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PubMed1987

Identification of two variants of troponin T in the developing chicken heart using a monoclonal antibody.

Troponin T (TNT) expressed in the developing chicken cardiac muscle was examined by immunoblotting combined with two-dimensional electrophoresis (2-D PAGE) and peptide mapping. When the whole lysate of the neonatal heart was examined by 2-D PAGE, two TNT variants were detected on the gel by monoclonal antibody to TNT. Expression of the two variants was developmentally regulated: one isoform (type I) was expressed from embryonic through neonatal stages, and the other (type II) from the late embryonic stage through adulthood during cardiac muscle development. The type-I isoform, but not type-II isoform, was also expressed transiently in chicken skeletal muscle at embryonic stages. As judged from the peptide maps, the two isoforms differed in the N-terminal region but not in the C-terminal region.

Aging↗
PubMed1985

Identification of multiple variants of fast muscle troponin T in the chicken using monoclonal antibodies.

Two monoclonal antibodies, T1/7 and T1/61, have been prepared which are specific for chicken fast muscle troponin T. Both are of the IgG gamma 1 subclass. Both antibodies cross-react strongly with human fast and chicken cardiac troponin T, but while T1/7 reacts weakly with rabbit fast troponin T, T1/61 does not. The antibodies can be used for fibre typing of both chicken and human muscle. The antibodies have been used to identify fast troponin T on two-dimensional maps of proteins from a variety of chicken muscles by electrophoretic transfer to nitrocellulose followed by immunoperoxidase staining. Using this technique five variant forms of fast troponin T have been identified. Two variants, fBT1 and fBT2, are expressed in breast muscle, while the other three, fLT1, fLT2 and fLT3 are expressed in leg muscle. Of the leg muscle variants, fLT1 and fLT2 correspond to the two forms described previously [Wilkinson, J.M., Moir, A.J.G. and Waterfield, M.D. (1984) Eur. J. Biochem. 143, 47-56]. The third variant, fLT3, has not been described before and is expressed in muscles which have a high content of slow fibres. In addition to these clearly defined variant forms immunostaining reveals multiple minor variants of troponin T present in leg muscle which may reflect complex RNA processing of the troponin T gene transcript.

Animals↗
PubMed1991

Isolation and identification of a variant of bluetongue virus serotype 11 from a ram in a bluetongue outbreak in western Texas.

A field strain of bluetongue virus was isolated from a blood sample of a ram during an outbreak of bluetongue in November 1985 in western Texas. In this bluetongue outbreak at least 25 of the 2,000 sheep were infected. Isolation was made by intravenous inoculation of 11-day-old embryonated chicken eggs. The serotype was identified as serotype 11 by serum neutralization tests. The genomic pattern on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of the new isolate is similar to that of bluetongue virus prototype 11. Comparisons were also made with proteins labeled in vivo with [3H]leucine and separated by SDS-PAGE. We conclude that this virus belongs to serotype 11, with slight differences in both genome and protein electrophoretic patterns.

Animals↗
PubMed1993

Complete nucleotide sequence of an Amerindian human T-cell lymphotropic virus type II (HTLV-II) isolate: identification of a variant HTLV-II subtype b from a Guaymi Indian.

The complete nucleotide sequence of a human T-cell lymphotropic virus type II (HTLV-II) isolate from a Panamanian Guaymi Indian was determined and analyzed. When this new viral isolate (HTLV-IIG12) was compared with prototypic HTLV-IIMoT, the overall nucleotide sequence similarity was 95.4%, while the predicted amino acid sequence similarity was 97.5%. Although the overall percentage of nucleotide and amino acid identity with prototypic HTLV-IIMoT (subtype a) was high, HTLV-IIG12 displayed several distinctive features that defined it as an HTLV-II subtype b. However, there were several characteristics unique to this isolate, which included a cluster of nucleotide substitutions in the pre-gag region and changes in restriction enzyme sites within the pre-gag region and the gag, pol, env, and pX genes. In addition, two nucleotide changes in the C terminus of the Tax protein coding sequence inserted an Arg residue for a stop codon and appeared to result in a larger tax gene product in HTLV-IIG12. Although the HTLV-IIG12 isolate appears to be a variant of the prototypic HTLV-IIb, this information represents the first complete nucleotide sequence of any HTLV-II subtype b. These data will allow further studies on the evolutionary relationships between the HTLV-II subtypes and between HTLV-I and HTLV-II.

Amino Acid Sequence↗
PubMed1993

Murine intestinal disaccharidases: identification of structural variants of sucrase-isomaltase complex.

This study was directed to determine the extent of variability in structure or expression of intestinal disaccharidase [gamma-glucoamylase (gamma-GA), sucrase-isomaltase (SI), and lactase] between different strains of mice. Reduced levels of sucrase activity (approximately 20 U/g of protein) were observed in three strains of mice belonging to the CBA/Ca lineage. Four other strains of mice analyzed exhibited higher levels of sucrase activity (approximately 50 U/g of protein). Decreased levels of sucrase in CBA/Ca mice were not associated with decreased levels of activity associated with the isomaltase subunit or with decreased levels of SI mRNA expression. High-performance liquid chromatographic gel filtration, heat inactivation, and kinetic analysis indicated that the differences between strains in sucrase activity might be attributed to structural differences in the sucrase subunit of the SI complex, thus rendering it more susceptible to cleavage and inactivation. However, no differences in kinetic properties of the sucrase subunit were observed between strains. Murine gamma-GA was found to account for a greater proportion of maltase activity (approximately 70%) than that observed in other species (i.e., approximately 20%). In addition, CBA/Ca mice were found to be deficient in intestinal maltase activity (approximately 60 U/g) compared with the other strains studied (approximately 300 U/g).

Analysis of Variance↗
PubMed1990

Identification of two variant short chain acyl-coenzyme A dehydrogenase alleles, each containing a different point mutation in a patient with short chain acyl-coenzyme A dehydrogenase deficiency.

Two distinct mutant alleles of the precursor (p) short chain acyl-CoA dehydrogenase (SCAD) gene were identified in a SCAD-deficient patient (YH2065) using the polymerase chain reaction to amplify cDNA synthesized from total RNA from her fibroblasts. Cells from this patient had previously been shown to synthesize a labile variant SCAD in contrast to the normal stability of variant SCADs in two other SCAD-deficient cell lines (Naito, E., Y. Indo, and K. Tanaka. 1989. J. Clin. Invest. 84:1671-1674). In the present study, both mutant alleles of YH2065 were found to contain a C----T transition, one at position 136 and the other at position 319 of the coding region of pSCAD cDNA. Clones of cDNA amplified from this region showed only one of the C----T transitions, indicating that each mutation was derived from different pSCAD alleles. Each of these mutations altered a known restriction endonuclease site, and restriction analysis of additional cDNA clones from amplified mutant cDNA and Southern blotting of mutant genomic DNA confirmed the presence of two unique mutant alleles in YH2065, indicating YH2065 is a compound heterozygote. These C----T transitions result in the substitution of Arg-22 and Arg-83 of the mature SCAD with Trp and Cys, respectively.

Acyl-CoA Dehydrogenases↗
PubMed1993

Two-step polymerase chain reaction for diagnosis of scrub typhus and identification of antigenic variants of Rickettsia tsutsugamushi.

Two-step polymerase chain reaction (nested PCR) method was examined for the diagnosis of scrub typhus. Primers were derived from the type-specific antigen (TSA) gene DNA sequences of Rickettsia tsutsugamushi, Gilliam strain. These primers served to produce rickettsia-specific products in the amplification of template DNA prepared from all serovariants, Gilliam, Karp, Kato, Kawasaki, Kuroki and Shimokoshi strains, and the fragments of product after digestion with several kinds of restriction endonuclease showed the respective patterns to strain in acrylamide or agarose gel electrophoresis. The rickettsia-specific DNAs were also derived, by this nested PCR, by amplifying DNA from patients' bloods and mites from endemic areas, and the serotype of rickettsiae infected to these hosts could be identified from fragment patterns of the amplified products observed after endonuclease treatment. These results indicate that this PCR is sensitive and specific method not only for detection of rickettsial DNA in patient specimens and in mites, but also for the typing of rickettsiae infected to these hosts.

Animals↗
PubMed2026

Identification of candidate variants in plasma associated with early versus late disease progression under anti-PD-1 therapy in metastatic NSCLC.

BACKGROUND: Immune checkpoint inhibitors (ICIs), including anti-programmed cell death protein 1 (anti-PD-1) antibodies, have significantly improved outcomes in patients with metastatic non-small cell lung cancer (mNSCLC). However, substantial heterogeneity exists in clinical benefit, with some patients exhibiting early progression (EP) and others late progression (LP). To date, no biomarkers of EP versus LP disease have been implemented in clinical practice. Circulating tumor DNA (ctDNA) analysis represents a minimally invasive strategy for identifying such biomarkers. In this proof-of-concept study, we evaluated the performance of the TruSight Oncology 500 ctDNA (TSO500 ctDNA) panel and explored its feasibility to identify candidate variants associated with early and late disease progression under anti-PD-1 therapy. METHODS: Baseline ctDNA from eight mNSCLC patients treated with pembrolizumab was extracted and sequenced using the TSO500 ctDNA assay, a 523-gene targeted next-generation sequencing panel. Patients were classified according to their response as LP or EP. Variant calling was performed using the DRAGEN Bio-IT platform, and variants were annotated and clinically interpreted using the Clinical Genomics Workspace (CGW; PierianDx) according to Association for Molecular Pathology (AMP)/American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines. Survival outcomes were assessed using Kaplan-Meier and log-rank tests. Performance of ctDNA variants was evaluated using receiver operating characteristic (ROC) curve analysis, and multi-gene models were assessed using leave-one-out cross-validation with penalized logistic regression. RESULTS: All patients harbored detectable variants, including SNVs (100%), MNVs (87.5%), deletions (75%), and insertions (62.5%). Tier I variants were identified in 37.5% of patients, while all cases showed tier II and multiple tier III alterations. TP53 variants were associated with poorer outcomes under anti-PD-1 therapy. Individual gene alterations in TP53, ERBB3, SMC1A or LATS1 showed moderate discriminatory performance between LP and EP patients; however, combination of mutated genes improved apparent discrimination. Notably, specific two-gene combinations (SMC1A + LATS1 or ERBB3 + LATS1) showed the highest discriminatory performance between LP and EP patients in this exploratory cohort. CONCLUSIONS: This study demonstrates the feasibility and analytical performance of the TSO500 ctDNA panel and provides hypothesis-generating evidence that plasma gene variants may be useful to evaluate early versus late disease progression in patients with mNSCLC receiving immunotherapy.

TruSight Oncology 500↗
PubMed1984

Identification of beta variant hemoglobins by DNA restriction endonuclease mapping.

An alternative method for identifying beta variant hemoglobins is described. Computer analysis of restriction sites was used to predict which beta variants could be detected by DNA mapping. 61 of 217 variants were shown to have changes in restriction fragment patterns which were useful markers for the abnormal hemoglobin. A further 25 could be identified by polyacrylamide electrophoresis. Implications of DNA analysis in diagnosis of variant hemoglobins are discussed.

Base Sequence↗
PubMed1986

Ultrathin-layer polyacrylamide gel isoelectric focusing for the identification of hemoglobin variants.

An ultrathin-layer polyacrylamide gel isoelectric focusing method for typing hemoglobin is described. This method, which uses Pharmalyte pH 6.7-7.7 and an electrode wick distance of 5.0 cm, clearly resolves the A, F, S, C and a number of rare variant allelic products. The technique is ideal for rapid screening of a large number of fresh blood and/or bloodstained samples.

Blood Protein Electrophoresis↗
PubMed1990

Identification of metaplastic variants generated by transfection of a nonmetastatic rat mammary epithelial cell line with DNA from a metastatic rat mammary cell line.

The rat mammary 37 epithelial cell line yields non-metastasizing adenomas in syngeneic rats. On cellular DNA transfection, a series of cell lines have been isolated that grow in drug-selective medium. Representative transfected cell lines all yield tumors in rats that consist predominantly of spindle cells, but two also contain epithelial-like cells and glandlike elements (C18P, C19P). Immunocytochemical staining for milk fat globule membrane antigens, human callus keratin, and laminin confirms the identity of the epithelial cells and suggests a (myo)epithelial origin for the spindle cells. Some of the transfected cell lines also generate well-differentiated metaplastic elements in their tumors. One cell line (CT4-41) produces rhabdomyoblastic and possibly smooth-muscle-related elements; two (C18P, C19P) produce squamous metaplasia and sebaceous elements; and two (CL1-31, C11P) produce cartilaginous elements. The identities of the heterologous elements are confirmed by immunocytochemical staining for myoglobin, actin (CT4-41), keratin (C18P, C19P), type II collagen, and type II keratan sulfate (CL1-31). Those cell lines that have acquired the ability to metastasize from subcutaneous sites (CT4-41, C18P) reproduce the same metaplastic elements in their metastases. Thus, a cloned mammary epithelial cell line can be made to generate many of the well-differentiated, heterologous elements observed in human breast carcinomas, and this change is often associated with the rat cells acquiring metastatic properties.

Animals↗
PubMedDate not supplied

Peptide mapping using thermospray LC/MS detection: rapid identification of hemoglobin variants.

A rapid and easily interpreted method for peptide mapping is demonstrated with hemoglobin A and three variants. Digests of the globin chains are generated in columns containing immobilized trypsin. The resulting protein fragments are resolved by reverse phase HPLC and then analyzed by thermospray mass spectrometry. The entire process is carried out on-line. The result is a chromatographic trace which is two dimensional; in addition to the standard elution pattern, the individual mass spectra collected for each peak contain information about their identity and their purity. In two of the variants used, hemoglobin C and hemoglobin Baylor, the exact nature of the amino acid substitution could be determined unambiguously in a single analysis, from the masses of the new tryptic peptides observed. In the third case, hemoglobin S, the mass of the peptide containing the amino acid replacement is consistent with two separate sites of substitution. This ambiguity was resolved in a second analysis, using immobilized carboxypeptidase Y, prior to mass spectral analysis. The resulting partial digest permits reconstruction of the critical sequence region and correct assignment of the allelic site.

Amino Acid Sequence↗
PubMed1987

Identification of molecular variants of p210bcr-abl in chronic myelogenous leukemia.

The aberrant abl protein product of a chronic myelogenous leukemia (CML) blast crisis cell line (K562) and of five Philadelphia chromosome-positive CML patients in blast crisis were analyzed by an immune complex kinase assay using two antipeptide sera generated against the hydrophilic domain of v-abl and a region within the third exon of the breakpoint cluster region (bcr) respectively. Both the anti-abl and anti-bcr sera detected a 210 kd band in extracts derived from K562 cells and from two CML patients with myeloid blast crisis. p210 was detected by the anti-abl but not the anti-bcr sera in three CML patients with myeloid (one patient) and lymphoid (two patients) blast crisis, indicating the absence of bcr exon 3 in this protein. Southern blot analysis on DNA derived from one of the patients in the latter group was consistent with the break on chromosome 22 occurring 5' to bcr exon 3. Our observations demonstrate that the Philadelphia translocation results in the generation of a chimeric bcr-abl protein with at least two molecular variants, both of which are enzymatically active as protein kinases.

Blast Crisis↗
PubMed1986

Inherited thrombocytopenia, elevated serum IgA and renal disease: identification as a variant of the Wiskott-Aldrich syndrome.

A kindred with X-linked hereditary thrombocytopenia in association with elevated serum IgA and a mild nephropathy is described. Thirteen males with thrombocytopenia were identified in three generations amongst 49 family members who were available for screening. Serious infective sequelae were absent but five patients had suffered from severe eczema since infancy. The platelet volume as measured by an automated counter and electron microscopy was reduced compared with normal and in vitro tests demonstrated minor abnormalities of immune function in three patients. The disorder is identified as a novel variant of the Wiskott-Aldrich syndrome and comparisons are made with previously described kindreds showing different patterns of expression.

Adult↗
PubMed1979

[A case of succinylcholine apnea. Identification of genetic variants].

A report of a case of prolonged apnoea after succicurarium in a female patient with abnormal pseudo-cholinesterases is followed by a presentation of a study of pseudo-cholinesterases in 12 members of the family. The value of the use of 4 tests of inhibition of cholinesterase activity in determining the genotype of an individual is emphasised.

Apnea↗
PubMed1984

Identification of genetic variants in erythrocyte lysate by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis followed by silver-staining has been employed to study 27 red cell lysates for genetic variation. Forty-six polypeptides selected without respect to variability were considered suitable for scoring. Only 23 of the total of 1,242 polypeptides could not be scored unambiguously. Of the remaining 1,219 polypeptides, 38 exhibited the combination of a normal and a variant polypeptide. All variants were present in either the father or the mother of the subjects. The observed index of heterozygosity was 3.1% +/- 0.5%.

Adult↗