The Role of Lamins in the Nucleoplasmic Reticulum, a Pleiomorphic Organelle That Enhances Nucleo-Cytoplasmic Interplay.

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer's disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.

Novel fungicidal benzylsulfanyl-phenylguanidines.

A series of substituted benzylsulfanyl-phenylamines was synthesized, of which four substituted benzylsulfanyl-phenylguanidines (665, 666, 667 and 684) showed potent fungicidal activity (minimal fungicidal concentration, MFC ≤ 10 µM for Candida albicans and Candida glabrata). A benzylsulfanyl-phenyl scaffold with an unsubstituted guanidine resulted in less active compounds (MFC=50-100 µM), whereas substitution with an unsubstituted amine group resulted in compounds without fungicidal activity. Compounds 665, 666, 667 and 684 also showed activity against single C. albicans biofilms and biofilms consisting of C. albicans and Staphylococcus epidermidis (minimal concentration resulting in 50% eradication of the biofilm, BEC50 ≤ 121 µM for both biofilm setups). Compounds 665 and 666 combined potent fungicidal (MFC=5 µM) and bactericidal activity (minimal bactericidal concentration, MBC for S. epidermidis ≤ 4 µM). In an in vivo Caenorhabditis elegans model, compounds 665 and 667 exhibited less toxicity than 666 and 684. Moreover, addition of those compounds to Candida-infected C. elegans cultures resulted in increased survival of Candida-infected worms, demonstrating their in vivo efficacy in a mini-host model.

A fungicidal piperazine-1-carboxamidine induces mitochondrial fission-dependent apoptosis in yeast.

To unravel the working mechanism of the fungicidal piperazine-1-carboxamidine derivative BAR0329, we found that its intracellular accumulation in Saccharomyces cerevisiae is dependent on functional lipid rafts. Moreover, BAR0329 induced caspase-dependent apoptosis in yeast, in which the mitochondrial fission machinery consisting of Fis1 (Whi2), Dnm1 and Mdv1 is involved. Our data are consistent with a prosurvival function of Fis1 (Whi2) and a proapoptotic function of Dnm1 and Mdv1 during BAR0329-induced yeast cell death.

Membrane rafts are involved in intracellular miconazole accumulation in yeast cells.

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.

Design and synthesis of a series of piperazine-1-carboxamidine derivatives with antifungal activity resulting from accumulation of endogenous reactive oxygen species.

In this study, we screened a library of 500 compounds for fungicidal activity via induction of endogenous reactive oxygen species (ROS) accumulation. Structure-activity relationship studies showed that piperazine-1-carboxamidine analogues with large atoms or large side chains substituted on the phenyl group at the R(3) and R(5) positions are characterized by a high ROS accumulation capacity in Candida albicans and a high fungicidal activity. Moreover, we could link the fungicidal mode of action of the piperazine-1-carboxamidine derivatives to the accumulation of endogenous ROS.

Re-expression of alpha skeletal actin as a marker for dedifferentiation in cardiac pathologies.

Differentiation of foetal cardiomyocytes is accompanied by sequential actin isoform expression, i.e. down-regulation of the 'embryonic' alpha smooth muscle actin, followed by an up-regulation of alpha skeletal actin (alphaSKA) and a final predominant expression of alpha cardiac actin (alphaCA). Our objective was to detect whether re-expression of alphaSKA occurred during cardiomyocyte dedifferentiation, a phenomenon that has been observed in different pathologies characterized by myocardial dysfunction. Immunohistochemistry of alphaCA, alphaSKA and cardiotin was performed on left ventricle biopsies from human patients after coronary bypass surgery. Furthermore, actin isoform expression was investigated in left ventricle samples of rabbit hearts suffering from pressure- and volume-overload and in adult rabbit ventricular cardiomyocytes during dedifferentiation in vitro. Atrial goat samples up to 16 weeks of sustained atrial fibrillation (AF) were studied ultrastructurally and were immunostained for alphaCA and alphaSKA. Up-regulation of alphaSKA was observed in human ventricular cardiomyocytes showing down-regulation of alphaCA and cardiotin. A patchy re-expression pattern of alphaSKA was observed in rabbit left ventricular tissue subjected to pressure- and volume-overload. Dedifferentiating cardiomyocytes in vitro revealed a degradation of the contractile apparatus and local re-expression of alphaSKA. Comparable alphaSKA staining patterns were found in several areas of atrial goat tissue during 16 weeks of AF together with a progressive glycogen accumulation at the same time intervals. The expression of alphaSKA in adult dedifferentiating cardiomyocytes, in combination with PAS-positive glycogen and decreased cardiotin expression, offers an additional tool in the evaluation of myocardial dysfunction and indicates major changes in the contractile properties of these cells.

Expression of retinoid-regulated genes in lamellar ichthyosis vs. healthy control epidermis: changes after oral treatment with liarozole.

Lamellar ichthyosis is a keratinization disorder caused by TGM1, Ichthyin and several other gene mutations. A new treatment option is liarozole, which blocks the cytochrome P450 (CYP26)-mediated catabolism of endogenous all-trans retinoic acid. This study focuses on the expression of retinoid-related genes in ichthyotic epidermis before and after treatment with oral liarozole. We first compared the mRNA expression of cellular retinoic acid binding protein II (CRABPII), keratin (KRT) 2 and 4, CYP26A1 and B1, and two markers of inflammation (interleukin-1alpha and tumours necrosis factor (TNF)-alpha) in shave biopsies from 11 genetically defined, untreated patients and 12 age- and sex-matched healthy controls, finding no overt differences between the groups, besides elevated CRABPII expression. We then studied the biomarkers before and after 4 weeks of treatment with liarozole (75 or 150 mg/day), which produced a better therapeutic response in patients with Ichthyin (n=3) than in those with TGM1 (n=6) mutations. A significant decrease in the mRNA expression of KRT2 and TNF-alpha, and trends toward increased expression of KRT4 and CYP26A1 were observed in liarozole-treated patients, consistent with an increased retinoid stimulation of epidermis. However, there were no dose-related responses and the results of the immunostaining did not always parallel the mRNA findings. The results suggest that liarozole exerts a therapeutic effect in lamellar ichthyosis by mildly affecting the expression of retinoid- regulated genes in epidermis.

Cardiotin localization in mitochondria of cardiomyocytes in vivo and in vitro and its down-regulation during dedifferentiation.

BACKGROUND: Cardiotin expression is observed in adult cardiac tissue. In the present study, we provide evidence for the specific localization of cardiotin in cardiac mitochondria and for its down-regulation during adaptive remodeling (dedifferentiation) of cardiomyocytes. METHODS: Immunocytochemistry was used to study cardiotin localization in adult rabbit papillary muscle, in late-stage embryonic rabbit left ventricular tissue, and in left ventricle samples of rabbits suffering from pressure and volume overload. Western blot analysis of cardiotin was performed in purified pig heart mitochondrial fractions. Cardiotin expression was monitored in vitro in isolated adult rat and rabbit left ventricular cardiomyocytes. RESULTS: Western blot analysis revealed the presence of cardiotin in the mitochondrial fractions of pig heart. Immunoelectron microscopy confirmed the presence of cardiotin in cardiac mitochondria of normal adult rabbits both in vivo and in vitro. Quantification of the localization of immunogold particles suggests an association of cardiotin with the mitochondrial inner membrane. Cardiotin expression is initiated in late-stage embryonic rabbit heart, whereas in adult ventricular tissue cardiotin clearly stained longitudinal arrays of mitochondria. Pressure- and volume-overloaded myocardium showed a reduction in cardiotin expression in dispersed local myocardial areas. Cell cultures of adult cardiomyocytes showed a gradual loss in cardiotin expression in parallel with a sarcomeric remodeling. CONCLUSIONS: Our results demonstrate the specific localization of cardiotin in adult cardiomyocyte mitochondria and propose its use as an early marker for cardiomyocyte adaptive remodeling and dedifferentiation.

Efficacy of a single oral dose of 200 mg pramiconazole in vulvovaginal yeast infections: an exploratory phase IIa trial.

Pramiconazole (R126638) is a novel azole with potent antifungal activity against yeasts, dermatophytes and many other fungal species. The aim of this study was to evaluate the efficacy and tolerance of a single oral dose of 200 mg pramiconazole in acute and recurrent vulvovaginal yeast infections. Thirty-two patients (15 acute and 17 recurrent cases) were KOH microscopy- and culture-positive at inclusion. Clinical cure was 53% at one week and 66% at one month. Mycological eradication was obtained in 88% at one week, whereas at one month 75% of the patients were still culture-negative. Effects in both acute and recurrent cases appeared to be similar for mycological cure. The composite sign and symptom score (sum of scores for oedema, erythema, excoriation pruritus, burning and irritation) had a median value of 7.5 (range 2-17) at inclusion. At one week this value was reduced to 1.0 (range 0-8) and at one month a further reduction to 0 (range 0-11) was seen. p-values compared with baseline at both follow-up visits were <0.001. The drug was well tolerated and the reported adverse events were rare and minimal. In conclusion, the results of this trial indicate that pramiconazole possesses properties that warrant further clinical studies in a larger number of patients with acute and recurrent vulvo notvaginal yeast infection to confirm its efficacy and tolerability.

Novelties in the multifaceted miconazole effects on skin disorders.

BACKGROUND: Miconazole nitrate is a time-honored antifungal of the imidazole class. OBJECTIVE: To revisit the various aspects of action of the drug in a dermatologic setting. METHOD: Review of the current peer-reviewed publications. RESULTS/CONCLUSION: Miconazole essentially inhibits 14alpha-demethylase, an enzyme required for the biosynthesis of ergosterol, which is the main sterol constituent of fungal cell membranes. Hence, toxic methylated sterols accumulate. Synthesis of triglycerides and phospholipids is also affected. In addition, miconazole also exhibits other ancillary mechanisms of action that probably participate in the therapeutic efficacy of the drug. The oxidative and peroxidative enzyme activities are altered leading to an intracellular build up of a toxic concentration of hydrogen peroxide. This may contribute to the deterioration of subcellular organelles and to cell necrosis. Farnesol synthesis is stimulated in Candida spp. leading to the prevention of yeast-to-mycelium formation. Overall, miconazole is fungistatic through its effect on ergosterol biosynthesis, but it may also have a fungicidal effect against a number of fungal species due to its effect on hydrogen peroxide accumulation. In addition, miconazole is active against a series of Gram-positive bacteria and has been shown to help the repair of the skin barrier function and to help mitigate some inflammatory cell reactions (such as in acne). To conclude, miconazole exerts multi-pronged effects both against pathogenic fungi and on skin physiology.

Retinoic acid metabolism blocking agents (RAMBAs): a new paradigm in the treatment of hyperkeratotic disorders.

Synthetic vitamin A derivatives, retinoids,have long been the mainstay of treatment for several disorders of keratinization, notably the ichthyoses and severe acne. Some forms of psoriasis also respond well. Their considerable power comes at a price. They have dose-limiting side effects and can be highly teratogenic, limiting their use in women of childbearing age.Thus, retinoids are used less often than their potential would warrant. However, the recent development of compounds that block the catabolism of endogenous vitamin A, called Retinioic Acid Metabolism Blocking Agents or RAMBAs, offers new possibilities. With these drugs, retinoid effects with less side effects and a reduction of the post-treatment teratogenicity period due to their favourable pharmacokinetic profile might be expected. In this review, we discuss how retinoids work, how they are metabolized and how RAMBAs influence this process. We also review the presently available data from clinical trials with RAMBAs.

The role of ketoconazole in seborrheic dermatitis.

Although the prominent broad-spectrum activity of ketoconazole was reported in the early 1980s, its effect against Malassezia species was most pronounced; thus, it was developed for the treatment of various skin infections in which a link with these fungal species was proposed. Later, a number of ancillary properties were described for ketoconazole that comprised its anti-inflammatory, antiseborrheic, and antiproliferative profile. The incorporation of ketoconazole in an adapted vehicle could further promote its efficacy. Recently, a new formulation-an anhydrous gel containing ketoconazole 2%-was launched in which all of the ancillary properties were optimized.

Miconazole induces changes in actin cytoskeleton prior to reactive oxygen species induction in yeast.

The antifungal compound miconazole inhibits ergosterol biosynthesis and induces reactive oxygen species (ROS) in susceptible yeast species. To further uncover the mechanism of miconazole antifungal action and tolerance mechanisms, we screened the complete set of haploid Saccharomyces cerevisiae gene deletion mutants for mutants with an altered miconazole sensitivity phenotype. We identified 29 S. cerevisiae genes, which when deleted conferred at least 4-fold hypersensitivity to miconazole. Major functional groups encode proteins involved in tryptophan biosynthesis, membrane trafficking including endocytosis, regulation of actin cytoskeleton, and gene expression. With respect to the antifungal activity of miconazole, we demonstrate an antagonism with tryptophan and a synergy with a yeast endocytosis inhibitor. Because actin dynamics and induction of ROS are linked in yeast, we further focused on miconazole-mediated changes in actin cytoskeleton organization. In this respect, we demonstrate that miconazole induces changes in the actin cytoskeleton, indicative of increased filament stability, prior to ROS induction. These data provide novel mechanistic insights in the mode of action of a ROS-inducing azole.

A pilot study on seborrheic dermatitis using pramiconazole as a potent oral anti-Malassezia agent.

BACKGROUND: Seborrheic dermatitis is considered to be a Malassezia-driven disease. Little objective information is available so far from biometrological quantitative assessments of this skin condition. Pramiconazole is a novel triazole with potent in vitro antifungal activity, especially against Malassezia spp. OBJECTIVE: To study the sequential effects of pramiconazole on Malassezia, inflammation and epidermal changes. METHOD: This study was performed in 2 groups of subjects suffering from seborrheic dermatitis. The first group (n = 17) remained untreated and was used as control. Clinical, mycological and biometrological assessments were performed at inclusion and during the following 2 weeks. The second group of subjects (n = 10) received a single 200-mg oral dose of pramiconazole at inclusion. Clinical, mycological and biometrological evaluations were performed before and during 1 month following the single antifungal intake. For both parts of the study, several parameters were assessed including yeast density, desquamation, erythema, itching and sebum excretion. RESULTS: In the control group, no significant changes were observed in any of the parameters during the observation period. The findings were markedly different in the pramiconazole-treated subjects. The yeast density was significantly improved on days 3, 7 and 28. Desquamation, erythema, itching, and the global clinical evaluation as assessed by the patients and investigators became significantly improved on days 7 and 28. A trend in decrease of scaliness was noted. No effect on sebum excretion was evidenced. In conclusion, a single 200-mg dose of pramiconazole exhibitsin vivo efficacy in controlling some important clinical aspects of seborrheic dermatitis. Following a reduction in the number of yeasts on day 3, a decrease in the severity of clinical signs and symptoms occurred from day 7 onwards. Sebum excretion appeared uninvolved in the clearing process of seborrheic dermatitis. CONCLUSION: A single 200-mg dose of pramiconazole appears to abate seborrheic dermatitis. The density in Malassezia present on lesional skin is first decreased, followed by clearing of the clinical signs.

Structural remodelling of cardiomyocytes in the border zone of infarcted rabbit heart.

Cardiomyocyte dedifferentiation, as detected in hibernating myocardium of chronic ischemic patients, is one of the characteristics seen at the border of myocardial infarcts in small and large animals. Our objectives were to study in detail the morphological changes occurring at the border zone of a rabbit myocardial infarction and its use as model for hibernating myocardium. Ligation of the left coronary artery (LAD) was performed on rabbit hearts and animals were sacrificed at 2, 4, 8 and 12 weeks post-infarction. These hearts together with a non-infarcted control heart were perfusion-fixed and tissue samples were embedded in epoxy resin. Hibernating cardiomyocytes were mainly distributed in the non-infarcted region adjacent to the border zone of infarcted myocardium but only in a limited number. In the border zone itself vacuolated cardiomyocytes surrounded by fibrotic tissue were frequently observed. Ultrastructural analysis of these vacuolated cells revealed the presence of a basal lamina inside the vacuoles adjacent to the surrounding membrane, the presence of pinocytotic vesicles and an association with cisternae of the sarcoplasmatic reticulum. Myocyte quantitative analyses revealed a gradual increase in vacuolar area/total cell area ratio and in collagen fibril deposition inside the vacuoles from 2 to 12 weeks post-infarction. Related to the remote zone, the increase in cell width of myocytes located in and adjacent to the border zone demonstrated cellular hypertrophy. These results indicate the occurrence of cardiomyocyte remodelling mechanisms in the border zone and adjacent regions of infarcted myocardium. It is suggested that the vacuoles represent plasma membrane invaginations and/or dilatations of T-tubular structures.

Absence of an active metabolite for the triazole antifungal pramiconazole.

Pramiconazole is an antifungal with high potential for the treatment of dermatophyte and yeast infections of the skin. It is currently not known whether pramiconazole is active alone as the parent agent or assisted by active metabolites. The in vitro metabolism as well as the metabolic stability of pramiconazole was investigated in subcellular liver fractions and isolated hepatocytes of several species. Results indicate that the metabolism of pramiconazole was slow, since the enzyme-mediated disappearance of pramiconazole was rather slow. To investigate whether pramiconazole was converted into an active metabolite in humans, serum samples from healthy volunteers receiving a daily dose of 100 or 200 mg pramiconazole for one week were assayed with an agar diffusion bioassay and liquid chromatography-tandem mass spectrometry. It was concluded that there was no active metabolite present in serum samples from healthy volunteers after oral dosing of pramiconazole.

In vitro activity of R126638 and ketoconazole against Malassezia species.

The in vitro activity of a new triazole R126638 against Malassezia yeasts was compared with that of ketoconazole. With the agar dilution technique, minimal inhibitory concentrations were lower for R126638 compared with ketoconazole against Malassezia globosa, M. obtusa, M. slooffiae, M. restricta and two strains of M. sympodialis. On human stratum corneum in vitro, both R126638 and ketoconazole were very effective in reducing the production of hyphae from 15% to 1% with R126638 and to 2% with ketoconazole. Scanning electron microscopy did not reveal obvious surface differences between untreated cultures and cultures exposed to ketoconazole or R126638 in the concentration range 0.01-1 microg/ml. However, transmission electron microscopy showed partial to complete necrosis of the cytoplasmic organelles of Malassezia yeasts. The combined scanning electron microscopy and transmission electron microscopy findings confirm earlier observations of the "mummifying" effect of azoles against Malassezia spp. In conclusion, R126638 is an interesting new triazole with high activity against the Malassezia yeasts, which are involved in pityriasis versicolor and seborrhoeic dermatitis.

Spatiotemporal pattern of neuroinflammation after impact-acceleration closed head injury in the rat.

Inflammatory processes have been implicated in the pathogenesis of traumatic brain damage. We analyzed the spatiotemporal expression pattern of the proinflammatory key molecules: interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and inducible nitric oxide synthase in a rat closed head injury (CHI) paradigm. 51 rats were used for RT-PCR analysis after CHI, and 18 for immunocytochemistry. We found an early upregulation of IL-1beta, IL-6, and TNF-alpha mRNA between 1h and 7h after injury; the expression of iNOS mRNA only revealed a significant increase at 4h. After 24h, the expression decreased towards baseline levels, and remained low until 7d after injury. Immunocytochemically, IL-1beta induction was localized to ramified microglia in areas surrounding the primary impact place as well as deeper brain structures. Our study shows rapid induction of inflammatory gene expression that exceeds by far the primary impact site and might therefore contribute to tissue damage at remote sites.

Effect of a single overnight topical application of miconazole nitrate paste on acne papules.

BACKGROUND: The classical management of acne calls for prolonged oral and/or topical treatments; however, some patients request a rapid effect to make the papules disappear within a few hours or days. OBJECTIVE: To test the effect of a single overnight application of a paste containing 0.25% miconazole nitrate on acne papules, and comparison with the effect of the same but unmedicated paste. METHOD: Narrow-band reflectance spectroscopy was used to assess the changes in E index (erythema) after overnight application of the pastes. In the first study, a total of 117 acne papules were assessed in 15 adolescents. Measurements of the E index were performed at 24-h intervals for 4 days on acne papules and the surrounding normal-looking skin. The last two assessments were performed the mornings preceding and following, respectively, a single application of the medicated paste. The second part of the study consisted of a double-blind, split-face study on 25 adolescents with acne. They applied each of the two formulations on a randomized part of the forehead. A total of 161 acne papules received the medicated paste and 160 received the same but unmedicated paste. Measurements were performed the mornings before and after the overnight treatment. RESULTS: In the first part of the study, no significant daily difference was observed between the three series of pretreatment E indices, on both acne papules and normal skin. The treatment did not affect the E index of normal skin. By contrast, it decreased significantly the E index of the acne papules. The second part of the study also showed that the medicated paste significantly decreased the E index of acne papules. This effect was significantly (P < 0.05) superior to that of the unmedicated paste. CONCLUSION: A single overnight application of miconazole nitrate paste appears to reduce the erythematous aspect of acne papules.

Structural adaptation in adult rabbit ventricular myocytes: influence of dynamic physical interaction with fibroblasts.

The mechanism of induction of cardiomyocyte (CM) dedifferentiation, as seen in chronic hibernating myocardium, is largely unknown. Recently, a cellular model was proposed consisting of long-term cocultures of adult rabbit CMs and cardiac fibroblasts in which typical structural characteristics of hibernation-like dedifferentiation could be induced. Only CMs in close contact with fibroblasts underwent these changes. In this study, we further investigated the characteristics of the fibroblast-CM interaction to seek for triggers and phenomena involved in CM dedifferentiation. Adult rabbit CMs were cocultured with cardiac or 3T3 fibroblasts. Heterocellular interactions and the structural adaptation of the CMs were quantified and studied with vital microscopy and electron microscopy. Immunocytochemical analysis of several adhesion molecules, i.e., N-cadherin, vinculin, beta1-integrin, and desmoplakin, were examined. Upon contact with CMs, fibroblasts attached firmly and pulled the former cells, resulting in anisotropic stretch. Quantification of the attachment sites revealed a predominant binding of the fibroblast to the distal ends of the CM in d 1 cocultures and a shift towards the lateral sides of the CMs on d 2 of coculture, suggesting a redistribution of CM membrane proteins. Immunocytochemical analysis of cell adhesion proteins showed that these were upregulated at the heterocellular contact sites. Addition of autologous and nonautologous fibroblasts to the CM culture similarly induced a progressive and accelerated structural adaptation of the CM. Dynamic passive stretch invoked by the fibroblasts and/or intercellular communication involving cell adhesion molecule expression at the interaction sites may play an important role in the induction of hibernation-like dedifferentiation of the cocultured adult rabbit CMs.