The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.     

Relationship of diaphragmatic contractility to diaphragmatic blood flow in newborn lambs

We determined the relationship of diaphragmatic contraction rate to diaphragmatic blood flow (Qdi), metabolism, and contractility in nine open-chested mechanically ventilated newborn lambs. The diaphragm was paced for 15 min at slow (20/min) and fast (100/min) contraction rates each followed by a 30-min rest period. There was a mild reduction in transdiaphragmatic pressure (Pdi) during the slow contraction period accompanied by a shift to the right of the curve relating stimulation frequency (10-100 Hz) to Pdi. Pdi returned to control at the start of the fast contraction period, but then fell by 30% within 2 min with continued fast contraction rates. The frequency-Pdi curve was significantly shifted to the right. Qdi, O2 transport, and O2 consumption increased during slow contraction and to an even greater extent during fast contraction. Fractional O2 extraction reached an apparent maximum during slow contraction. Lactate efflux from the right phrenic vein during slow contraction remained unchanged from control. During fast contraction lactate efflux rose proportionately more than did O2 consumption. We conclude that the energy demands at fast rates of diaphragmatic contraction in newborn lambs cannot be met by aerobic metabolism alone despite increasing O2 transport to the diaphragm.     

The cardiovascular effects of amodiaquine and structurally related antimalarials: An individual patient data meta-analysis

BackgroundAmodiaquine is a 4-aminoquinoline antimalarial similar to chloroquine that is used extensively for the treatment and prevention of malaria. Data on the cardiovascular effects of amodiaquine are scarce, although transient effects on cardiac electrophysiology (electrocardiographic QT interval prolongation and sinus bradycardia) have been observed. We conducted an individual patient data meta-analysis to characterise the cardiovascular effects of amodiaquine and thereby support development of risk minimisation measures to improve the safety of this important antimalarial.Methods and findingsStudies of amodiaquine for the treatment or prevention of malaria were identified from a systematic review. Heart rates and QT intervals with study-specific heart rate correction (QTcS) were compared within studies and individual patient data pooled for multivariable linear mixed effects regression.The meta-analysis included 2,681 patients from 4 randomised controlled trials evaluating artemisinin-based combination therapies (ACTs) containing amodiaquine (n = 725), lumefantrine (n = 499), piperaquine (n = 716), and pyronaridine (n = 566), as well as monotherapy with chloroquine (n = 175) for uncomplicated malaria. Amodiaquine prolonged QTcS (mean = 16.9 ms, 95% CI: 15.0 to 18.8) less than chloroquine (21.9 ms, 18.3 to 25.6, p = 0.0069) and piperaquine (19.2 ms, 15.8 to 20.5, p = 0.0495), but more than lumefantrine (5.6 ms, 2.9 to 8.2, p < 0.001) and pyronaridine (−1.2 ms, −3.6 to +1.3, p < 0.001). In individuals aged ≥12 years, amodiaquine reduced heart rate (mean reduction = 15.2 beats per minute [bpm], 95% CI: 13.4 to 17.0) more than piperaquine (10.5 bpm, 7.7 to 13.3, p = 0.0013), lumefantrine (9.3 bpm, 6.4 to 12.2, p < 0.001), pyronaridine (6.6 bpm, 4.0 to 9.3, p < 0.001), and chloroquine (5.9 bpm, 3.2 to 8.5, p < 0.001) and was associated with a higher risk of potentially symptomatic sinus bradycardia (≤50 bpm) than lumefantrine (risk difference: 14.8%, 95% CI: 5.4 to 24.3, p = 0.0021) and chloroquine (risk difference: 8.0%, 95% CI: 4.0 to 12.0, p < 0.001). The effect of amodiaquine on the heart rate of children aged <12 years compared with other antimalarials was not clinically significant. Study limitations include the unavailability of individual patient-level adverse event data for most included participants, but no serious complications were documented.ConclusionsWhile caution is advised in the use of amodiaquine in patients aged ≥12 years with concomitant use of heart rate–reducing medications, serious cardiac conduction disorders, or risk factors for torsade de pointes, there have been no serious cardiovascular events reported after amodiaquine in widespread use over 7 decades. Amodiaquine and structurally related antimalarials in the World Health Organization (WHO)-recommended dose regimens alone or in ACTs are safe for the treatment and prevention of malaria.

In this meta-analysis, Xin Hui Supanee Chan and colleagues investigate the cardiovascular effects of amodiaquine and structurally-related antimalarials using individual patient data from trials.     

Glutamine-dependent inhibition of pial arteriolar dilation to acetylcholine with and without hyperammonemia in the rat

Glutamine has been shown to influence endothelial-dependent relaxation and nitric oxide production in vitro, possibly by limiting arginine availability, but its effects in vivo have not been well studied. Hyperammonemia is a pathophysiological condition in which glutamine is elevated and contributes to depressed CO(2) reactivity of cerebral arterioles. We tested the hypothesis that acute hyperammonemia decreases pial arteriolar dilation to acetylcholine in vivo and that this decrease could be prevented by inhibiting glutamine synthetase with L-methionine-S-sulfoximine (MSO) or by intravenous infusion of L-arginine. Pial arteriolar diameter responses to topical superfusion of acetylcholine were measured in anesthetized rats before and at 6 h of infusion of either sodium or ammonium acetate. Ammonium acetate infusion increased plasma ammonia concentration from approximately 30 to approximately 600 microM and increased cerebral glutamine concentration fourfold. Arteriolar dilation to acetylcholine was intact after infusion of sodium acetate in groups pretreated with vehicle or with MSO plus methionine, which was coadministered to prevent MSO-induced seizures. In contrast, dilation in response to acetylcholine was completely blocked in hyperammonemic groups pretreated with vehicle or methionine alone. However, MSO plus methionine administration before hyperammonemia, which maintained cerebral glutamine concentration at control values, preserved acetylcholine dilation. Intravenous infusion of L-arginine during the last 2 h of the ammonium acetate infusion partially restored dilation to acetylcholine without reducing cerebral glutamine accumulation. Superfusion of 1 or 2 mM L-glutamine through the cranial window for 1 h in the absence of hyperammonemia attenuated acetylcholine dilation but had no effect on endothelial-independent dilation to nitroprusside. We conclude that 1) hyperammonemia reduces acetylcholine-evoked dilation in cerebral arterioles, 2) this reduction depends on increased glutamine rather than ammonium ions, and 3) increasing arginine partially overcomes the inhibitory effect of glutamine.     

Regional cerebral blood flow in cats with cross-linked hemoglobin transfusion during focal cerebral ischemia

The beneficial effect of hemodilution on cerebral blood flow (CBF) during focal cerebral ischemia is mitigated by reduced arterial oxygen content (CaO2). In anesthetized cats subjected to permanent middle cerebral artery occlusion, the time course of regional CBF was evaluated after isovolemic exchange transfusion with either albumin or a tetrameric hemoglobin-based oxygen carrier. The transfusion started 30 min after arterial occlusion. We tested the hypothesis that bulk oxygen transport (CBF x CaO2) to ischemic tissue is increased by hemoglobin transfusion at a hematocrit of 18% compared with albumin-transfused cats at a hematocrit of 18% or control cats at a hematocrit of 30% and equivalent arterial pressure. In the nonischemic hemisphere, CBF increased selectively after albumin transfusion, and oxygen transport was similar among groups. In the ischemic cortex, albumin transfusion increased CBF, but oxygen transport was not increased above that of the control group. Hemoglobin transfusion increased both CBF and oxygen transport in the ischemic cortex above values in the control group, but the increase was delayed until 4 h of ischemia. Consequently, acute injury volume measured at 6 h of ischemia was not significantly attenuated. In contrast to the cortex, CBF in the ischemic caudate nucleus was not substantially increased by either albumin or hemoglobin transfusion. Therefore, in a large animal model of permanent focal ischemia in which transfusion starts 30 min after ischemia, tetrameric cross-linked hemoglobin transfusion can augment oxygen transport to the ischemic cortex, but the increase can be delayed and not necessarily provide protection. Moreover, an end-artery region such as the caudate nucleus is less likely to benefit from hemodilution.     

Comparison of cerebrovascular effects of intravenous cocaine injection in fetal, newborn, and adult sheep

Cocaine may cause stroke, intracranial hemorrhage, seizures, and neurobehavioral abnormalities in fetuses, newborns, and adults, and there could be developmental and/or species differences in mechanisms for these cocaine-induced cerebrovascular effects. To evaluate developmental differences in responses to cocaine, we compared the cerebrovascular and metabolic responses to a 2 mg/kg iv cocaine dose in unanesthetized fetal (n = 8, previously reported, direct fetal injection), newborn (n = 6), and adult (n = 12) sheep. We measured cerebral blood flow, mean arterial blood pressure, and arterial and venous O(2) content, and we calculated cerebral O(2) consumption and cerebral vascular resistance at baseline and at 30 s and at 5, 15, and 60 min after cocaine injection. Cerebral blood flow increased 5 min after injection in the fetus and newborn, but not until 15 min in the adult. In the fetus, cocaine caused a transient cerebral vasoconstriction at 30 s; in all three groups, cocaine caused cerebral vasodilation, which was delayed in the adult. Cerebral metabolic O(2) consumption increased 5 min after injection in the fetus and newborn, but not until 15 min after injection in the adult. Arterial O(2) content decreased 5 min after injection in the fetus and 15 min after injection in the adult. We speculate that clinical differences in response to cocaine injection may be explained, in part, by these developmental differences in the cerebrovascular and metabolic responses to cocaine.     

Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits

We tested the hypothesis that intracarotid estrogen infusion increases cerebral blood flow (CBF) in a concentration-dependent manner and direct application of estrogen on pial arterioles yields estrogen receptor-mediated vasodilation. Rabbits of both genders were infused with estrogen via a branch of the carotid artery. Estrogen doses of 20 or 0.05 microg. ml(-1). min(-1) were used to achieve supraphysiological or physiological plasma estrogen levels, respectively. CBF and cerebral vascular resistance were determined at baseline, during the infusion, and 60-min postinfusion, and effects on pial diameter were assessed via a cranial window. Pial arteriolar response to estrogen alone and to estrogen after administration of tamoxifen (10(-7)), an antiestrogen drug that binds to both known estrogen receptor subtypes, was tested. No gender differences were observed; therefore, data were combined for both males and females. Systemic estrogen infusion did not increase regional CBF. Estradiol dilated pial arteries only at concentrations ranging from 10(-4)-10(-7) M (P < or = 0.05). Pretreatment with tamoxifen alone had no effect on arteriolar diameter but inhibited estrogen-induced vasodilation (P < 0.001). Our data suggest that estrogen does not increase CBF under steady-state conditions in rabbits. In the pial circulation, topically applied estradiol at micromolar concentrations dilates vessels. The onset is rapid and dependent on estrogen receptor activation.     

P-450 epoxygenase and NO synthase inhibitors reduce cerebral blood flow response to N-methyl-D-aspartate

Epoxyeicosatrienoic acids are cerebral vasodilators produced in astrocytes by cytochrome P-450 epoxygenase activity. The P-450 inhibitor miconazole attenuates the increase in cerebral blood flow (CBF) elicited by glutamate. We evaluated whether epoxygenase activity is involved in the CBF response to activation of the N-methyl-D-aspartate (NMDA) receptor subtype by using two structurally distinct inhibitors, miconazole and N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH), a selective epoxygenase substrate inhibitor. Drugs were delivered locally through microdialysis probes in striata of anesthetized rats. Local CBF was measured by hydrogen clearance and compared with CBF in contralateral striatum receiving vehicle. Microdialysis perfusion of NMDA doubled CBF and increased nitric oxide (NO) production estimated by recovery of labeled citrulline in the dialysate during labeled arginine infusion. Perfusion of miconazole or MS-PPOH blocked the increase in CBF without decreasing citrulline recovery. Perfusion of N(omega)-nitro-L-arginine decreased baseline CBF and inhibited the CBF response to NMDA. Perfusion of MS-PPOH did not inhibit the CBF response to sodium nitroprusside. We conclude that both the P-450 epoxygenase and NO synthase pathways are involved in the local CBF response to NMDA receptor activation, and that the signaling pathway may be more complex than simply NO diffusion from neurons to vascular smooth muscle.     

Clinical features of symptomatic patellofemoral joint osteoarthritis

Introduction

Patellofemoral joint osteoarthritis (OA) is common and leads to pain and disability. However, current classification criteria do not distinguish between patellofemoral and tibiofemoral joint OA. The objective of this study was to provide empirical evidence of the clinical features of patellofemoral joint OA (PFJOA) and to explore the potential for making a confident clinical diagnosis in the community setting.

Methods

This was a population-based cross-sectional study of 745 adults aged ≥50 years with knee pain. Information on risk factors and clinical signs and symptoms was gathered by a self-complete questionnaire, and standardised clinical interview and examination. Three radiographic views of the knee were obtained (weight-bearing semi-flexed posteroanterior, supine skyline and lateral) and individuals were classified into four subsets (no radiographic OA, isolated PFJOA, isolated tibiofemoral joint OA, combined patellofemoral/tibiofemoral joint OA) according to two different cut-offs: ''any OA'' and ''moderate to severe OA''. A series of binary logistic and multinomial regression functions were performed to compare the clinical features of each subset and their ability in combination to discriminate PFJOA from other subsets.

Results

Distinctive clinical features of moderate to severe isolated PFJOA included a history of dramatic swelling, valgus deformity, markedly reduced quadriceps strength, and pain on patellofemoral joint compression. Mild isolated PFJOA was barely distinguished from no radiographic OA (AUC 0.71, 95% CI 0.66, 0.76) with only difficulty descending stairs and coarse crepitus marginally informative over age, sex and body mass index. Other cardinal signs of knee OA - the presence of effusion, bony enlargement, reduced flexion range of movement, mediolateral instability and varus deformity - were indicators of tibiofemoral joint OA.

Conclusions

Early isolated PFJOA is clinically manifest in symptoms and self-reported functional limitation but has fewer clear clinical signs. More advanced disease is indicated by a small number of simple-to-assess signs and the relative absence of classic signs of knee OA, which are predominantly manifestations of tibiofemoral joint OA. Confident diagnosis of even more advanced PFJOA may be limited in the community setting.     

Distribution of MT1 Melatonin Receptor Promoter-Driven RFP Expression in the Brains of BAC C3H/HeN Transgenic Mice

The pineal hormone melatonin activates two G-protein coupled receptors (MT₁ and MT₂) to regulate in part biological functions. The MT₁ and MT₂ melatonin receptors are heterogeneously distributed in the mammalian brain including humans. In the mouse, only a few reports have assessed the expression of the MT₁ melatonin receptor expression using 2-iodomelatonin binding, in situ hybridization and/or polymerase chain reaction (PCR). Here, we described a transgenic mouse in which red fluorescence protein (RFP) is expressed under the control of the endogenous MT₁ promoter, by inserting RFP cDNA at the start codon of MTNR1a gene within a bacterial artificial chromosome (BAC) and expressing this construct as a transgene. The expression of RFP in the brain of this mouse was examined either directly under a fluorescent microscope or immunohistochemically using an antibody against RFP (RFP-MT₁). RFP-MT₁ expression was observed in many brain regions including the subcommissural organ, parts of the ependyma lining the lateral and third ventricles, the aqueduct, the hippocampus, the cerebellum, the pars tuberalis, the habenula and the habenula commissure. This RFP-MT₁ transgenic model provides a unique tool for studying the distribution of the MT₁ receptor in the brain of mice, its cell-specific expression and its function in vivo.