Pattern of expression of c-Myc, Max and Bin1 in human anagen hair follicles.

BACKGROUND: We recently reported the presence of c-Myc immunoreactivity in two distinct regions of the inner root sheath (IRS) of human anagen hair follicles; they corresponded to the regions where keratinocytes of Henle's and Huxley's layers enter the terminal differentiation phase that will lead to their exfoliation in the pilary canal. These regions were denoted lower (LR) ring and upper ring (UR). OBJECTIVES: To extend these observations to other genes connected to c-Myc and specifically to Max and Bin1. Max is the best known heterodimeric partner of c-Myc, interacting with its C-terminal domain, and Bin1 is an adaptor protein interacting with its N-terminal domain. METHODS: Human anagen hair follicles were processed for c-Myc, Max and Bin1 immunohistochemistry and immunofluorescence. The presence of different isoforms of Bin1 was evaluated by Western blot analysis. RESULTS: Analysis of sections cut in several planes, including tangential, demonstrated the presence of a third ring of c-Myc-positive cells (intermediate ring; IR) in the cuticle of the IRS corresponding to the region where this thin layer undergoes keratinization. Max immunoreactivity was observed in the three layers of the IRS starting in the lower bulbar region and ending in each of them at the level of the corresponding c-Myc-positive ring. Bin1 immunoreactivity was clearly distinguished only in Huxley's layer and in the cuticle, starting in some cells below the UR and terminating at the level of the latter. The companion layer of the outer root sheath was also labelled up to the infundibular region. Max and Bin1 immunostaining were less consistently observed in other skin adnexae and in the epidermis. CONCLUSIONS: The results indicate that the asynchronous differentiation along the axis of the hair follicle of the different layers of the IRS and of the companion layer involves the expression of different genes that are interrelated in the so-called 'Myc network'. The specific localization of c-Myc in the IRS only at the level of the discrete and limited regions of the three rings appears to be the hallmark of the switch from differentiation to terminal differentiation/cell deletion.

c-Myc expression in human anagen hair follicles.

The hair follicle represents a very attractive organ system for studying the precise balance between cell proliferation, growth, differentiation, and death of cells, because it periodically and regularly regenerates, retaining its morphogenetic signals throughout its life. One of the most intriguing oncogenes which is able to induce both cell growth and apoptosis, depending upon the environmental conditions, is c-myc. The aim of the present study was to investigate its presence and localization in human hair follicles by immunohistochemistry and immunofluorescence. Our observations demonstrated the consistent presence of two clusters of c-Myc-expressing cells in anagen follicles, located in two annular regions of the inner root sheath, at the border between cells characterized by putative trichohyalin granules and cells which are keratinized. The lower group belongs to Henle's layer, while the upper group belongs to Huxley's layer. c-Myc oncoprotein seems to favour apoptosis/differentiation and may be a marker for terminal differentiation of trichocytes, at least in the inner root sheath. Our findings agree with the interpretation that the complex morphology of the hair follicle reflects its complex function; the extrusion of a highly organized multicellular structure, the hair shaft, driven by another highly organized multicellular structure, the inner root sheath.

Structural and biochemical analysis of the parasite Gordius villoti (Nematomorpha, Gordiacea) cuticle.

The cuticle of the nematomorpha Gordius villoti is a proteinaceous extracellular structure that covers the body during the endoparasitic life in the hemocoelic cavity of insect hosts, and of the free-living adult animals. The ultrastructure of the cuticle has a complex spatial organization with several parallel layers of large diameter fibers, interposed thinner fibrous elements and honeycomb-shaped matrix surrounding the fibers. When adult isolated cuticles were partially solubilized by several compounds, the structure revealed a strong insolubility and the main fibers were always observable. HPLC and spectrophotometric assays carried out to investigate the presence of tyrosine cross-linking, indicated such a mechanism as a key-element in the hardening process of the cuticle. Such data strongly suggest that the Gordius cuticle contains dityrosine compounds, whose formation is probably mediated by endogenous peroxidase activity.

Genistein in the control of breast cancer cell growth: insights into the mechanism of action in vitro.

Genistein significantly inhibited cell growth (IC50 around 10 microM) of MCF-7, MDAMB-231 and HBL-100 cell lines, but not of skin-derived fibroblasts and counteracted the growth-stimulatory effects exerted by estradiol and growth factors. It abolished the paracrine stimulation observed in MCF-7 cells in co-culture with MDAMB-231 or fibroblasts. Genistein-treated cells accumulated in the S and G2/M phases of the cell cycle and underwent apoptosis. Genistein decreased tyrosine phosphorylation induced upon treatment with transforming growth factor-alpha. Finally, genistein bound the estrogen receptor (ER) (relative affinity constant Kd = 4 nM), induced pS2 and cathepsin-D transcription and increased nuclear ER levels.

Isolation and characterization of the gene coding for human cytidine deaminase.

The human gene coding for cytidine deaminase (CD), the enzyme which catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, was isolated and structurally characterized. CD is a single copy gene with a length of 31 kb and consists of four exons. Exon-intron junctions do not bracket functional domains of the encoded protein as the boundary between exons 2 and 3 interrupts the catalytically important zinc-finger domain, which is well conserved along phylogenesis. 5'-RACE and RNase mapping experiments identify one major and multiple other minor transcription initiation sites, which are present in placenta as well as in the myeloid cell lines, HL-60 and U937. The 5'-flanking region of the gene contains an orientation-dependent functional promoter and is characterized by the presence of several potential sites for the binding of known transcriptional factors.

Coronary angiography and aorto-coronary bypass surgery in type 2 diabetic patients.

Diabetic and non-diabetic subjects with angina who underwent angiography and were subsequently treated surgically or medically and followed up for 5 years were analysed in order to assess coronary angiographic findings, efficacy of coronary artery bypass grafting and prognostic criteria in Type 2 diabetic patients with angina as compared to non-diabetic subjects. A total of 1853 of non-diabetic and 145 diabetic subjects underwent angiography, including respectively 857 and 68 who had surgery. Perioperative mortality, survival, reinfarction and asymptomaticity rates were measured. Multivariate analysis of risk factors and clinical features was performed. Diabetic patients had a higher frequency of multi-vessel stenoses (p < 0.001), a greater diffusion of stenoses (p < 0.005) and worse left ventricular motion (p < 0.005). No differences were found in perioperative infarction and mortality. Operated diabetic patients had a higher survival rate (p < 0.001) and a longer symptom-free period (p < 0.05) than unoperated diabetic patients. Operated diabetic patients had similar survival and more frequent recurrence of angina (p < 0.05) than operated non-diabetic patients. Survival rate was lower for unoperated diabetic patients than unoperated non-diabetic patients (p < 0.05). Recurrence of angina was similar. Multivariate analysis did not indicate diabetes as a factor affecting survival. It is concluded that surgery for Type 2 diabetic patients with coronary artery disease is a suitable therapeutic option conferring a reduction in mortality regardless of the presence of diabetes.

Comparison of two intensified conventional insulin regimens with three and four daily injections.

When choosing an intensified conventional insulin therapy, no specific differentiation is made between the three-injection regimen (regular insulin at breakfast and lunch, and regular+intermediate-acting insulin at dinner) and the four-injection regimen (regular insulin at breakfast, lunch and dinner, and intermediate-acting insulin at bedtime). No published studies have evaluated to our knowledge the differences between these two regimens. In 1991, we proposed to 30 stable type 1 diabetic patients without residual insulin secretion a change from three to four daily injections: 7 refused, 4 were later excluded for intercurrent events; 19 followed the four daily injection regimen for 2 years. In these non randomized 19 patients, the Student's test for paired data was used to compare with a self-controlled study the 1989-90 three-daily injection period with the 1991-92 four-daily-injection period in order to evaluate any differences in daytime blood glucose values (bi-monthly out-patient measurements taken at 8 am, 10 am, 3 pm and 5 pm), nocturnal blood glucose levels at 3 am (bi-monthly patient self-monitoring by means of a blood glucose meter, for a total of 188 vs 188 measurements), HbA1c (a total of 203 vs 207 bi-monthly out-patient measurements), the number of nocturnal hypoglycaemic attacks, body weight and mean insulin requirement.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin polymerization in boar spermatozoa: fertilization is reduced with use of cytochalasin D.

The aggregational state of actin in boar spermatozoa after capacitation and the acrosome reaction has been examined by several methods. In vitro fertilization (IVF) experiments were conducted in the presence and absence of cytochalasin D (CD) to evaluate the role of actin polymerization in the events of fertilization. The fertilizing capacity was very high in controls, but, when CD (an inhibitor of the polymerization of actin) was added to the capacitation medium, there was a marked decrease in the fertilizing capacity of the boar spermatozoa. There was a further decrease when CD was present during both capacitation and fertilization processes. In addition to the IVF tests, biochemical and immunoelectron microscopic methods were used to analyze the state of aggregation of actin in boar spermatozoa after capacitation, and the acrosome reaction. By immunoelectron microscopy with a phalloidin probe, there were no gold particles, indicating the presence of F-actin on boar sperm heads capacitated and acrosome-reacted in media containing CD. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis there were differences in NP-40 solubility, reflecting actin polymerization, between CD-treated and untreated sperm. These results suggest that actin polymerizes during capacitation and the acrosome reaction and that this polymerization is essential to the fertilization process.

F-actin in acrosome-reacted boar spermatozoa.

Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F-actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric.

[The dietary protein contribution and hepatic encephalopathy in cirrhosis]. / Apporto proteico dietetico ed encefalopatia epatica nella cirrosi.

Hepatic encephalopathy is a neuropsychiatric syndrome, which can occur in the clinical course of acute (fulminant) or chronic hepatic failure of various aetiology; reversible metabolic abnormalities without neuronal structural changes are frequently found in this condition. High blood ammonia levels, an imbalance between plasma concentrations of branched-chain and aromatic amino acids, false neurotransmitters and neurotransmitters receptor changes in CNS are the commonly recognized pathogenetic mechanism of this syndrome. Protein malnutrition is a frequent occurrence in liver cirrhosis, especially of alcoholic aetiology. High protein diets may precipitate hepatic encephalopathy; protein restriction leads to malnutrition and enhances a negative nitrogen balance. Several clinical studies have shown that vegetable proteins are tolerated better than animal in patients with liver cirrhosis and chronic portal-systemic encephalopathy: encephalopathy index is usually lower after vegetable-protein than animal-protein diet. The favourable therapeutic effect of vegetable diets on nitrogen metabolism can be mainly accounted for by the increased intake of dietary fibers and increased incorporation and elimination of nitrogen in fecal bacteria. Mixture of amino acids enriched with branched-chain amino-acids may contribute to maintain a positive nitrogen balance and minimize muscle wasting in cirrhotics.

Electron microscopic localization of F-actin in acrosome reacted boar spermatozoa by means of a phalloidin-FITC complex.

An immunocytochemical study at the ultrastructural level has been performed in boar spermatozoa in order to clarify the aggregation state of actin before and after the acrosome reaction. A new phalloidin probe has been used to detect F-actin: a phalloidin derivative conjugated with FITC, followed by incubation with an anti-FITC antibody. The protein A-gold technique was then applied for the localization of the antigenic sites. Gold particles were localized on the sperm surface only after the acrosome reaction which was induced by the ionophore A23187.

Identification and characterization of the major components of the Oncorhynchus mykiss egg chorion.

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS-PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic-acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the "asparagine-linked" glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS-PAGE. These results suggest that the four main chorion polypeptides might share common structural features.

Functional significance of intermediate filament meshwork in annelid helical muscles.

A desmin-like protein of mol wt 54 kDa was identified in the body wall muscles of some Polychaeta, Oligochaeta, and Hirudinea utilizing SDS-PAGE followed by blot and screening with a vertebrate anti-desmin antibody. The pattern in immunofluorescence is compared to electron micrographs where several bundles of filamentous structures are clearly identifiable. These bundles are unevenly arranged in round or flattened circomyarian fibers and sometimes clearly connect Z elements with hemidesmosomes. The mechanism of intermediate filaments as a functional integration in muscle fibers is analyzed and a possible role as a block to superelongation typical of helical muscles is discussed.

Simultaneous estimation of epidermal growth factor receptors and steroid receptors in a series of 136 resectable primary breast tumors.

Human breast cancer cell lines, as well as human breast cancer biopsies, possess specific high-affinity epidermal growth factor receptors (EGF-r). However, reports on the presence of EGF-r in human breast cancer biopsies are still controversial, especially concerning the relationship between EGF-r and other biological variables whose prognostic relevance is well known, such as the estrogen (ER) and progesterone (PgR) receptors. In the present study, the EGF-r content was estimated in a series of 136 unselected breast cancer primaries along with cytoplasmic (ERc) and nuclear (ERn) ER and cytoplasmic PgR. EGF-binding activity consisted of a single class of high-affinity binding sites (Kd = 0.55 nM) and ranged from 0 to 275 fmol/mg protein. We observed a strong inverse association between EGF-r and ERc (p less than 0.001); in fact, about two thirds of the tumors were ERc-positive/EGF-r-negative or ERc-negative/EGF-r-positive. The same type of association was found between EGF-r and either ERn or PgR. Kendall's rank correlation test confirmed that the EGF-r concentrations were correlated with the levels of ERc (tau = -0.291, p less than 0.0001), ERn (tau = -0.27, p less than 0.0005) and PgR (tau = -0.162, p less than 0.01). The EGF-r content was significantly higher (p less than 0.0001) in the ERc-negative tumors (72.6 +/- 54.4 fmol/mg protein) as compared to the ERc-positive ones (33.0 +/- 37.4 fmol/mg protein). Similarly, the subset of PgR-positive tumors was characterized by lower EGF-r mean concentrations when compared to PgR-negative cases (35.4 +/- 54.4 vs. 63.8 +/- 54.4 fmol/mg protein). These results confirm the previously described inverse relationship between EGF-r and steroid receptors. Moreover, they suggest the existence of an interaction between steroid hormones and EGF and support the need for further studies to better understand their respective roles in modulating breast cancer growth.

Comparison of immunochemical and radioligand binding assays for estrogen receptors in human breast tumors.

We have compared a new enzyme immunoassay (EIA) for estrogen receptors (ER) with our conventional radioligand binding assays (multipoint dextran-coated charcoal assay for cytoplasmic ER and hydroxylapatite exchange assay for nuclear ER). Cytoplasmic ERs were measured in 76 human breast cancer specimens by EIA and by five-point Scatchard analysis. The correlation between the two assays yielded a straight line with a slope of 0.92 (r = 0.95; P less than 0.001); conversely, in 31 nuclear salt extracts, linear regression analysis of hydroxylapatite exchange assay data with EIA showed a clear correlation (r = 0.93; P less than 0.001) but a slope of 1.7, demonstrating that EIA detects more ER sites. The binding of the antibody to the cytoplasmic ER molecules was investigated by sucrose density gradient analysis, which showed that EIA recognizes both cytoplasmic forms (9 and 3S), but does not distinguish between them. Advantages and drawbacks of this method are discussed with respect to its application for routine receptor determination for clinical management of breast cancer patients.

Localization and distribution of actin in mammalian sperm heads.

Actin was identified in boar and mole spermatozoa by utilizing indirect immunofluorescence, immunoelectron microscopy, and SDS-PAGE, followed by blot and screening with an anti-actin monoclonal antibody. Actin was detected in two places in the sperm head: the equatorial segment of the acrosome and the postacrosomal region. The protein was present in a nonfilamentous form and was localized under the plasma membrane. A small amount of actin was also detected in the sperm tail. The function of actin in the sperm head is discussed.

A double-labeling assay for simultaneous estimation and characterization of estrogen and progesterone receptors using radioiodinated estradiol and tritiated Org 2058.

Estrogen (ER) and progesterone receptors (PgR) appear to be a prerequisite to elicit a biologic response by a hormone-target organ. Current methodologies for analysis of these proteins (e.g., dextran-coated charcoal, DCC) in single-label assay (SLA) require relatively large amounts of tissue material, time and laboriousness. Therefore, we have developed for breast cancer tissue an improved dual-label assay (DLA) for simultaneous titration (by DCC) and/or characterization (by sedimentation properties) of ER and PgR on the same sample, using 125I-E2 and 3H-Org 2058 as tracers. The interaction of 125I-E2 with ER and plasma proteins in comparison to 3H-E2 was studied in terms of specificity, time course, affinity binding and sedimentation pattern. 125I-E2 bound the same molecular forms displayed by 3H-E2 (9 and 3S) but with lower titers (about 1.3-fold), irrespective of the technique used, and did not bind to sex hormone-binding globulin. Simultaneous detection of 125I and 3H was achieved by use of a gamma counter plus a beta counter sequentially. ER and PgR titrations with DCC in DLA were in good agreement with those obtained with SLA, in terms of titers and Ka values. An analogous result was obtained with sucrose density gradient (SDG) analysis. Both the DLA methods were highly reproducible (CV less than 8.0%). Between the rotors available for SDG, the vertical one was preferable because of the larger number of samples processed and of less perturbation of sedimenting receptor molecules. Furthermore, a biochemical application of the method is described. In conclusion, the DLA procedure, by simplifying ER and PgR estimation, makes it possible to study, even on small tumor biopsies, the molecular properties of these proteins in relation to the clinical response of the disease.