Effects of high-probability requests on the acquisition and generalization of responses to requests in young children with behavior disorders.

The failure to respond to requests in young children often is maintained by the reactions of the adults that encounter this behavior. This failure to respond to requests has been identified as a primary reason for the children's exclusion from community, social, and instructional opportunities. Numerous interventions that target the failure to respond have consisted of punishment and reinforcement procedures. More recently, antecedent interventions have focused on changing the context in which a request is delivered. In the current study, high-probability requests were provided as an antecedent to delivering a low-probability request. The requests were delivered by multiple trainers in an attempt to produce generalized appropriate responding to adults who did not use the high-probability sequence. Results showed an immediate increase in appropriate responding in 2 children when the intervention was delivered. In addition, when the intervention was implemented by more than one adult, spontaneous increases in responding also were observed toward adults who had never implemented the request sequence. Improvements in responding to requests were maintained after the intervention was discontinued.


Formation of cholic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid in human skin fibroblasts.

Whether 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) was converted into cholic acid in human skin fibroblasts was examined. THCA was incubated with subcellular fractions of cultured skin fibroblasts in the presence of NAD+, ATP, CoA, and Mg2+. The reaction products were analyzed by thin-layer chromatography and high-performance liquid chromatography after p-bromophenacyl ester derivatization. The highest specific activity was found in the light mitochondrial fraction (2.71 nmol/mg protein/h). The specific activity was about 9-fold higher than that in heavy mitochondrial fraction. The peroxisomal fraction prepared from the light mitochondrial fraction by sucrose gradient centrifugation was also able to catalyze the conversion of THCA into cholic acid. The specific activity in this fraction was a further 2.2-fold higher than that in the light mitochondrial fraction. These results suggest that cultured human skin fibroblasts are able to convert THCA into cholic acid, and that the activity exists in peroxisomes.


Purification and properties of 4-hydroxybiphenyl UDP-glucuronyltransferase from bovine liver microsomes.

A UDP-glucuronyltransferase isoform glucuronizes phenolic xenobiotics such as 4-nitrophenol, and an isoform glucuronizing 4-hydroxybiphenyl has also been found in rat liver. We purified a UDP-glucuronyltransferase isoform glucuronizing 4-hydroxybiphenyl from bovine liver microsomes by solubilization with 0.7% sodium cholate followed by three column chromatographic separations using DEAE-Toyopearl 650S, UDP-hexanolamine Sepharose 4B, and hydroxyapatite. The purified bovine liver 4-hydroxybiphenyl UDP-glucuronyltransferase (named Bovine 4HBGT) had glucuronidation activities toward 4-hydroxybiphenyl and 4-methylumbelliferone but had little activity toward 4-nitrophenol and 1-naphthol. The apparent molecular mass of Bovine 4HBGT was 54,000 Da on SDS-PAGE, and this was decreased to 50,000 Da by digestion with endo-beta-N-acetylglucosaminidase H. These data suggest that Bovine 4HBGT consists of a 50,000 Da polypeptide and a high mannose type oligosaccharide chain(s) of about 4,000 Da. The NH2-terminal sequence of GT-3 was GKVLVWPVDFSXWINI. These properties of Bovine 4HBGT were very similar to those of rat UDP-glucuronyltransferase glucuronizing xenobiotics. However, the NH2-terminal sequence of Bovine 4HBGT had higher homology with that of rat liver 4-hydroxybiphenyl UDP-glucuronyltransferase than with that of rat liver 4-nitrophenol UDP-glucuronyltransferase.


Primary structure of the antihemorrhagic factor in serum of the Japanese Habu: a snake venom metalloproteinase inhibitor with a double-headed cystatin domain.

The complete amino acid sequence of an antihemorrhagic factor, HSF, in the serum of the Japanese Habu snake, Trimeresurus flavoviridis, has been determined. The protein is composed of 323 amino acid residues and contains three asparagine-linked oligosaccharide chains at positions 123, 185, and 263. The molecule contains two copies of the cystatin domain in the N-terminal portion up to position 240, and these domains show a remarkable sequence homology (about 50%) to those of plasma glycoproteins such as alpha 2-HS (human) and fetuin (bovine) and to a lesser extent to that of HRG (human). The amino acid sequence of the noncystatin region towards the C-terminus is unique, showing no significant homology with those of the corresponding regions of alpha 2-HS and fetuin. In spite of the presence of cystatin domains, HSF does not inhibit cysteine proteinases such as papain and cathepsin B but does inhibit several metalloproteases in Habu venom. The results suggest that HSF is the first protein found to be functionally related to metalloproteinase inhibitors among the structurally homologous proteins with a double-headed cystatin domain, and is a member of a novel family (family 4) with divergent functions of the cystatin superfamily proteinase inhibitors. Although HSF possesses similar physicochemical properties to those of oprin, a snake venom metalloproteinase inhibitor with antihemorrhagic activity isolated from opossum serum [Catanese & Kress (1992) Biochemistry 31, 410-418], its primary structure is strikingly different from that of oprin.


Hyperproduction of human interferon gamma by rat cells maintained in low-serum medium using the fibronectin gene promoter.

An expression vector, pF1900M, which expresses a cloned gene at a high level in quiescent mammalian cells was constructed using the rat fibronectin (FN) promoter. Human interferon gamma (HuIFN-gamma) cDNA inserted downstream of the FN promoter in pF1900M was introduced into rat 3Y1 cells and several IFN-producing cell lines were established. These cells secreted a low level of IFN when they were growing but secreted at a high level after they had reached confluence. The level was further increased when the confluent cells were maintained in low-serum medium and a cell line, I7, produced 4 x 10(5) IU/ml of IFN, comparable to that produced by genetically engineered Escherichia coli in 2 days. The IFN-producing ability of I7 cells could be maintained by successive replacements of low-serum medium for at least 2 weeks. HuIFN-gamma secreted into the medium had a molecular weight range of 22,000 to 25,000, similar to that of IFN-gamma produced by human lymphocytes. The N-linked glycosylation of HuIFN-gamma seemed to occur properly, since treatment of the IFN with N-glycanase resulted in a reduction of molecular weight to 17,000, which corresponds to that calculated from the deduced amino acid sequence of HuIFN-gamma.


Cloning and expression of the Klebsiella pneumoniae galactose operon.

The entire galactose (gal) operon of Klebsiella pneumoniae was isolated and functionally analyzed in Escherichia coli. The genes encoding galactokinase (galK), galactose-1-phosphate uridyltransferase (galT), and UDP-galactose-4-epimerase (galE) were mapped by complementation analysis. The gene order E-T-K was found to be identical to that of Salmonella spp. and E. coli. Analysis of the nucleotide sequence in the control region revealed significant homology with that of E. coli. Two major sites for transcriptional initiation, both mapped to a cytosyl residue, were identified by primer extension. When the operon is expressed in E. coli, the K. pneumoniae gal gene products make up about 30% of the total cellular proteins. The presence of a powerful promoter responsible for high level synthesis of the gal proteins was also demonstrated using beta-galactosidase as reporter.


A novel core protein as well as polymorphic epithelial mucin carry peanut agglutinin binding sites in human gastric carcinoma cells: sequence analysis and examination of gene expression.

The peanut agglutinin (PNA)-binding site is protein-bound Gal beta 1-->3GalNAc, and is a tumor-associated carbohydrate marker expressed in many human carcinomas. PNA-binding glycoproteins isolated from KATO-III human gastric carcinoma cells were deglycosylated by trifluoromethanesulfonic acid, and rabbit antibodies against the core proteins were used to screen a lambda gt11 expression library constructed from these cells. Two different core proteins were identified by this approach. One was polymorphic epithelial mucin (PEM), initially found in breast carcinomas. PEM mRNA was expressed in normal tissues of the stomach, colon, and lung, but not in the small intestine, thyroid, and spleen. High levels of PEM mRNA were detected in some nude mouse-transplanted carcinomas, i.e. colorectal, pancreatic, stomach, and lung carcinomas. The other core protein was a novel one called MGC-24, which has a molecular mass of 24 kDa, is rich in hydroxyl amino acids and cysteine, and lacks repeating motifs. The mature MGC-24 glycoprotein behaved as a high-molecular-mass one upon SDS-polyacrylamide gel electrophoresis even after neuraminidase treatment. Treatment with endo-alpha-N-acetylgalactosaminidase in the absence of neuraminidase significantly changed the staining pattern by anti-MGC-24, confirming that MGC-24 carried PNA-binding sites. MGC-24 mRNA was intensely expressed in normal tissues of the colon, small intestine and thyroid, and in some nude mouse-transplanted colorectal and pancreatic adenocarcinomas.


Modification of immunopharmacological activities of synthetic monosaccharide lipid A analogue, GLA60, by lysozyme.

Recent studies by our group suggested that lysozyme has a high affinity for bacterial lipopolysaccharide (LPS) of both the smooth and rough forms, and inhibits various immunomodulatory activities of LPS. GLA60 is a synthetic monosaccharide analogue of bacterial lipid A well known as having most of the activities of lipid A with very low toxicity. In this study, we characterized the interaction of lysozyme with GLA60 in comparison to that with Escherichia coli 0111 LPS (smooth form) by means of an immunopharmacological approach. Using dansylated lysozyme (DNS-LZM) as a probe, LZM was found to bind to GLA60. The mitogenic and polyclonal B-cell activating activities were significantly reduced by complex formation. However, there was no inhibitory effect on GLA60 induced production of IL-1 and TNF of macrophages. Interestingly, the activities of macrophages induced by the complex were found to be significantly higher than those induced by GLA60 itself. In contrast, the activities of 0111 LPS were significantly inhibited by LZM. Since the GLA60-LZM complex produced a turbid suspension but the 0111 LPS-LZM complex remained soluble, we consider that the activities of GLA60 alone were mediated by the common functional LPS receptor for dispersed form in both macrophages and B-lymphocytes, but activation of macrophages by the complex was mediated either by another LPS receptor not present in B-lymphocytes or through the phagocytic function of macrophages.


Regulation of myeloid-specific calcium binding protein synthesis by cytosolic protein kinase C.

Two calcium binding proteins, MRP-8 and MRP-14, are specifically synthesized in human myeloid cells. This paper shows that Me2SO, all-trans-retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), but not 12-O-tetradecanoyl phorbol-13-acetate (PMA) are potent inducers of MRP-8/14 protein complex in human leukemic cells. Transforming growth factor-beta 1 (TGF-beta 1) is shown to enhance the inductive effect of RA and 1 alpha,25(OH)2D3. We have examined the possibility that MRP expression is regulated through the protein kinase pathway. Both cytosolic and membrane-bound protein kinase C (PKC) activities increased during differentiation by RA and 1 alpha,25(OH)2D3. PMA-treatment led to a decrease of cytosolic PKC activity and an increase of membrane-bound PKC activity in the presence of these differentiation inducers, while PMA alone resulted in low cytosolic and high membrane-bound PKC activities. PKC inhibitor H7 inhibited MRP synthesis in HL-60 cells treated with RA and 1 alpha,25(OH)2D3. These results suggest that cytosolic PKC activity may be involved in a stimulatory pathway of MRP synthesis and that protein phosphorylation reactions may play important roles in MRP expression during myelocytic differentiation.


Molecular shape of dystrophin.

The molecular shape of dystrophin has been reported to be a 175 nm flexible rod [Pons, F. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 7851-7855] or a 120 nm dumbbell [Murayama, T. et al. (1990) Proc. Jpn. Acad. 66B, 96-99]. The present work revealed that 100 nm flexible rods with or without spheres were predominant in highly purified dystrophin preparations. When the sample was subjected to gel filtration, dystrophin oligomers were isolated just after the void volume and the fraction largely consisted of dumbbell-shaped molecules. From various rotary-shadowed images, it was suggested that dystrophin is a rod with spheres at both ends, approximately 110 nm long and 2 nm wide. It appeared that this monomer binds to another monomer in a staggered way, forming a dimer, and the dimers associate with each other side-by-side, forming a dumbbell-shaped tetramer, 130 nm long and 5 nm wide. The tetramers form an end-to-end aggregate. It seemed that the dumbbell structure was not affected by alkaline (pH 11) treatment to dissociate dystrophin associated glycoproteins, but was deteriorated by detergent, NP-40, Triton X-100, or CHAPS, used for solubilization of membrane-bound dystrophin.


Tetrahymena profilin is localized in the division furrow.

Localization of Tetrahymena profilin was examined by an immunofluorescence method. In interphase Tetrahymena cells, immunofluorescence for profilin was diffusely distributed in the cytoplasm, while in dividing cells, additional intense fluorescence was observed in the division furrow. From the result of immunofluorescence localization using cytoskeletal cell models, a significant fraction of profilin appeared to become insoluble in association with a cytoskeletal structure just beneath the division furrow during cytokinesis, although remaining profilin existed as a soluble form in the cytoplasm. Double immunofluorescence staining with anti-profilin and anti-actin antibodies revealed that the localization of profilin in the division furrow coincided with that of contractile ring microfilaments in terms of both position and timing. This is the first report describing the coexistence of profilin with actin filaments in the division furrow, implying the possible involvement of profilin in assembly and disassembly of contractile ring microfilaments in the process of cytokinesis.


Starch branching enzymes from immature rice seeds.

Four forms of branching enzyme, termed RBE1, RBE2 (a mixture of RBE2A and RBE2B), RBE3, and RBE4, were apparently separated by DEAE-cellulose column chromatography of soluble extract from immature rice seeds, and each of these four forms was further purified by gel-filtration. RBE1, RBE2A, and RBE2B were the predominant forms of the enzyme. The molecular size, amino-terminal amino acid sequence, and immunoreactivity with anti-maize branching enzyme-I (BE-I) antibody were identical among these three forms, except that the molecular mass of RBE2A was almost 3 kDa higher than those of RBE1 and RBE2B. These results indicate that RBE1, RBE2A, and RBE2B are the same (termed rice BE-I). The cDNA clones coding for rice BE-I have been identified from a rice seed library in lambda gt11, using the maize BE-I cDNA as a probe. The nucleotide sequence indicates that rice BE-I is initially synthesized as an 820-residue precursor protein, including a putative 64- or 66-residue transit peptide at the amino terminus. The rice mature BE-I contains 756 (or 754) amino acids with a calculated molecular mass of 86,734 (or 86,502) Da, and shares a high degree of sequence identity (86%) with the maize protein. The consensus sequences of the four regions that form the catalytic sites of amylolytic enzymes are conserved in the central region of the rice BE-I sequence. Thus, rice BE-I as well as the maize protein belongs to a family of amylolytic enzymes.


Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle.

A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.


Inhibition of phosphoenzyme formation from phosphate and sarcoplasmic reticulum Ca(2+)-ATPase by vanadate binding to high- or low-affinity site on the enzyme.

In the preceding paper, we suggested that 1 mol Ca(2+)-ATPase of sarcoplasmic reticulum (SR) contains 0.5 ml of high-affinity vanadate binding sites as well as 0.5 ml of low-affinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (Pi). When vanadate was added to the reaction medium in which the Ca(2+)-ATPase had been phosphorylated by Pi in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17 microM, and reached maximum at 50 microM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of E2P was competitively prevented by Pi with a dissociation constant of 7.4 mM, which was very close to Km for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with Pi at a phosphorylation site on the Ca(2+)-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca(2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)


Induction of vesicle-to-micelle transition by bile salts for DOPE vesicles incorporating immunoglobulin G.

The vesicle-to-micelle transition of immunoliposomes formed by dioleoylphosphatidyl-ethanolamine (DOPE) and palmitoyl-immunoglobulin G (p-IgG) was investigated in the presence of bile salts and conjugated bile salts. Turbidity and the release of calcein from liposomes were measured as a function of the amount of bile salts added and compared with the solubilizing profiles of the salts according to the number and configurational state of hydroxy groups in the cholate. The solubilizing phenomena by bile salts conjugated with glycine or taurine were investigated in comparison with non-conjugated bile salts. The solubilizing effect of bile salts on the bilayer of immunoliposomes increased remarkably with the number of hydroxy groups, but was not influenced by the configurational state of the hydroxy group. The half-maximal concentration of bile salts, defined as the concentration giving the half-maximum turbidity of liposome solutions, decreased with hydrophobicity in the phosphatidylcholine (PC) bilayer. The increase in the hydrophobicity of bile salts induces the ability to permeabilize and solubilize phospholipid vesicles. In the case of PC or PE liposome bilayers with inserted protein, bile salts conjugated with taurine or glycine had lower hydrophobicity than non-conjugated bile salts and showed a lower half-maximal concentration. The conjugated bile salts are believed to interact with lipids and solubilize the bilayers, while the head groups of bile salts interact with the inserted protein and extract it from the lipid bilayer.


Treatment of low density lipophorin with lipoprotein lipase: diacylglycerol content has no effect on dissociation of apolipophorin III from low-density lipophorin.

The mechanism of the conversion of low-density lipophorin (LDLp) to high-density lipophorin (HDLp) in long-distance flight insects was investigated using a lipoprotein lipase from a bacterium, Alcaligenes sp. Diacylglycerol of LDLp was steadily hydrolyzed in vitro by the lipase, resulting in a 90% loss of diacylglycerol from LDLp during incubation. The "lipase-treated LDLp" thus obtained still contained associated apolipophorin-III (apoLp-III). These data suggest that the dissociation of apoLp-III is independent of the depletion of diacylglycerol from LDLp, and that the decrease in particle diameter caused by the depletion of diacylglycerol does not force the dissociation of apoLp-III from the lipophorin particle. Some physico-chemical properties of the lipase-treated LDLp were measured.


pH-sensitive liposomes containing polymerized phosphatidylethanolamine and fatty acid.

With the ultimate aim of targeting cancer drugs to malignant tissues, liposomes containing polymeric phosphatidylethanolamine and a fatty acid were prepared. For this purpose diacetylenic phosphatidylethanolamine (DAPE), a phosphatidylethanolamine containing diacetylene, was synthesized. Liposomes containing DAPE, fatty acid, and either phosphatidylethanolamine (PE) or phosphatidylethanolamine-beta-oleoyl-gamma-palmitoyl (POPE) were then prepared. Polymerization of DAPE was effected by UV illumination. The polymeric liposomes so obtained were stable at physiological pH but became leaky below pH 6.5. Of various compositions studied, the greatest pH-sensitivity was found with liposomes composed of 35 mol% DAPE, 35 mol% POPE, and 30 mol% saturated fatty acid. The presence of blood plasma albumin decreased vesicle stability while apolipoprotein A-I (apo A-I) had the opposite effect and plasma as a whole had a slightly stabilizing effect.


Heat-induced aggregation of recombinant erythropoietin in the intact and deglycosylated states as monitored by gel permeation chromatography combined with a low-angle laser light scattering technique.

Aggregate formation of recombinant human erythropoietin (r-EPO) on heat-treatment was followed by gel permeation chromatography combined with a low-angle laser light scattering technique under various conditions with respect to pH and salt concentration in order to provide basic knowledge about the change strictly required to be monitored for medicinal proteins. When heated at 60 degrees C at neutral pH, an aggregate with a limited size consisting of about 20 r-EPO molecules was formed. On heating at 50 degrees C at acidic pH, aggregation was unlimited. The aggregation proceeded non-covalently in acidic pH, but in alkaline pH covalent bond formation was also involved. Increase in salt concentration enhanced the aggregation. Deglycosylation of the N-linked oligosaccharides made r-EPO remarkably susceptible to aggregation on heat-treatment, indicating that the carbohydrate chains are essential to the stability of r-EPO.


Primary structure of an inactive mutant of phospholipase A2 in the venom of Bungarus fasciatus (banded krait).

From the acidic components of Bungarus fasciatus venom, a very small amount (0.16%) of a novel phospholipase A2 was obtained. Both neurotoxicity and enzyme activity were found to be lacking. Amino acid sequence study showed that it has a normal backbone of group I snake venom phospholipase A2 with 118 amino acid residues. The lack of enzyme activity was attributed to its mutation of the indispensable Asp residue to an Ala residue, i.e., the usual His-Asp47 turned out to be His-Ala47. This is the eighth isoform of phospholipase A2 found from the venom of Bungarus fasciatus. Examination of structural homology with three other isoforms revealed 66% similarity at most.


Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis.

Anthranilate synthase I (ASI) of Bacillus caldotenax, a thermophilic bacterium, was purified from a plasmid-bearing Escherichia coli and characterized. The molecular weight determination under native and denaturing conditions revealed that it was a monomeric enzyme of M(r) = 54,000. The N-terminal amino acid sequence is the same as expected from DNA sequence of trpE except that the N-terminal methionine is lacking. All four cysteines in the molecule could be titrated with 5,5'-dithiobis (2-nitrobenzoic acid) in more than 8 M urea. The purified enzyme retained its full enzymatic activity after being heated at 60 degrees C. Six mutated genes for the ASI with histidine in place of each conserved arginine, Arg321, Arg353, Arg358, Arg416, Arg429, and Arg452, were prepared by site-directed mutagenesis. All the mutated genes except one, the gene encoding an ASI mutant with histidine in place of Arg452 (R452H ASI) complemented an E. coli (trpE). The mutated ASIs were purified and compared with the wild type ASI. No distinctive differences in enzymatic properties were found between the wild type and the enzymatically active mutated ASIs. R452H ASI was enzymatically inactive, though its conformation seemed to be unchanged after the substitution based on CD spectra and the SH titration curve.


Immunolocalization of kinesin in sea urchin coelomocytes. Association of kinesin with intracellular organelles.

We have recently used domain-specific monoclonal antibodies (mAbs) to immunofluorescently localize kinesin to vesicle-like structures in the cytoplasm of sea urchin coelomocytes. In order to characterize further these localization patterns we have examined the distribution of kinesin with respect to the arrangement of microtubules (MTs) and various organelles. In double-label experiments involving the immunofluorescent staining of kinesin (using a mixture of the mAbs SUK2, 4 and 5), MTs were labeled with an antiserum against sea urchin tubulin, the endoplasmic reticulum (ER) was labeled with an antiserum against a luminal calsequestrin-like protein, the Golgi apparatus was labeled with rhodamine-wheat germ agglutinin (WGA) or NBD-ceramide, mitochondria were labeled with rhodamine 123, endosomes were labeled with Texas Red-ovalbumin, and lysosomes were labeled with Lucifer yellow or acridine orange. Kinesin-labeled vesicle-like structures were found in the same regions of the cells as MTs and the ER, being widely distributed in motile cells, but restricted to the perinuclear regions of stationary cells. There also appeared to be a correlation between the distribution of endosomes and kinesin staining in a subpopulation of cells. The kinesin binding structures were found occasionally to align in linear arrays, consistent with the idea that kinesin may transport ER and endosomes along linear MT tracks. No clear correlations were observed between the kinesin staining and the distribution of mitochondria, the Golgi apparatus or lysosomes, suggesting that kinesin may specifically associate with only a subclass of organelles in coelomocytes.


Characterization of a 58 kDa cis-Golgi protein in pancreatic exocrine cells.

We have studied the biochemical characteristics and localization of a 58 kDa cis-Golgi marker protein (p58) in rat pancreatic exocrine cells. The protein remained associated with membranes after extraction at alkaline pH and was largely resistant to proteases, added to intact microsomes. By electrophoresis p58 could be resolved into two bands which in two-dimensional gels separated into several charge variants around pI 5.5. This size and charge heterogeneity of p58 did not appear to be due to acylation, glycosylation or phosphorylation. In non-reduced gels p58 migrated as two kinetically related, high relative molecular mass forms, apparently corresponding to disulfide-linked homo-dimers and -hexamers. Immuno-electron microscopy localized p58 to both the fenestrated cis-Golgi cisternae and small Golgi vesicles or buds as well as large, pleiomorphic structures, scattered throughout the cells and associated with distinct smooth ER (endoplasmic reticulum) clusters. These findings correlated with cell fractionation results showing the concentration of p58 in two microsomal subfractions, banding at intermediate densities between the rough ER and trans-Golgi in sucrose gradients. Our results indicate that p58 is a major component of pre- and cis-Golgi elements and could be part of the transport machinery that operates in these membranes. Together with results obtained with other cell types, these observations suggest that the peripheral smooth ER clusters are involved in the early stages of the secretory pathway in the pancreatic acinar cells.


Plasticity of early endosomes.

We observed that the structural organization of early endosomes was significantly modified after cell surface biotinylation followed by incubation in the presence of low concentrations of avidin. Under these conditions early endosomes increased in size to form structures which extended over several micrometers and which had an intra-luminal content with a characteristic electron-dense appearance. The modified early endosomes were not formed when either avidin or biotinylation was omitted, suggesting that they resulted from the cross-linking of internalized biotinylated proteins by avidin. Accumulation of a fluid-phase tracer was increased after the avidin-biotin treatment (145% after 45 min). Both recycling and transport to the late endosomes still occurred, albeit to a somewhat lower extent than in control cells. Quantitative electron microscopy showed that the volume of the endosomal compartment was increased approximately 1.5-fold but that the surface area of the compartment decreased relative to its volume after avidin-biotin treatment. Finally, overexpression of a rab5 mutant, which is known to inhibit early endosome fusion in vitro, prevented the formation of these structures in vivo and caused early endosome fragmentation. Altogether, our data suggest that early endosomes exhibit a high plasticity in vivo. Cross-linking appears to interfere with this dynamic process but does not arrest membrane traffic to/from early endosomes.


Proteolytic processing of secretory proteins in Paramecium: immunological and biochemical characterization of the precursors of trichocyst matrix proteins.

We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing. Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precursor serum after preadsorption no longer stained the central, paracrystalline region, but still stained the peripheral as well as the structureless region of the secretory granule. In trichocyst-developing mutants tl (trichless) and ftA (football A), the precursors for all four groups of mature proteins were present but their processing was affected: severely blocked in tl (which has no recognizable crystalline trichocyst matrix), and partially blocked in ftA (which has some abnormal trichocyst matrices with crystalline centers). These observations constitute further evidence that proteolytic processing of precursors occurs in parallel with crystallization.


The rhythm of protein synthesis does not depend on oscillations of ATP level.

A rhythm of the [3H]leucine incorporation rate with a period of about one hour (circahoralian rhythm) has been found in rat hepatocytes grown in vitro as a monolayer and in liver organ culture. The periodicity of the incorporation rate remained after correction for changes in leucine pool size. A similar periodicity of the leucine incorporation rate was detected in a cell-free system prepared from rat hepatocytes. We have also found circahoralian oscillations of the ATP level and similar oscillations of the leucine tRNA aminoacylation rate in a hepatocyte monolayer. The addition of 1 mM ADP to the culture resulted in a considerable increase in the ATP level in the cells, but the rhythm of protein synthesis was retained under these conditions. The conclusion that there is a flexible association between changes in the ATP and GTP levels on the one hand, and oscillations of the protein synthesis rate on the other, is also supported by experiments with a cell-free system, in which the rhythm of protein synthesis rate was observed in the presence of excess ATP and GTP. We propose an hypothesis to explain the fractal pattern of circahoralian metabolic rhythms.


Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum.

We reported previously that Ca2+ depletion of Dictyostelium discoideum cells severely inhibits extracellular cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further experiments were performed to learn more about the nature of this phenomenon. Examination of the polysomal distribution of PD transcripts in control cells and in cells depleted of Ca2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indicating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein synthesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellular levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins.


Cytostellin: a novel, highly conserved protein that undergoes continuous redistribution during the cell cycle.

Cytostellin, a 240 kDa protein, has been purified from mammalian cells by immunoaffinity chromatography using monoclonal antibody H5. Immunofluorescence microscopy shows diffuse and punctate cytostellin immunoreactivity in interphase nuclei. Nuclease digestion and salt extraction are not required to expose the epitope. The onset of prophase is marked by the appearance of multiple intensely immunofluorescent cytostellin-containing 'bodies' within the nucleus. Nuclear disassembly is heralded by the movement of cytostellin bodies from the nucleus to multiple positions throughout the cell. Cytostellin bodies in metaphase, anaphase and telophase cells are widely dispersed, including some in cell processes far removed from the mitotic spindle apparatus. However, a distinct subset of larger, more intensely staining bodies surrounds the mitotic spindle apparatus. Cytostellin bodies remain in the cytoplasm of the daughter cells and disappear after the appearance of nascent nuclei. Cytostellin is immunologically distinct from other nuclear and cytoplasmic proteins, and it has been detected by immunoblot analysis in all species tested from yeast to humans. Based upon these findings, we postulate that cytostellin has a cell cycle-dependent function which is conserved in higher and lower eukaryotic cells.


Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium.

Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions. When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.


Purification and immunological detection of pea nuclear intermediate filaments: evidence for plant nuclear lamins.

A major structural component of the inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of 1-3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization of putative plant lamin proteins from pea nuclei. We conclude that plant cells contain this ancestral class of intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.


Regulation of alternative splicing in the IIICS region of human fibronectin pre-mRNA encoding cell binding sites CS1 and CS5.

The cell binding sites CS1 and CS5 in the IIICS region of human fibronectin (FN) mediate the adhesion of specific cell types by interacting with the integrin alpha 4 beta 1. IIICS pre-mRNA is alternatively spliced via the use of three alternative splice acceptor sites and one alternative splice donor site. These alternative splicing pathways can potentially give rise to variant FN molecules which are CS1+,CS5+; CS1+,CS5-; CS1-,CS5+ or CS1-,CS5-. Here we show that selection of the acceptor site which incorporates mRNA encoding CS1 and CS5 is more frequent in foetal tissues compared to adult liver, whereas an alternative acceptor site and the alternative donor site, which exclude CS1 and CS5, are used at a higher level in adult liver compared to foetal tissue. All possible splice junctions were accurately processed, and selected at different levels in mRNA expressed from a IIICS minigene transiently transfected into a HeLa cell line which does not express FN, suggesting that all the cellular factors required for alternative processing of IIICS are present in this system. Furthermore, pre-mRNA expressed from a mutant construct lacking IIICS-1 intron sequence, was correctly processed in HeLa cells via selection of all possible splice sites. On the basis of our results we propose that regulation of splice site selection in IIICS and thus expression of CS1 and CS5 is achieved by subtle tuning of splicing systems involving the interaction of local cis elements and cellular factors which are not necessarily restricted developmentally or tissue-specifically, and that expression of CS1 and CS5 is independently regulated.


Variant choriocarcinoma (BeWo) cells that differ in adhesion and migration on fibronectin display conserved patterns of integrin expression.

Starting from the BeWo choriocarcinoma cell line, two stable variant cell lines (epi and lc) were isolated. Epi cells displayed an epithelioid colony morphology while lc were fibroblastoid. lc cells attached and spread on fibronectin-coated surfaces at significantly lower density of fibronectin than epi or the parent cell line. lc also migrated more efficiently to fibronectin in a trans-filter assay than either epi or parent cells. Integrin expression by the cell lines was investigated by flow cytometry and immunoprecipitation from surface-labelled cells with a panel of subunit-specific antibodies. Integrins alpha 2 beta 1, alpha 5 beta 1, alpha v beta 1 and alpha 6 beta 4 were detected in each case, and levels of expression were identical in the two variant lines. Anti-functional antibodies were used to probe the role of integrins in fibronectin- and vitronectin-mediated adhesion. Complete inhibition of adhesion to fibronectin was observed with anti-beta 1 antibody, and partial inhibition with anti-alpha 5, suggesting that integrin alpha 5 beta 1 is mainly responsible for the interaction. Adhesion to vitronectin was inhibitable using anti-alpha v and anti-beta 1 antibodies, suggesting that integrin alpha v beta 1 is active in these cells as a vitronectin receptor. There was a correlation between the altered morphology of the variant cells and alterations in the distribution of integrin alpha 6 beta 4 and laminin in monolayer cultures. The results support the idea that fibronectin may mediate the migratory behaviour of extravillous trophoblast in vivo. Switch to a more migratory phenotype may be mediated by the selective activation of integrins and altered interaction with basement membrane.


Attachment of human vascular smooth muscles cells to intact microfibrillar assemblies of collagen VI and fibrillin.

Human vascular smooth muscle cells have been used to assess the implied role of connective tissue microfibrils as cellular ligands. Preparations of intact high-M(r) microfibrillar assemblies of collagen VI and of fibrillin, respectively, were isolated from foetal bovine skin and used as ligands in cell attachment and spreading assays. Intact collagen VI microfibrils were capable of mediating cell attachment and partial spreading. Cell attachment assays using ligands composed of defined collagen VI fragments generated by pepsin or bacterial collagenase digestions demonstrated that both the triple-helical and non-collagenous domains of collagen VI had cell adhesion activity, although at reduced levels relative to intact microfibrils. Fibronectin was identified as a modulator of intact collagen VI microfibril-mediated cell attachment. These observations are indicative of complex multiple interactions between collagen VI microfibrils and smooth muscle cells. Purified fibrillin-containing microfibrils were also shown to support smooth muscle cell adhesion. Both pepsin-resistant and pepsin-sensitive domains of fibrillin exhibited some cell attachment activity, but at reduced levels relative to the intact fibrillin microfibrils. These data provide the first direct evidence of a physiological role for intact microfibrillar assemblies in cell-matrix interactions, and the involvement of integrin cell surface receptors containing the beta 1 subunit.


Capillary endothelial cells secrete a heparin-binding mitogen for pericytes.

The cells of the retinal microvasculature consist predominantly of mesodermally derived pericytes and endothelial cells, and the regulatory factors which govern their co-ordinated growth and define their phenotypic characteristics in vivo may be regarded as key elements of the angiogenic process. An investigation of these cells in co-culture experiments has led to the identification of a potent mitogen for pericytes in medium conditioned by retinal endothelial cells (EC-FBS). EC-FBS activity was shown to be non-dialyzable, and stable to both heat and acid treatment. EC-FBS was inactivated by passage over a heparin-Agarose column. The column-bound activity could be eluted as a single peak at approximately 1.0 M NaCl. Stimulation of pericyte growth was also achieved with platelet-derived growth factor (PDGF), acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) and could be blocked by using the appropriate antiserum (anti-PDGF or anti-aFGF). Neither antisera, however, blocked the activity of EC-FBS. The EC-FBS mitogen markedly altered the phenotypic behavior of pericytes compared with PDGF and the FGFs; yet, unlike them, it failed to stimulate the growth of smooth muscle cells (SMC) and Balb/c 3T3 cells.


Stabilization of intercellular contacts in MDCK cells during Ca2+ deprivation. Selective effects of monocarboxylic acids on desmosomes.

Short-chain monocarboxylic acids (MCAs) selectively protect desmosomal junctions of MDCK cells from disruption by chelating agents and low calcium medium. This effect occurs in the millimolar concentration range and increases inversely with carbon chain length (formate > acetate = propionate > butyrate > isobutyrate > isovalerate). The relative activity of MCAs does not correlate with their overall hydrophobicity or ability to chelate ions, or their effectiveness in lowering cytosolic pH. It exhibits chemical specificity and is dependent upon postconfluency culture age. MCAs also inhibit cell rounding produced by low concentrations of aminocarboxylate-chelating agents. Their effect on cell rounding, but not on desmosomes, can be antagonized by okadaic acid. The possibility is discussed that MCAs may produce their effects by binding specifically to protein(s) associated with the desmosome of mature, fully polarized MDCK monolayers.


Hemidesmosomes in the epithelial cell line 804G: their fate during wound closure, mitosis and drug induced reorganization of the cytoskeleton.

Recently, we identified a novel epithelial cell line, 804G, derived from rat bladder, which readily forms hemidesmosomes in vitro. One of the major structural components of the plaques of 804G cell hemidesmosomes is a 230 kDa antigen recognized by autoantibodies in the sera of patients with bullous pemphigoid (BP). An additional polypeptide of 180 kDa also localizes to the hemidesmosome plaque of 804G cells as determined by immunoelectron microscopy. Using confocal fluorescence/phase microscopy, we have employed both 230 kDa and 180 kDa antibody probes to monitor the fate of hemidesmosomes following closure of in vitro wounds, during mitosis, and following drug induced disruption of the cytoskeleton. The punctate cell-substratum associated staining generated by the hemidesmosomal antibodies in stationary unwounded 804G cell cultures is greatly diminished or even lost in cells which enter wound sites, presumably in response to enhanced cell motility. Few, if any hemidesmosomes are observed at the ultrastructural level in cells which have migrated into the wound area. However, as closure of the wound becomes complete, staining along the substratum attached surface of cells returns. During mitosis, there is no obvious loss of hemidesmosomal antigens along the basal surface of 804G cells, and formed hemidesmosomes can be observed in mitotic cells at the ultrastructural level. In 804G cells treated with colchicine, the typical subnuclear pattern of distribution of hemidesmosomal antigens is unaffected. In contrast, following treatment of 804G cells with cytochalasin D, hemidesmosomal antigens become concentrated at the cell periphery and no longer appear in the subnuclear region. Furthermore, formed hemidesmosomes are observed at the cell periphery of cytochalasin D-treated cells by electron microscopy. We suggest that hemidesmosomal plaques are mobile within the plasma membrane. We speculate that hemidesmosomal interactions with extracellular ligands are dynamic and we discuss a possible mechanism by which cytochalasin D induces reorganization of hemidesmosomes along the basal surface of 804G cells.


Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances.

A simplified model of TIRF optics was used to quantitate the relative membrane/substratum separation distances from the spatial pattern of TIRF image brightness. Phase-contrast and total internal reflection fluorescence microscopy (TIRFM) images were collected of bovine aortic endothelial cells (BAEC) plated onto glass microscope slides for 15 min, 30 min and 24 h. BAEC adherent for 15 min showed an absence of a focal contact morphology, with the region of closest apposition beneath the cell center. After 30 min, multiple contacts with the surface were established and the morphology became more irregular. BAEC attached for 24 h showed well-defined focal contact regions aligned in characteristically striated patterns. The relative distance between closest and farthest membrane/substratum separations are consistent with reported distance between focal and matrix contacts. Topographical maps of membrane/substratum separation distances over the entire ventral surface of the plated cells were constructed to demonstrate the utility of quantitative TIRF microscopy.


Comparative analysis of the effects of dimethyl sulfoxide and retinoic acid on the antigenic pattern of human ovarian adenocarcinoma cells.

HOC-7 malignant ovarian surface epithelial cells have been exposed to differentiation promoters like dimethyl sulfoxide (DMSO) and retinoic acid (RA) and the resulting cell phenotypes were characterized immunologically. Immunocytochemistry revealed that DMSO caused elevation of membrane-associated staining for epidermal growth factor-receptor (EGF-R) and for desmoplakins I and II (DPI+II). DMSO also stimulated cytoplasmic and surface labelling for CA 125 and extracellular deposition of fibronectin (FN). A fixed-cell ELISA system was used for quantification of these differentiation-like responses and revealed that DMSO efficiently induced expression of EGF-R, CA 125, FN and DPI+II in dose-dependent manner. Immunocytochemistry, ELISA and Western blotting additionally demonstrated that both DMSO and RA caused down-regulation of myc oncoproteins. Densitometer evaluation of electrophoresed proteins revealed a 50% DMSO- and a 25% RA-induced myc reduction. Apart from growth reduction, which was seen for both inducers, inhibition of myc gene expression was the only response of HOC-7 cells to RA-treatment. The extent of myc down-regulation seems, therefore, to be crucial for the initiation of maturational processes in the cells. Subsequent phenotypic differentiation of HOC-7 cells causes elevated levels of EGF-R, CA 125, FN and DPI+II. This cell model might be useful for the distinction between induced growth reduction and differentiation of ovarian cancer cells.


Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro.

Alpha-smooth muscle actin is considered a reliable marker for distinguishing between arterial smooth muscle and endothelial cells. Several authors have reported heterogeneity in the expression of this actin isoform in atherosclerotic lesions. Such heterogeneity appears to result from the presence of different smooth muscle cell phenotypes (contractile and synthetic) in these lesions. In the present study, we show that bovine aortic endothelial cells, which are characterised by the presence of Factor VIII-related antigen (FVIII) and by the absence of alpha-smooth muscle actin (alpha-SM actin) may be induced to express the latter when exposed to TGF-beta 1. FVIII was detected by immunofluorescence, alpha-SM actin was detected by immunofluorescence and immunoblotting. The number of cells expressing alpha-SM actin increased with time of incubation with TGF-beta 1, and this increase occurred concomitantly with a decrease in the expression of FVIII. Double immunofluorescence demonstrated the presence of cells that expressed both FVIII and alpha-SM actin after 5 days of incubation with TGF-beta 1. With longer incubation times (10-20 days) the loss of FVIII expression was complete and over 90% of the cells expressed alpha-SM actin. Ultrastructurally, cells in control cultures showed the typical features of endothelial cells. In the TGF-beta 1-treated cultures, cells which appeared indistinguishable from contractile and synthetic smooth muscle cells were observed. Withdrawal of TGF-beta 1 after 10 days incubation resulted in the re-appearance of polygonal cells which were FVIII-positive and alpha-SM actin-negative.(ABSTRACT TRUNCATED AT 250 WORDS)


Porcine smooth muscle cell-conditioned medium stimulates LDL receptor activity in Hep G2 cells.

Paracrine factors may modulate low density lipoprotein (LDL) receptor activity in hepatocytes. To study this the effect of conditioned medium prepared from a range of cell types on the binding and internalisation of 125I-LDL in Hep G2 cells was studied. Seven of the fourteen conditioned media tested, including those from P388D1, U937, porcine smooth muscle (Pc SMC) Swiss 3T3, STO, = 48 and MDCK cells, were found to increase the binding and internalisation of 125I-LDL at 37 degrees C by Hep G2 cells (P < 0.01). The largest increase in LDL receptor activity was produced by conditioned medium from Pc SMC cells and was, therefore, selected for further analysis. The Pc SMC-conditioned medium increased LDL receptor number in Hep G2 cells by three-fold but had no effect on LDL receptor activity in human skin fibroblasts. DNA synthesis and cholesterol synthesis by Hep G2 cells were inhibited by Pc SMC-conditioned medium. Preliminary characterisation of the Pc SMC-derived factor(s) suggests that it is a protein(s) of low relative molecular mass.


Induction of dystrophin localization in cultured Xenopus muscle cells by latex beads.

The distribution of dystrophin in Xenopus myotomal muscle cells was examined in conventional and confocal immunofluorescence microscopy. By labeling dissociated single muscle fibers with a monoclonal or a polyclonal antibody against dystrophin, we found that dystrophin is ten times more concentrated at the myotendinous junction (MTJ) than at the extrajunctional sarcolemma. At the MTJ, dystrophin lines the membrane invaginations where myofibrils attach to the membrane. It is colocalized with talin, but is not related to the distribution of acetylcholine receptors (AChRs) which are clustered at the postsynaptic membrane in the vicinity of the MTJ in these fibers. We found that the localization of dystrophin can be induced in cultured Xenopus myotomal muscle cells by treating them with polystyrene latex beads. Dystrophin is discretely localized at the bead-muscle contacts. With electron microscopy, a sarcolemma specialization with all the salient features of the MTJ, including basal lamina-lined membrane invaginations along which myofibrils make attachment. Although these beads also induce clustering of AChRs, the patterns of dystrophin and AChR localization are distinct. The appearance of dystrophin at the bead-contacted sarcolemma is coincident with the development of the membrane invaginations. This, together with its concentration along membrane invaginations at the MTJ in vivo, suggests a role for dystrophin in the formation of this junctional specialization. Since the signal for MTJ development can be presented to cultured muscle cells in a temporally and spatially controlled manner by beads, this system offers a simple model for analyzing the mechanism of this sarcolemma specialization.


Chemotactic properties, cellular binding and uptake of peptides and peptide derivatives: studies with Tetrahymena thermophila.

Receptor-mediated binding of leukocyte chemotactic peptide, N-formylMet-Leu-Phe (fMLP), occurs in the ciliated protozoon Tetrahymena thermophila. In vivo labelling of the cells with N-formylMet-Leu-[3H]Phe ([3H]fMLP) shows that the cells bind the ligand with high affinity (KD = 4 x 10(-9) M to 1 x 10(-8) M). Moreover, Scatchard transformations of the binding data show that there are about 5 x 10(5) binding sites per cell on the cell surface. Two fluorescent derivatives of leukocyte chemotactic peptide, N-dansylMet-Leu-Phe (dansMLP) and N-formylMet-Leu-Phe-(N-dansyl-)Lys (fMLPdanLys) compete for the N-formylMet-Leu-Phe (fMLP) binding sites on the cell surface. Moreover, both derivatives have retained significant chemoattracting potentials. Fluorescence from dansMLP, but not from fMLPdansLys and dansyl-beta-endorphin, is internalized preferentially into small vesicles. The differences may, however, reflect that the fluorescence from the dansyl group is strongly quenched by a hydrophilic microenvironment when using the two latter peptide derivatives. In contrast, the dansyl group from dansMLP must be assumed to be embedded in a hydrophobic microenvironment in the vesicular membrane or membrane protein. Rhodamine-labelled bovine serum albumin, egg albumin and cytochrome c as well as dansylated bovine serum albumin, which are poor chemoattractants, are preferentially seen to be internalized into large vesicles (food vacuoles).


Axonal growth on astrocytes is not inhibited by oligodendrocytes.

Axon growth in vitro may be inhibited by contact with oligodendrocytes, but most axons grow readily on the surface of astrocyte monolayers. Since both cell types are in close contact with one another in the damaged nervous system, we have examined the growth of axons on cultures which contain both astrocytes and oligodendrocytes. Cultures derived from neonatal rat forebrain develop with a monolayer of large flat astrocytes attached to the culture dish, and with many smaller cells of the oligodendrocyte lineage on their surface. Dorsal root ganglia placed on these cultures grow axons readily, the overall extent of growth being unaffected by the presence or absence of oligodendrocytes, many of which express galactocerebroside and the inhibitory molecule janusin. A previous set of experiments had shown that growth of these axons is inhibited by oligodendrocytes by themselves. Scanning electron microscopy coupled with silver-intensified immunostaining reveals that the axons grow on the surface of the astrocytic layer, underneath the oligodendrocytes, and are therefore in contact with both cell types as they grow. The presence of astrocytes therefore alters the results of axonal contact with oligodendrocytes.


Statistical evidence for a random commitment of pluripotent cephalic neural crest cells.

The neural crest (NC) of vertebrate embryos yields cell types belonging to the neural, melanocytic and mesectodermal lineages. To test the possibility that the precursors of these lineages segregate from pluripotent cells by a process involving stochastic determinants, we have analyzed with statistical methods the associations between six differentiated cell types in 201 clones obtained in vitro from migrating cephalic NC cells. Our analysis suggests that neuronal, adrenergic and Schwann cells are not randomly associated, whereas these neural cell types differentiate in the clones independently of both melanocytes and cartilage. These results raise the possibility that pluripotent NC progenitors give rise to the precursors of the major NC-derived lineages (neural, melanocytic and mesectodermal) by a process involving stochastic restrictions of their developmental potentialities.


Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments.

Multipotential stem cells in the neural tube give rise to the different neuronal cell types found in the brain. Abrupt changes in intermediate filament gene expression accompany this transition out of the precursor state: transcription of the intermediate filament nestin is replaced by that of the neurofilaments. In order to identify human neural precursor cells, and to learn more about the evolution of the intermediate filaments expressed in the central nervous system, we have isolated the human nestin gene. Despite considerable divergence between the human and rat nestin genes, in particular in the repetitive parts of the carboxy-terminal region, the positions of the introns are perfectly conserved. Two of the three intron positions are also shared by the neurofilaments, but not by other classes of intermediate filaments. This implies that nestin and the neurofilaments had a common ancestor after branching off from the other classes of intermediate filaments, and that nestin separated from the neurofilament branch before the different neurofilament genes diverged. The characterization of human nestin also facilitates the identification of human multipotential neural precursor cells. This will be of importance for central nervous system (CNS) tumor diagnosis and transplant-based clinical approaches to human neurodegenerative diseases.


Developmental stage- and tissue-specific expression of shibire, a Drosophila gene involved in endocytosis.

Dynamin, a microtubule-activated GTPase, has recently been identified as the product of the shibire gene in Drosophila. shi(ts) mutants are defective in synaptic vesicle recycling, which leads to rapid and reversible temperature-sensitive paralysis. In the present study, results from RNase protection assays and analysis of cDNA clones define a complex pattern of developmental- and tissue-specific alternative splicing at two sites within the coding region. This analysis is also supported by western blot analysis with two polyclonal antibodies. In situ hybridization data revealed a high concentration of shi transcripts in the central and peripheral nervous system throughout neuronal development. Other than the nervous system, shi transcripts are also expressed at a high level in early embryos, larval imaginal discs, and male and female gonads. These data provide a basis for interpreting the wide range of phenotypic effects of shi mutations in terms of the putative membrane-sorting properties of dynamin and for further functional study of different dynamin isoforms.


Localization of actin in the Tetrahymena basal body-cage complex.

In the ciliate cytoskeleton, basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. We have used two polyclonal anti-actin antibodies to localize actin within the basal body-cage complex of Tetrahymena. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked chicken muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the anti-oral apparatus antiserum. Agarose-linked chicken muscle actin was used to affinity-purify anti-chicken muscle actin antibodies from a commercially available antiserum against chicken muscle actin. Both affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. The anti-actin antibodies were localized to both somatic and oral basal bodies in Tetrahymena by immunofluorescence microscopy. At the ultrastructural level with the immunogold technique, these antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments and (d) cage wall filaments. In addition, the antibody labeled filament bundles that interconnect groups of basal bodies (membranelles) within the oral apparatus. Identical labeling patterns were observed with basal bodies in the isolated oral apparatus, basal bodies in the in situ oral apparatus and somatic basal bodies in situ. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. These results provide evidence for actin in the basal body-cage complex and raise the possibility of a contractile system associated with basal bodies.


Differential regulation of beta III and other tubulin genes during peripheral and central neuron development.

Mammalian peripheral and central neurons differ considerably in the composition and properties of their axonal cytoskeletons. Recent reports of the selective expression of a high molecular weight (HMW) tau protein in neurons with peripherally projecting axons have furthered the idea that the microtubules in central and peripheral neurons are disparate. In the present study, we examined the possibility that the various tubulin genes are differentially expressed in central versus peripheral neurons. To examine this, we compared the expression of four of the beta-tubulin mRNAs (classes beta I, beta II, beta III, beta IV) and the alpha 1-tubulin mRNA in rat dorsal root ganglion (DRG) neurons with their expression in cerebral cortex during postnatal development (P5-90), using northern blots and in situ hybridization. We document both similarities and differences in tubulin gene expression in these two regions of the neuraxis during postnatal development. In both DRG and cortex, the expression of the class beta I- and beta II-tubulin mRNAs and the alpha 1-tubulin mRNA was higher at earlier stages of postnatal development than in the adult. However, class beta IV-tubulin mRNA levels increased during cortical development but decreased during DRG postnatal development. The opposite pattern was found for the neuron-specific class beta III-tubulin gene, the mRNA levels of which were high in cortex, at birth and then decreased with increasing postnatal development. In DRG, the beta III-tubulin mRNA levels generally increased during postnatal development. Beta III-tubulin protein levels were examined qualitatively at different developmental stages (5-90 days) by immunoblotting and immunocytochemistry.(ABSTRACT TRUNCATED AT 250 WORDS)


A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton.

The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210,000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other proteins.


Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells.

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15-20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.


Continuous growth of vimentin filaments in mouse fibroblasts.

We have investigated the dynamics of intermediate filament assembly in vivo by following the fate of heterologous chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. Using RNase protection, metabolic protein pulse-chase and immunofluorescence microscopy, we have examined the fate of newly assembled subunits under physiological conditions in situ. Following induction and subsequent removal of inducer, chicken vimentin mRNA had a half-life of approximately 6 h while both chicken and mouse vimentin protein polymer had long half-lives--roughly equivalent to the cell generation time. Moreover, following deinduction, chicken vimentin immunolocalization progressed from a continuous (8-10 h chase) to a discontinuous (> or = 20 h chase) pattern. The continuous chicken vimentin staining reflects the uniform incorporation of chicken vimentin throughout the endogenous mouse vimentin network while the discontinuous or punctate chicken vimentin staining represents short interspersed segments of assembled chicken vimentin superimposed on the endogenous polymer. This punctate staining pattern of chicken vimentin was present throughout the entire array of intermediate filaments, with no bias toward the perinuclear region. These results are consistent with a continuous growth model of intermediate filament assembly, wherein subunit addition occurs at discrete sites located throughout the cytoskeleton.


Amphibian intestinal villin: isolation and expression during embryonic and larval development.

An actin-binding protein of M(r) 105,000 has been isolated from anuran amphibian intestinal mucosa. Polyclonal antibodies directed against chicken and pig intestinal villins and anti-porcine villin headpiece monoclonal antibody crossreact with the amphibian M(r) 105,000 protein. Furthermore, the latter possesses an NH2-terminal sequence that is very homologous to those of avian and mammalian villins. In addition, polyclonal antibodies directed against amphibian intestinal M(r) 105,000 protein crossreact with chicken and mouse intestinal epithelial cell villins. These data indicate that the amphibian intestinal M(r) 105,000 protein is immunologically and structurally related to villin, an actin-binding protein expressed in specific epithelial tissues in vertebrates. Morphological, immunocytochemical and immunoblotting techniques were then used to investigate the expression of villin during embryonic and larval intestinal development of Xenopus laevis. Villin is not found in the egg or the endoderm of the early embryo. It is first detected just before hatching in the apical domain of endodermal cells at a time when few surface microvilli are visible by transmission electron microscopy. In the newly hatched larva, villin accumulates as these cells differentiate. These results provide a detailed developmental profile of Xenopus intestinal villin expression and demonstrate that this protein is a useful marker for the presumptive intestinal endoderm.


Membrane-binding properties of filensin, a cytoskeletal protein of the lens fiber cells.

Filensin is a 100/110 kDa membrane-associated protein found in lens fiber cells. Previous studies have shown that this protein polymerizes in vitro and binds strongly to vimentin and to another 47 kDa lens membrane protein. Using cosedimentation assays, flotation assays and immunoelectron microscopy, we have examined the properties of purified filensin and measured its binding to lens membranes. Filensin behaves as a ureaextractable, hydrophilic protein which does not partition with Triton X-114 and is not affected by 1 M hydroxylamine at alkaline pH, an agent known to release fatty-acylated proteins from the membrane. Immunoblotting of urea-extracted lens membranes with two different affinity-purified antibodies reveals that, unlike intact filensin, a COOH-terminal filensin degradation product (51 kDa) remains tightly associated with the membranes. Purified filensin binds directly to urea-stripped lens membranes, but not to protein-free vesicles reconstituted from total lens lipids. The binding of filensin is not significantly influenced by the purified 47 kDa protein. Interestingly, the filensin-binding capacity of urea-extracted membranes is increased at least two-fold after trypsin treatment, which removes entirely the 51 kDa peptide from the membranes and presumably unmasks additional filensin-acceptor sites. Consistent with this, filensin binds to trypsinized and non-trypsinized membranes with similar affinities (2 x 10(-7) and 4 x 10(-7) M, respectively). Treatment of the membranes with thrombin, which also eliminates the 51 kDa peptide, does not increase their binding capacity, apparently because filensin-acceptor sites are also destroyed during proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)


Further characterisation of the talin-binding site in the cytoskeletal protein vinculin.

The cytoskeletal protein vinculin is a component of adherens-type junctions where it is one of a number of interacting proteins thought to link the cytoplasmic domain of adhesion receptors to F-actin. Vinculin has been shown to bind to at least three other cytoskeletal proteins, talin, paxillin and alpha-actinin. In this study, we further characterise the talin-binding domain in vinculin using a series of chick vinculin polypeptides expressed as glutathione-S-transferase fusion proteins in Escherichia coli. Thus 125I-talin bound to a fusion protein spanning residues 1-398, but not to those spanning residues 399-881 or 881-1066 in an SDS-PAGE gel-blot assay. We have previously characterised two chick vinculin cDNAs (2.89 kb cDNA and cVin5) which are identical in the region of overlap except that cVin5 lacks coding sequence for residues 167-207. Interestingly, a fusion protein spanning residues 1-398, but lacking residues 167-207, was unable to bind talin. However, further analysis showed that residues 167-207 are insufficient to support binding, and deletion of as few as 31 N-terminal residues abolished binding activity. The results of the gel-blot assay were essentially confirmed using purified fusion proteins adsorbed to glutathione-agarose beads. The smallest vinculin fusion protein able to bind talin contained residues 1-258. This fusion protein was as effective as whole vinculin in inhibiting the binding of 125I-vinculin to talin-coated microtitre wells. Interestingly, mutations which altered the charge characteristics of the highly conserved residues 178 and 181 abolished binding, whereas conservative substitutions were without effect. However, such mutations did not abolish the ability of mutant polypeptides spanning residues 1-398 to target to cell-matrix junctions in Cos cells. We have investigated the possible origin of the cDNA clone cVin5 by defining the structure of a 5' portion of the chicken vinculin gene, and by analysing vinculin transcripts in a variety of adult tissues and embryonic fibroblasts using reverse transcriptase and polymerase chain reaction. Although residues 167-207 are encoded on a separate exon, we have been unable to identify a tissue where this exon is alternatively spliced.


The tight association of the tyrosine kinase substrate annexin II with the submembranous cytoskeleton depends on intact p11- and Ca(2+)-binding sites.

Annexin II, a member of the annexin family of Ca(2+)- and lipid-binding proteins, is a major substrate of the pp60src kinase. It is unique within the annexin protein family, since it can form a tight heterotetrameric complex with the cellular protein ligand p11, a member of the S100 protein family. Within the cell, the annexin II2p11(2) complex is localized at the cytoplasmic surface of the plasma membrane in the submembranous cytoskeleton. This intracellular localization is thought to be the consequence of a typical annexin II property observed in vitro, its Ca(2+)-dependent binding to phospholipids and cytoskeletal elements (F-actin, non-erythroid spectrin). We employed site-directed mutagenesis to create mutant annexin II molecules with defects either in the p11-binding site or in the Ca(2+)-binding sites present in repeats 2, 3 and 4. The mutated annexin II derivatives were expressed in HeLa and RMCD cells by transfection of the appropriate DNA constructs in order to analyze the importance of p11- and Ca(2+)-binding for the intracellular localization of annexin II. Immunofluorescence microscopy with a monoclonal antibody that specifically detected the transfected annexin II derivatives indicated that the Ca(2+)-dependent incorporation of annexin II into the submembranous network depended on its ability to form the annexin II/p11 complex and on the presence of intact Ca(2+)-binding sites. Neither monomeric annexin II lacking an intact p11-binding site, nor the annexin II mutant with defects in the Ca(2+)-binding sites in repeats 2, 3 and 4 were associated with the Triton X-100-resistant network of the submembranous cytoskeleton.


Integrin expression and differentiation in transformed human epidermal cells is regulated by fibroblasts.

Normal human foreskin keratinocytes (HFKs) and transformed HFKs (FEPE1L-8 cells) generated by the introduction of cloned human papillomavirus type 16 sequences were compared for the expression and function of a family of adhesion receptors termed integrins. Initially, cells were examined in conventional monolayer cultures. FEPE1L-8s expressed integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 4 and beta 1 at comparable levels to HFKs. Further, these receptors were fully functional in mediating specific interactions with exogenously supplied ligands. However, FEPE1L-8s exhibited decreased synthesis of a number of extracellular matrix components, including laminin, fibronectin and epiligrin, compared to normal HFKs, which may be an alternate mechanism for regulating adhesion. Subsequently, organotypic cultures (OCs), which provide a suitable in vitro model system for the ordered stratification and differentiation of keratinocytes, were used to study the regulation of integrins and various epidermal markers in normal and transformed cells. OCs consisted of keratinocytes plated on a collagen gel containing primary human fibroblasts, grown at an air-medium interface. Unlike normal HFKs, the transformed FEPE1L-8 cells exhibited (a) disorganized stratification and limited differentiation capacity, (b) invasion into the collagen gel, and (c) unregulated expression of alpha 3 beta 1 and alpha 2 beta 1, and under-expression of alpha 6 beta 4 integrins. Ordered stratification and spatial regulation of integrin expression could be induced in the FEPE1L-8s by substituting Swiss 3T3 fibroblasts in the collagen gel. Further data indicate that the human fibroblasts induce the transformed HFKs to invade into the collagen gel.(ABSTRACT TRUNCATED AT 250 WORDS)


The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopic approach.

A key role in the regulation of membrane traffic is played by the rab proteins, members of a family of ras-related small GTP-binding proteins. This family comprises at least 25 identified members, the intracellular localization of only a few of which has been investigated. rab6p has been shown to be distributed along the exocytic pathway in association with the medial and trans regions of the Golgi apparatus. A confocal laser scanning microscopic (CLSM) approach coupled with image analysis was used to compare the localization of rab6p with selected reference Golgi markers by double immunofluorescence on culture cell lines. CLSM analysis shows that, under a set of well-defined conditions, one can investigate the possible colocalization of known markers of Golgi compartments and orientate a couple of labeled Golgi antigens with regard to the polarity of the Golgi apparatus. Thus, having validated the CLSM analysis, the localization of rab6p was studied and compared with some of these markers and the VSV-G protein in VSV (vesicular stomatitis virus)-infected cells blocked at 20 degrees C. rab6p is shown to be associated in all the cell lines used with the last cisternae of the Golgi apparatus and particularly with the trans-Golgi network (TGN), the site of protein sorting at the exit of the Golgi apparatus. These results were supported by an electron microscopic study using double-immunolabeled cryosections: rab6p was found in some flat cisternae of the Golgi stack and colocalized with the VSV-G protein in the TGN. Our results show that the small GTP-binding protein rab6p is distributed from medial Golgi to TGN along the exocytic pathway.


A novel vesicle-associated protein (VAP-1) in sea urchin eggs containing multiple RNA-binding consensus sequences.

We have identified a novel high molecular weight, vesicle-associated protein (VAP-1) in the eggs of the sea urchin Strongylocentrotus purpuratus. Biochemical fractionation and immunofluorescence analysis of unfertilized eggs indicate that VAP-1 is a peripheral membrane protein associated with microsomal membrane fractions. Sequence analysis of partial VAP-1 cDNA clones reveals that the protein contains at least four RNA-binding consensus sequences. The RNA-binding sequences are separated by several glycine rich domains and this organization, RNA-binding domains separated by glycine rich sequences, is common to several RNA-binding proteins including the heterogeneous ribonuclear protein A1 and nucleolin. The characteristics of VAP-1 suggest that the protein may function as a multidomain RNA-binding protein. The possibility that VAP-1 may play a role in nuclear RNA processing is also discussed.


A block in degradation of MHC class II-associated invariant chain correlates with a reduction in transport from endosome carrier vesicles to the prelysosome compartment.

Invariant chain (Ii) associated with MHC class II molecule is processed proteolytically via several distinct intermediates during its intracellular transport through endosomal compartments. Leupeptin added to the culture medium blocks processing of Ii, prevents its dissociation from the class II molecules and leads to an intracellular accumulation of a 22 kDa intermediate form of Ii. We show here that leupeptin has a very general effect on protein transport in the endocytic pathway. When added to Mel Juso cells leupeptin reduces the transport of endocytosed material from multivesicular body-like, endosome carrier vesicles (ECV) to the prelysosomal compartment (late endosome) and leads to a concomitant increase in the number of ECV. Our results argue that one effect of leupeptin, related to antigen processing and presentation, is to block transport of antigen and/or MHC class II molecules to prelysosomal compartments.


Fluorescent phospholipids preferentially accumulate in sub-tegumental cells of schistosomula of Schistosoma mansoni.

Schistosomes do not make sterols or fatty acids de novo and thus require host lipids for survival. The acquisition of host lipids may also be an important factor in the schistosome's defense from host immunity; however, little is known about the regulation of this process. Here we have examined binding of radiolabeled and fluorescently labeled liposomes to schistosomula, and followed incorporation of fluorescent phospholipids into the worm by both morphological and biochemical methods. Saturable binding of radiolabeled phosphatidylcholine containing liposomes was observed and epifluorescence microscopy showed binding of C6-NBD-phosphatidylcholine (C6-NBD-PC), C12-NBD-phosphatidylcholine (C12-NBD-PC) and C6-NBD-phosphatidyl-ethanolamine (C6-NBD-PE) containing liposomes on the surface of the parasite. Following back-exchange with unlabeled liposomes, NBD-PC and NBD-PE were observed to be preferentially incorporated into specific cell types within the worm. Furthermore, cells which had accumulated the fluorescent lipid formed an interconnecting cellular network immediately below the tegument, identified as cytons. By contrast, fluorescein-PE was found only on the surface of the parasite and in the gut but not in the cytons. Biochemical analysis demonstrated that > 90% of the C6-NBD-PC and C12-NBD-PC remained as the intact molecule after a one hour incubation with the parasite, but that greater than 70% of the NBD-PE was converted to other lipids. These studies demonstrate that incorporation of phospholipid analogs into schistosomula can be followed morphologically and biochemically. As there was little localization of NBD-PE or NBD-PC in the gut, these analogs must be assimilated by crossing the tegument.(ABSTRACT TRUNCATED AT 250 WORDS)


Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy.

The temporal and spatial progression of DNA replication in interphase nuclei of eukaryotic cells has been investigated. Application of a recently developed technique for the immunofluorescence double staining of cell nuclei labelled first with iododeoxyuridine (IdUrd) and subsequently with chlorodeoxyuridine (CldUrd) allows the visualization of two replication patterns in the same nucleus originating from two different periods of the S-phase. We have analysed changes in the three-dimensional replication patterns during the S-phase. To record dual colour three-dimensional images of doubly stained nuclei, a confocal microscope is used. This CSLM is equipped with a specific laser/filter combination to collect both fluorescence signals (FITC and Texas Red) in a single scan, thus precluding pixel shift between the images. A method for the quantitative evaluation of the degree of overlap between DNA regions replicated in two different periods of the S-phase is applied. The results confirm the generally accepted theory that DNA is replicated coordinately in a specific temporal order during the S-phase. The replication time of a DNA domain (i.e. the time between initiation and termination of DNA replication within a domain) at the very beginning of the S-phase was known to be one hour (Nakamura et al., 1986). Our observations show that in the rest of the S-phase, the replication time of a DNA region is also about one hour. We conclude that replicon clusters located in the same region are replicated in the same relatively short period of time. After this period there is no unreplicated DNA left in this region.


Expression of wild-type and nuclear localization-deficient human lamin A in chick myogenic cells.

Previous analysis of chick embryonic muscle (CEM) differentiation in vivo and in ovo demonstrated that lamin A accumulation to steady-state levels preceded the accumulation of muscle-specific proteins. These observations have suggested the appearance of A-type lamins may be important for differentiation. To test this hypothesis, we have temporally and quantitatively altered the expression of A-type lamins in CEM cells by transient transfection of wild-type (wt; pHLA) or nuclear localization-deficient (NLd; pHLA-del) human lamin A expression plasmids. Transfected CEM cells synthesized the wt and NLd human lamin As to high levels, both of which were resistant to high-salt extraction. The wt human lamin A localized to the nucleus, whereas the NLd protein showed cytoplasmic staining patterns, as well as time-dependent nuclear localization. The presence of endogenous chicken lamins A and B2 in NLd human lamin A cytoplasmic structures suggested the interspecies lamin copolymerization. Thus, this approach may provide a possible method for analysis of lamin-lamin or lamin-lamina component interactions in vivo. With regard to muscle differentiation, CEM cells transfected with either pHLA or pHLA-del demonstrated moderate and transient increased levels of the muscle-specific myosin heavy chain and creatine kinase activity. These increases appeared temporally and quantitatively to reflect the transient accumulation of the human lamin As. In contrast, beta-tubulin and skeletal tropomyosin protein accumulations appeared unaffected. On the basis of these results, we suggest that nuclear lamina content and structure may play a limited, permissive and indirect role in the temporally regulated expression of the myogenic phenotype.


Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity.

A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [32P]phosphorylated peptide, unreacted [gamma-32P]ATP, and 32P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.


Determination of the nicotine metabolite trans-3'-hydroxycotinine in urine of smokers using gas chromatography with nitrogen-selective detection or selected ion monitoring.

A gas chromatographic method for the determination of the nicotine metabolite trans-3'-hydroxycotinine is described. The method involves conversion of the metabolite to the tert.-butyldimethylsilyl derivative, chromatography on a fused-silica capillary column, and determination using nitrogen-phosphorus detection or electron ionization mass spectrometry with selected ion monitoring. A structural analogue, trans-3-hydroxy-1-methyl-5-(2-pyridyl)pyrrolidin-2-one (trans-3'-hydroxy-ortho-cotinine), was used as an internal standard. Using selected ion monitoring, good precision and accuracy were obtained for determination of trans-3'-hydroxycotinine in urine over the concentration range 10-10,000 ng/ml. There was a good correlation between concentrations determined by selected ion monitoring and by nitrogen-phosphorus detection in urine of smokers, although low concentrations determined using nitrogen-phosphorus detection tended to be somewhat higher, suggesting some interference from urinary constituents. Concentrations and 24-h excretion of trans-3'-hydroxycotinine in the urine of 22 cigarette smokers are reported and compared to concentrations and excretion of nicotine, cotinine, nicotine 1'-N-oxide, nornicotine, and cotinine N-oxide.


Specific and sensitive quantitation of 2,2'-dichlorodiethyl sulphide (sulphur mustard) in water, plasma and blood: application to toxicokinetic study in the rat after intravenous intoxication.

A sensitive and specific capillary gas chromatographic method has been developed to measure trace amounts of 2,2'-dichlorodiethyl sulphide (sulphur mustard) in environmental or biological samples. Sulphur mustard was isolated from water or plasma by a solid-phase extraction procedure and from blood by liquid-liquid extraction. The accuracy and precision of the methods were demonstrated using replicate analyses of spiked water, plasma or blood: within-run and between-run variabilities were less than 20%. These analytical methods were used to evaluate the rate of sulphur mustard degradation in water or plasma. Good linear calibration curves, with a detection limit of 45 ng/ml, were obtained for quantitation and determination of sulphur mustard in blood following its intravenous administration to rats. Initial toxicokinetic data were obtained.


Identification of a benzhydrolic metabolite of ketoprofen in horses by gas chromatography-mass spectrometry and high-performance liquid chromatography.

A benzhydrolic metabolite of ketoprofen, formed by reduction of the keto group of the drug, has been identified by gas chromatography-mass spectrometry in equine plasma and urine. After partial synthesis, its structure has been confirmed by UV, IR and 1H NMR spectroscopy. The kinetics of ketoprofen and this metabolite have been monitored in plasma by high-performance liquid chromatography. The two products were quantified in plasma up to 4 and 3 h, respectively, and were detected in urine up to 72 and 24 h, respectively, after a single intravenous administration to horses at the dose of 2.2 mg/kg. Simultaneous detection of both compounds increases the reliability of antidoping control analysis.


Gas chromatographic determination of 2- and 3-dechloroethylifosfamide in plasma and urine.

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 micrograms/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.


Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection.

An improved method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid (alendronate) in human urine and an assay in human plasma are described. The methods are based on co-precipitation of the bisphosphonate with calcium phosphates, automated pre-column derivatization of the primary amino group of the bisphosphonic acid with 2,3-naphthalene dicarboxyaldehyde (NDA)-N-acetyl-D-penicillamine (NAP) or cyanide (CN-) reagents, and high-performance liquid chromatography (HPLC) with electrochemical (ED) or fluorescence detection (FD). The feasibility of ED of the NDA-CN- derivative of aldendronate has been demonstrated, and a HPLC-ED assay in human urine has been validated in the concentration range 2.5-50.0 ng/ml. In order to eliminate the cyanide ion from the assay procedure, several other nucleophiles in the NDA derivatization reaction were evaluated. An NDA-NAP reagent was found to produce highly fluorescent derivatives of alendronate. The assay in urine based on NDA-NAP derivatization and HPLC-FD has been developed and fully validated in the concentration range 1-25 ng/ml. Based on the same NDA-NAP derivatization, an assay in human plasma with a limit of quantification of 5 ng/ml has also been developed. Both HPLC-FD assays were utilized to support various human pharmacokinetic studies with alendronate.


Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography.

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane-isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound alpha 1-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.


Simultaneous determination of sulphadiazine and trimethoprim in plasma and tissues of cultured fish for residual and pharmacokinetic studies.

Clean-up and high-performance liquid chromatographic methods for the simultaneous determination of sulphadiazine and trimethoprim in fish plasma and tissues have been developed. The average recovery of sulphadiazine varied from 74% in liver to 92% in plasma, whereas that of trimethoprim varied from 60% in liver to 97% in plasma. The sample pretreatment procedures were simple, selective and robust, having a limit of quantification of 250 ng/ml for trimethoprim and 50 ng/ml for sulphadiazine in plasma, 15 ng/g for sulphadiazine and 80 ng/g for trimethoprim in muscle, and 30 ng/g for sulphadiazine and 160 ng/g for trimethoprim in liver. The assay was tested on plasma from Atlantic salmon treated with Tribrissen.