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G Krystal

Publications and source records attributed to G Krystal.

At least 109 records · Page 6Linked to original sources
PubMed1988

The rat prepro gastrin releasing peptide gene is transcribed from two initiation sites in the brain.

The gastrin-releasing peptide (GRP) is a gastrointestinal hormone, a neuropeptide, and a growth factor. To study systematically GRP gene expression in embryonic and in adult mammals, we cloned the rat prepro-GRP gene from brain cDNA libraries. Analysis of these cDNA clones along with organ-specific mRNA studies show that there are prepro-GRP transcripts initiating in the brain, but not in the duodenum, from a novel promoter. The latter is located at least four hundred base pairs upstream from a second promoter that is active in both duodenum and in brain. In contrast to human prepro-GRP, there is no alternative processing of the 3'-region of rat brain and duodenum prepro-GRP transcripts; hence, these mRNAs encode a single 147 amino acid prepro-GRP hormone that is homologous to the third isoform of the human prepro-GRP hormone.

Amino Acid Sequence↗
PubMed1988

Activation of multiple hemopoietic growth factor genes in Abelson virus-transformed myeloid cells.

We have recently shown that Abelson murine leukemia (A-MuLV) virus can transform cells in large mixed colonies to give tumorigenic myeloid cell lines capable of autonomous growth in vitro. Initial studies revealed that granulocyte-macrophage colony-stimulating factor (GM-CSF) production was consistently activated in these cells. Using a sensitive S1 RNA mapping technique and additional bioassays, we have now obtained evidence of expression of other hemopoietic growth factor genes. Uniformly 32P-labeled, single-stranded DNA probes (greater than 4 x 10(8) cpm/micrograms) were generated for interleukin 3 (IL-3) and GM-CSF using pTZ based vectors. IL-3 mRNA was detected in four of four cloned transformants (from two different infections) at approximately 1% of the level seen in pokeweed mitogen (PWM)-stimulated spleen cells. GM-CSF mRNA was detected in the two clones that showed the highest IL-3 mRNA levels. Medium conditioned by these cells was able to stimulate IL-3-dependent 32D cells, and IL-3- and GM-CSF-dependent B6SUtA cells, and also supported the growth of a variety of single and multilineage colonies in assays of mouse marrow cells even in the presence of neutralizing antibodies to GM-CSF. Rearrangements of the IL-3 and GM-CSF genes were not apparent by Southern blot analysis. Additional bioassays revealed the presence of two other growth factors: IL-6 (hybridoma growth factor or Ifn-beta 2) assayed on B13.29 cells, a factor-dependent murine B-cell hybridoma; and a new pre-B-cell stimulatory factor different from any of the above. Elucidation of the mechanism underlying this phenomenon may provide important insights into the regulation of hemopoietic growth factor gene expression and the role such genes play in human leukemogenesis.

Abelson murine leukemia virus↗
PubMed1988

Partial characterization of a novel stromal cell-derived pre-B-cell growth factor active on normal and immortalized pre-B cells.

Characterization of three stromal cell lines that support the proliferation of murine pre-B cells showed that they all exhibit a fibroblast-like morphology, but express laminin and collagen type IV, and behave as pre-adipocytes. One of these lines was used as a feeder for the isolation of feeder-dependent (A8 cells) and feeder-independent (H9 cells) pre-B-cell subclones present in a spontaneously immortalized line of pre-B cells that originally arose in a long-term lymphoid marrow culture. Both pre-B subclones show the same JH gene rearrangement and are strongly BP-1 positive but differ from most pre-B-cell lines described to date in that they do not express immunoglobulin (Ig), B220, or T200. Cell density experiments confirmed that A8 cells do not proliferate (or survive) in the absence of an appropriate feeder layer when cultured alone at concentrations less than 10(5) cells/ml, but greater than 10(5) cells/ml can undergo a short-lived burst of proliferation. In contrast, H9 cells are readily maintained as an autonomous line at concentrations greater than 10(4) cells/ml, but their growth is suboptimal in the absence of stromal cells at lower concentrations, and at less than 3000 cells/ml, their ability to proliferate ceases. Additional experiments demonstrated that at least part of the supportive function of the stromal cells is due to their ability to produce a low molecular weight, heat-resistant factor that is active on normal pre-B cells as well as both A8 and H9 cells, and appears to be different from any previously described hemopoietic growth factor.

Animals↗
PubMed1988

Use of a sensitive bioimmunoabsorbent assay to isolate and characterize monoclonal antibodies to biologically active human erythropoietin.

At present, one of the most sensitive assays for human erythropoietin (Ep) is a bioassay that measures the Ep-dependent proliferation of spleen cells from phenylhydrazine-treated mice after 24 hours in culture. We describe how this assay can be used as the basis of a very sensitive method for detecting mouse antibodies to biologically active human Ep. In this procedure, microtiter wells are first coated with goat anti-mouse Ig antibody, then treated with mouse antibodies (serum or hybridoma culture supernatants), and finally incubated with a fixed amount of pure human Ep. Specific binding of anti-Ep antibodies is detected by adding spleen cells from phenylhydrazine-treated mice to the wells and measuring the ability of the cells to incorporate 3H-thymidine 24 hours later. This bioimmunosorbent assay (BISA) revealed the presence of anti-EP antibodies in sera from mice immunized with either pure human urinary Ep or a synthetic dodecapeptide corresponding to the aminoterminal region of Ep and in the culture supernatants from three of eight stable anti-Ep antibody-producing hybridoma cell lines that we have isolated. The three monoclonal antibodies showed similar reactivities in the BISA, but showed different affinities for Ep, with Kd values of approximately 0.7, 8, and 240 nmol/L, respectively. Further studies showed that all antibodies were capable of neutralizing Ep bioactivity and of binding 125I-labeled Ep in a radioimmunosorbent assay (RIA) but were virtually unreactive to Ep adsorbed to the bottom of enzyme-linked immunosorbent assay (ELISA) wells. Our results suggest that the BISA strategy may be an important complement to conventional RIA and ELISA techniques for identification of monoclonal antibodies specific for biologically active growth factors.

Antibodies, Monoclonal↗
PubMed1987

A silver-binding assay for measuring nanogram amounts of protein in solution.

We recently reported a highly sensitive assay for measuring protein in solution based on the capacity of glutaraldehyde-treated protein to bind silver. This assay has now been made more sensitive, with a lower limit of detection of 5 ng, and more reproducible by supplementing protein samples with sodium dodecyl sulfate (SDS) to reduce protein loss to glassware. Two procedures have been developed. In one, protein samples are supplemented with both SDS and Tween 20 to yield very steep protein dose-response curves, which allow for more precise protein determinations, and very stable color formation, permitting OD measurements to be made several hours after the assay has been completed. In the second procedure, protein samples are supplemented with SDS alone which results in a less steep dose-response curve and less stable color formation but makes the assay substantially more tolerant of interfering substances. Thus, proteins in most commonly used buffers can be assayed directly with the second procedure without the need for buffer exchange. The procedure of choice, therefore, depends on the type and concentration of interfering substance. Proteins in buffers totally incompatible with either assay procedure (e.g., those containing reducing agents) can be easily buffer exchanged by centrifugation through 0.2% SDS equilibrated, drained Bio-Gel P-2 beads. The clinical utility of this improved assay is demonstrated by the accurate quantitation of protein in 0.5 microliter of samples of human cerebral spinal fluid. This assay should therefore prove especially useful when a limited amount of protein is available for quantitation.

Chromatography, Gel↗
PubMed1987

Effects of granulocyte-macrophage colony-stimulating factor and erythropoietin on leukemic erythroid colony formation in human early erythroblastic leukemias.

Erythroid colonies from five patients with an early erythroblastic leukemia were obtained in "serum-free" cultures in the presence or absence of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and homogeneous native erythropoietin (Epo). Erythroid colonies with abnormal morphology and karyotype could be grown in different culture conditions. Their erythroid nature was ascertained by the presence of carbonic anhydrase I and glycophorin A. Leukemic erythroid progenitors strongly differed from normal progenitors in that spontaneous colonies were always obtained, sometimes with an extremely high plating efficiency (up to 5.7%). Colonies were found to be autonomous from exogenous hematopoietic growth factors because they were still obtained with a high plating efficiency at an average of one cell per culture in the absence of any added growth factor. No evidence for an autocrine secretion of Epo or GM-CSF emerged because Epo or GM-CSF could not be detected by biologic or radioimmunologic assays from the culture supernatant or cellular extracts of the leukemic cells and that Epo or GM-CSF antibodies did not block autonomous growth. In all cases, however, hematopoietic growth factors increased the plating efficiency of the abnormal erythroid progenitors. In the two "de novo" leukemias, leukemic erythroid progenitors responded primarily to Epo, whereas in the three other patients' (chronic myeloid leukemia) blast crisis they responded maximally to GM-CSF plus Epo. Recombinant erythroid-potentiating activity had no effect in any of these cases. These results suggest that the leukemic erythroid clonogenic cells arise from expansion of erythroid progenitors at different levels of differentiation (ie, CFU-E or BFU-E, depending upon the disease) and that autonomous growth is not related to a secretion of Epo or GM-CSF.

Cell Cycle↗
PubMed1986

Correction of defective IL 3 responses of T lymphocytes from autoimmune mice.

MRL-+ and MRL-lpr congenic mice differ by the presence and expression of the homozygous recessive lymphoproliferation (lpr) gene. One manifestation of this gene is a massive T cell proliferation that results in a generalized lymphadenopathy in older animals. Interleukin 3 (IL 3), a recently described lymphokine, has been shown to influence lymphocyte differentiation. It was possible that abberrant IL 3 production was the mechanism responsible for the lpr controlled lymphadenopathy. Consequently, in this paper we tested the MRL congenic mice for their ability to produce IL 3. We report that the T lymphocytes from MRL-+ and MRL-lpr could neither respond to pokeweed mitogen in the induction of proliferation nor produce IL 3. Moreover, IL 3 was not produced constitutively nor could be induced at any time period up to 5 days in vitro. This hyporesponsiveness was shown to be controlled at the accessory cell level. Addition of T cell-depleted peritoneal exudate cells from normal major histocompatibility complex (MHC) compatible mice was able to restore the ability to secrete IL 3 in response to pokeweed mitogen in MRL-+ and young MRL-lpr mice. The defect in the accessory cells could be overridden by two means: the incorporation of phorbol myristate acetate in the induction system and preincubation of the cells in tissue culture.

Animals↗
PubMed1986

Identification of the interleukin-3 receptor using an iodinatable, cleavable, photoreactive cross-linking agent.

Murine interleukin-3 (mIL-3) is a lymphokine that stimulates the proliferation and differentiation of both pluripotent hemopoietic stem cells and their committed progeny. However, very little is known about the mechanism by which this growth factor elicits its effects on responsive cell populations. To gain insight into early events following mIL-3 receptor interaction, we initiated studies to isolate the receptor and study its properties. In this report, we demonstrate the use of a new iodinatable, cleavable, photoreactive cross-linking agent, sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate to identify the mIL-3 receptor. These studies reveal the mIL-3 receptor to be a single polypeptide chain with a molecular weight of 67 kDa and an isoelectric point of approximately 6.2.

Animals↗
PubMed1986

Modulation by normal serum factors of Kirsten murine sarcoma virus-induced transformation in adult rat cells infected in early passage.

Adult rat adrenal cells, infected with Kirsten murine sarcoma virus in early passage, transform consistently in 10% fetal bovine serum (FBS)-supplemented medium. Substitution of 3% horse serum (HS) for FBS reverses early foci and delays transformation. The influence of the serum on DNA synthesis, anchorage dependence, tumorigenicity, and subcellular Mr 21,000 transforming protein (p21) distribution was followed from infection in passage 1 to complete transformation. In FBS, increased expression of p21 preceded other evidence of transformation. Subsequently, p21-positive cells transformed morphologically, but initially their growth parallelled that of coexisting untransformed cells. Foci formed at passages 5 to 10, and the cells became anchorage independent and tumorigenic at passages 10 to 20. As transformation in FBS progressed, p21 relocated from a diffuse distribution to sites of retraction from substrata and then to ruffles and lamellae on cellular processes. Early in transformation, HS-medium reduced proliferation of morphologically normal and morphologically transformed p21-positive cells. This effect was counteracted by the addition of FBS or the Mr 50,000 to 100,000 fraction of FBS. Fully transformed, tumorigenic cells grew rapidly in both sera but, if transferred from FBS to HS, became more anchorage and density dependent, and p21 relocated from cell processes to the cell bodies. In immortal lines, the substitution of HS for FBS accelerated rather than delayed the progression of Kirsten murine sarcoma virus-induced transformation. These results show that Kirsten murine sarcoma virus-induced transformation of adult presenescent cells is controlled by physiological factors to which immortal cells appear refractory. The changes in subcellular distribution of p21 during transformation parallel the expression of some, but not other, transformation parameters and suggest a possible association of p21 with increased membrane activity.

Adrenal Glands↗
PubMed1986

Purification of human erythropoietin to homogeneity by a rapid five-step procedure.

Human urinary erythropoietin (Ep) has been purified using a simple five-step procedure to yield preparations with potencies of 80,000 U/mg in 25% yield. The five steps involve: (1) affinity chromatography on CM Affi-Gel Blue, (2) chromatofocusing, (3) wheat germ lectin (or hydroxylapatite) chromatography, (4) reverse-phase high-performance liquid chromatography (HPLC) using a phenyl column, and (5) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ep activity was determined at each stage using a highly sensitive and specific in vitro assay that measures [3H]-thymidine incorporation into erythroid cells from spleens of phenylhydrazine-treated mice. The step 5 material was also tested with the in vivo polycythemic mouse assay procedure and was found to have a similar potency to that obtained in the [3H]-thymidine in vitro assay. SDS-PAGE analysis of the step 5 material revealed a single 38.5-kd band that comigrated with Ep bioactivity. Homogeneity was confirmed by amino acid sequence analysis. Starting with urine containing approximately 13 U/mg of protein, the cumulative degrees of purification achieved with each step were: step 1,25-fold; step 2, 75-fold; step 3, 300-fold; step 4, 1,500-fold; and step 5, 5,000-fold. Corresponding overall recoveries after each step were: greater than 100%, 70%, 45%, 30%, and 25%. These recoveries could be obtained when as little as 5,000 U of starting urinary Ep were processed because of the introduction of Tween 20 and SDS into buffers used at various stages of the purification procedure. In addition, a rapid method for determining Ep purity which involves reverse-phase HPLC of trypsinized 125I-labeled Ep is presented. This allows the establishment of purity with far less material than is required for amino acid sequencing.

Amino Acid Sequence↗
PubMed1985

A method for quantitating nanogram amounts of soluble protein using the principle of silver binding.

A highly sensitive and quantitative assay for measuring protein in solution based on the capacity of protein to bind silver is described. In this procedure, protein samples are first treated with glutaraldehyde and then exposed to ammoniacal silver. After 10 min, the reaction is terminated by the addition of sodium thiosulfate and the optical density measured at 420 nm. The useful range of the assay for the majority of standard proteins tested lies between 15 and 2000 ng. This represents a 100-fold increase in sensitivity over the Coomassie brilliant blue dye-binding procedure. There is little or no interference from carbohydrates, nonionic detergents, or ethanol, and pretreatment of protein samples with Bio-Gel P-2 to remove salts, thiol agents, EDTA, and sodium dodecyl sulfate makes this procedure compatible with most commonly used buffers. The cost in terms of silver utilization is nominal with a typical assay involving 10 samples tested in triplicate amounting to less than $0.02 U. S.

Animals↗
PubMed1984

CM Affi-Gel Blue chromatography of human urine: a simple one-step procedure for obtaining erythropoietin suitable for in vitro erythropoietic progenitor assays.

A method for both concentrating and purifying human urinary erythropoietin (Ep) using CM Affi-Gel Blue is described. We have found that up to 40 litres of urine can be processed on a 1 litre gel bed of this material. This gives a 25-50-fold purification of Ep with an apparent Ep recovery in excess of 100%. The high recovery of Ep is probably due, in part, to the removal of inhibitors present in the initial urine. By selecting urine that contains high levels of Ep (greater than 0.5 units/ml), it is possible with this method routinely to obtain preparations with specific activities of 100-300 units of Ep per mg protein. Such preparations are noninhibitory when assayed in either short-term suspension cultures or in longer-term methylcellulose cultures at concentrations up to 5-10 units/ml. Similar tests with these same bioassay systems have shown that other non-Ep stimulating factors (i.e. erythroblast enhancing factor (EEF), granulocyte/macrophage colony stimulating factor (GM-CSF) and burst promoting activity (BPA) ) are also not present at detectable levels. In this study we also show that the loss of biological activity which often occurs when partially purified Ep preparations are stored in solution is markedly reduced in the presence of either 1% bovine serum albumin or 0.1% sodium dodecyl sulphate.

Animals↗
PubMed1983

DNA repair by articular chondrocytes. I. Unscheduled DNA synthesis following ultraviolet irradiation in monolayer culture.

The hypothesis that aging of articular chondrocytes at a cellular level results from loss of DNA repair capability was studied by measuring unscheduled DNA synthesis (UDS). Cultured rabbit and human articular chondrocytes were irradiated with 254 nm ultraviolet light (20 J/m2) following treatment with 10 mM hydroxyurea. Neither the "in vitro senescence" nor spontaneous transformation that developed during serial passage of rabbit chondrocytes was accompanied by diminution of UDS. Synthesis of sulfated glycosaminoglycans declined more rapidly than the ability of the cells to divide. Levels of UDS by chondrocytes from old donors, rabbit or human, were comparable to those of younger individuals. UDS was greater in human than rabbit chondrocytes. Similar data have been reported previously for dermal fibroblasts but do not necessarily indicate that there is a direct or causative relationship between UDS capability and the longevity of mammalian species. X-Irradiation of rabbit chondrocytes or cartilage explants, in doses up to 40 000 rads, yielded no measurable UDS.

Adult↗
PubMed1983

Evidence for an erythroblast-enhancing factor (EEF) in human and mouse serum.

Evidence accumulated over the past 20 years suggests that factors besides erythropoietin (Ep) may play a significant role in regulating mammalian erythropoiesis in vivo. These include moieties with both stimulatory and inhibitory activities. In this report data are summarized that demonstrate the presence of an erythropoietic stimulator in human and mouse serum that is distinct from Ep, burst-enhancing factor, albumin, or iron-saturated transferrin. Like Ep, this factor causes a significant increase in the amount of 59Fe incorporation into heme in vitro when 24-hr cultures of mouse marrow cells are used, but, unlike Ep, it has no effect in stimulating 59Fe uptake into red blood cells in vivo when injected alone into polycythemic mice. This differential effect with the two assays appears to reflect differences in the makeup of the erythroid progenitor cell compartments in polycythemic and normal mouse bone marrow. However, in both the in vitro and in vivo assays this factor acts synergistically with Ep to cause a dramatic stimulation of 59Fe incorporation. This non-Ep stimulator has been partially purified and appears to be a 130K protein with an isoelectric point of 4.8. For maximal activity, it appears to require substantially higher levels of iron-saturated transferrin than Ep. In addition, colcemid and time-course studies suggest that it acts late in erythroid differentiation, primarily on erythroblasts; hence, the term erythroblast-enhancing factor (EEF) has been proposed. Finally, serum EEF levels were found to vary in both mice and man in a predictable fashion, reflecting physiologic requirements for mature red blood cells. These last findings suggest that EEF acts as a positive regulator of terminal erythropoiesis in vivo.

Animals↗
PubMed1983

Physical and biological characterization of erythroblast enhancing factor (EEF), a late acting erythropoietic stimulator in serum distinct from erythropoietin.

A factor with erythropoietic stimulating activity but distinct from erythropoietin (Ep) has been identified in normal human and mouse serum. Like Ep this factor causes a significant increase in the amount of 59Fe incorporated into heme in short term cultures of mouse marrow cells. However, dose response studies show that it acts synergistically rather than additively with erythropoietin. This factor has been partially purified using Sephadex G150 and chromatofocusing, and appears to be a protein with a molecular weight of 130,000 daltons and an isoelectric point of 4.8. Colcemid and time course studies indicate that this stimulator acts late in erythroid differentiation, primarily on erythroblasts. Benzidine staining studies suggest it may increase 59Fe uptake in hemoglobin-producing erythroblasts by enhancing their viability. Accordingly the term erythroblast enhancing factor (EEF) is proposed. Serum EEF levels have been found to be markedly reduced in polycythemic mice and EEF injected together with Ep into such animals has a synergistic effect in stimulating new red cell production. These findings strongly suggest that EEF acts as a positive regulator of terminal erythropoiesis in vivo.

Anemia↗
PubMed1983

A simple microassay for erythropoietin based on 3H-thymidine incorporation into spleen cells from phenylhydrazine treated mice.

Spleens taken from B6C3F1 mice on the third day following the second of 2 daily injections of phenylhydrazine HCl (60 mg/kg) contain approximately 10 times the number of cells (i.e., 10(9) cells) found in spleens from normal mice. More than 90% of these spleen cells are recognizable erythroid and form the basis of a new in vitro microassay for erythropoietin (Ep) which uses 3H-thymidine incorporation as an endpoint. This assay takes 24 h and can be carried out in the presence of either 20% FCS or a well-defined serum substitute. Under the conditions used, Ep levels from 30 mU/well down to as little as 0.2 mU/well can be accurately measured with a corresponding variation in counts of 50-100-fold. One spleen is sufficient for 1000 triplicate Ep determinations, and the use of this microassay procedure requires only very small (1-60) microliters) samples for evaluation. Preliminary studies with human plasma suggest that this assay may be more specific for Ep than established in vitro 59Fe bioassay methods.

Animals↗
PubMed1982

Stimulation of DNA synthesis by ascorbate in cultures of articular chondrocytes.

The addition of 0.2 mM Na L-ascorbate increased the incorporation of 3H-thymidine by rabbit articular chondrocytes in cell and organ culture. The stimulatory response of explants to ascorbate was potentiated by pretreatment of the cartilage with 0.2% clostridial collagenase (type 1) or trypsin for 15-30 minutes. In explants there was a latent period of 3 to 4 days before increased labeling of the nuclei could be detected. The effect was transient and declined after 8 days of culture. It was more evident in organ cultures of immature (3-month-old) than 2- to 3-year-old rabbits. Age differences were not detected in cell cultures. Explants of adult human articular cartilage were stimulated by ascorbate when the medium was supplemented with 10% fresh human serum but not by fetal bovine serum. The findings indicated that synthesis of DNA by articular chondrocytes in situ is regulated by responsiveness of the cells proper to compounds such as vitamin C, by properties of the extracellular matrix, and by factors in the serum. Ascorbate was cytotoxic at concentrations greater than 0.2 mM in the presence of certain batches of serum.

Animals↗