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J L Rothstein

Publications and source records attributed to J L Rothstein.

35 records · Page 2Linked to original sources
PubMed1995

Expression of the human Achaete-scute 1 gene in olfactory neuroblastoma (esthesioneuroblastoma).

Olfactory neuroblastoma (ONB) is a rare neuronal malignancy of the olfactory mucosal. Markers used in the diagnosis of ONB do not distinguish ONB from other neuronal tumors or tumors with neuroendocrine features thus making the diagnosis of ONB difficult. Using a modified RT-PCR technique, we show that the human homologue of the Drosophila achaete-scute gene HASH1 is expressed in 6 primary and one metastatic ONB specimens, whereas Olfactory Marker Protein (OMP) is not. Previous studies have shown that HASH1 is expressed in immature olfactory neurons and is required for their development. OMP, whose function is unknown, is expressed exclusively in mature olfactory neurons. Together, these data suggest that ONB is derived from immature olfactory neurons of neuroectodermal origin. Analysis of RNA expression in primary tumor specimens and in an established cell line make this an ideal system to study olfactory growth and differentiation. Furthermore, these studies represent the first molecular genetic analysis of this rare and unusual neuronal tumor.

Adolescent↗
PubMed1994

Expression of epidermal growth factor family gene members in early mouse development.

Transcription of four members of the epidermal growth factor (EGF) family, EGF, TGF-alpha, Amphiregulin, and Cripto, was investigated in the ovulated unfertilized egg and preimplantation embryo using cDNA libraries. EGF was present as a maternal message only, TGF-alpha was present at low levels in each library, Amphiregulin was not detected, and Cripto transcripts were first detected in the blastocyst cDNA library. In situ hybridization studies of the implanting embryo revealed Cripto expression localized to the entire ectoderm and then to the rapidly growing ectoplacental cone. At gastrulation, Cripto was detected in the primitive streak and developing mesoderm. During organogenesis, Cripto localized to the developing heart. Two Cripto transcripts were detected: one is confined to the early embryo and teratocarcinoma cells, and the other, first found in the fetus, is the major form detected in adult organs.

Amphiregulin↗
PubMed1994

Expression patterns of novel genes during mouse preimplantation embryogenesis.

Little is known about the repertoire of genes expressed following zygotic gene activation, which occurs during the two-cell stage in the mouse. As an initial attempt to isolate novel genes, we used previously prepared two-cell and two-cell subtraction cDNA libraries (Rothstein et al., Genes Dev 6:1190-1201, 1992) to isolate a panel of seven cDNA clones. Three cDNAs had no match in the current DNA sequence data banks and three others revealed sequence homology to portions of sequences in the data banks. One cDNA was 90% homologous to the ras-related gene Krev/rap 1A. The temporal patterns of expression of these genes during oocyte maturation and preimplantation development were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR) assay developed to measure relative levels of mRNAs. Three distinct temporal patterns of expression, designated Classes 1-3, were found. The two Class 1 genes displayed an actin-like pattern, with a gradual decline in expression during oocyte maturation and through the two-cell stage, followed by increases at the eight-cell and/or blastocyst stages. The four genes in Class 2 were expressed at relatively high levels during oocyte maturation and through the one-cell stage and then declined abruptly between the one- and two-cell stages; an increase then occurred at the eight-cell and/or blastocyst stages. The expression of the gene in Class 3 declined during oocyte maturation, but then showed a transient increase at the one-cell stage, with only a very slight increase in synthesis at either the eight-cell or blastocyst stage.

Actins↗
PubMed1993

Phenotypically diverse mouse thymic stromal cell lines which induce proliferation and differentiation of hematopoietic cells.

The heterogeneity of the thymic stroma has made careful characterization of particular thymic stromal cell types difficult. To this end, we have derived a panel of cloned thymic stromal cell lines from simian virus 40 T antigen (SV40-T antigen) transgenic mice. Based on their analysis with monoclonal antibodies that distinguish among subsets of thymic stroma cells, and on the morphology and ultrastructural features of the different clones, we suggest that our panel includes representatives of the thymic subcapsular cortex or thymic nurse cells (427.1), the deep cortex or cortical reticular cells (1308.1) and the medulla including medullary interdigitating (IDC)-like cells (6.1.1) and medullary epithelial cells (6.1.7). A fifth cell type of undesignated but apparent medullary origin (6.1.11) was also isolated. All of the cell lines constitutively express the SV40 T antigen transgene and the class I antigens of the major histocompatibility complex (MHC), and they can be induced to express MHC class II antigens upon stimulation with recombinant interferon-gamma (IFN-gamma). These cell lines elaborate a factor(s) that induces the proliferation of cells from the fetal liver and bone marrow, but not from the neonatal thymus. A factor(s) elaborated by the 1308.1 cell line also induces the proliferation of fetal thymocytes in the absence of mitogens, phorbol esters or calcium ionophore which is augmented with the addition of recombinant interleukin-2 (IL-2). Analysis by reverse transcription polymerase chain reaction with primers for some mouse cytokines reveals that each of these cell lines contain granulocyte-macrophage colony-stimulating factor (GM-CSF) transcripts and that 1308.1, 6.1.1 and 6.1.7 produce IL-6 mRNA. Cell lines 1308.1 and 6.1.1 also produce IL-7; 6.1.1 produces IL-1 beta and tumor necrosis factor (TNF)-alpha while the 427.1 cell line produces IL-5 and IFN-gamma mRNA. None of the cell lines tested express the IL-2 receptor, IL-2, IL-3, IL-4, TNF-beta or macrophage inflammatory proteins mRNA. Conditioned medium (CM) from 1308.1 and 6.1.11 induced differentiation of cells purified from the mouse fetal liver into granulocytes; 1308.1 CM also induced differentiation of the mouse hematopoietic stem cell line 32DCl3(G) suggesting that the CM contains granulocyte (G)-CSF activity. Each cell line produces GM-CSF but the greatest activity is associated with 1308.1 and 6.1.11 CM. The availability of these well-characterized, functional, cloned thymic stromal cells will allow a more detailed analysis of the role of each cell type in both myeloid and T cell development.

Animals↗
PubMed1993

Construction of primary and subtracted cDNA libraries from early embryos.

By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification and study of novel genes or gene families, and it is an important new tool for understanding the molecular control of mammalian development.

Animals↗
PubMed1992

Depressed serum high density lipoprotein cholesterol levels in veterans with spinal cord injury.

Cardiovascular diseases are the most frequent cause of death among persons with spinal cord injury (SCI), and these diseases are reported to occur prematurely in the disabled compared to the able bodied population. The mechanism of accelerated coronary heart disease (CHD) in persons with SCI may be partially explicable on the basis of the lipoprotein profile. We performed fasting lipoprotein determinations on 100 veterans with SCI, 50 with paraplegia and 50 with quadriplegia, and 50 veteran controls. The mean age of the subjects with SCI was 47.8 +/- 1.4 years with a duration of injury of 16.3 +/- 1.2 years. The mean serum high-density lipoprotein (HDL) cholesterol was depressed in subjects with paraplegia or quadriplegia compared to controls (37 +/- 1 or 40 +/- 1 versus 48 +/- 2 mg/dL, p < 0.0001). Although serum total cholesterol was lower (p < 0.01) in subjects with SCI than in controls, there was no significant difference in mean serum low-density lipoprotein (LDL) cholesterol. Thirty-seven percent of subjects with SCI have serum HDL cholesterol levels less than 35 mg/dL with no significant difference in lipoprotein distribution between high and low cord lesions. Eighteen percent of individuals with SCI have an absolute elevation of LDL cholesterol (greater than 160 mg/dL). About 40% of those with SCI and LDL cholesterol levels between 130 and 160 mg/dL also have serum HDL cholesterol values below 35 mg/dL, all of whom would have their serum HDL cholesterol level undetected if lipoprotein profiles were performed according to present recommendations--that is, only if the serum total cholesterol is elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Drug use and abuse in an urban veteran spinal cord injured population.

Substance abuse in the spinal cord injured (SCI) population has been addressed in a few controlled studies. These reports have led to the belief that many SCI patients were illicit drug users prior to their injury and that their drug abuse was a contributing cause of their accidents. One large study determined from answers obtained on a questionnaire that drug abuse, other than alcohol, was prevalent among injured veterans. Our study uses urine toxicology to determine the frequency of drug use and abuse, excluding alcohol, in 72 inpatients and 81 outpatients associated with an urban Veterans Affairs Medical Center. In a blinded experiment, urinary concentrations of opiates, barbiturates, amphetamines, methadone, benzodiazepines, and cocaine were determined with Syva reagents on an Abbott VP. Urinary cannabinoids were determined using Abbott TDX and the FPIA method. The use of illicit (unprescribed) drugs was surprisingly low (< or = 13 percent) for an urban medical center. Outpatient cannabinoid abuse was significantly more frequent than inpatient usage (p < 0.01). Barbiturates were not found in any patient. Benzodiazepines were taken most commonly (29 percent). Since benzodiazepine usage is the direct result of physician prescription, wide-spread usage of this agent is avoidable. Physicians should pay attention to the probable deleterious consequences of benzodiazepine addiction related to depressive effects on the central nervous system caused by its chronic use.

Adult↗
PubMed1992

Gene expression during preimplantation mouse development.

To develop a resource for the identification and isolation of genes expressed in the early mammalian embryo, large and representative cDNA libraries were constructed from unfertilized eggs, and two-cell, eight-cell, and blastocyst-stage mouse embryos. Using these libraries, we now report the first stages at which the cytokines interleukin (IL)-6, IL-1 beta, and interferon (IFN)-gamma are transcribed in the developing embryo and the presence of IL-7 transcripts in the unfertilized egg. Transcripts for IL-1 alpha, -2, -3, -4, or -5 were not detected at these stages. To identify novel genes expressed on activation of the embryonic genome, the egg and eight-cell stage-specific cDNA libraries were subtracted from the two-cell library, yielding a specialized cDNA library enriched for transcripts expressed at the two-cell stage. Sequence and Southern blot analysis of several of these cDNAs expressed predominantly at the two-cell stage of embryogenesis revealed them to be from novel genes, thereby providing the first molecular tools with which to approach the study of gene expression in the early mammalian embryo.

Animals↗
PubMed1988

Tumor necrosis factor/cachectin. Induction of hemorrhagic necrosis in normal tissue requires the fifth component of complement (C5).

TNF induces hemorrhagic necrosis (HN) when injected into skin exposed to bacterial agents but not when injected into normal skin. In this paper, we present several lines of evidence suggesting that TNF requires the fifth component of complement (C5) to induce HN in skin exposed to bacteria. First, mouse strains that do not have C5 did not develop HN after injection of TNF and bacteria into skin. Second, plasma from C5-sufficient mice could correct the defect in these C5-deficient mice. Third, heating at 56 degrees C for 30 min inactivated the capacity of plasma to reconstitute C5-deficient mice. Fourth, CVF, which is known to inactivate complement, abrogated the capability of C5-sufficient mice to respond. Fifth, depleting plasma of hemolytic activity while generating C5a did not affect the capacity of the activated plasma to reconstitute C5-deficient mice. Finally, only the plasma fraction containing molecules of the size range of C5a reconstituted C5-deficient mice. These findings indicate that C5a and not the membrane attack complex is required for HN. Although we do not know through which mechanism C5a participates in the development of HN, we propose that the described HN response is related to a local defense mechanism in which TNF and C5a lead to the disruption of capillaries in the direct vicinity of bacteria. By this mechanism the rapid spread of bacteria or their products into the circulation is prevented. Such a tissue response is consistent with the known higher susceptibility of C5-deficient mice to bacterial infections and provides a model with which to search for the multiple steps involved in this important local defense mechanism.

Animals↗
PubMed1988

Spontaneous production of tumor necrosis factor alpha by Kupffer cells of MRL/lpr mice.

We report that freshly isolated, unstimulated Kupffer cells (KC) from MRL/lpr female mice in short-term culture spontaneously produce high levels of TNF-alpha. TNF production was first detected in KC cultures at age 6 wk and increased with the age of the mice. Moreover, the levels of spontaneous TNF production by KC directly correlated with the age of the MRL/lpr mice. Although TNF production by KC could be induced with C. parvum in vivo or LPS in vitro in all nonautoimmune C3H/HeN, BALB/c, DBA/2, C57B16 mice, the only other strain in which spontaneous TNF production by KC was observed was MRL/++ mice greater than 10 mo old.

Aging↗
PubMed1988

Synergy between tumor necrosis factor and bacterial products causes hemorrhagic necrosis and lethal shock in normal mice.

We find a strong synergism between tumor necrosis factor (TNF) and bacteria or their products. Endotoxin-"free" recombinant TNF, even at very high doses (160 micrograms), did not alone cause hemorrhagic necrosis (HN) in the skin of normal mice. Similarly, TNF alone had a low systemic toxicity in tumor- and pathogen-free mice. However, TNF given intravenously with nanogram quantities of the endotoxin lipopolysaccharide caused lethal shock. Furthermore, subcutaneous injection of lipopolysaccharide made skin susceptible to subsequent induction of HN by TNF injected in the same site 24 hr later. Mycoplasma-infected cells or corynebacteria also synergized with TNF to cause HN or lethal shock. In addition, we find that lymphotoxin, a cytokine functionally and genetically related to TNF, also synergized with the bacteria to cause HN, whereas interleukin 1 alpha or interferon gamma did not. Together, the results indicate that a synergy between TNF and bacteria or their products causes HN and lethal shock in normal mice.

Animals↗
PubMed1988

Effect of macrophage activation on resistance of mouse peritoneal macrophages to infection with herpes simplex virus types 1 and 2.

To define the effect of heterogeneity of murine peritoneal macrophages (M phi) on intrinsic resistance to herpes simplex virus (HSV) infection, several M phi populations were characterized for their response to infection with HSV type 1 (HSV-1) and HSV-2. Steady-state resident M phi (Res M phi) were compared in parallel with M phi activated with Corynebacterium parvum (now designated Propionibacterium acnes) (CP M phi) and thioglycollate-elicited inflammatory M phi (TG M phi). Res M phi were completely non-permissive for productive virus infection and showed no c.p.e. The intrinsic resistance of CP M phi to HSV infection was similar to that of Res M phi, in that the infection was non-productive for infectious virus, but CP M phi showed marked c.p.e. TG M phi showed semi-permissiveness, with virus yields at least 10-fold higher than those in Res M phi and CP M phi, and marked c.p.e. The three distinct intrinsic response patterns were maintained regardless of whether M phi were derived from CD-1 or B6C3F1 mice, or whether the infecting virus was HSV-1 or HSV-2. To define the level at which M phi restrict HSV replication, immunofluorescence assays for viral antigens and hybridization analyses for viral DNA were performed. All M phi populations showed immediate early and early virus polypeptides. Res M phi and CP M phi showed no viral DNA replication, but TG M phi showed moderate levels of viral DNA synthesis that paralleled the infectious virus titres produced. Investigation of the mechanism for the heterogeneous intrinsic antiviral response among the M phi revealed that interferon was not involved, because antiserum to mouse alpha/beta interferon did not alter the intrinsic resistance patterns. Induction of c.p.e. in M phi required live, replication-competent HSV. The involvement of tumour necrosis factor (TNF) in c.p.e. was found to be unlikely; no significant amounts of TNF were detected in the culture medium of the M phi, and inclusion of anti-TNF antibody did not inhibit c.p.e.

Animals↗
PubMed1987

Relationship of tumour necrosis factor and endotoxin to macrophage cytotoxicity, haemorrhagic necrosis and lethal shock.

In this communication we discuss preliminary evidence suggesting a very strong synergism between tumour necrosis factor (TNF) and lipopolysaccharide (LPS) or between TNF and other bacteria in causing haemorrhagic necrosis and lethal shock. We found that TNF by itself does not cause haemorrhagic necrosis when injected into normal skin. TNF also had a rather low systemic toxicity when injected into disease-free, germfree-derived, defined-flora animals. On the other hand the addition of small amounts of LPS markedly raised the lethality of intravenous TNF treatments, and LPS injected into normal skin 'prepared' the site of injection for subsequent induction of haemorrhagic necrosis by locally injected TNF. Similar synergism was observed between TNF and mycoplasma. We suggest that the synergism between TNF and bacterial endotoxin (or other bacteria or bacterial products) may be part of an important defence mechanism against infections which is independent of specific immunity mediated by B and T cells. This synergism may be useful in increasing the therapeutic effects of TNF on tumours if the development of systemic toxicity in this treatment can be prevented.

Animals↗
PubMed1986

Highly malignant tumor variants retain tumor-specific antigens recognized by T helper cells.

We have studied the components of a complex of tumor-specific antigens to determine if all of the components of the complex were lost during progression from a rather benign regressor tumor to a highly malignant (HM) cancer. We find that the HM tumor cells have lost antigens recognized by CTL but retained antigens recognized by Th cells. Immunization with variants expressing Th-defined antigens induced tumor-specific immunity to challenge with a parental variant that expressed a CTL-recognized target antigen, but did not induce immunity to challenge with the variant that expressed the Th-defined antigen alone. Together, these findings suggested that Th cells fail to exert direct selective pressure upon the tumor, resulting in retention of "lineage-specific," Th-recognized antigens by highly immunoselected variants. Possible advantage could be taken of this fact for the development of specific immunotherapy.

Animals↗
PubMed1986

Tumor necrosis factor: a potent effector molecule for tumor cell killing by activated macrophages.

Activated macrophages (aM phi) destroy more effectively cancer cells than normal cells. The mechanism by which macrophages destroy cancer cells is not known. We report here that tumor cells susceptible to aM phi were killed by recombinant (r) tumor necrosis factor type alpha (TNF-alpha), whereas variant tumor cells resistant to aM phi after selection in vitro or in vivo were resistant to killing by rTNF-alpha. The converse selection for rTNF-alpha-resistant variants resulted in cells that were also resistant to killing by aM phi. The sensitivity of macrophage-resistant variants was not changed to other tumoricidal cells or soluble mediators, except that the macrophage-resistant variants were also resistant to the effects of another cytotoxic protein, B-cell lymphotoxin, which is structurally related to rTNF-alpha. Similar results were obtained regardless of whether short-term or long-term cytotoxic effects of aM phi were measured. Finally, it was shown that killing of tumor cells by murine aM phi was completely inhibited with a polyclonal antibody that neutralizes the effects of murine TNF-alpha. These results suggest a major role for TNF-alpha in tumor cell destruction by aM phi in vitro and in vivo.

Animals↗