ABSTRACT
BACKGROUND AND PURPOSE: Previous studies have suggested that elevated progesterone levels are associated with a slower disease course in amyotrophic lateral sclerosis (ALS). Given that the effects of progesterone are mediated in part by the classical progesterone receptor (PR), the expression and cellular localization of the A and B isoforms (PR-A and PR-B, respectively) of the PR in control (neuropathologically normal) and ALS-affected spinal cord (SC) were examined. METHODS: Semi-quantitative RT-PCR, immunohistochemistry and immunofluorescence analyses of the cervical and lumbar SC of post-mortem ALS patients (n = 19) and control subjects (n = 10) were performed. Primers and antibodies used allowed the detection of both PR-A and PR-B isoforms together (PR-A+B) or PR-B isoform alone. RESULTS: Lumbar PR-A+B and cervical PR-B mRNA expression were significantly higher in ALS than controls. In both ALS and controls, PR-A+B immunoreactivity (IR) was occasionally detected in motor neurons. In contrast, PR-A+B IR was prominent in axonal processes and vessels. This was more evident in nerve roots and large arteries in ALS compared with controls. Colocalization of PR-A+B with markers of neurons, axonal processes and vascular endothelium was also observed. CONCLUSIONS: Evidence that both PR-A and PR-B isoforms are expressed in the human SC is provided, with some regional variation in isoform expression between ALS and controls. The IR was more prominent in nerve roots and large arteries in ALS, suggesting a potential role in the degenerative process.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Motor Neurons/metabolism , Receptors, Progesterone/metabolism , Spinal Cord/metabolism , Adult , Aged , Aged, 80 and over , Amyotrophic Lateral Sclerosis/pathology , Female , Humans , Immunohistochemistry , Male , Middle Aged , Motor Neurons/pathology , Spinal Cord/pathology , Spinal Nerve Roots/metabolism , Spinal Nerve Roots/pathologyABSTRACT
The authors have characterized frontal cortical tau protein in cognitively intact (4) and cognitively impaired (ALSci, 4) ALS patients and compared it with control (2) or Alzheimer disease (AD, 1)- derived tau. The authors observed expression of both 3R and 4R tau isoforms; increased insoluble tau protein; phosphatase resistance; and hyperphosphorylation at T175, S208, and S210. Soluble tau from both AD and ALSci was also phosphorylated at S237. Tau hyperphosphorylation is associated with ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/complications , Amyotrophic Lateral Sclerosis/metabolism , Cognition Disorders/etiology , Cognition Disorders/metabolism , tau Proteins/analysis , tau Proteins/chemistry , Aged , Biomarkers/analysis , Biomarkers/chemistry , Female , Humans , Male , Middle Aged , PhosphorylationABSTRACT
Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.
Subject(s)
DNA-Directed RNA Polymerases/genetics , Epitopes/immunology , Gene Products, env/immunology , Genetic Vectors , HTLV-I Antigens/immunology , Human T-lymphotropic virus 1/immunology , Retroviridae Proteins, Oncogenic/immunology , Vaccinia virus/genetics , Animals , Antibodies, Monoclonal/immunology , Bacteriophage T7/genetics , Binding Sites, Antibody , Cloning, Molecular , Gene Expression , Gene Products, env/chemistry , Gene Products, env/genetics , Glycosylation , HTLV-I Antibodies/immunology , HTLV-I Antigens/chemistry , HTLV-I Antigens/genetics , HTLV-I Infections/blood , HTLV-I Infections/immunology , HeLa Cells , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 2/immunology , Humans , L Cells , Mice , Oligosaccharides/immunology , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Retroviridae Proteins, Oncogenic/chemistry , Retroviridae Proteins, Oncogenic/genetics , Simian T-lymphotropic virus 1/immunology , Structure-Activity Relationship , Tumor Cells, Cultured , Viral ProteinsABSTRACT
A local decrease in progesterone synthesis in the placenta and fetal membranes has long been proposed as a possible mechanism in the control of human labor. We have examined whether changes occur in the abundance of mRNA for 3 beta-hydroxysteroid dehydrogenase/delta 5-->delta 4 isomerase (3 beta-HSD), the enzyme which catalyzes the conversion of pregnenolone to progesterone in human placenta and fetal membranes, by Northern blot analysis using a cDNA probe to human placental type-I 3 beta-HSD, the predominant isoenzyme in the placenta. The abundance of 3 beta-HSD mRNA (1.7-kb transcript) was about 10-fold greater in term placenta than in chorio-decidua, but undetectable in total RNA from amnion. There was no change in the abundance of 3 beta-HSD mRNA in either placenta or chorio-decidua obtained after elective cesarean section at term, after preterm labor, or after term or postterm vaginal delivery. We conclude that the abundance of 3 beta-HSD mRNA does not change in the placenta or fetal membranes with labor, consistent with the view that changes in 3 beta-HSD gene expression and decreased progesterone production are unlikely to effect intrauterine paracrine/autocrine regulatory mechanisms leading to term or preterm labor in women.
Subject(s)
Extraembryonic Membranes/enzymology , Labor, Obstetric/metabolism , Multienzyme Complexes/genetics , Placenta/enzymology , Pregnancy/metabolism , Progesterone Reductase/genetics , RNA, Messenger/metabolism , Steroid Isomerases/genetics , Blotting, Northern , Chorion/metabolism , Decidua/metabolism , Female , Humans , Multienzyme Complexes/metabolism , Pregnenolone/metabolism , Progesterone/metabolism , Progesterone Reductase/metabolism , Steroid Isomerases/metabolismABSTRACT
A mutant of Escherichia coli K-12 lacking pyruvate dehydrogenase and phosphoenolpyruvate synthase was used to study the transport of pyruvate by whole cells. Uptake of pyruvate was maximal in mid-log phase cells, with a Michaelis constant for transport of 20 microM. Pretreatment of the cells with respiratory chain poisons or uncouplers, except for arsenate, inhibited transport up to 95%. Lactate and alanine were competitive inhibitors, but at nonphysiological concentrations. The synthetic analogs 3-bromopyruvate and pyruvic acid methyl ester inhibited competitively. The uptake of pyruvate was also characterized in membrane vesicles from wild-type E. coli K-12. Transport required an artificial electron donor system, phenazine methosulfate and sodium ascorbate. Pyruvate was concentrated in vesicles 7- to 10-fold over the external concentration, with a Michaelis constant of 15 microM. Energy poisons, except arsenate, inhibited the transport of pyruvate. Synthetic analogs such as 3-bromopyruvate were competitive inhibitors of transport. Lactate initially appeared to be a competitive inhibitor of pyruvate transport in vesicles, but this was a result of oxidation of lactate to pyruvate. The results indicate that uptake of pyruvate in E. coli is via a specific active transport system.
