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R K Liem

Publications and source records attributed to R K Liem.

At least 19 recordsLinked to original sources
PubMed1992

Primary structure of high molecular weight tau present in the peripheral nervous system.

The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in Alzheimer disease. A protein of higher molecular weight, immunologically related to tau, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence of the high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, including four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomologous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8- to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tau mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of high molecular weight tau protein also suggests that it, like low molecular weight tau, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the domains specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.

Amino Acid Sequence↗
PubMed1991

Structure of the gene for the neuronal intermediate filament protein alpha-internexin and functional analysis of its promoter.

We have isolated the gene encoding the neuronal intermediate filament protein alpha-internexin using low stringency hybridization conditions and an NF-M (neurofilament middle molecular weight subunit) cDNA probe. This gene consists of three exons and two introns. The sequence data and the exon-intron organization of the gene establish its classification as a type IV intermediate filament gene. Transient transfection experiments showed that up to 5 kilobases of the alpha-internexin promoter region transcribed equally well in both the alpha-internexin-expressing and -nonexpressing cell lines. The results also demonstrated that the region from -77 to +73 relative to the transcription start site was sufficient for accurate basal transcription, but inclusion of the -254 to -78 region was required for efficient transcription. Sequence analysis shows that the -254 to -78 region contains several potential positive regulatory elements.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of the rat chromosomal gene for a polypeptide (pS1) antigenically related to statin.

Increasing evidence shows the existence of nonproliferation-specific gene(s) whose expression is mostly present in growth-arrested cells. One member of this gene family has been identified by previous work as a nuclear protein of 57,000 Da, termed statin. Logical extensions of statin research are to identify the genomic and cDNA clones encoding for statin and to study the regulation of statin gene expression. During the search for the statin gene, we have identified a cDNA clone and a genomic clone named S1 and S10, respectively, by screening a rat brain lambda gt11 expression library with the statin antibody and subsequently using S1 cDNA as a probe to screen a rat genomic cosmid library. Here, we report the cloning and sequencing of the S1 cDNA and S10 genomic clones. Primary sequence analyses indicate that the derived amino acid sequence of S1 shares high homology (greater than 92.6%) with human elongation factor 1 alpha (EF-1 alpha), whereas the 5'- and 3'-untranslated regions are less than 20% homologous. Despite the unusually high degree of similarity between S1 and human EF-1 alpha at the amino acid sequence level, their protein products are different and immunologically distinct. The in vitro transcription and translation product of S1 (pS1), a 49,000-Da polypeptide, reacts only with the monoclonal antibody against statin; this antibody exhibits no antigenic reaction to the EF-1 alpha protein. Northern blot analysis shows that the S1 message is most abundant in G0 phase of 3T3 mouse fibroblasts, but becomes significantly reduced in G1 and S phase cells. EF-1 alpha messages do not show such dramatic changes during cell cycle phase transition. These findings suggest that the expression of the identified S1 cDNA clone is specific for nonproliferating cells and that the in vitro translation product of the S1 cDNA is recognized by the statin antibody. Genomic Southern blots indicate that S1 cDNA is encoded by a single copy gene in the rat genome and is a unique member of the EF-1 alpha/S1 supermultigene family. DNA sequence analysis demonstrates that the rat S1 transcription unit is 12 kilobase pairs in length and contains seven introns. The organization of exons is virtually identical between S1 and human EF-1 alpha. In contrast, neither a TATA box nor a CAAT box is located in the proximal 5'-flanking regions from positions -1 to -1359 of the S1 gene, where we could expect to find the regulatory region containing the elements controlling gene expression; no evident sequence homology to the human EF-1 alpha gene is detected in this region.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Astroglia in CNS injury.

The astroglial response to CNS injury is considered in the context of neuron-glial relationships. Although previous models suggested that astroglial cells present in "scars" impede axon regrowth owing to irreversible changes in the glial cell following injury, recent in vivo and in vitro studies indicate that astroglial cells exhibit considerable plasticity, elevating expression of the glial filament protein and altering expression of properties which support axons, including extracellular matrix components and cell surface adhesion systems. Both in vivo and in vitro studies on neuron-glia interactions in different brain regions suggest that glia express region-specific properties, including ion channels, neurotransmitter uptake and receptor systems, and cell surface adhesion systems. Together these findings suggest that a more detailed analysis of glial response to injury in different brain regions will lead to an appreciation of the diversity of the astroglial response to injury, and its regulation by neuron-glia relationships.

Animals↗
PubMed1991

Kinesin heavy chain from bovine brain and Drosophila appear to be highly homologous molecules.

A microtubule-enriched fraction was prepared from bovine white matter, and kinesin and other microtubule-associated proteins were extracted from taxol-stabilized microtubules by homogenization and ultracentrifugation in the presence of nucleotides (guanosine triphosphate and adenosine triphosphate). The kinesin-enriched fractions were subjected to preparative SDS-PAGE, and the band representing the kinesin heavy chain was excised, homogenized, and subjected to partial enzymatic digestion with Staphylococcus aureus V8 protease. Four peptides were selected for sequence analysis and compared to the previously published sequence for the Drosophila kinesin heavy chain (Yang JT, Laymon RA, Goldstein LSB, Cell 56:879-889, 1989). All four peptides matched closely with portions of the Drosophila sequence corresponding to the central, alpha-helical domain. Total amino acid composition analysis of bovine kinesin heavy chain also reveals a high degree of homology to the Drosophila sequence.

Adenosine Triphosphatases↗
PubMed1991

Characterization of a panel of neurofilament antibodies recognizing N-terminal epitopes.

Peptides corresponding to sequences from the amino-terminal "head" regions of the low, middle, and high molecular weight neurofilament proteins (NF-L, NF-M, and NF-H) were synthesized by a modification of the Merrifield solid-phase method, and a panel of polyclonal antibodies to these epitopes were prepared in rabbits by the injection of synthetic peptides conjugated to the carrier protein keyhole limpet hemocyanin (KLH). An additional, monoclonal antibody recognizing both glial fibrillary acidic protein (GFAP) and vimentin was also produced, by fusion of cells of the mouse myeloma line NS-1 with spleen cells from a mouse immunized with cytoskeletal extracts. Antibody specificities were confirmed by a combination of Western blotting against cytoskeletal extracts and immunofluorescence using both rat brain sections and fibroblasts transfected with fully encoding cDNAs for each neurofilament protein, driven by viral promoters.

Amino Acid Sequence↗
PubMed1991

Expression of alpha B-crystallin in the developing rat kidney.

The expression and cellular localization of alpha B-crystallin during rat renal development was studied by Northern blot analysis and by immunocytochemistry. Northern blotting of total RNA extracted from whole kidneys revealed that the messenger RNA for alpha B-crystallin rapidly increased after birth to reach adult levels by 20 days. At the same time, immunohistochemistry for alpha B-crystallin demonstrated that the prominent elongation of Henle's loop during the first 10 days of life was accompanied by increased alpha B-crystallin expression. Thus, the development of alpha B-crystallin expression is correlated temporally with the acquisition of tubule function in early post-natal life.

Animals↗
PubMed1991

Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons.

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.

Animals↗
PubMed1991

High molecular weight tau: preferential localization in the peripheral nervous system.

Using epitope mapping we have demonstrated that a high molecular weight protein (Mr approximately 115 x 10(3)) present in brain and spinal cord is a member of the tau family of microtubule-associated proteins. Antibodies directed against the amino-terminal, middle and carboxyl-terminal portions of tau recognize this protein. A limited survey of neuronal tissues has shown that this high molecular weight tau protein is present in brain, spinal cord, dorsal root ganglia, dorsal and ventral roots and peripheral nerves. High molecular weight tau protein is expressed at higher levels in spinal cord than in brain and is the only form of tau detected in the adult peripheral nervous system.

Animals↗
PubMed1991

Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts.

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

Animals↗
PubMed1990

Multiple mRNAs of rat brain alpha-crystallin B chain result from alternative transcriptional initiation.

Two major classes of mRNAs for the alpha-crystallin B chain (or alpha(B)crystallin), about 0.9 and 1.2 kilobases in length, are expressed in rat brain. To examine the structures of these mRNAs, we isolated cDNA clones from rat brain and genomic DNA from rat liver. Characterization of these clones as well as Northern blot analysis indicated that the various mRNAs differed in the lengths of their 5' leader sequences. RNase protection assays revealed that the gene for alpha-crystallin B chain contains multiple start sites. The transcriptional start sites of the longer mRNAs are preceded by a putative CAAT box and that of the shorter mRNA by a putative TATA box. The shorter mRNA encodes the alpha-crystallin B chain protein, whereas the longer mRNA contained three extra small open reading frames upstream of the AUG start codon for the protein. The shorter mRNA is abundant in lens, heart, muscle, and kidney, while the longer mRNAs are constitutively expressed at low levels in a wide variety of tissues. The shorter mRNA was increased by treatment with phorbol 12-myristate 13-acetate in rat C6 glioma cells. Since there is only a single copy of the alpha-crystallin B chain gene, our results indicate that the two classes of mRNAs are generated by alternative transcriptional initiation from different promoters and their expressions are regulated differentially.

Amino Acid Sequence↗
PubMed1990

C17, a retrovirally immortalized neuronal cell line, inhibits the proliferation of astrocytes and astrocytoma cells by a contact-mediated mechanism.

We have investigated the ability of various cell lines to effect contact-mediated inhibition of astrocytic cells. Of the lines tested, only C17, a mouse cell line from postnatal day 0 cerebellum immortalized by infection with a retroviral construct containing the avian myc gene, and U251, a human astrocytoma line, were able to inhibit the proliferation of astrocytic cells. When co-cultured with either primary astrocytes from rat cerebellum or the U251 line, the C17 cells induced a rapid cessation of glial cell division as well as complex astrocytic process extension. The effects on glial mitosis were cell-dose-dependent, with ten C17 cells/glial cell being the optimal ratio. At this ratio [3H]thymidine incorporation into the U251 cells was reduced by greater than 80% and there was a virtual stasis in glial cell number at 48 hours. Fixed C17 cells as well as partially purified C17 membranes were also potent inhibitors of astrocytic proliferation, suggesting that the gliastatic effect of the C17 cell line is membrane associated. However, neither of these preparations induced astrocytic process formation. We also confirmed earlier reports that U251 cells inhibited their own proliferation in a density-dependent manner but at a lower efficiency than the C17 cells.

Animals↗
PubMed1990

The predicted amino acid sequence of alpha-internexin is that of a novel neuronal intermediate filament protein.

Our laboratory recently isolated and began to characterize a 66 kd rat brain cytoskeletal protein, dubbed alpha-internexin for its interactions in vitro with several other cytoskeletal proteins. Although alpha-internexin bore several of the characteristics of intermediate filament (IF) proteins, including the recognition by an antibody reactive with all IF proteins, it did not polymerize into 10 nm filaments under the conditions tested. Here we show that the predicted amino acid sequence of a cDNA encoding alpha-internexin shows the latter to be an IF protein, probably most closely related to the neurofilament proteins. Northern blotting shows that alpha-internexin expression is brain specific, and that rat brain alpha-internexin mRNA levels are maximal prior to birth and decline into adulthood, while the converse is seen for NF-L, the low molecular weight neurofilament subunit, suggesting that these two proteins play different roles in the developing brain.

Amino Acid Sequence↗
PubMed1990

Alpha-internexin, a novel neuronal intermediate filament protein, precedes the low molecular weight neurofilament protein (NF-L) in the developing rat brain.

alpha-Internexin is a 66 kDa protein that copurifies with intermediate filaments (IF) from rat spinal cord and optic nerve. This protein is axonally transported in rat optic nerve along with the neurofilament triplet proteins in slow component a. Polymerization in vitro and distribution in vivo confirm that alpha-internexin is a neuronal IF. We raised 2 highly specific monoclonal antibodies to alpha-internexin which were applied to frozen rat brain sections and Western blots of cytoskeletal extracts. These results indicate that alpha-internexin is primarily an axonal protein found in most, if not all, neurons of the CNS. Immunoreactive proteins of similar molecular weight were found in cytoskeletal extracts of CNS tissue from several additional species, including mouse and cow. While the distribution of alpha-internexin as given by immunocytochemical methods is similar to that of low molecular weight neurofilament protein (NF-L) in the adult, its distribution in the embryo is far more extensive. At embryonic day 16, when the expression of NF-L is still limited to a relatively small number of cells and levels of expression are low, alpha-internexin is already found at much higher levels and in cells not yet expressing NF-L in detectable quantities. Similar results are found at embryonic day 12. These data suggest that neuronal IF in the developing nervous system contain a higher proportion of alpha-internexin than their adult counterparts, and that expression of alpha-internexin precedes that of NF-L in many or most neurons of the developing brain.

Aging↗
PubMed1990

Transfected rat high-molecular-weight neurofilament (NF-H) coassembles with vimentin in a predominantly nonphosphorylated form.

A fully encoding cDNA for the high-molecular-weight rat neurofilament protein (NF-H) has been isolated from a lambda gt11 library, sequenced and subcloned into eukaryotic expression vectors. Sequence analysis shows that rat NF-H has an overall homology of 72 and 88% with human and mouse NF-H, respectively. The head and rod domains are almost entirely identical, and the divergences are due to differences in the long C-terminal extensions of the molecule. The consensus phosphorylation sequence for neurofilaments Lys-Ser-Pro (KSP) is present 52 times. The predicted molecular mass of the protein is 115 kDa, 42% lower than that observed by SDS-PAGE. Upon transfection into vimentin-containing fibroblasts, such as L tk-, L929, and 3T6 cells, NF-H is seen distributed with vimentin by light and electron microscopic examinations indicating that copolymers of NF-H and vimentin are formed in these cells. Only a negligible proportion of the cells is positive when stained with a number of antibodies directed against phosphorylated NF-H epitopes. This is in contrast with the middle molecular weight NF protein (NF-M) transfected into L tk- and L929 cells, which can readily be detected by antibodies against phosphorylated neurofilament epitopes. The mobilities of the transfected protein on 1- and 2-dimensional gels confirm that NF-H is predominantly in a nonphosphorylated form. These results indicate that phosphorylation of NF-H, but not NF-M, on the KSP sequence is due to protein kinases, which are not present in fibroblasts and are presumably NF-H specific. The stable NF-H-expressing cell lines can therefore be used to study these putative neurofilament kinases in vitro and in vivo.

Amino Acid Sequence↗