Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease

We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimer''s disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function.     

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil

Background

Multiple congenital ocular anomalies (MCOA) syndrome is a hereditary congenital eye defect that was first described in Silver colored Rocky Mountain horses. The mutation causing this disease is located within a defined chromosomal interval, which also contains the gene and mutation that is associated with the Silver coat color (PMEL17, exon 11). Horses that are homozygous for the disease-causing allele have multiple defects (MCOA-phenotype), whilst the heterozygous horses predominantly have cysts of the iris, ciliary body or retina (Cyst-phenotype). It has been argued that these ocular defects are caused by a recent mutation that is restricted to horses that are related to the Rocky Mountain Horse breed. For that reason we have examined another horse breed, the Icelandic horse, which is historically quite divergent from Rocky Mountain horses.

Results

We examined 24 Icelandic horses and established that the MCOA syndrome is present in this breed. Four of these horses were categorised as having the MCOA-phenotype and were genotyped as being homozygous for the PMEL17 mutation. The most common clinical signs included megaloglobus, iris stromal hypoplasia, abnormal pectinate ligaments, iridociliary cysts occasionally extending into the peripheral retina and cataracts. The cysts and pectinate ligament abnormalities were observed in the temporal quadrant of the eyes. Fourteen horses were heterozygous for the PMEL17 mutation and were characterized as having the Cyst-phenotype with cysts and occasionally curvilinear streaks in the peripheral retina. Three additional horses were genotyped as PMEL17 heterozygotes, but in these horses we were unable to detect cysts or other forms of anomalies. One eye of a severely vision-impaired 18 month-old stallion, homozygous for the PMEL17 mutation was examined by light microscopy. Redundant duplication of non-pigmented ciliary body epithelium, sometimes forming cysts bulging into the posterior chamber and localized areas of atrophy in the peripheral retina were seen.

Conclusions

The MCOA syndrome is segregating with the PMEL17 mutation in the Icelandic Horse population. This needs to be taken into consideration in breeding decisions and highlights the fact that MCOA syndrome is present in a breed that are more ancient and not closely related to the Rocky Mountain Horse breed.     

Prevalence of pathogenic Yersinia enterocolitica strains in pigs in the United States

Yersinia enterocolitica is considered an important food-borne pathogen impacting the pork production and processing industry in the United States. Since this bacterium is a commensal of swine, the primary goal of this study was to determine the prevalence of pathogenic Y. enterocolitica in pigs in the United States using feces as the sample source. A total of 2,793 fecal samples were tested for its presence in swine. Fecal samples were collected from late finisher pigs from 77 production sites in the 15 eastern and midwestern pork-producing states over a period of 27 weeks (6 September 2000 to 20 March 2001). The prevalence of ail-positive Y. enterocolitica was determined in samples using both a fluorogenic 5' nuclease PCR assay and a culture method. The mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when both PCR- and culture-positive results were combined. Forty-one of 77 premises (53.25%) contained at least one fecal sample positive for the ail sequence. The PCR assay indicated a contamination rate of 12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method. Of the 345 PCR-positive samples, 252 were culture negative, while of the 114 culture-positive samples, 21 were PCR negative. Among 77 premises, the PCR assay revealed a significantly (P < 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). Thus, higher sensitivity, with respect to number of samples and sites identified as positive for the PCR method compared with the culture method for detecting pathogenic Y. enterocolitica, was demonstrated in this study. The results support the hypothesis that swine are a reservoir for Y. enterocolitica strains potentially pathogenic for humans.     

Identification and synthesis of novel alkaloids from the root system of Nicotiana tabacum: affinity for neuronal nicotinic acetylcholine receptors

A novel pyridine derivative, 3,5-bis-(1-methyl-pyrrolidin-2-yl)-pyridine, and a pair of diastereomers of 1,1'-dimethyl-[2,3']bipyrrolidinyl were isolated from the root of Nicotiana tabacum plants and identified as novel alkaloids by GC-MS analysis. The structures of these new alkaloids were confirmed by total synthesis. The affinities of these novel alkaloids, and other structurally related compounds for alpha4beta2*, alpha7* neuronal nicotinic acetylcholine receptors (nAChRs), and for nAChRs mediating nicotine-evoked dopamine release from rat striatum were also assessed. The results indicate that these compounds do not interact with alpha7* nAChRs, but inhibit [3H]nicotine binding to the alpha4beta2* nAChR subtype. The results also demonstrate that these compounds act as antagonists at nAChRs mediating nicotine-evoked dopamine release from rat striatum.     

Synthesis and evaluation of urea-based indazoles as melanin-concentrating hormone receptor 1 antagonists for the treatment of obesity

A series of urea-based N-1-(2-aminoethyl)-indazoles was synthesized and evaluated for melanin-concentrating hormone receptor 1 (MCHr1) antagonism in both binding and functional assays. Several compounds that acted as MCHr1 antagonists were identified, and optimization afforded a compound with excellent binding affinity, good functional potency, and oral efficacy in a chronic model for weight loss in diet-induced obese mice.     

Passive osmotic properties of in situ human articular chondrocytes within non-degenerate and degenerate cartilage

Osteoarthritis is characterized by many factors, including proteoglycan loss, decreased collagen stiffness, and increased cartilage hydration. Chondrocyte swelling also occurs, and correlates with the degree of osteoarthritis, however, the cause is unknown but might be related to alterations to their passive osmotic properties. We have used two-photon confocal laser scanning microscopy to measure the passive osmotic characteristics of in situ chondrocytes within relatively non-degenerate and degenerate human tibial plateau cartilage, and in chondrocytes isolated from relatively non-degenerate cartilage. Explants with bone attached were taken from a total of 42 patients undergoing arthroplasty and graded macroscopically and microscopically into two groups, grade 0 + 1 and grade 2 + 3. There was a significant increase in cartilage hydration between these two groups (P < 0.05), however, there was no change when medium osmolarity was varied over approximately 0-480 mOsm. The passive osmotic behavior of in situ chondrocytes (at 4 degrees C) was identical over a range of culture medium osmolarities ( approximately 0-515 mOsm), however, the maximum swelling of cells within degenerate cartilage and isolated chondrocytes was greater compared to those in non-degenerate cartilage. The swelling in the majority of in situ chondrocytes was accounted for by the reduced interstitial osmolarity occurring with cartilage degeneration. There was, however, a small population of in situ chondrocytes whose volume was in excess (>/=2,500 microm(3)) of that predicted from the decreased interstitial osmotic pressure. These results show that for the majority of cells studied, the differences in passive chondrocyte volume between relatively non-degenerate, degenerate, and isolated cells were entirely accounted for by changes to the extracellular osmolarity (180-515 mOsm).     

The genetic architecture of response to long-term artificial selection for oil concentration in the maize kernel

In one of the longest-running experiments in biology, researchers at the University of Illinois have selected for altered composition of the maize kernel since 1896. Here we use an association study to infer the genetic basis of dramatic changes that occurred in response to selection for changes in oil concentration. The study population was produced by a cross between the high- and low-selection lines at generation 70, followed by 10 generations of random mating and the derivation of 500 lines by selfing. These lines were genotyped for 488 genetic markers and the oil concentration was evaluated in replicated field trials. Three methods of analysis were tested in simulations for ability to detect quantitative trait loci (QTL). The most effective method was model selection in multiple regression. This method detected approximately 50 QTL accounting for approximately 50% of the genetic variance, suggesting that >50 QTL are involved. The QTL effect estimates are small and largely additive. About 20% of the QTL have negative effects (i.e., not predicted by the parental difference), which is consistent with hitchhiking and small population size during selection. The large number of QTL detected accounts for the smooth and sustained response to selection throughout the twentieth century.     

Enhanced toxicity and cellular binding of a modified amyloid beta peptide with a methionine to valine substitution

The amyloid beta peptide (Abeta) is toxic to neuronal cells, and it is probable that this toxicity is responsible for the progressive cognitive decline associated with Alzheimer's disease. However, the nature of the toxic Abeta species and its precise mechanism of action remain to be determined. It has been reported that the methionine residue at position 35 has a pivotal role to play in the toxicity of Abeta. We examined the effect of mutating the methionine to valine in Abeta42 (AbetaM35V). The neurotoxic activity of AbetaM35V on primary mouse neuronal cortical cells was enhanced, and this diminished cell viability occurred at an accelerated rate compared with Abeta42. AbetaM35V binds Cu2+ and produces similar amounts of H2O2 as Abeta42 in vitro, and the neurotoxic activity was attenuated by the H2O2 scavenger catalase. The increased toxicity of AbetaM35V was associated with increased binding of this mutated peptide to cortical cells. The M35V mutation altered the interaction between Abeta and copper in a lipid environment as shown by EPR analysis, which indicated that the valine substitution made the peptide less rigid in the bilayer region with a resulting higher affinity for the bilayer. Circular dichroism spectroscopy showed that both Abeta42 and AbetaM35V displayed a mixture of alpha-helical and beta-sheet conformations. These findings provide further evidence that the toxicity of Abeta is regulated by binding to neuronal cells.     

A rapid ATP affinity-based purification for the human non-receptor tyrosine kinase c-Src

The non-receptor tyrosine kinase c-Src plays a central role in a variety of cell signaling pathways that regulate cell growth, differentiation, apoptosis, and other important cellular processes. An 85-amino acid N-terminal deletion construct of c-Src (DeltaN85 c-Src) has been structurally characterized and used extensively in biochemical and biophysical studies. In this report, we have established a relatively rapid, simplified purification of DeltaN85 c-Src from recombinant baculovirus-infected insect cells. Q-Sepharose anion-exchange and aminophenyl-ATP affinity chromatography were used to isolate 5mg of >98% pure DeltaN85 c-Src from 900 mg of total soluble protein. The specific activity of DeltaN85 c-Src (20 U mg(-1)) was found to be >or = 5-fold greater than previously reported values. A lag in the autophosphorylation kinetics of DeltaN85 c-Src was observed, and the reaction occurred with observed first-order rate constants k1=0.20+/-0.01 min(-1) and k2=0.38+/-0.01 min(-1) under the experimental conditions used. Steady-state kinetic analysis of peptide phosphorylation by DeltaN85 c-Src gave Km values of 99+/-23 microM and 190+/-30 microM for the peptide and ATP substrates, respectively, and a value of k(cat)=17+/-2s(-1). Overall, we present a dramatically improved purification strategy that represents a simplified, relatively rapid protocol for the isolation of milligram quantities of DeltaN85 c-Src required for rigorous structure-function and inhibition studies that rely on a pre-steady-state kinetic approach.