A model for integrative study of human gastric acid secretion.

We have developed a unique virtual human model of gastric acid secretion and its regulation in which food provides a driving force. Food stimulus triggers neural activity in central and enteric nervous systems and G cells to release gastrin, a critical stimulatory hormone. Gastrin stimulates enterochromaffin-like cells to release histamine, which, together with acetylcholine, stimulates acid secretion from parietal cells. Secretion of somatostatin from antral and corpus D cells comprises a negative-feedback loop. We demonstrate that although acid levels are most sensitive to food and nervous system inputs, somatostatin-associated interactions are also important in governing acidity. The importance of gastrin in acid secretion is greatest at the level of transport between the antral and corpus regions. Our model can be applied to study conditions that are not yet experimentally reproducible. For example, we are able to preferentially deplete antral or corpus somatostatin. Depletion of antral somatostatin exhibits a more significant elevation of acid release than depletion of corpus somatostatin. This increase in acid release is likely due to elevated gastrin levels. Prolonged hypergastrinemia has significant effects in the long term (5 days) by promoting enterochromaffin-like cell overgrowth. Our results may be useful in the design of therapeutic strategies for acid secretory dysfunctions such as hyper- and hypochlorhydria.     

A Bacterial Phosphatase-Like Enzyme of the Malaria Parasite Plasmodium falciparum Possesses Tyrosine Phosphatase Activity and Is Implicated in the Regulation of Band 3 Dynamics during Parasite Invasion

Eukaryotic parasites of the genus Plasmodium cause malaria by invading and developing within host erythrocytes. Here, we demonstrate that PfShelph2, a gene product of Plasmodium falciparum that belongs to the Shewanella-like phosphatase (Shelph) subfamily, selectively hydrolyzes phosphotyrosine, as shown for other previously studied Shelph family members. In the extracellular merozoite stage, PfShelph2 localizes to vesicles that appear to be distinct from those of rhoptry, dense granule, or microneme organelles. During invasion, PfShelph2 is released from these vesicles and exported to the host erythrocyte. In vitro, PfShelph2 shows tyrosine phosphatase activity against the host erythrocyte protein Band 3, which is the most abundant tyrosine-phosphorylated species of the erythrocyte. During P. falciparum invasion, Band 3 undergoes dynamic and rapid clearance from the invasion junction within 1 to 2 s of parasite attachment to the erythrocyte. Release of Pfshelph2 occurs after clearance of Band 3 from the parasite-host cell interface and when the parasite is nearly or completely enclosed in the nascent vacuole. We propose a model in which the phosphatase modifies Band 3 in time to restore its interaction with the cytoskeleton and thus reestablishes the erythrocyte cytoskeletal network at the end of the invasion process.     

The cyclin-ubiquitin ligase activity of cyclosome/APC is jointly activated by protein kinases Cdk1-cyclin B and Plk

The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, we have purified cyclosome from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, we found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylated the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases was not similar. Each protein kinase could restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity was observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases.     

Arthropod toxins inhibiting Ca2+ and Na+ channels prevent AC‐1001 H3 peptide‐induced apoptosis

Peptide toxins of arthropods are one of the potential sources of bioactive substances. Toxins are able to bind to calcium channels and block them. Ca²⁺ ions play an important role in many cell processes, in particular, in apoptosis. In this work, we study the effect of some arthropod toxins on intracellular processes associated with the induction of apoptosis. Synthetic analogs of U₅‐scytotoxin‐Sth1a, ω‐hexatoxin‐Hv1a, ω‐theraphotoxin‐Hhn2a, and μ‐agatoxin‐Aa1a toxins—inhibitors of calcium L, P, and Q channels and sodium channels were used in the study. Apoptosis was induced by AC‐1001 H3 peptide. We study the effect of toxins on the level of apoptosis, ROS, mitochondrial potential, GSH, and ATP in CHO‐K1 cells. We show that all the tested toxins are able to dose dependently block the induction of apoptosis triggered by AC‐1001 H3 and reduce the level of natural apoptosis in CHO‐K1 cells. Cell incubation with apoptosis inducer AC‐1001 H3 in the presence and absence of toxins causes an increase in the intracellular concentrations of ROS, ATP, and mitochondrial potential and decreases the GSH concentration. The present study reveals the antiapoptotic effect of a number of arthropod peptide toxins. The toxins studied can represent a novel approach used in the treatment of pathologies associated with the activation of apoptotic mechanisms.     

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1^-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1^-/- tp53^-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1^-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.     

Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping

Many enzymes acting on DNA require Mg²⁺ ions not only for catalysis but also to bind DNA. Binding studies often employ Ca²⁺ as a substitute for Mg²⁺, to promote DNA binding whilst disallowing catalysis. The SfiI endonuclease requires divalent metal ions to bind DNA but, in contrast to many systems where Ca²⁺ mimics Mg²⁺, Ca²⁺ causes SfiI to bind DNA almost irreversibly. Equilibrium binding by wild-type SfiI cannot be conducted with Mg²⁺ present as the DNA is cleaved so, to study the effect of Mg²⁺ on DNA binding, two catalytically-inactive mutants were constructed. The mutants bound DNA in the presence of either Ca²⁺ or Mg²⁺ but, unlike wild-type SfiI with Ca²⁺, the binding was reversible. With both mutants, dissociation was slow with Ca²⁺ but was in one case much faster with Mg²⁺. Hence, Ca²⁺ can affect DNA binding differently from Mg²⁺. Moreover, SfiI is an archetypal system for DNA looping; on DNA with two recognition sites, it binds to both sites and loops out the intervening DNA. While the dynamics of looping cannot be measured with wild-type SfiI and Ca²⁺, it becomes accessible with the mutant and Mg²⁺.     

Melanoma central nervous system metastases: An update to approaches,challenges, and opportunities

In February 2018, the Melanoma Research Foundation and the Moffitt Cancer Center hosted the Second Summit on Melanoma Central Nervous System (CNS) Metastases in Tampa, Florida. In this white paper, we outline the current status of basic science, translational, and clinical research into melanoma brain metastasis development and therapeutic management. We further outline the important challenges that remain for the field and the critical barriers that need to be overcome for continued progress to be made in this clinically difficult area.     

Dual effect of nitric oxide on phenoloxidase-mediated melanization

The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO₂ at the active site of PO followed by oxidation of DOPA by NO₂. On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect’s haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.