Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Hypersensitivity and reduced inhibition of DNA synthesis in ataxia telangiectasia lymphoblasts treated with low levels of neocarzinostatin

The effects of neocarzinostatin (NCS) on lymphoblastoid cell lines (LCLs) established from ataxia telangiectasia (A-T) were determined. A-T lymphoblasts were found to be hypersensitive to low levels of NCS as measured by cell growth and cell survival. On the other hand, A-T lymphoblasts failed to postpone DNA synthesis to the same degree as normal lymphoblasts following treatment with NCS. LCLs established from Nijmegen breakage syndrome (NBS) could be distinguished from ataxia and normal cell lines by their intermediate level of survival following exposure to NCS.     

Deletions in the SV40 late polyadenylation region downstream of the AATAAA mediate similar effects on expression in various mammalian cell lines.

A series of deletions in the SV40 late polyadenylation region was assayed by transient expression in a hamster fibroblast cell line. Because of differences in expression data between our results and the published results of another laboratory using a similar set of deletions introduced into a monkey kidney cell line, we studied our deletions in cells of different tissue-types and species (1). Deletion of the SV40 late polyadenylation region to 49 nucleotides downstream of the hexanucleotide AATAAA showed a small effect on gene expression, while further truncation of the region to 6 nucleotides downstream of the AATAAA showed an 85% drop in marker enzyme activity, protein levels and steady-state message levels. Another deletion in the same region, from base pair 10 to 15 past the AATAAA, which removes the wild-type site of RNA cleavage, showed a 50% drop in marker gene expression. The effects of these mutants on gene expression were similar in all of the cell lines tested and agree with other studies that DNA downstream of the AATAAA plays a role in efficient gene expression.     

Demonstration of transthyretin mRNA in the brain and other extrahepatic tissues in the rat

Studies were conducted to ascertain if transthyretin mRNA was present in extrahepatic tissues of the rat. A trnasthyretin cDNA clone was isolated from a lambda gt11 human liver cDNA library by antibody screening and its identity was confirmed by nucleotide sequence analysis. This transthyretin cDNA clone was used to survey poly(A+) RNA isolated from 12 different rat tissues for transthyretin mRNA by Northern blot analysis. The liver contained the highest level of transthyretin mRNA and this level was not altered by the vitamin A status of the rat. A significant amount of transthyretin mRNA was found in the brain (30% of the level of the liver) which was localized in specific regions of the brain. In addition, detectable levels of transthyretin mRNA (1% to 2% of that of the liver) were observed in the stomach, heart, skeletal muscle, and spleen. Translation of brain poly(A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-transthyretin antiserum resulted in a protein band of the same size as liver pre-transthyretin. Liver pre-transthyretin was processed by the cotranslational addition of dog pancreas microsomal membranes to a protein that migrated coincidentally with monomeric serum transthyretin. These data suggest that transthyretin in the brain and the cerebrospinal fluid results from de novo synthesis and that transthyretin may play a significant physiological function, as yet unknown, within the nervous system.     

WI-38 cell long-term quiescence model system: A valuable tool to study molecular events that regulate growth

A number of cell culture model systems have been used to study the regulation of cell cycle progression at the molecular level. In this paper we describe the WI-38 cell long-term quiescence model system. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Through studies aimed at understanding the cause and molecular nature of the prolongation of the prereplicative phase, we have determined that gene expression plays an important role in establishing growth factor “competence” and that once the cell becomes “competent” there is a defined order to the molecular events that follow during the remainder of G-1. More specifically, we have determined that the prolongation represents a delay in the ability of long term quiescent cells to become fully “competent” to respond to growth factors which regulate progression through G-1 into S. This prolongation appears to occur as a result of changes during long term quiescence in the ability of immediate early G-1 specific genes (such as c-myc) to activate the expression of early G-1 specific genes (such as ornithine decarboxylase). While ODC is the first and thus far only growth associated gene identified as a target of c-myc (and the Myc/Max protein complex), it is likely that further studies in this model system will reveal other early G-1 growth regulatory genes. We anticipate that future follow-up studies in this model system will provide additional valuable information abuot the function of growth-regulatory genes in controlling growth factor responsiveness and cell cycle progression.     

An anionic synthetic sugar containing 6-SO(3) -NAcGlc mimics the sulfated cruzipain epitope that plays a central role in immune recognition

Cruzipain (Cz), the major cysteine proteinase of Trypanosoma?cruzi, is a glycoprotein that contains sulfated high-mannose-type oligosaccharides. We have previously determined that these sulfate groups are targets of specific immune responses. In order to evaluate the structural requirements for antibody recognition of Cz, a systematic structure-activity study of the chemical characteristics needed for antibody binding to the Cz sulfated epitope was performed by immunoassays. With this aim, different synthesized molecules were coupled to the proteins BSA and aprotinin and confronted with (a) mouse sera specific for Cz and its carboxy-terminal (C-T) domain, (b) antibodies raised in rabbits immunized with Cz and its C-terminal domain and (c) IgGs purified from human Chagas disease sera. Our results indicate that a glucosamine containing an esterifying sulfate group in position O-6 and an N-acetyl group was the preferred epitope for the immune recognition of sera specific for Cz and its C-T domain. Although to a minor extent, other anionic compounds bearing sulfate groups in different positions and number as well as different anionic charged groups including carboxylated or phosphorylated monosaccharides, disaccharides and oligosaccharides were recognized. In conclusion, we found that synthetic anionic sugar conjugates containing N-acetyl d-glucosamine-6-sulfate sodium salt (GlcNAc6S) competitively inhibit the binding of affinity purified rabbit anti-C-T IgG to the C-T extension of Cz. Extending these findings to the context of natural infection, immune assays performed with Chagas disease serum confirmed that the structure of synthetic GlcNAc6S mimics the N-glycan-linked sulfated epitope displayed in the C-T domain of Cz.     

Sequencing of the primary adhesion domain of bovine von Willebrand factor.

A cDNA library, constructed from bovine heart endothelial cell poly(A)+ RNA, was screened using a BstXI fragment of human von Willebrand and factor (vWF) cDNA as a probe. This probe codes for the major adhesion domain of vWF that includes the GPIb, collagen and heparin binding domains. Of the ten positive clones obtained, a clone that spanned the region of interest was sequenced by the dideoxynucleotide method yielding a sequence of 1550 bp. This region of the bovine cDNA codes for amino acids corresponding to #262 to #777 in human vWF and encompasses the entire pro adhesion domain. Both the nucleotide sequence and the deduced amino acid sequence are 82% homologous to those of human vWF. Cysteine residues #471, 474, 509 and 695, which form intrachain bonds in human vWF, are also present in the bovine vWF sequence.