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Biomedical subjects

D Amsterdam

Publications and source records attributed to D Amsterdam.

At least 55 records · Page 3Linked to original sources
PubMed1981

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↗
PubMed1980

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↗
PubMed1980

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↗
PubMed1980

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↗
PubMed1979

Simple inoculum standardizing system for antimicrobial disk susceptibility tests.

A collaborative study was undertaken to evaluate a simple, convenient device which expedites inoculum standardization for antimicrobial disk susceptibility tests. The Inocupac system (Minnesota Mining & Manufacturing [3M] Co., St. Paul, Minn.) was used to perform disk tests in parallel with the standard Bauer-Kirby method. Five investigators tested 100 selected isolates, each in triplicate. Inter- and intralaboratory precision of both disk procedures was essentially comparable. The Inocupac system failed to consistently provide satisfactory growth with some streptococci, but when testing common gram-negative bacilli, staphylococci, and most enterococci, the Inocupac system gave zones about the same size (+/-2 mm) as the Bauer-Kirby procedure. Interpretive agreement between the two test systems varied from 90 to 99% with different antimicrobial agents, and repeated tests with the Bauer-Kirby method demonstrated the same degree of interpretive agreement. The Inocupac system is a valid alternative method for inoculating disk susceptibility tests.

Anti-Bacterial Agents↗
PubMed1979

Cytology, growth characteristics and cellular alterations following SV40-induced transformation of human foetal brain cells derived from a Gm2 gangliosidosis and control.

Cell cultures were derived from the cerebra of a control and a Gm2 gangliosidosis (Tay-Sachs disease (TSD)) foetus. Both cell lines were identified as astrocytic, based on the ultrastructural demonstration of glial fibres. The control culture exhibited morphological differentiation when exposed to dibutyryl cAMP, a finding which was not observed with the TSD cells. The TSD culture demonstrated the pathobiological features of the disease, which included the absence of hexosaminidase A, increased concentration of Gm2 ganglioside and the detection of membranous cytoplasmic bodies by electron microscopy. Control and TSD cells were exposed to SV40 virus, resulting ultimately in the isolation of transformants which differed from the parental cell types in morphology, growth rate and greatly accelerated cell death. Both control and TSD cell lines have been in propagation for over 200 subcultures and the transformants were identified as astrocytic, based on the retention of characteristic glial fibres. The control culture demonstrated a chromosome range of 34--63, with a mean of 47. In contrast, the TSD transformants exhibited a range of 50--107 and a mean of 74. Transformed lines retained their parental hexosaminidase isoenzyme profiles; Hex A and B in control, and Hex B in TSD cells. Membraneous cytoplasmic bodies persisted in the TSD line. Neither line could be induced to differentiate after exposure to cAMP. Additionally, they had a population doubling time of under 85 h and failed to release infectious virus particles. Significant alterations in the total quantity and distribution profile of gangliosides were noted following viral transformation. A large increase in the percentage of Gm3 and a more modest increase in Gm2 were detected. In contrast, transformed lines were characterized by substantial reduction in the percentage of glucosamine-containing Gm2 and polysialoganglioside. Additionally, cultures exhibited a characteristic reduction in ganglioside content after transformation. The in vitro transformation of human brain cells has resulted in the derivation of permanent astrocytic lines which are, by virtue of their rapid growth rate and long-term survival, uniquely suited and adapted to the large scale in vitro production of substantial quantities of cells required for extensive biochemical study. Significantly, those characteristics which are unique to the Gm2 gangliosidosis storage disease have been retained in a permanent model CNS cell line.

Brain↗
PubMed1977

Light and electron microscopic studies of "nude" mice CNS after subcutaneous administration of the E variant of the encephalomyocarditis (EMC) virus.

Sixteen 3 month old "nude" mice, 24 of their litter mates and 30 Swiss mice were injected subcutaneously with 0.1 ml suspension of the E variant of the encephalomyocarditis (EMC) virus. While the mortality rate of the litter mates and Swiss mice during 5-7 days after inoculation was more than 40%, none of the "nude" mice died during the experiment. The surviving animals were sacrificed at 24 h intervals from day one to seven days after injection. Brain suspensions assayed for the presence of the virus yielded significant titers at 24 h in all groups, which increased during 7 days. The litter mates and Swiss mice showed proliferation of lymphocytes and microglial cells in the perivascular areas of the brain during the fifth to the seventh day. The "nude" mice, on the other hand, displayed no perivascular lymphocytic infilteration during the same periods. Ultrastructurally, all groups showed aggregates of ribosomes in the cytoplasmic matrix on the third day, which became enlarged in size on the 5th day. At 7 days, both litter mates and Swiss mice showed an increased number of necrotic cells, while these changes were not observed in the "nude" mice. These findings suggest that the high mortality rate in immunologically normal mice was related to the efforts of T cells to eliminate virus-infected cells and to produce extensive necrosis, while T cell-depleted animals showed good survival rates.

Animals↗
PubMed1977

API computer profiles: correlation of API 20E with API 10S.

A comparison of identifications of 201 clinical isolates with the 21-test API 20 Enteric kit and the subset of tests in API 10S indicated 85.6% agreement at the species level and 93.5% agreement at the genus level.

Bacteriological Techniques↗
PubMed1976

GM2 ganglioside in fetal Tay-Sachs disease brain cultures: a model system for the disease.

Cultured cells derived from Tay-Sachs disease (TSD) fetal cerebellum were shown to accumulate GM2 ganglioside when compared with control cultures. In contrast, fibroblasts derived from TSD fetal lung do not contain GM2. GM2 was identified by thin-layer chromatography (TLC) and confirmed by gas-liquid chromatography (GLC). Unlike fetal TSD brain, the cell cultures established from fetal TSD brain have high concentrations of globoside and GD3 and little or no asialo GM2(GA2). The GM2 and related glycosphingolipids in these cultured cells contain a high percentage of C24:0 and C24:1 fatty acids. In spite of these differences, this TSD cell strain is unique in that it accumulates GM2, and can therefore serve as a useful in vitro model for the study of TSD.

Cells, Cultured↗
PubMed1976

MORLUC numeric system for the identification of Enterobacteriaceae.

Foul hundred eighty-six members of the Enterobacteriaceae representing nine genera were identified by conventional methods, and the results were compared with MORLUC (Biotrol Company Inc., Jamaica, N.Y.). MORLUC, an acronym for melibiose, ONPG (o-nitrophenyl-beta-galactopyranoside), rhamnose, lysine decarboxylase, urease, and citrate, are six prepackaged reagent-impregnated paper loops which are sealed within a plastic packet. The hydrogen sulfide reaction obtained from a triple sugar iron slant is coupled with MORLUC results and is readily converted into a three-digit numerical code, which is referenced on a preprinted single page listing. Additionally, the triple sugar iron is used to confirm the glucose fermentation by an unknown isolate. Comparisons of individual MORLUC tests and standard methods results in a better than 92% agreement, except for unrease. Four hundred sixty-six of the 486 bacterial isolates, or 96% of the strains which were numerically identified by MORLUC, agreed with conventional diagnoses.

Carboxy-Lyases↗
PubMed1975

Ultrastructural alterations of tissue cultures from human fetal brain infected with the E variant of EMC virus.

Explant outgrowths from human fetal brain were infected with 104 plaque forming units of the E variant of the encephalomyocarditis virus. Ultrastructurally, the majority of the cultured cells were astrocytes containing a moderate amount of glial fibrils. The earliest alterations at 44 hrs after infection of the culture consisted of dilatation of the rough endoplasmic reticulum (ER) and moderate enlargement of the mitochondria with increased density of their matrix. Twenty hours thereafter, increased amounts of the rough ER and of free ribosomes were observed in the infected cells. This was followed by aggregates of larger dense particles which developed into a parallel lattice-like pattern within the cytoplasm, presumably presenting viral particles, without obvious cytonecrosis. The present observations of the continuous development of the crystalloid formation of the virus within the cytoplasmic matrix as well as the mode of formation of the free ribosomes adjacent to the ER support the previous hypothesis that, after the uptake of the virus into the cytoplasmic matrix, it associates with the ER, where production of viral RNA polymerase and of viral RNA is initiated.

Astrocytes↗
PubMed1975

Glucose tolerance and pancreatic ultrastructure in mice with long-term diabetes induced by EMC virus (M variant) (38518).

Of 27 young male CD-1 mice infected with the M variant of the encephalomycarditis (EMC) virus and surviving for 11 or 12 mo, all but three had normal glucose tolerance curves, and all displayed normal islet cell morphology, at the time of sacrifice, in spite of an initital hyperglycemic response. Three animals maintained diabetic glucose tolerance curves one year after infection but they, too, failed to show significant morphological alterations in their pancreatic beta cells.

Animals↗
PubMed1975

EMC virus and cultured human fetal pancreatic cells. Ultrastructural observations.

Tissue cultures of pancreatic acinar cells of seven human fetuses were infected with either the M or the E variant of the encephalomyocarditis (EMC) virus. Between the third and fifth days after infection, severe ultrastructural damage was noted. Margination, condensation, and fragmentation of the nuclear chromatin and the appearance of numerous cytoplasmic vesicles filled with finely granular material were the major changes observed. Viral particles were not identified. One hundred plaque-forming units (PFU) were sufficient to induce these alterations with the M variant while 10,000 PFU were needed to obtain the same results with the EMC-E virus. The described lesions are qualitatively similar to those induced in vivo in experimental CD-1 mice infected with the virus.

Cell Nucleus↗