Identification of the phosphorylation sites in the survival motor neuron protein by protein kinase A

The survival motor neuron (SMN) protein plays an essential role in the assembly of uridine-rich small nuclear ribonuclear protein complexes. Phosphorylation of SMN can regulate its function, stability, and sub-cellular localization. This study shows that protein kinase A (PKA) phosphorylates SMN both in vitro and in vivo. Bioinformatic analysis predicts 12 potential PKA phosphorylation sites in human SMN. Mass spectrometric analysis of a tryptic digest of SMN after PKA phosphorylation identified five distinct phosphorylation sites in SMN (serines 4, 5, 8, 187 and threonine 85). Mutagenesis of this subset of PKA-phosphorylated sites in SMN affects association of SMN with Gemin2 and Gemin8. This result indicates that phosphorylation of SMN by PKA may play a role in regulation of the in vivo function of SMN.     

Ultrastructural investigation on the cell membranes of the vomeronasal organ in the rat: A freeze-etching study

Summary The free surfaces and cell contacts in the epithelia of the vomeronasal organ of the rat were investigated by freeze-etching. The microvilli of receptor cells show a lower density of intramembranous particles (IMP) than the microvilli in the receptor-free epithelium. The ratio between the IMP on P and E-face is approximately 111 in the receptor terminals, and 3.51 in the cilia and microvilli of the receptor-free epithelium. Although atypical in length and only poorly equipped with rootlet fibers, the cilia of the receptor-free epithelium are furnished with typical ciliary necklace structures of up to 10 rows of membrane particles. Differences in the density of IMP on the P-faces of different cilia are probably due to continual ciliogenesis and also due to the different age of cilia in the receptor-free epithelium. Zonulae occludentes show different configurations in the neuroepithelium and in the receptor-free epithelium. In the former, they show a tendency to cross-link and form facet-like patterns, reflecting a constant morphology and relative stability for this apical region. In the receptor-free epithelium the junctional rows of zonulae occludentes display only loosely interconnected networks and a tendency to orient parallel to each other and to the free surface. In addition to zonulae occludentes, typical square aggregations of IMP are observed in the receptor-free epithelium. They are not exclusively restricted to the zone of intensive cell contacts by means of fine interdigitating cell processes, and their function has yet to be identified experimentally.This paper is dedicated to Dr. David G. MoultonPortions of this work are from a thesis in preparation by F.M. Supported by the Deutsche Forschungsgemeinschaft (SFB 114)     

Chiropteran vomeronasal complex and the interfamilial relationships of bats

Within the extant orders of living mammals, the distribution of the vomeronasal organ (VNO) and associated structures is very stable, being universally present in the vast majority or universally absent in cetaceans and sirenians. Chiroptera is the most noteworthy exception, with variation in the absence or presence of the vomeronasal complex occurring even at the species level in some instances. The VNO and/or its component structures, such as the accessory olfactory bulb, were studied in serially sectioned snouts and brains from 114 genera and 292 species representing all extant chiropteran families except Myzopodidae and Antrozoidae. Taxa were scored for the following characters: (1) degree of formation of the vomeronasal epithelial tube, (2) shape of the vomeronasal cartilage, (3) occurrence of the nasopalatine duct, and (4) occurrence of the accessory olfactory bulb. To reconstruct the evolutionary history of the bat vomeronasal complex, the distributions of these four characters were mapped, using the computer program MacClade, onto chiropteran phylogenies in the literature derived from other data sets. In all phylogenies, these four characters exhibit a high degree of homoplasy, only part of which is accounted for by several polymorphic taxa. However, perhaps the most remarkable result is that in the most parsimonious solutions the absence of the vomeronasal epithelial tube and accessory olfactory bulb is identified as primitive for Chiroptera, with both structures reevolving numerous times: such a scenario would be unique to bats among mammals. An alternative, though less parsimonious interpretation, which does not require reevolution of this very complex system, is that a well-developed vomeronasal epithelial tube is primitive for Chiroptera, as in nearly all other orders of mammals, but has been reduced or lost in the majority of families. Explication of the peculiar evolutionary history of the vomeronasal system in bats awaits studies on the adult morphology in the more than 630 species not yet examined and, in particular, on ontogeny, which to date is known for only a handful of taxa.A preliminary account of this research was presented at the Tenth International Bat Research Conference and Twenty-Fifth North American Bat Research Symposium held at Boston University, Massachusetts, on 6–11 August 1995.     

Isolation and characterization from porcine serum of a soluble sulfotransferase responsible for 6-O-sulfation of the galactose residue in 2′-fucosyllactose: Implications in the synthesis of the ligand for L-selectin

A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) to 2'-fucosyllactose (2'-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 microM for PAPS and 15.38 mM for 2'-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl LewisX (sLeX). Results indicated that 3'-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6'-sialyllactose and 6'-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2'-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin.     

Changes in serum leptin, insulin, androstenedione and luteinizing hormone during ovarian cycle in the bat, Taphozous longimanus

Detailed reproductive pattern and associated endocrine characteristics have been documented in only a few species of order Chiroptera. The aim of the present study was to examine the changes in body weight, serum insulin, leptin, androstenedione and luteinizing hormone (LH) concentrations during annual ovarian cycle in the sheath-tailed bat, Taphozous longimanus. Bats were sampled over three years. Leptin, a satiety hormone produced primarily by adipose tissue, provides information to feeding center of the brain about nutritional status, fat mass, appetite and energy expenditure. The circulating concentration of leptin begins to increase from October and attains a peak in December. The peak serum leptin concentration coincides with body weight in November before winter dormancy in December. The serum leptin levels dissociate from body weight during December. The other peaks of serum leptin levels coincide with late stages of the two successive pregnancies. The serum insulin concentration begins to increase from September and attains a peak during December. The insulin concentration remains low from January to August. The circulating androstenedione concentration begins to increase in October, reaching a peak in December. This increase in androstenedione concentration correlated with the period of heavy accumulation of abdominal fat and increase in body weight. There was a sharp decline in androstenedione concentration and body weight in January. The serum LH shows peaks, in November, coinciding with the peaked body weight, the other peaks in January and May, coinciding with ovulation for the two successive pregnancies. The high leptin and insulin levels might be responsible for the maintenance of reproductive response and gonadal function during adverse environmental condition in the winter, while high androstenedione, and associated body weight along with LH might be responsible for maintaining basal gonadal function. We conclude that high leptin, androstenedione and insulin serve, as signal for the reproductive functions in that sufficient body fat are available to meet the caloric demands and maintain normal function during adverse winter conditions.     

Poly-β-hydroxybutyrate (PHB) increases growth performance and intestinal bacterial range-weighted richness in juvenile European sea bass, Dicentrarchus labrax

The bacterial storage polymer poly-β-hydroxybutyrate (PHB) has the potential to be used as an alternative anti-infective strategy for aquaculture rearing. In this research, the effects of (partially) replacing the feed of European sea bass juveniles with PHB were investigated. During a 6-week trial period, the PHB showed the ability to act as an energy source for the fish. This indicated that PHB was degraded and used during gastrointestinal passage. The gut pH decreased from 7.7 to 7.2 suggesting that the presence of PHB in the gut led to the increased production of (short-chain fatty) acids. The diets supplemented with 2% and 5% PHB (w/w) induced a gain of the initial fish weight with a factor 2.4 and 2.7, respectively, relative to a factor 2.2 in the normal feed treatment. Simultaneously, these treatments showed the highest bacterial range-weighted richness in the fish intestine. Based on molecular analysis, higher dietary PHB levels induced larger changes in the bacterial community composition. From our results, it seems that PHB can have a beneficial effect on fish growth performance and that the intestinal bacterial community structure may be closely related to this phenomenon.     

Stepping, strain gating, and an unexpected force-velocity curve for multiple-motor-based transport

BACKGROUND: Intracellular transport via processive kinesin, dynein, and myosin molecular motors plays an important role in maintaining cell structure and function. In many cases, cargoes move distances longer than expected for single motors; there is significant evidence that this increased travel is in part due to multiple motors working together to move the cargoes. Although we understand single motors experimentally and theoretically, our understanding of multiple motors working together is less developed. RESULTS: We theoretically investigate how multiple kinesin motors function. Our model includes stochastic fluctuations of each motor as it proceeds through its enzymatic cycle. Motors dynamically influence each other and function in the presence of thermal noise and viscosity. We test the theory via comparison with the experimentally observed distribution of step sizes for two motors moving a cargo, and by predicting slightly subadditive stalling force for two motors relative to one. In the presence of load, our predictions for travel distances and mean velocities are different from the steady-state model: with high motor-motor coupling, we predict a form of strain-gating, where-because of the underlying motor's dynamics-the motors share load unevenly, leading to increased mean travel distance of the multiple-motor system under load. Surprisingly, we predict that in the presence of small load, two-motor cargoes move slightly slower than do single-motor cargoes. Unpublished data from G.T. Shubeita, B.C. Carter, and S.P.G. confirm this prediction in vivo. CONCLUSIONS: When only a few motors are active, fluctuations and unequal load sharing between motors can result in significant alterations of ensemble function.     

Development of a simple assay protocol for high-throughput screening of Mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Km of the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 microM, 5 microM, and 25 microM, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25 degrees C and 37 degrees C. The Z' factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-microl volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.     

Quantitative trait loci for growth trajectories in Populus

Growth trajectories are a biological process important to plant and animal breeding, and to evolutionary genetic studies. In this article, we report the detection of quantitative trait loci (QTLs) responsible for growth trajectories in poplars that are used as a model system for the study of forest biology. These QTLs were localized on a genetic linkage map of polymorphic markers using a statistical mapping method incorporating growth-curve models. The effects of the QTLs on growth are described as a function of age, so that age-specific changes in QTL effects can be readily projected throughout the entire growth process. The QTLs identified display increased effects on growth when trees age, yet the timing of QTL activation is earlier for stem height than diameter, which is consistent with the ecological viewpoint of canopy competition. The implications of the results for breeding and silviculture are discussed.