Saccharomyces boulardii Tht 500101 changes gut microbiota and ameliorates hyperglycaemia, dyslipidaemia, and liver inflammation in streptozotocin-diabetic mice.

Type 1 diabetes mellitus (T1DM) is a disorder resulting from chronic autoimmune destruction of insulin-producing pancreatic ß-cells, lack of insulin production and hyperglycaemia. The aim of this study was to evaluate the hypothesis that streptozotocin-diabetic mice treated with Saccharomyces boulardii THT 500101 strain present improvement of glucose and triglycerides metabolism, reduction of liver inflammation concomitant with a beneficial impact in the gut microbiota profile. C57BL/6 male mice were randomly assigned into three groups: Control, Diabetes, Diabetes+Probiotic, and were euthanised 8 weeks after probiotic chronic administration. Mice submitted to treatment presented reduced glycemia in comparison with the diabetic group, which was correlated with an increase in C-peptide level and in hepatic glycogen content. Fat metabolism was significantly altered in streptozotocin-induced diabetic group, and S. boulardii treatment regulated it, leading to a decrease in serum triglycerides secretion, increase in hepatic triglycerides storage and modulation of inflammatory profile. The phenotypic changes seen from chronic S. boulardii treatment were found to be broadly associated with the changes in microbioma of diabetic animals, with increased proportion in Bacteroidetes, Firmicutes and Deferribacteres, and a decreased proportion of Proteobacteria and Verrucomicrobia phylum. Thus, the data presented here show up a novel potential therapeutic role of S. boulardii for the treatment and attenuation of diabetes-induced complications.

Maternal antibiotic prophylaxis affects Bifidobacterium spp. counts in the human milk, during the first week after delivery.

Human milk is an important source of microorganisms for infant gut colonisation. Although the maternal antibiotic prophylaxis is an important strategy to prevent maternal/neonatal sepsis, it has to be investigated how it may affect the human milk microbiota, especially the genus Bifidobacterium, which has been associated to health benefits. Here, we investigated the impact of the maternal antibiotic prophylaxis on the human milk Bifidobacterium spp. and total bacteria counts, in the first week (short-term) and first month (medium-term) after delivery. Human milk samples were collected from 55 healthy lactating women recruited from the University Hospital of the University of São Paulo at days 7±3 and 30±4 after vaginal delivery. Twenty one volunteers had received maternal antibiotic prophylaxis (MAP group) and 34 had not received MAP (no-MAP group) during or after labour. Total DNA was isolated from milk samples, and the bacterial counts were estimated by quantitative PCR (qPCR). We found lower levels of Bifidobacterium in the MAP group in the first week after delivery (median = 2.1 vs 2.4 log of equivalent cells/ml of human milk, for MAP and no-MAP groups, respectively; P=0.01), although there were no statistical differences in total bacteria count. However, no differences were found in Bifidobacterium counts between the groups at day 30±4 (median = 2.5 vs 2.2 log of equivalent cells/ml of human milk, for MAP and no-MAP groups, respectively; P=0.50). Our results suggest that MAP has a significant impact on Bifidobacterium counts in human milk, reducing this population in the first week after delivery. However, throughout the first month after delivery, the Bifidobacterium counts tend to recover, reaching similar counts to those found in no-MAP group at day 30±4 after delivery.

From [11C]CO2 to [11C]amides: a rapid one-pot synthesis via the Mitsunobu reaction.

A novel amide synthesis methodology is described using amines, CO2 and Grignard reagents and Mitsunobu reagents. The method was applied to carbon-11 radiochemistry to label amides using cyclotron-produced [11C]CO2. The synthetic utility of the one-pot labelling methodology was demonstrated by producing [11C]melatonin. The incorporation of [11C]CO2 into [11C]melatonin was 36% - determined by radioHPLC 2 min post [11C]CO2 delivery.

Present situation and new perspectives for vaccination against Neisseria meningitidis in Tuscany, Central Italy.

BACKGROUND: In Italy one third of bacterial meningitis are caused by Neisseria meningitidis. In March 2005, the Regional Health Authority of Tuscany included the meningococcal serogroup C conjugate (MCC) vaccine in the recommended vaccination program with a schedule of three doses to all newborns at 3, 5 and 13 months of age (from 2008 amended to a single dose at 13 months) and a single catch-up dose until age 6. OBJECTIVE: To evaluate the impact of the current national and regional immunization strategies against N. meningitidis and to highlight new perspectives for meningococcal disease prevention with the existing tetravalent meningococcal vaccine (ACWY) and with the future incoming meningococcal B vaccines. METHODS: Meningitis incidence rates in Italy and in Tuscany were calculated for the period 1994-2011 and 2005-2011,respectively. Immunization coverage with MCC vaccine in Tuscany and vaccination status of meningitis cases were reported. Literature review on meningococcal conjugate vaccine use and recommendation was performed. RESULTS: A decrease in incidence rates of meningococcal meningitis was observed in all age groups involved in the immunization campaign. Immunization coverage with MCC increased progressively year by year in Tuscany. A herd immunity effect was measured in unvaccinated age groups. Since 2006 no cases of invasive meningococcal C infection in vaccinated subjects were observed in Tuscany. CONCLUSIONS: Implementation of MCC vaccination in Tuscany was effective in preventing meningococcal C disease, confirming the effectiveness of the vaccine. A new tetravalent (ACWY) conjugate vaccine is now available and its use in all Italian Regions should be considered.

The domain Archaea in human mucosal surfaces.

Archaea present distinct features from bacteria and eukaryotes, and thus constitute one of the branches of the phylogenetic tree of life. Members of this domain colonize distinct niches in the human body, arranged in complex communities, especially in the intestines and the oral cavity. The diversity of archaea within these niches is limited to a few phylotypes, constituted in particular by methane-producing archaeal organisms. Although they are possibly symbionts, methanogens may play a role in the establishment of mucosal diseases by favouring the growth of certain bacterial groups.

Polyelectrolyte multilayer film coating and stability at the surfaces of oral prosthesis base polymers: an in vitro and in vivo study.

A new type of coating involving a layer-by-layer technique has been recently reported. This coating is composed of a polyelectrolyte multilayer film that confers specific properties on surfaces to which it is applied. Here, we studied the applicability of such a technique to the coating of oral prostheses, by first testing the construction of polyelectrolyte multilayer films on several polymers used in oral prosthesis bases, and, subsequently, by studying the stability of these coatings in vitro, in human saliva, and in vivo in a rat model. We demonstrated that the multilayered films are able to coat the surfaces of all tested polymers completely, thus increasing their wettability. We also showed that saliva does not degrade the film after 7 days in vitro and after 4 days in vivo. Taken together, our results establish that the layer-by-layer technique is suitable for the coating of oral devices.

Multilayer polyelectrolyte films functionalized by insertion of defensin: a new approach to protection of implants from bacterial colonization.

Infection of implanted materials by bacteria constitutes one of the most serious complications following prosthetic surgery. In the present study, we developed a new strategy based on the insertion of an antimicrobial peptide (defensin from Anopheles gambiae mosquitoes) into polyelectrolyte multilayer films built by the alternate deposition of polyanions and polycations. Quartz crystal microbalance and streaming potential measurements were used to follow step by step the construction of the multilayer films and embedding of the defensin within the films. Antimicrobial assays were performed with two strains: Micrococcus luteus (a gram-positive bacterium) and Escherichia coli D22 (a gram-negative bacterium). The inhibition of E. coli D22 growth at the surface of defensin-functionalized films was found to be 98% when 10 antimicrobial peptide layers were inserted in the film architecture. Noticeably, the biofunctionalization could be achieved only when positively charged poly(l-lysine) was the outermost layer of the film. On the basis of the results of bacterial adhesion experiments observed by confocal or electron microscopy, these observations could result from the close interaction of the bacteria with the positively charged ends of the films, which allows defensin to interact with the bacterial membrane structure. These results open new possibilities for the use of such easily built and functionalized architectures onto any type of implantable biomaterial. The modified surfaces are active against microbial infection and represent a novel means of local host protection.

Use of bar-clip attachments to enhance the retention of a maxillofacial prosthetic obturator: a clinical report.

Precision attachments are commonly used in prosthetic dentistry but are still questioned in maxillofacial prosthetics. The aesthetic enhancement and functional rigidity provided by attachments are favourable features to many challenging clinical situations such as class-III defect (Aramany's maxillectomy classification). Surgical reconstruction is the standard treatment for this type of defect repair. However, owing to the need for dental rehabilitation and patient reserve, prosthetic rehabilitation was the modality chosen in this clinical case. The use of bar attachments is described in this paper as providing increased stability and retention of the prosthesis, and improved obturator water and airtightness.

Putative histidin-rich proteins in the epidermis of lizards.

In the stratum granulosum of mammalian epidermis, histidin-rich proteins (filaggrins) determine keratin clumping and matrix formation into terminal keratinocytes of the stratum corneum. The nature of matrix, interkeratin proteins in the epidermis of nonmammalian vertebrates, and in particular in that of reptilian, mammalian progenitors are unknown. The present biochemical study is the first to address this problem. During a specific period of the renewal phase of the epidermis of lizards and during epidermal regeneration, keratohyalin-like granules are formed, at which time they take up tritiated histidine. The latter also accumulate in cells of the alpha-keratin layer (soft keratin). This pattern of histidine incorporation resembles that seen in keratohyalin granules of the stratum granulosum of mammalian epidermis. After injection of tritiated histidine, we have analysed the distribution of the radioactivity by histoautoradiography and electrophoretic gel autoradiography of epidermal proteins. Extraction and electrophoretic separation of interfilamentous matrix proteins from regenerating epidermis 3-48 hours post-injection reveals the appearance of protein bands at 65-70, 55-58, 40-43, 30-33, 25-27, and 20-22 kDa. Much weaker bands were seen at 100, 140-160, and 200 kDa. A weak band at 20-22 kDa or no bands at all are seen in the normal epidermis in resting phase and in the dermis. In regenerating epidermis at 22 and 48 hours post-injection, little variation in bands is detectable, but low molecular weight bands tend to increase slightly, suggesting metabolic turnover. Using anti-filaggrin antibodies against rat, human, or mouse filaggrins, some cross-reactivity was seen with more reactive bands at 40-42 and 33 kDa, but it was reduced or absent at 140, 95-100, 65-70, 50-55, and 25 kDa. This suggests that different intermediate degradative proteins of lizard epidermis may share some epitopes with mammalian filaggrins and are different from keratins with molecular weight ranging from 40 to 65-68 kDa. The immunocytochemical observation confirms that a weak filaggrin-like immunoreactivity characterizes differentiating alpha-keratogenic layers in normal and regenerating tail. A weak filaggrin labeling is discernable in small keratohyalin-like granules but is absent from the larger granules and from mature keratinocytes. The present results indicate, for the first time, that histidine-rich proteins are involved in the process of alpha-keratinization in reptilian epidermis. The cationic, interkeratin matrix proteins implicated may be fundamentally similar in both theropsid-derived and sauropsid amniotes.

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula.

The distribution of three anti-cytokeratin (alpha-keratin) antibodies (AE1, AE2, AE3) in the epidermis of a lizard has been studied by immunocytochemistry at light and electron microscope and by immunoblot analysis. This study shows the expression of different keratins in the resting stage epidermis of the lizard Podarcis sicula. In this stage the epidermis has an external beta-layer, an underlying alpha-layer, some layers of living suprabasal cells and a basal stratum germinativum. The AE1 antibody is localized in the basal and suprabasal cells only in the outer scale surface, but is absent from the inner surface, the hinge region and from the keratinized beta- and alpha-layers. The AE2 antibody is mainly localized at the level of the hinge region and of the alpha-layer and gives a lower reaction in the beta-layer. The AE3 antibody is mainly localized in basal and suprabasal cells, lower in the alpha-layer, and absent from the beta-layer. The electron microscope shows that all the three antibodies immunolabel cytoplasmic fibrillar structures in the deep alpha-layers and that AE2 and AE3 antibodies label small electron-dense areas in the external dense beta-layer within the electron-lucid matrix. Immunoblot analysis of the keratins extracted and separated by gel electrophoresis demonstrates the presence of a band of high molecular weight (67-68 kDa) positive to all three antibodies. In addition AE1 antibody recognizes a 44-45 kDa band and a 57-58 kDa band, AE2 recognizes a 60-61 kDa band, and AE3 recognizes a 47 kDa and a 56-57 kDa band. The localization of the keratins identified by immunoblot analysis in the epithelial layers is discussed taking in account the immunolabeling at light and electron microscope. The present study suggests that also in the normal epidermis of this reptiles, in both the alpha- and the beta-layer, the molecular masses of keratins increase from the basal to the keratinized layers, a phenomenon which is generalized to adult and embryonic amniotes epidermis.

[Cost of ICU in Italy. Results from an empirical study on a sample of 12 hospitals]. / Il costo del reparto di terapia intensiva in Italia. Risultati da un'indagine empirica su un campione di 12 centri.

BACKGROUND: The Italian hospital payment system based on DRG doesn t properly include Intensive Care Units (ICU) costs. Since great emphasis has been recently given to rationing health care resources, assessing ICU costs seems to be dramatically relevant. Aim of the study was to assess the average yearly cost and the cost per diem of a sample of Italian multispecialistic ICU wards. METHODS: In September 1995, a questionnaire concerning data on variable and fixed cost was sent to 25 Italian ICU wards, 11 NHS hospital-based (Northern Italy: 5; Central Italy: 4; Southern Italy: 2) and 14 school of medicine-based (Northern Italy: 7; Central Italy: 5; Southern Italy: 2). Variable cost data included: disposable, drugs, blood and blood-derived products, physical tests, chemical and microbiological routines, instrumental diagnostic procedures and physiotherapy. Concerning fixed costs, data on personnel and equipment were requested. In addition, some hospital overheads data (utilities; power; heating; maintenance; cleaning; laundry; accounting; waste disposal; cafeteria) were collected. RESULTS: On the basis of the 12 questionnaires returned (Northern Italy: 9; Central Italy: 3; Southern Italy: 0), the yearly cost of an ICU ward is Liras 4,580,032,000 (range 2,739,277,000-7,704,292,000), whereas the average cost per diem is Liras 1,802,000 (range 1,234,000-3,179,000). Cost of personnel is about 61% of the above mentioned costs. CONCLUSIONS: Despite the lack of questionnaires from Southern Italy and the unavailability of some data concerning both the cost of equipment and the overheads, the remarkable average cost values obtained could support further research.

Drosophila vitelline membrane cross-linking requires the fs(1)Nasrat, fs(1)polehole and chorion genes activities.

Abstract. During the final step of Drosophila vitelline membrane formation, the structural proteins composing this layer become cross-linked by covalent bonds. In the present report, we analyzed the vitelline membrane cross-linking in mutants having defects either in this layer or in the chorionic layers. In the fs(1)Nasrat and fs(1)polehole mutant alleles conferring defects in vitelline membrane formation, disruption of vitelline membrane cross-linking was observed, indicating the involvement of these two genes in the process. On the contrary, in the fs(1)Nasrat and fs(1)polehole alleles showing defects only at the termini of the embryo the vitelline membrane is properly formed, confirming a multifunctional activity of their gene products. Altered vitelline membrane cross-linking was also detected in a mutant of the chorion protein gene Cp36and in the chorion amplification mutant fs(1)K1214, suggesting a role of the structural components of chorion layers in the process of vitelline membrane hardening.

Immunocytochemical and electrophoretic distribution of cytokeratins in the regenerating epidermis of the lizard Podarcis muralis.

Using immunocytochemistry at light- and electron-microscope levels, we studied the distribution of three monoclonal antibodies (AE1, AE2, AE3) specific for mammalian alpha-keratins in regenerating lizard epidermis. We also characterized the keratins expressed during this process by immunoblotting after electrophoretic separation. The AE1 antibody is localized in the basal and suprabasal layers of prescaling and scaling epidermis. During the first stages of scale neogenesis, the AE1 antibody also marks the differentiating oberhautchen and beta-layer, but it disappears from these layers as they mature. This antibody does not stain the prekeratinized and keratinized outermost layers in the hinge region. The AE2 antibody labels the superficial wound epidermis, prekeratinizing and keratinized beta- and alpha-layers, but not basal and suprabasal cells. The AE3 antibody labels all living and keratinized epidermal layers, although AE3 immunoreactivity decreases and disappears as the beta-layer matures. The ultrastructural study shows that the AE2 and AE3, but not the AE1, antibodies specifically label small electron-dense areas within the beta-layer, suggesting retention of alpha-keratins. In the stages of tail regeneration examined, immunoblotting with the three antibodies used for the immunolocalization gives a pattern similar to that of the normal epidermis, except distally, where the process of scale differentiation begins. In this region, in addition to the keratin forms discovered in the normal and in proximal regenerating epidermis, an intense low molecular weight band at 40-41 kDa, positive to all three antibodies, is clearly detectable. Furthermore, in the distal region AE1 and AE3 antibodies, but not the AE2, recognize a weak band at 77-78 kDa not present in the normal and proximal epidermis. The localization and the possible role of the different keratins in the regenerating epidermis is discussed.

Organization and characterization of the keratin cytoskeleton in the previtellogenic ovarian follicle of the lizard Podarcis sicula raf.

The cytokeratin (CK) cytoskeleton, previously described by immunofluorescence in the ovarian follicle of Podarcis sicula, at the electron microscope results constituted by bundles of 10 nm thick intermediate filaments containing keratin. These bundles are better evident in the cytoplasm of the pyriform cell apex pointed toward the oocyte surface and mostly in the intercellular bridges connecting fully differentiated pyriform cells to the oocyte. During the differentiation of pyriform cells, the intermediate filament bundles first appear inside the intercellular bridge, when the small follicle cells progressively enlarge after their fusion with the oocyte and assume a morphology of "intermediate" cells. The present study also reports a comparative analysis by immunolabeling, SDS-PAGE, and immunoblotting with anticytokeratins CK8, CK18, and CK19 antibodies of both the ovarian follicle and the intestine of Podarcis sicula. These antibodies, specific to the keratins of monolayered intestinal cells, react also against those expressed in the oocytes of Xenopus laevis. This study shows the presence in the ovarian follicle of this reptile only of keratin forms of homologues to the CK8 and CK18 of mammals and the lack of CK19. The same analysis carried out utilizing AE1 and AE3 antibodies, which recognize most of the acidic and basic keratins in mammals, has shown additional forms of keratins specifically expressed in the ovarian follicle (50 kDa) and in both the examined tissues (49 and 60 kDa). The reported results indicate that in the ovarian follicle of this reptile, keratins have peculiar characteristics that can be explained by the unique structural function of the cytoskeleton in this system.

The costs of pharmacological treatment for major depression. The Italian Prospective Multicentre Observational Incidence-Based Study.

OBJECTIVE: To describe the pharmacological treatment for major depression under the conditions of routine Italian public mental health facilities, assess its costs, and study its main predictors according to a societal perspective. DESIGN: This was a prospective multicentre observational study designed to evaluate the economics of treatment of major depression using a specifically designed 65-item questionnaire. Data on drug consumption were collected in a section of the questionnaire and are presented here. PATIENTS AND PARTICIPANTS: 60 mental health facilities were selected and 556 patients were enrolled and followed up for 15 months. RESULTS: Pharmacological treatment appears to be the most common treatment for major depression. 98% of patients were prescribed an antidepressant. Selective serotonin reuptake inhibitors (SSRIs) were the most prescribed antidepressants. Patients treated with SSRIs suffered from less severe depression than those treated with tricyclic antidepressants. Benzodiazepines were prescribed for 84% of patients enrolled. The total drug cost was 1,120,000 Italian lire ($US707) per patient (1995 values). Less than 20% of this cost was borne by the Italian National Health Service, as the majority of drugs used were not reimbursed. CONCLUSIONS: The costs of the most widespread approach to treating major depression (pharmacological treatment) are not currently covered by the Italian National Health Service. Prescribing of drugs seems to diverge from the standards of treatment indicated by the Italian Drug Committee.

Alpha1-antitrypsin phenotypes and anti-neutrophil cytoplasmic auto-antibodies in inflammatory bowel disease.

OBJECTIVES: Alpha1-antitrypsin (alpha1-AT) is encoded by a highly polymorphic gene with over 75 codominantly expressed alleles at the protease inhibitor (Pi) locus classified as normal, deficient, dysfunctional or null. The aim of this study was to determine in patients with inflammatory bowel disease: (i) the prevalence of anti-neutrophil cytoplasmic auto-antibodies (ANCA) and their antigen specificities; (ii) alpha1-AT Pi phenotypes; and (iii) possible associations between ANCA, disease activity and deficient alpha1-AT alleles. DESIGN: Study of 95 consecutive patients with ulcerative colitis (UC) and 63 patients with Crohn's disease (CD). METHODS: Diagnosis and disease activity were determined by clinical, endoscopic and histological criteria. ANCA by indirect immunofluorescence (IIF) and Pi phenotyping by isoelectric focusing were performed for all patients. Positive IIF sera were tested in antigen-specific enzyme-linked immunosorbent assay: proteinase 3 (PR3), myeloperoxidase (MPO), lactoferrin (LF), lysozyme, human leucocyte elastase (HLE), cathepsin G and bactericidal/permeability increasing protein (BPI). RESULTS: Sixty-one patients with UC (64.2%) and only 11 with CD (17.5%) had ANCA (P < 0.001). Antigen specificities were PR3 (7/61), MPO (3/61), LF (6/61), HLE (1/63) and BPI (10/61) in UC, and PR3 (2/11) and BPI (2/11) in CD. Three PiZ alleles were found, matching the prevalence in the normal French control population. No relationship was found between the presence of ANCA, antibody specificity, disease activity and deficient alpha1-AT alleles. CONCLUSION: ANCA are more frequent in UC than CD and do not correlate with disease activity. ANCA and protease/antiprotease imbalance do not appear to modulate the clinical course of inflammatory bowel disease.

Influence of age on outcome after percutaneous transluminal coronary angioplasty.

This study estimates the influence of age on outcomes (mainly survival) of 21,516 patients who underwent percutaneous transluminal coronary angioplasty (PTCA) between 1980 and 1996. We prospectively analyzed the patients in 5 age groups: <50, 50 to 59, 60 to 69, 70 to 79, and > or =80 years old. During the in-hospital period after PTCA, mortality increased from 0.28% in patients aged <50 to 3.45% in patients aged > or =80; Q-wave myocardial infarction was not significantly associated with age, and the 2 older groups were referred less often to coronary artery bypass graft surgery. During follow-up, lasting up to 10 years, the hazard of death was significantly influenced by age; Q-wave myocardial infarction was influenced by age, although the magnitude of the effect was relatively small and of questionable clinical significance; and coronary artery bypass graft surgery was performed less often in the 2 older age groups. Additional PTCA was similarly performed among the age groups. Age, diabetes mellitus, systemic hypertension, heart failure class, angioplasty in graft vessel, number of coronary vessels narrowed, and previous myocardial infarction were predictors of death over the 10-year follow-up. Age was the most important correlate of death after PTCA, with a 65% increase in the hazard of death for each 10-year increase in age. Age has an independent effect on early and late survival after PTCA.

Antibody response of children with enteropathogenic Escherichia coli infection to the bundle-forming pilus and locus of enterocyte effacement-encoded virulence determinants.

Enteropathogenic Escherichia coli (EPEC) express a plasmid-encoded type IV pilus termed bundle-forming pilus, which is associated with the formation of bacterial microcolonies on cultured epithelial cells. Bacterial attachment and effacement of the enterocyte brush border membrane is attributed to a surface outer membrane protein adhesin termed intimin and EPEC-secreted proteins EspA, EspB, and EspD. Except for intimin, production in vivo or antibody response against these virulence determinants during natural EPEC infections in young children has not been demonstrated. Antibody responses against BfpA, intimin, EspA, and EspB were investigated in Brazilian children naturally infected with EPEC. Generally, IgG antibodies against BfpA and EspB were the most commonly found, followed by anti-EspA and intimin antibodies. Thus, bundle-forming pilus and locus of enterocyte attachment-encoded products are produced in vivo during natural EPEC infections and elicit an immune response against heterologous EPEC virulence determinants. These findings have important implications in the immunoprophylaxis against EPEC infections.

Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster.

In Drosophila a remarkable feature of oogenesis is the regression of the nurse cells after dumping their cytoplasmic contents into the oocyte. We have studied the nature of this process at the late stages of egg chamber development. In egg chambers DAPI staining shows highly condensed chromatin from stage 12 and TUNEL labelling shows DNA fragmentation up to stage 14. Gel electrophoresis of the end-labelled DNA, extracted from isolated egg chambers at the same stages of development, shows a ladder typical of apoptotic nuclei. This provides evidence that, during Drosophila oogenesis, the nurse cells undergo apoptosis. Apoptotic nuclei have also been detected in dumping-defective egg chambers, indicating that the cytoplasmic depletion of nurse cells is concurrent with but apparently not the cause of the process.

Cytokeratin cytoskeleton in the differentiating ovarian follicle of the lizard Podarcis sicula Raf.

By immunoblotting and immunocytochemical techniques, we characterized the cytokeratins previously localized by us in the previtellogenic ovarian follicle of Podarcis sicula. Our results show that these cytokeratins correspond to those expressed in the monolayered epithelia. In fact, the immunoblotting analysis showed that the NCL-5D3 antibody, specific for human low molecular weight cytokeratins expressed in monolayered epithelia, reacted with the cytokeratins extracted both from the ovary and from the monolayered intestinal mucosa of Podarcis sicula. Furthermore, this antibody, in this reptile as in humans, clearly immunolabeled sections of corresponding tissues. The organization of the cytokeratin cytoskeleton in the main steps of the ovarian follicle differentiation was also clarified. The reported observations suggest that in Podarcis sicula, the cytokeratin cytoskeleton is absent in the early oocytes. It first appears in the growing oocytes as a thin cortical layer in concomitance with its becoming visible also in the enlarging follicle cells. In the larger follicles, this cytoskeleton appears well organized in intermediate cells and in particular in fully differentiated pyriform cells. In both these cells a cytokeratin network connects the cytoplasm to the oocyte cortex through intercellular bridges. At the end of the previtellogenic oocyte growth, the intense immunolabeling of the apex in the regressing pyriform cells suggests that the cytokeratin, as other cytoplasmic components, may be transferred from these follicle cells to the oocyte. At the end of the oocyte growth, in the larger vitellogenic oocytes surrounded by a monolayer of follicle cells, the cytokeratin constitutes a heavily immunolabeled cortical layer thicker than in the previous stages.