Looking back: menstrual disorders of school-girls in 1913.
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Adolescent↗
Biomedical subjects
E F Howard
Publications and source records attributed to E F Howard.
The NF-kappaB inhibitor diethyldithiocarbamate (DDTC) increases brain cell death in a transient middle cerebral artery occlusion model of ischemia.
A transient ischemic middle cerebral artery occlusion model of stroke was used to examine the role of the transcription factor NF-kappaB in cell death as measured by DNA fragmentation and infarction volume. The left middle cerebral artery was occluded for either 30 min or 2 h in rats. One set of animals was pretreated with diethyldithiocarbamate (DDTC), an inhibitor of NF-kappaB, 30 min prior to reperfusion. The animals were reperfused and allowed to survive for 2 or 7 days. DNA fragmentation was assayed by in situ end labeling in the stroke core and penumbral regions. Specific cortical and subcortical regions were measured using quantitative image analysis. DNA fragmentation was seen only on the ischemic side of the brains in all cases. Overall, the DDTC-treated groups showed significantly increased DNA fragmentation within the ischemic side compared to the saline control groups. DDTC treatment also caused an increase in stroke volume based on triphenyl tetrazolium chloride staining. Electrophoretic mobility shift assays showed NF-kappaB activation peaking 15 min following reperfusion and that this activation was blocked by the DDTC treatment. This study suggests that the use of NF-kappaB inhibitors to block cell death following stroke needs to be carefully examined because global inhibitors may not promote neuronal survival.
Animals↗
Is nuclear factor-kappaB a good treatment target in brain ischemia/reperfusion injury?
Nuclear factor-kappaB (NF-kappaB) is a transcription factor which resides in its unactivated form in the cytoplasm. Following activation subsequent to cerebral ischemia and reperfusion injury, NF-kappaB acts on genes for cytokines, adhesion molecules, nitric oxide synthase, cyclooxygenase-2, metalloproteinase-9, and perhaps apoptotic genes. These genes have a variety of positive and negative influences on the outcome of brain injury. The effect of manipulation of NF-kappaB needs to be examined critically, as it pertains to these reactive genes.
Animals↗
Induction of angiotensin II subtype 2 receptor-mediated blood pressure regulation in synthetic diet-fed rats.
OBJECTIVE: Chronic feeding of a purified synthetic diet induces renin-angiotensin system-dependent moderate high blood pressure in normal Sprague-Dawley rats. The present study was designed to characterize the angiotensin II (Ang II) receptor type 2 (AT2)-specific mechanism of blood pressure regulation in these rats. METHODS: The effect of the AT2 receptor antagonist PD123319 (PD) on blood pressure was examined in vivo in synthetic diet-fed rats. Ang II-dependent contraction of aortic rings prepared from the synthetic diet-fed rats was also investigated. RESULTS: After 8 weeks of feeding the synthetic diet, the mean arterial pressure (MAP) was significantly elevated above levels measured in control rats (117 +/- 2 versus 102 +/- 3 mmHg, P < 0.05). Intravenous administration of PD to conscious hypertensive rats elicited an immediate dose-dependent increase in MAP that was sustained for approximately 7.4 min with 3 mg/kg PD. The angiotensin converting enzyme inhibitor captopril, but not the Ang II type 1 receptor blocker losartan, significantly attenuated the effect of PD on blood pressure. PD did not increase the plasma level of catecholamines. The PD-dependent blood pressure increase was not observed in normotensive control rats. Aortic ring assays revealed that functional activation of the AT2 receptor occurs only in the hypertensive rats, and this AT2 response is abolished by indomethacin (5 micromol/l) but not by Nomega-nitro-L-arginine methyl ester (100 Fmol/l). CONCLUSION: These results clearly demonstrate that AT2 receptor-mediated blood pressure regulation is functional in this experimental model of hypertension. Furthermore, cyclooxygenase metabolites might be the key mediators for the AT2 receptor-mediated blood pressure-lowering action.
Angiotensin II↗
Lipopolysaccharides and cytokines downregulate the angiotensin II type 2 receptor in rat cardiac fibroblasts.
The present study examines the effect of lipopolysaccharides and proinflammatory cytokines on the expression of the second isoform of the angiotensin II receptor (AT(2)), which may have a role in lowering collagen deposition in cardiac tissue. Cardiac fibroblasts express high levels of both angiotensin II type 1 (AT(1)) and type 2 receptors. Incubation with lipopolysaccharides for 24 h dose- and time-dependently decreased angiotensin II AT(2) receptor expression with no apparent difference in the affinity. Actinomycin D, cycloheximide, N(omega)-nitro-L-arginine methyl ester and the protein tyrosine kinase inhibitor herbimycin A, but not the protein kinase C inhibitors bisindolylmaleimide and calphostin C, abolished the inhibitory action of lipopolysaccharides. The cytokines interleukin-1beta and tissue necrosis factor-alpha mimicked the effect of lipopolysaccharides. All three compounds induced inducible nitric oxide synthase (iNOS). The nitric oxide donor sodium nitroprusside and the cGMP analog 8-bromoguanosine cyclic monophosphate downregulated angiotensin II AT (2) receptor expression. The findings are consistent with the pathway in which lipopolysaccharides or cytokines induce iNOS. The data suggest that lipopolysaccharide- or cytokine-dependent induction of iNOS and resultant production of nitric oxide leads to the production of cGMP, which in turn downregulates expression of the angiotensin II AT (2) receptor in cardiac fibroblasts.
Animals↗
Brain actin-associated protein phosphatase 1 holoenzymes containing spinophilin, neurabin, and selected catalytic subunit isoforms.
We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1(A) holoenzymes selectively enriched in PP1gamma(1) over PP1beta isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956-9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470-3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H. , Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997) J. Cell Biol. 139, 951-961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427-470, or homologous neurabin residues 436-479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1gamma(1) from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1gamma(1) over PP1beta. Moreover, immunoprecipitation of PP1gamma(1) from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1beta immunoprecipitation did not. Thus, PP1(A) holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins.
Amino Acid Sequence↗
NF-kappa B is activated and ICAM-1 gene expression is upregulated during reoxygenation of human brain endothelial cells.
Reperfusion injury is mediated, in part, by the upregulated expression of genes in microvascular endothelial cells that encode for inflammatory cytokines and adhesion molecules. The redox-regulated transcription factor, nuclear factor kappa B (NF-kappaB), may play a major role in the induced expression of these genes. In this study we use cultured human brain microvascular endothelial cells (HBMEC) to investigate whether reoxygenation of hypoxic HBMEC results in the activation of NF-kappaB and the upregulation of the adhesion molecule, ICAM-1. When HBMEC were subjected to hypoxia followed by reoxygenation but not hypoxia alone, an NF-kappaB complex composed of p65 and p50 Rel proteins was rapidly activated within 15-30 min. Four hours later, expression of the ICAM-1 gene was significantly upregulated. The antioxidant pyrrolidine dithiocarbamate and the proteasome inhibitor, n-Tosyl-Phe-chloromethyl ketone, blocked both the activation of NF-kappaB and the upregulation of the ICAM-1 gene. These results indicate that NF-kappaB is activated in HBMEC by reoxygenation and may play a significant role in the upregulation of the ICAM-1 gene. Agents which inhibit NF-kappaB activation may be potential therapeutic agents in acute ischemic stroke.
Brain↗
Nuclear factor-kappa B activation during cerebral reperfusion: effect of attenuation with N-acetylcysteine treatment.
We examined activation of the transcription factor, nuclear factor-kappaB (NF-kappaB), which participates in the upregulation of endothelial cell adhesion proteins, during reperfusion after temporary middle cerebral artery occlusion (TMCAO). We hypothesized that N-acetylcysteine (NAC), an antioxidant which inhibits NF-kappaB activation, would alter events in brain reperfusion injury. We used a rat model of TMCAO. The left sides of the brains were rendered ischemic for 2 h, and then the area was allowed to reperfuse. The animals were treated with NAC (150 mg/kg) or saline placebo, sacrificed, and activated NF-kappaB was assessed in both the left and right hemispheres, all at varying intervals. Cerebral infarction volume was also measured in each of the hemispheres collected from a separate group of animals. Activated NF-kappaB, consisting of p65 and p50 Rel proteins, was significantly increased 15 min after reperfusion in the affected hemisphere. The activation at 15 min was completely abolished with NAC treatment. NAC treatment 1 h prior to the end of occlusion and at 24 h reduced the percentage infarction volume of the affected hemispheres from 35.5+/-2.8% (S.E.) to 18. 1+/-2.1% (p<0.01). NAC treatment at 1 h after the occlusion (after the NF-kappaB peak) and again at 24 h also significantly reduced the percentage infarction volume from 34.8+/-3.8% to 24.6+/-3.8% (p<0. 05). Thus, while NAC inhibited activation of NF-kappaB at 15 min after reperfusion, the drug acted to reduce cerebral infarction by additional, undefined mechanisms. These results bring into question the various roles of NF-kappaB in cerebral infarction followed by reperfusion.
Acetylcysteine↗
The relative levels of different types of beta-mRNA and beta-globin in BFU-E derived colonies from patients with beta chain variants; further evidence for somatic mosaicism in the Hb Costa Rica carrier [beta 77(EF1)His-->Arg].
We have identified and quantitated the different types of mRNA in single BFU-E derived colonies from Hb S and Hb Atlanta [beta 75 (E19)Leu-->Pro] heterozygotes and observed that the normal and mutated mRNAs were present in equal quantities. Similar studies for the different protein products gave less accurate data because high performance liquid chromatography methods were not sensitive enough for the analysis of a single colony, and as many as five colonies needed to be combined. The level of Hb S (approximately 40%) was the same as in red cell lysates of the Hb S heterozygote, while that of the unstable beta-Atlanta chain was lower than expected from the values observed in red cells. Similar studies for a carrier of the stable Hb Costa Rica [beta 77(EF1) His-->Arg] which was reported to be the result of a somatic cell mutation (1) gave quite different data. Dot-blot analysis with 32P-labeled probes and allele specific amplification methodology identified numerous colonies with beta A-mRNA only, while 12-15% of the colonies contained both beta A- and beta-Costa Rica-mRNA. This limited distribution of the beta-Costa Rica-mRNA was confirmed by hemoglobin analysis with anion exchange high performance liquid chromatography. These results are considered to provide additional and convincing evidence for a somatic mosaicism for the CAC-->CGC mutation at codon 77 of the beta gene which occurred during the development of the embryo and results in the presence of only some 6-8% of the abnormal Hb Costa Rica in the circulating red cells.
Erythroid Precursor Cells↗
Cell specific distribution of LH/hCG receptor messenger ribonucleic acid in rat testicular Leydig cells.
By using four different cell isolation procedures, we previously identified two morphologically and biochemically distinct Leydig cell populations in rat testis. The light cells were vacuolated and bound 125I-labeled human choriogonadotropin (hCG) with high affinity but upon hCG stimulation in vitro, cAMP and testosterone production by these cells were minimal. On the other hand, the heavier cells displayed typical Leydig cell morphology and bound very little hCG but vigorously produced cAMP and testosterone (Browne, E.S., Bhalla, V.K., 1991, J. Androl. 12:132-139). This study examines the distribution of LH/hCG receptor mRNAs in the two cell types. The light cell fraction contains larger transcripts of LH/hCG receptor but the heavier Leydig cells contain shorter transcripts. The observations raises the intriguing possibility that shorter rather than larger LH/hCG receptor transcripts are responsible for the induction of a biologically functional, G-protein coupled, LH/hCG receptor in Leydig cells.
Animals↗
Need for persuasion.
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Financing, Government↗
Functional expression of the intestinal peptide-proton co-transporter in Xenopus laevis oocytes.
The expression of the intestinal peptide-proton cotransporter was examined in Xenopus laevis oocytes by microinjection of poly(A)+ mRNA prepared from rabbit intestinal mucosal cells. The concomitant expression of the glucose-sodium co-transporter was used as the control for the effectiveness of the expression technique. There was significant endogenous activity of Gly-Sar uptake in water-injected oocytes, but the uptake activity increased nearly 3-fold in poly(A)+ mRNA-injected oocytes. The expression of the peptide transporter was time-dependent. There was no detectable expression on day 1 after injection. The expression became noticeable on day 2 and increased with time, reaching a maximum on day 4. There was no further change on days 5 and 6. The endogenous uptake rate measured in water-injected oocytes, on the contrary, showed a slight decrease during this time. The expressed peptide transporter retained its substrate specificity, having affinity for the dipeptides, Gly-Sar and Gly-Pro, and no or little affinity for the free amino acids, Gly and Sar. The expressed peptide transporter also showed a dependence on a transmembrane H+ gradient for maximal activity. These data demonstrate that the mammalian intestinal peptide-proton co-transporter can be successfully expressed in Xenopus laevis oocytes. This expression system can provide an effective assay procedure to clone the gene encoding the transporter.
Animals↗
Mandated coverage: federal activity.
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Cost Control↗
M3-muscarinic receptor subtype predominates in the bovine iris sphincter smooth muscle and ciliary processes.
The distribution of mRNAs encoding muscarinic acetylcholine receptor (mAChR) subtypes (M1, M2, M3, and M4) was investigated in the bovine iris-ciliary body by Northern blot hybridization with subtype-specific oligonucleotide probes that were complementary to unique regions of the M1, M2, M3, and M4 mAChRs. Whole rat brain RNA, which contains all four subtypes, was employed as a positive control. Both the iris sphincter and the ciliary processes were found to contain predominantly the M3 mAChR subtype and minor amounts of the M2 subtype. Traces of the M4 subtype were detected also in the ciliary processes.
Animals↗
Reduction of T-kininogen messenger RNA levels by dexamethasone in the adjuvant-treated rat.
When inflammation is induced in rats following injection of Freund's complete adjuvant, steady state levels of T-I and T-II kininogen mRNAs increase markedly as do plasma levels of T-I and T-II kininogens. When rats are additionally treated with dexamethasone, T-I and T-II steady state mRNA levels and plasma levels of T-kininogens are reduced. The results suggest that dexamethasone may affect the magnitude of T-kininogen gene induction caused by inflammation.
Animals↗
Transient chloramphenicol acetyltransferase expression of the G gamma globin gene 5'-flanking regions containing substitutions of C----T at position -158, G----A at position -161, and T----A at position -175 in K562 cells.
The expression of G gamma is affected by mutations that occur in promoter sequences located in the 5'-flanking region of the gene. We have assayed the promoter activity of G gamma genes that have mutations of C----T at position -158 or G----A at position -161. In addition, we determined the activity of a promoter fragment containing T----A at position -175 (in the octamer motif) in combination with a -158 C----T which was produced during the polymerase chain reaction amplification procedure. Constructs containing these fragments were transfected by electroporation into K562 cells and the promoter activity was measured as chloramphenicol acetyltransferase activity. The data show a 4-5-fold enhancement of activity for the -158 C----T and the -161 G----A promoters over the 'normal' G gamma promoter and an 8-fold increase in the activity of the promoter with the double mutation (-158 C----T and -175 T----A). These results are consistent with data involving the increase in G gamma production in patients heterozygous for these mutations.
Base Sequence↗
A C----T substitution at nt--101 in a conserved DNA sequence of the promotor region of the beta-globin gene is associated with "silent" beta-thalassemia.
Sequence analyses and dot-blot analyses with synthetic oligonucleotide probes have identified eight individuals in three Turkish families and one Bulgarian family with one chromosome having a C----T mutation at nucleotide position--101 relative to the Cap site of the beta-globin gene. This nucleotide is part of one of the conserved blocks of nucleotides within the promoter region; in vitro expression analyses with the chloramphenicol acetyltransferase system showed that this substitution will decrease the effectiveness of transcription. Five subjects had a thalassemia intermedia due to the additional presence of a known classical high hemoglobin (Hb) A2 beta-thalassemia mutation on the second chromosome; their hematologic condition was relatively mild. The three persons with a heterozygosity for the--101 C----T mutation had normal hematologic data without microcytosis but with high-normal levels of Hb A2 and a mild imbalance in chain synthesis. The newly discovered mutation is considered one of the silent types of beta-thalassemia. It is relatively rare because it was absent among several hundred normal and beta-thalassemia chromosomes.
Base Sequence↗
Metastasis-stimulating activity in the mouse uterus.
Mouse uterine luminal proteins are thought to play important roles in inducing diapausing blastocysts to implant into the uterine wall. Employing a syngeneic teratocarcinoma cell line (402AX), we demonstrate that neoplastic cells are better able to invade and metastasize if they are coinjected with uterine fluid from pregnant or estrogen-primed mice. This metastasizing activity of uterine fluid was partially purified by using disc polyacrylamide electrophoresis and gel filtration chromatography. Preliminary experiments indicate that the post-albumin and albumin bands contain most of the bioactivity. Furthermore, these bands contain smaller molecular weight proteins (less than 14,000) than can be separated by detergent and mild acetic acid (0.1 N) treatment.
Animals↗