Antitumour activities of levans produced by Zymomonas mobilis strains

Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Characterization of cell lines stably expressing human normal or mutated EGFP-tagged MC4R

The melanocortin receptor type 4 (MC4-R) is involved in food intake and represents a potential target for the treatment of some forms of obesity. The fluorescent protein EGFP was fused to the wild-type or mutated coding sequence of the human MC4-R. After transfection in HEK 293, clones stably expressing hMC4-R-EGFP were selected. Wild-type chimeric hMC4-R was well addressed to the cell membrane as demonstrated using confocal microscopy and displayed the same pharmacological characteristics as native hMC4R. NDP-alpha MSH induced a time-dependent internalization of MC4-R that was partially prevented by AgRP. The two mutated chimeric receptors studied here (CTCT-deleted and C271A) showed a high alteration of their response to ligand and were retained inside the cells. In conclusion, we have developed a model of clones stably expressing EGFP-tagged-hMC4-R. This is the only such model available to date and it provides a useful tool to follow the trafficking of MC4-R inside living cells.     

Extrasynaptic accumulations of acetylcholinesterase in the rat sternocleidomastoid muscle after neonatal denervation. Light and electron microscopic localization and molecular forms

Denervated neonatal rat sternocleidomastoid muscle has decreased levels of total AChE when compared to control muscle. Denervated versus control values of total muscle AChE present a three-phase curve in function of time after denervation. There is a rapid initial fall 0-3 days after denervation, an increase during about 2 weeks, then again a decrease in total AChE. Thus, there is a transitory net accumulation of AChE after the initial fall of activity in denervated developing muscle. Extrasynaptic areas of high AChE activity develop between 1 and 2 weeks after denervation and remain visible up to 1 month after denervation before vanishing. An electron microscope study shows that these accumulations are internal to the muscle fiber, close to a limited number of muscle nuclei and associated to the sarcoplasmic reticulum and nuclear envelope, but not to the T-tubule system. As found in adult rat muscle, the initial fall in AChE affects first the 16 S AChE form, and soon after, the 4 S and 10 S AChE forms. A main difference with adult muscle is the sudden increase and predominance over other forms of 10 S AChE 2 weeks after denervation at birth. Later, the decrease in AChE affects 16 S and 4 S AChE before 10 S AChE. The regions rich in extrasynaptic sites of AChE accumulation possess a very high proportion of 10 S AChE. Thus, the mechanisms of biosynthesis, intracellular transport and/or secretion of AChE may be very different in young, developing muscle compared to adult muscle.     

Pleiotrophin cellular localization in nerve regeneration after peripheral nerve injury.

Pleiotrophin (PTN) is a member of the family of heparin-binding growth factors that displays mitogenic activities and promotes neurite outgrowth in vitro. In vivo, PTN is widely expressed along pathways of developing axons during the late embryonic and early postnatal period. Although the level of PTN gene expression is very low during adulthood, activation of the gene may occur during recovery from injury and seems to play an important role in tissue regeneration processes. In this study, we investigated whether PTN was involved in the regenerative process of injured peripheral nerves. To refer localization of the fluorescent markers to myelinated axons, we developed a specific computer tool for colocalization of fluorescence images with phase contrast images. Immunohistochemical analysis showed PTN in different types of nonneural cells in distal nerve segments, including Schwann cells, macrophages, and endothelial cells, but not in axons. Schwann cells exhibited PTN immunoreactivity as early as 2 days after injury, whereas PTN-positive macrophages were found 1 week later. Strong PTN immunoreactivity was noted in endothelial cells at all time points. These findings support the idea that PTN participates in the adaptive response to peripheral nerve injury. A better understanding of its contribution may suggest new strategies for enhancing peripheral nerve regeneration.     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

Difference in the ability of neonatal and adult denervated muscle to accumulate acetylcholinesterase at the old sites of innervation

In adult rat sternocleidomastoid muscle, AChE is concentrated in the region rich in motor end-plates (MEP). All major AChE forms, "16 S," "10 S," and "4 S," are accumulated at high levels, and not only "16 S" AChE. After denervation, muscle AChE decreases; 2 weeks after denervation, low levels (20-40% of control) are reached for all forms. During the following weeks, a slow but steady increase in "10 S" and "16 S" AChE occurs in the denervated muscle. At this stage, all forms are again observed to be highly concentrated in the region containing the old sites of innervation. Thus, in adult rat muscle the structures able to accumulate "16 S," "10 S," and "4 S" AChE in the MEP-rich regions remain several months after denervation. In normal young rat sternocleidomastoid muscle at birth, all AChE forms are already accumulated in the MEP-rich region. After denervation at birth, the denervated muscle loses its ability to keep a high concentration of "4 S," "10 S," and "16 S" AChE in the old MEP-rich region. All AChE forms are still present 1 month after denervation, but they are decreased and diffusedly distributed over the whole length of the muscle. In particular, "16 S" AChE is detected in the same proportion (10-15%) all along the denervated muscle. Thus, the diffuse distribution of AChE, and especially "16 S" AChE, after neonatal denervation, contrasts with the maintained accumulation observed in adult denervated muscle. It seems that denervation of young muscle results in a specific loss of the muscle ability to concentrate high levels of all AChE forms at the old sites of innervation.     

Exogenous Pleiotrophin Applied to Lesioned Nerve Impairs Muscle Reinnervation

Pleiotrophin (PTN) is a heparin-binding growth factor involved in nerve regeneration after peripheral nerve injury. After crush injury, PTN is found in distal nerve segments in several non-neural cell types, including Schwann cells, macrophages, and endothelial cells, but not in axons. To further clarify the role for PTN in nerve regeneration, we investigated the effects of PTN applied to lesioned peripheral nerve in vivo. PTN in a dose of 1 mg/kg impaired muscle reinnervation. Thus, gastrocnemius muscle failed to recover its contractile properties as assessed by in situ maximal isometric tetanic force. PTN also decreased non-neural cell densities and delayed macrophage recruitment in the distal crushed nerve. These results are discussed in the light of recent evidence that PTN is a multifunctional polypeptide.     

Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.