Woodland caribou calving fidelity: Spatial location, habitat, or both?

Individuals that isolate themselves to give birth can use more than one strategy in choosing birth sites to maximize reproductive success. Previous research has focused on the consistency in the use of the same birth-site across years (i.e., spatial fidelity), but individuals alternatively may use similar habitat conditions across years (i.e., habitat fidelity). Using GPS telemetry, we determined whether woodland caribou expressed spatial or habitat fidelity during calving, and evaluated intrinsic and extrinsic factors associated with expressing either type of fidelity. We identified 56 individuals with ≥2 putative birth events, via a movement-based model, across northern Ontario between 2010 and 2014. Individuals were classified as expressing (1) spatial fidelity by comparing sequential calving locations to a random spatial distribution of available calving locations, (2) habitat fidelity using a logistic use model compared to a null (intercept only) model, (3) no fidelity (neither criterion met), or (4) both spatial and habitat fidelity (both criteria met). Across all individuals, 37% expressed no fidelity (36 of 98), 15% expressed only spatial fidelity (15 of 99), 35% expressed only habitat fidelity (34 of 98), and 14% expressed both spatial and habitat fidelity (14 of 98). Older individuals were more likely to express spatial fidelity, whereas lower availability of upland and lowland conifer forests without linear features increased the probability an individual expressed habitat fidelity. Our results indicate that managing for caribou calving needs to consider protecting both specific, known birthing sites, but also broad-scale areas of preferred habitat for calving. Understanding the mechanisms that influence caribou expressing calving fidelity, and associated fitness costs, is crucial for the conservation of the species.

Strategies for improving the emission performance of hybrid electric vehicles.

Low emission vehicle technologies need widespread adoption in the transport sector to overcome its significant decarbonisation challenges. Hybrid Electric Vehicles (HEVs) represent an intermediate technology between pure electric vehicles and internal combustion engines that have proven capability in reducing petroleum consumption. HEV customers often cite improved fuel economy as a major benefit from adopting this technology; however, outstanding questions remain regarding their respective emission levels. Through an extensive literature study, we show that several issues remain with HEV emissions performance which stem from frequent high-power cold starts, engine calibration issues and inefficient operating conditions for catalytic converters. HEVs have more NOx, HC, CO and particle number emissions compared to conventional vehicles by up to 21.0, 5.8, 9.0 and 23.3 times, respectively. Improved engine control algorithms, after-treatment design and thermal design of three-way catalysts emerge as research priorities for improving the emissions performance of HEVs.

Polyketide ß-branching: diversity, mechanism and selectivity.

Covering: 2008 to August 2020 Polyketides are a family of natural products constructed from simple building blocks to generate a diverse range of often complex chemical structures with biological activities of both pharmaceutical and agrochemical importance. Their biosynthesis is controlled by polyketide synthases (PKSs) which catalyse the condensation of thioesters to assemble a functionalised linear carbon chain. Alkyl-branches may be installed at the nucleophilic α- or electrophilic ß-carbon of the growing chain. Polyketide ß-branching is a fascinating biosynthetic modification that allows for the conversion of a ß-ketone into a ß-alkyl group or functionalised side-chain. The overall transformation is catalysed by a multi-protein 3-hydroxy-3-methylglutaryl synthase (HMGS) cassette and is reminiscent of the mevalonate pathway in terpene biosynthesis. The first step most commonly involves the aldol addition of acetate to the electrophilic carbon of the ß-ketothioester catalysed by a 3-hydroxy-3-methylglutaryl synthase (HMGS). Subsequent dehydration and decarboxylation selectively generates either α,ß- or ß,γ-unsaturated ß-alkyl branches which may be further modified. This review covers 2008 to August 2020 and summarises the diversity of ß-branch incorporation and the mechanistic details of each catalytic step. This is extended to discussion of polyketides containing multiple ß-branches and the selectivity exerted by the PKS to ensure ß-branching fidelity. Finally, the application of HMGS in data mining, additional ß-branching mechanisms and current knowledge of the role of ß-branches in this important class of biologically active natural products is discussed.

Clinicopathologic Features of Membranous-Like Glomerulopathy With Masked IgG Kappa Deposits.

INTRODUCTION: Ig deposits identified on renal biopsy samples by paraffin immunofluorescence that show negative staining by routine immunofluorescence on frozen tissue have become known as "masked" deposits. Membranous-like glomerulopathy with masked IgG kappa (κ) deposits is a recently recognized pattern of immune complex deposition characterized by masked deposits that show IgG κ restriction and are subepithelial and mesangial by electron microscopy. Based on the frequent presence of C3-only staining by routine immunofluorescence microscopy (IF), these cases could be misdiagnosed as C3 glomerulonephritis in the absence of paraffin immunofluorescence evaluation. METHODS: The clinicopathologic details of all cases of membranous-like glomerulopathy with masked IgG κ deposits diagnosed in our laboratory were included, beginning with the initial recognition of this entity in 2011 through the end of 2015. Inclusion was based on renal biopsy sample morphologic features including glomerular deposits that stain for IgG κ and have a staining intensity that is significantly brighter by paraffin IF than by routine IF on frozen tissue. RESULTS: This pattern of immune complex deposition has been seen in 41 patients in our laboratory over a 5-year period. The patients with these biopsy findings are most commonly young female individuals with a mean age of 27.5 years, with 88% being less than 40 years. All patients had proteinuria with a mean 24-hour urine protein of 3.5 g (range 0.5-12.8 years) and 35% with nephrotic-range proteinuria. Hematuria was present in 88% of patients, and 29% had elevated serum creatinine at presentation. Autoimmune serologic tests were positive in 55% of patients, with a weakly positive antinuclear antibody being most common. Despite this, only 1 patient (2%) fulfilled the diagnostic criteria for systemic lupus erythematosus. The outcome data were mixed, as some patients showed spontaneous remission and mild disease whereas others progressed to end-stage renal disease. There was no apparent correlation between the treatment used and outcome in this retrospective analysis. One patient underwent transplantation and developed biopsy-proven recurrence of disease in the graft 42 months posttransplantation. The etiology of this entity remains unknown. DISCUSSION: We provide an expanded case series detailing the clinicopathologic findings of patients with membranous-like glomerulopathy with masked IgG κ deposits. Patients are most commonly young female individuals <40 years of age and commonly have positive autoimmune serologic studies such as antinuclear antibody, although few carry a diagnosis of any well-defined autoimmune disease such as lupus. The outcome data were mixed, as some patients showed spontaneous remission and mild disease whereas others progressed to ESRD. There was no apparent correlation between the treatment used and outcome in this retrospective analysis.

Membranous nephropathy as a rare renal manifestation of IgG4-related disease.

IgG4-related disease, a newly described immune-mediated disorder with tissue infiltration of IgG4-positive plasma cells, has been reported in nearly every organ. In the kidney, it manifests as IgG4-related tubulointerstitial nephritis (TIN) but may also present as membranous nephropathy. We report a patient with IgG4 renal disease who had membranous nephropathy as well as TIN.

PLA2R-associated membranous glomerulopathy is modulated by common variants in PLA2R1 and HLA-DQA1 genes.

Membranous glomerulopathy (MG) is most commonly caused by autoantibodies directed against the podocyte phospholipase A2 receptor (PLA2R1) and common variants in this gene are associated with MG. Here for the first time, we carried out a large case-control association study (n=1512) of PLA2R-positive and -negative MG to determine the extent of association in these pathologic subtypes. We performed four separate sets of analyses to determine significance of the single-nucleotide polymorphisms (SNPs) and their haplotypes followed by joint analysis and trans-ethnic mapping to increase power. The PLA2R1 SNP rs35771982 was most strongly associated with PLA2R-positive MG (P=1.4 × 10(-14), odds ratio (ORGG)=1.98). The associations of other SNPs in PLA2R1 could be explained because of linkage disequilibrium with the G-allele. Haplotypes in PLA2R1 did not exceed the significance of rs35771982 even after 10 000 permutations. PLA2R1 variants were only associated with PLA2R-positive MG and predominantly in Caucasians. PLA2R1 variants did not associate with MG in African Americans (AA). There was strong epistasis between HLA-DQA1 SNP rs2187668 and the PLA2R1 variant rs35771982. Thus, common variants in the PLA2R1, particularly rs35771982, modulate PLA2R-positive MG with HLA-DQA1 in Caucasians. PLA2R-negative MG especially in AA, may provide a novel opportunity to discover new genes underlying MG.

The perichondrial cutaneous graft and "flip-flop" flap in facial reconstruction: a series of 41 cases.

Reconstruction of defects of the nose and eyelids can be challenging. The standard surgical options include full thickness skin grafts and local flaps. Another technique that offers a reliable and dimensionally stable one-stage reconstruction is the perichondrial cutaneous graft. The donor site is closed with the postauricular flip-flop flap. We have used these techniques in a series of 41 consecutive patients. Good or excellent aesthetic reconstructions were obtained in 39 cases, with 3 complications: 1 failed graft, and 2 small postauricular wound dehiscences. The perichondrial cutaneous graft can produce excellent cosmetic results when it is used to reconstruct complex defects of the nose and eyelid. Closure of the donor site in the conchal bowl with the flip-flop flap is a reliable and elegant technique.

Is IgA nephropathy the commonest primary glomerulopathy among young adults in the USA?

Over the past decade, focal segmental glomerulosclerosis (FSGS) has emerged as the most common primary glomerulopathy in adults in the USA. However in our practice, we became aware of increased numbers of patients with IgA nephropathy (IgAN). In order to further examine this, a retrospective analysis of renal biopsy diagnoses from adults was done from our biopsy database. Adult renal biopsies received from 3/1/2001 to 2/28/2005 were analyzed to determine the frequency of common primary glomerulopathies, which included FSGS, IgAN, membranous nephropathy (MN), minimal change disease, and membranoproliferative glomerulonephritis Type I (MPGN). The patients were grouped as all adults (>or=20 years) and young adults (20-39 years). The distribution of common primary glomerulopathies among the two age groups, expressed as a percentage of all non-transplant diagnoses (n = 4,504), was IgAN 6.9/3.4%, FSGS 9.6/3.2%, MN 6.8/1.6%, minimal change disease 2.5/0.9%, MPGN 1.2/0.2%. IgAN was as common as FSGS in young adults in our biopsy population (IgAN/FSGS 154/143 1.08:1). IgAN was the most common primary glomerulopathy in young adult Caucasians (IgAN/FSGS 2.1:1). IgAN was also the most common cause of end-stage renal disease (ESRD) in young adult Caucasians. In contrast, IgAN was rare in African Americans in whom FSGS remains more common (FSGS/IgAN 9.7:1). These findings from a large renal biopsy referral center serving 24 Midwestern and Southern states suggest that IgAN may be the most common primary glomerulopathy and the most common cause of ESRD in young adult Caucasians in the USA.

Failure of MK-801 to suppress D1 receptor-mediated induction of locomotor activity and striatal preprotachykinin mRNA expression in the dopamine-depleted rat.

N-methyl-D-aspartate receptor antagonism exerts suppressive influences over dopamine D1 receptor-mediated striatal gene expression and locomotor behavior in the intact rat. The present study examined the effects of the N-methyl-D-aspartate receptor antagonist MK-801 on locomotor activity and striatal preprotachykinin mRNA expression stimulated by the D1 agonist (+/-)6-chloro-7, 8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide in rats with bilateral dopamine lesions. Two months after neonatal dopamine lesions with 6-hydroxydopamine, rats were challenged with (+/-)6-chloro-7, 8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (1.0 mg/kg) 15 min after administration of the N-methyl-D-aspartate receptor antagonist MK-801 (0.1 mg/kg). In the intact rat, MK-801 prevented the induction of striatal preprotachykinin mRNA by D1 agonism. Similarly, direct infusion of (+/-)6-chloro-7, 8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (3.0 microg) into the intact striatum produced an increase in locomotor activity that was suppressed by MK-801 (1.0 microg) co-infusion. In the dopamine-depleted rat, MK-801 (0.1 mg/kg) administered prior to (+/-)6-chloro-7, 8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (1.0 mg/kg) increased, rather than suppressed, striatal preprotachykinin mRNA levels. Intrastriatal infusion of MK-801 (1.0 microg) failed to inhibit D1-mediated induction of motor activity in dopamine-depleted animals. Together, these data provide further support that N-methyl-D-aspartate receptor antagonists lose their ability to block D1-mediated behavioral activation following dopamine depletion. The activation, rather than suppression, of tachykinin neurons of the direct striatonigral pathway may play a facilitatory role in this mechanism.

Combined intrastriatal dopamine D1 and serotonin 5-HT2 receptor stimulation reveals a mechanism for hyperlocomotion in 6-hydroxydopamine-lesioned rats.

Loss of dopaminergic innervation to the striatum increases the sensitivity of dopamine (DA) D1 and serotonin (5-HT) 5-HT2 receptor signaling. Previous work from our laboratory has shown that systemic co-administration of D1 and 5-HT2 receptor agonists leads to the synergistic overexpression of striatal preprotachykinin mRNA levels in the DA-depleted, but not intact animals. In the present study, we examined this mechanism as related to locomotor behavior. Adult male Sprague-Dawley rats were subject to bilateral i.c.v. 6-hydroxydopamine (6-OHDA; 200 microg in 10 microl/side) or vehicle (0.9% saline and 0.1% ascorbic acid). After 3 weeks, rats were tested for locomotor responses to bilateral intrastriatal infusions of vehicle (0.9% NaCl), the D1 agonist SKF82958 [(+/-)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-(1H)-3-benzazepine hydrobromide; 0.1, 1.0 or 10.0 microg/side], the 5-HT2 agonist DOI [(+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane; 0.1, 1.0 or 10.0 microg/side] or subthreshold doses of DOI and SKF82958 (0.1 microg+0.1 microg in 0.8 microl/side). Rats with DA loss demonstrated supersensitive locomotor responses to SKF82958, but not DOI. Combined administration of subthreshold SKF82958 and DOI doses (0.1 microg+0.1 microg) synergistically increased locomotor behavior only in 6-OHDA-lesioned rats. These effects were blocked by either the D1 antagonist SCH23390 3-methyl-1-phenyl-2,3,4,5-tetrahydro-7-chloro-8-hydroxy-(1H)-3-benzazepine or the 5-HT2 antagonist ritanserin (each 1.0 microg in 0.8 microl/side). The results of this study suggest that the behavioral synergy induced by local co-stimulation of D1 and 5-HT2 receptors within the 6-OHDA-lesioned striatum may lead to hyperkinesias that can occur with continued pharmacological treatment of Parkinson's disease.

Polyoma viral infection in renal transplantation: the role of immunosuppressive therapy.

BACKGROUND: Polyoma virus infection in renal transplant recipients has been observed with increasing frequency in recent years. Renal allograft involvement in this condition may occur as a result of primary infection or secondary to reactivation of the latent virus. Interstitial nephritis, ureteric stenosis, rise in serum creatinine and allograft function loss have been attributed to this viral infection. METHODS: In this study we reviewed our experience with 8 patients who developed polyoma viral infection confirmed by allograft biopsy. All patients were receiving mycophenolate mofetil as part of the immunosuppression and 7 of the 8 patients were on tacrolimus. All patients have biopsy proven polyoma viral infection. The following therapeutic maneuvers were carried out following the diagnosis of polyoma viral infection: 1) stopping mycophenolate and 2) switching tacrolimus to cyclosporine or reducing the tacrolimus dose to adjust it at a lower therapeutic trough level. The clinical course and outcome of our patients were reviewed in relation to manipulation of immunosuppressive medications. RESULTS: The incidence of this infection in our transplant program in the last 3 yr was 5.3%. Seventy-five percent of the patients had at least one rejection episode and 63% had more than one rejection episode. The main risk factor for the development of polyoma viral infection was related to the intensity of immunosuppression. The use of antirejection therapy after histological diagnosis of polyoma virus infection was not associated with improvement of renal function despite the histological appearance of acute rejection. Thus, the interstitial nephritis associated with polyoma viral infection appears to be an inflammatory response to the virus rather than acute rejection. Six out of the 8 patients stabilized renal function with reduction in immunosuppression. CONCLUSIONS: Reduction in immunosuppression was associated with the stabilization of renal function when instituted early. However, these patients were left with a degree of allograft dysfunction and their outcome may be significantly compromised. The lack of effective antiviral therapy for polyoma virus may limit the use of newer and more potent immunosuppressive medications.

NMDA receptor antagonism modifies the synergistic regulation of striatal tachykinin gene expression induced by dopamine D(1) and serotonin(2) receptor stimulation following neonatal dopamine depletion.

Co-application of SKF-38393 (dopamine D(1) agonist; 1 mg/kg) and DOI (serotonin(2) agonist; 1 mg/kg) induced a synergistic increase in striatal preprotachykinin (PPT) mRNA levels in adult rats 60 days after neonatal intracerebroventricular injection of 6-hydroxydopamine. This magnitude of response was not observed in intact (vehicle-injected) rats and was restricted to the dorsomedial (DM, 333+/-25% of lesion) subregion of the anterior striatum, with smaller increases observed in the dorsolateral striatum (DL, 206+/-26% of lesion). A single i.p. injection of MK-801 (NMDA antagonist; 0.1 mg/kg) administered prior to dopamine D(1) (D(1)) and serotonin(2) (5-HT(2)) receptor co-stimulation suppressed the synergistic regulation of PPT mRNA expression in the DM striatum, but also produced a large increase in PPT message levels within the DL striatum (321+/-17% of lesion). These data suggest that the synergistic regulation of PPT mRNA within the DM striatum induced by D(1)/5-HT(2) receptor co-stimulation in the dopamine lesioned rat is dependent on NMDA receptor activity. However, MK-801 may simultaneously potentiate striatal PPT mRNA expression by a separate mechanism due to the changed environment of the dopamine-depleted basal ganglia.

Neonatal dopamine depletion reveals a synergistic mechanism of mRNA regulation that is mediated by dopamine(D1) and serotonin(2) receptors and is targeted to tachykinin neurons of the dorsomedial striatum.

It has been hypothesized that dopamine(D1) and serotonin(2) receptors become sensitized to agonist-mediated regulation of gene expression following loss of dopaminergic innervation to the striatum. We have previously demonstrated that the combined administration of dopamine(D1) and serotonin(2) receptor agonists to dopamine-depleted adult rats induced preprotachykinin mRNA expression within the periventricular rostral striatum to levels which were significantly different than what could be elicited by either agonist alone. In the present study, we have determined that this phenomenon is revealed only after dopamine depletion. In addition, it is targeted primarily to tachykinin producing neurons of the dorsomedial striatum and is dependent on both dopamine(D1) and serotonin(2) receptor activation. Preprotachykinin mRNA levels in the intact striatum were unaltered 4 h following an i.p. injection of either SKF-38393 (1 mg/kg, dopamine(D1) partial agonist) or (+/-)-1-(4-Iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI 1 mg/kg, serotonin(2) agonist). However, the combined application of both agonists increased (+44%) preprotachykinin message levels, but these changes were restricted to the dorsomedial striatum. In adult animals depleted of dopamine as neonates, striatal preprotachykinin mRNA expression was reduced by approximately 50%. From this lowered level of basal expression, DOI or SKF-38393 raised preprotachykinin mRNA levels within the dorsomedial, but not the dorsolateral striatum. Furthermore, co-stimulation of dopamine(D1) and serotonin(2) receptors produced a nearly four-fold induction of preprotachykinin message levels in the dorsomedial striatum that was significantly greater than either agonist alone. Application of both agonists also elevated preprotachykinin mRNA expression within the dorsolateral striatum, but to a lesser extent. All increases in preprotachykinin mRNA resulting from co-application of SKF-38393 and DOI were prevented by pretreatment with either SCH-23390 (1 mg/kg, dopamine(D1) antagonist) or ritanserin (1 mg/kg, serotonin(2) antagonist). Alternately, preproenkephalin mRNA expression was unaffected by dopamine(D1) receptor stimulation, but was slightly elevated by DOI or both agonists together (42-58%) in intact animals. However, neither agonist treatment in this experiment significantly altered preproenkephalin mRNA expression in the dopamine-depleted striatum which was elevated in response to dopamine lesion alone. Dopamine depletion appears to promote a synergistic interaction between dopamine(D1) and serotonin(2) receptors that leads to enhanced expression of striatal preprotachykinin mRNA levels. The localization of this phenomenon to tachykinin neurons of the direct striatonigral pathway specifically within the dorsomedial regions of the rostral striatum may be relevant to the problem of dyskinetic behaviors which arise during the pharmacological treatment of movement disorders.

Serotonin 2A receptor regulation of striatal neuropeptide gene expression is selective for tachykinin, but not enkephalin neurons following dopamine depletion.

Serotonin (5-HT) 2A receptor-mediated regulation of striatal preprotachykinin (PPT) and preproenkephalin (PPE) mRNAs was studied in adult rodents that had been subjected to near-total dopamine (DA) depletion as neonates. Two months following bilateral 6-hydroxydopamine (6-OHDA) lesion, PPT mRNA levels decreased 59-73% across dorsal subregions of the rostral and caudal striatum while PPE transcripts increased 61-94%. Four hours after a single injection of the serotonin 2A/2C receptor agonist, (+/-)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 1 mg/kg), PPT mRNA expression was significantly increased in DA-depleted rats across all dorsal subregions of the rostral and caudal striatum as compared to 6-OHDA-treated animals alone. In the intact rat, DOI did not influence PPT mRNA levels in the rostral striatum, but did raise expression in the caudal striatum where 5-HT2A receptors are prominent. DOI did not regulate PPE mRNA levels in any striatal sub-region of the intact or DA-depleted rat. Prior administration of the 5-HT2A/2C receptor antagonist, ritanserin (1 mg/kg) or the 5-HT2A receptor antagonist, ketanserin (1 mg/kg) completely blocked the DOI-induced increases in striatal PPT mRNA in both lesioned and intact animals. The ability of ketanserin to produce identical results as ritanserin suggests that 5-HT2A receptor-mediated regulation is selectively strengthened within tachykinin neurons of the rostral striatum which are suppressed by DA depletion. The selectivity suggests that 5-HT2A receptor upregulation following DA depletion is capable of regulating tachykinin biosynthesis without influencing enkephalin expression in striatal output neurons.

Distribution of serotonin 2A and 2C receptor mRNA expression in the cervical ventral horn and phrenic motoneurons following spinal cord hemisection.

Cervical spinal cord injury leads to a disruption of bulbospinal innervation from medullary respiratory centers to phrenic motoneurons. Animal models utilizing cervical hemisection result in inhibition of ipsilateral phrenic nerve activity, leading to paralysis of the hemidiaphragm. We have previously demonstrated a role for serotonin (5-HT) as one potential modulator of respiratory recovery following cervical hemisection, a mechanism that likely occurs via 5-HT2A and/or 5-HT2C receptors. The present study was designed to specifically examine if 5-HT2A and/or 5-HT2C receptors are colocalized with phrenic motoneurons in both intact and spinal-hemisected rats. Adult female rats (250-350 g; n = 6 per group) received a left cervical (C2) hemisection and were injected with the fluorescent retrograde neuronal tracer Fluorogold into the left hemidiaphragm. Twenty-four hours later, animals were killed and spinal cords processed for in situ hybridization and immunohistochemistry. Using (35)S-labeled cRNA probes, cervical spinal cords were probed for 5-HT2A and 5-HT2C receptor mRNA expression and double-labeled using an antibody to Fluorogold to detect phrenic motoneurons. Expression of both 5-HT2A and 5-HT2C receptor mRNA was detected in motoneurons of the cervical ventral horn. Despite positive expression of both 5-HT2A and 5-HT2C receptor mRNA-hybridization signal over phrenic motoneurons, only 5-HT2A silver grains achieved a signal-to-noise ratio representative of colocalization. 5-HT2A mRNA levels in identified phrenic motoneurons were not significantly altered following cervical hemisection compared to sham-operated controls. Selective colocalization of 5-HT2A receptor mRNA with phrenic motoneurons may have implications for recently observed 5-HT2A receptor-mediated regulation of respiratory activity and/or recovery in both intact and injury-compromised states.

MK-801 prevents dopamine D1 but not serotonin 2A stimulation of striatal preprotachykinin mRNA expression.

We examined dopamine (DA) and serotonin (5-HT) receptor-mediated influences on striatal preprotachykinin (PPT, tachykinin precursor) mRNA regulation in organotypic slice cultures. A 3 h exposure to SKF-38393 (10 microM, DA D1 agonist) or DOI (10 microM, 5-HT2 agonist) increased PPT mRNA levels to 196.4% and 154.0%, respectively. Responses to SKF-38393 were prevented by SCH-23390 (10 microM, D1 antagonist) whereas DOI-stimulated increases were prevented by ketanserin (10 microM, 5-HT2A antagonist). Since striatal tachykinin neurons also possess NMDA receptors that regulate gene expression, stimulation of PPT message levels was examined in the presence of MK-801, a non-competitive NMDA antagonist. Alone, MK-801 (10 nM) did not significantly alter basal PPT message levels. However, MK-801 prevented SKF-38393-stimulated increases in PPT mRNA expression while DOI-induced expression was not affected. These results provide evidence that D1 regulation of striatal tachykinin expression is dependent on NMDA-type glutamate neurotransmission while 5-HT2A regulation appears independent.

Striatal preprotachykinin mRNA levels are regulated by stimulatory agents and dopamine D1 receptor manipulation in rodent organotypic slice cultures.

We have utilized an organotypic slice culture system to determine factors which directly influence the expression of striatal preprotachykinin (PPT) mRNA. Striatal slices were generated from 3-day-old male rat pups and cultured on Millicell-CM inserts in serum-containing media. Under these conditions, striatal PPT mRNA levels fell significantly (-55.7+/-6.2%) in slices cultured for 2 days in vitro (2DIV) as compared to slices placed in culture for 3 h (0DIV). However, striatal PPT mRNA expression did not decline further in 4DIV cultured slices (-59.6+/-7.1%). When 2DIV slices were exposed to combined high potassium (K(+), 10 mM) and forskolin (10 microM) stimulation for 3 h, PPT mRNA levels were increased within areas of the brain normally associated with tachykinin production. Application of the dopamine (DA) D1 receptor agonist SKF-38393 (10 microM) at 2DIV for 3 h also increased (+162.9+/-28.9%) PPT mRNA expression, but increases were localized within the striatum. SKF-38393-stimulated increases were completely blocked by the D1 antagonist SCH-23390 (10 microM), which alone had no effect on mRNA levels. However, a 3-h incubation with SKF-38393 on 0DIV slice cultures did not affect PPT mRNA expression whereas SCH-23390 decreased PPT message levels (-24.5+/-5.4%). These findings indicate that tachykinin gene expression is inducible within slice culture preparations and that the maintenance of normal striatal PPT mRNA levels depends on DA D1 receptor tone.

Serotonin 2A and 2C receptor biosynthesis in the rodent striatum during postnatal development: mRNA expression and functional linkage to neuropeptide gene regulation.

The present study was designed to determine if there are region-specific differences in serotonin (5-HT) neurotransmission and 5-HT receptor expression that may limit the stimulatory effects of the 5-HT releaser p-chloroamphetamine (pCA) on striatal neuropeptide gene expression to the posterior striatum (P-STR) during postnatal maturation. Sprague-Dawley rat brains from postnatal days (PND) 1-35 were processed for 5-HT(2A) and 5-HT(2C) receptor mRNA expression by in situ hybridization and monoamine analysis by HPLC. Within the P-STR, 5-HT(2A) receptor mRNA expression reached young adult (PND 35) levels by PND 3, while levels in the A-STR were significantly less (range: 1.43 +/- 0.219-6. 36 +/- 0.478) than P-STR (5.36 +/- 0.854-12.11 +/- 1.08) at each respective age throughout the time course. 5-HT(2C) receptor mRNA expression reached young adult levels at PND 7 in the A-STR and by PND 3 in the P-STR. At each PND age 5-HT(2C) receptor mRNA levels within the P-STR were significantly less (6.23 +/- 1.02-12.32 +/- 0.427) than the A-STR (7.31 +/- 1.65-26.84 +/- 2.24). 5-HT content increased across the developmental time course within the P-STR (5.01 +/- 0.327-15.7 +/- 1.03 ng/mg protein) and A-STR (2.97 +/- 0. 223-11.2 +/- 0.701 ng/mg protein). Four hours following injection (i. p.) of pCA (10 mg/kg), preprotachykinin (PPT) mRNA levels increased 89% in the P-STR but not the anterior (A-STR) striatum of the 3-week-old rat, which were prevented by preinjection (30 min, i.p.) of the 5-HT(2) receptor antagonist ritanserin (1 mg/kg). Together, these data suggest that faster maturity of 5-HT(2A) receptor expression in the P-STR may be sufficient to convey the region-specific acute stimulatory effects of pCA on PPT mRNA transcription in the developing rodent striatum. These results provide further evidence that the influence of 5-HT on neuropeptide gene expression is far stronger in caudal vs. rostral striatal regions during postnatal development.

Acute rejection-associated tubular basement membrane defects and chronic allograft nephropathy.

BACKGROUND: Acute rejection is a major risk factor for chronic allograft nephropathy, although the link(s) between these events is not understood. The hypothesis of this study is that alterations in tubular basement membranes (TBMs) that occur during acute rejection may be irreversible and thereby play a role in the development of chronic allograft nephropathy. METHODS: Fourteen renal transplant patients were selected, each having had two or more biopsies performed (42 total). All biopsies were scored for acute and chronic rejection using Banff 1997 criteria. The initial biopsy showed only acute interstitial rejection (type I rejection). No biopsies contained significant chronic arterial lesions of chronic vascular rejection. The entire cortex was examined on Jones methenamine silver-stained sections at x400 for interruption in TBM staining. The number of tubules with TBM abnormalities was counted, and the renal cortical area was measured by image analysis. Periodic acid-Schiff/immunoperoxidase stain was performed on 12 acute rejection biopsies stained for laminin, cytokeratin 7, CD3, CD20, and CD68. Controls consisted of 11 biopsies (8 negative for rejection and 3 acute tubular necrosis). RESULTS: Numerous TBM alterations in silver staining were identified as being associated with acute rejection and tubulitis, consisting of abrupt TBM discontinuities and/or extreme attenuation with segmental or complete absence of TBM. A loss of TBM matrix proteins was confirmed by absent laminin staining in areas of acute rejection and tubulitis. There was herniation of tubular cells into the interstitium through TBM defects confirmed by cytokeratin staining. The TBM defects were spatially associated with inflammatory cells, particularly macrophages. When the biopsies were divided into two groups, <10 and> 10 TBM breaks/mm2, there were statistically significant morphologic and clinical correlations. The number of TBM disruptions correlated with the serum creatinine at the time of biopsy, a combined Banff t + i score, the difference in tubular atrophy between the initial and most recent biopsy and the difference between the nadir creatinine and most recent creatinine. CONCLUSION: Damage to TBM develops in acute rejection as a consequence of interstitial inflammation and tubulitis. These lytic events correlate with the later development of clinical and morphologic evidence of chronic injury in the absence of arterial injury of chronic rejection. We suggest that chronic allograft nephropathy may have an inflammatory interstitial origin.

Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: studies with the inducible nitric oxide synthase inhibitor L-N(6)-(1-Iminoethyl)lysine.

Reactive oxygen species are suggested to participate in ischemia-reperfusion (I-R) injury. However, induction of inducible nitric oxide synthase (iNOS) and production of high levels of nitric oxide (NO) also contribute to this injury. NO can combine with superoxide to form the potent oxidant peroxynitrite (ONOO(-)). NO and ONOO(-) were investigated in a rat model of renal I-R injury using the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine (L-NIL). Sprague-Dawley rats were subjected to 40 min of bilateral renal ischemia followed by 6 h of reperfusion with or without L-NIL administration. Control animals received a sham surgery and had plasma creatinine values of 0.4 +/- 0.1 mg/dl. I-R surgery significantly increased plasma creatinine levels to 1.9 +/- 0.3 mg/dl (P <.05) and caused renal cortical necrosis. L-NIL administration (3 mg/kg) in animals subjected to I-R significantly decreased plasma creatinine levels to 1.2 +/- 0.10 mg/dl (P <.05 compared with I-R) and reduced tubular damage. ONOO(-) formation was evaluated by detecting 3-nitrotyrosine-protein adducts, a stable biomarker of ONOO(-) formation. Immunohistochemistry and HPLC revealed that the kidneys from I-R animals had increased levels of 3-nitrotyrosine-protein adducts compared with control animals. L-NIL-treated rats (3 mg/kg) subjected to I-R showed decreased levels of 3-nitrotyrosine-protein adducts. These results support the hypothesis that iNOS-generated NO mediates damage in I-R injury possibly through ONOO(-) formation.