Biochemical characterization of the mammalian stress proteins and identification of two stress proteins as glucose- and Ca2+-ionophore-regulated proteins

Biochemical properties of the heat shock or stress proteins of mammalian cells have been investigated using two-dimensional gel electrophoresis and immunological techniques. Of the major mammalian stress proteins (Mr = 72,000, 73,000, and 90,000) and minor stress proteins (Mr = 80,000, 100,000, and 110,000), the 80- and 90-kDa proteins were found to be phosphoproteins in all cell types examined. The 100-kDa protein was found to incorporate phosphate in only some cell types examined. In studies of the metabolic incorporation of mannose into the stress proteins, only the 100-kDa protein was found to be a glycoprotein. Two of the stress proteins, the 80- and 100-kDa species, were found to be identical with the proteins induced in cells grown in the absence of glucose (i.e. the "glucose-regulated proteins"). These same two proteins also were induced in cells treated with the calcium ionophore A23187. To begin examining the intracellular location of these multiregulated proteins, immunofluorescence microscopy studies were carried out using a monoclonal antibody against the 100-kDa stress protein. The antigen was localized primarily with the Golgi apparatus and less prominently with the plasma membrane and nucleus. Heat shock treatment resulted in an increased number of the cells exhibiting a nuclear location of 100 kDa.     

Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Turnover of cytokeratin polypeptides in mouse hepatocytes

The turnover of cytokeratin polypeptides A (equivalent to No. 8 of the human cytokeratin catalog) and D (equivalent to human cytokeratin No. 18) of mouse hepatocytes was studied by pulse-labeling of mouse liver proteins after intraperitoneal injection of L-[guanido-14C]arginine and [14C]sodium bicarbonate. At various times after injection cytoskeletal proteins were prepared and separated by SDS-polyacrylamide gel electrophoresis, and the specific radioactivities of polypeptides recovered from excised gel slices were determined. With L-[guanido-14C]arginine a rapid increase in the specific radioactivity of both cytokeratins was observed which reached a plateau between 12 and 24 h. With [14C]sodium bicarbonate maximal specific radioactivity was obtained at 6 h followed by a rapid decrease to half maximum values within the subsequent 6 h and then a slower decrease. Half-lives were determined from the decrease of specific radioactivities after pulse-labeling by least-squares plots and found to be 84 h (for cytokeratin component A) and 104 h (component D) for arginine labeling. Values obtained after bicarbonate labeling were similar (95 h for A and 98 h for D). These results show that liver cytokeratins are relatively stable proteins and suggest that components A and D are synthesized and degraded at similar rates, probably in a coordinate way.     

Biochemical and immunological identification of human neutrophil elastase on nitrocellulose membranes.

Human neutrophil elastase (HNE) was analyzed for protein(s), antibody staining and activity staining, on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis followed by Western blotting. The HNE activity, which was identified with N-acetyl-D,L-alanine alpha-naphthyl ester as substrate, was well preserved in the presence of 0.1% LDS at 4 C during electrophoresis. As little as 0.1 microgram HNE was required for the activity staining. The HNE appeared to be three peptides having a major band at mass ratio 27,000, a second major band at mass ratio 28,000 with a minor protein band at mass ratio 29,000. On transfer to nitrocellulose, the mass ratio 28,000 band displayed poor immunoreactivity. This was the second most dense band with highest enzymatic staining. This procedure is a useful method and analytical tool to determine the correlation of enzymatically active proteins, subunits and immunoreactive protein(s) of elastase from various sources, including neutrophils.     

Biochemical and immunological relationships among fibronectin-like proteins from different sea urchin species

Fibronectin-like proteins were purified from ovaries of the sea urchin species, Paracentrotus lividus (PI), Sphaerechinus granularis (Sg), Arbacia lixula (Al), Pseudocentrotus depressus (Pd), and Anthocidaris crassispina (Ac), by gelatin-Sepharose affinity chromatography. The major component had a molecular mass of 180 kDa and was eluted by 1 M NaCl or 8 M urea, depending on the species used. By substrate adhesion assay, we tested the biological activity of the 180 kDa protein purified from Paracentrotus lividus (P1-180K) and showed that it promotes the adhesion of homologous embryonic cells to the substrate. An antiserum, developed against Temnopleurus hardwickii fibronectin-like protein (Th-180K), was used in Western blots of the proteins purified from the five species. The antibody cross-reacted with Pl-180K, Pd-180K and Ac-180K. A peptide map of P1-180K, obtained by V8 protease partial digestion, was compared with those obtained from the other four proteins and showed an homology between 40 and 56%. This report confirms that fibronectin-like proteins can be purified from sea urchins on the basis of their binding to gelatin-Sepharose; the proteins differ for their binding affinity to gelatin and share different epitopes, suggesting that they are members of a sea urchin fibronectin super family.     

Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells

The Bcl-2 family of proteins comprises both cell death inhibiting and cell death promoting members, generally believed to be cytoplasmic and predominantly membrane-associated. Like Bcl-2, many Bcl-2-related proteins contain a C-terminal membrane insertion domain and much research is aimed at evaluating the functional role of their localization to the outer membranes of mitochondria, the endoplasmic reticulum, and perinuclear membranes. However, confocal fluorescence microscopy of human breast cancer cells and rat colon cancer cells immunostained with commercial antibodies raised against different epitopes of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax protein revealed that these proteins are not only present in the cellular cytoplasm, but also within interphase nuclei. This was confirmed by Western blot analysis of isolated nuclei. In human cells, certain epitopes of Bcl-2, but not of Bax, were also found to be associated with mitotic chromatin. Anti-estrogen treatment of human breast cancer cells or transfection with antisense bcl-2 led to a reduction in both cytoplasmic and nuclear Bcl-2. Transfection of human bcl-2 and bax into rat cells resulted in cytoplasmic and nuclear Bcl-2 and Bax. This data seems in line with increasing evidence that the role of the Bcl-2 family of proteins should be extended to activities inside the nuclear compartment.     

Biochemical and immunological characterization of the duck hepatitis B virus envelope proteins.

To examine the envelope proteins of duck hepatitis B virus (DHBV), which are encoded by the pre-S/S open reading frame of the viral genome, an antiserum was raised in rabbits against a fusion protein comprising most of the pre-S coding segment. By using this antiserum, viral particles could be precipitated from serum, and two pre-S proteins with molecular sizes of approximately 35 and 37 kilodaltons were detected in the sera and livers of DHBV-infected ducks after Western blotting and after biosynthetic labeling of a primary duck liver cell culture. In serum, the pre-S proteins were shown to exist predominantly in DHBV-DNA-free particles associated with a 17-kilodalton protein which, by N-terminal amino acid sequence analysis, was shown to represent the viral S protein which is encoded by the 3' proximal segment of the DHBV pre-S/S open reading frame. To compare the immunogenic potential of the S and pre-S proteins, serum particles and gel-purified S protein were used to immunize rabbits. In neither case was a significant immune response against the DHBV S protein observed. However, a good antibody titer against DHBV pre-S was obtained even after immunization with small amounts of the pre-S antigen.     

In situ identification of cellular drug targets in mammalian tissue

1. Download : Download high-res image (166KB)
2. Download : Download full-size image

     

Transient expression of bile-duct-specific cytokeratin in fetal mouse hepatocytes

The differentiation of hepatocytes and biliary epithelial cells has been histochemically analyzed with anti-calf cytokeratin antiserum in the fetal mouse liver. Almost all young fetal hepatocytes transiently express bile-duct-specific cytokeratin; subsequently, the strong staining of the cytokeratin is confined to progenitor cells of intrahepatic biliary epithelial cells around portal veins. These results suggest that all fetal hepatocytes are bi-potent in terms of the differentiation of mature hepatocytes and intrahepatic bile-duct cells, and that the microenvironment around portal veins plays an important role in bile-duct differentiation. Large periportal hepatocytes continue to stain weakly for cytokeratin until 2 weeks after birth, although the number of positive hepatocytes decreases with development. The differentiation of bile ducts from periportal hepatocytes may continue for 2 weeks after birth.     

Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.     

A proteomic approach to identification of plutonium-binding proteins in mammalian cells

Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry was employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu⁴⁺-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore™ analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases.     

Monoclonal cytokeratin antibody recognizing a heterotypic complex: immunological probing of conformational states of cytoskeletal proteins in filaments and in solution

A novel type of monoclonal murine antibody (Ks18.18) directed against an epitope depending on human cytokeratin (CK) 18, a member of the acidic (type I) CK subfamily, is described. We show by SDS-PAGE immunoblots and dot-blot assays that this antibody is unreactive with both the denatured and the renatured individual polypeptides but binds strongly to heterotypic coiled-coil complexes of CK 18 with several members of the complementary basic (type II) CK subfamily, notably with CK 8; i.e., its most frequent natural partner. We also show that specific interactions between complementary CK polypeptides take place during the incubation steps of immunoblotting procedures as polypeptides, or fragments thereof, that detach from the substrate can bind to complementary polypeptides attached to the substratum, which may result in false assignments of antibody reactivities. The conformation-specific, CK 18-dependent epitope of Ks18.18 was detected in intermediate filaments (IFs) of cultured cells, simple epithelia, and many carcinomas and, surprisingly, also in the basal cells of some stratified epithelia. Ks18.18 also reacts with altered CK configurations as present in the spheroidal bodies of mitotic cells and in the Mallory bodies of hepatocytes intoxicated with certain drugs, thus indicating that the heterotypic CK complexes are maintained in these structures. We have also used antibody Ks18.18 to demonstrate the existence of heterotypic CK 8 and 18 complexes in a distinct soluble form among supernatant proteins from cell homogenates which is indistinguishable from the heterotypic tetramer obtained after experimental disintegration of IFs. The potential value of such IF conformation-specific antibodies in cell biological research and pathology is discussed.     

Biochemical correlates of the structural allometry and site-specific properties of mammalian adipose tissue

1. The maximum activities of the glycolytic enzymes hexokinase (HK) and phosphofructokinase (PFK) were measured in defatted homogenates of adipose tissue from nine homologous depots of 57 wild and captive mammals belonging to 17 species and eight orders and differing in body mass by six orders of magnitude. 2. Site-specific differences in the enzyme activities were similar in all terrestrial species and were not consistently related to adipocyte volume. 3. The specimen-mean maximum activities of HK and PFK did not correlate with body mass, body composition or natural diet. 4. When specimens of different body composition and body mass were compared, glycolytic enzyme activity per adipocyte was directly proportional to adipocyte volume. 5. Site-specific differences in collagen content of adipose tissue did not correspond to those adipocyte volume. When homologous depots of different specimens were compared, the collagen content of adipose tissue was directly proportional to body mass. 6. Adipose tissue of large cetaceans contains more collagen than predicted from the allometric equations fitted to the data from terrestrial mammals. 7. Neither the scaling of the collagen content with body mass nor the site-specific differences in its abundance are consistent with a role as protection or support for adjacent tissues. 8. There are consistent site-specific differences in the extracellular components of adipose tissue as well as in the structure and metabolism of the adipocytes. 9. Adipose tissue differs from most other tissues in that its maximum metabolic capacities do not scale to body mass. 10. Adjustment of the biochemical activity of adipose tissue to changes in body mass and body composition must depend upon neural and endocrine controls, not upon intrinsic differences in its metabolic capabilities.     

De novo identification of mammalian ciliary motility proteins using cryo-EM

1. Download : Download high-res image (226KB)
2. Download : Download full-size image

     

Biochemical and immunological adaptation in schistosome parasitism

The objective of this review is to clarify aspects of immunological and biochemical adaptations of schistosomes to their intermediate and final mammalian hosts. Adaptations to the mammalian hosts are traced in relation to cercarial penetration of mammalian skin, glucose transport and metabolism. The unusual ability of schistosome surface membrane to escape immune recognition and damage are reviewed. Moreover, the behavioural changes induced in the intermediate hosts by schistosomes are considered. The evolutionary adaptation to molluscan hosts aims to increase the probability of transmission of the parasite into its mammalian host. This review inspires more hope for further design of anti-schistosome drugs through disturbing aspects of biochemical and immunological adaptations in schistosome parasitism.     

Expression of nuclear matrix proteins in rat liver tissue

We have studied the synthesis of nuclear matrix proteins as it occurs in the rat liver. To investigate their kinetics in tissue, nuclear matrix proteins were prepared from liver of rats injected with radioactive methionine. Synthesis of lamins was not observed in quiescent hepatocytes although they were the principal proteins of this subcellular fraction, suggesting that lamins are very stable in the liver. When hepatocytes were stimulated to divide by partial hepatectomy, only synthesis of lamin B was initiated. Many proteins not visible on Coomassie blue-stained gels were detectable by autoradiography. In the nuclear matrix extracts of quiescent hepatocytes, one of the most prominently labeled ones was a protein of 70 kDa. After hepatectomy, an additional protein of 62 kDa was detectable. These proteins were visible 1 h after the injection of radioactivity, but were no longer observed in nuclear matrices prepared 24 h after injection. These experiments indicate that in addition to lamins, two nuclear matrix proteins are present in the rat liver that were not detected previously, perhaps because of their rapid turnover.     

A guide through present computational approaches for the identification of mammalian microRNA targets

Computational microRNA (miRNA) target prediction is a field in flux. Here we present a guide through five widely used mammalian target prediction programs. We include an analysis of the performance of these individual programs and of various combinations of these programs. For this analysis we compiled several benchmark data sets of experimentally supported miRNA-target gene interactions. Based on the results, we provide a discussion on the status of target prediction and also suggest a stepwise approach toward predicting and selecting miRNA targets for experimental testing.