Granular presence of terminin is the marker to distinguish between the senescent and quiescent states

We have previously identified statin, a nonproliferating-cell-specific nuclear protein of 57,000 dalton whose presence can be used to distinguish between growing and nongrowing cells. In this report we identify another protein, terminin, whose presence (by immunofluorescence microscopy) can be used to distinguish between temporarily and permanently growth-arrested cells. Thus terminin is a marker to separate the senescent from the quiescent state. By means of an unique monoclonal antibody (mAb1.2), the presence of terminin is recognized as granules in the cytoplasm of in vitro aged fibroblasts; these granules are not found in serum-starved, contact-inhibited, growing or transformed fibroblasts, except for those cells experiencing the initiation of apoptosis due to long-term deprivation of nutrients. Preliminary histochemical studies show that terminin is also found in the superficial epithelial layer of the esophagus, where terminal differentiation is followed by apoptosis and sloughing off into the lumen. Biochemical characterization by Western blot shows the terminin antibody recognizing a protein of 84 kilodalton (kDa) in growing and quiescent cells, whereas in senescent cells a protein of 57 kDa is recognized; this result suggests that a senescence-dependent protease may cleave the 84 kDa protein to 57 kDa. This proteolytic action seems to render the specific antigenic epitope exposed in its native state and accessible to the terminin antibody by immunofluorescence microscopy. It is this product of posttranslational modification in the form of a cytoplasmic 57 kDa protein that is the marker distinguishing between senescence and quiescence.     

Cross-Species Interaction between Rapidly Evolving Telomere-Specific Drosophila Proteins

Telomere integrity in Drosophila melanogaster is maintained by a putative multisubunit complex called terminin that is believed to act in analogy to the mammalian shelterin complex in protecting chromosome ends from being recognized as sites of DNA damage. The five proteins supposed to form the terminin complex are HP1-ORC associated protein, HP1-HOAP interacting protein, Verrocchio, Drosophila Telomere Loss/Modigliani and Heterochromatic Protein 1. Four of these proteins evolve rapidly within the Drosophila genus. The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation. However, terminin is not an experimentally proven entity, and no biochemical studies have been performed to investigate its assembly and action in detail. Motivated by these facts in order to initiate biochemical studies on terminin function, we attempted to reconstitute terminin by co-expressing its subunits in bacteria and investigated the possible role of the fast-evolving parts of terminin components in complex assembly. Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro. We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction.     

Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Neuregulin-1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-beta 1a or the III-beta 1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-beta 1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.     

Characterization of a cytoskeletal matrix associated with myelin from rat brain.

Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.     

Human micro-calpain: simple isolation from erythrocytes and characterization of autolysis fragments

Heterodimeric p-calpain, consisting of the large (80 kDa) and the small (30 kDa) subunit, was isolated and purified from human erythrocytes by a highly reproducible four-step purification procedure. Obtained material is more than 95% pure and has a specific activity of 6-7 mU/mg. Presence of contaminating proteins could not be detected by HPLC and sequence analysis. During storage at -80 degrees C the enzyme remains fully activatable by Ca2+, although the small subunit is partially processed to a 22 kDa fragment. This novel autolysis product of the small subunit starts with the sequence 60RILG and is further processed to the known 18 kDa fragment. Active forms and typical transient and stable autolysis products of the large subunit were identified by protein sequencing. In casein-zymograms only the activatable forms 80 kDa+30 kDa, 80 kDa+22 kDa and 80 kDa+18 kDa displayed caseinolysis.     

Isolation and some properties of bovine brain 100 kDa heat shock protein

1. The 100 kDa protein was purified from bovine brains. 2. The antibody against the 100 kDa brain protein was prepared and was monospecific to the antigen. 3. The antibody cross-reacted with HeLa cell HSP100 (100 kDa heat shock protein). 4. The physicochemical, immunochemical properties and a partially amino acid sequence indicated that the 100 kDa protein was HSP100. 5. Peptide mapping using Staphylococcus aureus V8 protease showed a core peptide with 10 kDa molecular mass common to both HSP100 and HSP90. 6. The amino acid sequence of the 10 kDa fragment of the 100 kDa protein showed a high homology with that of human HSP90 (38-60); the difference was only two of 23 amino acid residues determined.     

Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody.

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).     

Inhibition of Nuclear Protein Import by a Monoclonal Antibody against a Novel Class of Nuclear Pore Proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS--albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.     

G-proteins in skeletal muscle. Evidence for a 40 kDa pertussis-toxin substrate in purified transverse tubules.

In muscle, it has been established that guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, elicits a rise in tension in chemically skinned fibres, and that pretreatment with Bordetella pertussis toxin (PTX) decreases GTP[S]-induced tension development [Di Virgilio, Salviati, Pozzan & Volpe (1986) EMBO J. 5, 259-262]. In the present study, G-proteins were analysed by PTX-catalysed ADP-ribosylation and by immunoblotting experiments at cellular and subcellular levels. First, the nature of the G-proteins present in neural and aneural zones of rat diaphragm muscle was investigated. PTX, known to catalyse the ADP-ribosylation of the alpha subunit of several G-proteins, was used to detect G-proteins. Three sequential extractions (low-salt-soluble, detergent-soluble and high-salt-soluble) were performed, and PTX was found to label two substrates of 41 and 40 kDa only in the detergent-soluble fraction. The addition of pure beta gamma subunits of G-proteins to the low-salt-soluble extract did not provide a way to detect PTX-catalysed ADP-ribosylation of G-protein alpha subunits in this hydrophilic fraction. In neural as well as in aneural zones, the 39 kDa PTX substrate, very abundant in the nervous system (Go alpha), was not observed. We then studied the nature of the G alpha subunits present in membranes from transverse tubules (T-tubules) purified from rabbit skeletal muscle. Only one 40 kDa PTX substrate was found in T-tubules, known to be the key element of excitation-contraction coupling. The presence of a G-protein in T-tubule membranes was further confirmed by the immunoreactivity detected with an anti-beta-subunit antiserum. A 40 kDa protein was also detected in T-tubule membranes with an antiserum raised against a purified bovine brain Go alpha. The presence of two PTX substrates (41 and 40 kDa) in equal amounts in total muscle extracts, compared with only one (40 kDa) found in purified T-tubule membranes, suggests that this 40 kDa PTX substrate might be involved in excitation-contraction coupling.     

Surface proteins localized in ruffling membranes and filopodia of human glioma cells detected using Mab S-11E10

We prepared mouse monoclonal antibody (Mab S-11E10) for the surface antigen specifically distributed in ruffling membranes and filopodia of cultured human glioma cells. The antibody reacted to the specified structures of other glioma cells (U251MG, KNS 60) as well as those of KNS 42 cells, which were the immunizing source. The antigens, identified by Mab S-11E10, had molecular weights of 65 and 66 kDa. Immunohistochemically, the antibody reacted only to astrocytes in the human brain, but the cross-reactivity was noted in extraneural tissues such as lymphocytes and epithelium of the bowel ducts. In 5 out of 6 low grade astrocytomas, most of the tumor cells were strongly stained, while tumor cells of highly cellular areas of anaplastic astrocytomas and glioblastomas were either not stained or only weakly stained. The specific localization of 65-66 kDa doublet proteins may suggest relation to spreading and locomotion of cells, and may correlate to astrocytic differentiation and/or function.     

Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope.

A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein.     

Determination of copy number of c-Myc protein per cell by quantitative Western blotting

The protooncogene c-Myc plays a key role in growth control, differentiation, and apoptosis. An abnormally high expression of c-myc has been found to be associated with many neoplasms. c-Myc gene expression is usually measured at the mRNA level. Few studies have been published on quantitative Myc protein determination. A major drawback of ELISA (enzyme-linked immunosorbent assay) methods is the uncertainty of the specificity of the antibody reaction. In contrast, antibody specificity can be easily controlled by Western/immunoblotting. Here we describe a method to quantify c-Myc protein in primary human IMR90 lung fibroblasts based on Western blotting. Using a high-resolution polyacrylamide gel, we were able to differentiate the cellular c-Myc protein (64 kDa) from a c-Myc internal standard (65 kDa). We determined both the total c-Myc protein content per cell and its distribution in the cytoplasmic and nuclear fractions. About 4000 c-Myc protein molecules were detected in the cytoplasmic fraction and 29,000 copies in the nuclear fraction for proliferating human lung fibroblasts IMR90. The ratio of nuclear (active) to cytoplasmic (inactive) c-Myc protein changed from 17:1 for proliferating cells to 2.5:1 for confluent cells.     

Phosphorylation of calf uterine progesterone receptor by cAMP-dependent protein kinase

We have examined the potential for using calf uterine progesterone receptor (PR) as a substrate for phosphorylation by cAMP-dependent protein kinase (cAMP-PK), PR was found to interact with anti-PR monoclonal antibody alpha PR6 (Sullivan et al., 1986), which was to immunopurify the receptor. Protein staining of the purified preparation revealed the presence of two major bands corresponding to 114 kDa and 90 kDa peptides; only 114 kDa peptide could be photoaffinity-labeled with R5020. The 90 kDa peptide co-migrated with 90 kDa heat shock protein (hsp-90) precipitated by anti-hsp-90 monoclonal antibody AC88 (Riehl et al., 1985). Incubation of the immunopurified protein-A-Sepharose-adsorbed PR with the catalytic subunit of cAMP-PK in the presence of gamma-[32P]ATP and divalent cations resulted in a Mg++-dependent incorporation of 32P-radioactivity into both the 114 kDa and the hsp-90 peptides. Small 32P-incorporation was also seen in the 114 kDa peptide in the presence of Mn++. A 60 degrees C preincubation of immunopurified PR increased the extent of phosphorylation of the hsp-90 peptide. A pretreatment with alkaline phosphatase reduced the ability of PR to act as a substrate while the steroid occupancy of PR appeared to enhance the phosphorylation of the 114 kDa peptide. The differential cation requirement for the phosphorylation of 114 kDa and hsp-90 peptides and a selective hormone-dependent increase in the phosphorylation of the 114 kDa peptide suggest a possible role of phosphorylation in mediating progesterone action in the calf uterus.     

Purification and internal amino acid sequence of the 80 kDa protein kinase C substrate from Swiss 3T3 fibroblasts. Homology with substrates from brain

The acidic 80 kDa protein kinase C (PKC) substrate was purified from 2.3 x 10(10) Swiss 3T3 fibroblasts. Partial amino acid sequence data were obtained from five peptides generated by S. aureus V8 cleavage of the protein, enabling a total of 91 amino acid residues to be assigned. The sequences of these five peptides were compared to the deduced amino acid sequences of acidic 80-87 kDa PKC substrates from both actively proliferating A431 epidermal carcinoma cells, and fully differentiated neural tissue. Despite their similar physical properties, there was no homology between the peptides derived from the fibroblast 80 kDa protein and the PKC substrate from A431 cells. However, there was 66% homology with the 87 kDa bovine brain protein within the regions covered by the peptides about 30% of the total protein). Furthermore, comparison of the peptides from the fibroblast 80 kDa protein with proteolytic peptides derived from the acidic 80 kDa rat brain protein revealed an overall homology of 89%. These data provide the first direct evidence that the 80 kDa PKC substrate from Swiss 3T3 fibroblasts is closely related to the 80-87 kDa PKC substrates detected in fully differentiated neural tissue.     

Gastric parietal cell antigens of 60-90, 92, and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60-90-kDa component

Thirty-four human sera containing parietal cell autoantibodies (PCA) specifically immunoprecipitated two antigens, with apparent molecular masses of 60-90 kDa and 100-120 kDa under nonreducing conditions and 60-90 kDa and 120-150 kDa under reducing conditions, from porcine gastric membrane extracts. A third antigen of 92 kDa was only observed in immunoprecipitates analyzed under reducing conditions. By immunoblotting, 24 of the 34 PCA-positive sera reacted with only the 60-90-kDa antigen, three reacted with a broad 60-120-kDa smear, one reacted only with a 92-kDa antigen and six did not react. Reactivity with the 60-90-kDa antigen was observed with gastric membranes from dog, pig, rat, and rabbit. Twenty PCA-negative sera did not react with these components by immunoprecipitation or immunoblotting. PCA reactivity with the 60-90-kDa antigen was abolished when the gastric membranes were (a) digested with Pronase, (b) reduced with 100 mM dithiothreitol, (c) treated with sodium periodate, or (d) digested with N-glycanase. The 60-90-kDa and 100-120-kDa components were insensitive to neuraminidase treatment. N-glycanase digestion of 125I-labeled antigens purified by immunoprecipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis collapsed the 60-90-kDa antigen to a sharp 34-kDa band; the 100-120-kDa component was unaffected. These observations suggest that (i) parietal cell antigens comprise three components of 60-90, 92, and 100-120 kDa; (ii) the epitopes differ in conformational sensitivity; (iii) the 60-90-kDa antigen is a conserved molecule comprising a 34-kDa core protein extensively glycosylated with N-linked oligosaccharides; (iv) sialic acid residues are not present in the 60-90- and 100-120-kDa molecules, and (v) the carbohydrate and protein moieties of the 60-90-kDa molecule are required for antibody binding.     

The sequential appearance of components of the synaptonemal complex during meiosis of the female rat.

This paper describes the light microscopy (LM) and electron microscopy (EM) localization of synaptonemal complex (SC) antigens in oocytes of rats. For this purpose, we used monoclonal antibodies (Mabs) that recognize components of 30 + 33, 125, and 190 kDa antigens of SCs of rat spermatocytes. The LM localization was performed by immunofluorescence and the EM localization by immunogold staining. The reaction of the Mabs with oocytes was similar to the reaction with spermatocytes, but weaker. The 30 + 33 kDa as well as the 190 kDa antigens could always be demonstrated if axial elements of the SC were present, irrespective of whether these were paired or unpaired. Thus, these antigens could be detected from leptotene--early zygotene until diplotene. The 190-kDa antigen appeared in a diffuse manner just before the appearance of the 30 + 33 kDa antigens. The 30 + 33 kDa antigens were not only detected in the axial elements of SCs but also in characteristic aggregates, which appeared in zygotene and persisted until after the SCs had disappeared. Such aggregates had rarely been observed in spermatocytes. The 125 kDa antigen was only present in the tripartite segments of SCs, at the inner edge of the lateral elements. Thus, the reaction of the Mab against the 125 kDa antigen was detectable in zygotene, pachytene, and very early diplotene. It appeared later than 30 + 33 kDa and 190 kDa antigens and it disappeared earlier. We found that several steps of the immunostaining procedure could cause variation in the intensity of the Mab reaction.     

嗜线虫致病杆菌血腔毒素Tp40的纯化和特性分析

[目的]嗜线虫致病杆菌是一种昆虫病原线虫共生菌,它能够产生多种杀虫毒素.本研究旨在从嗜线虫致病杆菌Xenorhabdus nematophila HB310菌株的细胞内纯化新的杀虫蛋白毒素,并对其进行基因克隆和序列分析.[方法]应用盐析和制备型非变性凝胶电泳等方法纯化蛋白,再通过对5龄大蜡螟幼虫血腔注射进行活性筛选.对获得的目的蛋白与已知蛋白进行同源分析,克隆出该目的蛋白的基因序列,从而进行相应的基因和氨基酸序列分析.[结果]本研究纯化的Tp40蛋白对大蜡螟LD50为68.54 ng/头,其SDS-PAGE电泳图谱只显示出一条分子量约为42 kDa的多肽.Western印迹分析表明Tp40与已知的Txp40为同源蛋白,并且仅存在于细胞内.编码该蛋白的基因开放读码框全长1107bp(GenBank登录号:EU095326),编码368个氨基酸残基,预测分子量为41.5 kDa,等电点为8.66,与GenBank中的其余13株昆虫病原线虫共生菌所包含的相似基因核苷酸序列及推导的氨基酸序列比较,同源性分别为85%～99%和70%～99%.[结论]Tp40蛋白具有很高的血腔杀虫活性,其基因序列具有较强的保守性,是昆虫病原线虫共生菌复合体杀虫过程中的一种关键因子.     

Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.     

Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo

The membrane leakage caused by the cell penetrating peptide Tp10, a variant of transportan, was studied in large unilamellar vesicles with the entrapped fluorophore calcein. The vesicles were composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. A significant decrease in membrane leakage was found when the 55kDa streptavidin protein was attached to Tp10. When a 5.4kDa peptide nucleic acid molecule was attached, the membrane leakage was comparable to that caused by Tp10 alone. The results suggest that direct membrane effects may cause membrane translocation of Tp10 alone and of smaller complexes, whereas these effects do not contribute for larger cargoes.     

Differential heat shock response of primary human cell cultures and established cell lines

The influence of hyperthermia on the cellular growth and protein synthesis pattern from primary human brain tumour cells and skin fibroblasts was compared with established and experimentally transformed tumour cell lines. Primary cell cultures did not show any visible morphological changes after 42 degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized a protein with an apparent molecular mass of 70 kDa and an isoelectric pH of 7.0 as early as 3 h after the initial hyperthermal treatment.