia and to find the most useful parameter(s) for predicting the subsequent development of hypertension. SUBJECTS AND METHODS: The oral glucose (75 g) tolerance test (OGTT) was performed in 313 patients, who were divided into three groups according to glucose tolerance based on the WHO criteria: normal, borderline and diabetes mellitus. The fasting insulin (IRI) levels, sigmaIRI (the sum of the insulin levels 0, 30, 60 and 120 min after the OGTT), insulinogenic index and Homa's index, a candidate for the simple assessment of insulin sensitivity, of the normotensive and hypertensive subjects in each subgroup were compared. In addition, 145 normotensive subjects were followed up for over 3 years and observed for the development of hypertension. RESULTS: Hypertensive diabetic subjects had not only higher fasting IRI levels and sigmaIRI values, but they also had higher Homa's indices than normotensive diabetics. Normotensive subjects with normal glucose tolerance (n = 20) did not develop hypertension. However, 16 out of 94 patients with borderline glucose tolerance and five out of 31 diabetics became hypertensive. The incidence of hypertension in the group with fasting IRI > or = 15, sigmaIRI > or = 150 or Homa's index > or = 4 was between 5 and 9 times higher than that in the group with fasting IRI < 10, sigmaIRI < 100 or Homa's index < 2. This difference was still significant when multivariate analysis, including various factors such as age, body mass index (BMI) and sex, was performed. CONCLUSIONS: These results suggest that higher plasma IRI levels and/or insulin resistance are closely related to the pathogenesis of hypertension in patients with diabetes mellitus. Homa's index, fasting and sigmaIRI may be useful predictors of the subsequent development of hypertension.

Double-staining of horizontal and amacrine cells by intracellular injection with lucifer yellow and biocytin in carp retina.

Horizontal and amacrine cells in the isolated carp retina were impaled with micropipette electrode, identified by their characteristic light responses, and injected iontophoretically with markers for morphological study. Both Lucifer Yellow CH and biocytin were injected simultaneously. Lucifer Yellow was seen by its own fluorescence while biocytin was visualized by binding with Texas Red-linked or horseradish peroxidase-conjugated avidin. For cone-connected horizontal cells, biocytin-coupled cells were found to be approximately five-times more numerous than Lucifer Yellow-coupled cells. Coupling for both tracers was consistently hampered by intravitreally applied dopamine. In untreated retinas, the injected Lucifer Yellow was restricted within one rod-connected horizontal cell, while biocytin revealed several coupled neighbors. Amacrine cells, labeled by the tracers, were morphologically grouped into eight types, based on our earlier classification. Among them, amacrine cells, belonging to three types (Fnd, Pmb or Pma), were confirmed to be Lucifer Yellow-coupled, and the number of biocytin-coupled cells was more numerous (about 2.5 times) than that of Lucifer Yellow-coupled cells. Most amacrine cells (i.e. Pwd, Fnb and Fna) showed biocytin-coupling with no Lucifer Yellow-coupling. A few classified (i.e. Pwb and Fwa) and unclassified cells did not show any coupling. Since the tracer coupling takes place via gap junctions, the majority of amacrine cells, belonging to certain homologous types, appear to be functionally coupled with each other in the inner plexiform layer. However, dopamine did not influence the range of tracer coupling between amacrine cells in the carp retina under the present experimental conditions.

Functional and morphological correlates of amacrine cells in carp retina.

Experiments were conducted on isolated retinas of adult carp (Cyprinus carpio) to investigate correlations between photoresponses and morphological features of amacrine cells. The fluorescence dye Lucifer Yellow CH was iontophoretically injected into single cells which had been characterized electrophysiologically. Photoresponses were classified into two main types (transient ON-OFF and sustained), which were further subgrouped into "fast" and "slow" ON-OFF types and into ON-center and OFF-center types, respectively. In the spectral response curve, all the types dealt with showed a maximum response at 621 nm, indicating that main input signals derive from red-sensitive cones. The cells marked by intracellular injection of the dye showed a great variety in morphology. Cells were classified into 8 subtypes, based on soma shape (fusiform or pyriform), dendritic field area (narrow, less than 0.3 mm2; medium, 0.3-0.8 mm2; wide, greater than 0.8 mm2), and dendritic stratification in the inner plexiform layer (restricted to sublamina a or b, or distributed diffusely). In certain cases a given response type was correlated with a specific morphological type, while receptive field size was not strictly correlated with dendritic field areas. Long and fine peripheral "axon-like" processes were found to arise from the primary dendrites of most fusiform cells. Dye-coupling was found among cells which appear to belong to the same cell category.