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Biomedical subjects

A L Goldstein

Publications and source records attributed to A L Goldstein.

At least 19 recordsLinked to original sources
PubMed2004

Thymosin beta4 promotes angiogenesis, wound healing, and hair follicle development.

New blood vessel formation is important in many physiological process, including development, wound repair, and tumor growth. In aged animals, angiogenesis is reduced resulting in poor wound healing. We have identified a novel small molecule, thymosin beta(4), that promotes angiogenesis and wound repair in both normal and aged rodents. It also promotes hair growth in normal and aged rodents. It acts by increasing angiogenesis and cell migration and is currently in clinical trials for wound repair.

Aging↗
PubMed2001

Effect of peptide-carrier coupling on peptide-specific immune responses.

Synthetic peptides are covalently linked to immunogenic carrier proteins to enhance the anti-peptide immune response. To investigate whether the method of conjugation influences the immune response, we evaluated two distinctly different choices of linker for a peptide-carrier construct. HPG-30, a synthetic peptide derived from the p17 gag protein of human immunodeficiency virus 1, was covalently linked to keyhole limpet hemocyanin by either glutaraldehyde or a maleimide ester. Glutaraldehyde linkage enhanced the anti-peptide antibody and native protein response compared to maleimide. The maleimide-linked conjugate was more effective at inducing a peptide-specific cellular response. Thus, manipulation of the conjugation method can modify the magnitude and character of the immune response to a synthetic peptide vaccine.

Amino Acid Sequence↗
PubMed2001

Development of Saccharomyces cerevisiae as a model pathogen. A system for the genetic identification of gene products required for survival in the mammalian host environment.

Saccharomyces cerevisiae, a close relative of the pathogenic Candida species, is an emerging opportunistic pathogen. An isogenic series of S. cerevisiae strains, derived from a human clinical isolate, were used to examine the role of evolutionarily conserved pathways in fungal survival in a mouse host. As is the case for the corresponding Candida albicans and Cryptococcus neoformans mutants, S. cerevisiae purine and pyrimidine auxotrophs were severely deficient in survival, consistent with there being evolutionary conservation of survival traits. Resistance to the antifungal drug 5-fluorocytosine was not deleterious and appeared to be slightly advantageous in vivo. Of mutants in three amino acid biosynthetic pathways, only leu2 mutants were severely deficient in vivo. Unlike the glyoxylate cycle, respiration was very important for survival; however, the mitochondrial genome made a respiration-independent contribution to survival. Mutants deficient in pseudohyphal formation were tested in vivo; flo11Delta mutants were phenotypically neutral while flo8Delta, tec1Delta, and flo8Delta tec1Delta mutants were slightly deficient. Because of its ease of genetic manipulation and the immense S. cerevisiae database, which includes the best annotated eukaryotic genome sequence, S. cerevisiae is a superb model system for the identification of gene products important for fungal survival in the mammalian host environment.

Amino Acids↗
PubMed2001

Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR.

Candida albicans and Cryptococcus neoformans cause both superficial and disseminated infections in humans. Current antifungal therapies for deep-seated infections are limited to amphotericin B, flucytosine, and azoles. A limitation is that commonly used azoles are fungistatic in vitro and in vivo. Our studies address the mechanisms of antifungal activity of the immunosuppressive drug rapamycin (sirolimus) and its analogs with decreased immunosuppressive activity. C. albicans rbp1/rbp1 mutant strains lacking a homolog of the FK506-rapamycin target protein FKBP12 were found to be viable and resistant to rapamycin and its analogs. Rapamycin and analogs promoted FKBP12 binding to the wild-type Tor1 kinase but not to a rapamycin-resistant Tor1 mutant kinase (S1972R). FKBP12 and TOR mutations conferred resistance to rapamycin and its analogs in C. albicans, C. neoformans, and Saccharomyces cerevisiae. Our findings demonstrate the antifungal activity of rapamycin and rapamycin analogs is mediated via conserved complexes with FKBP12 and Tor kinase homologs in divergent yeasts. Taken together with our observations that rapamycin and its analogs are fungicidal and that spontaneous drug resistance occurs at a low rate, these mechanistic findings support continued investigation of rapamycin analogs as novel antifungal agents.

Antifungal Agents↗
PubMed2000

Thymosinalpha1 is chemopreventive for lung adenoma formation in A/J mice.

The effects of thymosin (THN) alpha1 were investigated using the urethane injection carcinogenesis A/J mouse model. Lung adenomas were observed 2.5, 3, and 4 months after urethane injection (400 mg/kg i.p.) into female A/J mice. Daily administration of THNalpha1 (0.4 mg/kg, s.c.) reduced lung adenoma multiplicity significantly, by approximately 45, 40, and 17%, respectively, 2.5, 3, and 4 months after urethane injection. Animals treated with THNalpha1 had a significantly greater white cell density than control A/J mice. Endogenous THNalpha1-like peptides were detected in the mouse lung. By radioimmunoassay and by Western blot, prothymosin alpha was detected in the mouse lung. By immunocytochemistry, THNalpha1-like peptides were detected in all lung compartments including the bronchus, adenoma, bronchioles, and alveoli. These results indicate that exogenous THNalpha1 prevents lung carcinogenesis in A/J mice.

Adenoma↗
PubMed2000

Thymosin alpha1 is a time and dose-dependent antagonist of dexamethasone-induced apoptosis of murine thymocytes in vitro.

It is well established that glucocorticoid hormones induce apoptosis in immature developing thymocytes. Thymocyte apoptosis can be modulated by growth factors, anti-oxidants and adhesion receptors. We have previously demonstrated that thymosin alpha1 (Talpha1) antagonizes dexamethasone-induced apoptosis of CD4+CD8+ thymocytes. In the present study, we further characterize the dose and time dependence of Talpha1's antagonism of dexamethasone-induced thymocyte apoptosis. Talpha1 is effective at concentrations ranging from 2 to 100 microg/10(6) thymocytes. Talpha1 pre-treatment is necessary to achieve its anti-apoptotic activity. Talpha1 provides temporary protection to thymocytes by slowing dexamethasone's apoptotic activity up to 12 h post dexamethasone treatment. Additionally, Talpha1's activity is not sensitive to cycloheximide treatment, suggesting Talpha1's activity is independent of protein synthesis. Finally, Talpha1 is unable to antagonize apoptosis induced by the reactive oxygen species, H2O2, suggesting Talpha1's antagonism of dexamethasone occurs at the early stages of dexamethasone-induced apoptosis, prior to the production of reactive oxygen species. This evidence suggests that Talpha1 may provide a mechanism to transiently extend the life of a thymocyte during thymic selection.

Adjuvants, Immunologic↗
PubMed1999

Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae.

Heterologous gene replacement cassettes are powerful tools for dissecting gene function in Saccharomyces cerevisiae. Their primary advantages over homologous gene replacement cassettes include reduced gene conversion (leading to efficient site-specific integration of the cassette) and greater independence of strain background. Perhaps the most widely used cassettes are the MX cassettes containing the dominant selectable kanamycin resistance gene (kanr), which confers resistance to G418 (Wach et al., 1994). One limitation of the kanMX cassettes is that they are not counterselectable and therefore not readily recyclable, which is important when constructing strains with more than one gene deletion. To address this limitation, and to expand the choices of heterologous markers, we have created two new MX cassettes by replacing the kanr ORF from plasmids pFA6-kanMX3 and pFA6-kanMX4 with the Candida albicans URA3 ORF. These plasmids, pAG60 (CaURA3MX4) and pAG61 (CaURA3MX3) are identical to the kanMX cassettes in all other respects but have the added advantage of being counterselectable and therefore readily recyclable in S. cerevisiae.

Candida albicans↗
PubMed1999

Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae.

Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.

Drug Resistance, Microbial↗
PubMed1999

Thymosin beta4 accelerates wound healing.

Angiogenesis is an essential step in the repair process that occurs after injury. In this study, we investigated whether the angiogenic thymic peptide thymosin beta4 (Tbeta4) enhanced wound healing in a rat full thickness wound model. Addition of Tbeta4 topically or intraperitoneally increased reepithelialization by 42% over saline controls at 4 d and by as much as 61% at 7 d post-wounding. Treated wounds also contracted at least 11% more than controls by day 7. Increased collagen deposition and angiogenesis were observed in the treated wounds. We also found that Tbeta4 stimulated keratinocyte migration in the Boyden chamber assay. After 4-5 h, migration was stimulated 2-3-fold over migration with medium alone when as little as 10 pg of Tbeta4 was added to the assay. These results suggest that Tbeta4 is a potent wound healing factor with multiple activities that may be useful in the clinic.

Animals↗
PubMed1998

Direct allelic variation scanning of the yeast genome.

As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).

Alleles↗
PubMed1998

Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export.

To identify Saccharomyces cerevisiae genes important for nucleocytoplasmic export of messenger RNA, we screened mutant strains to identify those in which poly(A)+ RNA accumulated in nuclei under nonpermissive conditions. We describe the identification of DBP5 as the gene defective in the strain carrying the rat8-1 allele (RAT = ribonucleic acid trafficking). Dbp5p/Rat8p, a previously uncharacterized member of the DEAD-box family of proteins, is closely related to eukaryotic initiation factor 4A(eIF4A) an RNA helicase essential for protein synthesis initiation. Analysis of protein databases suggests most eukaryotic genomes encode a DEAD-box protein that is probably a homolog of yeast Dbp5p/Rat8p. Temperature-sensitive alleles of DBP5/RAT8 were prepared. In rat8 mutant strains, cells displayed rapid, synchronous accumulation of poly(A)+ RNA in nuclei when shifted to the non-permissive temperature. Dbp5p/Rat8p is located within the cytoplasm and concentrated in the perinuclear region. Analysis of the distribution of Dbp5p/Rat8p in yeast strains where nuclear pore complexes are tightly clustered indicated that a fraction of this protein associates with nuclear pore complexes (NPCs). The strong mutant phenotype, association of the protein with NPCs and genetic interaction with factors involved in RNA export provide strong evidence that Dbp5p/Rat8p plays a direct role in RNA export.

Alleles↗
PubMed1998

Thymosin alpha 1 stimulates endothelial cell migration, angiogenesis, and wound healing.

In wound healing, lymphoid cells release soluble factors that attract fibroblasts and macrophages, initiating repair, endothelial cell migration, angiogenesis, and matrix production. We analyzed the effect of thymosin alpha1 (Talpha1) on endothelial cell migration, angiogenesis, and wound healing. Talpha1, a 28 amino acid peptide initially isolated from the thymus, enhanced the morphologic differentiation of endothelial cells and was a potent chemoattractant for endothelial cells and monocytes in vitro. In vivo, Talpha1 stimulated angiogenesis in a subcutaneous model. When given either topically or i.p., it accelerated wound healing in a punch model, demonstrating that Talpha1 promotes angiogenesis and wound healing.

Adjuvants, Immunologic↗
PubMed1998

Thymosin fraction 5 inhibits the proliferation of the rat neuroendocrine MMQ pituitary adenoma and C6 glioma cell lines in vitro.

Cytokines such as interleukin-1 (IL-1) and IL-6 stimulate the hypothalamic-pituitary-adrenal (HPA) axis. In addition, these proteins affect pituitary cell proliferation in vitro. Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus that enhances immune system functioning. Because TF5 similarly stimulates the HPA axis, we examined the effects of this preparation on neuroendocrine tumor cell proliferation. Cells of the PRL-secreting rat anterior pituitary adenoma, MMQ (5-50 x 10(3) cells/well), were exposed to vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (100-500 microg/ml) for up to 96 h and the proliferation of MMQ cells monitored using the MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). TF5-mediated inhibition of cell proliferation was dependent on both TF5 concentration and the initial MMQ cell number. Minimal reductions in optical densities resulted from exposure to 100 microg/ml TF5, whereas the highest concentration of this preparation (i.e. 500 microg/ml) completely blocked MMQ cell division. The concentration-dependent effects of TF5 were particularly striking at initial plating densities of 25 and 50 x 10(3) MMQ cells/well; in contrast, all concentrations of TF5 completely inhibited MMQ cell growth at 5 and 10 x 10(3) cells/well. The antiproliferative actions of TF5 on MMQ cells were demonstrable within 24 h and remained for up to 96 h as determined by the MTT assay and actual cell counts. Because the highest densities of MMQ cells were partially refractive to the antiproliferative effects of TF5, we examined the effects of PRL (1-1000 nM) and MMQ cell conditioned medium (50%) on TF5 inhibition of MMQ adenoma proliferation. The TF5 concentration-dependent inhibition of MMQ cell growth was largely reversed by the 50% conditioned medium, whereas PRL slightly potentiated the antiproliferative actions of TF5. The proliferation of the rat C6 glioma cell line (10-30 x 10(3) cells/well) demonstrated greater sensitivity to TF5: concentrations as low as 10 microg/ml TF5 inhibited C6 cell proliferation (P < 0.01), and near-maximal inhibition was noted at 200 microg/ml TF5. Significant reductions in MMQ and C6 cell viabilities accompanied decreases in cell number and morphological analysis indicated these cells were dying by apoptosis. The peptides thymosin alpha1 (T alpha1), thymosin beta4 (T beta4), MB35, and MB40 had no effect on either MMQ or C6 cell proliferation, indicating that these TF5 components are not the principle active peptides. Therefore, TF5 was further separated into 60 fractions by preparative reverse phase HPLC. HPLC fractions 17, 25, 26, and 27 significantly suppressed MMQ cell proliferation (P < 0.01) to the same extent as TF5; other HPLC fractions had no effect. These data demonstrate a new biological property of TF5: the inhibition of cell proliferation and the induction of apoptosis in neuroendocrine tumor cells. The proliferation effects were time and concentration dependent and could be partially reversed by an activity present in the MMQ cell conditioned medium. Thus, TF5 and cytokines have opposite effects on adenoma cells because IL-2 and IL-6 stimulate GH3 cell proliferation. We propose that circulating thymic peptides may act to prevent pituitary adenoma and glioma tumor formation, an action opposed by autocrine growth factors secreted by these tumors.

Adenoma↗
PubMed1997

Thymosin alpha 1 antagonizes dexamethasone and CD3-induced apoptosis of CD4+ CD8+ thymocytes through the activation of cAMP and protein kinase C dependent second messenger pathways.

It is well established that glucocorticoid hormones and anti-CD3 monoclonal antibodies induce apoptosis in immature developing thymocytes. This process can be modulated by soluble factors, anti-oxidants and adhesion receptors. Previously we have demonstrated that thymosin alpha 1 (T alpha 1), a 28-amino acid thymic peptide hormone, is a dose and time dependent antagonist of dexamethasone (DEX) and CD# induced DNA fragmentation of murine thymocytes in vitro. To further investigate the mechanism of T alpha 1 action we determined a T alpha 1 sensitive thymocyte population and examined some of the molecular events associated with T alpha 1 anti-apoptotic activity. Phenotypic analysis of the sub-populations of thymocytes, based on CD4 and CD8 expression, revealed that T alpha 1 exerts its effect on CD4+ CD8+ immature thymocytes. T alpha 1 treatment of thymocytes delays the production of free radicals and the subsequent consumption of glutathione, that is observed during both DEX and CD3 induced apoptosis. We further demonstrate that T alpha 1 stimulates the production of cAMP and activates PKC in thymocytes. These data suggest that T alpha 1 exerts an influence on the development of a population of immature T-cells in the thymus by effecting the sensitivity of thymocytes to apoptosis during the pre-selection stages of thymic development. Our studies also suggest that the mechanism of T alpha 1 action involves the induction of both cAMP and PKC dependent second messenger pathways.

Animals↗
PubMed1997

Biodistribution of synthetic thymosin beta 4 in the serum, urine, and major organs of mice.

Thymosin beta 4 (T beta 4) is a peptide of 43 amino acids that was first isolated from the thymus gland and subsequently found to be ubiquitous in nature. T beta 4 functions mainly as an actin-sequestering molecule in nonmuscle cells, where its primary role is to maintain the large pool of unpolymerized G-actin in the cell. Studies on the pharmacokinetics of T beta 4 in human and other mammals have not been reported so far. In the present study, we have measured T beta 4 concentrations in serum, urine, and 10 major organs of female Swiss-Webster mice following intraperitoneal administration of 400 micrograms synthetic T beta 4. Using a modified enzymatic immunoassay, our data show a significant increase of T beta 4 in serum starting 2 min after injection and lasting for 40 min (average: 2.34 +/- 0.54 micrograms/ml). High concentrations were found in urine (59.3 +/- 7.54 micrograms/ml) at three different points after injection (20 min, 40 min, and 2 h). Of the 400 micrograms T beta 4 administered to mice 83% was recovered at the end of the study, 44.6% of which corresponded to urine, 1.4% to serum, and 37.5% to the organs. In 50% of the tested organs, the wet weight concentrations of T beta 4 increased significantly from the first 40 min to 2 h after injection in comparison to their baseline wet weight concentrations. These organs were: the brain (72 micrograms/g), heart (80 micrograms/g), liver (15 micrograms/g vs 9 micrograms/g), kidneys (65 micrograms/g vs 28 micrograms/g), and peritoneal fat (47 micrograms/g vs 13 micrograms/g). Wet weight concentrations increased in the thymus (196 micrograms/g vs 147 micrograms/g) and muscle (45 micrograms/g vs 0 micrograms/g) after 6 h of injection. The spleen showed an increase in wet weight concentrations at the 2 min timepoint (267 micrograms/g vs 161 micrograms/g). Ovaries had a biphasic increase at 40 min (72 micrograms/g vs 62 micrograms/g) and 24 h (92 micrograms/g vs 62 micrograms/g) after T beta 4 administration. In lungs, the highest wet weight increase after injection (149 micrograms/g at timepoint 6 h) was not higher than its basal wet weight concentration (153 micrograms/g). These pharmacokinetic studies of T beta 4 in mice have established that high levels of T beta 4 are found in blood following I.P. administration and the kidney rapidly removes the peptide from the circulation. The kinetics of this response should help define the proper scheduling of administration of T beta 4 during clinical trials in disorders, such as the acute respiratory distress syndrome (ARDS), associated with actin toxicity.

Animals↗
PubMed1997

Biodistribution of synthetic thymosin alpha1 in the serum, urine and major organs of mice.

Thymosin fraction 5 (TF5), a thymic preparation, has been shown to be an immune-potentiating agent consisting of biologically active polypeptide components with hormone-like activities. Thymosin alpha1 (T alpha1) was the first biologically active polypeptide to be purified from TF5 and completely characterized. It is an acidic peptide with an isoelectric point of 4.2 and a molecular weight of 3108. T alpha1 is considered a biological response modifier which amplifies T-cell immunity. In the present study, we have studied some pharmacokinetic properties of T alpha1 by measuring its concentrations in serum, urine and ten major organs of female Swiss-Webster mice following administration of 500 microg T alpha1 intraperitoneally. Using a modified enzymatic immunoassay, our data show a significant increase of T alpha1 in serum 2 min after injection and lasting for 2 h (average: 1.55 +/- 0.27 microg/ml). In urine, at four different time points after injection (20 min, 40 min, 2 h, 6 h), increased concentrations of T alpha1 were found between 24.2 and 25.4 microg/ml (average: 25 +/- 0.47 microg/ml). Of the 500 microg T alpha1 administered to mice, 8.97% was recovered at the end of the study, of which 2% corresponded to urine, 1.25% to serum (2 ml of serum per mouse), and 5.72% to organs. Since the urine/day volume and the serum volume of any Swiss Webster mouse is ca 2 ml, additional extrapolation of the above mentioned values could show percentages of recovery close to 40% for urine and 2.5% for serum. In most of the organs, the wet weight concentrations of T alpha1 increased significantly during the first 40 min after injection in comparison to their baseline wet weight concentrations. These organs consisted of the following: thymus (33.1 +/- 3.5 microg/g vs 18 microg/g baseline); lungs (7.7 +/- 1.1 microg/g vs 1.9 microg/g baseline); spleen (15.6 +/- 0.7 microg/g vs 5.6 microg/g); kidneys (6.2 +/- 1.1 microg/g vs 3.9 microg/g); ovaries (9.2 +/- 1.4 microg/g vs 0 microg/g); and peritoneal fat (4 +/- 1 microg/g vs 0 microg/g). No significant increases were observed in the liver (1.7 +/- 0.1 microg/g vs 1.4 microg/g) and heart (0.7 +/- 0.5 microg/g vs 0 microg/g). Increased concentrations of T alpha1 were not detected in the brain and skeletal muscle tissues. These pharmacokinetic studies of T alpha1 in mice indicate that rapid renal excretion of T alpha1 represents a major source of humoral loss following I.P. administration. Recent preliminary studies in humans confirm that the kidney rapidly releases high levels of T alpha1 in urine in a time frame consistent with that observed in mice.

Adipose Tissue↗
PubMed1997

Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells.

Thymosin beta 4 (T beta 4) is a 4.9 kDa polypeptide that interacts with G-actin and is thought to be an important mediator in cell proliferation, migration, and differentiation. T beta 4 has been identified as a factor involved in the differentiation of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel. Here we have used various in vitro and in vivo migration assays to demonstrate the role of T beta 4 in endothelial cell migration. Our results demonstrate that T beta 4 acts as a chemoattractant for endothelial cells, stimulating the migration of HUVECs in Boyden chambers four- to sixfold over that observed with media alone. Of the primary cell types tested, only human coronary artery cells responded to T beta 4 treatment, suggesting that the migration activity of T beta 4 was endothelial cell-specific. T beta 4 significantly accelerated the rate of migration into the scratch wounded area of a HUVEC monolayer. T beta 4 treatment also increased the production of matrix metalloproteinases that may degrade the basement membrane during angiogenesis. Additional experiments using subcutaneously implanted Matrigel showed that T beta 4 stimulated cell migration in vivo. These results provide the first direct evidence that T beta 4 has chemoattractive activity and promotes angiogenesis by stimulating the migration of endothelial cells.

Cell Movement↗