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Biological Science Disciplines↗
Biomedical subjects
C M Kane
Publications and source records attributed to C M Kane.
Role of angiotensin II in altered baroreflex function of conscious rabbits during late pregnancy.
OBJECTIVE: Pregnancy alters baroreflex control of heart rate in conscious rabbits, but the mechanism for this action is unknown. This study tested the hypothesis that endogenous angiotensin II is the mediator. STUDY DESIGN: To test this hypothesis the baroreflex relationship between arterial pressure and heart rate in conscious rabbits was determined before and after administration of the angiotensin II AT1 receptor antagonist losartan (n = 7) before pregnancy and at the end of gestation. RESULTS: Pregnancy decreased mean arterial pressure, increased heart rate, and modified the reflex by shifting the mean arterial pressure-heart rate relationship to a lower pressure level, by increasing minimum heart rate, and by decreasing baroreflex gain (P < .05). Before pregnancy, losartan decreased baroreflex gain but had no other effect on reflex function. In contrast, during late gestation losartan further decreased mean arterial pressure, further decreased reflex gain, decreased maximum heart rate, and shifted the curve to a lower mean arterial pressure level (P < .05). CONCLUSION: These results suggest that in conscious rabbits during pregnancy endogenous angiotensin II contributes to hypotension-induced tachycardia but does not decrease reflex gain or elevate minimum heart rate.
Angiotensin II↗
Structural basis for the species-specific activity of TFIIS.
Many proteins involved in eukaryotic transcription are similar in function and in sequence between organisms. Despite the sequence similarities, there are many factors that do not function across species. For example, transcript elongation factor TFIIS is highly conserved among eukaryotes, and yet the TFIIS protein from Saccharomyces cerevisiae cannot function with mammalian RNA polymerase II and vice versa. To determine the reason for this species specificity, chimeras were constructed linking three structurally independent regions of the TFIIS proteins from yeast and human cells. Two independently folding domains, II and III, have been examined previously using NMR (). Yeast domain II alone is able to bind yeast RNA polymerase II with the same affinity as the full-length TFIIS protein, and this domain was expected to confer the species selectivity. Domain III has previously been shown to be readily exchanged between mammalian and yeast factors. However, the results presented here indicate that domain II is insufficient to confer species selectivity, and a primary determinant lies in a 30-amino acid highly conserved linker region connecting domain II with domain III. These 30 amino acids may physically orient domains II and III to support functional interactions between TFIIS and RNA polymerase II.
Amino Acid Sequence↗
Expression of a renin/GFP transgene in mouse embryonic, extra-embryonic, and adult tissues.
A reporter construct was assembled with 4-kb of renin 5'-flanking sequence fused to humanized green fluorescent protein (GFP) cDNA. Transgenic mice carrying this construct were produced and assayed for GFP expression. In the adult, expression was detected in juxtaglomerular (JG) cells of the kidney and granular convoluted tubular cells of the submandibular gland. Furthermore, treatment of mice with captopril induced GFP expression in renal vascular smooth muscle cells. During embryogenesis, GFP expression was first detected at embryonic day E13 in the adrenal gland and Wolffian duct. Expression was also seen in the developing renal vasculature as early as E14 and remained detectable through birth. Renal GFP expression became restricted to JG cells in adults. Fetal adrenal and gonadal arteries also expressed GFP. In the placenta, GFP was observed in giant cell trophoblasts, consistent with reports of renin expression in chorionic cells of both humans and mice. We conclude that 4 kb of renin 5' flank is sufficient to direct multiple known renin expression patterns. Furthermore, the renin-GFP construct characterized here will provide a useful vital reporter for renin expression.
Aging↗
Genetic interactions between TFIIS and the Swi-Snf chromatin-remodeling complex.
The eukaryotic transcript elongation factor TFIIS enables RNA polymerase II to read through blocks to elongation in vitro and interacts genetically with a variety of components of the transcription machinery in vivo. In Saccharomyces cerevisiae, the gene encoding TFIIS (PPR2) is not essential, and disruption strains exhibit only mild phenotypes and an increased sensitivity to 6-azauracil. The nonessential nature of TFIIS encouraged the use of a synthetic lethal screen to elucidate the in vivo roles of TFIIS as well as provide more information on other factors involved in the regulation of transcript elongation. Several genes were identified that are necessary for either cell survival or robust growth when the gene encoding TFIIS has been disrupted. These include UBP3, KEX2, STT4, and SWI2/SNF2. SWI1 and SNF5 disruptions were also synthetically lethal with ppr2Delta, suggesting that the reduced ability to remodel chromatin confers the synthetic phenotype. The synthetic phenotypes show marked osmosensitivity and cytoskeletal defects, including a terminal hyperelongated bud phenotype with the Swi-Snf complex. These results suggest that genes important in osmoregulation, cell membrane synthesis and integrity, and cell division may require the Swi-Snf complex and TFIIS for efficient transcription. The detection of these genetic interactions provides another functional link between the Swi-Snf complex and the elongation machinery.
Adenosine Triphosphatases↗
Regional conductance changes during hemorrhage in pregnant and nonpregnant conscious rabbits.
Late pregnant (P) conscious rabbits are less able to maintain arterial pressure during hemorrhage than nonpregnant (NP) animals. This study tested the hypothesis that the difference is due in part to less reflex vasoconstriction when the rabbits are P. Rabbits (n = 14) were instrumented with arterial and venous catheters as well as ultrasonic flow probes around the superior mesenteric, renal, and/or terminal aortic arteries. Pregnancy increased (P < 0.05) blood volume [235 +/- 5 (P) vs. 171 +/- 3 (NP) ml], terminal aortic conductance [1.88 +/- 0.11 (P) vs. 0.98 +/- 0.06 (NP) ml. min(-1). mmHg(-1)], mesenteric conductance [1.20 +/- 0.19 (P) vs. 0.80 +/- 0. 05 (NP) ml. min(-1). mmHg(-1)], and heart rate [191 +/- 4 (P) vs. 162 +/- 3 (NP) beats/min] and decreased arterial pressure [59 +/- 1 (P) vs. 67 +/- 2 (NP) mmHg; P < 0.05]. Renal conductance was unaltered. The rabbits were bled in both the NP and P states at 2% of the initial blood volume per minute until arterial pressure fell below 45 mmHg. Arterial pressure fell with less blood loss in P rabbits [28 +/- 2% (P) vs. 39 +/- 2% (NP) of initial blood volume; P < 0.001]. Terminal aortic conductance decreased (P < 0.001) before the pressure fall in both groups, but the response was reduced in P rabbits. Mesenteric and renal conductances did not change in either group before the blood pressure fall. During the pressure fall, terminal aortic conductance increased (P < 0.05) only in NP rabbits. Mesenteric conductance increased in both groups. In summary, rabbits in late gestation are less able to maintain arterial pressure during hemorrhage, at least in part because of reduced vasoconstriction in tissues perfused by the terminal aorta.
Animals↗
Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through.
The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.
Models, Molecular↗
Hemodynamic and hormonal responses to hemorrhage in conscious rabbits at mid- and late gestation.
This study tests the hypothesis that conscious rabbits late in pregnancy (P), but not at midgestation (MP), are less able to maintain arterial pressure during hemorrhage. Blood volume (BV) was elevated (P < 0.05) by an average of 13 +/- 4 (MP) and 35 +/- 3% (P). Rabbits were bled in both the nonpregnant (NP) and P state at 2% of the initial BV per minute. The hemorrhage was stopped after arterial pressure decreased. In NP rabbits, arterial pressure was well maintained near control pressures of 70 +/- 2 mmHg until 38 +/- 2% of the initial BV was removed and then rapidly fell to reach a nadir at 35 +/- 2 mmHg. In contrast, in P rabbits, basal arterial pressure was lower (61 +/- 2 mmHg; P < 0.05) and gradually decreased to below control after <25% of the initial BV was removed. Moreover, the rapid hypotensive phase was triggered with a lower percent BV removal (33 +/- 2%; P < 0.05). Basal heart rate was higher during P (149 +/- 5 vs. 189 +/- 9 beats/min; P < 0.05), and reflex increases were delayed. The slope of the relationship between arterial pressure and vasopressin was not modified during P, although the line was shifted to a lower pressure (P < 0.05). Larger increases in plasma renin activity and ANG II concentration were produced during hemorrhage in P rabbits. In contrast, no differences in the changes in arterial pressure, heart rate, and vasopressin were found between NP and MP rabbits during hemorrhage, although increases in renin and ANG II were greater at MP (P < 0.05). In summary, although P conscious rabbits are less able to maintain blood pressure during hemorrhage, this change is not evident at MP. These data suggest that the factors that mediate the P-induced alterations in arterial pressure regulation are not operative until late in gestation.
Angiotensin II↗
An essential component of a C-terminal domain phosphatase that interacts with transcription factor IIF in Saccharomyces cerevisiae.
One of the essential components of a phosphatase that specifically dephosphorylates the Saccharomyces cerevisiae RNA polymerase II (RPII) large subunit C-terminal domain (CTD) is a novel polypeptide encoded by an essential gene termed FCP1. The Fcp1 protein is localized to the nucleus, and it binds the largest subunit of the yeast general transcription factor IIF (Tfg1). In vitro, transcription factor IIF stimulates phosphatase activity in the presence of Fcp1 and a second complementing fraction. Two distinct regions of Fcp1 are capable of binding to Tfg1, but the C-terminal Tfg1 binding domain is dispensable for activity in vivo and in vitro. Sequence comparison reveals that residues 173-357 of Fcp1 correspond to an amino acid motif present in proteins of unknown function predicted in many organisms.
Amino Acid Sequence↗
Genomic characterization of a testis-specific TFIIS (TCEA2) gene.
There is a family of genes encoding TFIIS-related proteins in human cells. We have focused upon the genomic organization of one family member expressed primarily in the testis. This gene encodes a transcription elongation factor similar to but distinct from that encoded by a previously reported TFIIS gene. Also in contrast to the previously reported TFIIS gene, the testis gene contains introns. All exon-intron junction sequences match the consensus GT/AG rule. The gene consists of seven exons and six introns with a total size of approximately 7 kb. The nucleotide sequence of the 5 flanking region of the testis TFIIS gene contains several potential regulatory factor-binding sites, not all of which are present in the TFIIS gene, whose expression is nearly ubiquitous. Elucidation of the full structure of the testis TFIIS gene should be useful for determining its chromosomal localization and its potential role in the regulation of gene expression in human tissues.
Animals↗
Transcription elongation through DNA arrest sites. A multistep process involving both RNA polymerase II subunit RPB9 and TFIIS.
The role of yeast RNA polymerase II (pol II) subunit RPB9 in transcript elongation was investigated by examining the biochemical properties of pol II lacking RPB9 (pol IIDelta9). The maximal rate of chain elongation was nearly identical for pol II and pol IIDelta9. By contrast, pol IIDelta9 elongated more efficiently through DNA sequences that signal the elongation complex to pause or arrest. The addition of purified recombinant RPB9 to pol IIDelta9 restored the elongation properties of the mutant polymerase to those of the wild-type enzyme. Arrested pol IIDelta9 complexes were refractory to levels of TFIIS that promoted maximal read-through with pol II. However, both pol II and pol IIDelta9 complexes stimulated with TFIIS undergo transcript cleavage, confirming that transcript cleavage and read-through activities can be uncoupled. Our observations suggest that both TFIIS and RPB9 are required to stimulate the release of RNA polymerase II from the arrested state.
Cloning, Molecular↗
Nutritional factors modify the inhibition of CNS development by combined exposure to methadone and ethanol in neonatal rats.
The consequences resulting from the combined exposure to methadone and ethanol during a time period equivalent to the third trimester brain growth spurt was the purpose of this study. Rat pups were treated on postnatal days 6-10 and sacrificed on postnatal day 11. Body weight along with the heart, liver, kidneys, whole brain, cerebrum, cerebellum, and brain stem weights were measured. The impact of nutritional factors were identified by delivery of the drug solutions in one of two intubation vehicles differing in both caloric density and composition. Ethanol and methadone in combination result in significantly increased detrimental effects compared to methadone alone only when possible nutritional compromise was present. The combined effect of both drugs significantly inhibited body growth and the development of all brain regions studied. Neither drug alone, nor in combination, produced significant inhibition of growth in the liver, heart, or kidney. The nutritional status of the pup, as represented by vehicle composition, was able to modify the specific drug effects and suggests that nutritional status can mask or enhance the determination of specific drug effects.
Animal Nutritional Physiological Phenomena↗
Basic mechanisms of transcript elongation and its regulation.
Ternary complexes of DNA-dependent RNA polymerase with its DNA template and nascent transcript are central intermediates in transcription. In recent years, several unusual biochemical reactions have been discovered that affect the progression of RNA polymerase in ternary complexes through various transcription units. These reactions can be signaled intrinsically, by nucleic acid sequences and the RNA polymerase, or extrinsically, by protein or other regulatory factors. These factors can affect any of these processes, including promoter proximal and promoter distal pausing in both prokaryotes and eukaryotes, and therefore play a central role in regulation of gene expression. In eukaryotic systems, at least two of these factors appear to be related to cellular transformation and human cancers. New models for the structure of ternary complexes, and for the mechanism by which they move along DNA, provide plausible explanations for novel biochemical reactions that have been observed. These models predict that RNA polymerase moves along DNA without the constant possibility of dissociation and consequent termination. A further prediction of these models is that the polymerase can move in a discontinuous or inchworm-like manner. Many direct predictions of these models have been confirmed. However, one feature of RNA chain elongation not predicted by the model is that the DNA sequence can determine whether the enzyme moves discontinuously or monotonically. In at least two cases, the encounter between the RNA polymerase and a DNA block to elongation appears to specifically induce a discontinuous mode of synthesis. These findings provide important new insights into the RNA chain elongation process and offer the prospect of understanding many significant biological regulatory systems at the molecular level.
Animals↗
Altered heart rate baroreflex during pregnancy: role of sympathetic and parasympathetic nervous systems.
Two studies were performed to determine whether the attenuation of baroreflex control of heart rate during late pregnancy in conscious rabbits is due to changes in parasympathetic (Para) or sympathetic (Sym) control of the heart. In the first, baroreflex relationships between arterial pressure and heart rate were generated before and after treatment with propranolol (Pro) to block Sym or with methscopolamine (Meth) to block Para. Each rabbit was studied in both the pregnant and nonpregnant state. Pregnancy decreased maximum baroreflex gain from 14.9 +/- 4.0 to 4.8 +/- 0.9 beats.min-1.mmHg-1 (P < 0.01) and decreased heart rate range from 177 +/- 6 to 143 +/- 10 beats/min (P < 0.01), primarily by increasing minimum heart rate (114 +/- 6 to 134 +/- 8 beats/min; P < 0.01). The difference between pregnant and nonpregnant rabbits in baroreflex gain was not altered by Meth but was abolished by Pro, suggesting that it is due to decreased Sym control of the heart. The elevated minimum heart rate of pregnancy persisted after Pro, but was abolished by Meth, suggesting that it is mediated by decreased Para control of the heart. In the second study, isolated buffer-perfused hearts from pregnant and nonpregnant rabbits were treated with increasing doses of isoproterenol (0.3-300 mM) or acetylcholine (0.3-10,000 microM), and the heart rate responses were determined. Hearts from pregnant rabbits were more sensitive to isoproterenol (P < 0.05), but less responsive to acetylcholine (P < 0.05). In conclusion, pregnancy-induced decreases in cardiac reflex gain and range appear to be mediated by alterations in Sym and Para, respectively. The change in Sym occurs proximal to the heart, whereas the decreased contribution of Para may be due, at least in part, to decreased sensitivity of the heart to acetylcholine.
Acetylcholine↗
Downregulation of renin gene expression by interleukin-1.
The As4.1 cell line was established from a mouse kidney tumor by transgene-targeted tumorogenesis. These cells express high levels of renin mRNA from their endogenous renin gene and release approximately eightfold-more prorenin than active renin in culture. Levels of renin mRNA in As4.1 cells are decreased in a dose-dependent manner by the addition of physiological concentrations of cytokine interleukin-1 to the media. Stability of renin mRNA and initial rates of release of active renin and prorenin were not significantly altered by interleukin-1. In contrast, transcription initiated from a construct that consisted of 4.1 kilobases of renin 5' flanking sequence fused to a reporter gene (chloramphenicol acetyltransferase) was markedly inhibited by interleukin-1. On the basis of our findings, we conclude that downregulation of renin synthesis caused by interleukin-1 occurs primarily at the level of transcription and that DNA sequence or sequences mediating that effect are positioned within 4.1 kilobases upstream of the renin gene. The physiological relevance of this regulation is related to the events that occur during septic shock, characterized by hypotension, cardiovascular collapse, multiple organ failure, and high mortality. Unexpectedly, hypotension associated with septic shock does not lead to activation of the renin-angiotensin system. The hypotension in septicemia is believed to be mediated by the combined action of many modulators including cytokines, and data presented here suggest direct involvement of interleukin-1 in this process.
Animals↗
Purification and characterization of an RNA polymerase II phosphatase from yeast.
RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by >30,000-fold. The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa. The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells. These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid. Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases. Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical. The activity of the highly purified CTD phosphatase is stimulated >300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity. These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa.
Chromatography, Ion Exchange↗
Isolation and characterization of the Schizosaccharomyces pombe gene encoding transcript elongation factor TFIIS.
A gene designated tfs1 has been isolated from Schizosaccharomyces pombe based on its similarity to genes encoding transcription elongation factor TFIIS. The nucleotide sequence of the tfs1 gene predicts a polypeptide with similarity to mammalian. Drosophila and Saccharomyces cerevisiae TFIIS. A haploid Sz. pombe strain with tfs1 deleted from the genome is viable. Thus, tfs1 is not essential for viability. However, deletion of tfs1 results in slow growth and increased sensitivity to the drug 6-azauracil, a phenotype similar to that of a S. cerevisiae strain deleted for the gene encoding TFIIS. The DNA sequence of tfs1 has been deposited in GenBank under Accession Number U20526.
Base Sequence↗
Identifying regulators of transcript elongation in eukaryotes.
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Animals↗