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In this study the ultrastructure of Rana esculenta skin is described.Cytochemical methods were used to localize guanylate cyclase in the presenceof atrial natriuretic peptide and immunocytochemical methods showed the presenceof the atrial natriuretic peptide in various levels of skin. The peptide ismainly found in the epithelium and in the lymph sacs of the tela subcutanea.Its receptors are located in the same zones and are indicated by guanylatecyclase activity. We demonstrate that frog skin is a target organ for atrialnatriuretic peptide and propose that, at this level, the peptide carries out animportant osmoregulatory role. [ABSTRACT FROM AUTHOR]

Background/objectives: Glaucoma is the leading cause of irreversible blindness, disproportionately affecting individuals of African ancestry. Limited research has examined the impact of neighbourhood quality and socioeconomic factors on primary open-angle glaucoma (POAG) risk in this population. This study aims to address these gaps by evaluating associations between ocular health and neighbourhood characteristics using geospatial data., Subjects/methods: We conducted a case-control study with 5192 African ancestry individuals from the Philadelphia area using data from the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study. Geocoded U.S. Census data were merged with individual-level demographics and neighbourhood-level measures, including air quality, food accessibility, and socioeconomic indicators, to assess their association with glaucoma risk and severity., Results: The study included 3039 controls (58.5%) and 2153 POAG cases (41.5%). Higher POAG risk was associated with older age (OR 1.72 per 10-year increase, p < 0.001), male gender (OR 2.04, p < 0.001), lower BMI (OR 0.87 per 10 kg/m 2 increase, p = 0.003), and nonuse of alcohol (OR 0.56 for alcohol use, p < 0.001). Low food access was more common in controls (OR 0.86, p = 0.03), and severe POAG cases were associated with lower homeownership rates (OR 0.95 per 10% increase, p = 0.049). However, most socioeconomic and environmental factors (air quality, education, income, occupation, family structure) were not significantly linked to POAG risk or severity., Conclusion: Socioeconomic status did not significantly protect against POAG in African ancestry individuals. Individual factors were more influential, suggesting neighbourhood and socioeconomic factors may have a lesser impact than previously hypothesised., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: The study protocol and the consent statement were approved by the University of Pennsylvania institutional review board (IRB) (IRB protocol #812036), and the research adhered to the tenets of the Declaration of Helsinki. All subjects provided informed consent., (© 2024. The Author(s).)

Purpose: Investigate the relationship between body mass index (BMI) and primary open-angle glaucoma (POAG) in an African ancestry cohort from the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study., Design: Retrospective, cross-sectional "case-control" comparison study., Methods: A total of 6,634 POAAGG study subjects were eligible: 2,977 cases and 3,657 controls. Ocular and demographic data were collected from on-site exams, standardized interviews, and electronic medical records. BMI was calculated: weight(kg)/height(m) 2 , and categorized as low (<18.5), moderate (18.5-24.9), high (25.0-29.9), or very high (≥30). Structural and functional POAG progression were assessed by annual rate of change in retinal nerve fiber layer thickness and visual field mean deviation, respectively, using a linear mixed-effects model. Regression analyses evaluated associations of BMI with POAG status, phenotype, and progression., Results: Lower BMI was associated with increased POAG risk (aOR[95% confidence interval], 1.02 [1.007,1.023] per kg/m 2 decrease in BMI, p = .0003). In cases, low BMI was associated with larger cup-to-disc ratio (p = .007) and worse visual acuity (p = .04). Fast functional POAG progressors had a significantly lower mean BMI than slow progressors (25.7 vs. 30.0 kg/m 2 , p = .04)., Conclusions: In this African ancestry cohort, low BMI was associated with increased POAG risk. POAG cases with low BMI were more likely to have larger cup-to-disc ratios, worse visual acuity, and faster functional progression, indicating more severe glaucoma., (Copyright © 2024. Published by Elsevier Inc.)

Objective: To investigate outcomes of trabeculectomy ab externo in African ancestry primary open-angle glaucoma (POAG) patients, and to analyze the impact of demographic and phenotypic factors on surgical success and complication rates., Patients and Methods: A retrospective case-control study enrolled 63 eyes of 55 POAG cases who underwent trabeculectomy ab externo. Data on demographics, family glaucoma history, surgical specifics, and pre/postoperative measures (intraocular pressure, visual acuity, visual field, medication usage, complications within one year) were collected. Analysis included linear/logistic regression models adjusting for inter-eye correlation., Results: Trabeculectomy yielded success without additional medication in 46%, qualified success with medication in 22%, and surgical failure necessitating further intervention in 32% within one year. Subjects experienced reduction in intraocular pressure (IOP) (46%), daily glaucoma medication (73%), and eye drop usage (67%) 1-year post-trabeculectomy (all P <0.001). However, there was a postoperative decline of 56% in visual acuity (VA) ( P <0.001) and a significant worsening of visual field parameters, including a 14% decrease in mean deviation ( P =0.02) and a 19% decrease in visual field index ( P =0.004). Top of FormBottom of Form59% of patient eyes experienced complications within one year of surgery. Univariate analysis of predictive factors for surgical outcomes revealed that younger age at surgery ( P =0.01) and family history of glaucoma ( P =0.046) were predictive of lower rates of surgical success. Multivariable analysis revealed worse preoperative VA (OR 0.79 per 0.1 LogMAR increases, P =0.02) was associated with lower likelihood of surgical success., Conclusion: This study underscores the low rates of trabeculectomy success and high rates of complications in an African ancestry population with POAG. While the procedure exhibited positive effects on IOP control and medication reduction, our analysis found that multiple factors, particularly age, family history, and worse preoperative VA play crucial roles in influencing surgical success., Prcis: Trabeculectomy in African ancestry individuals with primary open-angle glaucoma shows a 46% success rate and frequent complications, indicating that younger age and family history are significant predictors of surgical failure in this high-risk population., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Glaucoma is the leading cause of irreversible blindness, and its pathophysiology includes neuroinflammatory changes. The present therapies for glaucoma target pressure-lowering mechanisms with limited success, making neuroinflammation a target for future interventions. This review summarizes the neuroinflammatory pathways seen in glaucoma and their interplay with stress. Glucocorticoids have been shown to activate proinflammatory glial cells, contributing to the neuroinflammation in glaucoma. Glucocorticoids have also been shown to increase the IOP directly. Stress-associated autonomic dysfunction can affect the vascular homeostasis in the retina and create oxidative stress. Diabetes, hyperglycemic-mediated endothelial damage, and vascular inflammation also play important roles in the neuroinflammation in glaucoma and diabetic retinopathy. Psychosocial stress has been implicated in an increased IOP and glaucoma outcomes. People who experience maladaptive chronic stress suffer from a condition known as allostatic load, which describes pathologic neuroendocrine dysregulation. The effects of allostatic load and chronic stress have been studied in patients affected by a lower socioeconomic status (SES) and marginalized racial identities. A lower SES is associated with higher rates of glaucoma and also affects the access to care and screening. Additionally, people of African ancestry are disproportionately affected by glaucoma for reasons that are multifactorial. In conclusion, this review explores neuroinflammation in glaucoma, highlighting opportunities for future investigation.

Background/aims: Primary open-angle glaucoma (POAG) disproportionately affects individuals of African ancestry. In these patients' eyes, a large cup-to-disc ratio (LCDR > 0.90) suggests greater retinal ganglion cell loss, though these patients often display varied visual ability. This study investigated the prevalence and risk factors associated with LCDR in African ancestry individuals with POAG and explored the differences between blind (>20/200) and not blind (≤20/200) LCDR eyes., Methods: A case-control methodology was used to investigate the demographic, optic disc, and genetic risk factors of subjects in the Primary Open-Angle African American Glaucoma Genetics Study. Risk factors were analyzed using univariable and multivariable logistic regression models with inter-eye correlation adjusted using generalized estimating equations., Results: Out of 5605 eyes with POAG, 1440 eyes (25.7%) had LCDR. In the multivariable analysis, LCDR was associated with previous glaucoma surgery (OR = 1.72), increased intraocular pressure (OR = 1.04), decreased mean deviation (OR = 1.08), increased pattern standard deviation (OR = 1.06), thinner retinal nerve fiber layer (OR = 1.05), nasalization of vessels (OR = 2.67), bayonetting of vessels (OR = 1.98), visible pores in the lamina cribrosa (OR = 1.68), and a bean-shaped cup (OR = 2.11). Of LCDR eyes, 30.1% were classified as blind (≤20/200). In the multivariable analysis, the statistically significant risk factors of blindness in LCDR eyes were previous glaucoma surgery (OR = 1.72), increased intraocular pressure (OR = 1.05), decreased mean deviation (OR = 1.04), and decreased pattern standard deviation (OR = 0.90)., Conclusions: These findings underscore the importance of close monitoring of intraocular pressure and visual function in African ancestry POAG patients, particularly those with LCDR, to preserve visual function.

The growing summer drought stress is affecting the nutritional value of pastures, no longer sufficient to support the nutritional status of sheep in extensive rearing. Adipokines affect organ and tissue functionality can be useful to evaluate animal welfare and prompt an improvement in the management of the grazing animals. Leptin (Lep) is an adipokine mainly produced by adipose tissue that regulates food intake by an anorexigenic action. Lep has also been detected in the human and rat gastrointestinal tract, where it regulates the rate of gastric emptying. In this study, Lep system was evaluated in the abomasum of 15 adult sheep reared on Apennine pastures and subjected to different diets. Until the maximum pasture flowering (MxF group), the sheep fed on fresh forage; from that moment until the maximum pasture dryness (MxD group), the experimental group (Exp group) received a feed supplementation in addition to MxD group feeding. The Lep system was investigated in the abomasum samples by immunohistochemistry (IHC) and RT-qPCR. Double-label localisation of Lep and leptin receptor (LepR) with neuroendocrine hormones was conducted to distinguish the gland cell types. The analysis performed revealed the presence of Lep and LepR in the chief and neuroendocrine cells of the fundic glands of the abomasum. RT-qPCR evidenced the transcript for Lep and LepR also identifying the long isoform (LepRb). No significant differences were observed among the three groups of sheep subjected to different diets. The abundant immunostaining observed in the fundic glands suggests that the Lep intervenes in the regulation of abomasum in sheep with a similar pattern to monogastric species while long term food supplementation seems do not influence the local function of the Lep system. A better understanding of the gastrointestinal system can contribute to improving sheep management and optimising the sustainability of livestock production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Background/aims: To investigate the rates of structural and functional progression of primary open-angle glaucoma in an African ancestry cohort and identify risk factors for progression., Methods: This retrospective study included 1424 eyes from glaucoma cases in the Primary Open-Angle African American Glaucoma Genetics cohort, with ≥2 visits for retinal nerve fibre layer (RNFL) thickness and mean deviation (MD) measurements over ≥6-month follow-up. The rates of structural progression (change in RNFL thickness/year) and functional progression (change in MD/year) were calculated from linear mixed effects models, accounting for intereye correlation and longitudinal correlation. Eyes were categorised as slow, moderate or fast progressors. Risk factors for progression rates were assessed using univariable and multivariable regression models., Results: The median (interquartile) rates of progression were -1.60 (-2.05 to -1.15) µm/year for RNFL thickness and -0.40 (-0.44 to -0.34) decibels/year for MD. Eyes were categorised as slow (structural: 19%, functional: 88%), moderate (structural: 54%, functional: 11%) and fast (structural: 27%, functional: 1%) progressors. In multivariable analysis, faster RNFL progression was independently associated with thicker baseline RNFL (p<0.0001), lower baseline MD (p=0.003) and beta peripapillary atrophy (p=0.03). Faster MD progression was independently associated with higher baseline MD (p<0.0001), larger cup-to-disc ratios (p=0.02) and lower body mass index (p=0.0004)., Conclusion: The median rates of structural and functional progression in this African ancestry cohort were faster than the rates reported from previously published studies in other ethnic groups. Higher baseline RNFL thickness and MD values were associated with faster progression rates. Results highlight the importance of monitoring structural and functional glaucoma progression to provide timely treatment in early disease., Competing Interests: Competing interests: JMO’B reports income from consulting from Atheneum Partners (New York, New York), Cerner Enviza (Kansas City, Missouri), Calico (San Francisco) and Kantar Health (New York, New York). AR reports a financial relationship with Gyroscope Therapeutics., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Allergic rhinitis and asthma are the most common causes of chronic inflammation of the upper and lower airways in childhood. However, a nasal biomarker that can link to pulmonary inflammation is yet to be found. The present paper aims to investigate the possible role in inflammation of two inducible 70-kDa Heat Shock Proteins (HSP70) members, HSPA1A/B and HSPA6, in nasal mucosa cells of allergic children through their mRNA expression analysis, and their correlation to both spirometric and FeNO values. The relationship between FeNO in lower airways and ∆Cts of HSPA1A/B in nasal mucosa seems to be influenced by clinical symptoms regardless of age, sex, and sensitization patterns. Therefore, HSP70 expression, as well as FeNO levels, could have a predictive capability to identify lower airways inflammation and thus to recognize rhinitic children having a potential risk of asthma development., (© 2022. The Author(s).)

Amphibian parasites of the genus Amphibiocystidium are members of the class Ichthyosporea (=Mesomycetozoea), within the order Dermocystida. Most of the species in the Dermocystida fail to grow in ordinary culture media, so their life cycle has only been partially constructed by studies in host tissues. However, to date, there have been few reports on the life cycle of Amphibiocystidium parasites with respect to the developmental life stages of both Dermocystidium and Rhinosporidium parasites. In this study, we provide light and electron microscopic findings of developmental phenotypes of Amphibiocystidium sp., a parasite previously characterized in the Italian stream frog (Rana italica), which has caused an ongoing infection in a natural population of Central Italy. These phenotypes exhibited distinct morphological characteristics that were similar to A. ranae from the skin of R. temporaria, but showed histochemical properties particularly comparable with those of maturing phenotypes of Rhinosporidium seeberi, and compatible with fungal-like parasites. Therefore, for Amphibiocystidium sp. phenotypes, we suggest adopting the terminology used for maturing stages of R. seeberi, such as juvenile sporangia, early mature sporangia and mature sporangia. The characterization of these developmental stages will be useful to increase the understanding of the life cycle of parasites of the genus Amphibiocystidium and of the interactions with their amphibian hosts., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Small Heat Shock Proteins (sHSP) are molecular chaperones that play an essential role in maintaining protein homeostasis and promoting cell survival. In this work, for the first time, multiple cDNAs encoding for small Hsp27 and Hsp30, designed, respectively, as PbHsp27-(1-2) and PbHsp30-(1-5), were cloned and characterized in the amphibian Pelophylax bergeri, which is a suitable model for studying biological responses to environmental perturbations. Domain architecture analysis showed that PbHsp27 and PbHsp30 cDNAs displayed the typical signature motifs of the sHSP family such as the conserved α-crystallin domain flanked by variable N-terminal and C-terminal regions. Phylogenetic analysis revealed that PbHsp27 and PbHsp30 clustered, respectively, with Hsp27 and Hsp30 members of other vertebrates, but more closely with amphibians. Overall PbHsp27 and PbHsp30 transcriptional activity, analyzed by qRT-PCR, evidenced that, in ex vivo skin exposed to thermal shock and cadmium treatment, PbHsp27 and PbHsp30 mRNAs were inducible and regulated differently. This study provides the basis for future research on the potential use of PbHsp27 and PbHsp30 as biomarkers of proteotoxic stress in amphibians., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mesomycetozoean-induced infections (order Dermocystida, genus Amphibiocystidium) in European and North American amphibians are causing alarm. To date, the pathogenicity of these parasites in field conditions has been poorly studied, and demographic consequences on amphibian populations have not been explored. In this study, an Amphibiocystidium sp. infection is reported in a natural population of the Italian stream frog (Rana italica) of Central Italy, over a 7-year period from 2008 to 2014. Light and electron microscope examinations, as well as partial 18S rDNA sequence analysis were used to characterize the parasite. Moreover, a capture-mark-recapture study was conducted to assess the frog demographics in response to infection. Negative effects of amphibiocystidiosis on individual survival and population fitness were absent throughout the sampling period, despite the high estimates of disease prevalence. This might have been due to resistance and/or tolerance strategies developed by the frogs in response to the persistence of Amphibiocystidium infection in this system. We hypothesized that in the examined R. italica population, amphibiocystidiosis is an ongoing endemic/epidemic infection. However, ecological and host-specific factors, interacting in a synergistic fashion, might be responsible for variations in the susceptibility to Amphibiocystidium infection of both conspecific populations and heterospecific individuals of R. italica.

The rock partridge, Alectoris graeca, is a polytypic species declining in Italy mostly due to anthropogenic causes, including the massive releases of the closely related allochthonous chukar partridge Alectoris chukar which produced the formation of hybrids. Molecular approaches are fundamental for the identification of evolutionary units in the perspective of conservation and management, and to correctly select individuals to be used in restocking campaigns. We analyzed a Cytochrome oxidase I (COI) fragment of contemporary and historical A. graeca and A. chukar samples, using duplicated analyses to confirm results and nuclear DNA microsatellites to exclude possible sample cross-contamination. In two contemporary specimens of A. graeca, collected from an anthropogenic hybrid zone, we found evidence of the presence of mtDNA heteroplasmy possibly associated to paternal leakage and suggesting hybridization with captive-bred exotic A. chukar. These results underline significant limitations in the reliability of mtDNA barcoding-based species identification and could have relevant evolutionary and ecological implications that should be accounted for when interpreting data aimed to support conservation actions., Competing Interests: The authors have declared that no competing interests exist.

There have been a few studies on the negative effects of pollutants on amphibian skin, the first structural barrier that interacts with the environment and its potential contaminants. In this study an ex vivo skin organ culture from the amphibian Pelophylax bergeri was used to evaluate cell stress responses induced by short-term exposure to cadmium (Cd), a toxic heavy metal known to be an environmental hazard to both humans and wildlife. Histopathological studies were carried out on skin explants using light microscopy and changes in the expression of stress proteins, such as Metallothionein (MT) and Heat shock proteins (HSPs), were investigated by Real-time RT-PCR. Results revealed that amphibian skin reacts to Cd-induced stress by activating biological responses such as morphological alterations and dose- and time-dependent induction of Mt and Hsp70 mRNA expression, suggesting their potential role as biomarkers of exposure to Cd. This work provides a basis for a better understanding of the tissue-specific responses of amphibian skin as a target organ to Cd exposure and its in vitro use for testing potentially harmful substances present in the environment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In human and wildlife populations, the natural microbiota plays an important role in health maintenance and the prevention of emerging infectious diseases. In amphibians, infectious diseases have been closely associated with population decline and extinction worldwide. Skin symbiont communities have been suggested as one of the factors driving the different susceptibilities of amphibians to diseases. The activity of the skin microbiota of amphibians against fungal pathogens, such as Batrachochytrium dendrobatidis, has been examined extensively, whereas its protective role towards the cutaneous infectious diseases caused by Amphibiocystidium parasites has not yet been elucidated in detail. In the present study, we investigated, for the first time, the cutaneous microbiota of the Italian stream frog (Rana italica) and characterized the microbial assemblages of frogs uninfected and infected by Amphibiocystidium using the Illumina next-generation sequencing of 16S rRNA gene fragments. A total of 629 different OTUs belonging to 16 different phyla were detected. Bacterial populations shared by all individuals represented only one fifth of all OTUs and were dominated by a small number of OTUs. Statistical analyses based on Bray-Curtis distances showed that uninfected and infected specimens had distinct cutaneous bacterial community structures. Phylotypes belonging to the genera Janthinobacterium, Pseudomonas, and Flavobacterium were more abundant, and sometimes almost exclusively present, in uninfected than in infected specimens. These bacterial populations, known to exhibit antifungal activity in amphibians, may also play a role in protection against cutaneous infectious diseases caused by Amphibiocystidium parasites.

Heat-Shock Protein 70 (HSP70) is a class of highly conserved proteins which is involved in essential functions as molecular chaperones and in the acquired tolerance processes. In this work, two cDNAs encoding a constitutive Hsc70 and an inducible Hsp70 from the water frog Rana (Pelophylax) lessonae, belonging to the Rana (P.) esculenta complex of central Italy, have been isolated and characterized. The two cDNA clones, named Rl-Hsc70 and Rl-Hsp70, encode 646 and 640 amino acid proteins respectively, which present extremely conserved functional domains characteristic of cytosolic members of the HSP70 family. Comparative studies of the amino acid sequences showed that Rl-Hsc70 and Rl-Hsp70 had the highest homology with constitutive and inducible HSP70 members of other amphibian species. The phylogenetic analysis also demonstrated a separate clustering of the Rl-Hsc70 and Rl-Hsp70 with constitutive and inducible members from other vertebrate species. Heat-inducibility assays performed during embryogenesis showed that the two isolated mRNAs displayed different expression profiles. Rl-Hsp70 was induced only in heat shock-treated embryos, whereas Rl-Hsc70 transcript levels, which were constitutively modulated in non-stressed embryos, did not increase following the heat treatment. In situ hybridization experiments demonstrated that both transcripts showed a tissue-specific enrichment in the central nervous system and in the somites., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Pounds et al. argue that global warming contributes to amphibian declines by encouraging outbreaks of the chytrid fungus Batrachochytrium dendrobatidis. Although our findings agree with the climate-linked epidemic hypothesis, this pathogen is probably not the only proximate factor in such cases: in the Trasimeno Lake area of Umbria in central Italy, for example, the water frog Rana lessonae first declined in the late 1990s, yet chytridiomycosis was not observed until 2003 (refs 5, 6). Here we show that the chytrid was common there throughout 1999-2002, in a previously unknown form that did not cause disease. We therefore think that the focus by Pounds et al. on a single pathogen is hard to justify because the host-parasite ecology is at present so poorly understood.

The pathogen of frogs Amphibiocystidium ranae was recently described as a new genus. Due to its spherical shape, containing hundred of endospores, it was thought to be closely related to the pathogens of fish, mammals, and birds known as Dermocystidium spp., Rhinosporidium seeberi, and Sphaerothecum destruens in the Mesomycetozoea, but further studies were not conducted to confirm this relationship. To investigate its phylogenetic affinities, total genomic DNA was extracted from samples collected from infected frogs containing multiple cysts (sporangia) and endospores. The universal primers NS1 and NS8, used to amplify the 18S small-subunit rRNA by PCR, yielded approximately 1,770-bp amplicons. Sequencing and basic local alignment search tool analyses indicated that the 18S small-subunit rRNA of A. ranae from both Rana esculenta and Rana lessonae was closely related to all of the above organisms. Our phylogenetic analysis placed this pathogen of frogs as the sister group to the genus Dermocystidium and closely related to Rhinosporidium. These data strongly supported the placement of the genus Amphibiocystidium within the mesomycetozoeans, which is in agreement with the phenotypic features that A. ranae shares with the other members of this class. Interestingly, during this study Dermocystidium percae did not group within the Dermocystidium spp. from fish; rather, it was found to be the sister group to Sphaerothecum destruens. This finding suggests that D. percae could well be a member of the genus Sphaerothecum or perhaps represents a new genus.

The atrial natriuretic peptide (ANP)-system was examined in Rana esculenta lung with cytochemical and immunocytochemical methods. The results showed a cellular type that synthesizes the hormone and the presence of receptors in various components of the lung. The lung, therefore, produces ANP and at the same time is a target organ for the hormone. ANP may play an important physiological role in pulmonary function and in protecting against pulmonary edema., (Copyright 2004 Wiley-Liss, Inc.)

Actin is a highly conserved cytoskeletal protein that is ubiquitous in all eukaryotes. Little is known about actin expression in amphioxus, the closest living relative of the vertebrates. In the present study, involving Western blotting and indirect immunofluorescence, we report the characterization and localization of various actin isoforms in amphioxus (Branchiostoma lanceolatum) tissues. Three antibodies against vertebrate actins were used: a polyclonal antibody recognizing beta-cytoplasmic actin (anti-beta actin), a monoclonal antibody against sarcomeric actins (anti-alphaSR-1), and a monoclonal antibody specific for alpha-smooth actin (anti-alphaSM-1). Western blot analysis of amphioxus extracts immunodecorated with these antibodies showed a 43-kDa-positive band co-migrating with respective controls. The amphioxus isoactin expression patterns recognized by these antibodies were similar to those of vertebrates, i.e., anti-beta actin showed positive staining mainly in non-muscle cells, anti-alphaSR-1 labelled dorsolateral myotomal muscles, and anti-alphaSM-1 stained ventral muscles. These results demonstrate that at least two muscle actins are present in amphioxus, suggesting that muscle actin gene duplication events began before vertebrate divergence from the amphioxus lineage.

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes were studied. The Cu2+ exposure of mussels caused severe perturbations in haemocyte actin and fibronectin organization with respect to non-exposed organisms. Cytoskeletal actin was analysed by indirect immunofluorescence, using an antitotal actin monoclonal antibody, and by rhodamine-conjugated phalloidin. The majority of haemocytes from Cu(2+)-exposed mussels displayed a round morphology, with short and blunt filopodia; they lacked the polarized phenotype which was typical in control samples. The cytoskeleton alteration, more evident after phalloidin staining, resulted in the disappearance of filamentous actin. The actin cortical meshwork also appeared disorganized. The cytoskeletal morphology studied by transmission electron microscopy after negative staining of Triton X-100-treated haemocytes confirmed these observations. The structural organization of actin when analysed by Western blotting showed a larger number of Triton-soluble actin pools in treated mussel haemocytes. Fibronectin was studied by indirect immunofluorescence using a polyclonal antiserum directed against mussel fibronectin. In treated mussels, fibronectin appeared to be strongly disorganized and its levels decreased in both haemocytes and haemolymph. The mechanism(s) of the copper-induced alterations on actin and fibronectin organization in mussel immunocytes is discussed.

alpha-Smooth muscle (alpha SM) actin of endothermic vertebrates is selectively recognized by the monoclonal antibody anti-alpha SM-1. Immunoreactivity to this antibody has been shown to be localized in the NH2-terminal sequence Ac-EEED (Chaponnier et al. 1994). Among terrestrial ectothermic vertebrates, two amphibian (Triturus vulgaris, Rana esculenta) and three reptilian species (Pseudemys scripta elegans, Natrix natrix, Podarcis sicula) were screened to investigate if their vascular and visceral smooth muscles were stained by anti-alpha SM-1. In all the specimens tested, Western-blot analysis of tissue extracts immunodecorated with anti-alpha SM-1 revealed a single polypeptide chain having the same electrophoretic mobility as bovine alpha SM actin. The binding to amphibian and reptilian tissue extracts was inhibited by the synthetic peptide Ac-EEED, but not Ac-DEED, as occurs in mammals. alpha SM actin expression was found in vascular and visceral smooth muscle cells of the species tested. The media of small and large blood vessels was labelled by anti-alpha SM-1. In the stomach and intestine the outer longitudinal and inner circular layers of the muscularis and of the muscularis mucosae were stained. In addition, myofibroblasts of the subepithelial layer were labelled. A more restricted expression of this isoactin was detected in turtle (P. scripta elegans) visceral smooth muscle cells, which may be related to the involvement of the digestive system in respiratory activity. These data suggest that in vertebrate evolution alpha SM actin arose earlier than previously proposed.

Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.

The presence of an alpha-smooth muscle (alpha-sm) actin-like protein in planaria (Dugesia lugubris s.l.) is reported. The protein shows a 42 kDa molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and is specifically recognized by the mammalian anti alpha-sm actin monoclonal antibody. When a planarian is induced to regenerate by head amputation, the immunostaining of the alpha-sm actin-like molecule becomes important in the area of growing blastema, reaching a maximum between 70-120 hours after injury. Conventional electron microscopy at the 4-day-regeneration stage shows that blastema-forming cells are a homogeneous population whose morphological features resemble those of migrating mesenchyme-like cells; only the myoblasts show a recognizable phenotype. The immunocytochemical localization of alpha-sm actin-like molecule by immunoperoxidase (light microscopy) and immunogold stains (electron microscopy) was carried out on both intact and injured worms. The antigen was localized mainly at the basal portion of the epidermal cells and in the undifferentiated mesenchyme-like cells. Myoblasts, but not differentiated myofibers, were also labelled by this antibody. The results indicate that in the lower Eumetazoan planarians, as well as in vertebrates, the alpha-sm actin can be considered to be a marker for myoid differentiation. The suggestion that alpha-sm actin can be used as a marker for mesenchyme-like cells in vertebrates and in invertebrates is also discussed.

1. A comparative study of multiple forms of acid phosphatase (AcPase) in various organs of mammals was carried out. 2. These studies indicated that the high-molecular weight AcPase is preferentially expressed by tissues which undergo cell proliferation such as epithelial tissues; on the contrary, the low-molecular weight enzyme seems to be characteristic of highly differentiated tissues such as nervous, muscle and blood erythrocytes. 3. The existence of a new AcPase activated by Zn2+ ions was observed in all tissues studied with the exception of erythrocytes. 4. The enzyme shows a molecular weight of 57 kDa, is insensitive to NaF, hydrolyzes p-nitro-phenylphosphate and o-c-phenylphosphate; ATP, a-naphthyl-phosphate and beta-glycerolphosphate are also dephosphorylated.

Actin- and fibronectin-like proteins were characterized in the planarian, Dugesia lugubris s.l., by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analysis using antisera to vertebrate actin and fibronectin. These antisera recognized protein bands of 42 kDa and 220 kDa, respectively. In addition, the immunohistochemical distribution of both actin- and fibronectin-like material was examined by using immuno-electron microscopy. Actin-like protein was localized in myofibrils in various differentiation stages, and in the peripheral cytoplasm and lamellipodia of cells that were migrating. The fibronectin-like component was associated with the extracellular matrix in the fibrillar structures and with the surface of the migrating cells. Our data suggest that similar cellular and molecular mechanisms are involved in cell-matrix interactions and in the morphogenesis of living organisms at different evolutionary levels.

1. The presence of high-Mr and low-Mr acid phosphatases [orthophosphoric-monoester phosphohydrolase, (acid optimum), EC 3.1.3.2] in the skeletal muscle of frog Rana esculenta was reported. 2. The subcellular localization and some characteristics of both enzymes were also described. 3. The low-Mr AcPase was purified to homogeneity. The enzyme did not absorb on Concanavalin A-Sepharose 4B indicating that this was not a glycoprotein. 4. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr 13.7 +/- 0.8 kDa in the presence of sodium dodecyl sulphate. 5. The Mr of the native enzyme was 14.0 +/- 1.1 kDa as determined by gel filtration on a Sephadex G-100 column. The isoelectric point was 6.02. 6. The enzyme was strongly inhibited by 1 mM Ag+, Hg2+, Sn2+ and Cu2+ while other cations both at 10(-2) and 10(-3) M showed little or no effect. 7. The enzyme was insensitive to NaF and tartrate but was strongly deactivated by formaldehyde, PMB, Iodoacetamide and Triton X-100. Phosphate was a competitive inhibitor (k1 = 0.83 mM). 8. The best substrate for the enzyme was p-nitrophenylphosphate but phenylphosphate, flavin mononucleotide and o-P-tyrosine were also hydrolyzed, though at different rates. 9. The enzyme activity was enhanced in the presence of methanol, ethanol, acetone and glycerol indicating a phosphotransferase activity.

Particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing] has been cytochemically evidentiated in the cells which make-up the lung air-blood barrier. The cytochemical procedure utilized demonstrates the presence of membrane-bound guanylate cyclase activity through precipitation of lead pyrophosphate in tissues incubated with GTP or with guanylyl imidodiphosphate. Electron microscopic examination reveals that guanylate cyclase (GC) is localized, as micropinocytic vesicles, within endothelial components of small blood vessels, in basal lamina and in the flat alveolar cells. The secretory alveolar cells also exhibit the positive GC reactivity in their peripheric cytoplasm and in their microvilli. The observations support that GC and cGMP are involved in cellular transport phenomena. The enzyme might play a role in the secretion process of surface active material. Positive staining has been found also in other types of cells, namely alveolar macrophages and fibroblasts. A biochemical evaluation of GC activity shows that about 30-40% of this activity is associated with the particulate fraction, which justifies its abundance in the cytochemical reports shown in the paper.