Reaching out to hospitals and physicians.
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Economic Competition↗
Biomedical subjects
L Hellman
Publications and source records attributed to L Hellman.
Cloning and structural analysis of MMCP-1, MMCP-4 and MMCP-5, three mouse mast cell-specific serine proteases.
Here we present the cloning of three novel mouse mast cell-specific serine proteases, MMCP-1, MMCP-4 and MMCP-5. A region of approximately 4 kb covering the five exons and 930 bp 5' and 280 bp 3' flanking sequences of the gene for MMCP-1 was characterized by nucleotide sequence analysis. A comparison with the corresponding region of the rat mucosal mast cell-specific protease RMCP-II is presented. cDNA clones for the mast cell proteases MMCP-4 (950 bp) and MMCP-5 (1098 bp) were isolated from a cDNA library of a connective tissue mast cell-like mouse mastocytoma cell line. All three proteases were found to belong to the family of chymotrypic serine proteases as deduced from the absence of the Asp 189 which is characteristic for all serine proteases having cleavage specificities similar to pancreatic trypsin. The active polypeptides, excluding possible post-translational glycosylations, have an Mr of 25-26 kDa. Analysis of the amino acid composition reveals a positive net charge for all three proteases MMCP-1 +3, MMCP-4 +18 and MMCP-5 +12). Based on their high sequence identity (88%) and high positive net charges (+18 and +18, respectively) we assume that the MMCP-4 is the mouse homolog to rat RMCP-I. Probes specific for each of these three highly homologous protease genes have been generated by subcloning of fragments of approximately 100 bp in length, originating from the 3' ends of the mRNA into plasmid vectors. Northern blot analysis of mRNA from a number of murine cell lines shows gene expression of these proteases to be specific for the differentiation stage of the mast cell. The MMCP-1 is expressed only at the mucosal mast cell stage and 5 only in mast cells of the connective tissue mast cell stage. These serine proteases may serve as highly specific markers in the analysis of mast cell heterogeneity, differentiation and function.
Amino Acid Sequence↗
Enhancement of IgE synthesis in the human myeloma cell line U-266 with an IgE binding factor from a human T-cell line.
An IgE-binding factor(s) (IgE-BF(s] was partially purified from the supernatant of human HTLV-II carrying T-cell line MO. This IgE-BF(s) was shown to increase the IgE synthesis in the human myeloma cell line U-266, but did not affect its viability or growth. The effect of the IgE-BF(s) was dose-dependent and selective for IgE protein synthesis as beta 2-microglobulin synthesis in the U-266 and the immunoglobulin production in the U-1958 IgG-secreting human myeloma cell line were unaffected. The IgE-BF(s) increased the production of the epsilon heavy chain but not the lambda light chain production. The IgE-BF(s) was distinct from IL-1 beta, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-alpha, -beta, -gamma, M-CSF, and fragments of CD23.
Cytokines↗
Cloning and structural analysis of a gene encoding a mouse mastocytoma proteoglycan core protein; analysis of its evolutionary relation to three cross hybridizing regions in the mouse genome.
Serglycin (SGC) is a Ser-Gly-repeat-containing protein, used as a proteoglycan core protein in the parietal yolk sac and in mast cells, where glycosaminoglycan side chains are attached to the serine residues of the repeat region. In this article, the structure of the gene SGC encoding mouse SGC is reported. The gene is divided into three exons, which are all contained within a region of approximately 13 kb. Nucleotide (nt) sequence analysis was carried out on a region of 1.2 kb upstream from the first exon. The region containing the two promoters (active in parietal yolk sac and in mast cells, respectively) was analyzed for the presence of recognition sites for known DNA-binding proteins. A number of sequences closely related to known recognition sites were found in both promoters, and one consensus octamer-binding site could be identified in the putative yolk-sac promoter. Multiple regions in the mouse genome hybridizing with DNA fragments covering the Ser-Gly repeat region have previously been described, and it has been suggested that these loci may represent other proteoglycan core proteins. Analysis of nt sequence was carried out on three out of the more than 15 of these regions present in the mouse genome. However, none of the clones analyzed was found to have any open reading frame in the region of cross-hybridization which possibly could code for a SGC protein. Instead, one of the clones was found to contain an exon encoding a highly basic protein, unrelated to SGC. Hence, no evidence was found for a multigene family of Ser-Gly-repeat-containing proteoglycan-encoding genes.
Aggrecans↗
Sodium-24 studies in postmastectomy lymphedema.
Seven patients were studied with 24Na to determine the relative disappearance time of the isotope from the postmastectomy lymphedematous arm as compared to the normal side. The results tend to confirm previously held convictions that the edema is usually confined to the subcutaneous fat and skin. The disappearance time (T1/2) of the radioactive sodium from the muscle of the edematous side was usually comparable to that of the control side. The data also indicate that the impairment of fluid drainage from other areas, such as the lateral chest wall, that normally drain into the axilla, is impaired similarly to that of the subcutaneous fat of the arm. Operative procedures designed to relieve the edema of the arm by providing an alternate route of drainage should provide a conduit for the fluid to an area that does not normally drain to the axilla of the affected side.
Drainage↗
Primary structure of a mouse mastocytoma proteoglycan core protein.
The complete nucleotide sequence of a mouse mastocytoma proteoglycan core protein mRNA was determined. The mRNA, estimated to contain 1.1 kb, encodes a protein with an Mr of 16715. A 21-amino acid-residue region of the protein is composed of alternating serine and glycine residues. Southern-blot analysis of mouse genomic DNA with cDNA containing sequences corresponding to the Ser-Gly repeat region revealed more than 15 gene fragments. Hybridization with a probe corresponding to the N-terminal portion of the core protein identified two fragments, and cDNA covering the C-terminal part of the core protein and the 3' untranslated part of the mRNA hybridized to a single fragment. Antibodies against the core protein, obtained after immunization of rabbits with a fusion protein, reacted with both chondroitin sulphate proteoglycans and heparin proteoglycans produced by the tumour. In immunoblotting of a microsomal fraction from the mastocytoma, the antiserum recognized a single protein (Mr 17,000), which probably represents the core protein before glycosylation.
Aggrecans↗
Secretion of coagulant factor VIII activity and antigen by in vitro cultivated rat liver sinusoidal endothelial cells.
Different types of liver cells and a few extrahepatic cell types were analysed for the presence and production of factor VIII activity (VIII:C). Only freshly prepared suspensions of rat liver sinusoidal cells and pure monolayer cultures of rat liver endothelial cells (LEC) were found to contain and secrete detectable amounts of the coagulation factor. Secretion of VIII:C by cultured LEC was inhibited by cycloheximide and by monensin. Constant levels of VIII:C were produced for at least 48 h suggesting continuous synthesis rather than a burst release of stored material. VIII:C, as measured spectro-photometrically by conversion of X to Xa, was inhibited by anti-human VIII:C antiserum. Indirect immunocytochemistry using this antiserum gave positive staining only with LEC. Immunoprecipitation of metabolically labelled proteins in conditioned rat LEC medium with the anti human VIII:C antiserum revealed the presence of proteins of similar sizes to those reported for human VIII:C. These results indicate that rat LEC are an important site for production and secretion of procoagulant factor VIII and are not only a site for storage and release of the factor. The established conditions for synthesis of VIII:C in in vitro cultivated rat LEC should provide the means to study the regulation of VIII:C synthesis.
Animals↗
Comparative activity in vitro of cilofungin (LY 121019) with other agents used for treatment of deep-seated Candida infections.
Cilofungin (LY 121019) is a semi-synthetic lipopeptide antifungal agent that inhibits beta-1,3-D-glucan synthase activity in the cell wall of yeasts. Clinical strains of Candida species were tested for susceptibility to cilofungin and seven other antifungal agents. Candida albicans and Candida tropicalis were susceptible to cilofungin with mean MICs of 0.3 and 0.08 microgram/ml respectively. For most other species the mean MICs were greater than 1-2 micrograms/ml of cilofungin. Studies on the paradoxical growth effect observed with cilofungin in Sabouraud broth showed that, at high concentrations of cilofungin, sufficient damage occurred to make damaged cells highly susceptible to killing by fresh cilofungin. The damaged cells also had increased susceptibility to other antifungal agents to which they were normally resistant. These observations indicate that cilofungin may be a useful agent for the treatment of many invasive Candida infections, either alone or in combination with certain other antifungal agents.
Antifungal Agents↗
A rapidly evolving region in the immunoglobulin heavy chain loci of rat and mouse: postulated role of (dC-dA)n.(dG-dT)n sequences.
The nucleotide sequences of the introns that are located between the C4 exon and the first membrane exon of mouse and rat immunoglobulin epsilon-chain genes have been determined. The rat intron sequence was found to contain four separate clusters of repetitive sequences all of which consisted of (dC-dA)n.(dG-dT)n dinucleotide repeats. A comparison between this chromosomal region in mouse and rat revealed four deletions or duplications, three of which have occurred inside or at the borders of the CA clusters. Rearrangements have occurred inside or at the borders of all four repeats after the evolutionary separation of mouse and rat. The sequence comparison reveals in addition a duplication, connected to the CA repeats, which has occurred early in evolution, before the evolutionary divergence of mouse and rat. These findings suggest that (dC-dA)n.(dG-dT)n sequences are potential targets for recombination events.
Animals↗
Immunoglobulin synthesis in the human myeloma cell line U-266; expression of two immunoglobulin heavy chain isotypes (epsilon and alpha) after long-term cultivation in vitro.
A human IgE-producing myeloma has been cultivated in vitro as a continuous cell line (U-266) since 1968. Analysis of immunoglobulin production during early passages of the cell line demonstrated a high synthesis rate of monoclonal IgE. Analysis of late passages, cultivated after 1980, revealed a 3-6-fold lower rate of IgE secretion. This decrease was accompanied by the appearance of small amounts of IgA in the culture medium together with IgE. RNA was extracted from a late passage of U-266 and analyzed by Northern blotting, using epsilon and alpha-specific oligonucleotides as hybridization probes. The results showed the presence of epsilon as well as alpha-specific mRNA. Moreover the results demonstrated that the latter mRNA was derived from the alpha 2 locus and that the epsilon and the alpha 2-specific mRNA contained the same V region sequences. Southern blot analysis of DNA from the late passage of the U-266 cell line failed to reveal a recombinatory switch from the epsilon locus to the alpha 2 locus. The expression of alpha 2 is thus likely to be caused by differential splicing rather than by an isotype switch at the DNA level.
Cell Line↗
The NF-kappa B-binding site mediates phorbol ester-inducible transcription in nonlymphoid cells.
The mouse immunoglobulin kappa light-chain enhancer can interact with at least three independent nuclear proteins. One of these proteins, NF-kappa B, is constitutively present only in nuclear extracts derived from B cells and plasma cells. A DNA-binding protein with the same sequence specificity (and therefore presumed to be NF-kappa B itself) can be induced in pre-B cells, T cells, and nonlymphoid cells by phorbol 12-acetate-13-myristate (PMA); however, it is not clear whether the induced factor can activate transcription in nonlymphoid cells as NF-kappa B does in B cells. In this paper we show that multimerization of a fragment of the mouse kappa enhancer that carried only the binding site for NF-kappa B behaved like a B-cell-specific regulatory element. Furthermore, this unit served to activate transcription in nonlymphoid cells after treatment with PMA (but not with cyclic AMP derivatives), and the kinetics of transcription activation correlated well with the kinetics of factor induction. Thus, the induced DNA-binding activity appeared to be functionally indistinguishable from that of NF-kappa B.
B-Lymphocytes↗
The immunoglobulin lambda locus in rat consists of two C lambda genes and a single V lambda gene.
The immunoglobin lambda locus of the rat has been studied. Germ-line V lambda and C lambda genes were isolated from recombinant-phage libraries and characterized by nucleotide sequencing. The results showed that the lambda locus of the rat contains one single V lambda gene and two C lambda genes, thus representing one of the least complex lambda loci so far characterized. The two C lambda genes are separated by a spacer approx. 3 kb long. Two J segments are located at the 5' side of each C lambda gene. One of the C lambda genes (C lambda 1) probably represents a pseudogene, as the J lambda 1 segments have non-functional recombination and splice signals. The organization of the rat lambda locus resembles that of mouse, except that only one cluster is present in the rat. Thus since the evolutionary separation of the rat and mouse species ten MYR ( = 10(6) years) ago, either one cluster has been lost from the rat, or duplicated in the mouse.
Animals↗
TPA-induced differentiation of chronic lymphocytic leukemia cells: studies on mu-chain expression.
The regulation of IgM synthesis and secretion was studied in chronic lymphocytic leukemia cells, with a phenotype roughly similar to peripheral resting B cells, during phorbol ester (12-O-tetradecanoylphorbol-13-acetate)-induced differentiation. TPA treatment caused a 20 times increase in total RNA synthesis and 20 to 50 times increase in the protein synthesis as compared to control cells. Morphologically, 70-90% of the cells reached the lympho- or plasmablast stage of differentiation. In control culture cells, approximately equal amounts of mRNA coding for secretory (s) and membrane (m) mu-chains were found. The micron message was translated as surface IgM expression was detected. A posttranscriptional regulation of microsecond synthesis appears to exist, since only low amounts of cytoplasmic mu-chains were detected by immunoprecipitation and SDS-PAGE, and no secretion of pentameric IgM was detected as measured by an RIA. TPA induction caused a relative increase in the microseconds to microns mRNA ratio, demonstrating differentiation associated control mechanisms operating at the level of mRNA processing. The high levels of cytoplasmic microsecond-chain precursor and the efficient secretion of pentameric IgM in TPA-induced chronic lymphocytic leukemia cells indicated the presence also of posttranscriptional controls.
Aged↗
Expression of c-myc and c-fos during phorbol ester induced differentiation of B-type chronic lymphocytic leukemia cells.
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B-Lymphocytes↗
Structure and expression of kappa-chain genes in two IgE-producing rat immunocytomas.
The light chain expression in two IgE-producing rat immunocytomas, IR2 and IR162, was studied. Both immunocytomas produce light chains of the kappa type. The kappa chains were characterized at the protein level by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and amino acid (aa) sequencing. cDNA clones corresponding to the kappa-chain mRNA were also prepared and sequenced. The results showed that rat kappa chains have the same structure as their mouse counterparts with respect to signal sequence cleavage, somatic mutations in the V-J region and invariance of all the aa positions which are strongly conserved in the frame work regions of mouse V kappa chains (greater than 95% conservation). Results from studies on kappa-chain transcription lend support to the allelic exclusion model with only one functionally expressed light chain in each immunocytoma.
Amino Acid Sequence↗
Nonfunctional immunoglobulin light chain transcripts in two IgE-producing rat immunocytomas; implications for the allelic exclusion and transcription activation processes.
The rearrangement and expression of immunoglobulin light-chain genes have been studied in two IgE-producing immunocytomas, IR2 and IR162. In the IR2 tumor only one of the kappa-chain alleles is rearranged, expressing a full-length kappa-chain polypeptide. In IR162 one of the kappa-chain alleles is functionally rearranged, expressing a 1200-nucleotide (nt) long mRNA, which encodes a functional 23-kDal kappa-chain polypeptide. The second kappa-chain allele is aberrantly rearranged; i.e., a different V region is connected to a position that is located between the J cluster and the C kappa exon. Two mRNAs which are 750 and 850 nt are transcribed from the aberrantly rearranged allele, both of which appear to encode a 12-kDal polypeptide consisting of a signal sequence that is connected directly to the C region. The levels of expression from the two kappa-chain alleles are approximately the same, suggesting that no specific mechanism exists to suppress expression of a nonfunctional allele. The rat genome contains a single lambda-chain locus which includes two C-region exons. Although this locus remains in the germ-line configuration in the IR2 and the IR162 tumors, transcripts from the C lambda I and C lambda II regions were detected at a low level in both tumors. These transcripts were detected in RNA from the immunocytomas but not in rat liver RNA indicating that expression is tissue-specific. They lacked V-region sequences and resemble so-called sterile transcripts which are expressed at a low level from unrearranged mu- and kappa-chain genes.
Alleles↗
Rat immunoglobulin E heavy chain locus.
A 2100 base-pair long sequence has been established which covers all four constant domains of the rat epsilon-chain. An analysis of messenger RNA from an immunoglobulin E producing rat immunocytoma revealed two separate epsilon-chain mRNA species, 2.3 X 10(3) and 2.8 X 10(3) base-pairs long. The latter mRNA encodes the membrane binding form of the epsilon-chain. The membrane exons which are located approximately 2 X 10(3) base-pairs away from the 3'-side of the CH4 exon were also sequenced. A comparison between the rat and mouse epsilon-chains at the protein sequence level revealed an overall homology of 80% which, as expected, is considerably higher than the homology found between rat and human epsilon-chains. The fourth constant domain together with the two membrane exons exhibited the highest degree of homology, 81 to 89%. Only two differences were found when the epsilon-chains from LOU and Sprague Dawley rats were compared. The most striking difference at the nucleotide sequence level between the rat, mouse, and human epsilon genes was found within the first intron. The mouse genome contains a unique 366 base-pair long sequence in this region. The inserted sequence is repetitive and present in approximately 100 copies in the mouse genome. It is flanked by 22 base-pair long direct repeats and contains also 14 base-pair long inverted repeats, thus having properties in common with transposable elements.
Animals↗
A novel chromatographic procedure for purification of bacterial plasmids.
A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up.
Bacteria↗