Calf thymus high mobility group proteins are nonenzymatically glycated but not significantly glycosylated

Over the past decade, there have been many reports suggesting the presence of complex carbohydrates on nuclear and cytoplasmic proteins in mammalian cells. Some of the most often cited of these reports deal with the glycosylation of the high mobility group (HMG) proteins. These are relatively abundant chromosomal proteins that are known to be associated with nucleosomes and actively transcribed regions of chromatin. The original report describing HMG protein glycosylation presented several lines of evidence suggesting that these proteins are glycosylated, including carbohydrate compositional analysis and periodic-acid Schiff staining. We have attempted to repeat these observations with more highly purified protein than was utilized in the original study. Using carbohydrate compositional analysis performed by high pH anion exchange chromatography coupled to pulsed-amperometric detection, we saw no evidence for significant glycosylation of these proteins. In addition, we found no evidence for the presence of O- GlcNAc, a well known form of nuclear glycosylation. The HMG proteins did react with periodate, suggesting the presence of a modification containing cis-diols on the protein. Several tryptic peptides isolated from HMG 14 and 17 which retained the periodate reactivity had in common lysine residues, suggesting a potential modification of the straightepsilon-amino groups of lysines such as nonenzymatic glycation. Western blot analysis of the HMG proteins using anti-advanced glycation endproducts (AGE) antibodies confirmed the presence of glycation products on the HMG proteins.      

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.     

Divergent calcium signaling in RBCs from <Emphasis Type="Italic">Tropidurus torquatus</Emphasis> (Squamata – Tropiduridae) strengthen classification in lizard evolution

Background  

We have previously reported that a Teiid lizard red blood cells (RBCs) such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na⁺/H⁺ ionophore monensin, K⁺/H⁺ ionophore nigericin and the H⁺ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca²⁺ from intracellular stores upon ATP addition.     

Obtaining of fluorescent-labeled nodule bacteria strains of wild legumes for their detection in vive and in vitro

A series of expression vectors containing genes of fluorescent proteins TurboGFP and TurboRFP under the phage T5 constitutive promoter regulation, intended for lifetime marking of nodule bacteria is created: a series of vectors based on a broad-host-range replicon BBRI, for marking strains with an expression of reporter gene from a transformed plasmid and a series of vectors based on a plasmid pRL765gfp for marking strains by introduction genes of fluorescent proteins in a bacterial chromosome. It was shown that transformation is the most preferable method of constructions transfer in nodule bacteria cells, as in the presence of mob locus in the vectors necessary for conjugation, exists the possibility of occasional plasmid mobilization and its transition from marked strain cells in other soil bacteria. With application of the created vector constructions we obtained fluorescent tagged strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., Agrobacterium sp. Also their suitability for experiments in vivo and in vitro is shown.     

Marine Heterotrophic Amoebae, Flagellates and Heliozoa From Belize (Central America) and Tenerife (Canary Islands), With Descriptions of New Species, Luffisphaera Bulbochaete N. Sp., L. Longihastis N. Sp., L. Turriformis N. Sp. and Paulinella Intermedia N. Sp.

ABSTRACT. Thirty four taxa of heterotrophic protists (amoebae, flagellates and heliozoa) were encountered in cultures established from marine samples from Belize (Central America) and Tenerife (Canary Islands). Most species are flagellates drawn from the choanoflagellates, the cryptophyceans, the euglenids, the kinetoplastids, the bicosoecids, the chromulinids, the pedinellids and a variety of laxa of uncertain affinities (Protista incertae sedis). the identity of the thecate choanoflagellates Salpingoeca ringens Kent, 1880, and S. tuba Kent, 1880, is discussed, and four new species of heterotrophic protists are described: one new species of the amoeba genus Paulinella (Paulinella intermedia n. sp.) and three new species of the incertae sedis genus Luffisphaera Belcher & Swale, 1975 ( Luffisphaera bulbochaete n. sp.; L. longihastis n. sp.; L. turriformis n. sp.).     

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.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.      

New Thioureas and Related Substances Intended for Melanoma Targeting

Thiouracil and a few related drugs are known to be melanoma-seeking agents owing to specific incorporation into nascent melanin. The melanin-affinic properties are apparently due to binding to intermediates, preferably dopaquinone, produced in the melanin synthetic pathway by tyrosinase-catalysed oxidation of tyrosine. In the present paper, in vitro screening methods have been used for the identification of possible melanoma seekers according to the above principle. The binding of test substance to dopaquinone suppresses dopachrome formation by the withdrawal of dopaquinone from the reaction mixture, and the decrease in dopachrome concentration was monitored spectrophotometrically at 475 nm. In order to eliminate false results caused by tyrosinase inhibition, which also will decrease the dopachrome concentration, the oxygen consumption was followed potentiometrically. To avoid the effect of tyrosinase inhibition on dopachrome formation, additional experiments with autoxidation of L-dopa in the presence of test substance were performed. Of the 22 substances (mainly thioureylenes and thioamides) studied, 4,5,6-triamino-2(H)-pyrimidinehtionsulfate, trithiocyanuric acid, 2-thiouracil, 6-methyl-2-thiouracil, and 4-amino-2-mercaptopyrimidine most effectively decreased the dopachrome formation with no or little inhibition of tyrosinase activity. They should therefore be regarded as potential melanoma seekers. In a complementary autoradiographic study on the uptake of the potent tyrosinase inhibitor mercaptobenzothiazole (MBT) in B 16 melanoma, transplanted to mice, it was found that strong tyrosinase inhibition seems to decrease incorporation into melanin in vivo. MBT was partly accumulated in restricted areas of the tumor, which may be explained by the small molar dose injected.     

Evolution and phylogenetic utility of the period gene in Lepidoptera

Evolution and phylogenetic utility of the period gene are explored through sequence analysis of a relatively conserved 909-bp fragment in 26 lepidopteran species. Taxa range from tribes to superfamilies, primarily within the putative clade Macrolepidotera plus near outgroups, and include both strongly established and problematic groupings. Their divergence dates probably range from the late Cretaceous through much of the Tertiary. Comparisons within the same set of closely related species show that amino acid substitutions in period occur 4.9 and 44 times as frequently as they do in two other nuclear genes--dopa decarboxylase and elongation factor-1 alpha, respectively. In contrast, rates of observed synonymous substitution are within 60% of each other for these three genes. Synonymous changes in period approach saturation by the family level, whereas nonsynonymous and amino acid divergences across the Macrolepidoptera are less than half the maximal values reported for this gene. Phylogenetic analyses of period strongly supported groupings at the family level and below. In contrast to previous analyses at this level with other nuclear genes, much of the information lies in nonsynonymous change. Relationships up to the superfamily level were recovered with decreasing effectiveness, and little, if any, signal was apparent regarding relationships among superfamilies. This could reflect rapid radiation of the superfamilies, however, rather than saturation in the period locus; thus, period, in combination with other genes, remains a plausible candidate for approaching the difficult problems of lepidopteran family and superfamily relationships.