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Thermal carbonization of porous silicon (PS) at 820 °C under acetylene atmosphere is an appropriate method for humidity sensing purposes. It produces stable and hydrophilic surface still maintaining originally large specific surface area of PS. We report the temperature dependence of various electrical param- eters measured for the thermally-carbonized PS humidity sensor. Capacitance of the sensor in dry air (6 RH%) is almost constant at various temperatures, whereas in higher relative humidity values, the temperature dependence becomes evident. The resistance variation of the sensor is less dependent on RH as the temperature increases. While the capacitance showed linear behavior as a function of temperature, the resistance had a clear non-linear temperature dependence. In order to get information about the effects of frequency on capacitance values, we measured a phase angle and admittance of the sensor as a function of frequency at three different temperatures in low and high humidity. According to these results, it is preferable to operate this sensor construction using low frequency (<1 kHz). (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Different kind of drugs can be loaded into the porous silicon microparticles for oral dosing. In cases where the drug is in its crystalline form in the pores, the amount of the loaded drug can be determined accurately using a calorimetric method, thermoporometry. Even if the drug substance is not in crystalline form a sophisticated estimation can be given. In this work ibuprofen, antipyrine, and ranitidine have been studied. Ibuprofen and antipyrine were easily detected and quantified, but ranitidine, which does not penetrate into the PSi microparticles in its crystalline form, could only be qualitatively determined. The possibility to quantify this kind of drug substance is also discussed. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Much progress has recently been obtained in the classification and characterization of RCC by using cytogenetic, gene microarray and proteomic techniques. Pivotal for the understanding of the progression of malignancy of clear cell renal cell carcinomas are findings connecting its biology to inactivation of the von Hippel-Lindau tumour suppressor gene product (VHLp), found in most CC-RCCs. Disruption of VHLp function appears to be involved in altered cell cycle control, resistance to hypoxia, hyperangiogenesis and changes in the organization of cytoskeletal and extracellular matrix proteins in RCC. These changes are reflected in the overexpression of the vascular endothelial growth factor (VEGF) and the subunits of hypoxia-inducible factor (HIF), and other angiogenetic and metastasis-promoting factors. Other changes related to progression of malignancy in RCC are the upregulation of proinflammatory cytokines and changes in cell adhesion proteins.

Our aim was to evaluate bursal involvement at different stages of the impingement syndrome as judged by conventional histopathological examination and expression of tenascin-C, which is known to reflect active reparative processes in different tissues and disorders.Samples of subacromial bursa were taken from 33 patients with tendinitis, 11 with a partial tear and 18 with a complete tear of the rotator cuff, and from 24 control shoulders. We assessed the expression of tenascin-C, the thickness of the bursa, and the occurrence and degree of fibrosis, vascularity, haemorrhage and inflammatory cells.The expression of tenascin-C was significantly more pronounced in the complete tear group (p < 0.001) than in the partial tear, tendinitis or control groups. It was more pronounced in the tendinitis group than in the control group (p = 0.06), and there was more fibrosis in all the study groups than in the control group. The changes in the other parameters were not equally distinctive. Expression of tenascin-C did not correlate with the conventional histopathological parameters, suggesting that these markers reflect different phases of the bursal reaction.Tenascin-C seems to be a general indicator of bursal reaction, being especially pronounced at the more advanced stages of impingement and this reaction seems to be an essential part of the pathology of impingement at all its stages.

Several lactose samples containing various amounts of amorphicity were studied with an isothermal microcalorimetric technique, which allow to detect the heat and the quantity of water sorption simultaneously. As interaction with vapor is characteristic of different surfaces, the samples were easy to be discriminated from each other by studying sorption behavior. With the crystalline lactose samples, the amount of sorbed water was too minor to be detected reliably with the technique, but differences were found when the energy values (J g−1) were compared. In the future work, the measurement set-up will be improved so that sorption rates less than 0.1 nmol s−1can be measured repeatably and reliably.

Thermally-carbonized porous silicon films have been prepared by exploiting the dissociation of acetylene. Thermoanalytical methods have been used to study the oxidation behavior of these films in different oxidizing ambients. The results have been compared to other stabilization methods. Due to enhanced adsorption and only slightly reduced specific surface area, the carbonization of porous silicon was found to be an attractive treatment for sensing applications. The possible post-treatments are also discussed.

The molecular basis of the selectivity and the details of the vesicle formation in endocytic and secretory pathways are still poorly known and most probably involve as yet unidentified components. Here we describe the cloning, expression, and tissue and cell distribution of a novel protein of 67 kDa (called Vear) that bears homology to several endocytosis-associated proteins in that it has a VHS domain in its N terminus. It is also similar to gamma-adaptin, the heavy subunit of AP-1, in having in its C terminus a typical "ear" domain. In immunofluorescence microscopy, Vear was seen in the Golgi complex as judged by a typical distribution pattern, a distinct colocalization with the Golgi marker gamma-adaptin, and a sensitivity to treatment of cells with brefeldin A. In cell fractionation, Vear partitioned with the post-nuclear membrane fraction. In transfection experiments, hemagglutinin-tagged full-length Vear and truncated Vear lacking the VHS domain assembled on and caused compaction of the Golgi complex. Golgi association without compaction was seen with the ear domain of Vear, whereas the VHS domain alone showed a diffuse membrane- and vesicle-associated distribution. The Golgi association and the bipartite structure along with the differential targeting of its domains suggest that Vear is involved in heterotypic vesicle/suborganelle interactions associated with the Golgi complex. Tissue-specific function of Vear is suggested by its high level of expression in kidney, muscle, and heart.

The oxidation of porous silicon has been studied using differential scanning calorimeter. The oxidation was found to consist of two parts with different activation energies. This indicates the existence of two different reaction mechanism. The results from the hydrogen desorption measurements have been used to study the different oxidation behaviour of the n- and p+-type porous silicon. The results show that the dihydride structure dominates on the surface of the n-type porous silicon, contrary to p+-type porous silicon, where the monohydride is the major structure. Explanations of these features are discussed. Using the activation energy, the surface termination effects are investigated. The best improvement in the activation energy was observed in the sample, whose surface was partially stabilized by ammonium groups.

A well-known photolabile substance, nifedipine, was used as a sample material to test self-constructed irradiation cells and demonstrate their usefulness in photostability studies. The devices were made as accessories for a commercial isothermal microcalorimeter. Several powder samples containing various amounts of moisture were irradiated with monochromatic light as a scan measurement from 700 to 280 nm, and the heat flow evolved in the photodegradation of nifedipine was determined. According to the results, light does not affect the nifedipine molecule directly, but the photodegradation is a result of the combined effects of moisture and light.

Fodrin (nonerythroid spectrin) is a membrane skeletal protein that plays an important role in the establishment and maintenance of the cell shape and polarity. We have identified in alpha-fodrin an src homology 3 (SH3)-related region, a small domain that is present in a large number of proteins that are involved in signal transduction, cell polarization and membrane-cytoskeleton interactions. In this study we have explored the function of the alpha-fodrin SH3 by incubating fixed and permeabilized cultured chicken fibroblasts with the alpha-fodrin SH3 peptide, expressed in bacteria as a fusion protein with glutathione S-transferase. Immunofluorescence and immunoelectron microscopy showed that alpha-fodrin SH3 binds to the cytoplasmic face of the plasma membrane in the leading lamellae and the pseudopodial lobes of the spreading and locomoting cells. No, or only minimal, binding was seen in immotile cells, or in the stationary trailing ends of the locomoting cells. SH3 binding was also seen in cytochalasin-D-treated cells, suggesting that actin filaments are not responsible for the binding. These findings suggest that alpha-fodrin SH3 interacts with plasma membrane components that are present in the leading lamellae exclusively or are modulated in a manner specific to the leading lamellae.

Summary: Cystatin A is the major cysteine proteinase inhibitor in human squamous epithelia. We investigated the occurrence of cystatin A in normal, condylomatous, and dysplastic lesions of the cervix with or without human papillomavirus (HPV) infection. Cystatin A was detected by immunohistochemistry and HPV infection by DNA hybridization techniques. In the normal uterine cervix, cystatin A was seen throughout the epithelium, except in the basal and parabasal cell layers. In condylomatous lesions, the staining intensity was similar to that in normal epithelium. In low-grade cervical intraepithelial neoplasias (CINs), reduced staining was seen in the lower third of the epithelium; in high-grade CINs, a reduction in staining intensity was also seen in the middle and upper thirds. Cystatin A staining in epithelia and nuclei was negative in highly cellular and poorly differentiated CIN III. The cytoplasmic staining of cystatin A did not correlate with presence or type of HPV DNA. In the highgrade CINs infected with HPV types 16 and 18, however, cystatin A staining was more often confined to the nuclear compartment.

The distribution of saccharide moieties in human interfollicular epidermis was studied with fluorochrome-coupled lectins. In frozen sections Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin I (RCAI), and wheat germ agglutinin (WGA) stained intensively both dermis and viable epidermal cell layers, whereas peanut agglutinin (PNA) bound only to living epidermal cell layers. Ulex europaeus agglutinin I (UEAI) bound to dermal endothelial cells and upper cell layers of the epidermis but left the basal cell layer unstained. Dolichos biflorus agglutinin (DBA) bound only to basal epidermal cells, whereas both soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) showed strong binding to the spinous and granular cell layers. On routinely processed paraffin sections, a distinctly different staining pattern was seen with many lectins, and to reveal the binding of some lectins a pretreatment with protease was required. All keratin-positive cells in human epidermal cell suspensions, obtained with the suction blister method, bound PNA, whereas only a fraction of the keratinocytes bound either DBA or UEAI. Such a difference in lectin binding pattern was also seen in epidermal cell cultures both immediately after attachment and in organized cell colonies. This suggests that in addition to basal cells, more differentiated epidermal cells from the spinous cell layer are also able to adhere and spread in culture conditions. Gel electrophoretic analysis of the lectin-binding glycoproteins in detergent extracts of metabolically labeled primary keratinocyte cultures revealed that the lectins recognized both distinct and shared glycoproteins. A much different lectin binding pattern was seen in embryonic human skin: fetal epidermis did not show any binding of DBA, whereas UEAI showed diffuse binding to all cell layers but gave a bright staining of dermal endothelial cells. This was in contrast to staining results obtained with a monoclonal cytokeratin antibody, which showed the presence of a distinct basal cell layer in fetal epidermis also. The results indicate that expression of saccharide moieties in human epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different terminal saccharide moieties. The results also suggest that the emergence of a mature cell surface glycoconjugate pattern in human epidermis is preceded by the acquisition of cell layer-specific, differential keratin expression.

Abstract: The morphology of esophagitis, both in the presence and absence of acid, was studied by light microscopy and transmission and scanning electron microscopy. For this purpose the rabbit esophagus was isolatedin situ and perfused with agents known to cause esophageal mucosal damage (HCl, pepsin, taurocholate, and deoxycholate). In addition, changes in the permeability of the plasma membrane of the esophageal epithelial cells were assessed by staining the esophageal epithelium with trypan blue and antinuclear antibodies. The results indicate that HCl alone causes relatively few changes in the esophageal epithelium. However, when combined with pepsin or taurocholate, severe ulcerative changes were caused within an hour. Deoxycholate, which is formed in the upper gastrointestinal tract under nonacidic conditions, also causes severe damage. Further, it was shown that the esophagitis caused by pepsin and bile salts are clearly morphologically different. Bile salts affect primarily the cell membrane and intracellular organelles, while pepsin seems to affect the intercellular substance causing the epithelial cells to be shed. In contrast, the presence or absence of acidper se does not seem to influence the nature of the epithelial damage, since the lesions caused by the two bile salts (deoxycholate vs taurocholate + HCl) were morphologically similar.

Src-homology 3 (SH3) domain is a 60-70-amino acid motif present in a large variety of signal transduction and cytoskeletal proteins. We used reverse transcriptase-polymerase chain reaction with degenerate and specific primers and chicken brain mRNA to clone a cDNA that codes for a novel SH3 domain-containing protein. The sequence predicts a 448-amino acid polypeptide with a molecular mass of 51, 971 daltons. In the amino terminus, it shows a very high propensity for alpha-helicity, suggesting coiled-coil and possibly a higher order oligomeric arrangement. In the carboxyl terminus, there is a unique SH3 sequence. In Northern blotting, a major 3.7-kilobase and a minor 7.2-kilobase transcript was detected in most chicken tissues. In immunofluorescence microscopy and immunoelectron microscopy on cultured chicken fibroblasts, the protein was localized to focal adhesions in which it showed a distinct codistribution with the focal adhesion proteins vinculin, talin, and paxillin. Phosphoamino acid analysis showed that in cultured chicken heart fibroblasts, the protein contains phosphoserine, but no phosphothreonine or phosphotyrosine, and that the phosphorylation is not dependent on fibronectin. We propose this protein the name FAP52, for Focal Adhesion Protein of 52 kDa, and suggest that it forms part of the multimolecular complex constituting focal adhesion sites.

In our previous study on fixed tissue blocks, we reported a high apoptotic rate in patients with operated small cell lung carcinomas. In addition to tumour cells, numerous apoptotic bodies could also be found within alveolar macrophages within and close to tumour tissue. In order to test if such cells could be found in sputum smears and if their presence could be utilized as a marker in tumour diagnosis, we analyzed the occurrence of alveolar macrophages with apoptotic bodies (AMWABs) in a set of sputum smear and BAL samples from patients with and without a pulmonary malignancy. An increased amount of AMWABs in the cytoplasm could be found in sputum and BAL samples from patients with lung cancer. Interestingly, AMWABs could also be seen in patients with a histologically confirmed pulmonary malignancy, but with no detectable tumour cells in their sputum smear. Thus, the presence AMWABs in sputum smears could serve as a more sensitive marker of pulmonary malignancy than the prese nce of malignant cells per se. This is the first report describing apoptotic bodies in macrophages and the utility of their detection in cancer diagnosis.

In this study we investigated tumour growth in relation to the immunohistochemical expression of p53 and bcl-2 and to patient survival data in 33 operated hepatocellular carcinomas (HCCs). In order to estimate the growth, a growth index, based on the degree of cell proliferation, apoptosis and necrosis, was calculated for each tumour. Cell proliferation was determined immunohistochemically by the number of proliferating cell nuclear antigen (PCNA)-positive cells in tumours, the extent of apoptosis was determined by counting the number of cells labelled by the in situ 3'-end labelling technique and tumour necrosis was estimated as the percentage of necrotic areas in haematoxylin--eosin-stained tissue sections. In our analysis we found that the survival of patients with HCCs showing a high growth index (i.e. tumours showing a high proliferation and simultaneously a low degree of apoptosis and necrosis) was significantly shorter than with other patients (P = 0.004, log-rank test). When analysed separately, cell proliferation, apoptosis or necrosis did not show any significant association with survival. p53 positivity was found in 8/33 (24%) of tumours. There were significantly more p53-positive cases in tumours with a high growth index (P = 0.01, Fisher's exact test) suggesting that dysfunction of the p53 gene may affect tumour growth. p53-positive cases did not, however, have a significantly shorter survival time than p53-negative cases (P = 0.3, log-rank test). bcl-2 positivity was found in only 1/33 (3%) of the HCCs. Thus bcl-2 overexpression does not seem to play an important role in hepatocellular carcinogenesis. In summary, our results suggest that in HCCs a compound score based on the evaluation of the degree of cell proliferation, apoptosis and necrosis is a biologically more relevant prognostic indicator than any of its composite parameters alone.

The specific heat capacities (cp) for the brain and muscle equivalent liquids were determined with isothermal heat conduction microcalorimetry (IMC) and differential scanning calorimetry (DSC). IMC was found to afford an accurate technique to measure cpfor solid and liquid samples, when an appropriate reference is employed. The accuracy of obtained cpvalues was estimated to be better than 0.7% with the equivalent liquids. Intercomparison with a conventional isoperibolic calorimeter showed an excellent agreement within the estimated uncertainty of the isoperibolic calorimeter (±3%). Additionally, suitability of different kinds of IMC sample vessels was tested, and the standard electrical calibration procedure of IMC was evaluated through the determination of cpwith and without a reference material.

The quantitative x-ray diffraction analysis offers often an exact and practical technique to determine the relative amounts of the phases of the solid in question. The sample may contain only one amorphous phase.The purpose of this paper is to estimate the suitability of this technique to quantify the phase fractions of pharmaceutical substances. Three different method were used to determine the relative amounts of phases. Methods were compared and the major sources of error were estimated. The measurements and calculations are carried out using selegilin hydrochloride, mannitol and modified starch as an example. The results show that the amount of about 2 % of selegilin hydrochloride can be observed reliably from among the mannitol (crystalline substance) or modified starch (amorphous substance). During the work it became obvious that the accuracy of these calculations can essentially be improved planning the measurements carefully.

Antibodies against different cytoskeletal proteins were used to study the cytoskeletal organization of human spermatozoa. A positive staining with actin antibodies was seen in both the acrosomal cap region and the principal piece region of the tail. However, no staining was obtained with nitrobenzoxadiazol-phallacidin, suggesting that most of the actin was in the nonpolymerized form. Most of the myosin immunoreactivity was confirmed to a narrow band in the neck region of spermatozoa. Tubulin was located to the entire tail, whereas vimentin was only seen in a discrete band-like structure encircling the sperm head, apparently coinciding with the equatorial segment region. Surface staining of the spermatozoa with fluorochrome-coupled Helix pomatia agglutinin revealed a similar band-like structure that co-distributed with the vimentin-specific staining. Instead, other lectin conjugates used labeled either the acrosomal cap region (peanut and soybean agglutinins), both the acrosomal cap and the postacrosomal region of the head (concanavalin A), or the whole sperm cell surface membrane (wheat germ and lens culinaris agglutinins and ricinus communis agglutinin l). In lectin blotting experiments, the Helix pomatia agglutinin-binding was assigned to a 80,000-mol-wt polypeptide which, together with vimentin, also resisted treatment with Triton X-100. Only the acrosomal cap and the principal piece of the tail were decorated with rabbit and hydridoma antibodies against an immunoanalogue of erythrocyte alpha-spectrin (p230). p230 appeared to be the major calmodulin-binding polypeptide in spermatozoa, as shown by a direct overlay assay of electrophoretic blots of spermatozoa with 125I-calmodulin. The results indicate that spermatozoa have a highly specialized cytoskeletal organization and that the distribution of actin, spectrin, and vimentin can be correlated with distinct surface specializations of the sperm cells. This suggest that cytoskeleton may regulate the maintenance of these surface assemblies and, hence, affect the spermatozoan function.

The effects of epidermal growth factor (EGF) on the cytokeratin filaments of cultured murine epithelial cells were studied by the indirect immunofluorescence technique with affinity-purified antibodies. Mouse epithelial cells (MMC-E), grown on glass cover slips, and viewed by immunofluorescence microscopy, showed keratin-specific fluorescence as typical bright perinuclear aggregates corresponding to dense paracrystalline granules seen in electron microscopy. Within minutes after an exposure to EGF, the keratin granules in the MMC-E cells decreased. After 10 min of incubation, the cells had spread fibrillar keratin. Such an effect could not be found after a similar exposure to insulin, dexamethasone, dibutyryl cyclic AMP, or antimitotic drugs. EGF, therefore, has a relatively direct effect on the cytoskeletal organization of cultured epithelial cells. These rapid effects on the keratin filaments may explain the simultaneous EGF-induced ultrastructural surface changes of the cells. EGF may thus function as a regulatory factor in the migration of epithelial cells and in the mobility of their cell membranes. The epithelial cell line, MMC-E, should prove a useful model for studies on the action of EGF on nontransformed epithelial cells in vitro.

Human malignant mesotheliomas were studied by electron microscopy. Three main types of cells were seen—submesothelial epithelioid cells, epithelial lining cells and fibroblast-like cells. In submesothelial epithelioid cells prominent arrays of intermediate (10 nm) filaments were often seen attached to plasma membrane, mitochondria, nuclei and concentric whorls of rough endoplasmic reticulum. The other types of cell found in the tumors, epithelial lining cells and fibroblast-like cells, lacked such distinct filaments. The intermediate filaments were especially abundant in cells with extensive whorling of endoplasmic reticulum. The association of intermediate filaments with such deranged cytoplasmic organization suggests that they play a role in the altered behavior of malignant cells.

The room temperature oxidation of porous silicon was studied using isothermal methods. The oxidation was found to depend on the type of the porous silicon. The microcalorimetric signals from the oxidation of the p+- and n-type porous silicon in dry air were different. In humid air the signals from the oxidation could not be distinguished from the strong signal due to adsorption of water vapour, but when the samples were placed in water similar differences were observed. The reason for differences in reactions is discussed. The oxidation in different liquids was also studied. The signal from reactions in methanol and ethanol were found to be 100 times higher than in water. In FTIR studies the reaction gas produced by reactions between alcohols and the porous silicon, silane(SiH4) was found in the gas. Traces of SiOCH3and SiOC2H5groups were also found in FTIR spectra indicating SiOCxHypassivation of the surface.

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.

Pure adult human kératinocyte cultures were raised from suction-blister roof epidermis and cultured in MCDB-151 medium. In primary culture the epidermal cells rapidly adhered, spread and began to proliferate on collagen-coated growth substrata but not on uncoated plastic or glass substrata. A fibrillar keratin-specific fluorescence, showing a typical cell-cell arrangement, was seen in all cells in indirect immunofluorescence microscopy, whereas only some cells also showed vimentin-specific staining. A fine fibrillar fibronectin-specific surface staining was seen at the margin of attaching cells and in marginal cells of spreading cell islands, whereas no fluorescence could be seen in epidermal cells, with antibodies against type IV collagen or laminin. Interestingly, the marginal cells also showed intracellular fibronectin. The synthesis of fibronectin in epidermal cell cultures could also be revealed by metabolic labelling experiments with [35S]methionine. In contrast to primary cultures, subcultivated kératinocytes also adhered to uncoated plastic and glass substrata. After subcultivation, keratin and surface fibronectin distribution remained unaltered but after some subcultivations, most of the cells also showed fibrillar vimentin and expressed fibronectin intracellularly. The results show that the suction-blister method provides an easy way to obtain pure epidermal cell cultures without contaminating mesenchymal cells. Our results also suggest a direct role for fibronectin but not for collagen type IV or laminin in adhesion and spreading of epidermal cells in vitro.

Further studies are presented on the intracellular localization of the amyloid-related serum protein SAA previously shown to be produced by embryonal fibroblasts. In cultured embryonal fibroblasts, the fine fibrillar cytoplasmic immunofluorescence obtained by anti-SAA was distinguished from that of microfilaments and microtubules. By using electron microscopy and cells treated with drugs known to specifically alter intracellular fibrils, SAA was localized to 10-nm intermediate size filaments. These filaments form characteristic perinuclear bundles upon treatment with drugs such as demecolcine or vinblastine which disrupt micotubules. The results indicate that SAA is a constituent of the intracellular cytoskeleton.

Antibodies were raised against a cytoskeleton-associated, nonphosphorylated, 230,000-dalton bovine lens polypeptide (designated p230), and rendered monospecific by using a novel immunoaffinity technique. In immunofluorescence and electron microscopy of cultured fibroblasts, as well as of various other cells (endothelial, epithelial, lenticular, monocytes, neuroblastoma cells) and tissues (human kidney and liver), p230 was localized as a distinct subplasmalemmal layer in the peripheral cytoplasm of the cells. It constituted less than 0.3% of the total cellular protein in cultured fibroblasts and was not extractable with Triton X-100. In detergent-extracted cytoskeletal preparations of cultured fibroblasts, p230 remained as an elaborate peripheral network that showed a distribution distinctly different from that of the major cytoskeletal structures, stress fibers, cortical myosin, vinculin, and intermediate filaments (IF). The distribution was not dependent on the presence of intact stress fibers or microtubules, as shown by double-fluorescence microscopy of cells exposed to cytochalasin B or cultured in the presence of monensin and of cold-treated cells. Upon demecolcine-induced reorganization of intermediate filaments, however, the localization of p230 was rapidly altered to a dense plaque underneath the perinuclear aggregate of intermediate filaments. On the other hand, p230 seemed to colocalize with the detergent-resistant cell surface lamina, visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin-lectin. The results suggest that p230 is part of a cell surface- and cytoskeleton-associated subplasmalemmal structure that may play an important role in cell surface-cytoskeleton interaction in various cells both in vitro and in vivo.

Anchoring complex component laminin-5 and its subunits laminin (Ln)-alpha3 and Ln-beta3 chains, Type VII collagen, and integrin chains alpha3, alpha6, and beta4 were studied in developing and adult human intestine and compared with findings on Ln-alpha1 and Ln-alpha2 chains. In adult human duodenum, jejunum, and ileum, Ln-5 detected with a polyclonal antiserum and Ln-alpha3 and Ln-beta3 chains, detected with monoclonal antibodies (MAbs), were restricted to the epithelial basement membranes (BMs) of villi, whereas Ln-alpha2 chain was seen only focally in crypt bottoms. In double labeling experiments, the stretch of crypt BM corresponding to the proliferative cell compartment was found to be devoid of both Ln-alpha3 and Ln-alpha2 chains. Double labeling for Ln-5 and proliferating cell nuclear antigen also showed an abrupt onset of Ln-5 expression exactly at the upper edge of the proliferative cell compartment. Type VII collagen was negligible in duodenum and showed a rising duodenal-ileal gradient localizing to villar BMs. Double labeling for Ln-5 and Type VII collagen, however, indicated only partial co-distribution in the intestine. Electron microscopy of ileum revealed both anchoring filaments and anchoring fibrils but no hemidesmosomal plaques. Our results demonstrate the expression of Ln-5 in BMs outside of stratified epithelia and indicate that Ln-5 in the intestine is associated with the compartment of migrating and differentiating enterocytes. Absence of hemidesmosomes and the presence of other anchoring complex components, such as Ln-5, Type VII collagen, and integrin chains alpha3, alpha6, and beta4, suggests unique properties for epithelial cell attachment in the intestine.

The pericellular glycoprotein fibronectin, which has been localized in the pericellular matrix and at the adhesion sites of cells1–5, has been considered to be important in cell attachment to other cells and to surfaces1–8. Using the ionophore monensin, which blocks the secretion of endogenous fibronectin9,10, we have now shown that human fibroblasts do not require endogenous or exogenous fibronectin for adhesion and spreading, although its absence does prevent formation of the microfilament–vinculin system and the focal adhesion sites.

The sensitivity of human papillomavirus (HPV) detection was compared by colposcopy, histology and DNA hybridization among 304 women with abnormal Papanicolaou (Pap) smear. Colposcopically directed biopsies revealed HPV infection in 71% of cases, DNA hybridization in 35%, and both together in 78%. DNA hybridization detected HPV in 24% of the 84 benign cases with no histological signs of HPV, in 32% of the 133 condylomas verified by biopsies and in 51% of the 85 cases with intraepithelial neoplasia, 95% of which presented histological signs of HPV. The pattern of occurence of different HPV-types resembled findings in earlier reports. HPV infection is common with abnormal Pap smears and it can be identified relatively reliably by means of cytology, colposcopy and histology. DNA hybridization serves as a complementary technique which may reveal the oncological potential of the virus.

Specimens obtained from the middle ear during surgery for cholesteatoma showed either abrupt junctions without metaplasia or junctions of varying width with metaplastic epithelium. Another form of junctional area showed pronounced inflammatory cell reaction with squamous epithelium projections growing into the stroma under the columnar epithelium. Immunofluorescent staining for cytokeratins showed distinct decoration of the columnar and metaplastic epithelia as well as of the basal cells of squamous epithelium. Prekeratin staining showed decoration of both squamous and metaplastic epithelium and no staining of the columnar epithelium. The findings suggest that metaplasia forms part of the migrating front of the squamous epithelium and alone plays no major role in cholesteatoma genesis.

We describe the cloning and characterization of a new cytoplasmic protein designated epidermal growth factor receptor-associated protein with SH3- and TAM domains (EAST). It contains an Src homology 3 domain in its midregion and a tyrosine-based activation motif in its COOH terminus. Antibodies to EAST recognize a 68-kDa protein that is present in most chicken tissues. An epidermal growth factor (EGF)-dependent association between the EGF receptor (EGFR) and EAST was shown by reciprocal immunoprecipitation/immunoblotting studies with specific antibodies. Activated EGFR catalyzed the tyrosine phosphorylation of EAST, as judged by an in vitro kinase assay with both immunoprecipitated and purified EGFR. Immunoprecipitation/immunoblotting experiments also demonstrated an association between EAST and eps15, an EGFR substrate associated with clathrin-coated pits and vesicles, which is essential in the endocytotic pathway. The association between EAST and eps15 was not affected by EGF treatment. In immunofluorescence microscopy, EAST was shown to partially colocalize with clathrin. The sequence of the NH2-terminal portion of EAST shows a high degree of similarity with a group of proteins involved in endocytosis or vesicle trafficking. Thus, EAST is a novel signal transduction component probably involved in EGF signaling and in the endocytotic machinery.

We have determined the nucleotide sequence coding for the chicken brain alpha-spectrin. It is derived both from the cDNA and genomic sequences, comprises the entire coding frame, 5' and 3' untranslated sequences, and terminates in the poly(A)-tail. The deduced amino acid sequence was used to map the domain structure of the protein. The alpha-chain of brain spectrin contains 22 segments of which 20 correspond to the repeat of the human erythrocyte spectrin (Speicher, D. W., and V. T. Marchesi. 1984. Nature (Lond.). 311:177-180.), typically made of 106 residues. These homologous segments probably account for the flexible, rod-like structure of spectrin. Secondary structure prediction suggests predominantly alpha-helical structure for the entire chain. Parts of the primary structure are excluded from the repetitive pattern and they reside in the middle part of the sequence and in its COOH terminus. Search for homology in other proteins showed the presence of the following distinct structures in these nonrepetitive regions: (a) the COOH-terminal part of the molecule that shows homology with alpha-actinin, (b) two typical EF-hand (i.e., Ca2+-binding) structures in this region, (c) a sequence close to the EF-hand that fulfills the criteria for a calmodulin-binding site, and (d) a domain in the middle of the sequence that is homologous to a NH2-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. These regions are good candidates to carry some established as well as some yet unestablished functions of spectrin. Comparative analysis showed that alpha-spectrin is well conserved across the species boundaries from Xenopus to man, and that the human erythrocyte alpha-spectrin is divergent from the other spectrins.

INTEREST in cytoplasmic intermediate (10 nm) filaments has grown since the successful immunohistochemical differentiation of these filaments from microtubules and microfilaments1,2. Despite the widespread occurrence of intermediate filaments in various cell types, little is known about their function1,3,4. A cytoskeletal nucleus-anchoring role has been suggested based on their intracellular distribution, their connections to the Plasma membrane and nucleus, and their marked insolubility in detergents and salt solutions5,6. The presence of autoantibodies against intermediate filaments in the sera of patients7,8suggests that these filaments can be involved in pathogenetic processes. We report here that in studies on possible pathogenetic mechanisms involving intermediate filaments, antibody-independent binding of complement components to intermediate filaments has been observed. Initial observations were made using cultured human embryonal fibroblasts, as cytoplasmic filaments present in these cells have been characterised by using both immunological markers and drugs capable of selectively altering the morphology of microfilaments, intermediate filaments and microtubules1,7. Subsequent studies showed complement binding to similar cytoplasmic structures of other cultured cells and also cells in tissue sections.

Carcinoid tumor of the colon was studied in electron microscope. In cytoplasm, prominent intermediate-sized filaments were seen frequently attaching to nucleus and mitochondria. Direct contacts of intermediate filaments with secretory granules were not observed.