Cloning and recombinant expression of active full-length xylosyltransferase I (XT-I) and characterization of subcellular localization of XT-I and XT-II

Xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins. This is the first and apparently rate-limiting step in the biosynthesis of the tetrasaccharide linkage region in glycosaminoglycan-containing proteoglycans. The XYLT-II gene codes for a highly homologous protein, but its physiological function is not yet known. Here we present for the first time the construction of a vector encoding the full-length GFP-tagged human XT-I and the recombinant expression of the active enzyme in mammalian cells. We expressed XT-I-GFP and various GFP-tagged XT-I and XT-II mutants with C-terminal truncations and deletions in HEK-293 and SaOS-2 cells in order to investigate the intracellular localization of XT-I and XT-II. Immunofluorescence analysis showed a distinct perinuclear pattern of XT-I-GFP and XT-II-GFP similar to that of alpha-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and alpha-mannosidase II could also be demonstrated in untransfected cells. Using brefeldin A, we could also show that both xylosyltransferases are resident in the early cisternae of the Golgi apparatus. For its complete Golgi retention, XT-I requires the N-terminal 214 amino acids. Unlike XT-I, for XT-II, the first 45 amino acids are sufficient to target and retain the GFP reporter in the Golgi compartment. Here we show evidence that the stem regions were indispensable for Golgi localization of XT-I and XT-II.     

Size matters for the tripeptidylpeptidase II complex from Drosophila: The 6-MDa spindle form stabilizes the activated state

Tripeptidylpeptidase II (TPP II) is an exopeptidase of the subtilisin type of serine proteases, a key component of the protein degradation cascade in many eukaryotes, which cleaves tripeptides from the N terminus of proteasome-released products. The Drosophila TPP II is a large homooligomeric complex (approximately 6 MDa) that is organized in a unique repetitive structure with two strands each composed of ten stacked homodimers; two strands intertwine to form a spindle-shaped structure. We report a novel procedure of preparing an active, structurally homogeneous TPP II holo-complex overexpressed in Escherichia coli. Assembly studies revealed that the specific activity of TPP II increases with oligomer size, which in turn is strongly concentration-dependent. At a TPP II concentration such as prevailing in Drosophila, equilibration of size and activity proceeds on a time scale of hours and leads to spindle formation at a TPP II concentration of > or =0.03 mg/ml. Before equilibrium is reached, activation lags behind assembly, suggesting that activation occurs in a two-step process consisting of (i) assembly and (ii) a subsequent conformational change leading to a switch from basal to full activity. We propose a model consistent with the hyperbolic increase of activity with oligomer size. Spindle formation by strand pairing causes both significant thermodynamic and kinetic stabilization. The strands inherently heterogeneous in length are thus locked into a discrete oligomeric state. Our data indicate that the unique spindle form of the holo-complex represents an assembly motif stabilizing a highly active state.     

Signaling alkaline pH stress in the yeast Saccharomyces cerevisiae through the Wsc1 cell surface sensor and the Slt2 MAPK pathway

Alkalinization of the external environment represents a stress situation for Saccharomyces cerevisiae. Adaptation to this circumstance involves the activation of diverse response mechanisms, the components of which are still largely unknown. We show here that mutation of members of the cell integrity Pkc1/Slt2 MAPK module, as well as upstream and downstream elements of the system, confers sensitivity to alkali. Alkalinization resulted in fast and transient activation of the Slt2 MAPK, which depended on the integrity of the kinase module and was largely abolished by sorbitol. Lack of Wsc1, removal of specific extracellular and intracellular domains, or substitution of Tyr(303) in this putative membrane stress sensor rendered cells sensitive to alkali and considerably decreased alkali-induced Slt2 activation. In contrast, constitutive activation of Slt2 by the bck1-20 allele increased pH tolerance in the wsc1 mutant. DNA microarray analysis revealed that several genes encoding cell wall proteins, such as GSC2/FKS2, DFG5, SKT5, and CRH1, were induced, at least in part, by high pH in an Slt2-dependent manner. We observed that dfg5, skt5, and particularly dfg5 skt5 cells were alkali-sensitive. Therefore, our results show that an alkaline environment imposes a stress condition on the yeast cell wall. We propose that the Slt2-mediated MAPK pathway plays an important role in the adaptive response to this insult and that Wsc1 participates as an essential cell-surface pH sensor. Moreover, these results provide a new example of the complexity of the response of budding yeast to the alkalinization of the environment.     

Fine needle aspiration cytology of dedifferentiated acinic cell carcinoma of the parotid gland: a case report

BACKGROUND: Dedifferentiation of acinic cell carcinoma (ACC) to undifferentiated carcinoma occurs rarely and entails a poor prognosis. Most cases of dedifferentiation occur as recurrences of a previously excised ACC. More rarely the neoplasm presents with areas of well-differentiated ACC coexisting with dedifferentiated ones. CASE: An 85-year-old man presented with 2 nodular areas in the right parotid area. Fine needle aspiration of both nodules was performed. In both cases cytology revealed a double epithelial component in similar proportions. The first one corresponded to cohesive groups of small to intermediate-sized, polygonal cells with round, monomorphic nuclei. They were distributed in small and larger, branching groups with acinic morphology. Intermixed with this population, irregular groups of larger, pleomorphic cells with irregular nuclei, prominent nucleoli and scarce cytoplasm were present. In addition, smears showed an abundant lymphoid background. A cytologic diagnosis of "salivary carcinoma with coexisting areas of acinic cell differentiation and high grade, undifferentiated carcinoma" was given. Histopathology revealed a well-differentiated ACC with areas of high grade undifferentiated carcinoma (dedifferentiated ACC). CONCLUSION: The current case expands the cytomorphologic spectrum of ACC. Cytology may permit the preoperative recognition of dedifferentiation, allowing a more sound therapeutic approach.     

Phenotypic evolution of human craniofacial morphology after admixture: a geometric morphometrics approach

An evolutionary, diachronic approach to the phenotypic craniofacial pattern arisen in a human population after high levels of admixture and gene flow was achieved by means of geometric morphometrics. Admixture has long been studied after molecular data. Nevertheless, few efforts have been made to explain the morphological outcome in human craniofacial samples. The Spanish-Amerindian contact can be considered a good scenario for such an analysis. Here we present a comparative analysis of craniofacial shape changes observed between two putative ancestor groups, Spanish and precontact Aztecs, and two diachronic admixed groups, corresponding to early and late colonial periods from the Mexico's Central Valley. Quantitative shape comparisons of Amerindian, Spanish, and admixed groups were used to test the expectations of quantitative genetics for admixture events. In its simplest form, this prediction states that an admixed group will present phenotypic values falling between those of both parental groups. Results show that, in general terms, although the human skull is a complex, integrated structure, the craniofacial morphology observed fits the theoretical expectations of quantitative genetics. Thus, it is predictive of population structure and history. In fact, results obtained after the craniofacial analysis are in accordance with previous molecular and historical interpretations, providing evidence that admixture is a main microevolutionary agent influencing modern Mexican gene pool. However, expectations are not straightforward when moderate shape changes are considered. Deviations detected at localized structures, such as the upper and lower face, highlight the evolution of a craniofacial pattern exclusively inherent to the admixed groups, indicating that quantitative characters might respond to admixture in a complicated, nondirectional way.     

Nano-mechanical exploration of the surface and sub-surface of hydrated cells of Staphylococcus epidermidis

The surface of hydrated cells of Staphylococcus epidermidis has been probed using an atomic force microscope. While local force measurements over the surface of bacteria reveal a heterogeneous chemical surface, with heterogeneous mechanical properties, different kinds of force curves appear with high frequency, and are thought to provide information on features contributing strongly to the overall mechanical and surface behaviour of the cell. Force curves often present two different mechanical regimes, being the first one (outer) of about 48 nm thick, and presenting a local relative elasticity of about 0.08 N/m, which is about a third of the relative elasticity of the inner part of the cell wall, harder, with a relative elasticity of about 0.24 N/m, in water. Both regimes appears as straight lines in the force versus distance curves (the ‘corresponding’ stress–strain curves in contact mechanics), but hysteresis is observed between the approach and the retraction line in the inner regime, indicating a degree of viscoelasticity. No viscoelasticity is observed in the outer regime, however, which presents quite linear and juxtaposed approach-retraction lines. These kinds of force curves do not present measurable pull-off forces nor snap-in forces, which indicates an almost null interaction between tip and bacterial surface, which could be in agreement with the measured very high hydrophobicity of this strain. Another kind of force curve has been observed recurrently, showing peaks in the retraction curves. Adhesive pull-off forces were measured giving an average of about 2 nN. Interestingly, however, these force curves appear only when quite irregular and wavy retraction curves are present, from the very beginning of its trace (maximum indentation). This leads us to think that these pull-off forces measured by our AFM do not give information on surface forces-unbinding events at the surface of the bacteria, but could be related to events at the sub-surface of the cell surface. Oscillations seen in the retraction curve in the portion corresponding to the contact with the bacteria surface could be due to rupture phenomena within the multilayered cell wall architecture expected in Gram-positive bacteria as Staphylococcus epidermidis, which could result in local irreversible deformations of the cell surface. Imaging with a sharp tip in contact mode sometimes leads to surface damage. Force curves recorded over damaged parts of the cell surface showed a completely different behaviour, in many cases with two well-defined high-adhesion peaks, and also interestingly, with snap-in forces of about 0–2 nN, which seems to indicate a completely different electrical/hydrophobicity state only a few nanometers down from the surface. Similar indentation effects can occur in the contact of a bacterial cell with a solid surface, even when showing only atomic-molecular-scale roughness, thus interacting not only with the very surface of the cell, especially when soft layers are present in the outer. Our results highlight the importance of the cell surface mechanical properties and their interplay with purely surface properties when analyzing cell–material interaction, and show the AFM as a useful method for investigating this.     

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.