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Biomedical subjects

J J Greene

Publications and source records attributed to J J Greene.

At least 19 recordsLinked to original sources
PubMed2000

Purified dengue 2 virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice.

The full-length dengue 2 virus envelope glycoprotein (Egp) was expressed in insect cells by recombinant (r) baculovirus and found to form multimeric aggregates that were recovered in the void volume of gel filtration columns and by ultracentrifugation. An immunoblot confirmed that rEgp aggregrates disrupted with SDS sample buffer released a monomeric form that migrated with a molecular weight similar to native dengue 2 virus Egp on polyacrylamide gels. The rEgp aggregates reacted strongly with a panel of monoclonal antibodies specific for the native Egp and which identify critical structural and functional epitopes. The rEgp aggregates were purified by ultracentrifugation through 30% sucrose, and were shown to be the major protein band on a polyacrylamide gel and corresponding immunoblot. Purified rEgp aggregates in combination with aluminum hydroxide induced high titer neutralizing antibodies in adult mice. The generation of full-length dengue 2 rEgp aggregates in insect cells facilitated development of a simple, effective procedure for purification of the recombinant protein, and represents a good approach for producing highly immunogenic dengue 2 rEgp as a component of a subunit vaccine.

Adjuvants, Immunologic↗
PubMed2000

Influence of the cellular redox state on NF-kappaB-regulated gene expression.

The redox state has been shown to regulate a variety of biochemical functions including cellular proliferation. Previous studies from our laboratory and others have shown that the binding of many transcription factors to their cognate DNA sequences is sensitive to the redox environment. Therefore, it is likely that redox status serves as an additional regulatory control for the activity of transcription factors and that this may mediate the redox regulation of proliferation. To assess this possibility, the influence of altering the redox state on NF-kappaB-regulated gene expression was studied. A more-reducing environment favored higher levels of expression of gro, an endogenous gene associated with proliferation, when the redox levels were changed either naturally by altering culture density or chemically by treatment with modulators of glutathione synthesis. Furthermore, nuclear runoff studies showed that a more-reducing redox increased transcription of gro. In order to ascertain the singular effect of the redox state on the activity of NF-kappaB, expression of a secreted alkaline phosphatase (SEAP) reporter gene solely under the control of an NF-kappaB response element was measured under varying redox conditions. Changes in the redox state modulated the expression of this reporter system. Taken together, these results suggest the involvement of a redox mechanism regulating signaling events operating through the control of gene expression by transcription factors.

Acetylcysteine↗
PubMed2000

Balance as a predictor of ankle injuries in high school basketball players.

OBJECTIVE: The purpose of this study was to determine if a preseason measurement of balance while in a unilateral stance could predict susceptibility to ankle injury in a cohort of high school basketball players. Predicting risk for ankle injury could be important in helping to reduce the risk of these injuries and furthermore save health care costs. DESIGN: Cohort study. SETTING: Data were collected at five high schools during the first 2 weeks of the 1997-1998 and 1998-1999 basketball seasons. SUBJECTS: 210 (119 male, age = 16.1 +/- 1.1 yr; height = 182.98 +/- 7.4 cm; weight = 76.4 +/- 10.9 kg; and 91 female, age = 16.3 +/- 1.3 yr; height = 170.9 +/- 7.8 cm; weight = 63.4 +/- 8.4 kg) high school basketball players who did not sustain a time loss ankle or knee injury within the previous 12 months served as subjects. Subjects did not use prophylactic ankle taping or bracing during the season. ASSESSMENT OF RISK FACTORS: Balance was quantified from postural sway scores measured while subjects performed unilateral balance tests with eyes both open and closed. Logistic regression analysis was carried out to determine if gender, dominant leg, and balance scores were related to ankle sprain injuries. In addition, Fischer's exact test was used to determine if the rate of ankle injuries was the same whether the subject had poor, average, or good balance. Balance was assessed by measuring postural sway with the NeuroCom New Balance Master version 6.0 (NeuroCom International, Clackamas, OR, U.S.A.). Testing to determine postural sway consisted of having subjects stand on one leg for three trials of 10 seconds with their eyes open, then repeated with their eyes closed. Subjects then underwent the same assessment while standing on the other leg. Postural sway was defined as the average degrees of sway per second (degrees S/S) for the 12 trials producing a compilation (COMP) score. OUTCOME MEASURES: Ankle injury resulting in missed participation. RESULTS: Subjects who sustained ankle sprains had a preseason COMP score of 2.01 +/- 0.32 (Mean +/- SD), while athletes who did not sustain ankle injuries had a score of 1.74 +/- 0.31. Higher postural sway scores corresponded to increased ankle sprain injury rates (p = 0.001). Subjects who demonstrated poor balance (high sway scores) had nearly seven times as many ankle sprains as subjects who had good balance (low sway scores) (p = 0.0002.) CONCLUSION: In this cohort of high school basketball players, pre-season balance measurement (postural sway) served as a predictor of ankle sprain susceptibility.

Adolescent↗
PubMed2000

Redox regulation of casein kinase II autophosphorylation and its effect on Jun-DNA binding.

In previous studies we showed that an oxido-reductase is involved in the restoration of contact inhibition in fibrosacoma cells, implicating the redox state in growth control. In the present report we demonstrate that autophosphorylation of casein kinase II (CKII) is redox-dependent. We have also shown that phosphorylation of the transcription factor Jun by CKII is regulated by the redox state. In vitro kinase assays revealed that CKII-catalyzed phosphorylation increased the affinity of Jun for the DNA. In conformational analyses, CKII maintained an intact structure only within the redox range permissive to its autophosphorylation. Collectively, these data suggest that the redox state profoundly influences the ability of CKII to phosphorylate its substrate Jun and may do so by affecting the autophosphorylation as well as the structure of CKII. To demonstrate the biological relevance of these observations, redox potentials of fibroblast nuclei from different stages of growth were measured. Results indicated that as cell density increased, the intranuclear environment gradually became less reducing. Redox-dependent behaviors of growth-associated proteins such as Jun and CKII, together with evidence for in vivo changes in the nuclear redox state suggest that a redox mechanism may be involved in the regulation of cell growth.

Casein Kinase II↗
PubMed1997

Redox state changes in density-dependent regulation of proliferation.

The ability of certain transcription factors to bind to DNA has been demonstrated to be influenced by the redox environment. Therefore, fluctuations in the redox state of the cell may regulate the transcription of genes which control proliferation. To assess whether changes in the redox state may be related to proliferation, levels of oxidized (GSSG) and reduced (GSH) glutathione, the primary modulators of the redox state, were measured in cultures of varying densities of normal human fibroblasts which exhibit contact inhibition of proliferation, as well as fibrosarcoma cells, which lack this mechanism of growth control. Redox potentials calculated from normal, proliferating fibroblasts were found to be -34 mV more reducing than confluent, contact-inhibited cells. However, fibrosarcoma cells did not demonstrate this modulation in redox state. Further, to delineate whether these redox changes were the consequence or the cause of contact inhibition, cultures of subconfluent proliferating fibroblasts were treated with modulators of glutathione synthesis. Buthionine sulfoximine, an inhibitor of GSH synthesis, induced a less reducing redox state and decreased proliferation. In contrast, GSH synthesis precursors caused a more reduced redox state and increased proliferation. Collectively, these results suggest an interrelationship between redox state and growth control.

Acetylcysteine↗
PubMed1997

Medical coverage analysis for Wisconsin's Olympics: the Badger State Games.

The objective of this analysis is to determine the prevalence and severity of injuries encountered during the 1994-96 Badger State Summer Games Finals. Allocation of available medical personnel can be determined with this information. Medical contact with an athlete required an evaluation form to be completed by the health care professional covering the event. Information was compiled and analyzed to determine injury frequency and severity. Of the 31,580 athletes competing over the three year period in the 11 sports provided with medical personnel, 285 suffered a reportable injury. Soccer and basketball had the highest number of reported injuries with 68 and 65 injuries respectively. Basketball (2.00%), cycling (1.59%), wrestling (1.50%) and roller hockey (1.24%) had the highest injury rates. Severity of injury determined by the number of injuries transported to a medical facility found wrestling (23), soccer (22), basketball (11), and cycling (6) with the highest numbers of severe injuries. Wrestling (1.27%), basketball (.34%), soccer (.32%), and cycling (.21%) had the highest rate of severe injury. The most common sustained injuries were found to be sprains, strains, skin wounds, and contusions. These four types of injuries made up 70.18% of the injuries sustained. In conclusion, non-physician medical presence may be adequate coverage in most venues at multi-sport athletic competitions like the Badger State Games because of the relatively low frequency of severe injuries.

Athletic Injuries↗
PubMed1996

Cooperation of protein disulfide isomerase and redox environment in the regulation of NF-kappaB and AP1 binding to DNA.

Most transcription factors are multimeric complexes whose subunits depend on strict conformation requirements to form the active unit. Among these requirements is the presence of appropriate sulfhydryl interactions that are critical to transcription factor binding to cognate DNA recognition sites. Our experiments now suggest that modulation of these sulfhydryls may involve the action of thiol-modifying oxido-reductases such as protein disulfide isomerase (PDI). Electrophoretic mobility shift titration experiments incorporating different ratios of GSH:GSSG indicated that changes in GSH and GSSG concentrations corresponding to redox potential differences of as little as +/- 15 mV enabled or abolished binding of NF-kappaB and AP1 to their cognate DNA sites. Moreover, this binding range was modulated significantly by the addition of purified protein disulfide isomerase (PDI). Collectively, these results suggest that a reversible oxidation/reduction signalling pathway may exist in the cell whereby localized changes in redox potentials and/or oxido-reductase activity can be functionally relevant in the regulation of critical gene expression events.

Adaptor Protein Complex 1↗
PubMed1995

Induction of protein disulfide isomerase during proliferation arrest and differentiation of SH5Y neuroblastoma cells.

The relationship of genes associated with contact inhibition of cell growth and the commitment for differentiation was studied in the human neuroblastoma cell line SH5Y. These cells could be induced to differentiate in vitro into neuronal-like cells upon incubation with retinoic acid, an event that was accompanied by an enhancement in levels of neuron-specific acetylcholinesterase. The kinetics of differentiation, based on morphology and acetylcholinesterase levels, showed that proliferation arrest always preceded differentiation and may be a prerequisite for differentiation. To determine if this growth arrest is mediated by the same pathway underlying contact inhibition of proliferation, the expression of a gene associated with the induction of contact inhibition, protein disulfide isomerase (PDI), was quantified by Northern blot analysis and enzymatic activity after retinoic acid treatment. Retinoic acid caused a significant elevation of PDI-mRNA within 24 hrs. after treatment with a corresponding increase in enzyme activity which immediately preceded proliferation arrest and differentiation. Bacitracin, a specific inhibitor of PDI, abrogated the ability of retinoic acid to induce differentiation. However, treatment with interferon also increased PDI activity and caused proliferation arrest and SH5Y differentiation but into a fibroblastoid cell without neurite outgrowth. These results suggest that the commitment for differentiation of SH5Y cells involves a form of proliferation arrest in which activation of PDI activity is a required and early event but one that does not determine the final differentiation pathway.

Cell Differentiation↗
PubMed1994

Association of protein disulfide isomerase activity and the induction of contact inhibition.

Interferon-beta (IFN-beta) used as a biological response modifier can cause a wide array of effects, including the restoration of contact inhibition in HT1080 fibrosarcoma cells normally lacking this control. Sequence analysis and characterization of a cDNA whose expression has been shown to be transiently induced by IFN-beta now identifies this cDNA as corresponding to the gene for the multifunctional polypeptide protein disulfide isomerase (PDI). Cell proliferation studies using bacitracin as an inhibitor of PDI demonstrated that contact inhibition could be suppressed in human fibroblasts which normally exhibit this control--allowing attainable cell saturation densities to be significantly greater than those of control cells. PDI enzyme activity assays indicated that 1 mM bacitracin inhibited PDI intracellular activity up to 60%. Moreover, evaluations of PDI levels in normal fibroblasts of increasing densities suggested that enzyme levels correlate with density-dependent growth control, showing a transient increase in intracellular activity as cells approach confluency. Together these results suggest that induction of PDI activity is not only involved in the antiproliferative actions of IFN-beta, but it is critical to the regulation of contact inhibition growth control in normal fibroblasts.

Bacitracin↗
PubMed1994

Measurement of gene-specific transcription by nuclease protection of pulse-labeled nuclear RNA.

A gene-specific transcription assay was developed that is based on pulse-labeled incorporation of [3H]uridine into nuclear RNA. Transcription is quantified by scintillation counting of [3H]uridine incorporated into nuclear RNA that is protected from S1 nuclease digestion by hybridization with cold gene probes. This assay was dependent upon partial degradation of nuclear RNA and optimization of hybridization and nuclease digestion conditions. To validate this assay, transcription of beta-actin and c-myc genes was measured in two different human cell lines using the incorporation assay in parallel with the nuclear run-off assay. Transcription kinetics of the beta-actin and c-myc genes in serum-stimulated fibrosarcoma HT-1080 cells determined by [3H]uridine incorporation were comparable to that determined by the nuclear run-off method. For beta-actin, there was an approximate 2-fold increase in transcription rate within two hours of stimulation that declined to basal levels by 20 h. The c-myc gene response followed a similar kinetics as for the beta-actin gene except that maximal enhancement was greater at 6-9-fold. The relative transcriptional activities of the beta-actin gene to that of the c-myc gene were virtually identical using the two assay methods. Comparable transcription results using both methods were also observed when beta-actin and c-myc gene transcription were measured in log-phase HL-60 leukemia cells.

Actins↗
PubMed1993

Gene-specific modulation of RNA synthesis and degradation by extremely low frequency electromagnetic fields.

Pulse-labeling studies from our laboratory and others have shown that extremely low frequency (ELF) electromagnetic fields can produce a transient increase in gene transcription. In this study, the synthesis, degradation and processing, and steady state levels of specific RNA species during exposure to ELF radiation were determined in human leukemia HL-60 cells. The overall steady state RNA levels, assessed by continuous and equilibrium labeling with 3H-uridine, were not affected by ELF exposure. Northern blot analysis using probes specific for c-myc, beta-actin, and 45S ribosomal RNA gene products revealed that ELF did not alter the steady state levels of these RNAs. Examination of gene-specific transcription by a novel nuclease protection assay revealed that while ELF did not substantially alter the transcription rates for c-myc and beta-actin, transcription of the 45S ribosomal RNA gene was increased by 40-50%. To explain the observed increase in the synthesis of 45S ribosomal RNA without an associated increase in its steady state level, the degradation and processing of the ribosomal gene transcript in the presence and absence of an ELF field were followed by pulse-chase 3H-uridine labeling. This revealed that ELF radiation accelerated both the processing and degradation of the ribosomal RNA transcript. During ELF exposure, the half-life of the 45S ribosomal RNA was decreased from 115 min. to 85 min. These results show that ELF can selectively affect RNA levels by modulating either the transcription rate and/or RNA post-transcriptional processing and turnover.

Actins↗
PubMed1992

Regulation of C-myc and C-Ha-ras oncogene expression by cell shape.

The influence of cell shape on the expression of proto-oncogenes was examined in normal and malignant human cells that varied in their sensitivities to contact-inhibition of proliferation. Cells were constrained into varying degrees of roundness by plating onto culture surfaces coated with different concentrations of poly(2-hydroxyethyl methacrylate) (poly[HEMA]) and assayed for proliferation capacity and levels of c-myc, c-ras, c-fos, and c-fes mRNAs. Proliferation of contact-inhibited normal CUA-1 fibroblasts and the variant HT-IFNr cells was highly coupled to cell shape. As these cells became more rounded, a critical degree of roundness was reached at which proliferation ceased. In contrast, proliferation of non-contact-inhibited malignant HT-1080 cells was independent of cell shape. Northern analysis revealed that expression of c-myc and c-ras was highly sensitive to cell shape in the normal CUA-1 cells but not in the malignant HT-1080 or variant HT-IFNr cells. Levels of c-myc and c-ras mRNAs declined to nearly undetectable levels in CUA-1 cells at degrees of roundness that correlated with loss of proliferative ability. Expression of c-fos and c-fes oncogenes were independent of cell shape in all cells tested. Quantification of transcription rates by the nuclear run-off assay showed that shape modulation of c-myc and c-ras oncogene expression occurred at the transcriptional level. These data suggest that changes in cell shape can modulate expression of certain oncogenes and that these changes correlate with the cell's ability to proliferate. Moreover, inability to regulate c-myc and c-ras oncogene expression is associated with loss of shape-dependent growth controls and contact inhibition but that loss of this regulation alone is not sufficient to release cells from contact-inhibited controls.

Cell Division↗
PubMed1991

Delineation of electric and magnetic field effects of extremely low frequency electromagnetic radiation on transcription.

The relative effects of the electric and magnetic field components of extremely low frequency electromagnetic radiation (ELF) on transcription were examined in human leukemia HL-60 cells. Delineation of the individual field contributions was achieved by irradiating cells in separate concentric compartments of a culture dish within a solenoid chamber. This exposure system produced a homogeneous magnetic field with a coincident electric field whose strength varied directly with distance from the center of the culture dish. Irradiation of HL-60 cells with sine wave ELF at 60 Hz and a field strength of 10 Gauss produced a transient increase in the transcriptional rates which reached a maximum of 50-60% enhancement at 30-120 minutes of irradiation and declined to near basal levels by 18 hours. Comparison of transcription responses to ELF of cells in different concentric compartments revealed that the transcriptional effects were primarily the result of the electric field component with little or no contribution from the magnetic field.

Cell Line↗
PubMed1987

Identification of interferon-modulated proliferation-related cDNA sequences.

To identify genes mediating the antiproliferative action of interferon (IFN), two cDNA libraries were constructed with mRNA from IFN-treated and untreated human fibrosarcoma (HT1080) cells previously shown to be highly sensitive to the antiproliferative effects of IFN. Differential screening of these two libraries identified cloned sequences whose expression was either induced or repressed with IFN treatment. Rescreening of these sequences with cDNA probes constructed from proliferating or quiescent cells led to the identification of one IFN-induced and three IFN-repressed sequences whose expressions also appeared to be modulated by cell proliferation. Blot-hybridization analysis revealed that RNA levels corresponding to the three repressed genes decreased when HT1080 cells were treated with IFN or when proliferation of normal CUA foreskin fibroblast cells became naturally arrested by contact inhibition. Levels of RNA corresponding to the induced gene increased in HT1080 cells within 24 hr after IFN-treatment but declined below basal levels by 48 hr. Expression of these genes was unaffected or only slightly affected by IFN treatment in variant cells resistant to the antiproliferative effects of IFN. Collectively, these results suggest that the identified cDNAs correspond to genes that are involved in the antiproliferative action of IFN. Moreover, these results also suggest that IFN's antiproliferative action may be exerted through genes that contribute to arresting cell proliferation during contact inhibition.

Cell Division↗
PubMed1986

Preferential modulation of embryonic cell proliferation and differentiation by embryonic interferon.

Embryonic-interferon (E-IFN), a novel species of interferon (IFN) produced solely by embryo cells, inhibited the proliferation of embryo cells in early gestation preferentially vis-à-vis fetal cells in late gestation but had little effect on the in vitro differentiation of embryonic pre-adipocytes to adipocytes. In contrast, mature-interferon (M-INF) did not exhibit this preferential inhibition of cell proliferation and did inhibit pre-adipocyte differentiation. These results suggest that the function of E-INF is different from that of M-IFN and that this function may involve modulation of the developmental process.

Adipose Tissue↗
PubMed1986

Shape-dependent regulation of proliferation in normal and malignant human cells and its alteration by interferon.

The relationship between cell morphology, proliferation, and contact inhibition was studied in normal and malignant human cells which varied in their sensitivity to contact inhibition. Their ability to proliferate was examined under conditions where the cells were constrained into different shapes by plating onto plastic surfaces coated with poly(2-hydroxyethyl methacrylate). Poly(2-hydroxyethyl methacrylate) can precisely vary the shape of cells without toxicity. Cell proliferation was quantitated by cell counts and labeling indices were determined by autoradiography. The normal JHU-1 foreskin fibroblasts and IMR-90 lung fibroblasts exhibited contact-inhibited growth with a saturation density of 2.9 X 10(5) and 2.0 X 10(5) cells/cm2, respectively. These cells also exhibited stringent dependency on cell shape with a mitotic index of less than 3% at poly(2-hydroxyethyl methacrylate) concentrations at which the cells were rounded versus a labeling index of 75-90% when the cells were flat. The malignant bladder carcinoma line RT-4 exhibited partial contact-inhibited growth. Its dependency on cell shape was less stringent than that of normal cells with a mitotic index of 37-40% when rounded and 79% when flat. The malignant fibrosarcoma line, HT1080, was not contact inhibited and was entirely shape independent with a mitotic index of 70-90% regardless of cell shape. Treatment of HT1080 cells with low concentration of human fibroblast interferon (less than 40 units/ml) restored shape-dependent proliferation while having little effect on normal cells. Subantiproliferative doses of interferon were also shown to restore contact-inhibited proliferation control to malignant cells previously lacking it. The concordant restoration of contact inhibition and shape-dependent proliferation in malignant cells by interferon suggest that these two types of proliferation controls represent a manifestation of common regulatory mechanisms. However, since these effects occurred at interferon concentrations below that required to produce an antiproliferative effect, these actions of interferon may be distinct from the antiproliferative action.

Cell Communication↗
PubMed1985

Biological and morphological characteristics of phenotypic revertants appearing in interferon-treated mouse cells transformed by a human oncogene.

Phenotypic revertants appearing in interferon-treated mouse cells that had been transformed by an activated human c-Ha-ras1 oncogene (cell line RS485) were tested for several biological properties. The cloned revertants regained the growth characteristics of the untransformed parental NIH 3T3 cell line; unlike RS485 cells the revertants failed to form colonies in soft agar or to form rapidly growing tumors in nude mice. Animals inoculated with RS485 cells developed tumors within one week. In contrast, revertant clonal line 4C3 failed to form tumors four months after transplantation. Revertant 4C8 cells were tumorigenic; however, the developing tumors had increased latency, slower growth rate, and remained smaller than tumors of RS485 cells. Histopathological analysis revealed that revertant-associated fibrosarcomas were less anaplastic, less cellular and had relatively infrequent mitotic figures as compared to fibrosarcomas of RS485 cells. The result suggest the IFN-induced revertants exhibit a significantly less malignant phenotype than their parental transformed cells and that the biological differences are maintained after IFN treatment is discontinued.

Animals↗