The prevalence and purification of hepatoproliferin: a liver regeneration factor from rat hepatocytes

Hepatoproliferin (HPF), a liver regeneration factor that was able to augment the growth of hepatocytes in the presence of EGF, was produced by young rat livers and hepatectomized adult livers (70%), but not by adult intact livers. Therefore only growing and regenerating livers produce HPF. This growth factor was purified into two homogeneous bioactive species having different single SDS-PAGE bands at 18.5 and 14 kDa, and different single pI-bands at pH 4.3 and 8.7, respectively. HPF was synthesized de novo by hepatocytes in the liver as shown by the in vivo incorporation of radiolabeled 35S-sulfate and 14C/3H-glucosamine. This radioactive HPF was secreted ex vivo by hepatocytes, probably to act as an autocrinal hepatomitogen since 90% was found in the growth medium. HPF was neither a classical peptido-mitogen nor a heparin binding growth factor, but a liver-originated non-proteinaceous factor, which probably contains sulfonated saccharides such as glucosamine sulfate. HPF was neither a polyglycan nor a glycopeptide nor a peptidoglycan.     

Mechanism of interference mediated by human parainfluenza virus type 3 infection

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.     

The effect of heparin and a human heparin precipitable plasma fraction on antibody producing cells in vitro]

A study was made of the effect of heparin and heparin-precipitable fractions of human blood plasms (HPF) on the capacity of the cells of mouse spleen immunized with sheep erythrocytes to plaque-formation in vitro. It was found that heparin and HPF produced an inhibitory effect on the reaction of plaque formation as a result of interaction with lymphocytes. It is supposed that a possible point of application of heparin and HPF action are surface cell membranes of the antibody-forming cells.     

Hepatocyte proliferation factors from neonatal pig liver: purification and characterization

Two factors were found in the condition medium of neonatal pig liver fragments, which were capable of stimulating DNA synthesis in primary hepatocytes. They were named hepatocyte proliferation factor (HPF)-1 and HPF-2 and purified 1,025- and 2,580-fold, respectively. Both HPF-1 and HPF-2 seem to be anionic at pH 8.0 judged from the elution pattern of DEAE (DE52) column chromatography. HPF-1 was recovered as a non-adsorbed fraction in blue Sepharose and heparin Sepharose columns, and had a molecular weight of 26-31 kDa as estimated by gel filtration in high salt condition. Purified HPF-1 stimulated DNA synthesis of primary rat hepatocytes, but suppressed that of HepG2 cells. HPF-2 strongly bound to blue Sepharose and heparin Sepharose columns, and had a molecular weight of 71-90 kDa as estimated by SDS-PAGE under non-reduced condition. Purified HPF-2 stimulated DNA synthesis of primary rat hepatocytes dose dependently but did not suppress that of HepG2 cells. From further biological and chemical characteristics studied in this paper, HPF-1 and HPF-2 may be novel stimulating proteins for hepatocyte proliferation, although the possibility that they are already known growth factors can not be excluded without complete purification and its cloning.     

肝细胞表面去唾液酸糖蛋白受体的流式细胞分析

建立肝细胞表面去唾液酸糖蛋白受体（ASGPR）的流式细胞分析方法（FCM）,对正常及损伤鼠肝细胞、肝癌细胞（BEL-7402）表面的ASGPR作同步比较分析.以异硫氰酸荧光素标记的新半乳糖白蛋白（FITC-NGA）为ASGPR的特异性配体,以培养的正常肝细胞（L-02）为靶细胞,建立肝细胞表面ASGPR的FCM.测定并计算正常及损伤鼠肝细胞,BEL-7402细胞与同一浓度的FITC-NGA同步反应后的平均荧光强度（MIF）值.FITC-NGA与L-02细胞表面ASGPR趋近饱和结合的浓度为0.4 mg/L,该浓度下正常及损伤鼠肝细胞,BEL-7402细胞的MIF值分别为228.7、5.81、1.13.该结合可以被至少50倍于FITC-NGA的NGA或10 mmol/L的EDTA完全抑制.FCM能够良好地揭示FITC-NGA同ASGPR之间的受配体结合特性.该方法证实BEL-7402细胞表面几乎没有ASGPR,损伤鼠肝细胞表面ASGPR的数量较正常鼠肝细胞显著减少.     

Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips ofNicotiana andArabidopsis

Summary To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips ofNicotiana andArabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more numerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) larger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.Abbreviations CF chemical fixation - ER endoplasmic reticulum - FF freeze fracture - FS freeze substitution - HPF high pressure freezing Dedicated to the memory of Professor Oswald Kiermayer     

Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice

Cholestasis-induced liver injury during bile duct obstruction causes an acute inflammatory response. To further characterize the mechanisms underlying the neutrophil-induced cell damage in the bile duct ligation (BDL) model, we performed experiments using wild-type (WT) and ICAM-1-deficient mice. After BDL for 3 days, increased ICAM-1 expression was observed along sinusoids, along portal veins, and on hepatocytes in livers of WT animals. Neutrophils accumulated in sinusoids [358 +/- 44 neutrophils/20 high-power fields (HPF)] and >50% extravasated into the parenchymal tissue. Plasma alanine transaminase (ALT) levels increased by 23-fold, and severe liver cell necrosis (47 +/- 11% of total cells) was observed. Chlorotyrosine-protein adducts (a marker for neutrophil-derived hypochlorous acid) and 4-hydroxynonenal adducts (a lipid peroxidation product) were detected in these livers. Neutrophils also accumulated in the portal venules and extravasated into the portal tracts. However, no evidence for chlorotyrosine or 4-hydroxynonenal protein adducts was detected in portal tracts. ICAM-1-deficient mice showed 67% reduction in plasma ALT levels and 83% reduction in necrosis after BDL compared with WT animals. The total number of neutrophils in the liver was reduced (126 +/- 25/20 HPF), and 85% of these leukocytes remained in sinusoids. Moreover, these livers showed minimal staining for chlorotyrosine and 4-hydroxynonenal adducts, indicating a substantially reduced oxidant stress and a diminished cytokine response. Thus neutrophils relevant for the aggravation of acute cholestatic liver injury in BDL mice accumulate in hepatic sinusoids, extravasate into the tissue dependent on ICAM-1, and cause cell damage involving reactive oxygen formation.     

Role of HPF (hibernation promoting factor) in translational activity in Escherichia coli

During the stationary phase of growth in Escherichia coli, ribosome modulation factor (RMF) and hibernation promoting factor (HPF) dimerize most 70S ribosomes to form 100S ribosomes. The process of 100S formation has been termed 'ribosomal hibernation'. Here, the contributions of HPF to 100S formation and translation were analysed in vitro. HPF bound to, but did not dimerize the 70S ribosome. RMF dimerized and formed immature 90S ribosomes. Binding of both HPF and RMF converted 90S ribosomes to mature 100S ribosomes, which is consistent with the in vivo data. The role of HPF in in vitro translation also was investigated. In an artificial mRNA poly (U)-dependent phenylalanine incorporation assay, HPF bound to ribosomal particles and inhibited translation. In contrast, in a natural MS2 mRNA-dependent leucine incorporation assay, bound HPF was removed and hardly inhibited normal translation. Multiple alignment and phylogenetic analyses indicates that the hibernation system mediated by the HPF homologue, RMF and 100S ribosome formation may be specific to the proteobacteria gamma group. In contrast, most bacteria have at least one HPF homologue, and these homologues can be classified into three types, long HPF, short HPF and YfiA.     

Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species

We designed and synthesized 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical (*OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite ((-)OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H(2)O(2)), nitric oxide (NO), and superoxide (O2*-) by using HPF or APF alone, but (-)OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2',7'-dichlorodihydrofluorescein, and for the first time we could visualize (-)OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and (-)OCl in many biological and chemical applications.     

Arsenic trioxide induces human pulmonary fibroblast cell death via the regulation of Bcl-2 family and caspase-8

Arsenic trioxide (ATO; As₂O₃) can induce apoptotic cell death in various cancer cells including lung cancer cells. However, little is known about the toxicological effects of ATO on normal primary lung cells. In this study, we investigated the cellular effects of ATO on human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition and death. ATO inhibited HPF cell growth with an IC₅₀ of approximately 30–40 μM at 24 h and induced cell death accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). Thus, HPF cells were considered to be very resistant to ATO insults. ATO increased the expression of p53 protein and decreased that of Bcl-2 protein. This agent activated caspase-8 but not caspase-3 in HPF cells. Z-VAD (a pan-caspase inhibitor; 15 μM) did not significantly decrease cell growth inhibition, death and MMP (ΔΨ_m) loss by ATO. Moreover, administration of Bax or casase-8 siRNA attenuated HPF cell death by ATO whereas p53 or caspase-3 siRNAs did not affect cell death. In conclusion, HPF cells were resistant to ATO and higher doses of ATO induced the growth inhibition and death in HPF cells via the regulation of Bcl-2 family and caspase-8.     

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fusion properties of cells persistently infected with human parainfluenza virus type 3: participation of hemagglutinin-neuraminidase in membrane fusion.

Cells persistently infected with human parainfluenza virus type 3 (HPF3) exhibit a novel phenotype. They are completely resistant to fusion with each other but readily fuse with uninfected cells. We demonstrate that the inability of these cells to fuse with each other is due to a lack of cell surface neuraminic acid. Neuraminic acid is the receptor for the HPF3 hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. Uninfected CV-1 cells were treated with neuraminidase and then tested for their ability to fuse with the persistently infected (pi) cells. Neuraminidase treatment totally abolished cell fusion. To extend this result, we used a cell line deficient in sialic acid and demonstrated that these cells, like the neuraminidase-treated CV-1 cells, were unable to fuse with pi cells. We then tested whether mimicking the agglutinating function of the HN molecule with lectins would result in cell fusion. We added a panel of five lectins to the neuraminic acid-deficient cells and showed that binding of these cells to the pi cells did not result in fusion; the lectins could not substitute for interaction of neuraminic acid with the HN molecule in promoting membrane fusion. These results provide compelling evidence that the HN molecule of HPF3 and its interaction with neuraminic acid participate in membrane fusion and that cell fusion is mediated by an interaction more complex than mere juxtaposition of the cell membranes.     

Fusion properties of cells infected with human parainfluenza virus type 3: receptor requirements for viral spread and virus-mediated membrane fusion.

Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion.     

Human Parainfluenza Virus Type 3 HN-Receptor Interaction: Effect of 4-Guanidino-Neu5Ac2en on a Neuraminidase-Deficient Variant

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4 degrees C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37 degrees C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.     

Control of the ability of profilin to bind and facilitate nucleotide exchange from G-actin

A major factor in profilin regulation of actin cytoskeletal dynamics is its facilitation of G-actin nucleotide exchange. However, the mechanism of this facilitation is unknown. We studied the interaction of yeast (YPF) and human profilin 1 (HPF1) with yeast and mammalian skeletal muscle actins. Homologous pairs (YPF and yeast actin, HPF1 and muscle actin) bound more tightly to one another than heterologous pairs. However, with saturating profilin, HPF1 caused a faster etheno-ATP exchange with both yeast and muscle actins than did YPF. Based on the -fold change in ATP exchange rate/K(d), however, the homologous pairs are more efficient than the heterologous pairs. Thus, strength of binding of profilin to actin and nucleotide exchange rate are not tightly coupled. Actin/HPF interactions were entropically driven, whereas YPF interactions were enthalpically driven. Hybrid yeast actins containing subdomain 1 (sub1) or subdomain 1 and 2 (sub12) muscle actin residues bound more weakly to YPF than did yeast actin (K(d) = 2 microm versus 0.6 microm). These hybrids bound even more weakly to HPF than did yeast actin (K(d) = 5 microm versus 3.2 microm). sub1/YPF interactions were entropically driven, whereas the sub12/YPF binding was enthalpically driven. Compared with WT yeast actin, YPF binding to sub1 occurred with a 5 times faster k(off) and a 2 times faster k(on). sub12 bound with a 3 times faster k(off) and a 1.5 times slower k(on). Profilin controls the energetics of its interaction with nonhybrid actin, but interactions between actin subdomains 1 and 2 affect the topography of the profilin binding site.     

Regulation of asialoglycoprotein receptor expression in the proliferative state of hepatocytes

It is necessary to proliferate hepatocytes and to increase the number of hepatocytes for development of bioartificial liver (BAL) and reconstitutive therapy. But usually the cell has a precarious balance between proliferation and differentiation: as the cell proliferation increases, functional differentiation decreases. Therefore, it is desirable for the hepatocytes to be functional by differentiation as a material for such clinical use not to be proliferative. In this study, we investigated the background of hepatocyte proliferation for the springboard of control between proliferation and differentiation of hepatocytes, and we focused attention to the asialoglycoprotein receptors (ASGP-R) of the hepatocytes. Partially hepatectomized (PH) rats were used as a model animal. When the isolated hepatocytes were plated onto the artificial extracellular matrix of poly-(N-p-vinylbenzyl-O-beta-d-galactopyranosyl-d-gluconamide) (PVLA) having galactose residues as cell-specific ligand, the rate of adhesion was decreased along with liver regeneration. Interestingly, the release of the ASGP-R from hepatocytes in serum after PH in vivo and reduction of ASGP-R of the hepatocytes in the proliferative state occurred due to cell growth in vitro. It is suggested that the ASGP-R on the hepatocyte surface during the differentiation was released in the proliferative state.     

人脐带间充质干细胞微囊减轻小鼠急性肾损伤的研究

目的探讨人脐带间充质干细胞（MSCs）源性细胞外囊泡Oct-4 mRNA对受损的肾小管上皮细胞修复的作用及相关机制。 方法将培养的缺氧损伤肾小管上皮细胞置于含有人脐带MSCs细胞外囊泡及不同对照培养液的培养腔室玻片上孵育48?h,应用BrdU及TUNEL染色,检测各组细胞增殖或凋亡情况。将急性肾损伤模型小鼠分为4组：空白组、EVs组、Oct-4过表达组、Oct-4低敲组。并按照分组分别注射磷酸盐缓冲液（Vehicle）,人脐带MSCs细胞外囊泡（EVs）,过表达Oct-4基因的人脐带MSCs细胞外囊泡（EVs?+?Oct-4）及敲除Oct-4基因的人脐带MSCs外囊泡（EVs-Oct-4）,并在注射48?h及2周后采血测肌酐（Crea）及尿素氮（BUN）,了解肾功能变化;对各组上述处理后的肾组织应用TUNEL与增殖细胞核抗原表达量检测各组肾脏细胞凋亡与增殖情况;通过Masson染色检测了各组肾脏纤维化程度;通过PCR探索肾损伤后肾组织细胞Snail基因的表达变化。数据分析采用方差分析和SNK-q检验。 结果EVs?+ Oct-4处理缺氧的肾小管上皮细胞48?h后,TUNEL染色显示具有最少的凋亡细胞数（0~1）/?HPF,BrdU显示有最多的增殖细胞（7±2）/HPF。EVs,EV-Oct-4以及Vehicle对体外缺氧肾小管上皮细胞的上述作用依次减弱（P?相似文献   

Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects

The translocation of occupied surface insulin receptors to the nuclei of isolated hepatocytes was studied using the biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). When hepatocytes were photolabeled at 4 degrees C, extensively washed, and then further incubated at 37 degrees C for 1 h, photolabeled insulin receptors, which were initially localized to the cell surface, accumulated in the subsequently isolated nuclei. When the isolated nuclei were solubilized and subjected to polyacrylamide gel electrophoresis and radioautography, labeled proteins with Mr identical to the cell surface insulin receptor were detected. Light microscopic radioautography of nuclei isolated from cells incubated for 1 ha at 37 degrees C demonstrated that 28% of these nuclei were specifically labeled with one or more grains. Electron microscopic radioautography of intact cultured hepatocytes, incubated 60 min at 37 degrees C, revealed that 26% of the thin-sectioned nuclei contained at least a single grain and 8.3% of the total cell-associated associated grains were located over the nuclei. Only 1.6% of grains were localized to lysosomes. In contrast, if photolabeled hepatocytes were incubated at 4 degrees C for up to 2 h, negligible accumulation of nuclear radioactivity was observed by polyacrylamide gel electrophoresis on light or electron microscopic radioautography. Conclusions are as follows. Occupied cell surface insulin receptors can internalize and translocate to the nucleus of intact hepatocytes by a time- and temperature-dependent mechanism. Accumulation and possible degradation of insulin receptors in lysosomes involves only a small percentage of the receptors internalized. Nuclear translocation of occupied cell surface insulin receptors may be a mechanism which mediates insulin's long term effects.     

The serine protease Esperase HPF inhibits the formation of multispecies biofilm

The antifouling (AF) potential of the serine protease Esperase HPF (subtilisin) was evaluated for the ability to prevent the formation of a four-species bacterial biofilm. The effects of enzyme activity, time and application of the enzyme were tested on the density and the oxidative metabolism of biofilm developed in microtiter wells. Esperase HPF did not inhibit the oxidative metabolism of the bacterial biofilm or planktonic growth, but the enzyme inhibited biofilm formation by its proteolytic activity as inactivated enzyme had no effect. The effective enzyme concentration was determined over a period of 72 h, as by then all the tested concentrations inhibited biofilm formation maximally. The effective concentrations of the enzymes in solution were the same regardless of time of application (ie before or after biofilm formation), but immobilisation of the enzymes caused a lower effective concentration. Esperase HPF is an attractive alternative to the biocidal compounds used in AF coatings today.     

Effects of MCI-186 upon neutrophil-derived active oxygens

Reactions of 3-methyl-1-phenyl-2-pyrazoline-5-one (MCI-186) with hypochlorous acid and superoxide were analysed by spectrophotometry and mass spectrometry. The results were applied to the neutrophil system to evaluate the scavenging activity of neutrophil-derived active oxygen species by MCI-186. MCI-186 reacted rapidly with hypochlorous acid (1 x 10(6) M(-1)s(-1)) to form a chlorinated intermediate, followed by a slow conversion to a new spectrum. MCI-186 consumed 3 moles of hypochlorous acid and did not react with superoxide. The newly synthesized fluorescence probes, 2-[6-(4'-amino)-phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) and 2-[6-(4'-hydroxy)phenoxy-3H-anthen-3-on-9-yl]benzoic acid (HPF) successfully detected neutrophil-derived active oxygens (Setsukinai K, Urano Y, Kakinuma K, Majima HJ, Nagano T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 2003; 278: 3170-3175). The rate constants for the reaction of hypochlorous acid with MCI-186 and fluorescence probes was in the order of MCI-186 > APF > HPF. Fluorescence due to the oxidation of APF and HPF was observed with the stimulated neutrophils. The result that the intensity from APF oxidation was higher than that from HPF oxidation is compatible with reports that APF selectively reacts with hypochlorous acid. Fluorescence due to oxidation of both APF and HPF decreased when the reactions were carried out in the presence of a fluorescence probe and MCI-186 in a dose-dependent manner. These results indicate that MCI-186 effectively scavenges neutrophil-derived hypochlorous acid and other active oxygens.