Distribution of E-cadherin and β-catenin in relation to cell maturation and cell extrusion in rat and mouse small intestines

In the small intestines, cell renewal from stem cells present in the crypts is balanced by cell extrusion from the tips of the villi. The mechanism by which extrusion occurs is unknown. Recent in vitro data suggested that loss of E-cadherin could contribute to cell extrusion and induction of programmed cell death (PCD) in mouse small intestinal epithelium. We have studied if this also occurs in the intact rodent small intestine. Our results confirm that extruded cells are negative for E-cadherin. However, loss of the E-cadherin-interacting protein β-catenin preceded both extrusion and loss of E-cadherin. Thus, all extruded cells as well as all cells in the process of extrusion lacked staining for β-catenin. Moreover, almost 80% of all cells undergoing programmed cell death, as detected by the TUNEL reaction, lacked β-catenin whereas over 70% of such cells were positive for E-cadherin. However, most cells lacking β-catenin did not display signs of PCD as detected by the TUNEL method or by staining for active caspase-3. Therefore, these results suggest that loss of β-catenin precedes the onset of programmed cell death, loss of E-cadherin and extrusion from the villi.     

Effect of cold adaptation of α-and β-adrenergic responses of blood pressure in hindlimb and small intestine in situ and systemic blood pressure in rabbits

Changes in the main parameters of α-and β-adrenergic responses, sensitivity to agonists (EC ₅₀) and maximum response (P _m) of hindlimb and small intestinal blood pressure in situ and systemic blood pressure were studied in rabbits adapted to cold for 1–30 days (daily exposures to ?10°C for 6 h). The responses to phenylephrine, noradrenaline, adrenaline, clonidine (α-agonists), and isopropylnoradrenaline (β-agonist) corresponded to the equation p = (P _m A ⁿ )/(EC ₅₀ ⁿ + A ⁿ ) (1) with n = 1 and n = 2, respectively. Cold adaptation induced reciprocal changes in the response of both EC ₅₀ and P _m to α-agonists and in the response of P _m alone to isopropylnoradrenaline. The significant differences of the parameters from control observed during the first 5 days of adaptation gradually decreased by day 30. After 10 days of adaptation, the efficiency (E = P _m/2EC ₅₀) of response to α-and β-agonists of adrenoceptors significantly increased.     

Do small and large luteal cells of the sheep interact in the production of progesterone?

Corpora lutea from cyclic ewes were dissociated by collagenase and trypsin/EGTA treatments, and enriched fractions of small and large luteal cells were prepared on gradients of Ficoll. These fractions were incubated separately or remixed before incubation. Colchicine, cytochalasin B and the calcium channel-blocker verapamil significantly reduced progesterone production by both small and large luteal cell fractions, while isoprenaline stimulated an increase in progesterone production by large luteal cell fractions only. When fractions of small and large luteal cells were remixed, no more and no less progesterone was produced than would have been predicted from equivalent fractions incubated separately. There was therefore no evidence of synergism between small and large luteal cells in the production of progesterone. Prostaglandin F-2 alpha, which can inhibit LH-stimulated progesterone production by ovine luteal tissue in vitro, had no effect on LH-stimulated progesterone production by small luteal cell fractions, but significantly inhibited that by enriched fractions of large luteal cells. Since large luteal cell fractions were contaminated with small luteal cells, which are probably responsible for the progesterone-secretory response of these fractions to LH, it was concluded that the inhibition of LH-stimulated progesterone production by small luteal cells is dependent on the presence of large luteal cells. Oxytocin added to large and small luteal cell fractions did not affect progesterone production by either fraction. It was therefore concluded that the inhibitory action of PGF-2 alpha on LH-stimulated progesterone production may require the interaction of large and small luteal cells, but that oxytocin is not likely to be an intermediary in this interaction.     

Detection of trypanosomes in small ruminants and pigs in western Kenya: important reservoirs in the epidemiology of sleeping sickness?

Background

Trypanosomosis is a major impediment to livestock farming in sub-Saharan Africa and limits the full potential of agricultural development in the 36 countries where it is endemic. In man, sleeping sickness is fatal if untreated and causes severe morbidity. This study was undertaken in western Kenya, an area that is endemic for both human and livestock trypanosomosis. While trypanosomosis in livestock is present at high levels of endemicity, sleeping sickness occurs at low levels over long periods, interspersed with epidemics, underscoring the complexity of the disease epidemiology. In this study, we sought to investigate the prevalence of trypanosomes in small ruminants and pigs, and the potential of these livestock as reservoirs of potentially human-infective trypanosomes. The study was undertaken in 5 villages, to address two key questions: i) are small ruminants and pigs important in the transmission dynamics of trypanosomosis? and ii), do they harbour potentially human infective trypanosomes? Answers to these questions are important in developing strategies for the control of both livestock and human trypanosomosis.

Results

Eighty-six animals, representing 21.3% of the 402 sampled in the 5 villages, were detected as positive by PCR using a panel of primers that identify trypanosomes to the level of the species and sub-species. These were categorised as 23 (5.7%) infections of T. vivax, 22 (5.5%) of T. simiae, 21 (5.2%) of the T. congolense clade and 20 (5.0%) of T. brucei ssp. The sheep was more susceptible to trypanosome infection as compared to goats and pigs. The 20 T. brucei positive samples were evaluated by PCR for the presence of the Serum Resistance Associated (SRA) gene, which has been linked to human infectivity in T. b. rhodesiense. Three samples (one pig, one sheep and one goat) were found to have the SRA gene. These results suggest that sheep, goats and pigs, which are kept alongside cattle, may harbour human-infective trypanosomes.

Conclusion

We conclude that all livestock kept in this T. b. rhodesiense endemic area acquire natural infections of trypanosomes, and are therefore important in the transmission cycle. Sheep, goats and pigs harbour trypanosomes that are potentially infective to man. Hence, the control of trypanosomosis in these livestock is essential to the success of any strategy to control the disease in man and livestock.

     

Spatial and temporal diversity of small shallow waters in river Lužnice floodplain

To propose a concept of their mutual diversity, twenty-nine permanent shallow floodplain pools and oxbows in the river Lužnice floodplain were analysed for area, depth, shape, flooding, and shading by terrestrial vegetation, and sampled in all seasons for their water chemistry, phytoplankton composition and biomass, and zooplankton composition. The sites are regularly flooded, eutrophic, and often shaded by surrounding vegetation. Cryptophyceae, Chrysophyceae and Euglenophyceae dominated the phytoplankton, while Cyanophytes were rare. Within the rich zooplankton assemblage (63 species), cladocerans and rotifers dominated. Correlation matrices and multivariate analyses indicated that shaded and relatively deeper sites had lower oxygen saturation and higher concentrations of PO₄–P and NH₄–N. Shade and relative depth correlated negatively with phytoplankton biomass and number of phytoplankton taxa, and positively with Cryptophytes and large cladocerans—thus indicating poor mixing, poor light availability and low fish pressure on herbivores. Decomposition of leaf litter increased oxygen consumption, while shade from terrestrial vegetation restricted photosynthesis and decreased oxygen production. Larger sites were more species-rich in phytoplankton and supported Euglenophyceae, green algae and rotifers.     

Structural and mechanistic implications of metal binding in the small heat-shock protein αB-crystallin

The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.     

7,7′-Diazaindirubin—A small molecule inhibitor of casein kinase 2 in vitro and in cells

Aza- and diaza-bisindoles were synthesized by coupling of 7-azaisatin, 7-azaoxindol, 7-azaindoxyl acetate, and their non-aza counterparts, respectively. Whereas 7,7′-diazaindigo (10) and 7,7′-diazaisoindigo (11) did not show antiproliferative activity in several human tumor cell lines up to 100 μM, 7-azaindirubin (12) and 7′-azaindirubin (13) were more active than the parent molecule, indirubin, in LXFL529L cells (human large cell lung tumor xenograft), and 7,7′-diazaindirubin (14) was exhibiting substantially enhanced growth inhibitory activity in these cells. In the NCI 60 cell line panel, 14 displayed antiproliferative activity preferentially in certain melanoma and non-small cell lung cancer cells. In contrast to the potent serine/threonine/tyrosine kinase inhibition observed for indirubins, kinase inhibition profiling of 14 in 220 kinases revealed largely a loss of kinase inhibitory activity towards most kinases, with retained inhibitory activity for just a few kinases. At 1 μM concentration, especially casein kinases CK1γ3, CK2α, CK2α2, and SIK were inhibited by more than 50%. In cell-based assays, 14 markedly affected CK2-mediated signaling in various human tumor cells. In MCF7 cells, 14 induced cell cycle arrest at G1 and G2/M and apoptosis, whereas CK2-deficient MCF7 cells were resistant. These findings reveal a novel key mechanism of action for 14, suggesting primarily CK2 inhibition to be causally related to growth inhibition of human tumor cells.     

Postnatal development of β-galactosidase activity in the small intestine of the rat. Effect of adrenalectomy and diet

1. β-Galactosidase activity was studied in homogenates of the proximal and distal thirds of the small intestine from adult and infant rats. o-Nitrophenyl β-d-galactoside served as the substrate. 2. Activity in suckling rats is highest in the distal part of the small intestine. 3. The pH optimum was 3·5 in the distal third of the small intestine in rats aged 5 and 15 days, whereas in the proximal third the maximum was not clearly defined. 4. Activity was higher in both thirds in newborn than in adult rats, expressed per wet wt. or per wt. of protein. In the proximal third activity continually decreases with age, whereas in the distal part there is a rise up to day 15 and then a sudden decrease. Total β-galactosidase activity changes very little in the proximal third during postnatal development; the greatest changes occur in the distal third. 5. Adrenalectomy performed on day 15 postnatally slows down the decrease in β-galactosidase activity, particularly in the distal part. 6. Feeding a lactose diet to infant rats from day 14 postnatally in the presence of the mother rat also slows down the decrease in β-galactosidase activity and this is not found with a diet containing glucose and galactose instead of lactose.     

Identification and characterization of small cells in the adult pancreas: potential progenitor cells?

In this report we describe the identification of a novel cell type in human and canine pancreas using tissue culture techniques. These cells, representing less than 1% of total islet cells, are of a small size (7-10 microm) and highly quiescent. They display a fairly immature morphology, which is characterized by a weakly developed protein synthesis machinery, a few mitochondria and a small number of neuroendocrine granules. These cells, which we have termed "small cells," are usually organized into small clusters, which can be identified within the islets of predominantly small size. They can also be collected as separate structures from preparations of freshly isolated islets. Immunohistochemically, small cells are positive for PDX-1, synaptophysin, insulin, glucagon, somatostatin, pancreatic polypeptide, alpha-fetaprotein and Bcl-2 and negative for cytokeratin 19 and nestin. Insulin secretion studies demonstrated that these cells secrete insulin in a glucose-responsive fashion, although do not respond to secretagogues such as IBMX and arginine as do mature beta cells. Although this study does not provide evidence of the proliferative and differentiation potential of small cells, their immature morphology, along with a small size and quiescence, let us hypothesize that these cells may serve as progenitors contributing to the islet growth.     

Rates of change in physical and chemical lake variables – are they comparable between large and small lakes?

Changes over time in 16 physical and chemical variables were analysed and compared between Sweden’s largest lakes, Vättern and Vänern, and 48 smaller Swedish reference lakes during spring over the period 1984–2003. The rates of changes varied substantially among lakes and among variables, and they were clearly influenced by changes in both climate and atmospheric deposition. Rates of change of variables associated with atmospheric deposition such as sulphate concentrations were dependent on lake morphometry. This also applied to the rates of change of variables associated with climate change effects in the catchment such as calcium and magnesium concentrations. However, climate change effects could also be comparable between large and small lakes. Rates of change in physical and chemical variables directly influenced by the climate via the lake water surface, e.g., surface water temperature, and variables associated with the spring phytoplankton development such as phosphate–phosphorus and nitrate–nitrogen concentrations, were similar and therefore independent of lake morphometry. This study shows that climate change effects that act via the lake surface can be of the same order of magnitude among large and small lakes, but climate change effects that act via the catchment differ substantially in large lakes. It is essential to differentiate between these two types of climate effects in order to assess the impacts of climate change and the adaptation and vulnerability of lake ecosystems.     

Mesenchymal stem cell-derived small extracellular vesicles in the treatment of human diseases:Progress and prospect

Mesenchymal stem cells(MSCs)are self-renewing,multipotent cells that could differentiate into multiple tissues.MSC-based therapy has become an attractive and promising strategy for treating human diseases through immune regulation and tissue repair.However,accumulating data have indicated that MSC-based therapeutic effects are mainly attributed to the properties of the MSC-sourced secretome,especially small extracellular vesicles(sEVs).sEVs are signaling vehicles in intercellular communication in normal or pathological conditions.sEVs contain natural contents,such as proteins,mRNA,and microRNAs,and transfer these functional contents to adjacent cells or distant cells through the circulatory system.MSC-sEVs have drawn much attention as attractive agents for treating multiple diseases.The properties of MSC-sEVs include stability in circulation,good biocompatibility,and low toxicity and immunogenicity.Moreover,emerging evidence has shown that MSC-sEVs have equal or even better treatment efficacies than MSCs in many kinds of disease.This review summarizes the current research efforts on the use of MSC-sEVs in the treatment of human diseases and the existing challenges in their application from lab to clinical practice that need to be considered.     

The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins

Small heat-shock proteins (sHsps), such as αB-crystallin, are one of the major classes of molecular chaperone proteins. In vivo, under conditions of cellular stress, sHsps are the principal defence proteins that prevent large-scale protein aggregation. Progress in determining the structure of sHsps has been significant recently, particularly in relation to the conserved, central and β-sheet structured α-crystallin domain (ACD). However, an understanding of the structure and functional roles of the N- and C-terminal flanking regions has proved elusive mainly because of their unstructured and dynamic nature. In this paper, we propose functional roles for both flanking regions, based around three properties: (i) they act in a localised crowding manner to regulate interactions with target proteins during chaperone action, (ii) they protect the ACD from deleterious amyloid fibril formation and (iii) the flexibility of these regions, particularly at the extreme C-terminus in mammalian sHsps, provides solubility for sHsps under chaperone and non-chaperone conditions. In the eye lens, these properties are highly relevant as the crystallin proteins, in particular the two sHsps αA- and αB-crystallin, are present at very high concentrations.     

Paedomorphosis in two small species of toothed whales (Odontoceti): how and why?

To evaluate and assess the ontogenetic background for paedomorphosis in phocoenids, samples of 144 harbour porpoises, 81 white‐beaked dolphins, and 130 Commerson's dolphins were compared in terms of the development of epiphyseal fusion, cranial suture fusion, and ontogeny of cranial shape. Harbour porpoises and Commerson's dolphins terminated growth and development of all investigated traits sooner than white‐beaked dolphins, leading to lesser degrees of fusion of skeletal elements and less postnatal allometric development. The latter occurred even though shape in the two paedomorphic species developed at twice the rate relative to the size of white‐beaked dolphins. These observations imply that progenetic evolution has occurred convergently in phocoenid and Cephalorhynchus ancestors. The truncated ontogenies allow sexual maturity to be attained earlier and provide a greater reproductive potential. Both species inhabit similar temperate productive habitats and, hence, ecological factors are proposed to have supplied the selection pressures leading to progenesis. Constant prey availability must be a prerequisite for the observed phenomena because frequent food‐intake is necessitated by the limited capacity for energy storage and high heat‐loss entailed by the resulting small body sizes. Progenesis has rarely been proposed in mammal species. This may reflect rarity or that mammalian expressions of progenesis are less obvious. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 278–295.     

Effects of AlF4 – and ATP on intracellular calcium dynamics of crypt epithelial cells in mouse small intestine

Previous digital imaging analysis of intracellular calcium ion ([Ca2+]i) dynamics in the crypt of the small intestine showed little response by most columnar cells to cholinergic and adrenergic agonists. The objective of the present study was to demonstrate whether G-protein activators and other transmitters elicit [Ca2+]i changes in crypt cells. We used digital imaging to analyze spatiotemporal dynamics of [Ca2+]i in Fura-2/AM-loaded isolated crypts of mouse duodenum and ileum. A1F4- increased [Ca2+]i in crypt columnar cells. In many cases, we observed [Ca2+]i oscillations, which were synchronized throughout the entire crypt. The oscillations were blocked by octanol. ATP, but not adenosine, caused a [Ca2+]i increase in middle crypt-regions of the duodenum and upper regions of the ileum, and the [Ca2+]i wave propagated towards the crypt bottom. The ATP-induced [Ca2+]i increase was prevented by pretreatment with thapsigargin or suramin, but not by La3+ or an extracellular Ca(2+)-free environment. Neither dopamine, 5-hydroxytryptamine (5-HT), histamine, vasoactive intestinal peptide, substance P. cholera toxin, nor guanylin had significant effects. The [Ca2+]i dynamics of Paneth cells were independent of the AlF4(-)-induced synchronous oscillations of columnar cells and of the ATP-induced [Ca2+]i wave. In conclusion, crypt columnar cells have [Ca2+]i-dependent intracellular signaling mechanisms that are linked with G proteins, and by which the cells communicate with each other. ATP elicited [Ca2+]i mobilization from columnar cells via P2 receptors, although some regional differences were noted between the duodenum and ileum.     

The inhibition of hexose transport and metabolism by small amounts of adenosine 5′-triphosphate in isolated rat adipocytes

The effects of ATP on glucose transport and metabolism were studied in rat adipocytes. Over a concentration range of 10–250 μm, ATP was found to inhibit several aspects of adipocyte glucose metabolism, particularly when stimulated by insulin. Much of the effect of ATP on glucose metabolism appeared related to impairment of glucose transport, reflected by inhibition of both basal and insulin-stimulated rates of 3-O-methylglucose transport. ATP inhibited the V of insulin-stimulated 3-O-methylglucose transport, but had no effect on the K_m. The inhibitory effects of ATP were much less apparent when cells were preincubated with insulin, suggesting that ATP inhibited only the components of hexose transport not yet activated by the hormone. At very high medium glucose concentrations, where transport was no longer rate limiting for metabolism, there was no inhibition of glucose oxidation by 250 μm ATP. However, when hexose transport was blocked with cytochalasin B (50 μm), a small inhibitory effect of ATP persisted on basal and insulin-stimulated glucose and fructose oxidation, suggesting that intracellular metabolism was impaired. The mechanism of the intracellular effect did not appear to be caused by uptake of exogenous ATP. These studies provide further evidence that energy metabolism may play an important role in the regulation of facilitated glucose transport.     

Populations and production of fish in two small tributaries of the Paraná River,Paraná, Brazil

Mean biomass (153-1) and production (P) of fish in two small tributaries of the Paraná River (Paraná, Brazil) were 61 kg ha^–1 and 48 kg ha^–1 yr^–1 in the Caracu River and 29 kg ha^–1 and 26 kg ha^–1 yr^–1 in the Agua do Rancho River, respectively. Matrix correlation analysis revealed high positive correlations of both 153-2 and P to maximum depth and hiding places and, at a lower level of significance, to mean depth, pH and oxygen level. Lower 153-3 and P values were found in the Agua do Rancho River, whose valley has retained a more natural character, rich canopy and scarcity of macrophytes, but also lower conductivity and nitrogen and phosphate levels than those in the Caracu River.Address for correspondence     

Comparison of the homologous carboxy-terminal domain and tail of α-crystallin and small heat shock protein

The C-terminal domain and tail, which is the most conserved region of the -crystallin/small heat shock protein (HSP) family, was obtained from rat A-crystallin, bovine B-crystallin and mouse HSP25. All three domains have primarily -sheet conformation and less than 10% of -helix, like the proteins from which they are derived. Whereas the C-terminal part of A-crystallin forms dimers or tetramers, the corresponding regions of B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the -crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.Abbreviations A2Dt residues 64–173 of rat -crystallin - B2Dt residues 70–175 of bovine B-crystallin - bp base pair - HSP2Dt residues 92–209 of HSP25 - HSP(s) heat shock protein(s) - HSP25 mouse small HSP - PCR polymerase chain reaction - PMSF phenylmethylsulfonyl chloride - SDS sodium dodecyl sulfate; polyacrylamide - WSF water-soluble fraction     

The effect of fetal hypophysectomy and fetal death in gilts with small litters on concentrations of relaxin,progesterone and estradiol-17β

During late pregnancy concentrations of relaxin, progesterone (P) and estradiol-17β (E) in maternal plasma were measured in gilts with small litters of intact, hypophysectomized, partially hypophysectomized or dead fetuses and in gilts with litters of normal fetuses and numbers. To achieve a small litter size at parturition all but one or two fetuses were killed at surgery at Day 30 to 40 of gestation. Fetal hypophysectomy or sham procedures were attempted on Day 90 to 95. Gestation was prolonged in gilts carrying hypophysectomized or partially hypophysectomized fetuses (P<0.01). Lactation and farrowing did not occur if hypophysectomy was complete. Basal concentrations of E in plasma were lower (P<0.01), basal P appeared higher and basal relaxin was unchanged in gilts carrying hypophysectomized or dead fetuses as compared to gilts with intact fetuses. Near the end of pregnancy the concentration of E was 119.8 pg/ml in gilts with the normal number of fetuses, 32.6 pg/ml in the group with hypophysectomized fetuses, and 7.3 pg/ml in gilts with dead fetuses. The relaxin peak occurred near term in control pigs and was delayed in the groups with hypophysectomized and partially hypophysectomized fetuses. The concentration of relaxin at the peak in gilts with normal sized litters was 181.4±75.8 ng/ml as compared with 25.3±16.0 ng/ml in gilts with partially hypophysectomized fetuses and was 9.5±1.4 ng/ml in gilts with hypophysectomized fetuses and 10.6±3.3 ng/ml in gilts with one or two fetuses. In gilts with intact or partially hypophysectomized fetuses, or litters containing both types, which came into labor, the patterns of P and E were similar. In gilts with hypophysectomized fetuses, P and E at term showed little change from basal concentrations. The results confirm that the fetus influences basal concentrations of E and possibly P in late normal gestations. In addition, the presence of the fetal pituitary is associated with the peak in relaxin expected at term. These associations are likely to be related to pituitary function and/or the mass of the conceptus. Fetal hypophysectomy is clearly associated with maternal concentrations of P and E at Day 114 that are different from those in normal sows, suggesting that these two hormones may have an effect on the initiation of parturition in the pig.