On approaches to the functional restoration of salivary glands damaged by radiation therapy for head and neck cancer,with a review of related aspects of salivary gland morphology and development

Radiation therapy for cancer of the head and neck can devastate the salivary glands and partially devitalize the mandible and maxilla. As a result, saliva production is drastically reduced and its quality adversely altered. Without diligent home and professional care, the teeth are subject to rapid destruction by caries, necessitating extractions with attendant high risk of necrosis of the supporting bone. Innovative techniques in delivery of radiation therapy and administration of drugs that selectively protect normal tissues can reduce significantly the radiation effects on salivary glands. Nonetheless, many patients still suffer severe oral dryness. I review here the functional morphology and development of salivary glands as these relate to approaches to preventing and restoring radiation-induced loss of salivary function. The acinar cells are responsible for most of the fluid and organic material in saliva, while the larger ducts influence the inorganic content. A central theme of this review is the extent to which the several types of epithelial cells in salivary glands may be pluripotential and the circumstances that may influence their ability to replace cells that have been lost or functionally inactivated due to the effects of radiation. The evidence suggests that the highly differentiated cells of the acini and large ducts of mature glands can replace themselves except when the respective pools of available cells are greatly diminished via apoptosis or necrosis owing to severely stressful events. Under the latter circumstances, relatively undifferentiated cells in the intercalated ducts proliferate and redifferentiate as may be required to replenish the depleted pools. It is likely that some, if not many, acinar cells may de-differentiate into intercalated duct-like cells and thus add to the pool of progenitor cells in such situations. If the stress is heavy doses of radiation, however, the result is not only the death of acinar cells, but also a marked decline in functional differentiation and proliferative capacity of all of the surviving cells, including those with progenitor capability. Restoration of gland function, therefore, seems to require increasing the secretory capacity of the surviving cells, or replacing the acinar cells and their progenitors either in the existing gland remnants or with artificial glands.     

Anticancer effects and lysosomal acidification in A549 cells by Astaxanthin from Haematococcus lacustris

Astaxanthin (AXN) is known to have health benefits by epidemiological studies. Therefore, it is of interest to assess the effect of AXN (derived from indigenous unicellular green alga Haematococcus lacustris) to modulate cell cycle arrest, lysosomal acidification and eventually apoptosis using in vitro in A549 lung cancer cells. Natural extracts of astaxanthin were obtained by standardized methods as reported earlier and characterized by standard HPLC and MS. Treatment of A549 cells with AXN (purified fraction) showed significant reduction in cell viability (about 50%) as compared to crude extract at 50µM concentration. Thus, we show the anticancer effects and lysosomal acidification in A549 cells by Astaxanthin from Haematococcus lacustris for further consideration. Together, our results demonstrated the anticancer potential of AXN from Haematococcus lacustris, which is found to be mediated via its ability to induce cell cycle arrest, lysosomal acidification and apoptotic induction.     

Self-association of the Lentivirus protein, Nef

Background

The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.

Results

By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.

Conclusions

We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.     

Cucurbit[7]uril encapsulated cisplatin overcomes cisplatin resistance via a pharmacokinetic effect

The cucurbit[n]uril (CB[n]) family of macrocycles has been shown to have potential in drug delivery where they are able to provide physical and chemical stability to drugs, improve drug solubility, control drug release and mask the taste of drugs. Cisplatin is a small molecule platinum-based anticancer drug that has severe dose-limiting side-effects. Cisplatin forms a host-guest complex with cucurbit[7]uril (cisplatin@CB[7]) with the platinum atom and both chlorido ligands located inside the macrocycle, with binding stabilised by four hydrogen bonds (2.15-2.44 ?). Whilst CB[7] has no effect on the in vitro cytotoxicity of cisplatin in the human ovarian carcinoma cell line A2780 and its cisplatin-resistant sub-lines A2780/cp70 and MCP1, there is a significant effect on in vivo cytotoxicity using human tumour xenografts. Cisplatin@CB[7] is just as effective on A2780 tumours compared with free cisplatin, and in the cisplatin-resistant A2780/cp70 tumours cisplatin@CB[7] markedly slows tumour growth. The ability of cisplatin@CB[7] to overcome resistance in vivo appears to be a pharmacokinetic effect. Whilst the peak plasma level and tissue distribution are the same for cisplatin@CB[7] and free cisplatin, the total concentration of circulating cisplatin@CB[7] over a period of 24 hours is significantly higher than for free cisplatin when administered at the equivalent dose. The results provide the first example of overcoming drug resistance via a purely pharmacokinetic effect rather than drug design or better tumour targeting, and demonstrate that in vitro assays are no longer as important in screening advanced systems of drug delivery.     

Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcγ receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcγ receptor-mediated phagocytosis to enhance the innate immune response.Receptors that interact with the constant region of IgG (FcγR)⁴ mediate the recognition and elimination of soluble immune complexes and particles coated (opsonized) with immunoglobulins. Clustering of FcγR on the surface of leukocytes upon attachment to multivalent ligands induces their activation and subsequent internalization. Soluble immune complexes are internalized by endocytosis, a clathrin- and ubiquitylation-dependent process (1). In contrast, large, particulate complexes like IgG-coated pathogens are ingested by phagocytosis, a process that is contingent on extensive actin polymerization that drives the extension of pseudopods (2). In parallel with the internalization of the opsonized targets, cross-linking of phagocytic receptors triggers a variety of other responses that are essential components of the innate immune response. These include degranulation, activation of the respiratory burst, and the synthesis and release of multiple inflammatory agents (3, 4).Like T and B cell receptors, FcγR possesses an immunoreceptor tyrosine-based activation motif that is critical for signal transduction (3, 4). Upon receptor clustering, tyrosyl residues of the immunoreceptor tyrosine-based activation motif are phosphorylated by Src family kinases, thereby generating a docking site for Syk, a tyrosine kinase of the ZAP70 family (3, 4). The recruitment and activation of Syk in turn initiates a cascade of events that include activation of Tec family kinases, Rho- and ARF-family GTPases, phosphatidylinositol 3-kinase, phospholipase Cγ (PLCγ), and a multitude of additional effectors that together remodel the underlying cytoskeleton, culminating in internalization of the bound particle (5, 6).Phosphoinositide metabolism is thought to be critical for FcγR-induced phagocytosis (7, 8). Highly localized and very dynamic phosphoinositide changes have been observed at sites of phagocytosis: phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) undergoes a transient accumulation at the phagocytic cup, which is rapidly superseded by its complete elimination from the nascent phagosome (7). The secondary disappearance of PtdIns(4,5)P₂ is attributable in part to the localized generation of phosphatidylinositol 3,4,5-trisphosphate, which has been reported to accumulate at sites of phagocytosis (9). Activation of PLCγ is also believed to contribute to the acute disappearance of PtdIns(4,5)P₂ in nascent phagosomes. Indeed, the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate has been detected by chemical means during FcγR-evoked particle ingestion (10, 11). Moreover, imaging experiments revealed that DAG appears at the time and at the precise site where PtdIns(4,5)P₂ is consumed (7).Two lines of evidence suggest that the DAG generated upon engagement of phagocytic receptors modulates particle engulfment. First, antagonists of PLC severely impair phagocytosis by macrophages (7, 12). This inhibition is not mimicked by preventing the associated [Ca²⁺] transient, suggesting that DAG, and not inositol 1,4,5-trisphosphate, is the crucial product of the PLC (13). Second, the addition of exogenous DAG or phorbol esters, which mimic the actions of endogenous DAG, augment phagocytosis (14, 15).Selective recognition of DAG by cellular ligands is generally mediated by specific regions of its target proteins, called C1 domains (16). Proteins bearing C1 domains include, most notably, members of the classical and novel families of protein kinase C (PKC), making them suitable candidates to account for the DAG dependence of phagocytosis. Indeed, PKCα, a classical isoform, and PKCϵ and PKCδ, both novel isoforms, are recruited to phagosomes (12, 15, 17, 18). Although the role of the various PKC isoforms in particle engulfment has been equivocal over the years, Cheeseman et al. (12) convincingly demonstrated that PKCϵ contributes to particle uptake in a PLC- and DAG-dependent manner.PKCs are not the sole proteins bearing DAG-binding C1 domains. Similar domains are also found in several other proteins, including members of the RasGRP family, chimaerins, and Munc-13 (19–21). One or more of these could contribute to the complex set of responses elicited by FcγR-induced DAG production. The RasGRP proteins are a class of exchange factors for the Ras/Rap family of GTPases (22). There are four RasGRP proteins (RasGRP1 to -4), and emerging evidence has implicated RasGRP1 and RasGRP3 in T and B cell receptor signaling (23–27).The possible role of DAG-mediated signaling pathways other than PKC in phagocytosis and the subsequent inflammatory response has not been explored. Here, we provide evidence that DAG stimulates Ras and Rap1 at sites of phagocytosis, probably through RasGRPs. Last, the functional consequences of Ras and Rap1 activation were analyzed.     

Genome-wide end-sequenced BAC resources for the NOD/MrkTac☆ and NOD/ShiLtJ☆☆ mouse genomes

Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting β-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome, the reduced X chromosome coverage being due to the use of a male donor for this library. Clones from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841 single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29. These BAC libraries represent a powerful resource for functional studies, such as gene targeting in NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments.     

Inhibitor profiles of alkaline phosphatases in bovine preattachment embryos and adult tissues.

The alkaline phosphatases are a small family of isozymes. Bovine preattachment embryos transcribe mRNA for two tissue-specific alkaline phosphatases (TSAP2 and TSAP3) beginning at the 4- and 8-cell stages. Whereas no mRNA has been detected in oocytes, there is maternally inherited alkaline phosphatase activity. It is not known which isozyme(s) is responsible for the maternal activity or when TSAP2 and TSAP3 form functional protein. No antibodies are available that recognize the relevant bovine alkaline phosphatases. Therefore, sensitivity to heat and chemical inhibition was used to separate the different isozymes. By screening tissues, it was determined that the bovine tissue-nonspecific alkaline phosphatase (TNAP) is inactivated by low temperatures (65C) and low concentrations of levamisole (<1 mM), whereas bovine tissue-specific isozymes require higher temperatures (90C) and levamisole concentrations (>5 mM). Inhibition by L-homoarginine and L-phenylalanine was less informative. Cumulus cells transcribe two isozymes and the pattern of inhibition suggested heterodimer formation. Inhibition of alkaline phosphatase in bovine embryos before the 8-cell stage indicated the presence of only TNAP. At the 16-cell stage the pattern was consistent with TNAP plus TSAP2 or -3 activity, and in morulae and blastocysts the pattern indicated that the maternal TNAP is fully supplanted by TSAP2 or TSAP3.     

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.     

No correlation between germline mutation at repeat DNA and meiotic crossover in male mice exposed to X-rays or cisplatin

To test the hypothesis that mouse germline expanded simple tandem repeat (ESTR) mutations are associated with recombination events during spermatogenesis, crossover frequencies were compared with germline mutation rates at ESTR loci in male mice acutely exposed to 1 Gy of X-rays or to 10 mg/kg of the anticancer drug cisplatin. Ionising radiation resulted in a highly significant 2.7–3.6-fold increase in ESTR mutation rate in males mated 4, 5 and 6 weeks after exposure, but not 3 weeks after exposure. In contrast, irradiation had no effect on meiotic crossover frequencies assayed on six chromosomes using 25 polymorphic microsatellite loci spaced at approximately 20 cM intervals and covering 421 cM of the mouse genome. Paternal exposure to cisplatin did not affect either ESTR mutation rates or crossover frequencies, despite a report that cisplatin can increase crossover frequency in mice.

Correlation analysis did not reveal any associations between the paternal ESTR mutation rate and crossover frequency in unexposed males and in those exposed to X-rays or cisplatin. This study does not, therefore, support the hypothesis that mutation induction at mouse ESTR loci results from a general genome-wide increase in meiotic recombination rate.