Subject(s)
Alcohol Drinking/adverse effects , Attention , Ethanol/poisoning , Executive Function , Intelligence , Adult , Child, Preschool , Female , Humans , PregnancyABSTRACT
Little is known about the neurochemical effects accompanying the high-concentration inhalant exposures characteristic of binge solvent abuse. In adult animals, prior studies with other patterns of exposure indicate that toluene, a commonly abused household and industrial solvent, has significant effects on the glutamatergic and GABAergic neurotransmitter systems and on other neurotransmitter systems as well. In the current investigation, high-resolution "magic angle" spinning proton magnetic resonance spectroscopy (HR-MAS (1)H-MRS) was used to assess the effect of acute binge toluene inhalation on regional brain concentrations of various neurochemicals including glutamate (GLU), GABA, and glutamine (GLN) in juvenile male and female rats. Acute toluene (8000 ppm or 12,000 ppm) significantly reduced levels of hippocampal GABA (-12%) and GLU (-8%), and the GLU/GLN ratio, an index of glutamatergic tone, was significantly reduced (-22%) in the dorsal anterior striatum, driven largely by a 28% increase in GLN. Significant increases in alanine and lactate in several brain regions after acute toluene may be indicative of altered oxygen-dependent metabolism associated with the inhalation of higher concentrations of toluene (e.g., >5000 ppm). Other components of the MR-visible neurochemical profile, such as N-acetylaspartate (NAA), myo-inositol, creatine, and various choline containing compounds, were unchanged by acute toluene. The results are consistent with the notion that binge toluene exposure affects juvenile neurochemistry in systems mediating the rewarding and emotional aspects of substance abuse. Moreover the results provide a framework to understand further (1)H-MRS studies in clinical populations.
Subject(s)
Brain Chemistry/drug effects , Glutamic Acid/metabolism , Magnetic Resonance Spectroscopy , Toluene/toxicity , gamma-Aminobutyric Acid/metabolism , Analysis of Variance , Animals , Animals, Newborn , Behavior, Animal/drug effects , Dose-Response Relationship, Drug , Embryo, Mammalian , Female , Glutamine/metabolism , Humans , Inhalation , Male , Pregnancy , Rats , Rats, Sprague-Dawley , Solvents/toxicityABSTRACT
We present a simple 3D event position-estimation method using raw list-mode acquisition and maximum-likelihood estimation in a modular gamma camera with a thick (25mm) monolithic scintillation crystal. This method involves measuring 2D calibration scans with a well-collimated 511 keV source and fitting each point to a simple depth-dependent light distribution model. Preliminary results show that angled collimated beams appear properly reconstructed.
ABSTRACT
Although the incidence of primary cardiac tumors is approximately 0.28%, they are encountered in every age group and their clinical presentation usually resembles that of other cardiac or systemic diseases. The clinical symptoms of primary cardiac tumors are usually few until they are at an advanced critical stage making early diagnosis and treatment problematic. However, there is a subgroup of patients who may present with extracardiac systemic, endocrinologic or cutaneous manifestations and familiar inheritance allowing for early detection. This article describes the diagnosis and treatment of a young female who presented with an acute thromboembolic complication of an intracardiac myxoma.
Subject(s)
Heart Atria , Heart Neoplasms/diagnosis , Myxoma/diagnosis , Pulmonary Embolism/diagnosis , Adult , Diagnosis, Differential , Echocardiography , Female , Heart Neoplasms/surgery , Humans , Myxoma/surgery , SyndromeABSTRACT
The specific aim of the current study of 133 women with at least 1 pregnancy and measures of hypofibrinolytic and thrombophilic gene mutations was to determine retrospectively whether the mutations were associated with adverse pregnancy outcomes including prematurity, miscarriage, stillbirth, intrauterine growth retardation (IUGR), eclampsia, and abruptio placentae. Four gene mutations (factor V Leiden, methylenetetrahydrofolate reductase [MTHFR], prothrombin, and 4G/5G polymorphism of the plasminogen activator inhibitor type 1 [PAI-1] gene) were assessed by polymerase chain reaction (PCR). One hundred twenty-two women were genotyped for all 4 genes and divided into gene mutation (n = 68) and non-gene (n = 54) groups. The gene mutation group included those with at least 1 thrombophilic mutation (heterozygous for factor V Leiden, heterozygous for prothrombin, and homozygous for MTHFR), or hypofibrinolysis with homozygosity for the 4G polymorphism of the PAI-1 gene. The non-gene mutation group included those with no mutation for all 4 genes (wild-type normal) or who were wild-type normal for the prothrombin and factor V Leiden mutations and heterozygous for MTHFR and/or 4G/5G for the PAI-1 gene, neither heterozygosity associated with coagulation abnormalities. The 68 women with gene mutations, versus 54 in the non-gene mutation group, has more prematurity (10% v 4%, chi2 = 5.4, P = .021), more IUGR (3% v 0%, P = .035), and more total complications of pregnancy (37% v 21%, chi2 = 11.6, P = .001). The number of pregnancies (P = .0001) and 4G/4G polymorphism of the PAI-1 gene (P = .029) were positively associated with complications of pregnancy by stepwise logistic regression when the age, number of pregnancies, and all 4 gene mutations were the explanatory variables. Heritable hypofibrinolysis, mediated by 4G/4G homozygosity for the PAI-1 gene, is an independent significant, potentially reversible risk factor for pregnancy complications, probably acting through thrombotic induction of placental insufficiency.
Subject(s)
Plasminogen Activator Inhibitor 1/genetics , Polymorphism, Genetic , Pregnancy Complications/etiology , Antibodies, Anticardiolipin/blood , Female , Homocysteine/pharmacology , Humans , Methylenetetrahydrofolate Reductase (NADPH2) , Mutation , Oxidoreductases Acting on CH-NH Group Donors/genetics , Pregnancy , Retrospective Studies , Risk FactorsABSTRACT
In 41 women with at least one pregnancy drawn from a group of 149 (108 never-pregnant) women with polycystic ovary syndrome (PCOS), our specific aim was to determine whether hypofibrinolysis mediated by high plasminogen activator inhibitor activity (PAI-Fx) is an independent risk factor for miscarriage. The 41 women had 77 total pregnancies with 34 miscarriages (44%) and 42 live births (55%). There were 12 women with at least one pregnancy, at least one miscarriage, and no live births (16 pregnancies and 16 miscarriages). There were 15 women with at least one pregnancy, no miscarriages, and at least one live birth (25 pregnancies and 28 live births). Of 12 women with only miscarriages and no live births, 67% had PAI-Fx greater than 16.4 U/mL (normals' 95th percentile), versus 29% of 15 women with no miscarriages and all live births (chi2 = 3.8, P = .052). By stepwise logistic regression, the number of pregnancies (P = .0001) and PAI-Fx (P = .016) were significant positive explanatory variables for the number of miscarriages. Age, 4G/5G polymorphisms of the PAI gene, factor V Leiden, methylenetetrahydrofolate reductase (MTHFR) gene mutations, androstenedione, testosterone, sex hormone-binding globulin, the Quetelet index, and fasting serum insulin and glucose were not significant variables in the logistic regression model. In a separate stepwise logistic regression, three nonoverlapping groups of women (12 with > or = 1 pregnancy, > or = 1 miscarriage, and 0 live births, 10 with > or = 1 pregnancy, > or = 1 miscarriage, and > or = 1 live births, and 15 with > or = 1 pregnancy, 0 miscarriages, and > or = 1 live births) were the dependent variables. PAI-Fx was positively associated (P = .05) with the group with the worst pregnancy outcome (> or = 1 pregnancy, > or = 1 miscarriage, and 0 live births). The 41 women with PCOS and at least one pregnancy were more likely than healthy normal controls to have heterozygosity and homozygosity for the 4G/5G polymorphism of the PAI-1 gene (P = .028), but did not differ from normals for factor V Leiden (P > .10) or MTHFR (P > .09) mutations. PAI-Fx is a predominant independent significant positive reversible risk factor for miscarriage in women with PCOS.
Subject(s)
Abortion, Spontaneous/etiology , Plasminogen Inactivators/blood , Polycystic Ovary Syndrome/complications , Pregnancy Complications , Pregnancy/blood , Birth Rate , Female , Fibrinolysis/physiology , Humans , Polycystic Ovary Syndrome/blood , Risk FactorsABSTRACT
The class of 90 kDa heat shock proteins (Hsp90) is among the most abundant heat shock proteins (Hsps) in eukaryotic cells. In vertebrates, Hsp90 is encoded by two distinct gene families giving rise to products of 84 and 86 kDa. In mice the expression of these two genes, hsp84 and hsp86, vary with respect to each other in responses to stress, and also in response to signals for growth and development. Therefore, as a step towards understanding the molecular basis for the differential regulation of these two genes, we have isolated and characterized genomic clones of the murine hsp86 gene and its 5' flanking region. The gene is composed of eleven exons interrupted by 10 introns. The 5' region contains consensus TATA, several stimulatory protein-1 binding site (SP1) elements as well as six consensus heat shock elements (HSE) 5' of the transcription start site. An 806 bp fragment of the 5' promoter region conferred constitutive expression upon a reporter gene and this expression was increased upon heat shock.
Subject(s)
HSP90 Heat-Shock Proteins/genetics , Heat-Shock Proteins/genetics , Promoter Regions, Genetic/genetics , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , Mice , Molecular Sequence Data , Sequence Analysis, DNA , Transcription, Genetic/geneticsABSTRACT
The quantitative relationship between chemical structure and biological activity has received considerable attention in the fields of pharmacology and drug development. More recently, quantitative structure-activity relationships (QSARs) have been used for predicting chemical toxicity. It has been proposed that alcohols may elicit their toxic effects through hydrophobic interactions with the cellular membrane. The objective of this study was to evaluate the role of hydrophobicity in the loss of membrane integrity following acute exposure to short-chain aliphatic alcohols in rat liver epithelial cells in vitro. The series of alcohols studied included methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 2-butanol, 2-methyl-1-propanol, and 2-methyl-2-propanol. The lactate dehydrogenase (LDH) assay was used to quantify membrane integrity. The logarithm of the octanol/water partition coefficient (log P) was used to quantify hydrophobicity. LDH50 values, representing alcohol concentrations yielding a 50% increase in LDH release relative to untreated controls (i.e., mild disruption of membrane integrity), and EC50 values, representing alcohol concentrations yielding 50% of the maximal release of LDH (i.e., moderate disruption of LDH release), were experimentally determined for each alcohol. The LDH50 and EC50 values were then used to derive the QSAR relationship. The aqueous alcohol concentrations yielding LDH50 or EC50 values ranged from 8.9 x 10(-4) m (LDH50 for octanol) to 3.5 m (EC50 for methanol), and the log P of the alcohols ranged from -0.77 (methanol) to 3.00 (octanol). From these data, we have derived two QSAR equations describing the role of hydrophobicity in the release of LDH from rat liver epithelial cells following a 1-hr alcohol exposure. The QSAR equation for LDH50 values, log (1/LDH50) = 0.896 log P + 0.117 (n = 11, SD = 0.131), was nearly identical to the QSAR equation for EC50 values, log (1/EC50) = 0.893 log P + 0.101 (n = 11, SD = 0.133], suggesting that similar structure-activity relationships exist at both mild and moderate levels of membrane disruption. Our data indicate that an increase in LDH release was positively and linearly correlated with the hydrophobicity (r = 0.993). These data may help predict the potential biological effects of other, as yet untested, aliphatic alcohols and aliphatic alcohol-like compounds (e.g., anesthetics) on the plasma membrane.
Subject(s)
Alcohols/toxicity , Cell Membrane/drug effects , Alcohols/chemistry , Animals , Cell Membrane/physiology , Cells, Cultured , Epithelial Cells , L-Lactate Dehydrogenase/drug effects , L-Lactate Dehydrogenase/metabolism , Liver/cytology , Liver/drug effects , Rats , Solvents , Structure-Activity RelationshipABSTRACT
Although acquired mutations in the human p53 gene occur in many tumor types, germline mutations are rare. An exception is the occurrence of germline p53 mutations in a fraction of families afflicted with the Li-Fraumeni syndrome (LFS). Previous studies from our laboratory demonstrated increased levels of wild type p53 protein in skin fibroblasts (SF) of patients from heritable cancer syndrome, including familial adenomatous polyposis (FAP), neurofibromatosis type 1 (NF1), and bilateral retinoblastoma (bRB) (Kopelovich and DeLeo, 1984,1986). Here, we further address the association between germline p53 alterations and genetic predisposition to cancer in the SBLA syndrome and in FAP. DNA sequencing and single-stranded conformational polymorphism analysis (SSCP) were utilized to screen for the presence of mutations within exons 5-9 of the p53 gene in SF and in benign tumors. Thus we observed no germline mutations in exons 5-9 of the p53 gene in SF from SBLA or FAP patients, including the Gardner variant. In addition, we observed no acquired mutations in exons 5-9 of the p53 gene in benign tumors from FAP patients. In conclusion, we found no association between germline p53 mutations and SBLA or FAP. How mechanisms that involve nonmutational activation of the p53 protein might affect genetic predisposition to cancer remains to be established.
Subject(s)
Adenomatous Polyposis Coli/genetics , Exons , Genes, p53 , Li-Fraumeni Syndrome/genetics , Mutation , Adenomatous Polyposis Coli/pathology , Adult , Biopsy , Child, Preschool , DNA Primers , DNA, Neoplasm/analysis , Female , Fibroblasts/pathology , Humans , Introns , Li-Fraumeni Syndrome/pathology , Male , Middle Aged , Pedigree , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Skin/pathologyABSTRACT
The 90-kDa heat-shock (HS) proteins (HSP90) are members of the HSP family. Their synthesis is inducible by HS and a variety of stress signals. HSP90 is also abundant under normal physiological conditions and its synthesis can be regulated during growth and differentiation. Therefore, HSP90 is speculated to have important biological functions, in addition to its role in mediating stress responses. However, the mechanism(s) regulating hsp90 gene expression in nonstressed cells is poorly understood. As a prerequisite towards understanding the basis for hsp90 regulation, we have cloned and characterized the 5' flanking region of murine hsp84, one of two genes which code for HSP90 proteins. Full basal promoter activity of hsp84 was found to be associated with a 627-bp region immediately upstream from the transcription start point (tsp). Sequence analysis revealed several putative regulatory elements, including a HS element (HSE), an AP1-binding site (AP1), a cyclic AMP response element (CRE), and four stimulatory protein-1-binding sites (SP1). HS inducibility required the HSE which was bound by HS transcription factor-1(HSF-1) present in extracts prepared from cells exposed to HS. The HSE was not required for basal (non-HS) expression, but, interestingly, two protein-HSE complexes, devoid of HSF-1 and HSF-2, were formed under these conditions. The potential significance of these findings to the expression of hsp84 under normal physiological conditions is discussed.
Subject(s)
HSP90 Heat-Shock Proteins/genetics , Promoter Regions, Genetic , 3T3 Cells , Amino Acid Sequence , Animals , Cloning, Molecular , Gene Expression Regulation , Heat-Shock Response , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Sequence Homology, Nucleic AcidABSTRACT
1. In the marine mollusk Aplysia, egg laying is a complex behavior that lasts for up to several hours. We used behavioral and electrophysiological methods to determine how egg laying occurs in groups of animals and how it is related to other aspects of reproductive behavior. 2. Prolonged contact with an existing egg mass by the lips and tentacles of an animal is a sign stimulus for release of egg-laying behavior and two other fixed action patterns in the same individual, mating as a female during egg laying and mating as a male after egg laying. 3. Prolonged contact with the egg mass initiated repetitive spike activity in bag cell neurons, which are part of a peptidergic neural system that modulates neuronal activity in the CNS for up to several hours. The sign stimulus thus activates the neuromodulatory system, which may serve as an innate releasing mechanism, and an associated internal drive, for control of the behavioral sequence.
Subject(s)
Aplysia/physiology , Neurons/physiology , Neuropeptides/physiology , Sexual Behavior, Animal/physiology , Animals , Female , Gonadal Steroid Hormones/metabolism , Gonadal Steroid Hormones/physiology , Instinct , Male , Neuropeptides/metabolism , Oviposition/physiologyABSTRACT
RATIONALE AND OBJECTIVES: Little research has explored the use of radiologic services by emergency departments and the factors that influence use. This study aimed to identify and characterize these factors. METHODS: A total of 13,228 consecutive patient emergency charts from a large university emergency department were reviewed, and multiple parameters were entered into a database. The database was studied and statistical testing was done to identify significant parameters for patients who required imaging studies (X-ray group) and those who did not (non-X-ray group). RESULTS: Factors such as age, diagnosis, urgency of illness, and illness severe enough to require hospitalization were statistically significant in determining the need for a radiologic evaluation in the emergency setting. When these factors were equalized for the X-ray and non-X-ray groups by multivariate linear regression analysis, male sex was also found to be statistically significant. Factors such as race and presence or absence of health insurance were not statistically influential on multivariate analysis. CONCLUSIONS: Older age, diagnosis, and factors related to severity of illness affected the use of radiologic services in the emergency setting. Sex differences were also detected.
Subject(s)
Emergency Service, Hospital/statistics & numerical data , Hospitals, Teaching/statistics & numerical data , Hospitals, Urban/statistics & numerical data , Radiology Department, Hospital/statistics & numerical data , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Hospital Bed Capacity, 500 and over , Humans , Infant , Infant, Newborn , Male , Middle Aged , Multivariate Analysis , Ohio , Retrospective StudiesABSTRACT
Isolates of Streptococcus pneumoniae in cultures of blood from 258 adults seen in 10 Franklin County, Ohio, hospitals from 1991 and 1992 were serotyped. Most strains (230 [89.2%]) belonged to serotypes that are included in the current pneumococcal vaccine. An additional 16 isolates (6.2%) were immunologically related to strains with serotypes that are included in the vaccine. Only 12 isolates (4.6%) were not covered by the vaccine. The rate of mortality from pneumococcal bacteremia in adults remains high (20%). While recent studies have documented the efficacy of the pneumococcal vaccine for preventing pneumococcal bacteremia (56 to 70%), use of the pneumococcal vaccine in susceptible patients by physicians remains low (19% in Franklin County). Additional efforts need to be expended to increase the use of the pneumococcal vaccine.
Subject(s)
Bacteremia/microbiology , Community-Acquired Infections/microbiology , Streptococcal Infections/microbiology , Streptococcus pneumoniae/classification , Adult , Bacteremia/epidemiology , Bacteremia/mortality , Bacterial Vaccines , Community-Acquired Infections/epidemiology , Community-Acquired Infections/mortality , Humans , Ohio/epidemiology , Seasons , Serotyping , Streptococcal Infections/epidemiology , Streptococcal Infections/mortality , Streptococcus pneumoniae/isolation & purification , Vaccination/statistics & numerical dataABSTRACT
The objective of these experiments was to quantify the pattern of change in arterial blood pressure (BP) during a discriminative aversive classical conditioning paradigm in rat using a new "high resolution" computer analysis. Sprague-Dawley rats (n = 5) were restrained in a soft, conical cloth pouch and conditioned using a 6 sec. pulsed tone (CS+) followed by a 0.5 sec. tail shock; a steady tone, never followed by shock, served as a CS-. BP peaked at 16.4 +/- 6.5 mm Hg (mean +/- SD) above control at 1.5 +/- 0.1 sec. after onset of CS+. This "first component" ("C1") also occurred during CS- (12.1 +/- 3.8 mm Hg), although the magnitudes of the two were significantly (p < 0.05) different. Another group of rats (n = 8) was treated identically except the tones were 15 seconds long. The conditional BP response consisted of two components. C1 was reminiscent of that seen using the short tone: for CS+ a peak of 13.6 +/- 5.6 mm Hg at 1.5 sec. or, for CS-, of 10.0 +/- 4.3 at 1.3 sec. (p < 0.05). In CS+ trials BP peaked again ("C2," 7.4 +/- 2.5 mm Hg) at 8.3 +/- 1.2 sec. There was no statistically significant C2 for CS- trials, clearly demonstrating discrimination between tones. The unconditional BP response in both groups consisted of two large, closely spaced peaks in BP. Respiration was recorded in 3 additional rats. After shock delivery these subjects often showed a sudden shift between (1) a regular respiratory pattern with moderate chest excursion and (2) apneic episodes interspersed with single, deep breaths. This latter pattern was associated with large, low frequency fluctuations in BP. Continued development of the rat conditioning paradigm is especially warranted because of the ability to record sympathetic nerve activity in intact, awake subjects and the large number of readily available genetic strains, which model human pathological states.
Subject(s)
Arousal , Blood Pressure , Conditioning, Classical , Fear , Animals , Association Learning , Attention , Rats , Rats, Sprague-Dawley , RespirationABSTRACT
Enhanced cell kill has been observed when experimental tumors were treated with alkylating agents in combination with 2-nitroimidazoles (2-NI). In this study, modification of the cell kill induced by cyclophosphamide (CY) and an analog, ifosfamide (IFO), by two radiation sensitizers, misonidazole (MISO) and etanidazole (SR-2508), was measured. Three important parameters were determined: (a) the necessity for hypoxic reduction of the 2-NI to achieve an increase in tumor cell kill, (b) the optimal timing for administration of the alkylating agents and the 2-NI, and (c) the degree of enhancement of the CY- and IFO-induced cell kill. The subcutaneous (sc) 9L tumor model in male Fisher 344 rats was used in these experiments, and the endpoint measured was clonogenic cell survival 18-20 hr after treatment. Under hypoxic conditions, MISO potentiated both CY- and IFO-induced cell kill with a sensitizer enhancement ratio of approximately 1.3 and 1.5, respectively, at the 10(-3) survival level. This enhancement was seen when CY was administered simultaneously or 1.5 hr prior to MISO administration. A similar enhancement of CY-induced cell kill was measured under hypoxic conditions when SR-2508 was used. Enhanced IFO-induced cell kill was measured under hypoxic conditions only when the IFO was given 1 hr before MISO administration. No enhancement of the IFO-induced cell kill was observed when SR-2508 was used instead of MISO. Increased normal tissue damage (i.e., hemorrhagic cystitis) was observed when the MISO was administered along with CY or IFO. Four conclusions can be drawn from these data. Metabolism of the 2-NI by hypoxic cells is necessary for potentiation of CY- or IFO-induced cell kill. Only MISO can potentiate the cell kill induced by IFO. The timing of administration of the alkylating agents and the 2-NI is a critical determinant of the extent of the cell kill obtained. Cell kill induced by IFO appears to be enhanced by MISO to a greater extent than the cell kill induced by CY.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Neoplasms, Experimental/drug therapy , Animals , Cell Hypoxia/drug effects , Cell Survival/drug effects , Cyclophosphamide/administration & dosage , Etanidazole , Ifosfamide/administration & dosage , Male , Misonidazole/administration & dosage , Neoplasm Transplantation , Nitroimidazoles/administration & dosage , Radiation-Sensitizing Agents/therapeutic use , Rats , Rats, Inbred F344ABSTRACT
Induction of unrepairable DNA damage, accumulation of misrepaired DNA damage, and generation of imbalances in competing biochemical and/or metabolic processes have been proposed to explain the relationship between radiation-induced DNA damage and cell lethality. Theoretically, the temperature dependence of the critical DNA repair process(es) should be 1) either independent of or identical to the temperature dependence of cell killing if the first two hypotheses are correct, and 2) different if the third hypothesis is correct. To test this, exponentially growing rat 9L brain tumor cells were left at 37 degrees C or equilibrated for 3-14 h at 20 degrees C before irradiation. Cells were irradiated and allowed to repair at either 20 degrees C or 37 degrees C. Alternatively, the cells were irradiated at one of these temperatures and immediately shifted to the other temperature for repair. DNA damage was assessed by the alkaline elution technique; cell kill was assessed by a clonogenic assay. 9L cells maintained at 20 degrees C or 37 degrees C sustained the same amount of DNA damage as measured by alkaline elution. DNA repair instantaneously assumed the rate characteristic of the postirradiation temperature. For 9L cells equilibrated, irradiated, and repaired at 20 degrees C, the half-time of the fast phase of the DNA repair decreased by a factor of approximately 2 and the half-time of the slow phase decreased by a factor of approximately 5 over that measured in cells incubated, irradiated and repaired at 37 degrees C. Although the rate of DNA repair decreased substantially at 20 degrees C, the survival of 9L cells that were equilibrated and irradiated at 20 degrees C was greater (p less than 10(-4)) than those incubated and irradiated at 37 degrees C, when assayed by an immediate plating protocol. In addition, the survival of 9L cells equilibrated and irradiated at 20 degrees C and then shifted to 37 degrees C immediately after irradiation was greater (p less than 10(-2)) than that obtained with any other delayed plating protocol. Thus, the temperature dependence of the DNA repair processes measured by alkaline elution was different from the temperature dependence of cell killing measured either by an immediate or delayed plating protocol. These data support the hypothesis that many irradiated 9L tumor cells die because of imbalances in sets of competing biochemical and/or metabolic processes.
Subject(s)
DNA Damage/physiology , DNA Repair/physiology , Animals , Cell Death/genetics , Cell Death/radiation effects , Cell Survival/radiation effects , Colony-Forming Units Assay , Gamma Rays , Temperature , Tumor Cells, CulturedABSTRACT
The HSP86 gene family in BALB/c, AKR/J, C58/J, and NFS/N inbred mice comprises an intron-containing expressed gene and, depending on the strain, two to four other HSP86-related members that are apparently processed pseudogenes. The expressed gene locus, Hsp86-1, was identified by its sequence identity with the mouse HSP86 cDNA coding region together with the presence of an intron at the same position as in the homologous human gene. Hsp86-1 was mapped 11.6 cM from the immunoglobulin heavy chain gene IgH on Chromosome 12 using an intersubspecies backcross. Two of the other loci that were common to all inbred strains tested, designated Hsp86-ps1 and Hsp86-ps2, were mapped to positions on Chromosomes 11 and 3, respectively. An HSP86-related locus specific to NFS/N and C58/J mice, designated Hsp86-ps3, was mapped on Chromosome 9. Also, an HSP86-related locus that was unique to NFS/N mice, designated Hsp86-ps4, was mapped to Chromosome 4.
Subject(s)
Chromosome Mapping , Genes , Heat-Shock Proteins/genetics , Mice, Inbred Strains/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Chromosome Deletion , DNA/genetics , DNA/isolation & purification , Embryo, Mammalian , Genomic Library , Humans , Introns , Mice , Molecular Sequence Data , Molecular Weight , Polymerase Chain Reaction , Pseudogenes , Recombination, Genetic , Restriction Mapping , Sequence Homology, Nucleic AcidABSTRACT
The nucleotide sequence of a 6-kb region containing the gene for mouse 84-kD heat shock protein, HSP84, was determined. The hsp84 gene codes for a 5,500-base transcript and consists of 11 exons and 10 introns, ranging in length from 94 to 357 bp and 85 to 1,271 bp, respectively. One of the exons codes for a stretch of highly charged amino acids with two known phosphorylation sites. The presence of numerous introns in the hsp84 gene suggests that synthesis of the HSP84 protein would be precluded during severe heat shock, since such conditions interfere with splicing. The first intron, which is the largest, is located at the exact boundary between the 5'-untranslated region and the coding region and contains a sequence homologous to the heat shock element (HSE), an enhancer that is a characteristic feature of heat-inducible genes. A 71% homology was found between a 569-bp stretch within the first intron of the hsp84 gene, which includes the HSE-like sequence, and a portion of the first intron of the previously reported sequence of the human hsp89 beta gene. The promoter region of the hsp84 gene contained G + C-rich upstream sequences, potential binding sites for transcription factor Sp1, and a canonical TATA box. The hsp84 gene family includes at least six different hsp84-related pseudogenes, which arose about 2-3 million years ago.
Subject(s)
Heat-Shock Proteins/genetics , Multigene Family , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Blotting, Southern , Cloning, Molecular , DNA/genetics , DNA/isolation & purification , Exons , Genes , Introns , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Phosphorylation , Polymorphism, Restriction Fragment Length , Pseudogenes , Sequence Homology, Nucleic Acid , Sp1 Transcription Factor/metabolism , Transcription, GeneticABSTRACT
The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.
Subject(s)
DNA, Neoplasm/genetics , Receptors, Immunologic/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cell Line , Cell Membrane/immunology , DNA, Neoplasm/isolation & purification , Gene Library , Humans , Interferon-gamma/metabolism , Mice , Molecular Sequence Data , Receptors, Immunologic/isolation & purification , Receptors, Immunologic/metabolism , Receptors, Interferon , Restriction Mapping , Sequence Homology, Nucleic Acid , Thymoma , Thymus Neoplasms , TransfectionABSTRACT
Murine uterine steady-state protein levels of the 90-kilodalton heat shock protein (HSP90) have been demonstrated recently to be increased by estrogen in a target tissue- and steroid-specific manner (C. Ramachandran, M.G. Catelli, W. Schneider, and G. Shyamala, Endocrinology 123:956-961, 1988). We now report that this regulation occurred with both the HSP86 and HSP84 forms of HSP90 as well as with the 94-kilodalton glucose-regulated protein. At the mRNA level, this response was greatest for HSP86 (15-fold). In contrast, estradiol had no significant effect on HSP70.