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Conditions underlying balance impairment should be identified to improve knowledge regarding clinical interventions for frail older adults. This study aims to explore the relationship between balance functions and frailty by using the brief balance evaluation systems test (BESTest), which can assess biomechanical constraints, stability limits/verticality, anticipatory postural adjustments (APAs), reactive postural responses, sensory orientation and stability in gait. A total of 75 community-dwelling older women were included in this cross-sectional study. We evaluated frailty by using the Kihon checklist and assessed the participants' balance functions by using the Brief BESTest. We performed the Mann-Whitney U test and receiver operating characteristic curve analysis to compare each balance function between frail and nonfrail participants. Twenty-two of the 75 (29.3%) participants were included in the frailty group. We noted significant differences between the frailty and nonfrailty groups with regard to stability limit, APAs, sensory orientation, and stability in gait (P = 0.010, 0.001, 0.008 and <0.001, respectively). In terms of determining frailty and nonfrailty, APAs and stability in gait were moderately accurate (the area under the curve = 0.730 and 0.713, respectively). APAs showed the highest sensitivity (0.864), whereas stability limits, sensory orientation, and stability in gait showed the highest specificity (0.943, 0.849 and 0.868, respectively). Thus, frail and nonfrail older adults showed significantly different balance functions, such as stability limits, APAs, sensory orientation and stability in gait. The Brief BESTest is useful for evaluating balance functions in relation to frailty., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Endothelin (ET)-like immunoreactivity has been observed not only in mammals, but also in amphibians. The biological actions of ET are similar in amphibians and mammals, and amphibian ET-related receptors have been cloned and characterized. The cDNA sequences of mature and precursor forms of ET-related peptides, however, have not been reported in any amphibian until now. To identify the ET-related peptides, we screened the Xenopus laevis intestine cDNA library using the rapid amplification of cDNA ends method and cloned cDNAs encoding preproendothelin-1. The deduced amino acid sequence of X. laevis preproendothelin-1 comprises 223 amino acids, including a putative signal sequence of 19 amino acids, a mature ET-1 of 21 amino acids, as well as big ET-1 and ET-1-like sequences. X. laevis ET-1 is identical to mammalian ET-1 as well as ET-1 peptide, recently purified from the stomach of the European green frog, Rana ridibunda. This is the first report describing the cDNA encoding preproendothelin-1 in an amphibian species.

To compare the structure of the precursor polypeptide of dog endothelin-3, preproendothelin-3 (PPET-3), with the PPET-3 of other mammals, we cloned dog cDNA from lung tissue using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. An open reading frame encoding a 198-amino-acid polypeptide was found in the cDNA. Regions corresponding to a bioactive mature endothelin-3 peptide, an intermediate form known as big-endothelin-3 and an endothelin-3- like peptide were observed in the putative PPET-3. Comparative analysis showed that the similarity of the dog open reading frame sequence with those from human hypothalamus, mouse intestine, and rat eye is 76.2%, 69.5% and 66.3%, respectively, and that the similarity at the amino acid level is 65.6%, 59.8% and 58.8%, respectively. RT-PCR demonstrated significant elevated expression of PPET-3 mRNA in the kidney of dog with interstitial nephritis.

Endothelin (ET)-related receptors homologous to mammalian receptors have been cloned from Xenopus laevis, indicating that ET-related ligands may be present in this species. Here we cloned cDNAs encoding preproendothelin-3 (PPET-3) from the X. laevis intestinal cDNA library. X. laevis ET-3 cDNA encodes 201 amino acids, including a 20-amino-acid putative signal sequence, as well as mature ET-3, big ET-3, and ET-3-like sequences. X. laevis ET-3 differs by one amino acid from mammalian ET-3, and is identical to frog ET-3 recently purified from Rana ridibunda. This sequence together with other published PPET sequences were used to analyze the phylogenetic relationship among all ET family genes. This is the first report of the cDNA encoding the precursor protein of ET-3 in a non-mammalian species.

The endothelin-B (ETB) receptor is a G-protein-coupled receptor that binds endothelin ligands and is essential for the development of epidermal melanocytes and enteric neurons. Recent reports indicate that ETB is localized to nuclei in cardiac ventricular myocytes, although it has been thought that ETB is localized mainly on the plasma membrane. It remains unknown, however, whether this unique distribution of ETB occurs in other tissues. To elucidate the subcellular distribution of ETB in the intestine, we performed immunofluorescence of ETB in mouse intestine using a specific antibody. ETB-like immunoreactivity was detected in both the mucosal and muscle layers. In the mucosal layer, villous epithelial cells, stromal cells of the lamina propria, and cryptic cells were immunostained. Subcellularly, ETB is localized mainly to the nuclei of villous epithelial cells. In the muscle layer, immunoreactivity of ETB was localized to the myenteric plexus. These findings suggest that ETB may function as an "intracrine" receptor for intracellular endothelin ligands in villous epithelial cells and may regulate intestinal function.

The amino acid sequences of two of the three endothelin (ET) family peptides, ET-1 and ET-3, are identical among mammals, whereas for the other family member, ET-2 or vasoactive intestinal contractor (VIC), the mouse and rat sequences differ from the human counterpart ET-2 by one amino acid residue. To examine more deeply the structural diversity among ET-2/VIC orthologs (EDN2), we screened porcine ET-2/VIC-like cDNAs using the 5' rapid amplification of cDNA ends (RACE) method with degenerate primers based on ET-2/VIC mature peptides. Sequence analysis of the cDNAs showed that ET-2 is present in pig. The full-length cDNA sequence, produced by combining 5' RACE and 3' RACE products, revealed the porcine precursor protein of ET-2 (PPET-2). Porcine PPET-2, made up of 214 amino acids, includes a 26-residue putative signal sequence, big ET-2, mature ET-2, and ET-2-like peptide. The percent sequence identity of porcine PPET-2 with human PPET-2, and rat or mouse precursor protein of VIC runs between approximately 70% and 74%. ET-2, although expressed in intestine, has no anti-microbial activity.

We cloned and characterized horse preproendothelin-2 (PPET-2) cDNA from intestinal tissue. The cDNA encoded 178 amino acids of the PPET-2 polypeptide, in which a 21-amino-acid mature endothelin-2 peptide and a 16-amino acid endothelin-2-like peptide were found. For the open reading frame the correspondence of horse PPET-2 cDNA with those of the ferret, human, dog, mouse and rat was 85.1%, 84.9%, 82.1%, 77.8% and 77.2%, respectively. Analysis of the organ distribution of PPET-2 mRNA by reverse transcription-polymerase chain reaction demonstrated that the kidney, stomach and small intestine are major sites of expression of the PPET-2 gene. Surprisingly, the mRNA is not detected in the large intestine, where high expression is demonstrated in the mouse and rat. This difference may result from the underlying functional differences of the large intestine between a herbivore (horse) and an omnivore (mouse and rat).

A rapid analysis method for murine endothelin-A (ETA) and endothelin-B (ETB) receptor gene expression levels was established using real-time quantitative reverse transcriptase-polymerase chain reaction. We designed primer pairs and TaqMan probes specific for the two cDNAs and available for mouse and rat systems. The standard curve method was used to examine relative expression. The gene expression levels of ETA and ETB were estimated as gene expression rates by normalizing to the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. To examine the reproducibility of this assay system, we calculated the intra-assay and interassay coefficients of variation of the gene expression rate and found that a greater than 1.6-fold increase in relative gene expression is detectable as a significant change. ETA and ETB receptor gene expression was found in all 16 organs of mouse and rat examined, and high levels of expression were observed in the lung, uterus, ovary, intestine, and cerebellum. The gene expression patterns essentially agreed with those determined by RNase protection assay, Northern blot, and conventional endpoint polymerase chain reaction. These results show that this new rapid, sensitive, and semi-automated method is accurate, quantitative, and reproducible. This method is also useful for examining regulation of hormone receptor gene expression under physiological conditions in organs.

In order to understand the physiological roles of vasoactive intestinal contractor (VIC)/endothelin-2 (ET-2), we examined the expression of this peptide by specific reverse transcriptase polymerase chain reaction (RT-PCR) analysis and found that PC12 rat pheochromocytoma cells express the VIC gene. The 5'-flanking 1.0 kilo base pair (kb) region of the mouse VIC gene is sufficient to express a secreted alkaline phosphatase (SEAP) reporter gene in transiently transfected PC12 cells. The 1.0 kb promoter region may contain cis-acting elements that determine the rate of the VIC gene transcription in PC12 cells.

Endothelin (ET)-related peptide hormones, ET-1, vasoactive intestinal contractor (VIC), ET-2 and ET-3, have multiple physiological roles including vasoconstriction. To reveal the structural diversity of the precursor proteins of the ET family, cDNAs, cross-hybridizing with ET-1 and VIC probes, were cloned from the mouse intestine library. The deduced protein is a 214-amino acid precursor of mouse ET-3, preproendothelin-3 (PPET-3), which is the counterpart of the hypothalamus-, not placenta-, derived human PPET-3. Sequence identities of PPET-3 amino acids of mouse with human and rat are 65% over 194 amino acids and 65% over 214 amino acids. Phylogenetic analysis of the precursor proteins for ET-1, VIC and ET-3 suggest that members of the ET family are distantly related and probably descended from a common ancestral gene.

We established a real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) system for the analysis of rat endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 gene expression. We used this technique to examine the expression levels in rat in 16 different organs. ET-1 gene expression was observed in all organs examined, while VIC mRNA was detected in some organs such as heart, lung, ovary, stomach, and intestine. Ovary and intestine express both ET-1 and VIC mRNA at high levels, suggesting the importance of both peptides in these organs. In addition, we examined the gene expression levels in intestinal epithelial and mesenchymal tissues from rat fetuses at 16.5 and 19.5 days postcoitus (E16.5 and E19.5). We observed distinct differences in the temporal gene expression patterns for ET-1 and VIC in fetal intestinal epithelial tissue. In fetal mesenchymal tissue the expression level of ET-1 is significantly higher than that of VIC, and the levels of both genes remain unchanged over the time period observed. These findings suggest distinct biological roles and gene regulation mechanisms for ET-1 and VIC in intestinal epithelial and mesenchymal tissues.

Vasoactive intestinal contractor (VIC) is a member of the endothelin (ET) peptide family, which evokes a strong contractile response in the ileum, its gene being expressed only in the intestine. Using dot blot analysis, we carried out an interspecies comparison of the nucleotide and deduced amino acid sequences of the precursor for VIC with those of ET-1 and ET-3 to investigate the physiological significance of processing of the precursor for VIC. The highly conserved amino acid sequence was observed between the big form region (big VIC, big ET-1, and big ET-3) of about 40 amino acids and the like peptide region (VIC-like peptide, ET-1-like peptide, and ET-3-like peptide) of 15 amino acids downstream from the big form region. Sequence identity of amino acids of the precursors of ET-1 and ET-3 with that of VIC was 29 and 28%, respectively. Thus, the precursors for the three peptides might have arisen from a common progenitor gene. However, apparent cleavage sites of the like peptide regions are rather unique in the VIC-like peptide, i.e., it had dibasic amino acids at the amino and carboxy termini. Therefore, we suggest that the VIC-like peptide might be liberated from its precursor protein and play some role in the intestine in vivo.

We have examined contractions and 45Ca fluxes induced by norepinephrine (NE) and 80 mM potassium (high K) depolarization and their inhibition by diltiazem in rabbit mesenteric resistance vessels. Contraction induced by both NE and high K depended almost completely on extracellular Ca. Dose-response curves for diltiazem inhibition of NE (10(-5) M) and high K contractions showed ED50 values of 1 X 10(-8) and 6 X 10(-7) M, respectively, indicating that the receptor-operated channel (ROC) was more sensitive than the potential-operated channel (POC) to the action of diltiazem. Diltiazem (10(-6) M) was shown to inhibit NE- and 80 mM K-stimulated 45Ca influx effectively by 87 +/- 15 and 85 +/- 10%, respectively. Comparison of these data to those obtained from aorta suggest that although the sensitivity of the POC is approximately the same in aorta and mesenteric resistance vessels, the sensitivity of the ROC is much greater in the latter. This increased sensitivity is paralleled by a greatly decreased role of intracellular Ca release in NE contraction in mesenteric resistance vessels.

Exogenous GTP was required for the induction of Ca2+ release from smooth muscle SR by IP3 if endogenous GTP was depleted. NaN3 could function as a partial substitute for GTP as a cofactor for the IP3-induced Ca2+ release from the SR. In contrast to the IP3-induced Ca2+ release, caffeine-induced Ca2+ release from the SR did not require GTP. Pertussis toxin inhibited the IP3-induced Ca2+ release from the SR, whereas it had no effect on caffeine-induced Ca2+ release. These results indicate that in smooth muscle two different Ca2+ release-channels exist in the SR: (a) activated by IP3, and (b) activated by caffeine or Ca2+.

We studied a 59-year-old woman with dementia, retinitis pigmentosa, sensorimotor neuropathy, and attacks of vomiting associated with blood pressure lability and loss of consciousness. Abnormalities included CT evidence of cerebral atrophy, low IQ, slow central and peripheral nerve conduction velocities, axonal degeneration in sural nerve biopsy, and elevated levels of catecholamines and slow waves in EEG during attacks. Her sister, two brothers, and daughter also had progressive muscle weakness, visual disturbance, and similar vomiting attacks. The hereditary nervous system disorder does not fit any previously described condition.

The relationship between axonal degeneration and primary demyelination was studied in isolated rat sciatic nerve fibers previously exposed to antiserum from rabbits with either experimental allergic neuritis or experimental allergic encephalomyelitis, or immunized with antigalactocerebroside antiserum. Continuous demyelination over one to eight or more internodes was seen in association with phagocytic cells or, later, with increased numbers of Schwann cells. Paranodal demyelination was prominent proximal ahd distal to the zone of continuous demyelination. Axonal degeneration affected 5 to 15% of myelinated fibers exposed to antiserum and was not related to the length of demyelination must proximal to the axonal changes. At times, there were seven or eight consecutive demyelinated internodes with no distal axonal degeneration; in contrast, one demyelinated internode was often associated with axonal degeneration beginning just distally. The inflammatory reaction could account for axonal degeneration in antiserum-mediated demyelination.

We studied the effects of Ca++ antagonists on intact and skinned muscles of rabbit mesenteric artery. Intact muscle contractions were inhibited by 10(-6) M diltiazem, whereas greater levels were required to abolish contractions in skinned muscle fibers. In contrast, nisoldipine had no effect on skinned muscle contractions, although it inhibited, almost completely, the contraction of intact muscle at concentrations below 10(-6) M. In the presence of EGTA, norepinephrine-induced contractions result from a release of Ca++ from an intracellular store. Diltiazem inhibited these contractions at concentrations between 10(-6) and 10(-4) M. Higher doses were required in studies with skinned muscle preparations. Unlike diltiazem, nisoldipine only partially inhibited the Ca++-free norepinephrine-induced contractions in the range of 10(-7) to 10(-5) M. From these results, we assumed that at low concentrations (below 10(-6) M), diltiazem induced relaxation by blocking Ca++ influx, whereas at relatively high concentrations (above 10(-6) M), an inhibition of Ca++ release from an intracellular store also occurred. A similar conclusion was reached regarding the mechanism whereby nisoldipine inhibits force developments.

In rabbit aorta and superior mesenteric artery, application of NE causes release of Ca2+ from an intracellular Ca2+ store, probably SR. The amount of released Ca2+ is sufficient to activate the myofilaments submaximally. It is suggested that NE mediates Ca2+ release by a rapid increase in the free Ca2+ concentration near the SR (Ca2+-induced Ca2+ release). The uptake of Ca2+ into the store and the Ca2+-induced Ca2+ release is modulated by c-AMP.

The primary stimulus for activation of vascular smooth muscle is an increase in the cytosolic free Ca2+ concentration. The level of activating Ca2+ is determined by a variety of Ca2+ homeostatic mechanisms. Ca2+ entry from the extracellular space occurs through the resting Ca2+ leak and the excitable Ca2+ channels: viz. voltage-gated, receptor-operated and stretch-activated channels. Ca2+ release from sarcoplasmic reticulum is induced by inositol triphosphate (IP3) and, possibly, by Ca2+ itself. Activating Ca2+ binds to calmodulin, forming a complex which induces myosin light chain phosphorylation and initiates smooth muscle contraction. The continuous Ca2+ entry together with the higher Ca2+ sensitivity of the contractile apparatus can then maintain smooth muscle tension. Ca2+ buffering by the sarcoplasmic reticulum and Ca2+ extrusion by Ca2+ pumps serve to lower the cytosolic free Ca2+ concentration. These Ca2+-lowering mechanisms are possibly regulated by cyclic nucleotides.