Effects of hyperbaric oxygen therapy on the viability of irradiated soft head and neck tissues in mice.

OBJECTIVES: Hyperbaric oxygen therapy (HBOT) is used clinically in irradiation-induced injury to healthy tissues, but the effectiveness and working mechanism remain unclear. This study examined the effects of HBOT on irradiated salivary glands and tongue in a mouse model. MATERIALS AND METHODS: Mice were irradiated with a single dose (15 Gy) in the head and neck region and subjected to HBOT, either before or after irradiation. During the course of the treatments, salivary flow rates were measured and at different time points after radiation (2, 6, 10 and 24 weeks), salivary glands and tongue were harvested and (immuno) histochemically analysed. RESULTS: Proliferation and blood vessel density in salivary glands were enhanced by HBOT in the medium term (10 weeks after irradiation), while salivary flow rates were not influenced. In the long term, irradiation-induced proliferation in the muscle tissue of the tongue was decreased by HBOT. CONCLUSION: Hyperbaric oxygen therapy (HBOT) appears to stimulate regeneration or protection of salivary gland tissue following radiation therapy. Possible implications of the effect of HBOT on muscle tissue of the tongue for the prevention of dysphagia and trismus are discussed. This study provides insights on the cellular changes after HBOT and encourages further research on this topic to achieve a better implementation of the therapy in humans.

A central role for P48/45 in malaria parasite male gamete fertility.

Fertilization and zygote development are obligate features of the malaria parasite life cycle and occur during parasite transmission to mosquitoes. The surface protein PFS48/45 is expressed by male and female gametes of Plasmodium falciparum and PFS48/45 antibodies prevent zygote development and transmission. Here, gene disruption was used to show that Pfs48/45 and the ortholog Pbs48/45 from a rodent malaria parasite P. berghei play a conserved and important role in fertilization. p48/45- parasites had a reduced capacity to produce oocysts in mosquitoes due to greatly reduced zygote formation. Unexpectedly, only male gamete fertility of p48/45- parasites was affected, failing to penetrate otherwise fertile female gametes. P48/45 is shown to be a surface protein of malaria parasites with a demonstrable role in fertilization.

Dissociation of the complex between the neuroendocrine chaperone 7B2 and prohormone convertase PC2 is not associated with proPC2 maturation.

7B2 is a highly conserved neuroendocrine protein that is associated with the proform of the prohormone convertase PC2 in the early stages of the secretory pathway in intermediate pituitary cells of Xenopus laevis. Subsequent processing of 7B2 and dissociation of the 7B2/proPC2 complex is thought to be associated with the conversion of proPC2 to the mature enzyme. Here, we report that, in both Xenopus and mouse intermediate cells, proPC2 maturation does not take place when the proenzyme is associated with the 7B2 precursor and that, in contrast to the previous notion, dissociation of the complex between proPC2 and the N-terminal 7B2 fragment precedes, and is thus not directly linked to, proPC2 maturation. In vitro, conversion of newly synthesized proPC2 was efficiently blocked by recombinant 7B2 and studies with truncation mutants indicated that a short segment in the C-terminal region of 7B2 is necessary and sufficient for this inhibitory effect. Our results indicate that, after 7B2 precursor processing and dissociation of the N-terminal fragment, the C-terminal fragment of 7B2 may remain associated with proPC2, thereby preventing autocatalytic conversion of the proenzyme until the appropriate site for activation in the secretory pathway is reached.

Structural organization of the gene encoding the neuroendocrine chaperone 7B2.

The neuroendocrine-specific polypeptide 7B2 is a constituent of the regulated secretary pathway. Recently, 7B2 was found to function as a molecular chaperone for prohormone convertase PC2. This report describes the genomic organization of the 7B2 gene which consists of six exons. Exon I corresponds to the 5'-untranslated mRNA region, while exons 2 and 3 encode the signal peptide and the amino-terminal half of the 7B2 protein that is distantly related to a subclass of molecular chaperones. The carboxy-terminal half of 7B2, responsible for its inhibitory action on PC2, is encoded by exons 4-6. Primer-extension analysis showed that the human 7B2 gene is transcribed from multiple transcription-initiation sites. The human 7B2 gene promoter contains a cAMP-responsive element, an AP-1 site, and several Pit-1/GHF-1-binding domains and heat-shock-element-like sequences but no obvious TATA or CAAT boxes. Of further interest is the finding of two DNA elements which are common to the promoter regions of the 7B2 gene and other genes selectively expressed in neuroendocrine tissues.

The neuroendocrine chaperone 7B2 can enhance in vitro POMC cleavage by prohormone convertase PC2.

We previously showed that the neuroendocrine polypeptide 7B2 transiently interacts with prohormone convertase PC2 in the secretory pathway of neuroendocrine cells. Here we demonstrate that the processed, but not the intact, form of 7B2 can enhance the in vitro cleavage of newly synthesized prohormone proopiomelanocortin (POMC) in lysates of Xenopus intermediate pituitary cells. PC2 is presumably the cleavage enzyme involved since intact 7B2 abolishes the enhancing effect of processed 7B2 and is known to act as a specific inhibitor of PC2. Furthermore, processed 7B2 stimulates in vitro POMC cleavage by immunopurified Xenopus PC2. Our results indicate that 7B2 can display chaperone activity towards PC2.

7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway.

The neuroendocrine polypeptide 7B2 is a highly conserved secretory protein selectively present in prohormone-producing cells equipped with a regulated secretory pathway. We find that the amino-terminal half of 7B2 is distantly related to chaperonins, a subclass of molecular chaperones. When incubated in vitro with newly synthesized pituitary proteins, recombinant 7B2 specifically associates with prohormone convertase PC2. Metabolic cell labeling combined with coimmunoprecipitation studies showed that, in vivo, the precursor form of 7B2 interacts with the proform of PC2. Pulse-chase analysis revealed that this association is transient in that it commences early in the secretory pathway, while dissociation in the later stages appears to coincide with the cleavages of 7B2, proPC2, and prohormone. Our results suggest that 7B2 is a novel type of molecular chaperone preventing premature activation of proPC2 in the regulated secretory pathway.

The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2.

The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells. Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2. Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors). Our findings indicate that 7B2 is a physiological inhibitor of PC2 and may provide alternative avenues for the manipulation of peptide hormone levels.

Structure and expression of Xenopus prohormone convertase PC2.

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including alpha-melanophore-stimulating hormone (alpha-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to alpha-MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85-87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28-36%) and the region amino-terminal to the catalytic domain (59-60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black- and white-adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to alpha-MSH.