Light Chain Messenger RNA Research Articles

Decreased epileptogenesis in mice lacking the System xc - transporter occurs in association with a reduction in AMPA receptor subunit GluA1.

SummaryObjectiveAlthough the cystine/glutamate antiporter System xc − (Sxc −) plays a permissive role in glioma‐associated seizures, its contribution to other acquired epilepsies has not been determined. As such, the present study investigates whether and how Sxc − contributes to the pentylenetetrazole (PTZ) chemical kindling model of epileptogenesis.MethodsMale Sxc − null (sut/sut) mice and their wild‐type littermates were administered PTZ (i.p.) daily for up to 21 days (kindling paradigm). Seizure severity was scored on a 5‐point behavioral scale. Mossy fiber sprouting, cellular degeneration, and Sxc − light chain (xCT) messenger RNA (mRNA) were explored using Timm staining, thionin staining, and real‐time quantitative polymerase chain reaction (qPCR), respectively. Levels of reduced and oxidized glutathione and cysteine were determined via high‐performance liquid chromatography (HPLC). Plasma membrane protein levels of glutamate and γ‐aminobutyric acid (GABA) receptor subunits as well as the K+/Cl− co‐transporter KCC2 were quantified via western blot analysis.ResultsRepeated administration of PTZ produced chemical kindling in only 50% of Sxc − null mice as compared to 82% of wild‐type littermate control mice. Kindling did not result in any changes in xCT mRNA levels assessed in wild‐type mice. No cellular degeneration or mossy fiber sprouting was discernible in either genotype. Except for a small, but significant, decrease in oxidized cysteine in the hippocampus, no other change in measured redox couples was determined in Sxc − null mice. Cortical levels of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subunit GluA1 were decreased in Sxc − null mice as compared to wild‐type littermates, whereas all other proteins tested showed no difference between genotypes.SignificanceThis study provides the first evidence that Sxc − signaling contributes to epileptogenesis in the PTZ kindling model of acquired epilepsy. Further data indicate that a reduction in AMPA receptor signaling could underlie the resistance to PTZ kindling uncovered in Sxc − null mice.

Microbiome-Derived Lipopolysaccharide (LPS) Selectively Inhibits Neurofilament Light Chain (NF-L) Gene Expression in Human Neuronal-Glial (HNG) Cells in Primary Culture.

The remarkable co-localization of highly pro-inflammatory lipopolysaccharide (LPS) with sporadic Alzheimer’s disease (AD)-affected neuronal nuclei suggests that there may be some novel pathogenic contribution of this heat stable neurotoxin to neuronal activity and neuron-specific gene expression. In this communication we show for the first time: (i) the association and envelopment of sporadic AD neuronal nuclei with LPS in multiple AD neocortical tissue samples; and (ii) a selective repression in the output of neuron-specific neurofilament light (NF-L) chain messenger RNA (mRNA), perhaps as a consequence of this association. The down-regulation of NF-L mRNA and protein is a characteristic attribute of AD brain and accompanies neuronal atrophy and an associated loss of neuronal architecture with synaptic deficits. To study this phenomenon further, human neuronal-glial (HNG) cells in primary culture were incubated with LPS, and DNA arrays, Northern, Western, and ELISA analyses were used to quantify transcription patterns for the three member neuron-specific intermediate filament-gene family NF-H, NF-M, and NF-L. As in sporadic AD limbic-regions, down-regulated transcription products for the NF-L intermediate filament protein was significant. These results support our novel hypothesis: (i) that internally sourced, microbiome-derived neurotoxins such as LPS contribute to a progressive disruption in the read-out of neuron-specific genetic-information; (ii) that the presence of LPS-enveloped neuronal nuclei is associated with a down-regulation in NF-L expression, a key neuron-specific cytoskeletal component; and (iii) this may have a bearing on progressive neuronal atrophy, loss of synaptic-contact and disruption of neuronal architecture, all of which are characteristic pathological features of sporadic-AD brain. This is the first report that provides evidence for a neuron-specific effect of a human GI-tract microbiome-derived neurotoxin on decreased NF-L abundance in both sporadic AD temporal lobe neocortex in vivo and in LPS-stressed HNG cells in vitro.

Branched-chain in situ hybridization for κ and λ light chains: A powerful ancillary technique for determining B-cell clonality in cytology samples.

Current immunohistochemical and in situ hybridization (ISH) assays are generally inconclusive for clonality unless plasmacytic differentiation is present. This study examined a series of cytology specimens and explored the ability of a branched-chain RNA (bRNA) ISH assay for immunoglobulin κ constant (IGKC) and immunoglobulin λ constant (IGLC) to detect a clonal population of B lymphocytes. Pathology databases were used to identify fine-needle aspiration biopsies (n = 28) and exfoliative cytology samples (n = 20). Demographic, flow cytometry, and excision biopsy results were recorded. bRNA ISH was performed on the Leica Bond platform with the following probes: IGKC, IGLC, immunoglobulin λ-like polypeptide 5 (IGLL5), and a housekeeping gene (HKG). The bRNA ISH assay was validated with 30 surgical biopsies. On bRNA ISH, a clonal B-cell population (light-chain ratio > 10:1) was detected in 22 of 28 cases with a final diagnosis of lymphoma. In 2 cases, a κ predominance was present, although the ratio was <10:1. Eleven of the 17 κ-clonal lymphomas also expressed IGLL5, the latter recognized by the presence of an intranuclear signal. Two B-cell lymphomas lacked IGKC and IGLC, whereas 2 cases were negative for the HKG. In 12 of the 20 cases with reactive lymphoid tissue, bRNA ISH identified a polyclonal lymphoid population. No light-chain messenger RNA was detected in 6 cases (typically those associated with very few B cells). The automated bRNA ISH platform is a robust technique for detecting a clonal B-cell population in cytology material.

Comparison of in situ hybridisation and polymerase chain reaction in the diagnosis of B cell lymphoma.

AIM: To compare the sensitivity of the detection of immunoglobulin light chain messenger RNA (mRNA) restriction by in situ hybridisation (ISH) and clonal immunoglobulin heavy chain gene rearrangements by polymerase...

Origin of nodular lymphocyte-predominant Hodgkin's disease from a clonal expansion of highly mutated germinal-center B cells.

The atypical cells of nodular lymphocyte-predominant Hodgkin's disease, designated lymphocytic and histiocytic (L&H) cells, have a B-cell phenotype. To clarify the clonality of these cells, we studied rearranged immunoglobulin genes for the variable region of the heavy chain (V[H] genes) in individual L&H cells from 11 patients with nodular lymphocyte-predominant Hodgkin's disease. We also studied the expression of immunoglobulin light chains by those cells in six of the same patients. Single CD20+ L&H cells were isolated from frozen sections by a technique of micromanipulation. The rearranged V(H) genes of these cells were amplified by the polymerase chain reaction (PCR), sequenced, and compared with germ-line V(H) genes. Immunoglobulin light-chain messenger RNA (mRNA) was detected by in situ hybridization. Of 615 L&H cells isolated from all the frozen sections, 160 yielded PCR products. In each of the 11 patients, the L&H cells that could be evaluated had identically rearranged V(H) genes, whether they were isolated from the same nodule, different nodules, or different blocks of tissue. All the V(H) sequences derived from the L&H cells were highly mutated (7.5 to 27.2 percent). In two cases the coding capacity of the V(H) genes was completely or partially disrupted by mutations. Intraclonal diversity was found in six cases, and monotypic immunoglobulin light-chain mRNA was found in six. The L&H cells of nodular lymphocyte-predominant Hodgkin's disease represent a monoclonal expansion of B cells. The high load of V(H) gene mutations and signs of intraclonal diversity suggest a relation between L&H cells and germinal-center B cells at the centroblastic stage of differentiation.

ReactiveVersusNeoplastic Monocytoid B-Cell Proliferations:In Situ Hybridization Study of Immunoglobulin Light Chain mRNA

To distinguish reactive versus neoplastic monocytoid B-cell (MBC) proliferations, the clonality of MBC was examined in paraffin-embedded tissues by in situ hybridization (ISH) of immunoglobulin (Ig) light chain messenger RNA (mRNA) with sensitive oligonucleotide probes in 26 cases. They included 13 cases of lymphadenitis with MBC reaction and 13 cases of nodal (n = 8) and extranodal (n = 5) monocytoid B-cell lymphoma (MBCL). Two cases represented a composite lymphoma showing a centroblastic-centrocytic and MBCL component. The clonality of MBC infiltrates could be demonstrated in 16 of 26 (61.5%) cases by immunostaining for Ig light chains and in all (100%) cases by ISH. Neoplastic MBC usually expressed a faint-to-moderate light chain restriction of mRNA, whereas some MBC (10% to 30% of total MBCL population) showed a strong positivity irrespective of plasmacytoid differentiation as indicated by Ig immunostaining (present in 9 of 13 cases). Reactive MBC expressed a faint kappa and lambda light-chain mRNA positivity. Five percent to 20% of total reactive MBC showed also a strong positivity for both Ig light chain mRNA, although only a minor part of these cells (7 of 13 cases) expressed polyclonal Ig by immunohistochemistry. These results indicate that (1) both reactive and neoplastic MBC can differentiate into plasma cells; and (2) a relatively high percentage of reactive and neoplastic MBC show a detectable mRNA transcription, but not a corresponding Ig synthesis. Either the Ig detection is not sensitive enough or these cells might be in an early differentiation phase, where the Ig production has not yet started.

Demonstration of light chain mRNA in Hodgkin's disease.

The lineage of Hodgkin and Reed-Sternberg cells is still unclear. Detection of both immunoglobulin light chains in Hodgkin and Reed-Sternberg cells by immunohistochemistry is a well-known phenomenon. However, up to now, in situ hybridization techniques have failed to demonstrate light chain messenger(m) RNA in Hodgkin and Reed-Sternberg cells. In this investigation, we have analysed 26 cases of Hodgkin's disease (nodular lymphocyte predominant Hodgkin's disease, mixed cellularity, and nodular sclerosis type) using digoxigenin-labelled oligonucleotide probes for kappa and lambda light chains by in situ hybridization. In nearly half of the cases of nodular lymphocyte predominant Hodgkin's disease and in one case of mixed cellularity type, mRNA for only one light chain could be clearly demonstrated in the lymphocytic and histiocytic cells, Hodgkin, and Reed-Sternberg cells. These results support the idea that at least some cases of Hodgkin's disease are B-cell neoplasms.

Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization

In situ hybridization techniques were used to detect immunoglobulin light chain messenger RNA (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism.

Expression of ventricular-type myosin light chain messenger RNA in spontaneously hypertensive rat atria.

Using cloned DNA probes specific for two isoforms of cardiac myosin light chains (MLCs), nonphosphorylatable MLC1 and phosphorylatable, regulatory MLC2, we have observed that the MLC1 messenger RNA of ventricular type does not appear in detectable amounts in atrial cells of either normotensive Wistar-Kyoto rat strain (WKY) or spontaneously hypertensive rat strain (SHR). The messenger RNA of regulatory isoform of ventricular MLC2, on the other hand, is found in threefold excess in atria of SHR relative to that of age-matched WKY. The increased level of MLC2 messenger RNA is present even in 6-week-old SHR atria where there is no established overloading of the heart. Thus, it appears that the increased expression of the regulatory MLC2 gene in SHR atrial cells is a predetermined event, which, most likely, participates in functional adaptation of the myocardium in response to pressure overload and subsequent hypertrophy.

Molecular events during maturation of the immune response to oxazolone.

Sequence analysis of the heavy- and light-chain messenger RNA of hybridomas immunized with a specific hapten yields important clues about the interplay between genetic and selective events during the onset and maturation of the immune response. The maturation of the primary response to the hapten 2-phenyl-5-oxazolone is characterized by a drift to higher-affinity somatic variants of a germline-encoded basic sequence, whereas hybridomas from the secondary response demonstrate a further maturation dominated by a shift to alternative germline combinations.

Unrearranged immunoglobulin variable region genes have a functional promoter.

We have tested whether immunoglobulin light chain variable region genes are capable of directing initiation of transcription without undergoing the DNA rearrangement which creates a complete immunoglobulin gene. Two human Vk genes specifically initiated transcription in vitro at a site which is approximately 30 bases downstream of a TATA box and 20 bases upstream of the initiation codon. A Vk pseudogene which lacks a TATA box at an homologous position was not transcribed to a detectable extent in the in vitro system. One of the Vk genes was injected into Xenopus oocytes and it initiated transcription at precisely the same position as in the HeLa cell extract. It is suggested that the promoter which we have identified upstream of unrearranged Vk genes operates in lymphocytes after V-J joining has occurred to initiate transcription of light chain messenger RNA.

Nuclear RNA precursors in the processing pathway to MOPC 21 κ light chain messenger RNA

We have carried out pulse-chase and u.v. transcription mapping experiments which establish that immunoglobulin κ light chain messenger RNA in MOPC 21 myeloma cells is derived from a 10.6 × 10 3 base nuclear RNA precursor. This large nuclear RNA precursor is polyadenylated and appears to be processed into other polyadenylated κ-specific nuclear RNA species at 7.8, 5.6 and 4.8 × 10 3 bases. These are shown to be obligatory precursors to κ mRNA by glucosamine-mediated pulse-chase experiments in which virtually all of the κ sequences in these large nuclear RNAs are converted into κ mRNA. This kinetic evidence constitutes formal proof of the precursor-product relationship between these nuclear RNAs and cytoplasmic κ mRNA. In addition, the κ nuclear RNAs all show identical u.v. target sizes as expected for processing intermediates derived from the 10 × 10 3 base κ transcription unit. The major κ nuclear RNA intermediate at 4.8 × 10 3 bases appears to contain the intervening sequence separating the variable (V) and constant (C) regions in the active MOPC 21 κ gene. This 4.8 × 10 3 base species is shown directly to give rise to κ mRNA. Accordingly, we propose that V → C joining is the final splicing step in the nuclear RNA processing pathway for κ light chain mRNA.

Isolation and cell-free translation of a human immunoglobulin light chain messenger RNA

Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas.

Recombinant DNA clones constructed from immunoglobulin kappa light chain messenger RNA.

Recombinant DNA clones have been generated from mouse myeloma MOPC 21 immunoglobulin kappa light chain mRNA. Complementary DNA (cDNA) synthesized on kappa light chain mRNA by reverse transcriptase was made double stranded and inserted into the bacterial plasmid vector, pMB9. Approximately 70 tetracycline-resistant transformed colonies containing kappa light chain mRNA sequences were identified by colony hybridization. Five of these recombinant clones were selected and characterized. Three clones contain both kappa light chain constant and variable region sequences. Two of these three recombinant clones have been shown to include all of the kappa light chain constant and variable region coding sequences. Another of the five selected recombinant clones contain kappa light chain constant region sequences. The remaining characterized clone appears to be derived from sequences at the 5'-end of kappa light chain mRNA, possibly extending to the terminal cap structure.

Distribution of MPOC-315 light chain messenger RNA in free and membrane-bound polyribosomes

Summary To measure the distribution of immunoglobulin light chain mRNA, polyadenylated mRNA was isolated from membrane-bound polysomes, free polysomes and 100,000 × g supernatant of MOPC-315 plasmacytoma. Each fraction was hybridized with cDNA specific for purified light chain mRNA. Comparison of hybridization kinetics indicated that membrane-bound polysomal mRNA contained approximately three to four times more MOPC-315 light chain mRNA (L315 mRNA) per mg of mRNA than the other two fractions. Furthermore, because there are more free than membrane-bound polysomes, membrane-bound polysomes of the MOPC-315 plasmacytoma contain only about twice as much of the L 315 mRNA as do the free polysomes.

Purification of Immunoglobulin Light Chain Messenger RNA by Immunoprecipitation from Mouse Myeloma Tumor, MOPC-31C1

Polysomes producing IgGl(kappa) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410.000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25,000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cell-free system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Crt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4,4-fold during the immunoprecipitation of polysomes.

Sequence analysis of the 3′ non-coding region of mouse immunoglobulin light chain messenger RNA

Using an oligonucleotide d(pT10-C-A) as primer, cDNA has been transcribed from the 3' non-coding region of mouse immunoglobulin light chain mRNA and sequenced by a modification1 of the 'plus-minus' gel method2. The sequence obtained has partially corrected and extended a previously obtained sequence3. The new data contains an unusual sequence in which a trinucleotide is repeated seven times.

Existence of both kappa and lambda light chain messenger RNA sequences in mouse myeloma, MOPC-104E, known as a lambda chain producer

A mouse myeloma, MOPC-104E, which is known to synthesize and secrete λ type light chain protein as a constituent of immunoglobulin M, was shown to contain mRNA sequences coding for κ as well as λ type light chain protein. Light chain mRNA sequences were quantitated by nucleic acid hybridization reaction using radioactive DNA complementary to light chain mRNAs which had been purified from other myelomas. The amount of κ type light chain mRNA present in MOPC-104E is almost equivalent to that of λ type light chain mRNA. κ chain mRNA was not separated from λ chain mRNA either by centrifugation in sucrose density gradient or by polyacrylamide gel electrophoresis in formamide.

Demonstration that a mouse immunoglobulin light chain messenger RNA hybridizes exclusively with unique DNA

32P-labeled light chain messenger RNA was prepared from mouse MOPC 21 myeloma cells. The messenger RNA was hybridized to purified repetitive nuclear DNA and both the hybridized (repetitive 32P-RNA) and nonhybridized (nonrepetitive 32P-RNA) fractions were isolated. Only the nonhybridized RNA gave a T1 ribonuclease fingerprint showing oligonucleotides derived from the variable and constant regions of the light chain messenger RNA. In addition, this fingerprint showed oligonucleotides derived from the untranslated regions of the light chain messenger RNA. The nonrepetitive 32P-RNA was shown to rehybridize only with the unique fraction of total nuclear DNA. The rapidly hybridizing part of the unfractionated 32P-RNA preparation, therefore, is not a component of the light chain messenger RNA itself. Complementary DNA was prepared with reverse transcriptase using unlabeled light chain messenger RNA as template, and the transcripts were fractionated into various size classes. Complementary DNA molecules greater than 900 bases in length hybridized with both the initial messenger RNA and with the nonrepetitive 32P-RNA but failed to hybridize with excess purified repetitive 32P-RNA. The rapidly hybridizing component of the messenger RNA fraction, therefore, does not appear to be transcribed by reverse transcriptase. It is concluded that, under the experimental conditions used, the light chain messenger RNA hybridizes exclusively with unique DNA.

Partial amino acid sequence of the precursor of immunoglobulin light chain programmed by messenger RNA in vitro.

The five proteins programmed in a cell-free system by a mouse kappa light chain messenger RNA were labeled with [3H]leucine and subjected to amino acid sequence analyses. In all five proteins, 20 amino acid residues precede the amino terminus of the mature protein, indicating that there is one major point for the initiation of messenger RNA translation. The abundance (30 percent) of leucine residues in the extra piece (leucine at positions 6, 7, 8, 11, 12, and 13) indicates that this moiety is hydrophobic. Furthermore, it seems that the precursor may have an additional extra piece at the carboxyl terminus.