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Biomedical subjects

A L Goldstein

Publications and source records attributed to A L Goldstein.

At least 37 records · Page 2Linked to original sources
PubMed1997

C-terminal truncations of the yeast nucleoporin Nup145p produce a rapid temperature-conditional mRNA export defect and alterations to nuclear structure.

A screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective in nucleocytoplasmic trafficking of poly(A)+ RNA has identified an allele of the NUP145 gene, which encodes an essential nucleoporin. NUP145 was previously identified by using a genetic synthetic lethal screen (E. Fabre, W. C. Boelens, C. Wimmer, I. W. Mattaj, and E. C. Hurt, Cell 78:275-289, 1994) and by using a monoclonal antibody which recognizes the GLFG family of vertebrate and yeast nucleoporins (S. R. Wente and G. Blobel, J. Cell Biol. 125:955-969, 1994). Cells carrying the new allele, nup145-10, grew at 23 and 30 degrees C but were unable to grow at 37 degrees C. Many cells displayed a modest accumulation of poly(A)+ RNA under permissive growth conditions, and all cells showed dramatic and rapid nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. The mutant allele contains a nonsense codon which truncates the 1,317-amino-acid protein to 698 amino acids. This prompted us to examine the role of the carboxyl half of Nup145p. Several additional alleles that encode C-terminally truncated proteins or proteins containing internal deletions of portions of the carboxyl half of Nup145p were constructed. Analysis of these mutants indicates that some sequences between amino acids 698 and 1095 are essential for RNA export and for growth at 37 degrees C. In these strains, nuclear accumulation of poly(A)+ RNA and fragmentation of the nucleolus occurred rapidly following a shift to 37 degrees C. Constitutive defects in nuclear pore complex distribution and nuclear structure were also seen in these strains. Although cells lacking Nup145p grew extremely slowly at 23 degrees C and did not grow at 30 degrees C, efficient growth at 23 or 30 degrees C occurred as long as cells produced either the amino 58% or the carboxyl 53% of Nup145p. Strains carrying alleles of NUP145 lacking up to 200 amino acids from the carboxy terminus were viable at 37 degrees C but displayed nucleolar fragmentation and some nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. Surprisingly, these strains grew efficiently at 37 degrees C in spite of a reduction in the level of synthesis of rRNAs to approximately 25% of the wild-type level.

Biological Transport↗
PubMed1996

Adjuvant properties of montanide CSA 720 with a recombinant HIV P17 gag protein and synthetic peptide antigens.

The adjuvant properties of Montanide CSA 720 were assessed in a comparison with alum. BALB/c mice were immunized with recombinant HIV-1 gag protein p17 administered in either of the two adjuvants. The serum antibody response to p17 with Montanide CSA 720 appeared faster and reached a higher titre than with alum. The serum antibody response to p17 in Montanide CSA 720 was further characterized by a higher titre antibody directed against a 30 amino acid segment from the entire protein. The Montanide CSA 720 adjuvant was sufficiently strong to induce an antibody response against a weak synthetic peptide immunogen after two immunizations, while immunization with the peptide in alum generated no detectable serum antibody. The p17-specific proliferative response of splenocytes from animals immunized with recombinant protein in either adjuvant was similar.

AIDS Vaccines↗
PubMed1996

Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p. In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.

Alleles↗
PubMed1996

Enzyme-linked immunosorbent assay quantification of cytokine concentrations in human meningiomas.

OBJECTIVE: To gain insight into the network of cytokine gene expression in the brain tumor microenvironment, we investigated the presence of the following cytokines in freshly excised brain tumors: interleukin (IL)-1 beta, IL-2, IL-4, and IL-6. METHODS: Tumor specimens from nine meningiomas were grown as tissue explants. The supernatants from the explants were tested for the presence of the aforementioned cytokines via the enzyme-linked immunosorbent assay method. RESULTS: IL-6, which is thought to stimulate acute protein phase synthesis, neovascularization, and cell proliferation, was found in all of the samples in greater concentrations than the other cytokines tested. IL-1 beta, another stimulatory cytokine thought to be involved in acute protein phase synthesis and cell proliferation, was also found in 100% of the samples tested, in concentrations significantly lower than those of IL-6. As expected, the presence of IL-2 and IL-4 was not detectable in any of the samples. CONCLUSION: This study is the first to clearly determine the relative concentrations of IL-1 beta and IL-6, using enzyme-linked immunosorbent assay quantification. These findings are an important precursor to future studies using antibodies to IL-1 beta and IL-6 and antibodies to IL-6 receptors to modulate neoplastic growth both in vitro and in vivo.

Cell Division↗
PubMed1995

Matrigel induces thymosin beta 4 gene in differentiating endothelial cells.

We performed differential cDNA hybridization using RNA from endothelial cells cultured for 4 hours on either plastic or basement membrane matrix (Matrigel), and identified early genes induced during the morphological differentiation into capillary-like tubes. The mRNA for one clone, thymosin beta 4, was increased 5-fold. Immunostaining localized thymosin beta 4 in vivo in both growing and mature vessels as well as in other tissues. Endothelial cells transfected with thymosin beta 4 showed an increased rate of attachment and spreading on matrix components, and an accelerated rate of tube formation on Matrigel. An antisense oligo to thymosin beta 4 inhibited tube formation on Matrigel. The results suggest that thymosin beta 4 is induced and likely involved in differentiating endothelial cells. Thymosin beta 4 may play a role in vessel formation in vivo.

Base Sequence↗
PubMed1994

A distinctive form of soluble CD8 is secreted by stimulated CD8+ cells in HIV-1-infected and high-risk individuals.

The aim of the present study was to investigate the biochemical structure and pathogenic significance of the soluble CD8 (sCD8) present in the serum of HIV-1-infected individuals. In a longitudinal study of a cohort of HIV-infected homosexuals and the amount of sCD8 detected in the plasma was correlated with changes in lymphocyte subsets and with the clinical course of HIV infection. The level of sCD8 in the plasma, the percentage, and the absolute number of CD8+CD38+ cells were increased in HIV-seronegative, high-risk homosexuals and in seropositive HIV+ individuals. The plasma concentration of serum sCD8 showed a significant correlation with the absolute number of CD8+ and CD8+CD38+ cells in HIV+ homosexuals. In addition to a molecule with a molecular weight (m.w.) of 30 kDa, sCD8 isolated from the plasma of HIV-1-infected individuals and of healthy controls was found to consist of two molecules, one with a m.w. of 57 to 62 kDa and another with a m.w. of 66 to 70 kDa. The former was the predominant molecule in normal individuals, while the latter was the predominant molecule in HIV-negative high-risk homosexuals and in HIV-infected individuals. The latter molecule, secreted by chronically stimulated CD8+ cells, seems to be present in the circulation as a dimer. While it was previously shown that CD8 can be shed from the cell membrane in vitro, the present study indicates that in vivo-stimulated CD8+ cells release a distinctive form of soluble CD8.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Corticotropin-releasing factor stimulates cyclic AMP, arachidonic acid release, and growth of lung cancer cells.

The effects of corticotropin-releasing factor (CRF) on human lung cancer cell lines was investigated. Corticotropin-releasing factor increased the cAMP levels in a dose-dependent manner; CRF (100 nM) elevated the cAMP levels approximately eleven-fold using NCI-H345 cells and increased the gastrin-releasing peptide (GRP) secretion rate by approximately 70%. Similarly, sauvagine, a structural analogue of CRF, elevated the cAMP levels with a half-maximal effective dose (ED50) of 20 nM. The increase in cAMP caused by CRF and sauvagine was reversed by alpha-helical CRF(9-41). Corticotropin-releasing factor had no effect on cytosolic calcium but stimulated [3H]arachidonic acid release from NCI-H1299 cells with an ED50 of 30 nM. The increase in [3H]arachidonic acid release caused by 100 nM CRF was significantly reversed by 1 or 10 microM alpha-helical CRF(9-41). Also, CRF stimulated the clonal growth of NCI-H345 and H720 cells and the growth increase caused by CRF was reversed by alpha-helical CRF(9-41). These data suggest that CRF may be a regulatory peptide in lung cancer.

Amphibian Proteins↗
PubMed1993

Thymosin alpha 1 down-regulates the growth of human non-small cell lung cancer cells in vitro and in vivo.

The effect of thymosin alpha 1 (THN alpha 1) and its NH2-terminal fragment (THN1-14) and COOH-terminal fragment (THN15-28) on non-small cell lung cancer (NSCLC) growth was evaluated. Using an anti-THN alpha 1 antibody, receptors were identified on NSCLC cells that were pretreated with 10(-6) M THN alpha 1. [3H]Arachidonic acid was readily taken up by NSCLC cells and THN alpha 1 significantly increased the rate of arachidonic acid release. THN1-15 slightly stimulated but THN15-28 and THN beta 4 did not alter arachidonic acid release from NCI-H1299 cells. In clonogenic growth assays in vitro, THN alpha 1 (10(-6) M) significantly decreased NSCLC colony number whereas THN1-14, THN15-28, and THN beta 4 were less potent. Using growth assays in vivo, THN alpha 1 (10 micrograms s.c./day) but not THN1-14, THN15-28, or THN beta 4 inhibited significantly NSCLC xenograft formation in nude mice. These data suggest that biologically active THN alpha 1 receptors are present on NSCLC cells and that native THN alpha 1 inhibits the growth of human NSCLC.

Animals↗
PubMed1993

Para-aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme.

The Saccharomyces cerevisiae gene for para-aminobenzoate (PABA) synthase has been identified based upon its ability to confer sulfonamide resistance when present on a multicopy episomal vector. The 3840 bp DNA sequence fragment reported here contains a 2199 bp open reading frame encoding a 733 amino acid protein with similarity to the two components of PABA synthase described for prokaryotes (Escherichia coli PabA and PabB), suggesting that PABA synthase is bifunctional in yeast. The cloned sequence was confirmed to be PABA synthase by gene disruption. Chromosome gel analysis places the gene for PABA synthase on chromosome XIV.

Amino Acid Sequence↗
PubMed1992

Thymosin beta 4 (Fx peptide) is a potent regulator of actin polymerization in living cells.

Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.

3T3 Cells↗
PubMed1992

Combination therapy with thymosin alpha 1 potentiates the anti-tumor activity of interleukin-2 with cyclophosphamide in the treatment of the Lewis lung carcinoma in mice.

In this study we have investigated the effects of thymosin alpha 1 (T alpha 1) and interleukin-2 (IL-2), singly or in combination with cyclophosphamide (CY), on tumor growth, survival and cytotoxicity in C57Bl/6NCrlBR mice with Lewis lung carcinoma (3LL). Combined administration of T alpha 1 plus IL-2, after CY treatment, was much more effective than use of each biological response modifier (BRM) alone, and induced complete tumor regression in all of the mice studied. Combination immunotherapy alone without CY only slightly reduced the rate of tumor growth, and these results are in accordance with previous studied which showed that the 3LL carcinoma is resistant to cytokines. Combined chemo-immunotherapy also increased the cytotoxicity of spleen cells and markedly enhanced long-term survival in all treated animals. Depletion of immune cells, using either total-body sub-lethal irradiation (400 rads) or antibodies directed against T-cell (anti-CD4 and CD8) or NK-cell (anti-asialo GM1) populations, abolished the positive response to combination therapy. Histological analysis of the tumors obtained from mice treated with combination chemo-immunotherapy revealed a high number of infiltrating lymphoid cells surrounding a well-circumscribed area of necrosis consisting solely of dead cells. Our studies show that T alpha 1 potentiates IL-2-induced cytotoxic activities in vitro as well in vivo, and that these compounds have a powerful anti-tumor action when associated with chemotherapy.

Animals↗
PubMed1992

Identification of immunoreactive forms of thymosin alpha 1 in serum and supernatants by combining HPLC and RIA.

Thymosin alpha 1 (T alpha 1) is a biologically active peptide, originally isolated from the thymus and currently undergoing clinical trials as an immunomodulator in cancer patients, in individuals with chronic active hepatitis, and as an immunoenhancer of vaccines in immunocompromised individuals. Absorption of rabbit antibody to thymosin alpha 1 with a synthetic C-14 fragment of T alpha 1 results in an antiserum with increased affinity for the amino terminal region of T alpha 1 and the precursor protein prothymosin alpha (ProT alpha). Using HPLC methodologies, the predominant form of immunoreactivity in serum and thymus was T alpha 1 not the precursor. Using this assay we detected a decline in mouse serum T alpha 1 following irradiation but not thymectomy, an observation consistent with the existence of an important radiation sensitive lymphoid source of serum T alpha 1. The secretion of authentic T alpha 1 but not the precursor into culture medium by thymic epithelial cells as well as in mitogen-stimulated peripheral blood lymphocytes was also demonstrated by HPLC/RIA. HPLC analysis by molecular weight sizing columns demonstrated that unlike thymic epithelial cells or peripheral blood lymphocytes, the immunoreactive T alpha 1 (IRT alpha 1) form in the supernatants from tumor cells such as MCF-7 breast carcinoma was of a lower molecular weight than authentic T alpha 1. These studies suggest that the authentic form of T alpha 1 is the major immunoreactive form in normal serum and that it is secreted by the medullary thymic epithelial cells as well as by peripheral blood lymphocytes. An additional immunoreactive form, secreted by tumor cells has also been identified and is the subject of future studies.

Adolescent↗
PubMed1992

Cell-mediated immunity against HGP-30, a group-specific peptide of HIV p17 in individuals infected with the AIDS virus.

HGP-30, the synthetic peptide analogue and active component in an HIV-1 (human immunodeficiency virus, type 1) p 17 core-based experimental vaccine, has previously been shown to induce cytotoxic and helper T-lymphocyte responses. In order to further define the T-helper cell responses which are known to play a role in enhancing the immunological response to foreign antigens, we studied the response of individuals infected with HIV to HGP-30 at various stages of disease progression. We have investigated the proliferative cellular response of peripheral blood mononuclear cells (PBMCs) derived from individuals infected with HIV-1 to HGP-30. We have found a PBMC proliferative response to HGP-30 in 40% of the healthy seroconverted patients, in 35% of the CDC stage III patients and in 18% of the CDC stage IV patients. There was no correlation between the proliferative response to HGP-30 and other antigens such as HIV-like proteins or tetanus toxoid not to CD4 cell count. HLA-DR typing revealed the possible presentation of HGP-30 by several different class II molecules. Since these class II molecules occur frequently in the general population, HGP-30 appears to contain broadly reactive epitopes and thus is not restricted as are many peptide vaccines. Due to its broad reactivity and extreme conservation in many HIV-1 strains. HGP-30 is one of the promising candidates for inclusion as a subunit vaccine against HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1992

A phase I study of HGP-30, a 30 amino acid subunit of the human immunodeficiency virus (HIV) p17 synthetic peptide analogue sub-unit vaccine in seronegative subjects.

HGP-30-KLH vaccine in alum at doses of 10, 25, 50, and 100 micrograms/kg administered intramuscularly at weeks 0, 4, and 10 appear well-tolerated clinically. Local pain at the injection site, appears to be the main clinical toxicity. Laboratory parameters are not affected by administration of the vaccine candidate except for perhaps mild urinalysis abnormalities at the highest dose. This vaccine candidate has no apparent immunotoxicity and does not appear to affect lymphocyte populations or T-cell functional studies. Low levels and transient antibodies develop in a minority of subjects early after immunization with the vaccine candidate. These responses were observed in the lowest dose range. Higher doses, and longer follow-up will be needed to confirm this observation. T-cell proliferative responses to KLH and KLH-HGP-30 are consistent and may not be dose dependent, but the proliferative responses are variable and more data need to be accumulated. Preliminary, there appears to be an HGP-30-induced CTL response of HGP-30-coated EBV-transformed autologous B cell lines. This study was approved under an IND for the California Department of Health Services' Food and Drug Branch. They have provided excellent support and regulatory guidelines for this project. Future work will extend and confirm these initial observations.

AIDS Vaccines↗
PubMed1992

Thymosin alpha-1 enhances the fertilizing capacity of human sperm cell: implication in diagnosis and treatment of male infertility.

The effects of synthetic thymosin peptides (T alpha 1 and T beta 4) and their antibodies on the fertilizing capacity of human sperm cells were investigated. T alpha 1, but not the T beta 4, significantly (p < 0.001) increased the human sperm penetration rates in sperm penetration assay (SPA). Antibodies to both T alpha 1 and TB4, which predominantly bound to the acrosomal region of human sperm cell in the indirect immunofluorescence technique (IFT), also significantly (p < 0.001) increased (up to 4.7-fold) the human sperm penetration rates in SPA. The T alpha 1 and antibodies to both T alpha 1 and T beta 4 enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of T alpha 1 and antibodies to T alpha 1 and T beta 4 on percent sperm motility, although they significantly affected various motility characteristics such as velocity, amplitude of lateral head displacement (ALH), and beta frequency--the motility parameters involved in hyperactivation phenomenon of sperm cells. Both T alpha 1 and T beta 4 were detected in the seminal plasma of fertile men, and the levels of T alpha 1 were significantly (p = 0.002) lower in the seminal plasma of infertile men having defective sperm function. These results indicate that the thymosin molecules, especially T alpha 1, may have a role in human sperm capacitation leading to acrosome reaction. These findings also suggest that the T alpha 1 may find clinical applications in the specific diagnosis and treatment of male infertility in humans.

Acrosome↗
PubMed1992

Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.

We have combined techniques of genetics and histochemistry to identify genes required for the nucleocytoplasmic export of mRNA in the budding yeast Saccharomyces cerevisiae. We adapted in situ hybridization using a digoxigenin-labeled oligo(dT)50 probe to localize poly(A)+ RNA in fixed yeast cells and used yeast strains carrying the rna1-1 mutation to develop an assay. The rna1-1 mutation is the only previously described mutation that causes defects in mRNA export. As visualized with this RNA localization assay, rna1-1 strains accumulated poly(A)+ RNA at the nuclear periphery at the nonpermissive temperature. This was in contrast to the RNA localization pattern of wild-type cells or rna1-1 cells grown at permissive temperature. Wild-type cells showed bright uniform cytoplasmic staining with little detectable RNA in the nuclei. We used this RNA localization assay to screen a bank of temperature-sensitive yeast strains for mutants with inducible defects in mRNA trafficking. Strains identified in this manner are designated RAT mutants for ribonucleic acid trafficking. The rat1-1 allele conferred temperature-sensitive accumulation of poly(A)+ RNA in one to several intranuclear spots that appear to lie at the nuclear periphery. RNA processing was unaffected in rat1-1 strains, except for an inducible defect in trimming the 5' end of the 5.8S rRNA. The wild-type RAT1 gene was cloned by complementation; it encodes an essential 116-kD protein with regions of homology to the protein encoded by SEP1 (also known as DST2, XRN1, KEM1, and RAR5). Sep1p is a nucleic acid binding protein, a 5'----3' exonuclease, and catalyzes DNA strand transfer reactions in vitro. We discuss the possible significance of the Rat1p/Sep1p homology for RNA trafficking. We also discuss the potential of this RNA localization assay to identify genes involved in nuclear structure and RNA metabolism.

Amino Acid Sequence↗