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L M Hoffman

Publications and source records attributed to L M Hoffman.

At least 37 records · Page 2Linked to original sources

Vectors for in vitro synthesis of poly(A)+RNA transcripts.

An RNA expression plasmid containing a maize zein storage protein gene was modified by the addition of an oligodeoxynucleotide duplex containing oligo(A30) in the RNA sense strand to the 3' flank of the gene. By linearizing the plasmid at either side of the oligo(A) region, transcripts with or without a poly(A) tail may be synthesized. Poly(A) addition dramatically increases the translation of SP6-transcribed zein transcripts in Xenopus laevis oocytes. We describe a derivative of pSP64, pSP64A, containing the oligo(A) region, which may be useful for the transcription of polyadenylated RNAs from any template DNA.

DNA Restriction Enzymes↗

Induction of lymphomas and fibrosarcomas in nude mice after implantation of simian virus 40-transformed human meningioma.

Human cells transformed by Simian virus 40 (SV40) usually show little or no tumor formation after implantation in nude mice. In long-term studies, however, we have observed that 127 to 366 days after subcutaneous injection of SV40-transformed human meningioma cells (KJ-M2-T) into nude mice (Nu/Cox), 6 of 15 animals developed lymphomas or fibrosarcomas, usually at the site of inoculation. The induced tumors were of murine origin and were positive for SV40-T antigen. Chromosome analysis and G11 staining revealed no evidence of hybridization between human and mouse cells. No spontaneous shedding of SV40 was noted with KJ-M2-T cells in vitro; however, SV40 could be rescued after fusion of KJ-M2-T with BS-C-1 monkey kidney cells, but not with L929 mouse fibroblasts. A parallel study using SV40-transformed human fetal brain cells failed to induce tumors in nude mice despite the demonstration that infectious SV40 could also be rescued from this line after fusion with BS-C-1 but not with L-929. Subcutaneous injection of 5 X 10(3) TCID's of SV40 (strain J436) into nude mice resulted in the induction of fibrosarcomas at the injection site in 6 of 15 mice after 273 to 396 days. The induction of malignant lymphomas after implantation of SV40 transformed cells contrasted with the development of fibrosarcomas after injection of free virus. This study suggests that after subcutaneous implantation into nude mice, some SV40-transformed human tumor cell lines can serve as vectors for transmitting SV40 to murine cells causing transformation and tumor development in the host animal.

Animals↗

Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds.

The maize 15-Kd zein structural gene was placed under the regulation of French bean beta-phaseolin gene flanking regions. Agrobacterium tumefaciens-mediated transformation was used to insert the chimeric phaseolin-zein gene into the tobacco genome. Transgenic plants synthesized zein in a tissue-specific manner during the latter half of seed development. Transcription of the chimeric gene was initiated in phaseolin-derived sequences, and was terminated within the phaseolin gene 3' flanking region. Both zein- and phaseolin-derived polyadenylation signals were used in the processing of zein RNA in transgenic plant seeds. Zein accumulation, though subject to an 80-fold variation among 19 plants tested, could reach as much as 1.6% of the total seed protein in several plants. In developing tobacco seeds, zein was correctly processed by the removal of a 20-amino-acid signal peptide. Electron microscope immunogold localization of the zein expressed in embryo and endosperm tissue indicates that the monocot protein accumulates in the crystalloid component of vacuolar protein bodies. The density of gold label over the protein bodies is several fold greater in the embryo than the endosperm. Zein is found in roots, hypocotyls and cotyledons of germinating transgenic tobacco seeds.

Journal Article↗

Nucleotide sequences from phaseolin cDNA clones: the major storage proteins from Phaseolus vulgaris are encoded by two unique gene families.

The nucleotide sequences of eight partial and five full-length phaseolin cDNA clones show that phaseolin polypeptides are encoded by two distinct gene families which differ in their coding regions by the presence or absence of two different size direct repeats. The alpha-type phaseolin polypeptides are encoded by genes containing direct repeats which encode 14 additional amino acids. Aside from these differences, the alpha-and beta-type phaseolin genes show a high degree of homology (98%) which is consistent with these genes being derived from a common ancestral gene. Much of the heterogeneity found in the phaseolin polypeptides appears to be due to post-translational processing. Nucleotide sequence analysis demonstrates that the alpha-type genes contain only a few amino acid replacement substitutions and that the beta-type genes appear to contain no amino acid replacement substitutions. S1 nuclease mapping shows a complex pattern for transcriptional initiation of phaseolin mRNA. Hydropathy analysis shows that phaseolin polypeptides are predominately hydrophilic, and that the two N-glycosyl recognition sites are located in different hydropathic environments.

Amino Acid Sequence↗

Characterization of two Phaseolus vulgaris phytohemagglutinin genes closely linked on the chromosome.

A lambda 1059 library of Phaseolus vulgaris cv. 'Tendergreen' DNA was screened with a cloned lectin-like cDNA. Among the phages selected was clone lambda B10 containing two complete lectin genes in the same orientation approximately 4 kb apart. The DNA sequences of the lectin genes and their flanking regions have been determined and their transcriptional initiation sites were located by S1 nuclease mapping. On the basis of the deduced amino acid sequences and compositions and the mol. wts. of their encoded glycoproteins, the genes, dlec1 and dlec2, are predicted to encode erythro- and leucoagglutinating phytohemagglutinins (PHA-E and PHA-L), respectively. The mRNA coding regions of dlec1 and dlec2 are 90% homologous, suggesting an origin involving duplication of an ancestral gene. Both lectin genes are intronless and have at least two ATG codons in a short (11-14 bp) 5'-untranslated region. Most of their 5'-untranslated regions consist of alternating pyrimidines and purines (RY repeats). Upstream sequences are also highly conserved between dlec1 and dlec2, including stretches of nine or more alternating R and Y residues. RY repeats of such length are not found within the protein coding portion of dlec1, dlec2 or a Phaseolus lectinlike gene previously described. Overlapping double (dlec1) or triple (dlec2) polyadenylation addition signals are found and there is an unusually high degree of homology (84%) between their 3'-untranslated regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Structure of a chromosomal Phaseolus vulgaris lectin gene and its transcript.

A genomic library of Phaseolus vulgaris DNA was screened using a lectin cDNA clone that includes the entire coding region to obtain a lambda clone containing the corresponding nuclear lectin gene. Comparison of the cDNA and genomic nucleotide sequences showed that the gene has no intervening sequences. The 5' untranslated region was found to be only 10-16 base pairs long by S1 nuclease mapping. The lectin transcriptional unit is flanked by sequences which are exceptionally high (72-75%) in A + T content and its 5' flanking region contains potential transcription control elements.

Amino Acid Sequence↗

Molecular cloning of Phaseolus vulgaris lectin mRNA and use of cDNA as a probe to estimate lectin transcript levels in various tissues.

The complete amino acid sequence of a Phaseolus vulgaris cv. Tendergreen lectin was deduced from the nucleotide sequence of a cDNA plasmid. The lectin appears to be synthesized as a prepeptide consisting of a signal sequence of at least 20 residues and a mature protein of 223 amino acids. The 5' region of the lectin cDNA contains four AUG codons, one of which may initiate a 58 amino acid peptide out of phase with lectin translation. Lectin transcripts 1.1 kb long begin accumulating in cotyledons by the 9 mm stage, increasing in amount until about the 16 mm stage, after which their levels dramatically decrease. In addition, larger lectin-specific transcripts are detected in mid-maturation phase cotyledons. A 1.1 kb RNA species in leaves from plants filling their pods hybridizes with lectin cDNA, but no hybridizing RNA is found in primary leaves or roots.

Amino Acid Sequence↗

Tay-Sachs disease and normal cerebellar cells in culture: elevated levels of lysosomal enzymes in Tay-Sachs disease cells.

Tay-Sachs disease and normal fetal cerebellar cells were maintained in culture for up to four weeks. Elevated levels of the lysosomal enzymes hexosaminidase B, beta-galactosidase, and acid phosphatase were observed in the Tay-Sachs cells in long term cultures although elevated lysosomal enzyme levels were not observed in the normal cultures. Similarly, elevated lysosomal enzyme activities were observed in Tay-Sachs cultures which had been treated with Concanavalin A.

Acid Phosphatase↗

Housestaff activism: the emergence of patient-care demands.

There have been two trends within the physician housestaff movement: increased acceptance of collective bargaining and unions, and a shift from narrower economic to broader political demands, including some involving patient care. Case studies of politically active housestaff associations in New York, Chicago, and Los Angeles are used to examine the emergence of "patient-care" demands and their compatibility with collective bargaining frameworks. As house-staff have become principal providers of care to indigent populations in public hospitals, and economic cutbacks have endangered service as well as the positions of physicians, patient-care demands arise and become infused with demands for participation and control in decision-making. Common factors in the politicization of housestaff have been the contribution of activists of the sixties as leaders, and the impact of fiscal crisis and economic retrenchment in the seventies. However, the emergence and resolution of these issues has differed depending upon legal, political, historical, and organizational variations. In general, patient-care issues are supported by housestaff when they dovetail with housestaff interests. However, physician interests can diverge from those of patients, as in the case of manpower redistribution. On the whole, wages and benefits have done better than educational or patient-care demands. Educational demands have met with counterattack, and patient care, limited by the traditional scope of collective bargaining, has had to evolve indirectly, and has been hurt by long-term economic trends. Finally, national housestaff organization is limited by the wide-ranging politics and ideas of diverse regional organizations which represent different types of training institutions and career orientations.

California↗

Probable nuclear precursors of preprolactin mRNA in rat pituitary cells.

In the rat pituitary, several species of preprolactin (pPrl) mRNA larger than the mature mRNA have been reported (Maurer, R. A., Gubbins, E. J., Erwin, C. R., and Donelson, J. E. (1980) J Biol. Chem. 255, 2243-2246). We examined the size distribution of pPrl transcripts in nuclear, cytoplasmic, and nuclear poly(A)-containing RNA from rat pituitary tumor MtTW10 and from the pituitaries of diethylstilbestrol-treated Fischer 344 X Holtzman rats. RNA was electrophoresed in agarose/2.2 M formaldehyde horizontal slab gels, transferred to diazobenzyloxymethyl paper or to nitro-cellulose paper, and hybridized with a nick-translated recombinant DNA probe containing rat pPrl cDNA sequences. Up to 8 putative precursor mRNA molecules were detected which were from 2 to 13 times the length of mature pPrl mRNA. Three of these RNA species were larger than the maximum size of putative precursors previously reported. These probable precursors were nuclear in origin and polyadenylated, at least in the case of pituitary tumor cells. The largest molecule, 14 kilobase pairs long, may approximate the size of the initial transcript of the prolactin gene.

Animals↗

Long-term intracellular retention of hexosaminidase A by Tay-Sachs disease brain and lung cells in vitro.

Enzyme-replacement treatment for metabolic storage disorders has been widely studied using model cell culture systems. This study determines the long-term fate of human hexosaminidase A supplied to Tay-Sachs disease brain and lung cells. Hex A retention studies showed that the incorporated Hex A is retained in undiminished quantity by TSD lung cells maintained in stationary culture for 14 days. Tay-Sachs disease brain cells similarly followed for 28 days in stationary culture showed an initial reduction in Hex A for 3 days, after which the Hex A level stabilized and remained relatively constant for the next 25 days. Hexosaminidase B isoenzyme was found to accumulate in both cell lines during extended cultivation, despite the observation that significant amounts were excreted into the extracellular environment. The demonstration of long-term intracellular retention of exogenously supplied therapeutic enzyme by the target cells offers additional evidence for the feasibility of an enzyme-replacement approach for study and treatment of lysosomal storage disorders.

Brain↗

Tay-Sachs disease brain cells in culture: mobilization of stored GM2 after concanavalin A-mediated uptake of hexosaminidase A.

A human Tay-Sachs disease (TSD) fetal-brain-cell line is a useful model for the disease since the cells lack hexosaminidase A and accumulate the ganglioside, GM2. This brain-cell line was used to assess the effect of hexosaminidase A treatment on GM2 storage material. Entry of placental hexosaminidase A into the cells was obtained by pretreatment of the cultures with concanavalin A. Cells were analyzed periodically during six days. During the course of the experiment, GM2 in the cells decreased by approximately 50%. A substantial amount of hexosaminidase A was maintained in the cultures throughuot the experiment. This strategy was successful in mobilizing stored GM2 in TSD brain-cell cultures. Therefore, the activating factor needed for hexosaminidas A activity must be present in TSD-cultured brain cells.

Cell Line↗

Enzyme replacement treatment for Tay-Sachs disease brain cells in culture utilizing concanavalin A-mediated hexosaminidase A uptake: biochemical and morphological evidence of GM2 mobilization.

When Concanavalin A (Con A) is bound to the cell membrane, it functions as an artificial enzyme receptor, mediating the binding and intracellular incorporation of significant amounts of exogenous hexosaminidase A (Hex A) into Tay-Sachs disease (TSD) glia cells. The treated cells retained almost 50% of incorporated Hex A activity after 3 days incubation in Hex A free medium. Hex A was released from Con A within the cell and was available as free enzyme. Biochemical analysis of gangliosides in Con A and Hex A treated cells depicted a greater than 50% reduction in stored GM2 ganglioside and a fourfold reduction in GM2 label (14C) when compared to controls. Ultrastructural evidence of GM2 breakdown is presented which supports the biochemical and labeling studies.

Cell Membrane↗

Gangliosides containing glucosamine and galactosamine in transformed Tay-Sachs disease and normal human brain cell lines.

Human SV-40 transformed brain cell lines derived from Tay-Sachs disease (TSD) and normal fetal cerebra were grown in culture and analyzed for their ganglioside content. Both the TSD and normal cells contained GM3, GM2, and a novel triheoxyl N-acetylglucosamine-containing ganglioside. In order to increase tumorigenicity, the cells were cloned on soft agar. The cloned cells still contained GM3, GM2, and the N-acetylglucosamine-containing ganglioside. The per cent distribution of gangliosides in the TSD and normal SV-40 transformed cell lines was surprisingly similar despite the fact that the TSD transformed cells still lacked hexosaminidase A, the isoenzyme which is required to break down GM2.

Animals↗

Differential activities of glycolipid glycosyltransferases in Tay-Sachs disease: studies in cultured cells from cerebrum.

Four different glycolipid:glycosyltransferase activities involved in the biosynthesis in vitro of gangliosides and blood group-related glycosphingolipids have been tested in a simian virus 40-transformed glial cell culture derived from the cerebrum of a fetus with Tay-Sachs disease (TSD). The TSD cultured brain cells contained little activity of either UDP-Gal:GM2(beta 1-3)galactosyltransferase (GalT-3; EC 2.4.1.62), which catalyzes the formation of GM1a from GM2 (tay-Sachs) ganglioside, or GDP-Fuc:nLcOse4Cer (alpha 1-2)fucosyltransferase (FucT-2; EC 2.4.1.89), which catalyzes the formation of H1 glycolipid from nLcOse4Cer. These cells contained a potent inhibitor of the second reaction (catalyzed by a Golgi-rich membrane fraction from bovine spleen), whereas no inhibition of the first reaction (catalyzed by a membrane fraction from 14-day-old embryonic chicken brain) was observed. The activity of UDP-Gal:LcOse3Cer(beta 1-4)galactosyltransferase (GalT-4; EC 2.4.1.86) was 30- to 80-fold higher than the activity of GalT-3. The presence of CMP-AcNeu:nLcOse4Cer sialyltransferase activity and the absence of either GalT-3 or FucT-2 suggested a probable pathway for the synthesis of sialylneolactotetraosylceramide [GM1b(GlcNAc)] in addition to a specific blockage of GM1a ganglioside synthesis from GM2 in these TSD transformed cells.

ABO Blood-Group System↗