Dental anthropology of Central-Southern,Iron Age Italy: The evidence of metric versus nonmetric traits

Discrete and metric dental traits are used to assess biological similarities and differences among 13 bioarchaeological populations located on each side of the Apennine mountains in central-southern Italy and dated to the first millennium BC . An initial hypothesis, that the mountain chain might provide a significant geographical barrier for population movement (resulting in greater biological affinities among those groups on the same side), is not supported. Instead, the samples appear to cluster more on the basis of time than geography. Archaeological evidence, however, supports an association between populations on opposite sides of the mountains and thus is in accord with the dental data. As anticipated, discrete dental traits appear to be more useful than metric dental traits in assessing such population affinities. This research represents a beginning to a better comprehension of the complexity of the biological and cultural dynamics of Italian populations during recent millennia. Am J Phys Anthropol 107:371–386, 1998. © 1998 Wiley-Liss, Inc.     

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.     

Role of glycosphingolipid SSEA‐3 and FGF2 in the stemness and lineage commitment of multilineage differentiating stress enduring (MUSE) cells

ObjectivesMultilineage differentiating Stress Enduring (MUSE) cells are endogenous, stress‐resistant stem cells, expressing pluripotency master genes and able to differentiate in cells of the three embryonic sheets. Stage‐Specific Embryonic Antigen 3 (SSEA‐3), a glycosphingolipid (GSL), is the marker for identifying MUSE cells and is used to isolate this population from mesenchymal stromal cells. GSLs modulate signal transduction by interacting with plasma membrane components. The growth factor FGF2, important for MUSE cells biology, may interact with GSLs. Specific cell surface markers represent an invaluable tool for stem cell isolation. Nonetheless their role, if any, in stem cell biology is poorly investigated. Functions of stem cells, however, depend on niche external cues, which reach cells through surface markers. We addressed the role of SSEA‐3 in MUSE cell behaviour, trying to define whether SSEA‐3 is just a marker or if it plays a functional role in this cell population by determining if it has any relationship with FGF2 activity.ResultsWe evidenced how the SSEA‐3 and FGF2 cooperation affected the self‐renewal and clonogenic capacity of MUSE cells. The block of SSEA‐3 significantly reduced the multilineage potential of MUSE cells with production of nullipotent clones.ConclusionsWe contributed to dissecting the mechanisms underlying MUSE cell properties for establishing successful stem‐cell‐based therapies and the promotion of MUSE cells as a tool for the in vitro disease model.

We demonstrated that SSEA‐3, a glycosphingolipid (GSL), may act as an FGF‐2 co‐receptor in MUSE cell, this activates the FGF2 signalling cascade mainly through PI3K mediation. Our study is one of the first evidence attributing GSL a functional role in governing stem cell biology. (A) Experimental plan. (B) Effect of cultivation of MUSE cells with SSEA3 inhibition. (C) SSEA‐3—FGF2 interaction and pathway.     

Split Gp41-1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41-1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N-terminal split intein along with the N-moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C-terminal of split intein, the C-moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans-splicing was verified by transfecting both N-terminal and C-terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.     

Leishmania tarentolae and Leishmania infantum in humans,dogs and cats in the Pelagie archipelago,southern Italy

Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean basin with most of the infected human patients remaining asymptomatic. Recently, the saurian-associated Leishmania tarentolae was detected in human blood donors and in sheltered dogs. The circulation of L. infantum and L. tarentolae was investigated in humans, dogs and cats living in the Pelagie islands (Sicily, Italy) by multiple serological and molecular testing. Human serum samples (n = 346) were tested to assess the exposure to L. infantum by immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) and to L. tarentolae by IFAT. Meanwhile, sera from dogs (n = 149) and cats (n = 32) were tested for both Leishmania species by IFAT and all blood samples, including those of humans, by specific sets of real time-PCR for L. infantum and L. tarentolae. The agreement between serological tests performed for human samples, and between serological and molecular diagnostic techniques for both human and animal samples were also assessed.Overall, 41 human samples (11.8%, 95% CI: 8.9–15.7) were positive to L. infantum (5.2%, 95% CI: 3.3–8.1), L. tarentolae (5.2%, 95% CI: 3.3–8.1) and to both species (1.4%, 95% CI: 0.6–3.3) by serology and/or molecular tests. A good agreement among the serological tests was determined. Both Leishmania spp. were serologically and/or molecularly detected in 39.6% dogs and 43.7% cats. In addition to L. infantum, also L. tarentolae circulates in human and animal populations, raising relevant public health implications. Further studies should investigate the potential beneficial effects of L. tarentolae in the protection against L. infantum infection.     

Protein unfolding is not a prerequisite for endoplasmic reticulum-to-cytosol dislocation

We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ER in vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chains in vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.     

Technical difficulties and solutions of direct transesterification process of microbial oil for biodiesel synthesis

Microbial oils are considered as alternative to vegetable oils or animal fats as biodiesel feedstock. Microalgae and oleaginous yeast are the main candidates of microbial oil producers’ community. However, biodiesel synthesis from these sources is associated with high cost and process complexity. The traditional transesterification method includes several steps such as biomass drying, cell disruption, oil extraction and solvent recovery. Therefore, direct transesterification or in situ transesterification, which combines all the steps in a single reactor, has been suggested to make the process cost effective. Nevertheless, the process is not applicable for large-scale biodiesel production having some difficulties such as high water content of biomass that makes the reaction rate slower and hurdles of cell disruption makes the efficiency of oil extraction lower. Additionally, it requires high heating energy in the solvent extraction and recovery stage. To resolve these difficulties, this review suggests the application of antimicrobial peptides and high electric fields to foster the microbial cell wall disruption.     

The human melanoma associated protein melanotransferrin promotes endothelial cell migration and angiogenesis in vivo

Melanotransferrin is a member of the transferrin family, which is comprised of serum transferrin, lactoferrin and ovotransferrin, and is highly expressed on melanoma cells compared to normal melanocytes. Since melanoma is an highly vascularized tumour that expresses melanotransferrin at high levels, we tested purified recombinant melanotransferrin for its capability to induce angiogenesis in the chick chorioallantoic membrane. Macroscopic and microscopic evaluation of the vascular density demonstrated that melanotransferrin exerts an angiogenic response quantitatively similar to that elicited by fibroblast growth factor-2. Overexpression of vascular endothelial growth factor-receptor-2 was observed in newly formed vessels, suggesting that the angiogenic activity of melanotransferrin may depend on activation of endogenous vascular endothelial growth factor. In addition, when antibodies against vascular endothelial growth factor were included in the assay, the angiogenic response was inhibited by 50%. In a Boyden chamber assay purified recombinant melanotransferrin induced chemotactic migration of vascular cells, which was decreased in the the presence of anti-vascular endothelial growth factor antibodies suggesting an involvement of vascular endothelial growth factor present in endothelial cells also in this assay. However, melanotransferrin was found not to directly bind to integrin alphavbeta3 or the vascular endothelial growth factor-receptor-2 as assessed in a BlAcore assay. A possible correlation between vascularization occurring during melanoma progression and the expression of melanotransferrin and vascular endothelial growth factor was established by immunolocalization of the two factors in sections of melanoma at different clinical steps of melanoma progression. These latter data strongly imply that melanotransferrin may participate in the vascularization of solid tumours and that inhibition of melanotransferrin could form the basis for intervention in tumours which use this pathway.     

The depolarization-induced outflow of d-[H]aspartate from rat brain slices is modulated by metabotropic glutamate receptors

Rat brain slices were used to study the effects of different metabotropic glutamate receptor ligands on (i) the depolarization (30 mM KCl)-induced outflow of previously taken up d-[³H]aspartate; (ii) the inhibition of forskolin (30 μM)-induced cyclic AMP accumulation; and (iii) the hydrolysis of phosphoinositides. In addition, the localization of mRNAs coding for different metabotropic glutamate receptor subtypes was detected using in situ hybridization. (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (30–300 μM), a non selective metabotropic glutamate receptor agonist, significantly increased the KCl-induced output of radioactivity from cortical slices, whereas it inhibited the output from striatal slices. Conversely, (1S,3S,4S)-carboxycyclopropylglycine (0.1–1 μM), a relatively selective agonist of the mGluR2 metabotropic glutamate receptor subtype, had an inhibitory effect on the output of d-[³H]aspartate from both cortical and striatal slices and proved to be the most potent metabotropic glutamate receptor agonist in inhibiting cyclic AMP accumulation, but not in stimulating phosphoinositide hydrolysis. Since 2-amino-4-phosphonobutyrate (a mGluR4, mGluR6 and mGluR7 agonist) was not active in any of the assays tested, we hypothesized that the mGluR2 subtype could be involved in these events. Accordingly, mGluR2 mRNA expression was abundant in cortical neurons projecting to the striatum. Our experiments suggest that the stimulation of metabotropic glutamate receptors may either decrease or increase transmitter release depending on the subtype that prevails in the region under study.