Diet-independent remodeling of cellular membranes precedes seasonally changing body temperature in a hibernator

Polyunsaturated fatty acids (PUFA) have a multitude of health effects. Their incorporation into membrane phospholipids (PL) is generally believed to depend directly on dietary influx. PL influence transmembrane protein activity and thus can compensate temperature effects; e.g. PL n-6 PUFA are thought to stabilize heart function at low body temperature (T(b)), whereas long chain (>C18) n-3 PUFA may boost oxidative capacity. We found substantial remodeling of membranes in free-living alpine marmots which was largely independent of direct dietary supply. Organ PL n-6 PUFA and n-6 to n-3 ratios were highest at onset and end of hibernation after rapid increases during a brief transitional period prior to hibernation. In contrast, longer chain PL n-3 PUFA content was low at end of summer but maximal at end of hibernation. After termination of hibernation in spring, these changes in PL composition were rapidly reversed. Our results demonstrate selective trafficking of PUFA within the body, probably governed by a circannual endogenous rhythm, as hibernating marmots were in winter burrows isolated for seven months from food and external cues signaling the approaching spring. High concentrations of PL n-6 PUFA throughout hibernation are in line with their hypothesized function of boosting SERCA 2a activity at low T(b). Furthermore, we found increasing rate of rewarming from torpor during winter indicating increasing oxidative capacity that could be explained by the accumulation of long-chain PL n-3 PUFA. It may serve to minimize the time necessary for rewarming despite the increasing temperature range to be covered, because rewarming is a period of highest metabolic rate and hence production of reactive oxygen species. Considering the importance of PUFA for health our results may have important biomedical implications, as seasonal changes of T(b) and associated remodeling of membranes are not restricted to hibernators but presumably common among endothermic organisms.     

Imaging long-term fate of intramyocardially implanted mesenchymal stem cells in a porcine myocardial infarction model

The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [(18)F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [(18)F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33-35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC-associated [(18)F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [(18)F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI.     

Treatment of ocular motility disturbances in Graves' disease with botulinum toxin A

The aim of this study was to evaluate the efficacy of botulinum toxin-A injections on motility disturbance in patients with dysthyroid eye disease. In 36 patients (52 orbits) with active phase of Grave's ophthalmopathy with motility disturbance, botulinum toxin-A injections were applied. Ocular motility was measured before and after treatment in four main directions (elevation, depression, abduction and adduction) with F?rster perimeter. In all patients 20 units of botulinum toxin-A in one single injection was applied in the projection of the inferior rectus muscle. Statistically, the degree of upgaze increased notably in all three groups, but mostly in the first group where it amounted up to 5.8-6.0 (z = 10.0;p = 0.68). We could not prove notable increase of the motility grade in the adduction in none of the groups of the tested patients. Due to the fact that eyeball motility can be objectively measured, effect of the applied therapy can be clearly evaluated.     

Changes in testicular and nipple volume related to age and seasonality in Japanese macaques (Macaca fuscata), especially in the pre- and post-pubertal periods

We investigated, longitudinally and cross-sectionally, age and seasonal change in both the testis and nipple volume of Japanese macaques (Macaca fuscata) in relation to concentration profiles of gonadal steroids: testosterone (T) in males and progesterone (P) in females. Testicular volume (TV) and nipple volume (NV) showed rapid growth at puberty, 4.5 and 3.5 years of age in males and females, respectively, but in both sexes there were precocious individuals. The testis as a whole matures at about 10 years of age. TV change is closely related to T concentration profile. The pattern of TV change is composed of maturation and seasonal effects, with individual variation evident mainly in the latter. Some individuals show a simple pattern consisting of one peak in the breeding season (from summer to winter) and one trough in the non-breeding season. Other individuals exhibit a more complicated pattern composed of two or more peaks and troughs before and during the breeding season. The nipple matures at about 7 years but it is difficult to determine the exact maturational age as there are many confounding factors relating to NV. NV shows seasonal fluctuations similar to that of TV. Many animals have periods of substantial growth whereas others do not. The NV in adults from 10 to 25 years does not appear to change much with age, but animals older than 25 years of age have significantly smaller nipples. Seasonal fluctuation in NV mirrors that of the P level. Considered to be controlled by estrogen and P, the NV is a good indicator of the physiological status of reproduction, with its peak about 2 weeks earlier than that of P, that is, at the mid-follicular phase. NV and P level show a similar pattern in pregnancy; from conception, indicated by a P peak, NV and P concentration first decrease, then they increase until peri-parturition and slowly decrease again until the next breeding season.This revised version was published online in April 2005 with corrections to the cover date of the issue.     

Ethiopian mitochondrial DNA heritage: tracking gene flow across and around the gate of tears

Approximately 10 miles separate the Horn of Africa from the Arabian Peninsula at Bab-el-Mandeb (the Gate of Tears). Both historic and archaeological evidence indicate tight cultural connections, over millennia, between these two regions. High-resolution phylogenetic analysis of 270 Ethiopian and 115 Yemeni mitochondrial DNAs was performed in a worldwide context, to explore gene flow across the Red and Arabian Seas. Nine distinct subclades, including three newly defined ones, were found to characterize entirely the variation of Ethiopian and Yemeni L3 lineages. Both Ethiopians and Yemenis contain an almost-equal proportion of Eurasian-specific M and N and African-specific lineages and therefore cluster together in a multidimensional scaling plot between Near Eastern and sub-Saharan African populations. Phylogeographic identification of potential founder haplotypes revealed that approximately one-half of haplogroup L0–L5 lineages in Yemenis have close or matching counterparts in southeastern Africans, compared with a minor share in Ethiopians. Newly defined clade L6, the most frequent haplogroup in Yemenis, showed no close matches among 3,000 African samples. These results highlight the complexity of Ethiopian and Yemeni genetic heritage and are consistent with the introduction of maternal lineages into the South Arabian gene pool from different source populations of East Africa. A high proportion of Ethiopian lineages, significantly more abundant in the northeast of that country, trace their western Eurasian origin in haplogroup N through assorted gene flow at different times and involving different source populations.     

Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics

Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C(18)) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C(18)-SCX-C(18) stacked disks significantly improved protein identification by LC-MS/MS for an E. coli protein digest and by MALDI-MS for a 12 standard protein digest. Sequential fractionation based on C(18)- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C(18)-SCX-StageTips into a 96-well plate (StagePlate). Fractions were collected into other C(18)-StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrospray-MS/MS or MALDI-MS.     

Engineering of Substrate Selectivity for Tissue Factor·Factor VIIa Complex Signaling through Protease-activated Receptor 2

The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF·FVIIa complex and by FXa in the ternary TF·FVIIa·FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF·FVIIa-Xa complex normally but were severely impaired in binary TF·FVIIa·PAR2 signaling. The residues identified were located in the model-predicted S2′ pocket of FVIIa, and complementary PAR2 P2′ Leu-38 replacements demonstrated that the P2′ side chain was indeed crucial for PAR2 cleavage by TF·FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF·FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactor-associated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.     

Sleep Profile and Longevity in Three Generations of a Family of Captive Bolivian Aotus

The daily total sleep time (TST) of the only nocturnal simian primate, Aotus spp., remains little studied under controlled conditions. We conducted 3 experiments in 4 owl monkeys (Aotus sp.), aged 1–27+ yr, to determine the daily TST. We housed 3 their—a nuclear family—in 1 cage and the remaining senescent female in an adjacent separate cage. We monitored their activity-sleep pattern longitudinally for 15–20 d via actigraphy: by tagging an acclerometer-type miniature transmitter (Actiwatch-MINIMITTER) sensitive to omnidirectional movement, to the owl monkey's neck. The TST (9.5–12.5 h) was ca. 4.5–7 h less than the 17 h Perachio reported for owl monkeys in 1971 by polysomnography, under similar 12-h-light; 12-h dark conditions. Our finding corroborates well with the TST for other nonhuman primates. Four members of the Aotus colony at our facility reached 20 yr in captivity; the oldest (wild-born female) is still living at >27 yr, and the second oldest (captive-born male), is 23 years now.