Preparation and Pharmacokinetic Characterization of an Anti-Virulence Compound Nanosuspensions.

Antibiotic resistance has become a worldwide public health threat due to the rapid evolution and spread of antibiotic-resistant bacteria. CCG-211790 is a novel anti-virulence compound that does not kill bacteria but could ameliorate human diseases by inhibiting expression of virulence factors, thereby applying less selection pressure for antibiotic resistance. However, its potential clinical use is restricted because of its poor aqueous solubility, resulting in formulation challenges. Nanosuspension technology is an effective way to circumvent this problem. Nanosuspensions of CCG-211790 with two different particle sizes, NanoA (315 ± 6 nm) and NanoB (915 ± 24 nm), were prepared using an antisolvent precipitation-ultrasonication method with Tween 80 as the stabilizer. Particle and pharmacokinetics (PK) of CCG-211790 nanosuspensions were characterized. Both NanoA and NanoB demonstrated remarkable increases in dissolution rate compared with the bulk compound. The PK parameters of NanoA were comparable to those of CCG-211790 solution formulation in intravenous or oral administration, suggesting that CCG-211790 nanosuspensions with smaller particle size improved oral bioavailability and drug exposure compared to traditional formulations of drug candidates.

Physicochemical properties and formulation development of a novel compound inhibiting Staphylococcus aureus biofilm formation.

The emergence of antibiotic resistance over the past several decades has given urgency to new antibacterial strategies that apply less selective pressure. A new class of anti-virulence compounds were developed that are active against methicillin-resistant Staphylococcus aureus (MRSA), by inhibiting bacterial virulence without hindering their growth to reduce the selective pressure for resistance development. One of the compounds CCG-211790 has demonstrated potent anti-biofilm activity against MRSA. This new class of anti-virulence compounds inhibited the gene expression of virulence factors involved in biofilm formation and disrupted the biofilm structures. In this study, the physicochemical properties of CCG-211790, including morphology, solubility in pure water or in water containing sodium dodecyl sulfate, solubility in organic solvents, and stability with respect to pH were investigated for the first time. Furthermore, a topical formulation was developed to enhance the therapeutic potential of the compound. The formulation demonstrated acceptable properties for drug release, viscosity, pH, cosmetic elegance and stability of over nine months.

Challenges and New Therapeutic Approaches in the Management of Chronic Wounds.

Chronic non-healing wounds are estimated to cost the US healthcare $28-$31 billion per year. Diabetic ulcers, arterial and venous ulcers, and pressure ulcers are some of the most common types of chronic wounds. The burden of chronic wounds continues to rise due to the current epidemic of obesity and diabetes and the increase in elderly adults in the population who are more vulnerable to chronic wounds than younger individuals. This patient population is also highly vulnerable to debilitating infections caused by opportunistic and multi-drug resistant pathogens. Reduced microcirculation, decreased availability of cytokines and growth factors that promote wound closure and healing, and infections by multi-drug resistant and biofilm forming microbes are some of the critical factors that contribute to the development of chronic non-healing wounds. This review discusses novel approaches to understand chronic wound pathology and methods to improve chronic wound care, particularly when chronic wounds are infected by multi-drug resistant, biofilm forming microbes.

In Vitro Assessment of Microbial Barrier Properties of Cyanoacrylate Tissue Adhesives and Pressure-Sensitive Adhesives.

Background: Despite advances in incision care and surgical dressings, surgical site infections remain a common complication. Post-operative contamination of a surgical site is believed to play a role in many of these infections. Most surgical dressings adhere to the skin with pressure-sensitive adhesives. Cyanoacrylate tissue adhesives bond to skin with much greater strength and have inherent antimicrobial properties. This study was designed to compare the microbial barrier properties of common pressure-sensitive adhesives to medical-grade cyanoacrylate tissue adhesives (2-octyl cyanoacrylate and N-butyl cyanoacrylate). Methods: Samples of cyanoacrylate tissue adhesives and pressure-sensitive adhesives were placed on solid culture media. Five common bacterial pathogens were used to contaminate 50 cyanoacrylate samples and 150 pressure-sensitive adhesive samples. Each plate was evaluated for bacterial growth underneath the adhesive sample daily for a total of 72 hours. Results: No penetration was seen through any of the cyanoacrylate adhesive samples at 72 hours. In sharp contrast, bacteria penetrated 99.3% of the pressure-sensitive adhesive samples at 72 hours. Conclusions: Medical grade cyanoacrylate tissue adhesives provide a superior microbial barrier compared with common pressure-sensitive adhesives. Consideration could be given to the use of these adhesives for the securement of surgical dressings.

The attention set-shifting test is sensitive for revealing sex-based impairments in executive functions following developmental lead exposure in rats.

The literature on lead (Pb) exposure has focused in large part on hippocampal-based learning and memory deficits, although frontoexecutive dysfunctions are known to exist in Pb-exposed humans. This study examined the effects of perinatal (PERI) and early postnatal (EPN) developmental low-level Pb-exposures in rats on frontoexecutive functions, using the Attention Set-Shift Test (ASST). Control males and females performed the ASST similarly. Male EPN rats had difficulty with simple discrimination (SD) of odors and failed to complete the compound discrimination (CD) stage of the ASST. All other Pb-exposed rats completed the training and testing. Male PERI rats performed worse on the SD, intradimensional (ID), and intradimensional-reversal (ID-Rev) ASST stages when compared to male Control rats. Female EPN rats performed similar to Controls on the ID-Rev rats, whereas PERI rats performed better the trials-to-criterion on the ID-Rev than EPN and Control rats. Pb-exposed female rats had significant difficulty performing the ED/ED-Rev stages, with the number of trials-to-criterion double that required by Pb-exposed and Control male rats and Control female rats. Together, the ASST results showed that developmental Pb-exposure induces frontoexecutive dysfunction that persists into adulthood, with different sex-based vulnerabilities dependent upon the time-period of neurotoxicant exposure.

Pseudotumor from Metal-on-Metal Total Hip Arthroplasty Causing Unilateral Leg Edema: Case Presentation and Literature Review.

Metal-on-metal (MoM) total hip arthroplasty (THA) can be associated with adverse metal reactions, including pseudotumors. This case report describes a 58-year-old female with an MoM THA-related pseudotumor that caused unilateral leg edema from compression of her external iliac vein. After thorough preoperative workup to rule out infection and deep vein thrombosis and consultation with a vascular surgeon, the patient underwent revision THA and excision of her pseudotumor. She had complete resolution of her swelling at 4 years after surgery. Review of all available case reports for this rare complication revealed that almost all patients were female. All patients underwent revision THA, with resolution of their symptoms. Literature review demonstrates that women are disproportionally affected by complications associated with MoM THA. We recommend close monitoring of patients with MoM THA, particularly women, for development of adverse metal reactions.

Developmental Lead Exposure and Prenatal Stress Result in Sex-Specific Reprograming of Adult Stress Physiology and Epigenetic Profiles in Brain.

Developmental exposure to lead (Pb) and prenatal stress (PS) both impair cognition, which could derive from their joint targeting of the hypothalamic-pituitary-adrenal axis and the brain mesocorticolimbic (MESO) system, including frontal cortex (FC) and hippocampus (HIPP). Glucocorticoids modulate both FC and HIPP function and associated mediation of cognitive and other behavioral functions. This study sought to determine whether developmental Pb ± PS exposures altered glucocorticoid-related epigenetic profiles in brain MESO regions in offspring of female mice exposed to 0 or 100 ppm Pb acetate drinking water from 2 mos prior to breeding until weaning, with half further exposed to prenatal restraint stress from gestational day 11-18. Overall, changes in females occured in response to Pb exposure. In males, however, Pb-induced neurotoxicity was modulated by PS. Changes in serum corticosterone levels were seen in males, while glucocorticoid receptor changes were seen in both sexes. In contrast, both Pb and PS broadly impacted brain DNA methyltransferases and binding proteins, particularly DNMT1, DNMT3a and methyl-CpG-binding protein 2, with patterns that differed by sex and brain regions. Specifically, in males, effects on FC epigenetic modifiers were primarily influenced by Pb, whereas extensive changes in HIPP were produced by PS. In females, Pb exposure and not PS primarily altered epigenetic modifiers in both FC and HIPP. Collectively, these findings indicate that epigenetic mechanisms may underlie associated neurotoxicity of Pb and of PS, particularly associated cognitive deficits. However, mechanisms by which this may occur will be different in males versus females.

Unsuspected Malignancies in Routine Femoral Head Histopathologic Examination During Primary Total Hip Arthroplasty: Cost-Effectiveness Analysis.

BACKGROUND: Routine femoral head histopathology during primary total hip arthroplasty (THA) has been recently reported as a potentially useful screening tool for bone- and bone marrow-associated malignancies. However, cost-effectiveness of routine histopathology during THA remains unclear due to low prevalence of significant medical findings which alter patient management. The aim of this study was to evaluate the cost-effectiveness of routine histopathology in diagnosing unsuspected malignancy in patients undergoing primary THA. METHODS: From 1993 to 2011, we retrospectively analyzed routine histopathologic findings of 3200 femoral head specimens from 2725 patients that underwent primary THA. Preoperative and postoperative diagnoses were classified into concordant (clinical diagnosis concurred with pathologic diagnosis), discrepant (differing diagnosis with no resultant impact on patient management), and discordant (differing diagnosis with subsequent change in patient management). Cost-effectiveness analysis was performed using the incremental cost-utility ratio. RESULTS: A total of 3055 of 3200 pathologic samples were concordant with the preoperative diagnosis (95.4%), 140 of 3200 were discrepant (4.4%), and 5 of 3200 were discordant (0.2%). Routine histopathology revealed 1 unsuspected malignancy out of 640 (5 of 3200) femoral heads. The total cost of histopathologic screening was $614,664.80. The average cost to identify a discrepant case was $4390.46, and the cost to identify a discordant case was $122,932.96. The incremental cost-utility ratio was $49,569.74 per quality-adjusted life year (QALY) gained. CONCLUSION: Our study indicates routine femoral head histopathology may be cost-effective in diagnosing unsuspected malignancy at $49,569.74/QALY gained (less than World Health Organization recommended threshold $159,000/QALY gained), providing useful clinical information for surgeons considering the value of routine femoral head histopathology in patients undergoing THA.

Osteomyelitis: Recent advances in pathophysiology and therapeutic strategies.

This review article summarizes the recent advances in pathogenic mechanisms and novel therapeutic strategies for osteomyelitis, covering both periprosthetic joint infections and fracture-associated bone infections. A better understanding of the pathophysiology including the mechanisms for biofilm formation has led to new therapeutic strategies for this devastating disease. Research on novel local delivery materials with appropriate mechanical properties, lower exothermicity, controlled release of antibiotics, and absorbable scaffolding for bone regeneration is progressing rapidly. Emerging strategies for prevention, early diagnosis of low-grade infections, and innovative treatments of osteomyelitis such as biofilm disruptors and immunotherapy are highlighted in this review.

Sex-dependent effects of lead and prenatal stress on post-translational histone modifications in frontal cortex and hippocampus in the early postnatal brain.

Environmental lead (Pb) exposure and prenatal stress (PS) are co-occurring risk factors for impaired cognition and other disorders/diseases in adulthood and target common biological substrates in the brain. Sex-dependent differences characterize the neurochemical and behavioral responses of the brain to Pb and PS and sexually dimorphic histone modifications have been reported to occur in at-risk brain regions (cortex and hippocampus) during development. The present study sought to examine levels and developmental timing of sexually dimorphic histone modifications (i.e., H3K9/14Ac and H3K9Me3) and the extent to which they may be altered by Pb±PS. Female C57/Bl6 mice were randomly assigned to receive distilled deionized drinking water containing 0 or 100ppm Pb acetate for 2 months prior to breeding and throughout lactation. Half of the dams in each group were exposed to restraint stress (PS, three restraint sessions in plastic cylindrical devices 3×/day at for 30min/day (1000, 1300, and 1600h)) from gestational day 11-19 or no stress (NS). At delivery (PND0) and postnatal day 6 (PND6), pups were euthanized and frontal cortex and hippocampus were removed, homogenized, and assayed for levels of H3K9/14Ac and H3K9Me3. Sex-dependent differences in both levels of histone modifications as well as the developmental trajectory of changes in these levels were observed in both structures and these parameters were differentially affected by Pb±PS in a sex and brain-region-dependent manner. Disruptions of these epigenetic processes by developmental Pb±PS may underlie some of the sex-dependent neurobehavioral differences previously observed in these animals.

Retinal pathology detected by optical coherence tomography in an animal model of Parkinson's disease.

BACKGROUND: Optical coherence tomography (OCT) is a noninvasive procedure for analysis of retinal morphology. Significant changes in the thickness of the peripapillary retinal nerve fiber layer (RNFL) in Parkinson's disease (PD) have been reported, and the current study was performed to examine whether such changes can also be detected in an animal model of PD. METHODS: Optical coherence tomography measurements of peripapillary RNFL thickness, macula volume, and foveal thickness were obtained from 10 normal and five 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgus monkeys with stable Parkinsonian signs. RESULTS: Average RNFL thickness was significantly decreased in Parkinsonian monkeys compared with controls, with statistically significant RNFL thinning found in nasal and inferior quadrants. Macula volume and foveal thickness were also significantly reduced in Parkinsonian animals compared with controls. CONCLUSIONS: As described in PD, RNFL thinning, reduced macula volume, and reduced foveal thickness also occurs in monkeys with MPTP-induced Parkinsonism. These findings pave the way for additional studies in which OCT may be used to track changes in the retina that might be present very early in the PD pathological process, perhaps preceding the onset of motor signs.

Genetic diversity influences the response of the brain to developmental lead exposure.

Although extrinsic factors, such as nutritional status, and some intrinsic genetic factors may modify susceptibility to developmental lead (Pb) poisoning, no studies have specifically examined the influence of genetic background on outcomes from Pb exposure. In this study, we used gene microarray profiling to identify Pb-responsive genes in rats of different genetic backgrounds, including inbred (Fischer 344 (F344)) and outbred (Long Evans (LE), Sprague Dawley (SD)) strains, to investigate the role that genetic variation may play in influencing outcomes from developmental Pb exposure. Male and female animals received either perinatal (gestation through lactation) or postnatal (birth through weaning) exposure to Pb in food (0, 250, or 750 ppm). RNA was extracted from the hippocampus at day 55 and hybridized to Affymetrix Rat Gene 1.0 ST Arrays. There were significant strain-specific effects of Pb on the hippocampal transcriptome with 978 transcripts differentially expressed in LE rats across all experimental groups, 269 transcripts differentially expressed in F344 rats, and only 179 transcripts differentially expressed in SD rats. These results were not due to strain-related differences in brain accumulation of Pb. Further, no genes were consistently differentially regulated in all experimental conditions. There was no set of "Pb toxicity" genes that are a molecular signature for Pb neurotoxicity that transcended sex, exposure condition, and strain. These results demonstrate the influence that strain and genetic background play in modifying the brain's response to developmental Pb exposure and may have relevance for better understanding the molecular underpinnings of the lack of a neurobehavioral signature in childhood Pb poisoning.

Effects of developmental lead exposure on the hippocampal transcriptome: influences of sex, developmental period, and lead exposure level.

Developmental lead (Pb) exposure has profound effects on cognition and behavior. Much is known about effects of Pb on hippocampal-mediated behaviors, but little is known about the molecular consequences of Pb exposure and the influences of developmental timing of exposure, level of exposure, and sex as effect modifiers of Pb exposure on the brain. The aim of this study was to examine the effects of different levels of Pb exposure (250 and 750 ppm Pb acetate) during perinatal (gestation/lactation) and postnatal (through postnatal day 45) periods on the hippocampal transcriptome in male and female Long Evans rats. Total RNA was extracted from hippocampus from four animals per experimental condition. RNA was hybridized to Affymetrix Rat Gene RNA Arrays using standard methods. Pb exposure per se influenced the expression of 717 transcripts (328 unique annotated genes), with many influenced in a sex-independent manner. Significant differences in gene expression patterns were also influenced by timing and level of exposure, with generally larger effects at the lower level of exposure across all groups. Statistically enriched biological functions included ion binding, regulation of RNA metabolic processes, and positive regulation of macromolecule biosynthetic processes. Processes of regulation of transcription and regulation of gene expression were preferentially enriched in males, regardless of timing or amount of Pb exposure. The effect on transcription factors and the diverse pathways or networks affected by Pb suggest a substantial effect of developmental Pb exposure on plasticity and adaptability, with these effects significantly modified by sex, developmental window of exposure, and level of Pb exposure.

Prominin-1 localizes to the open rims of outer segment lamellae in Xenopus laevis rod and cone photoreceptors.

PURPOSE: Prominin-1 expresses in rod and cone photoreceptors. Mutations in the prominin-1 gene cause retinal degeneration in humans. In this study, the authors investigated the expression and subcellular localization of xlProminin-1 protein, the Xenopus laevis ortholog of prominin-1, in rod and cone photoreceptors of this frog. METHODS: Antibodies specific for xlProminin-1 were generated. Immunoblotting was used to study the expression and posttranslational processing of xlProminin-1 protein. Immunocytochemical light and electron microscopy and transgenesis were used to study the subcellular distribution of xlProminin-1. RESULTS: xlProminin-1 is expressed and is subject to posttranslational proteolytic processing in the retina, brain, and kidney. xlProminin-1 is differently expressed and localized in outer segments of rod and cone photoreceptors of X. laevis. Antibodies specific for the N or C termini of xlProminin-1 labeled the open rims of lamellae of cone outer segments (COS) and the open lamellae at the base of rod outer segments (ROS). By contrast, anti-peripherin-2/rds antibody, Xper5A11, labeled the closed rims of cone lamellae adjacent to the ciliary axoneme and the rims of the closed ROS disks. The extent of labeling of the basal ROS by anti-xlProminin-1 antibodies varied with the light cycle in this frog. The entire ROS was also faintly labeled by both antibodies, a result that contrasts with the current notion that prominin-1 localizes only to the basal ROS. CONCLUSIONS: These findings suggest that xlProminin-1 may serve as an anti-fusogenic factor in the regulation of disk morphogenesis and may help to maintain the open lamellar structure of basal ROS and COS disks in X. laevis photoreceptors.

Functional significance of aldehyde dehydrogenase ALDH1A1 to the nigrostriatal dopamine system.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is a member of a superfamily of detoxification enzymes found in various tissues that participate in the oxidation of both aliphatic and aromatic aldehydes. In the brain, ALDH1A1 participates in the metabolism of catecholamines including dopamine (DA) and norepinephrine, but is uniquely expressed in a subset of dopaminergic (DAergic) neurons in the ventral mesencephalon where it converts 3,4-dihydroxyphenylacetaldehyde, a potentially toxic aldehyde, to 3,4-dihydroxyphenylacetic acid, a non toxic metabolite. Therefore, loss of ALDH1A1 expression could be predicted to alter DA metabolism and potentially increase neurotoxicity in ventral mesencephalic DA neurons. Recent reports of reduced levels of expression of both Aldh1a1 mRNA and protein in the substantia nigra (SN) of Parkinson's disease patients suggest possible involvement of ALDH1A1 in this progressive neurodegenerative disease. The present study used an Aldh1a1 null mouse to assess the influence of ALDH1A1 on the function and maintenance of the DAergic system. Results indicate that the absence of Aldh1a1 did not negatively affect growth and development of SN DA neurons nor alter protein expression levels of tyrosine hydroxylase, the DA transporter or vesicular monoamine transporter 2. However, absence of Aldh1a1 significantly increased basal extracellular DA levels, decreased KCl and amphetamine stimulated DA release and decreased DA re-uptake and resulted in more tyrosine hydroxylase expressing neurons in the SN than in wildtype animals. These data suggest that in young adult animals with deletion of the Aldh1a1 gene there is altered DA metabolism and dysfunction of the DA transporter and DA release mechanisms.

Postoperative cervical myelopathy and cord compression associated with the use of recombinant bone morphogenetic protein-2 in posterior cervical decompression, instrumentation, and arthrodesis: a report of two cases.

STUDY DESIGN: Case report. OBJECTIVE: Two cases are presented in which the use of recombinant bone morphogenetic protein-2 (rh-BMP-2) in a posterior cervical decompression and instrumented arthrodesis may have contributed to seroma formation and cord compression. SUMMARY OF BACKGROUND DATA: The use of rh-BMP-2 has been proven effective in promoting bone formation in anterior lumbar spine arthrodesis. Whether rh-BMP-2 is safe and/or effective in the cervical spine has not been determined. Adverse effects when it is used for anterior cervical fusion procedures have been reported but its role in posterior cervical decompression and instrumented fusions has yet to be determined. METHODS: We report on two cases. The first is a 68-year-old man presenting with a substantial decline in his neurologic status approximately 2 weeks after surgery. The second is a 44-year-old man presenting with a substantial decline in his neurologic status approximately 5 days after surgery. Both complications occurred after a posterior cervical laminectomy and instrumented arthrodesis when rh-BMP-2 was used as a bone graft substitute. RESULTS: Both patients were found to have a moderate-to-large seroma causing severe compression on the spinal cord and were urgently taken to an operating room for evacuation of the seromas. Both showed improvement of their neurologic status immediately after surgery. As rh-BMP-2 is known to occasionally cause seroma formation it is postulated that it may have been the cause of the seromas. CONCLUSION: Caution should be exercised with rh-BMP-2 use in posterior cervical applications when a laminectomy has been performed. The safe and effective dose and technique for application have yet to be determined. Seroma formation is possible, which can cause acute stenosis with cord compression and neurologic compromise.

Novel artificial urinary sphincter in the canine model: the tape mechanical occlusive device.

OBJECTIVES: To assess the functionality, occlusive efficiency, and biocompatibility of a novel artificial urinary sphincter, the tape mechanical occlusive device (TMOD), after implantation in a live canine model, as well as its occlusive efficiency and sizing parameters in human cadavers. METHODS: Three female canines underwent implantation of the TMOD at the level of the bladder neck. Functionality was assessed starting at 2 weeks after implantation and continued for ≤9 weeks. The TMODs were activated at 2 weeks and then deactivated for 3, 30-minute sessions daily to permit voiding. The urethral occlusion pressures and biocompatibility for systemic toxicity and the local tissue response were examined. Additionally, the TMOD was inserted in 3 male cadavers to determine the sizing parameters and to assess the urethral occlusion pressures using pressure profilometry. RESULTS: In the canine model, the urethral occlusion pressures increased from a range of 9-42 cm H(2)O with the TMOD deactivated to a range of 57-82 cm H(2)O with the TMOD activated. Pathologic examination revealed unremarkable pseudocapsular tissues surrounding the device. No histologic or structural evidence of systemic toxicity was observed. Sizing parameters similar to those of other urologic implants were confirmed in the male cadavers, and the urethral occlusion pressures increased from 24 to 30 cm H(2)O with the device deactivated to 61-105 cm H(2)O with the device activated. CONCLUSIONS: The TMOD meets the current standards for an artificial urinary sphincter in terms of functionality, biocompatibility, and achieving desired occlusion pressures following chronic implantation. Additional testing in male canines followed by early human clinical trials is being contemplated.

Towards biomolecular assembly employing extended native chemical ligation in combination with thioester synthesis using an N-->S acyl shift.

The advent of kinetically controlled Native Chemical Ligation has permitted more efficient sequential Native Chemical Ligation reactions to take place in one pot where one or more of the peptide fragments contains an N-terminal cysteine residue and a C-terminal thioester. The reactivity of the thioester component can dictate how fragments behave through careful choice of leaving group (alkyl or aryl thiol) and the C-terminal amino acid residue. Although thioester reactivity is exquisitely controlled, reactivity of the N-terminal cysteine residue has been curbed using protecting groups, usually the thiazolidine-4-carboxo (Thz) group as it can be removed in the presence of the thioester at acidic pH. Only recently has the concept of orthogonal ligation been extended to thiol auxiliary mediated Native Chemical Ligation (a.k.a. Extended Ligation) which, owing to their inherent difference in reactivity, have allowed peptides to be selectively extended at the C-terminus without recourse to protecting groups on the N-terminus. Herein we explored the compatibility of acyl transfer auxiliaries with peptide thioester production via an N-->S acyl shift for this purpose.

Evidence for the location of the allosteric activation switch in the multisubunit phosphorylase kinase complex from mass spectrometric identification of chemically crosslinked peptides.

Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.

Neuroprotection in Parkinson models varies with toxin administration protocol.

Numerous factors contribute to substantia nigra pars compacta (SNc) dopamine (DA) neuron death in Parkinson's disease (PD), thus complicating the search for effective neuroprotective agents for this disease. Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse has been widely used for assessing neuroprotective agents for PD, the pathological processes resulting from MPTP exposure can vary greatly depending upon the MPTP administration protocol. This study assessed the degree to which the neuroprotective efficacy of particular agents may depend upon the MPTP administration protocol (i.e. acute vs. subacute toxin administration). Endpoints analysed were changes in tyrosine hydroxylase (TH) and NeuN cell numbers in the SNc, striatal DA and metabolite levels, and striatal TH+ fiber density. The efficacy of putative neuroprotective agents [i.e. LIGA 20, nicotinamide and pramipexole (PPX)] varied depending upon the MPTP administration protocol. LIGA 20 spared striatal DA levels in both MPTP models, while nicotinamide was only effective in the acute toxin administration model and PPX was only effective in the subacute model. In both MPTP models, LIGA 20 and nicotinamide significantly spared DAergic neurons; PPX only spared DAergic neurons in the subacute model. Only acute MPTP-treated mice that received nicotinamide had a significant sparing of striatal DAergic fibers. These results underscore the need to assess putative neuroprotective agents for PD in multiple animal models using multiple endpoints. This strategy may better identify compounds with broad neuroprotective/neurorestorative profiles that may be more likely to be clinically effective.