Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode''s medium, albumin- and bicarbonate-free) or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate). The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the success of sperm capacitation.     

Inappropriateness of Cardiovascular Radiological Imaging Testing; A Tertiary Care Referral Center Study

Aims

Radiological inappropriateness in medical imaging leads to loss of resources and accumulation of avoidable population cancer risk. Aim of the study was to audit the appropriateness rate of different cardiac radiological examinations.

Methods and Principal Findings

With a retrospective, observational study we reviewed clinical records of 818 consecutive patients (67±12 years, 75% males) admitted from January 1-May 31, 2010 to the National Research Council – Tuscany Region Gabriele Monasterio Foundation cardiology division. A total of 940 procedures were audited: 250 chest x-rays (CXR); 240 coronary computed tomographies (CCT); 250 coronary angiographies (CA); 200 percutaneous coronary interventions (PCI). For each test, indications were rated on the basis of guidelines class of recommendation and level of evidence: definitely appropriate (A, including class I, appropriate, and class IIa, probably appropriate), uncertain (U, class IIb, probably inappropriate), or inappropriate (I, class III, definitely inappropriate). Appropriateness was suboptimal for all tests: CXR (A = 48%, U = 10%, I = 42%); CCT (A = 58%, U = 24%, I = 18%); CA (A = 45%, U = 25%, I = 30%); PCI (A = 63%, U = 15%, I = 22%). Top reasons for inappropriateness were: routine on hospital admission (70% of inappropriate CXR); first line application in asymptomatic low-risk patients (42% of CCT) or in patients with unchanged clinical status post-revascularization (20% of CA); PCI in patients either asymptomatic or with miscellaneous symptoms and without inducible ischemia on non-invasive testing (36% of inappropriate PCI).

Conclusion and Significance

Public healthcare system – with universal access paid for with public money – is haemorrhaging significant resources and accumulating avoidable long-term cancer risk with inappropriate cardiovascular imaging prevention.     

Temporal variation in epidermal flavonoids due to altered solar UV radiation is moderated by the leaf position in Betula pendula

The physiological mechanisms controlling plant responses to dynamic changes in ambient solar ultraviolet (UV) radiation are not fully understood: this information is important to further comprehend plant adaptation to their natural habitats. We used the fluorimeter Dualex to estimate in vivo the epidermal flavonoid contents by measuring epidermal UV absorbance (A(375) ) in Betula pendula Roth (silver birch) leaves of different ages under altered UV. Seedlings were grown in a greenhouse for 15 days without UV and transferred outdoors under three UV treatments (UV-0, UV-A and UV-A+B) created by three types of plastic film. After 7 and 13 days, Dualex measurements were taken at adaxial and abaxial epidermis of the first three leaves (L1, L2 and L3) of the seedlings. After 14 days, some of the seedlings were reciprocally swapped amongst the treatments to study the accumulation of epidermal flavonoids in the youngest unfolded leaves (L3) during leaf expansion under changing solar UV environments. A(375) of the leaves responded differently to the UV treatment depending on their position. UV-B increased the A(375) in the leaves independently of leaf position. L3 quickly adjusted A(375) in their epidermis according to the UV they received and these adjustments were affected by previous UV exposure. The initial absence of UV-A+B or UV-A, followed by exposure to UV-A+B, particularly enhanced leaf A(375) . Silver birch leaves modulate their protective pigments in response to changes in the UV environment during their expansion, and their previous UV exposure history affects the epidermal-absorbance achieved during later UV exposure.     

Lipid Rafts and Cytoskeletal Proteins in Placental Microvilli Membranes from Preeclamptic and IUGR Pregnancies

Intrauterine growth restriction (IUGR) and preeclampsia (PE) are leading causes of perinatal and maternal morbidity and mortality. Previously we reported the expression of lipid rafts in classical microvillous membrane (MVM) and light microvillous membrane (LMVM), two subdomains in apical membrane from the human placental syncytiotrophoblast (hSTB), which constitute the epithelium responsible for maternal–fetal transport. Here the aim was to study the raft and cytoskeletal proteins from PE and IUGR. Microdomains from MVM and LMVM were tested with raft markers (placental alkaline phosphatase, lipid ganglioside, and annexin 2) and a nonraft marker (hTf-R). No changes were detected with those markers in whole purified apical membranes in normal, PE, and IUGR pregnancies; however, their patterns of distribution in lipid rafts were different in PE and IUGR. Cholesterol depletion modified their segregation, confirming their presence in lipid rafts, although unlike normal placenta, in these pathologies there is only one type of microdomain. Additionally, the cytoskeleton proteins actin, ezrin, and cytokeratin-7 showed clear differences between normal and pathological membranes. Cytokeratin-7 expression decreased to 50% in PE, and the distribution between LMVM and MVM (~43 and 57%, respectively) changed in both PE and IUGR, in contrast with the asymmetrical enrichment obtained in normal LMVM (~62%). In conclusion, lipid rafts from IUGR and PE have different features compared to rafts from normal placentae, and this is associated with alterations in the expression and distribution of cytoskeletal proteins.     

FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk

Coordination between functionally related adjacent tissues is essential during development. For example, formation of trunk neural crest cells (NCCs) is highly influenced by the adjacent mesoderm, but the molecular mechanism involved is not well understood. As part of this mechanism, fibroblast growth factor (FGF) and retinoic acid (RA) mesodermal gradients control the onset of neurogenesis in the extending neural tube. In this paper, using gain- and loss-of-function experiments, we show that caudal FGF signaling prevents premature specification of NCCs and, consequently, premature epithelial-mesenchymal transition (EMT) to allow cell emigration. In contrast, rostrally generated RA promotes EMT of NCCs at somitic levels. Furthermore, we show that FGF and RA signaling control EMT in part through the modulation of elements of the bone morphogenetic protein and Wnt signaling pathways. These data establish a clear role for opposition of FGF and RA signaling in control of the timing of NCC EMT and emigration and, consequently, coordination of the development of the central and peripheral nervous system during vertebrate trunk elongation.     

Differences in the substrate binding regions of renal organic anion transporters 1 (OAT1) and 3 (OAT3)

This study examined the selectivity of organic anion transporters OAT1 and OAT3 for structural congeners of the heavy metal chelator 2,3-dimercapto-1-propanesulfonic acid (DMPS). Thiol-reactive reagents were also used to test structural predictions based on a homology model of OAT1 structure. DMPS was near equipotent in its ability to inhibit OAT1 (IC(50) = 83 μM) and OAT3 (IC(50) = 40 μM) expressed in Chinese hamster ovary cells. However, removal of a thiol group (3-mercapto-1-propanesulfonic acid) resulted in a 2.5-fold increase in IC(50) toward OAT1 vs. a ～55-fold increase in IC(50) toward OAT3. The data suggested that compound volume/size is important for binding to OAT1/OAT3. The sensitivity to HgCl(2) of OAT1 and OAT3 was also dramatically different, with IC(50) values of 104 and 659 μM, respectively. Consistent with cysteines of OAT1 being more accessible from the external medium than those of OAT3, thiol-reactive reagents reacted preferentially with OAT1 in cell surface biotinylation assays. OAT1 was less sensitive to HgCl(2) inhibition and less reactive toward membrane-impermeant thiol reactive reagents following mutation of cysteine 440 (C440) to an alanine. These data indicate that C440 in transmembrane helix 10 of OAT1 is accessible from the extracellular space. Indeed, C440 was exposed to the aqueous phase of the presumptive substrate translocation pathway in a homology model of OAT1 structure. The limited thiol reactivity in OAT3 suggests that the homologous cysteine residue (C428) is less accessible. Consistent with their homolog-specific selectivities, these data highlight structural differences in the substrate binding regions of OAT1 and OAT3.     

Mechanisms involved in the long-term modulation of tyrosine hydroxylase by endothelins in the olfactory bulb of normotensive rats

The olfactory bulbs play a relevant role in the interaction between the animal and its environment. The existence of endothelin-1 and -3 in the rat olfactory bulbs suggests their role in the control of diverse functions regulated at this level. Tyrosine hydroxylase, a crucial enzyme in catecholamine biosynthesis, is tightly regulated by short- and long-term mechanisms. We have previously reported that in the olfactory bulbs endothelins participate in the short-term tyrosine hydroxylase regulation involving complex mechanisms. In the present work we studied the effect of long-term stimulation by endothelins on tyrosine hydroxylase in the rat olfactory bulbs. Our findings show that endothelin-1 and -3 modulated catecholaminergic transmission by increasing enzymatic activity. However, these peptides acted through different receptors and intracellular pathways. Endothelin-1 enhanced tyrosine hydroxylase activity through a super high affinity ET(A) receptor and cAMP/PKA and CaMK-II pathways, whereas, endothelin-3 through a super high affinity atypical receptor coupled to cAMP/PKA, PLC/PKC and CaMK-II pathways. Endothelins also increased tyrosine hydroxylase mRNA and the enzyme total level as well as the phosphorylation of Ser 19, 31 and 40 sites. Furthermore, both peptides stimulated dopamine turnover and reduced its endogenous content. These findings support that endothelins are involved in the long-term regulation of tyrosine hydroxylase, leading to an increase in the catecholaminergic activity which might be implicated in the development and/or maintenance of diverse pathologies involving the olfactory bulbs.