Fracture incidence and fracture-related mortality decreased with decreases in population mobility during the early days of the COVID-19 pandemic: an epidemiological study.

INTRODUCTION: We investigated the impact of coronavirus disease 2019 (COVID-19) social distancing measures on fracture incidence and fracture-related mortality, as well as associations with population mobility. METHODS: In total, 47 186 fractures were analysed across 43 public hospitals from 22 November 2016 to 26 March 2020. Considering the smartphone penetration of 91.5% in the study population, population mobility was quantified using Apple Inc's Mobility Trends Report, an index of internet location services usage volume. Fracture incidences were compared between the first 62 days of social distancing measures and corresponding preceding epochs. Primary outcomes were associations between fracture incidence and population mobility, quantified by incidence rate ratios (IRRs). Secondary outcomes included fracture-related mortality rate (death within 30 days of fracture) and associations between emergency orthopaedic healthcare demand and population mobility. RESULTS: Overall, 1748 fewer fractures than projected were observed during the first 62 days of COVID-19 social distancing (fracture incidence: 321.9 vs 459.1 per 100 000 person-years, P<0.001); the relative risk was 0.690, compared with mean incidences during the same period in the previous 3 years. Population mobility exhibited significant associations with fracture incidence (IRR=1.0055, P<0.001), fracture-related emergency department attendances (IRR=1.0076, P<0.001), hospital admissions (IRR=1.0054, P<0.001), and subsequent surgery (IRR=1.0041, P<0.001). Fracture-related mortality decreased from 4.70 (in prior years) to 3.22 deaths per 100 000 person-years during the COVID-19 social distancing period (P<0.001). CONCLUSION: Fracture incidence and fracture-related mortality decreased during the early days of the COVID-19 pandemic; they demonstrated significant temporal associations with daily population mobility, presumably as a collateral effect of social distancing measures.

Bulimia in college women: incidence and recovery rates.

In a longitudinal survey of college freshmen, the incidence of bulimia nervosa was 4.2 cases per 100 women per year. Prevalence remained stable (2.9%-3.3%) as new cases were offset by partial remissions. Some women continued bulimic behaviors without meeting the DSM-III-R criteria.

Eating pathology and DSM-III-R bulimia nervosa: a continuum of behavior.

This longitudinal survey study of 557 college women used the new Eating Pathology Scale to classify respondents as nondieters, casual dieters, intensive dieters, dieters at risk, and bulimic. Shifts in the severity of dieting behavior over a 6-month period occurred primarily between adjacent scale categories. While new cases of bulimia were drawn from intensive dieters and dieters at risk, those women who no longer met DSM-III-R criteria for bulimia nervosa continued to engage in bulimic behavior.

Regulation of calcium uptake in synaptosomes from rat brain by DL-2-amino-5-phosphonovaleric acid.

DL-2-Amino-5-phosphonovaleric acid stimulated calcium influx into synaptosomes isolated from rat brain. The increase in Ca2+ permeability of the synaptosomal membranes induced by this amino acid is not markedly dependent on the membrane potential or Na+ concentration. It is postulated that 2-amino-5-phosphonovaleric acid is an agonist for a receptor(s) which regulates intraneuronal free calcium concentrations by modulating a selective calcium channel. The observed stimulation of calcium uptake may provide an assay system for purification of the endogenous ligand for this receptor and for characterization of its physiological role in neuronal function.

Role of the amino terminus in ligand binding for the angiotensin II type 2 receptor.

Key amino terminal residues in type 1 (AT1) angiotensin II (AngII) receptors are not conserved within type 2 (AT2) receptors. We therefore characterized amino terminal mutants that are transiently expressed in COS-3 membranes. AT2 amino terminal deletion drastically reduced affinity for AngII, suggesting its importance for this subtype. AT1-AT2 amino terminal exchanges retained wild type AngII affinities (Kd ranging from 3-5 nM), indicating compensation despite substantial sequence dissimilarities. Finally, binding of AT2 selective ligands (CGP42112A and PD123319) was not dependent on the amino terminus.

Mutation of a conserved fifth transmembrane domain lysine residue (Lys215) attenuates ligand binding in the angiotensin II type 2 receptor.

A fifth transmembrane domain lysine residue is conserved in both the type 1 (AT1) and type 2 (AT2) angiotensin II (AngII) receptors. This lysine (Lys199) is believed to play a critical role in peptide binding for the AT1 receptor. To evaluate its possible role in the AT2 receptor, the analogous AT2 residue (Lys199) was changed to glutamine. This mutation greatly reduced the affinity for both 125I-AngII and 125I-Sar1,Ile8-AngII and abolished binding to the non-peptide 125I-PD122979. These data indicate that despite a relatively low homology of 34%, some commonalities in the binding mechanism for AngII may exist between the two subtypes.

Cloning and expression of angiotensin II type 2 (AT2) receptors from murine neuroblastoma N1E-115 cells: evidence for AT2 receptor heterogeneity.

Homology-based PCR was used to isolate angiotensin II type 2 (AT2) receptor cDNA from murine neuroblastoma N1E-115 cells. Despite subtle differences in the nucleotide sequence (the N1E-115 clone coded for Phe133 as TTC and Gln326 as CAG; base substitutions are in bold-italics), the AT2 receptor protein was identical to other reported murine AT2 clones. When transfected into COS-1 cells, the expressed AT2 receptor displayed high affinity for AngII and for AT2-selective compounds, GTP gamma S-insensitive agonist binding and enhanced agonist binding by dithiothreitol. Previously, we have demonstrated that N1E-115 cells possess two distinct subpopulations of AT2 receptors, defined as peak I and peak III receptors, that can be separated by heparin-sepharose chromatography. The two subpopulations differ pharmacologically, biochemically and immunologically. The binding properties of the cloned AT2 receptor closely resembled that of peak III receptors. Moreover, antisera raised against peak I AT2 receptors failed to immunoreact to either peak III receptors or cloned AT2 receptors expressed in COS-1 cells. Collectively, these data suggest that the cloned AT2 receptor is identical to peak III receptors from N1E-115 cells and that a novel AT2 receptor (peak I) remains to be cloned.

Angiotensin II stabilizes a multimeric type 2 (AT2) receptor complex in murine neuroblastoma N1E-115 cells.

Previous work has demonstrated that crosslinking of [125I]AngII to CHAPS solubilized angiotensin Type 2 receptors (AT2) in N1E-115 neuroblastoma cells identifies two radiolabeled proteins of 110 and 66 kDa. Similarly, affinity purification of AT2 receptors using AngII yields two proteins of 110 and 66 kDa. In the present study, anti-AT2 receptor antisera were used to examine the relationship between these two proteins. Agonist treatment (AngII) of intact cells increased the 110 kDa band while decreasing the 66 kDa protein. In intact or solubilized membranes, the ratio of 110 kDa/66 kDa proteins was significantly higher in the presence of an agonist and substantially lower with the antagonist Sar1,Ile8-AngII, suggesting that AngII stabilizes a large 110 kDa multimeric complex that may include the 66 kDa protein. To directly examine this hypothesis, anti-AT2 antisera were further purified against either the 110 or 66 kDa proteins. Both purified antibodies displayed crossreactivity with the two proteins. Moreover, when harshly reduced and denatured, the 110 kDa protein released a prominent immunoreactive 66 kDa protein, as well as other smaller proteins. Collectively, these results suggest that the 110 kDa protein consists, in part, of the 66 kDa protein and, as such, that an AT2 receptor subtype may exist as a multimeric complex that is stabilized by agonist occupancy.

Mutational analysis of the angiotensin II type 2 receptor: contribution of conserved extracellular amino acids.

While much work has been done examining the ligand-binding characteristics of the AT1 receptor, very little attention has been focused on the AT2 receptor. Both receptors bind angiotensin II (AngII) with identical affinities, but share only 34% homology. Although it is tempting to assume that conserved residues between the two subtypes are responsible for the binding of AngII, there is little data to support this view. To determine the commonalities in ligand binding of the AT1 and AT2 receptors, we have chosen several conserved extracellular amino acids which have been shown to be important in AngII binding [1,2] to the AT1 receptor for mutational studies of the AT2 receptor. Specifically, we have mutated tyrosine108 in extracellular loop 1 (ECL1), arginine182 in ECL2, and aspartate297 in ECL3 of the AT2 receptor in order to determine their contribution to AngII binding. In the AT2 receptor, mutation of tyrosine108 to an alanine resulted in a receptor with wild-type binding for AngII, while mutation of either arginine182 or aspartate297 drastically impaired AngII binding ( > 100 nM). These results demonstrate both similarities as well as clear differences between receptor subtypes in the contributions to AngII binding of several conserved extracellular amino acid residues.

Mutational analysis of the angiotensin type 2 receptor: contribution of conserved amino acids in the region of the sixth transmembrane domain.

Angiotensin II (AngII) mediates its physiological actions through two receptor subtypes: the Type 1 (AT1) and Type 2 (AT2) receptors. The subtypes have identical affinities for AngII, while sharing only 34% homology. Mutagenesis has focused mainly on the AT1 receptor, identifying residues important for AngII binding. In contrast, relatively little is known of the binding mechanism of the AT2 receptor. It has been hypothesized that residues that are conserved between the two subtypes that have been shown to be important in the AT1 receptor may also contribute to AngII binding in the AT2 receptor as well. To test this hypothesis, the role of two conserved residues in the sixth transmembrane domain of the AT2 receptor in ligand binding were investigated: tryptophan 269 and aspartate 279. In contrast to the AT1 receptor, mutation of Trp269 in the AT2 receptor to an alanine had no effect on AngII binding, while mutation of Asp279 to alanine similarly impaired AngII binding in both receptors. However, the more sterically conservative substitution of Asp279 to asparagine in the AT2 receptor showed near wild type affinity. Based on this finding, we mutated Asp263 in the AT1 receptor to asparagine. Subsequent studies indicated that this more conservative mutation had no effect on AngII binding to the AT1 receptor. Collectively, these results demonstrate that although there may be commonalities in ligand binding between the AT1 and AT2 AngII receptors, there are also clear differences.

Immunohistochemical mapping of angiotensin type 2 (AT2) receptors in rat brain.

Recently developed antisera selective for angiotensin Type 2 (AT2) receptors were used to localize AT2 receptors in rat brain by immunohistochemistry. While the results from these experiments were largely consistent with previous autoradiographic and radioligand binding analyses of AT2 receptor populations in brain, there were also some notable differences in the distribution of immunoreactivity. More specifically, in agreement with previous studies, AT2 antisera detected apparent receptor populations in the locus coeruleus and the bed nucleus of the accessory olfactory tract, whereas AT2 receptor-immunoreactivity in the cerebellum was primarily associated with the Purkinje cell layer and the deep cerebellar nuclei rather than the molecular layer as has been previously reported in autoradiographic studies. Other regions with prominent immune-staining included all subfields of the hippocampus, which had been previously reported to contain exclusively AT1 receptors. Limbic structures such as the amygdala, thalamic areas such as the rhomboid thalamic nucleus, the paraventricular thalamic nucleus, hypothalamic areas such as the paraventricular hypothalamic nucleus, and the supraoptic nucleus also exhibited prominent AT2-immunoreactivity. In the paraventricular hypothalamic nucleus, AT2 receptor staining appeared to be associated primarily with the magnocellular neurons. In all regions examined, AT2 receptor immunoreactivity was associated with the cytoplasm and cell membrane and was not localized within the nucleus. Collectively, these results confirm and extend the neuroanatomical resolution of previous autoradiographic studies as well as identify new AT2 receptor populations in rat brain.

Oxidative stress and HNE conjugation of GLUT3 are increased in the hippocampus of diabetic rats subjected to stress.

Recent studies demonstrate that cellular, molecular and morphological changes induced by stress in rats are accelerated when there is a pre-existing strain upon their already compromised adaptive responses to internal or external stimuli, such as may occur with uncontrolled diabetes mellitus. The deleterious actions of diabetes and stress may increase oxidative stress in the brain, leading to increases in neuronal vulnerability. In an attempt to determine if stress, diabetes or stress+diabetes increases oxidative stress in the hippocampus, radioimmunocytochemistry was performed using polyclonal antisera that recognize proteins conjugated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Radioimmunocytochemistry revealed that HNE protein conjugation is increased in all subregions of the hippocampus of streptozotocin (STZ) diabetic rats, rats subjected to restraint stress and STZ diabetic rats subjected to stress. Such increases were not significant in the cortex. Because increases in oxidative stress may contribute to stress- and diabetes-mediated decreases in hippocampal neuronal glucose utilization, we examined the stress/diabetes mediated HNE protein conjugation of the neuron specific glucose transporter, GLUT3. GLUT3 immunoprecipitated from hippocampal membranes of diabetic rats subjected to stress exhibited significant increases in HNE immunolabeling compared to control rats, suggesting that HNE protein conjugation of GLUT3 contributes to decreases in neuronal glucose utilization observed during diabetes and exposure to stress. Collectively, these results demonstrate that the hippocampus is vulnerable to increases in oxidative stress produced by diabetes and stress. In addition, increases in HNE protein conjugation of GLUT3 provide a potential mechanism for stress- and diabetes-mediated decreases in hippocampal neuronal glucose utilization.

Modulation of catecholamine metabolism in synaptosomes by a neuroregulatory factor from mammalian brain.

Incubation of synaptosomes from rat brain with bovine brain extract caused inhibition of oxidative deamination of dopamine, decreased formation of 3,4-dihydroxyphenylacetic acid, increased formation of norepinephrine and its N-methyl derivatives and increased release of catecholamines. Omission of Ca2+ from the extrasynaptosomal medium completely blocked the brain extract induced release of [3H]catecholamines and decreased, by about 80%, the effect on changes in catecholamine metabolism. Chromatography of the brain extract on Sephadex-G25 columns resulted in an active compound eluting at the position expected for compounds with molecular weights in the region of 1500 to 2500 Da.

Purification of neurocatin, a neuroregulatory factor from brain.

A neuroregulatory factor (neurocatin) has been isolated from bovine brain. Neurocatin is a powerful affector of catecholamine metabolism in synaptosomes isolated from rat brain, causing increased formation of norepinephrine (NE) and decreased formation of 3,4-dihydroxyphenylacetic acid (DOPAC). The ratio NE/DOPAC in synaptosomes has been used to measure the neurocatin content of samples during isolation. Neurocatin has now been purified to a single chromatographic peak by high-resolution HPLC and a preliminary amino acid content measured. It appears to be a small peptide with a molecular weight in the range of 2,000-2,500 Da.

Involvement of HMO-based pharmacists in clinical rounds at contract hospitals.

The contributions of managed care pharmacists who participate in clinical rounds at contract hospitals are described. A program in which pharmacists employed by Kaiser Permanente go on rounds at two contract hospitals as part of a multidisciplinary Kaiser team was implemented in January 1995. The rounding pharmacist reviews patients' charts, assesses optimal drug therapy, intervenes proactively, identifies candidates for home i.v. therapy, facilitates patient discharge, and provides on-the-spot drug information. Other services include therapeutic drug monitoring, dosage-adjustment recommendations, counseling, consultations, detecting adverse drug reactions, and education. The pharmacist documents all services and interventions, and their impact. At one hospital, a 500-bed institution, there were 1097 recommendations for intervention between April 1995 and May 1996. More than 99% of these recommendations were accepted. Over the 14-month period, the Kaiser team saved an estimated $523,907 and cost an estimated $57,643 (not counting benefits) in pharmacist participation. Managed care pharmacists on rounds at contract hospitals influenced enrolled patients' pharmacotherapy and reduced the plan's costs.

The angiotensin type 1 and type 2 receptor families. Siblings or cousins?

The diverse actions of angiotensin II (AngII) are mediated by cell surface receptors. Molecular cloning techniques have identified two distinct subtypes of AngII receptors referred to as AT1 and AT2. It is now well accepted that multiple forms of the AT1 receptor exist, but similar diversity of the AT2 subtype has not been conclusively demonstrated. Nonetheless, several converging lines of evidence do suggest that multiple AT2 receptors may be present in brain and cultured neuron-like cells lines. For instance, some AT2 receptors are regulated by guanine nucleotides and sulfhydryl-reducing agents, whereas others are insensitive. AT2 receptor populations also exhibit differing pharmacological profiles particularly with respect to their affinity for peptidic and non-peptidic ligands. Moreover, a recently developed anti-AT2 polyclonal antisera reveals a unique pattern of immunohistochemical staining in brain and it does not immunoreact with the recently cloned AT2 receptor. Collectively, these results support the hypothesis of multiple AT2 receptors at least within the CNS. Future studies should reveal whether these putative AT2 receptor subtypes result from unique genes or cell-specific post-translational modifications of a single gene product.

Heterogeneity of angiotensin type 2 (AT2) receptors.

Evidence continues to accumulate that strengthens the proposal of heterogeneity within both the AT1 and the AT2 receptor subtypes. Pharmacologic, biochemical and immunological studies of AT2 receptors expressed in N1E-115 cells strengthen the hypothesis of AT2 receptor heterogeneity. However, it is important to reassess these studies, especially in terms of how these results correlate with other reports of AT2 receptor heterogeneity. For example, AT2 receptor immunoreactivity was absent in some neuronal regions which have previously been proposed to express the AT2 receptor subtype. In particular, AT2 receptor staining was not seen in the inferior olive, a region which is known to express a high density of AT2 receptors. Upon first examination, these results were somewhat troubling. However, when compared with earlier reports, these results should not have been unexpected. For instance, Tsutsumi and Saaverdra previously have shown that AT2 receptors in the locus coeruleus are sensitive to the actions of guanine nucleotides, while AT2 receptors in the inferior olive are insensitive (21). These antisera were raised against a population of AT2 receptors which are sensitive to GTP gamma S and therefore, the lack of AT2 receptor staining in the inferior olive, as well as the presence of AT2 receptor immunoreactivity in the locus coeruleus, confirms and extends these earlier reports. In addition the AT2 receptors expressed in the locus coeruleus have been shown to be functionally distinct from AT2 receptors in the inferior olive. In this regard, Ang II has been shown to depress glutamate-induced EPSPs in the locus coeruleus, an effect which is mediated through the AT2 receptor (19). Conversely, AT2 receptors have been shown to increase the firing rate of neurons in the inferior olive (20). Collectively, these results would predict that staining should be absent in the inferior olive using these AT2-directed antisera. Indeed, in view of these earlier physiological and pharmacological studies, the presence of AT2 receptor immunoreactivity in the inferior olive would have been surprising. The most convincing example of AT2 receptor heterogeneity is the characterization of AT2 receptors present in N1E-115 cells. Separation of solubilized N1E-115 membranes by heparin-Sepharose chromatography generates two populations of AT2 receptors which are pharmacologically and biochemically distinct. In particular, CGP42112A was approximately 2 orders of magnitude more selective for Peak III AT2 receptors than was PD123319. Binding activity of Peak I and Peak III AT2 receptor populations also differed in their responses to GTP gamma S and DTT treatment. Lastly, the AT2-directed antisera, raised against the Peak I population of AT2 receptors, were not able to immunodetect the Peak III population of AT2 receptors in immunoblot analysis, or immunoprecipiatate AT2 binding activity from Peak III material. Pharmacological, biochemical and immunological analysis of the AT2 receptor clone isolated from N1E-115 cells revealed that it has the identical characteristics or properties of the Peak III receptor. The AT2 receptor isolated from N1E-115 cells exhibited a similar pharmacology as the Peak III AT2 receptor, in that CGP42112A was more effective at displacing 125I-Ang II binding activity than was PD123319. The AT2 receptor clone was also shown to be insensitive to the actions of GTP gamma S, as well as demonstrated increased binding activity in the presence of DTT, identical to the Peak III AT2 receptor. Lastly, immunoblot analysis of membranes prepared from COS-1 cells transfected with the AT2 receptor cDNA from N1E-115 cells did not demonstrate any immune-specific bands with the AT2-directed antisera. Characterization of an AT2 receptor cDNA isolated from N1E-115 cells reveals that this clone is identical to the Peak III type of AT2 receptor.

Endogenous angiotensin II-induced p44/42 mitogen-activated protein kinase activation mediates sodium appetite but not thirst or neurohypophysial secretion in male rats.

The renin-angiotensin-aldosterone system makes a critical contribution to body fluid homeostasis, and abnormalities in this endocrine system have been implicated in certain forms of hypertension. The peptide hormone angiotensin II (AngII) regulates hydromineral homeostasis and blood pressure by acting on both peripheral and brain targets. In the brain, AngII binds to the angiotensin type 1 receptor (AT1R) to stimulate thirst, sodium appetite and both arginine vasopressin (AVP) and oxytocin (OT) secretion. The present study used an experimental model of endogenous AngII to examine the role of p44/42 mitogen-activated protein kinase (MAPK) as a signalling mechanism to mediate these responses. Animals were given a combined treatment of furosemide and a low dose of captopril (furo/cap), comprising a diuretic and an angiotensin-converting enzyme inhibitor, respectively, to elevate endogenous AngII levels in the brain. Furo/cap induced p44/42 MAPK activation in key brain areas that express AT1R, and this effect was reduced with either a centrally administered AT1R antagonist (irbesartan) or a p44/42 MAPK inhibitor (U0126). Additionally, furo/cap treatment elicited water and sodium intake, and irbesartan markedly reduced both of these behaviours. Central injection of U0126 markedly attenuated furo/cap-induced sodium intake but not water intake. Furthermore, p44/42 MAPK signalling was not necessary for either furo/cap- or exogenous AngII-induced AVP or OT release. Taken together, these results indicate that p44/42 MAPK is required for AngII-induced sodium appetite but not thirst or neurohypophysial secretion. This result may allow for the discovery of more specific downstream targets of p44/42 MAPK to curb sodium appetite, known to exacerbate hypertension, at the same time as leaving thirst and neurohypophysial hormone secretion undisturbed.

Men and body image: are males satisfied with their body weight?

Dissatisfaction with body image is thought to be a key factor in the etiology of eating disorders among women. In contrast, men are reported to be generally satisfied with their body weight and body shape. The present survey study examined the relative desire for thinness or weight gain among 226 male and female freshman students. Most 18-year-old women (85%) wished to lose weight. Men expressed conflicting views regarding desire for thinness and were almost evenly split between those who wanted to lose weight (40%) and those who wished to gain weight (45%). The proportion of men and women who expressed no desire for weight change was comparable. Men and women who wished to lose weight shared negative body perceptions: both groups viewed themselves as overweight, and both expressed dissatisfaction with body shape. However, men used exercise for weight control while women resorted to restricted calorie diets. A key risk factor for eating disorders may be dieting itself.

Chimeric AT1/AT2 receptors reveal functional similarities despite key amino acid dissimilarities in the domains mediating agonist-dependent activation.

Chimeric AT1/AT2 angiotensin II (AngII) receptors in which the sixth and/or seventh transmembrane-spanning domains of the AT2 receptor were substituted into the AT1 receptor were used to investigate the activation mechanisms of the two receptor subtypes. Numerous reports have identified amino acid residues in the sixth and seventh transmembrane-spanning domains of the AT1 receptor involved in the intrareceptor activation mechanism following agonist binding. Many of these residues are not conserved in the AT2 receptor; the corresponding AT2 receptor residues are, in fact, disruptive of AngII-dependent activation when substituted into the AT1 receptor. Surprisingly, the chimeric AT1/AT2 receptors--which also lack these crucial AT1 residues--exhibited AngII-induced activation of phosphoinositide hydrolysis with efficacies and potencies similar to the wild-type AT1 receptor. Consistent with earlier reports, a AT1[Y292F] point mutant demonstrated greatly decreased agonist-induced activation of phosphoinositide hydrolysis. However, a AT1[Y292F/N295S] double-point mutant allowed for normal agonist-induced activation with a pharmacodynamic profile indistinguishable from the wild-type receptor. Despite amino acid dissimilarities, the same corresponding domains and even the same residue loci in both of the AngII receptor subtypes are equally able to mediate agonist-induced receptor activation. This suggests that these corresponding domains in the AT1 and the AT2 receptors are crucial to the activation mechanism, demonstrating greater structural flexibility than previously believed regarding AT1 receptor activation and supporting the possibility of a common activation mechanism for the two receptor subtypes.