Biosynthesis of the human transferrin receptor in cultured cells

The biosynthesis and degradation of the cell surface transferrin receptor has been investigated. The receptor is a glycoprotein, and evidence is presented that the mature receptor contains both complex and high mannose N-asparagine-linked oligosaccharides that are synthesized via a common high mannose intermediate as previously described for other glycoproteins. It is shown that fatty acid is associated with only the mature form of the receptor and that addition of fatty acid to the receptor and that addition of fatty acid to the receptor can occur as long as 48 h after synthesis. Glycosylation is not an absolute requirement for the receptor to act as acceptor for fatty acid, nor for transport to the cell surface, although the efficiency of both processes may be reduced in tunicamycin-treated cells. The protein moiety of the transferrin receptor is degraded with a half-life of approximately 60 h.     

Raf-1 activation disrupts its binding to keratins during cell stress

Keratins 8 and 18 (K8/18) heteropolymers may regulate cell signaling via the known K18 association with 14-3-3 proteins and 14-3-3 association with Raf-1 kinase. We characterized Raf-keratin-14-3-3 associations and show that Raf associates directly with K8, independent of Raf kinase activity or Ras-Raf interaction, and that K18 is a Raf physiologic substrate. Raf activation during oxidative and toxin exposure in cultured cells and animals disrupt keratin-Raf association in a phosphorylation-dependent manner. Mutational analysis showed that 14-3-3 residues that are essential for Raf binding also regulate 14-3-3-keratin association. Similarly, Raf phosphorylation sites that are important for binding to 14-3-3 are also essential for Raf binding to K8/18. Therefore, keratins may modulate some aspects of Raf signaling under basal conditions via sequestration by K8, akin to Raf-14-3-3 binding. Keratin-bound Raf kinase is released upon Raf hyperphosphorylation and activation during oxidative and other stresses.     

Phorbol acetate enhances the phosphorylation of cytokeratins 8 and 18 in human colonic epithelial cells.

The phosphorylation of epithelial-specific cytokeratin (CK) 8 and 18 was studied in the human colonic cell line HT29. Metabolic labelling of cells with orthophosphate resulted in phosphorylation of cytokeratins 8/18 on serine residues. When phorbol acetate was added to labelled cells, a 2.2-fold increase in CK8/18 phosphate labelling was noted, whereas increasing intracellular cAMP levels using forskolin or 8-Br-cAMP showed no significant change in CK phosphorylation. CKs8/18 were also phosphorylated by added PKC in the presence of [gamma-32P]ATP. Tryptic peptide map analysis of the phosphorylated CK8 species showed that treatment of cells with 8-Br-cAMP or phorbol acetate generated a phosphopeptide not seen in control cells. In contrast, tryptic peptide maps of phosphorylated CK18 showed no discernable differences. Our results support a role for PKC in the phosphorylation of epithelial cytokeratins, with some phosphorylation sites being modulated by cAMP dependent protein kinase.     

"Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights

Intermediate filaments (IFs) are major components of the mammalian cytoskeleton. They are among the most abundant cellular phosphoproteins; their phosphorylation typically involves multiple sites at repeat or unique motifs, preferentially within the "head" or "tail" domains. Phosphorylation and dephosphorylation are essential for the regulation of IF dynamics by modulating the intrinsic properties of IFs: solubility, conformation and filament organization, and, in addition, for the regulation of other IF post-translational modifications. These phosphorylation-regulated properties dictate generalized and context-dependent IF functions that reflect their tissue-specific expression. Most important among IF phosphorylation-mediated functions are the regulation of IF cellular or subcellular compartmentalization, levels and turnover, binding with associated proteins, susceptibility to cell stresses (including apoptosis), tissue-specific functions and IF-associated disease pathogenesis (where IF hyperphosphorylation also serves as a tissue-injury marker).     

Enhancing Maternal and Perinatal Health in Under-Served Remote Areas in Sub-Saharan Africa: A Tanzanian Model

Background

In Tanzania, maternal mortality ratio (MMR), unmet need for emergency obstetric care and health inequities across the country are in a critical state, particularly in rural areas. This study was established to determine the feasibility and impact of decentralizing comprehensive emergency obstetric and neonatal care (CEmONC) services in underserved rural areas using associate clinicians.

Methods

Ten health centres (HCs) were upgraded by constructing and equipping maternity blocks, operating rooms, laboratories, staff houses and installing solar panels, standby generators and water supply systems. Twenty-three assistant medical officers (advanced level associate clinicians), and forty-four nurse-midwives and clinical officers (associate clinicians) were trained in CEmONC and anaesthesia respectively. CEmONC services were launched between 2009 and 2012. Monthly supportive supervision and clinical audits of adverse pregnancy outcomes were introduced in 2011 in these HCs and their respective district hospitals.

Findings

After launching CEmONC services from 2009 to 2014 institutional deliveries increased in all upgraded rural HCs. Mean numbers of monthly deliveries increased by 151% and obstetric referrals decreased from 9% to 3% (p = 0.03) in HCs. A total of 43,846 deliveries and 2,890 caesarean sections (CS) were performed in these HCs making the mean proportion of all births in EmONC facilities of 128% and mean population-based CS rate of 9%. There were 190 maternal deaths and 1,198 intrapartum and very early neonatal deaths (IVEND) in all health facilities. Generally, health centres had statistically significantly lower maternal mortality ratios and IVEND rates than district hospitals (p < 0.00 and < 0.02 respectively). Of all deaths (maternal and IVEND) 84% to 96% were considered avoidable.

Conclusions

These findings strongly indicate that remotely located health centres in resource limited settings hold a great potential to increase accessibility to CEmONC services and to improve maternal and perinatal health.     

Protective effects of agomelatine on testicular damage caused by bortezomib

Bortezomib is a chemotherapeutic agent used to treat several cancers; however, it exhibits severe side effects in testicular tissue. We investigated the use of agomelatine to prevent testicular tissue damage caused by bortezomib. We used 36 male Sprague-Dawley rats divided randomly into six equal groups: group 1, no treatment control; group 2, agomelatine treatment only; group 3, bortezomib treatment only for 48 h; group 4, bortezomib + agomelatine treatment for 48 h; group 5, bortezomib treatment only for 72 h; and group 6, bortezomib + agomelatine treatment for 72 h. After treatments, the rats were sacrificed and testicular tissue was harvested. Lipid oxidation (LPO) and superoxide dismutase (SOD) levels in the tissues were determined using biochemical methods. Tissue samples also were examined using histopathological and immunohistochemical techniques. The LPO level was increased, while the SOD level was decreased in the bortezomib treated groups. We found that agomelatine treatment normalized LPO and SOD activities in the bortezomib treated groups. In the spermatogonia and Sertoli cells, the staining density of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and caspase 3 were decreased in the bortezomib + agomelatine groups at both 48 and 72 h compared to bortezomib only treated groups. We observed maturation arrest, basal membrane thickening, increase in inflammatory cells and connective tissue, and edema between germ cells in the bortezomib only treated groups. By contrast, normal basal membrane, less edema and more normal maturation were observed in the bortezomib + agomelatine groups at 48 and 72 h. We found that agomelatine reduced the damaging effects of bortezomib. The use of agomelatine to prevent bortezomib induced testicular tissue damage in human patients should be investigated further.     

Functional analysis of the human papillomavirus type 16 E1=E4 protein provides a mechanism for in vivo and in vitro keratin filament reorganization

High-risk human papillomaviruses, such as human papillomavirus type 16 (HPV16), are the primary cause of cervical cancer. The HPV16 E1=E4 protein associates with keratin intermediate filaments and causes network collapse when expressed in epithelial cells in vitro. Here, we show that keratin association and network reorganization also occur in vivo in low-grade cervical neoplasia caused by HPV16. The 16E1=E4 protein binds to keratins directly and interacts strongly with keratin 18, a member of the type I intermediate-filament family. By contrast, 16E1=E4 bound only weakly to keratin 8, a type II intermediate-filament protein, and showed no detectable affinity for the type III protein, vimentin. The N-terminal 16 amino acids of the 16E1=E4 protein, which contains the YPLLXLL motif that is conserved among supergroup A viruses, were sufficient to target green fluorescent protein to the keratin network. When expressed in the SiHa cervical epithelial cell line, the full-length 16E1=E4 protein caused an almost total inhibition of keratin dynamics, despite the phosphorylation of keratin 18 at serine 33, which normally leads to 14-3-3-mediated keratin solubilization. Mutant 16E1=E4 proteins which lack the LLKLL motif, or which have lost amino acids from their C termini, and which were compromised in the ability to associate with keratins did not disturb normal keratin dynamics. 16E1=E4 was found to exist as dimers and hexamers, whereas a C-terminal deletion mutant (16E1=E4Delta87-92) existed as monomers and formed multimeric structures only poorly. Considered together, our results suggest that by associating with keratins through its N terminus, and by associating with itself through its C terminus, 16E1=E4 may act as a keratin cross-linker and prevent the movement of keratins between the soluble and insoluble compartments. The increase in avidity associated with multimeric binding may contribute to the ability of 16E1=E4 to sequester its cellular targets in the cytoplasm.     

Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells

Sphingosylphosphorylcholine (SPC) is a naturally occurring bioactive lipid that is present in high density lipoproteins (HDL) particles and found at increased levels in blood and malignant ascites of patients with ovarian cancer. Here, we show that incubation of human epithelial tumour cells with SPC induces a perinuclear reorganization of intact keratin 8-18 filaments. This effect is specific for SPC, largely independent of F-actin and microtubules, and is accompanied by keratin phosphorylation. In vivo visco-elastic probing of single cancer cells demonstrates that SPC increases cellular elasticity. Accordingly, SPC stimulates migration of cells through size-limited pores in a more potent manner than lysophosphatidic acid (LPA). LPA induces actin stress fibre formation, but does not reorganize keratins in cancer cells and hence increases cellular stiffness. We propose that reorganization of keratin by SPC may facilitate biological phenomena that require a high degree of elasticity, such as squeezing of cells through membranous pores during metastasis.     

Glucosylated N-acetyllactosamine O-antigen chain in the lipopolysaccharide from Helicobacter pylori strain UA861

The O-antigen chain from the lipopolysaccharide of Helicobacter pylori strain UA861 was determined to be composed of an elongated type 2 N - acetyllactosamine backbone, -[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1- ]n-->, with approximately half of the GlcNAc units carrying a terminal alpha-d-Glc residue at the O -6 position. The O-chain of H.pylori UA861 was terminated by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D- GlcNAc] (LacNAc) epitope and did not express terminal Lewis X or Lewis Y blood-group determinants as previously found in other H.pylori strains. The absence of terminal Lewis X and Lewis Y blood-group epitopes and the replacement of Fuc by Glc as a side chain in the O- chain of H.pylori UA861 represents yet another type of lipopolysaccharide structure from H.pylori species. These structural differences in H.pylori lipopolysaccharide molecules carry implications with regard to possible different pathogenic events between strains and respective hosts.