Aint/Tacc3 is highly expressed in proliferating mouse tissues during development, spermatogenesis, and oogenesis.

Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.     

Beta-galactosidase in the seminal plasma and reproductive organs of the bull

The distribution of beta-galactosidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of beta-galactosidase was found in testis and in different parts of the epididymis, where the activity seemed to be partly in secretory (cauda secretion) and partly in non-secretory, bound form (caput to cauda epididymidis). Gel filtration on Sepharose 6B at pH 7.0 revealed two beta-galactosidase forms (GF-1, Mr approximately 500,000-600,000 and GF-2, Mr approximately 190,000-220,000) in reproductive organs and seminal plasma. The pH-optimum of both beta-galactosidase forms was about 3.75-4.75. Hg2+ and p-chloromercuribenzoate inhibited strongly these activities. Further, form GF-2 seemed to be slightly more sensitive to the thermal inactivation at 50-70 degrees C than form GF-1. In chromatofocusing beta-galactosidase activities in bull seminal plasma coeluted with those of the cauda epididymidis (pI-values 7.5-6.4). On the contrary, prostate, Cowper's gland, testis, ampulla and seminal vesicles had enzyme activities eluting at lower pI-values (6.3-4.2). Thus, the seminal plasma activity is mainly an indicator for the function of the epididymal cauda.     

Determination of human apolipoprotein C-II by electroimmunoassay. Studies on standardization and determination before and after physical training

1. Human VLDL and HDL were fractionated by sequential ultracentrifugation until free of contaminant plasma proteins. 2. Column chromatofocusing method was used to isolate apolipoprotein C-II from apoVLDL and apo HDL. C-apoprotein peak was rechromatofocused and the second peak was the apo C-II (pI 4.7, homogeneous band on SDS slab gel). 3. New Zealand white rabbits were immunized with apo C-II. Antiserum gave a single precipitate are of identity between whole serum, apoVLDL, apoHDL and apo C-II. 4. Apo C-II concentration was measured by electroimmunoassay method. During standardization 1% Triton X-100 improved the rocket shapes and contours. Total delipidation did not affect the assay system and so the antigenic determinants of apo C-II are all available to antiserum. The lowest concentration of apo C-II possible to determine with this method was 70 ng/sample well. 5. There was no difference between the apo C-II values before (39.8 +/- 7.1 mg/l, n = 19) and after (41.6 +/- 6.4 mg/l, n = 19) moderate physical training among normolipemic subjects. 6. Specific immunoprecipitation technique was also used to determine apo C-II content in standard pool serum.     

Influence of Developmental Conditions on Immune Function and Dispersal-Related Traits in the Glanville Fritillary (Melitaea cinxia) Butterfly

Organisms in the wild are constantly faced with a wide range of environmental variability, such as fluctuation in food availability. Poor nutritional conditions influence life-histories via individual resource allocation patterns, and trade-offs between competing traits. In this study, we assessed the influence of food restriction during development on the energetically expensive traits flight metabolic rate (proxy of dispersal ability), encapsulation rate (proxy of immune defence), and lifespan using the Glanville fritillary butterfly, Melitaea cinxia, as a model organism. Additionally, we examined the direct costs of flight on individual immune function, and whether those costs increase under restricted environmental conditions. We found that nutritional restriction during development enhanced adult encapsulations rate, but reduced both resting and flight metabolic rates. However, at the individual level metabolic rates were not associated with encapsulation rate. Interestingly, individuals that were forced to fly prior to the immune assays had higher encapsulation rates than individuals that had not flown, suggesting that flying itself enhances immune response. Finally, in the control group encapsulation rate correlated positively with lifespan, whereas in the nutritional restriction group there was no relationship between these traits, suggesting that the association between encapsulation rate on adult lifespan was condition-dependent. Thus stressful events during both larval development (food limitation) and adulthood (forced flight) induce increased immune response in the adult butterflies, which may allow individuals to cope with stressful events later on in life.     

Insect oviposition preference between Epichloë‐symbiotic and Epichloë‐free grasses does not necessarily reflect larval performance

Variation in plant communities is likely to modulate the feeding and oviposition behavior of herbivorous insects, and plant‐associated microbes are largely ignored in this context. Here, we take into account that insects feeding on grasses commonly encounter systemic and vertically transmitted (via seeds) fungal Epichloë endophytes, which are regarded as defensive grass mutualists. Defensive mutualism is primarily attributable to alkaloids of fungal origin. To study the effects of Epichloë on insect behavior and performance, we selected wild tall fescue (Festuca arundinacea) and red fescue (Festuca rubra) as grass–endophyte models. The plants used either harbored the systemic endophyte (E+) or were endophyte‐free (E?). As a model herbivore, we selected the Coenonympha hero butterfly feeding on grasses as larvae. We examined both oviposition and feeding preferences of the herbivore as well as larval performance in relation to the presence of Epichloë endophytes in the plants. Our findings did not clearly support the female's oviposition preference to reflect the performance of her offspring. First, the preference responses depended greatly on the grass–endophyte symbiotum. In F. arundinacea, C. hero females preferred E+ individuals in oviposition‐choice tests, whereas in F. rubra, the endophytes may decrease exploitation, as both C. hero adults and larvae preferred E? grasses. Second, the endophytes had no effect on larval performance. Overall, F. arundinacea was an inferior host for C. hero larvae. However, the attraction of C. hero females to E+ may not be maladaptive if these plants constitute a favorable oviposition substrate for reasons other than the plants' nutritional quality. For example, rougher surface of E+ plant may physically facilitate the attachment of eggs, or the plants offer greater protection from natural enemies. Our results highlight the importance of considering the preference of herbivorous insects in studies involving the endophyte‐symbiotic grasses as host plants.     

A novel familial case of diffuse leukodystrophy related to NDUFV1 compound heterozygous mutations

NDUFV1 mutations have been related to encephalopathic phenotypes due to mitochondrial energy metabolism disturbances. In this study, we report two siblings affected by a diffuse leukodystrophy, who carry the NDUFV1 c.1156C>T (p.Arg386Cys) missense mutation and a novel 42-bp deletion. Bioinformatic and molecular analysis indicated that this deletion lead to the synthesis of mRNA molecules carrying a premature stop codon, which might be degraded by the nonsense-mediated decay system. Our results add information on the molecular basis and the phenotypic features of mitochondrial disease caused by NDUFV1 mutations.     

Genetic Loci Associated with Alzheimer’s Disease and Cerebrospinal Fluid Biomarkers in a Finnish Case-Control Cohort

Objectives

To understand the relation between risk genes for Alzheimer’s disease (AD) and their influence on biomarkers for AD, we examined the association of AD in the Finnish cohort with single nucleotide polymorphisms (SNPs) from top AlzGene loci, genome-wide association studies (GWAS), and candidate gene studies; and tested the correlation between these SNPs and AD markers Aβ_1–42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF).

Methods

We tested 25 SNPs for genetic association with clinical AD in our cohort comprised of 890 AD patients and 701-age matched healthy controls using logistic regression. For the correlational study with biomarkers, we tested 36 SNPs in a subset of 222 AD patients with available CSF using mixed models. Statistical analyses were adjusted for age, gender and APOE status. False discovery rate for multiple testing was applied. All participants were from academic hospital and research institutions in Finland.

Results

APOE-ε4, CLU rs11136000, and MS4A4A rs2304933 correlated with significantly decreased Aβ_1–42 (corrected p<0.05). At an uncorrected p<0.05, PPP3R1 rs1868402 and MAPT rs2435211 were related with increased t-tau; while SORL1 rs73595277 and MAPT rs16940758, with increased p-tau. Only TOMM40 rs2075650 showed association with clinical AD after adjusting for APOE-ε4 (p = 0.007), but not after multiple test correction (p>0.05).

Conclusions

We provide evidence that APOE-ε4, CLU and MS4A4A, which have been identified in GWAS to be associated with AD, also significantly reduced CSF Aβ_1–42 in AD. None of the other AlzGene and GWAS loci showed significant effects on CSF tau. The effects of other SNPs on CSF biomarkers and clinical AD diagnosis did not reach statistical significance. Our findings suggest that APOE-ε4, CLU and MS4A4A influence both AD risk and CSF Aβ_1–42.     

Tailor-made approach for selective isolation and elution of low-density lipoproteins by immunoaffinity sorbent on silica

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.     

ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response

High density lipoproteins (HDL) mediate reverse cholesterol transport as well as the clearance of oxidation products or inflammatory mediators, thereby contributing to tissue integrity. The decrease in HDL in inflammation has been attributed to decreased lecithin:cholesterol acyltransferase activity, whereas the role of phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein has not been analyzed in detail. We have studied the activities of HDL-modifying proteins and the heterogeneity of HDL in healthy control subjects and three groups of postsurgery patients: no bacterial infection (group 1), bacterial focus and systemic inflammatory response (group 2), and severe sepsis (group 3). For all patients, a decrease in total HDL could be demonstrated, with a loss of mainly large, apolipoprotein A-I (apoA-I) HDL particles, an almost total loss of apoC-I, and an increase in apoE HDL (200-500 kDa), which did not contain significant amounts of apoA-I, apoA-II, or apoC-I. PLTP activity was increased in patients of groups 2 and 3, paralleled by a redistribution of PLTP into a population of small (120- to 200-kDa) particles, probably representing PLTP homodimers or lipid-complexed PLTP.In summary, the increase in apoE HDL and PLTP activity may improve the delivery of energy substrates and phospholipids to tissues that must maintain cellular membrane homeostasis under conditions of inflammatory stress.     

Mutations linked to leukoencephalopathy with vanishing white matter impair the function of the eukaryotic initiation factor 2B complex in diverse ways

Leukoencephalopathy with vanishing white matter (VWM) is a severe inherited human neurodegenerative disorder that is caused by mutations in the genes for the subunits of eukaryotic initiation factor 2B (eIF2B), a heteropentameric guanine nucleotide exchange factor that regulates both global and mRNA-specific translation. Marked variability is evident in the clinical severity and time course of VWM in patients. Here we have studied the effects of VWM mutations on the function of human eIF2B. All the mutations tested cause partial loss of activity. Frameshift mutations in genes for eIF2Bepsilon or eIF2Bbeta lead to truncated polypeptides that fail to form complexes with the other subunits and are effectively null mutations. Certain point mutations also impair the ability of eIF2Bbeta or -epsilon to form eIF2B holocomplexes and also diminish the intrinsic nucleotide exchange activity of eIF2B. A point mutation in the catalytic domain of eIF2Bepsilon impairs its ability to bind the substrate, while two mutations in eIF2Bbeta actually enhance eIF2 binding. We provide evidence that expression of VWM mutant eIF2B may enhance the translation of specific mRNAs. The variability of the clinical phenotype in VWM may reflect the multiple ways in which VWM mutations affect eIF2B function.