The Val103Ile Polymorphism of Melanocortin‐4 Receptor Regulates Energy Expenditure and Weight Gain

Objective: The melanocortin‐4 receptor (MC4R) regulates energy intake. On the basis of animal studies, it may also regulate energy expenditure. Research Methods and Procedures: The effect of the Val103Ile polymorphism of the MC4R gene on energy metabolism was studied in 229 middle‐aged nondiabetic subjects (Group 1, age 51.2 ± 9.8 years, BMI 26.8 ± 4.5 kg/m²) and on weight gain in 1013 elderly subjects (Group 2, age 69.9 ± 2.9 years, BMI 27.4 ± 4.1 kg/m²) during a 3.5‐year follow‐up study. In Group 1, insulin sensitivity, energy expenditure, and substrate oxidation were measured with the hyperinsulinemic euglycemic clamp combined with indirect calorimetry. Results: In Group 1, the Val103Ile genotype was associated with high rates of energy expenditure (63.42 ± 13.40 in eight subjects with the Val103Ile genotype vs. 59.86 ± 7.33 J/kg per minute in 221 subjects with the Val103Val genotype, p = 0.007), high rates of glucose oxidation (8.90 ± 6.15 vs. 6.07 ± 4.38 μmol/kg per minute, p = 0.020), and low levels of free fatty acids (0.45 ± 0.18 vs. 0.56 ± 0.23 mM, p = 0.029) in the fasting state, and with high rates of glucose oxidation during the clamp (18.88 ± 4.63 vs. 17.60 ± 3.24 μmol/kg per minute, p = 0.031). In Group 2, the 103Ile allele was associated with an increase in weight gain during the follow‐up (0.78 ± 3.98 vs. ?0.82 ± 3.98 kg, p = 0.038). Discussion: The Val103Ile polymorphism of the MC4R gene is associated with energy expenditure in humans. Furthermore, it may associate with glucose oxidation, free fatty acid levels, and weight gain.     

Habitat utilisation by fish community in a short-term regulated river reservoir

Short-term streamflow regulation (hydropeaking) affects the ecology of regulated rivers. We examined the longitudinal and temporal changes occurring in fish assemblages in a hydropeaking single river reservoir between two power plants by using electrofishing along the shoreline, hydroacoustics and test fishing in the open water. A longitudinally changing fish community was found among bottom-dwelling fish in the fast-flowing and highly disturbed upstream part of the reservoir progressing to generalists and pelagic fish in the lentic and most stable environment at the downstream end. The fish assemblage showed temporal patterns as fish density increased during night-time darkness and also towards autumn. Our work provides evidence for gradient effects of flow regulation and contributes to awareness of the effects of disturbance (flow/habitat variability) on biological systems.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350–400 and 400–600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L⁻¹ production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4-tert-butylcatechol. Because no activity was detected on L-tyrosine and on l-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60°C. The UV–visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.     

Effects of mean luminance changes on human contrast perception: contrast dependence, time-course and spatial specificity

Background

When we are viewing natural scenes, every saccade abruptly changes both the mean luminance and the contrast structure falling on any given retinal location. Thus it would be useful if the two were independently encoded by the visual system, even when they change simultaneously. Recordings from single neurons in the cat visual system have suggested that contrast information may be quite independently represented in neural responses to simultaneous changes in contrast and luminance. Here we test to what extent this is true in human perception.

Methodology/Principal Findings

Small contrast stimuli were presented together with a 7-fold upward or downward step of mean luminance (between 185 and 1295 Td, corresponding to 14 and 98 cd/m²), either simultaneously or with various delays (50–800 ms). The perceived contrast of the target under the different conditions was measured with an adaptive staircase method. Over the contrast range 0.1–0.45, mainly subtractive attenuation was found. Perceived contrast decreased by 0.052±0.021 (N = 3) when target onset was simultaneous with the luminance increase. The attenuation subsided within 400 ms, and even faster after luminance decreases, where the effect was also smaller. The main results were robust against differences in target types and the size of the field over which luminance changed.

Conclusions/Significance

Perceived contrast is attenuated mainly by a subtractive term when coincident with a luminance change. The effect is of ecologically relevant magnitude and duration; in other words, strict contrast constancy must often fail during normal human visual behaviour. Still, the relative robustness of the contrast signal is remarkable in view of the limited dynamic response range of retinal cones. We propose a conceptual model for how early retinal signalling may allow this.     

CIP2A promotes proliferation of spermatogonial progenitor cells and spermatogenesis in mice

Protein phosphatase 2A (PP2A) is a critical regulator of protein serine/threonine phosphorylation. However, the physiological and developmental roles of different PP2A complexes are very poorly understood. Here, we show that a newly characterized PP2A inhibitory protein CIP2A is co-expressed with ki-67 and with self-renewal protein PLZF in the spermatogonial progenitor cell (SPC) population in the testis. CIP2A and PLZF expression was shown also to correlate Ki-67 expression in human testicular spermatogonia. Functionally, CIP2A mutant mouse testes exhibited smaller number of PLZF-positive SPCs and reduced sperm counts. Moreover, seminiferous tubuli cells isolated from CIP2A mutant mice showed reduced expression of Plzf and other renewal genes Oct-4 and Nanog at mRNA level. However, PLZF-deficient testes did not show altered CIP2A expression. Importantly, spermatogonia-specific restoration of CIP2A expression rescued PLZF expression and sperm production defects observed in CIP2A mutant mice. Taken together, these results reveal first physiological function for an emerging human oncoprotein CIP2A, and provide insights into maintenance of PLZF-positive progenitors. Moreover, demonstration that CIP2A expression can be systematically inhibited without severe consequences to normal mouse development and viability may have clinical relevance regarding targeting of oncogenic CIP2A for future cancer therapies.     

Long Telomeres in Blood Leukocytes Are Associated with a High Risk of Ascending Aortic Aneurysm

Ascending aortic aneurysm is a connective tissue disorder. Even though multiple novel gene mutations have been identified, risk profiling and diagnosis before rupture still represent a challenge. There are studies demonstrating shorter telomere lengths in the blood leukocytes of abdominal aortic aneurysm patients. The aim of this study was to measure whether relative telomere lengths are changed in the blood leukocytes of ascending aortic aneurysm patients. We also studied the expression of telomerase in aortic tissue samples of ascending aortic aneurysms. Relative lengths of leukocyte telomeres were determined from blood samples of patients with ascending aortic aneurysms and compared with healthy controls. Telomerase expression, both at the level of mRNA and protein, was quantified from the aortic tissue samples. Mean relative telomere length was significantly longer in ascending aortic aneurysm blood samples compared with controls (T/S ratio 0.87 vs. 0.61, p<0.001). Expressions of telomerase mRNA and protein were elevated in the aortic aneurysm samples (p<0.05 and p<0.01). Our study reveals a significant difference in the mean length of blood leukocyte telomeres in ascending aortic aneurysm and controls. Furthermore, expression of telomerase, the main compensating factor for telomere loss, is elevated at both the mRNA and protein level in the samples of aneurysmal aorta. Further studies will be needed to confirm if this change in telomere length can serve as a tool for assessing the risk of ascending aortic aneurysm.     

Treatment of bran containing bread by baking enzymes; effect on the growth of probiotic bacteria on soluble dietary fiber extract in vitro

Different ways of treating bran by baking enzymes prior to dough making and the baking process were used to increase the amount of water-soluble dietary fiber (DF) in wheat bread with added bran. Soluble DF was extracted from the bread with water and separated from the digestible material with gastrointestinal tract enzymes and by solvent precipitation. The baking enzyme mixtures tested (xylanase and glucanase/cellulase, with and without lipase) increased the amounts of soluble arabinoxylan and protein resistant to digestion. The isolated fiber was used as a growth substrate for 11 probiotic and intestinal Bifidobacterium strains, for commensal strains of Bacteroides fragilis and Escherichia coli, and for potential intestinal pathogenic strains of E. coli O157:H7, Salmonella typhimurium, and Clostridium perfringens. Fermentation analyses indicated that the tested strains had varying capacity to grow in the presence of the extracted fiber. Of the tested probiotic strains B. longum species generally showed the highest ability to utilize the fiber extracts, although the potential pathogens tested also showed an ability to grow on these fiber extracts. In sum, the enzymes used to improve the baking process for high-fiber bread can also be used to produce in situ soluble fiber material, which in turn can exert prebiotic effects on certain potentially beneficial microbes.     

Microbial Respiration in Arctic Upland and Peat Soils as a Source of Atmospheric Carbon Dioxide

Knowledge on soil microbial respiration (SMR) rates and thus soil-related CO₂ losses from Arctic soils is vital because of the crucial importance of this ecosystem within the global carbon (C) cycle and climate system. Here, we measured SMR from various habitats during the growing season in Russian subarctic tundra by applying two different approaches: ¹⁴C partitioning approach and root trenching. The variable habitats encompassed peat and mineral soils, bare and vegetated surfaces and included both dry and moist ones. The field experiment was complemented by laboratory studies to measure bioavailability of soil carbon and identify sources of CO₂. Differences in bioavailability of soils, measured in the laboratory as basal soil respiration rates, were generally greater than inter-site differences in SMR rates measured in situ, suggesting secondary constraints at field conditions, such as soil C content. There was a tendency towards lower SMR in vegetated peat plateaus compared to upland mineral tundra (on average 137 vs. 185 g CO₂ m^?2 growing season^?1, respectively), but no significant differences were found. Surprisingly, the bare surfaces (peat circles) with 3500-year-old C at the surface exhibited about the largest SMR among all sites as shown by both methods. This was related to the general development of peat plateaus in the region, and uplifting of deeper peat with high C content to the surface during the genesis of peat circles. This observation is particularly relevant for decomposition of deeper peat in vegetated peat plateaus, where soil material similar to the bare surfaces can be found. The data indicate that the large stocks of C stored in permafrost peatlands are principally available for decomposition despite old age.     

The Association of Genotype-Based Inbreeding Coefficient with a Range of Physical and Psychological Human Traits

Across animal species, offspring of closely related mates exhibit lower fitness, a phenomenon called inbreeding depression. Inbreeding depression in humans is less well understood because mating between close relatives is generally rare and stigmatised, confounding investigation of its effect on fitness-relevant traits. Recently, the availability of high-density genotype data has enabled quantification of variation in distant inbreeding in ‘outbred’ human populations, but the low variance of inbreeding detected from genetic data in most outbred populations means large samples are required to test effects, and only a few traits have yet been studied. However, it is likely that isolated populations, or those with a small effective population size, have higher variation in inbreeding and therefore require smaller sample sizes to detect inbreeding effects. With a small effective population size and low immigration, Northern Finland is such a population. We make use of a sample of ∼5,500 ‘unrelated’ individuals in the Northern Finnish Birth Cohort 1966 with known genotypes and measured phenotypes across a range of fitness-relevant physical and psychological traits, including birth length and adult height, body mass index (BMI), waist-to-hip ratio, blood pressure, heart rate, grip strength, educational attainment, income, marital status, handedness, health, and schizotypal features. We find significant associations in the predicted direction between individuals'' inbreeding coefficient (measured by proportion of the genome in runs of homozygosity) and eight of the 18 traits investigated, significantly more than the one or two expected by chance. These results are consistent with inbreeding depression effects on a range of human traits, but further research is needed to replicate and test alternative explanations for these effects.