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J Roth

Publications and source records attributed to J Roth.

At least 361 records · Page 20Linked to original sources
PubMed1991

Ligase-free subcloning: a versatile method to subclone polymerase chain reaction (PCR) products in a single day.

Often, it is convenient to subclone polymerase chain reaction (PCR) products into a plasmid vector for subsequent replication in bacteria, but conventional subcloning methods often fail. We report a rapid and versatile method to subclone PCR products directionally into a specific site of virtually any plasmid vector. The procedure requires only four primers, does not require DNA ligase, and may be accomplished in a single day. Ligase-free subcloning is performed by incorporating into the PCR primers sequences at the 5' ends that result in PCR products whose 3' ends are complementary to the 3' ends of the recipient linearized plasmid. The PCR product and the linearized plasmid are spliced together in a second PCR reaction in which Taq polymerase extends the complementary overlapping 3' ends (ligation by overlap extension). Denaturation followed by heterologous reannealing and cyclization results in a cyclic recombinant plasmid with two nicks that may be used directly to transform competent Escherichia coli. In our hands, ligase-free subcloning is rapid, and offers many advantages over existing strategies.

Bacteriophage lambda↗
PubMed1991

Expression of polysialylated N-CAM during rat heart development.

Developmental patterns of immunoreactivity for the neural cell adhesion molecule (N-CAM) and alpha 2.8-linked polysialic acid (PSA) were identified in embryonic and postnatal rat heart by immunocytochemistry and immunoblotting. Polyclonal antibodies against N-CAM and a monoclonal antibody which recognises only polymers of PSA with a chain length greater than eight units were used. Gold- and alkaline-phosphatase-labelled antibodies were used for detection. The N-CAM polypeptide isoform pattern seen by immunoblotting after endoneuraminidase treatment changed as development progressed. During embryonic development a 160-kDa polypeptide isoform was predominant. Around birth, 130-, 160- and 170-kDa polypeptide isoforms were found. The expression of the 130- and 170-kDa isoforms diminished until finally, in the adult, weak immunoreactivity for bands of 120-, 130- and 160-kDa was seen. In general the extent and intensity of PSA and N-CAM immunostaining in rat heart increased until birth and declined thereafter. Early in development prominent immunostaining for PSA and N-CAM was seen in the epicardium while later in development this area was only weakly stained. Initially myocardial cells, endocardial cells and some cells in the atrioventricular cushions were immunoreactive for both PSA and N-CAM. Later in development N-CAM immunostaining was more prominent than PSA immunoreactivity, reflecting a decrease in N-CAM polysialylation, which was also seen by immunoblotting. During innervation of the heart, nerve fibres were strongly immunostained for PSA and N-CAM, and this was the only immunostaining seen in adult heart.

Animals↗
PubMed1991

Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.

Adenocarcinoma↗
PubMed1991

Normal coding sequence of insulin gene in Pima Indians and Nauruans, two groups with highest prevalence of type II diabetes.

The nucleotide sequence of the insulin gene was determined in American Pima Indians and Micronesian Nauruans, two populations in whom the prevalence of non-insulin-dependent (type II) diabetes mellitus is the highest in the world. The insulin gene was amplified by the polymerase chain reaction to generate single-stranded DNA suitable for direct sequencing. The nucleotide sequences of the coding and adjacent regions of the insulin gene in six Pima Indians and two Nauruans with type II diabetes were identical to previously published insulin gene sequences of nondiabetic subjects.

Adult↗
PubMed1991

Polysialic acid of the neural cell adhesion molecule distinguishes small cell lung carcinoma from carcinoids.

The neural cell adhesion molecule (NCAM) exists in various types of neuroendocrine cells and their tumors. A typical feature of NCAM is polysialic acid, of which the chain length is developmentally regulated. The authors have performed a comparative immunohistochemical study on small cell lung carcinomas and bronchial as well as gastrointestinal carcinoids with the monoclonal antibody (MAb) 735 reactive with the long-chain form of polysialic acid. The small cell lung carcinomas, irrespective of their histological type, were positive for polysialic acid. Metastatic tumor cell complexes also exhibited immunostaining. The tumor cell-surface-associated immunostaining for polysialic acid was sensitive to endoneuraminidase. The mature and atypical bronchial and gastrointestinal carcinoids were not immunoreactive for polysialic acid. Cytoplasmic staining in groups of cells of carcinoids (2 of 28 cases) was due to nonspecific antibody binding, which could be prevented by increased ion strength. These data indicate that neuroendocrine tumors of the lung can be distinguished by their content of highly sialylated NCAM.

Antibodies, Monoclonal↗
PubMed1991

Polysialic acid as a marker of both immature and mature neural tissue in human teratomas.

The neural cell adhesion molecule (NCAM) is involved in cell-cell interaction during neural development. We employed a monoclonal antibody directed against the long chain polysialic acid moiety of NCAM to evaluate its usefulness as a marker of primitive neural elements in teratomas. This marker was compared with other neural markers, S-100, glial fibrillary acidic protein (GFAP), neurofilament protein (NFL), nerve growth factor receptor (NGFR), as to its effectiveness in labeled neural tissue in human teratomas. The anti-polysialic acid antibody was the only reagent that consistently marked all types of neural tissue, both mature and immature in these lesions. Immature neural elements alone have prognostic significance in teratomas. Our results indicate that anti-polysialic acid antibodies are the most sensitive and useful markers of immature neural elements in these lesions.

Antibodies, Monoclonal↗
PubMed1991

Expression of alpha 2,6-linked sialic acid residues in neoplastic but not in normal human colonic mucosa. A lectin-gold cytochemical study with Sambucus nigra and Maackia amurensis lectins.

Increased sialylation of cell surface glycoconjugates has been demonstrated in malignant tumors and shown to be correlated with the invasive and metastatic growth of colon carcinoma cells. The authors have applied the Maackia amurensis lectin, which interacts with alpha 2,3-linked sialic acid, and the Sambucus nigra I lectin specific for alpha 2,6-linked sialic acid. In human colon, alpha 2,3-linked sialic acid was detectable in normal and transitional mucosa as well as in adenomas with different degrees of dysplasia and in carcinoma. In contrast, alpha 2,6-linked sialic acid as visualized with Sambucus nigra I lectin was found only in severe dysplasia and carcinoma. Thus expression of binding sites for Sambucus nigra I lectin was associated with the occurrence of histologic features of malignancy. It is concluded that malignant transformation in human colonic epithelium is accompanied by the de novo expression of an alpha 2,6 sialyl-transferase. These findings provide the basis for more detailed studies of the possible role of cell surface glycoconjugates bearing alpha 2,6-linked sialic acid in growth behavior of human colonic epithelial cells.

Adenocarcinoma↗
PubMed1991

Cellular events associated with inflammatory angiogenesis in the mouse cornea.

The aim of this study was to establish an angiogenesis model in the mouse and to define immunohistochemically the cellular events that precede angiogenesis. After chemical cauterization of the murine cornea, neovascularization was observed within 36 hours. The cellular infiltrate was analyzed by using antibodies on cryostat and paraffin sections and by histochemical staining for mast cells. It was found that neither T lymphocytes nor mast cells nor macrophages in a more mature stage of development were part of the infiltrate that preceded the ingrowth of new blood vessels. Instead, the infiltrating cells appearing from 3 hours on were granulocytes and inflammatory monocytes, as detected by an antibody against the calcium-binding protein MRP14. The authors conclude that the induction of angiogenesis during nonspecific inflammation is associated with the early influx of myelomonocytic cells, but not with the infiltration of mature macrophages, T lymphocytes, or mast cells. This study shows that immunohistochemical analysis of cauterized murine corneas presents a useful tool for further studies on cells and cell products involved in the angiogenic process.

Animals↗
PubMed1991

RNA template-specific PCR: an improved method that dramatically reduces false positives in RT-PCR.

We report a novel modification of the reverse transcription PCR method, designated RNA template-specific PCR. With this approach, the 5' end of the first strand is tagged with a unique nucleotide sequence during reverse transcription that may then be exploited to amplify preferentially RNA-derived sequences. In our hands, RNA template-specific PCR retains the sensitivity of the traditional method, but greatly reduces the frequency of false positives, and virtually eliminates carryover contamination from PCR products amplified in previous experiments.

Animals↗
PubMed1991

Embryonic chicken lens cells cultured in reconstituted basement membrane: an experimental model to maintain the epithelial phenotype in culture.

The action of a reconstituted basement membrane has been studied on primary cultures of embryonic lens cells. When a solution of this matrix (Matrigel) was included in the culture medium, a high percentage of cells maintained the epithelial phenotype, judged by electron microscopy criteria, in contrast to the differentiated state induced by serum. Complete matrix stimulated by 6-fold the incorporation of 3H-thymidine into the cells, while one of its defined components, laminin, only had a 2-fold stimulatory effect. Thus, the basement membrane may stimulate mitogenesis and play a role complementary to that of growth factors in development.

Animals↗
PubMed1991

[Studies on the influence of air cavities during irradiation with high energy electrons].

Radiation therapy of tumors is often performed with high energy electrons. In certain situations air cavities in the irradiation volume can change the dose distribution. The effects of air cavities on the dose distribution are investigated. The results calculated with a therapy planning system and measured in a phantom are described and compared with one another and with the literature.

Humans↗
PubMed1990

RNA template-specific polymerase chain reaction (RS-PCR): a novel strategy to reduce dramatically false positives.

We report a novel modification of the reverse transcription-polymerase chain reaction method that we have dubbed RNA template-specific PCR (RS-PCR). With this approach, the 5' end of the first strand is tagged with a unique nucleotide sequence during reverse transcription which may then be exploited to amplify preferentially RNA-derived sequences. In our hands, RS-PCR greatly reduces the frequency of false positives and virtually eliminates carryover contamination from DNA fragments amplified in previous experiments.

Animals↗