Diminished injury in hypotransferrinemic mice after exposure to a metal-rich particle

Using the hypotransferrinemic (Hp) mouse model, we studied the effect of altered iron homeostasis on the defense of the lung against a catalytically active metal. The homozygotic (hpx/hpx) Hp mice had greatly diminished concentrations of both serum and lavage fluid transferrin relative to wild-type mice and heterozygotes. Fifty micrograms of a particle containing abundant concentrations of metals (a residual oil fly ash) was instilled into wild-type mice and heterozygotic and homozygotic Hp animals. There was an oxidative stress associated with particle exposure as manifested by decreased lavage fluid concentrations of ascorbate. However, rather than an increase in lung injury, diminished transferrin concentrations in homozygotic Hp mice were associated with decreased indexes of damage, including concentrations of relevant cytokines, inflammatory cell influx, lavage fluid protein, and lavage fluid lactate dehydrogenase. Comparable to other organs in the homozygotic Hp mouse, siderosis of the lung was evident, with elevated concentrations of lavage fluid and tissue iron. Consequent to these increased concentrations of iron, proteins to store and transport iron, ferritin, and lactoferrin, respectively, were increased when assayed by immunoprecipitation and immunohistochemistry. We conclude that the lack of transferrin in Hp mice did not predispose the animals to lung injury after exposure to a particle abundant in metals. Rather, these mice demonstrated a diminished injury that was associated with an increase in the metal storage and transport proteins.     

Glutamine synthetase in the phloem plays a major role in controlling proline production

To inhibit expression specifically in the phloem, a 274-bp fragment of a cDNA (Gln1-5) encoding cytosolic glutamine synthetase (GS1) from tobacco was placed in the antisense orientation downstream of the cytosolic Cu/Zn superoxide dismutase promoter of Nicotiana plumbaginifolia. After Agrobacterium-mediated transformation, two transgenic N. tabacum lines exhibiting reduced levels of GS1 mRNA and GS activity in midribs, stems, and roots were obtained. Immunogold labeling experiments allowed us to verify that the GS protein content was markedly decreased in the phloem companion cells of transformed plants. Moreover, a general decrease in proline content in the transgenic plants in comparison with wild-type tobacco was observed when plants were forced to assimilate large amounts of ammonium. In contrast, no major changes in the concentration of amino acids used for nitrogen transport were apparent. A (15)NH(4)(+)-labeling kinetic over a 48-hr period confirmed that in leaves of transgenic plants, the decrease in proline production was directly related to glutamine availability. After 2 weeks of salt treatment, the transgenic plants had a pronounced stress phenotype, consisting of wilting and bleaching in the older leaves. We conclude that GS in the phloem plays a major role in regulating proline production consistent with the function of proline as a nitrogen source and as a key metabolite synthesized in response to water stress.     

Pheomelanin as a Binding Site for Drugs and Chemicals

Certain drugs and chemicals, such as chloroquine, chlorpromazine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), are bound to melanin and retained in pigment cells for long periods. This specific retention in pigmented tissues can cause adverse effects in the skin, eye, inner ear, and pigmented nerve cells of the substantia nigra of the brain. To date, all studies have been focused on eu- and neuromelanin. In the present study, we show that chloroquine, chlorpromazine, chlomipramine, paraquat, acridine orange, and nickel, which are bound to eumelanin, also bind to synthetic pheomelanin, but the binding to pheomelanin is lower. The binding varied with the cysteine content and pH, and the results indicate that the binding is complex and includes ionic interactions. In addition, we have shown that these substances also bind to synthetic thiourea-containing melanin, but to quite a low extent. We also present a microautoradiographic study on the binding of ¹⁴C-chloroquine to natural pheomelanin in vivo in yellow mice C57BL (A^y/a). Black (C57/BL) and albino (NMRI) mice were used as controls. The autoradiography demonstrated a pronounced uptake of chloroquine in the hair follicles and the dermal melanocytes in the ear of yellow mice, which was comparable to the corresponding accumulation of label in black mice. In the albino mouse, the uptake was lower and more homogeneously distributed in the skin. These results suggest that the toxicologi-cal risks of melanin-related adverse effects are applicable to persons with a high content of pheomelanin in the skin and hair.     

SV40 large T antigen is modified with O-linked N-acetylglucosamine but not with other forms of glycosylation

SV40 large T antigen has been reported to be modified with several different sugars including N-acetylglucosamine, galactose, and mannose. In this report we have reexamined the glycosylation of T antigen and found that while we could detect modification with N-acetylglucosamine, we could not detect any other sugars on the protein. Surprisingly, even though [3H]galactose could be metabolically incorporated into the protein, analysis showed that all of the radioactivity in T antigen had been converted to other species. The N-acetylglucosamine was demonstrated to be linked to the protein in the form of O-linked N- acetylglucosamine, the best characterized form of nuclear and cytoplasmic glycosylation in mammalian systems. We have localized the major site of glycosylation to the amino terminal portion of the molecule. Analysis of mutated T antigen where serines 111/112 were substituted with alanine suggest that these residues constitute a glycosylation site on the protein. These two serines fall within a typical O-linked N-acetylglucosamine glycosylation site (PSS) and are also known to be phosphorylated. Thus, it is likely that competition between phosphorylation and glycosylation occurs at this site.      

Solid tumor preparation for flow cytometry using a standard murine model

The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well-differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3-methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanol-water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.     

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.     

Synchronization of human diploid fibroblasts at multiple stages of the cell cycle

Because of the scarcity of techniques for synchronizing the growth of cultured human diploid fibroblasts at multiple stages within the cell cycle, efforts were expended in this report to establish a set of protocols that would permit synchronization of cells at several different points throughout the cycle. The protocols that were developed to synchronize the growth of HSF-24 and HSF-55 cells, human foreskin-derived fibroblast cultures, were modifications of procedures employed to synchronize the growth of cultured rodent cells. Optimization of synchrony induction was directed by consideration of both the biochemical properties of the synchronized populations (determined via three-parameter flow cytometric measurements of DNA, RNA, and protein contents) and their kinetic behavior following reversal of the synchronization-inducing blockade (determined via combined flow cytometric analysis of DNA content, [3H]thymidine autoradiography, and measurement of increase in cell number). The conditions judged to yield the best results for studying events associated with production of a G0 block or for maintaining cells for prolonged periods in G0 were those in which the cells were grown to confluency in D-MEM supplemented with 10% fetal bovine serum. Procedures producing the best results for studying processes associated with the G0 to G1 transition, G1 events, and operations accompanying the transition from G1 to S, employed subconfluent growth for 48 h in alpha-MEM + 0.1% fetal bovine serum (alpha-MEM0.1F) followed by resuspension in alpha-MEM containing 10% fetal bovine serum (alpha-MEM10F). When the goal was to obtain cells in which to study very early S-phase events, satisfactory results were achieved by combining a 48-h period of subconfluent growth in alpha-MEM0.1F, followed by treatment for 24 h in alpha-MEM10F containing 5 micrograms/ml aphidicolin. For study of events occurring in mid- to late-cycle, acceptable results were achieved by combining a 48-h block in alpha-MEM0.1F with resuspension for 24 h in alpha-MEM10F containing 10(-3) M hydroxyurea followed by resuspension in drug-free alpha-MEM10F. The best results were obtained with these latter synchronization procedures (i.e., low-serum/high-serum + APC or HU/high serum) when the fetal calf serum was replaced with heat-inactivated calf serum. The success achieved in synchronizing the growth of these human diploid fibroblasts compared favorably/exceeded the results obtained with synchronized cultures of Chinese hamster ovary cells.     

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.