Crystal structure of the Tp34 (TP0971) lipoprotein of treponema pallidum: implications of its metal-bound state and affinity for human lactoferrin

The Tp34 (TP0971) membrane lipoprotein of Treponema pallidum, an obligate human pathogen and the agent of syphilis, was previously reported to have lactoferrin binding properties. Given the non-cultivatable nature of T. pallidum, a structure-to-function approach was pursued to clarify further potential relationships between the Tp34 structural and biochemical properties and its propensity to bind human lactoferrin. The crystal structure of a nonacylated, recombinant form of Tp34 (rTp34), solved to a resolution of 1.9A(,) revealed two metaloccupied binding sites within a dimer; the identity of the ion most likely was zinc. Residues from both of the monomers contributed to the interfacial metal-binding sites; a novel feature was that the delta-sulfur of methionine coordinated the zinc ion. Analytical ultracentrifugation showed that, in solution, rTp34 formed a metal-stabilized dimer and that rTp34 bound human lactoferrin with a stoichiometry of 2:1. Isothermal titration calorimetry further revealed that rTp34 bound human lactoferrin at high (submicromolar) affinity. Finally, membrane topology studies revealed that native Tp34 is not located on the outer surface (outer membrane) of T. pallidum but, rather, is periplasmic. How propensity of Tp34 to bind zinc and the iron-sequestering lactoferrin may relate overall to the biology of T. pallidum infection in humans is discussed.     

Association of ALOX5AP with ischemic stroke: a population-based case-control study

Arachidonate 5-lipoxygenase activating protein (ALOX5AP) has been reported to demonstrate linkage and association with ischemic stroke and myocardial infarction. However, replication studies have been conflicting and to date, a significant proportion of blacks have not been studied. We prospectively recruited cases of ischemic stroke from all 16 hospitals in the Greater Cincinnati/Northern Kentucky region and demographically matched them to stroke-free population-based controls. Single nucleotide polymorphisms (SNPs) were selected based on association with ischemic stroke in prior studies. Allelic, genotypic and haplotypic association testing was performed using HAPLOVIEW. Multiple logistic regression was used to control for the presence of traditional risk factors including hypertension, diabetes, hypercholesterolemia and smoking. A total of 357 cases and 482 controls were genotyped. The SNPs, rs9579646 and rs4769874 were found to be significantly associated at both allelic (P = 0.019 and P < 10⁻⁴, respectively) and genotypic level with ischemic stroke among whites after correction for multiple testing. Haplotype association was identified with ischemic stroke as well as ischemic stroke subtypes among whites. Although an overall haplotype association with ischemic stroke was identified among blacks no evidence of association among individual haplotypes, alleles or genotypes were observed. Allele frequencies for the SNPs examined were markedly different among whites and blacks. In conclusion, we report significant association of variants of ALOX5AP with ischemic stroke and ischemic stroke subtypes among whites. No significant association was identified among blacks.     

Frequency and rationale of fine needle aspiration biopsy conversion to core biopsy as a result of onsite evaluation

OBJECTIVE: To measure the frequency and analyze the rationale and potential diagnostic benefits of converting the fine needle aspiration (FNA) procedure to core biopsy. STUDY DESIGN: The frequency of conversion to core biopsy was calculated over 13 months. Analysis of these cases was conducted in regard to the appropriateness for conversion and whether the core biopsy provided additional specific diagnostic information. RESULTS: During this period, the onsite triaging pathologist recommended FNA conversion to core biopsy in 31 of 821 procedures (3.7%). In 3 instances, the core biopsy could not be performed. The rationale for conversion in the remaining 28 cases (3.4%) included either scant aspirated material in 9 cases (32%) or an anticipated need for additional histologic material to further characterize the lesion in the other 19 (68%). In 27 cases (96%), the rationale for conversion was considered to be appropriate, and in 3 of these (11%) the core provided a change in diagnosis. Additional useful diagnostic information was identified in 12 cases (44%). CONCLUSION: Conversion to core biopsy during FNA is infrequent but justified in most cases. Appropriate utilization of this approach is helpful and may be cost effective.     

GSK-3β Protein Phosphorylates and Stabilizes HLXB9 Protein in Insulinoma Cells to Form a Targetable Mechanism of Controlling Insulinoma Cell Proliferation

Insulinomas (pancreatic islet β cell tumors) are the most common type of functioning pancreatic neuroendocrine tumors that occur sporadically or as a part of the MEN1 syndrome that is caused by germ line mutations in MEN1. Tissue-specific tumor predisposition from germ line mutations in ubiquitously expressed genes such as MEN1 could occur because of functional consequences on tissue-specific factors. We previously reported the proapoptotic β cell differentiation factor HLXB9 as a downstream target of menin (encoded by MEN1). Here we show that GSK-3β inactivates the proapoptotic activity of HLXB9 by phosphorylating HLXB9 at Ser-78/Ser-80 (pHLXB9). Although HLXB9 is found in the nucleus and cytoplasm, pHLXB9 is predominantly nuclear. Both pHLXB9 and active GSK-3β are elevated in β cells with menin knockdown, in MEN1-associated β cell tumors (insulinomas), and also in human sporadic insulinomas. Pharmacologic inhibition of GSK-3β blocked cell proliferation in three different rodent insulinoma cell lines by arresting the cells in G₂/M phase and caused apoptosis. Taken together, these data suggest that the combination of GSK-3β and pHLXB9 forms a therapeutically targetable mechanism of insulinoma pathogenesis. Our results reveal that GSK-3β and pHLXB9 can serve as novel targets for insulinoma treatment and have implications for understanding the pathways associated with β cell proliferation.     

Energy Metabolites,Lipid Variables and Lactation Performance of Periparturient Murrah Buffaloes (Bubalus bubalis) Fed on Diet Supplemented with Inorganic Chromium

The aim of this study was to elucidate the effects of chromium (Cr) supplementation as inorganic Cr (CrCl₃?·?6H₂O) on energy balance, lipid peroxidation, and lactation performance in periparturient Murrah buffaloes. Twenty-four multiparous Murrah buffaloes according to lactation, parity, body mass, and expected calving date were divided equally. Experimental buffaloes were randomly assigned to four treatment diets: a control diet and three diets with an inorganic Cr supplementation at 0.5, 1.0, and 1.5 mg of Cr/kg dry matter (DM), respectively from 60 days before expected calving date until 60 days of lactation. Milk productions of buffaloes were recorded every day until 60 days in milk. Blood samples were collected at days ?60, ?45, ?30,?21, ?15, ?7, ?3, 0, 7, 15, 21, 30, 45, and 60 days relative to actual calving for determination of plasma glucose, nonesterified fatty acid (NEFA), thiobarbituric acid reactive substance (TBARS), total cholesterol, total protein, albumin, blood urea nitrogen (BUN), and minerals. Adding inorganic Cr to the diet of Murrah buffaloes increased milk yield. Percentage of fat and total solid yield increased significantly through the experiment in the Cr-supplemented group. At the day of calving, buffaloes showed a decrease in dry matter intake (DMI), plasma glucose, and zinc (Zn) and Cr concentrations. In contrast, plasma NEFA, TBARS, and copper (Cu) levels were found highest at the day of calving among all groups. Cr supplementation increased peripheral blood glucose concentration while decreased level of NEFA and TBARS was recorded in Cr-fed buffaloes. Supplemental Cr had no effect on plasma cholesterol, total protein, albumin, and BUN in periparturient period. Dietary Cr supplementation had positive effect on plasma Cr concentration, but the plasma concentration of Cu, Zn, and iron (Fe) was not affected by different dietary Cr level supplementation. The results suggest that dietary inorganic Cr supplementation improved milk yield by reducing negative energy balance and lipid peroxidation in buffaloes during periparturient period.     

DNAJB6 is a peptide-binding chaperone which can suppress amyloid fibrillation of polyglutamine peptides at substoichiometric molar ratios

Expanded polyglutamine (polyQ) stretches lead to protein aggregation and severe neurodegenerative diseases. A highly efficient suppressor of polyQ aggregation was identified, the DNAJB6, when molecular chaperones from the HSPH, HSPA, and DNAJ families were screened for huntingtin exon 1 aggregation in cells (Hageman et al. in Mol Cell 37(3):355–369, 2010). Furthermore, also aggregation of polyQ peptides expressed in cells was recently found to be efficiently suppressed by co-expression of DNAJB6 (Gillis et al. in J Biol Chem 288:17225–17237, 2013). These suppression effects can be due to an indirect effect of DNAJB6 on other cellular components or to a direct interaction between DNAJB6 and polyQ peptides that may depend on other cellular components. Here, we have purified the DNAJB6 protein to investigate the suppression mechanism. The purified DNAJB6 protein formed large heterogeneous oligomers, in contrast to the more canonical family member DNAJB1 which is dimeric. Purified DNAJB6 protein, at substoichiometric molar ratios, efficiently suppressed fibrillation of polyQ peptides with 45°Q in a thioflavin T fibrillation. No suppression was obtained with DNAJB1, but with the closest homologue to DNAJB6, DNAJB8. The suppression effect was independent of HSPA1 and ATP. These data, based on purified proteins and controlled fibrillation in vitro, strongly suggest that the fibrillation suppression is due to a direct protein–protein interaction between the polyQ peptides and DNAJB6 and that the DNAJB6 has unique fibrillation suppression properties lacking in DNAJB1. Together, the data obtained in cells and in vitro support the view that DNAJB6 is a peptide-binding chaperone that can interact with polyQ peptides that are incompletely degraded by and released from the proteasome.     

Finding Missing Heritability in Less Significant Loci and Allelic Heterogeneity: Genetic Variation in Human Height

Genome-wide association studies (GWAS) have identified many common variants associated with complex traits in human populations. Thus far, most reported variants have relatively small effects and explain only a small proportion of phenotypic variance, leading to the issues of ‘missing’ heritability and its explanation. Using height as an example, we examined two possible sources of missing heritability: first, variants with smaller effects whose associations with height failed to reach genome-wide significance and second, allelic heterogeneity due to the effects of multiple variants at a single locus. Using a novel analytical approach we examined allelic heterogeneity of height-associated loci selected from SNPs of different significance levels based on the summary data of the GIANT (stage 1) studies. In a sample of 1,304 individuals collected from an island population of the Adriatic coast of Croatia, we assessed the extent of height variance explained by incorporating the effects of less significant height loci and multiple effective SNPs at the same loci. Our results indicate that approximately half of the 118 loci that achieved stringent genome-wide significance (p-value<5×10⁻⁸) showed evidence of allelic heterogeneity. Additionally, including less significant loci (i.e., p-value<5×10⁻⁴) and accounting for effects of allelic heterogeneity substantially improved the variance explained in height.