Antifungal Resistance and Virulence Among <Emphasis Type="Italic">Candida</Emphasis> spp. from Captive <Emphasis Type="Italic">Amazonian manatees</Emphasis> and West Indian Manatees: Potential Impacts on Animal and Environmental Health

This work aimed at evaluating the antifungal susceptibility and production of virulence factors by Candida spp. isolated from sirenians in Brazil. The isolates (n = 105) were recovered from the natural cavities of Amazonian and West Indian manatees and were tested for the susceptibility to amphotericin B, itraconazole, and fluconazole and for the production of phospholipases, proteases, and biofilm. The minimum inhibitory concentrations (MICs) for amphotericin B ranged from 0.03 to 1 µg/mL, and no resistant isolates were detected. Itraconazole and fluconazole MICs ranged from 0.03 to 16 µg/mL and from 0.125 to 64 µg/mL, respectively, and 35.2% (37/105) of the isolates were resistant to at least one of these azole drugs. Concerning the production of virulence factors, phospholipase activity was observed in 67.6% (71/105) of the isolates, while protease activity and biofilm production were detected in 50.5% (53/105) and 32.4% (34/105) of the isolates, respectively. Since the natural cavities of manatees are colonized by resistant and virulent strains of Candida spp., these animals can act as sources of resistance and virulence genes for the environment, conspecifics and other animal species, demonstrating the potential environmental impacts associated with their release back into their natural habitat.     

The initial events in myelin synthesis: orientation of proteolipid protein in the plasma membrane of cultured oligodendrocytes

Proteolipid protein (PLP) is the most abundant transmembrane protein in myelin of the central nervous system. Conflicting models of PLP topology have been generated by computer predictions based on its primary sequence and experiments with purified myelin. We have examined the initial events in myelin synthesis, including the insertion and orientation of PLP in the plasma membrane, in rat oligodendrocytes which express PLP and the other myelin-specific proteins when cultured without neurons (Dubois-Dalcq, M., T. Behar, L. Hudson, and R. A. Lazzarini. 1986. J. Cell Biol. 102:384-392). These cells, identified by the presence of surface galactocerebroside, the major myelin glycolipid, were stained with six anti-peptide antibodies directed against hydrophilic or short hydrophobic sequences of PLP. Five of these anti-peptide antibodies specifically stained living oligodendrocytes. Staining was only seen approximately 10 d after PLP was first detected in the cytoplasm of fixed and permeabilized cells, suggesting that PLP is slowly transported from the RER to the cell surface. The presence of PLP domains on the extracellular surface was also confirmed by cleavage of such domains with proteases and by antibody-dependent complement-mediated lysis of living oligodendrocytes. Our results indicate that PLP has only two transmembrane domains and that the great majority of the protein, including its amino and carboxy termini, is located on the extracellular face of the oligodendrocyte plasma membrane. This disposition of the PLP molecule suggests that homophilic interactions between PLP molecules of apposed extracellular faces may mediate compaction of adjacent bilayers in the myelin sheath.     

Effects of chromium(VI) and chromium(III) on energy charge and oxygen consumption in rat thymocytes

The cytotoxic effects of chromium compounds in two oxidation states have been studied in rat thymocytes. endogenous nucleotide levels and oxygen consumption were examined as relevant parameters of the physiological state of the cell. Incubation of rat thymocytes with Cr(VI) produced a marked unbalance of endogenous purine nucleotide pool and a parallel decrease in oxygen consumption. A close correlation between the reduction of oxygen consumption and ATP level in rat thymocytes treated with increasing concentrations of Cr(VI) has been found. In rat thymocytes permeabilized with digitonin and in isolated rat liver mitochondria both Cr(VI) and Cr(III) showed, at different range of concentrations, a marked inhibition of maximal oxygen consumption rate (uncoupled respiration). The effects observed were depending on chromium oxidation state and on different mitochondrial sites of substrate oxidation.     

Contrasting levels of DNA polymorphism at the autosomal and X-linked visual color pigment loci in humans and squirrel monkeys

The X-linked color pigment (opsin) locus is known to be highly polymorphic in the squirrel monkey and other New World monkeys. To see whether this is also the case for the autosomal (blue) opsin locus, we obtained 32 squirrel monkey and 30 human blue opsin gene sequences. No amino acid polymorphism was found in either the squirrel monkey sample or the human sample, contrary to the situation at the X-linked opsin locus. This sharp contrast in the level of polymorphism might be due to differences in gene expression between the autosomal and the X-linked loci. At the X-linked locus, heterozygote advantage can occur because, owing to X-inactivation, the two alleles in a heterozygote are expressed in different cone cells, producing two types of cone cell, whereas at the autosomal locus, heterozygote advantage cannot occur because the two alleles in a heterozygote are expressed in the same cone cells, producing only one type of cone cell (i.e., phenotypically a homozygote). From the sequence data, the levels of nucleotide diversity (pi, i.e., the number of nucleotide differences per site) are estimated: for the human sample, pi = 0.00% per nondegenerate site, 0.00% per twofold degenerate site, and 0.04% per fourfold degenerate site in the coding regions and 0.01% per site in intron 4; for the squirrel monkey sample, pi = 0.00% per nondegenerate site, 0.00% per twofold degenerate site, and 0.15% per fourfold degenerate site in the coding regions and 0.17% per site in intron 4. The blue opsin genes from the common and pygmy chimpanzees, the gorilla, the capuchin, and the howler monkey were also sequenced. Features critical to the function of the opsin are well conserved in all known mammalian sequences. However, the interhelical loops are, on average, actually more conservative than the transmembrane helical regions. In addition, these sequence data and those from some other genes indicate that the common and pygmy chimpanzees are not closely related, their divergence data being from one third to one half the date of the human-chimpanzee divergence.      

Absence of the Mid-sized Neurofilament Subunit Decreases Axonal Calibers,Levels of Light Neurofilament (NF-L), and Neurofilament Content

Neurofilaments (NFs) are prominent components of large myelinated axons and probably the most abundant of neuronal intermediate filament proteins. Here we show that mice with a null mutation in the mid-sized NF (NF-M) subunit have dramatically decreased levels of light NF (NF-L) and increased levels of heavy NF (NF-H). The calibers of both large and small diameter axons in the central and peripheral nervous systems are diminished. Axons of mutant animals contain fewer neurofilaments and increased numbers of microtubules. Yet the mice lack any overt behavioral phenotype or gross structural defects in the nervous system. These studies suggest that the NF-M subunit is a major regulator of the level of NF-L and that its presence is required to achieve maximal axonal diameter in all size classes of myelinated axons.Neurofilaments (NFs)¹ are the most prominent cytoskeletal components in large myelinated axons and probably the most abundant and widely expressed of neuronal intermediate filament (IF) proteins. In mammals, NFs are composed of three proteins termed light (NF-L), mid-sized (NF-M), and heavy (NF-H) NFs. These proteins are encoded by separate genes (17, 21, 27) and have apparent molecular weights of ∼68,000, 150,000, and 200,000, respectively, when separated on SDS-PAGE gels.Like all IFs, NF proteins contain a relatively well-conserved α helical rod domain of ∼310 amino acids with variable NH₂-terminal and COOH-terminal regions (33). In NFs, the COOH-terminal domains are greatly extended relative to other IFs and contain a glutamic acid–rich region of unknown significance and in NF-M and NF-H a series of lysine-serine-proline-valine (KSPV) repeats (21, 27) which are major sites of phosphorylation in both proteins. In axons, NFs form bundles of 10-nm diameter “core filaments” with sidearms consisting of phosphorylated COOH-terminal tail sequences of NF-M and NF-H (12, 13, 26, 29) that have been thought to extend and maintain the spacing between filaments (4). Similar sidearm extensions are not found in IFs composed of other IF proteins such as desmin, glial fibrillary acidic protein, or vimentin. In NFs assembled in vitro, all three subunits appear to be incorporated into core filaments (12, 26). Thus, current models of NF assembly suggest that NF-M and NF-H are the major components of sidearm extensions and are anchored to a core of NF-L via their central rod domains.Although much is known about NF structure and assembly, questions remain concerning NF function. A primarily structural role for NFs is suggested by their prominence in large axons (41). Small unmyelinated axons contain few NFs (9) and some small neurons lack morphologically identifiable NFs (3, 32, 38). Most dendrites contain few NFs and only in dendrites of large neurons such as motor neurons are NFs numerous (41).A role for NFs as a major determinant of axonal diameter has long been suspected from the correlation between NF content in axonal cross sections and axonal caliber (16). This correlation persists during axonal degeneration and regeneration (14) and changes in NF transport correlate temporally with alterations in the caliber of axons in regenerating nerves (15). Additionally, fewer NFs occur at nodes of Ranvier where axonal diameter is reduced (1), and certain NF epitopes are found only in regions where maximal axonal caliber has developed (6).Several animal models have supported a role for NFs in establishing axonal diameter. One is a Japanese quail (Quiverer) with a spontaneous mutation in NF-L that generates a truncated protein incapable of forming NFs (31). Homozygous mutants contain no axonal NFs and exhibit a mild generalized quivering. In these animals, radial growth of myelinated axons is severely attenuated (44) with a consequent reduction in axonal conduction velocity (37). In transgenic mice, Eyer and Petersen (8) expressed an NF-H/β-galactosidase fusion protein in which the COOH terminus of NF-H was replaced by β-galactosidase. NF inclusions were found in the perikarya of neurons and the resulting NF aggregates blocked all NF transport into axons resulting in axons with reduced calibers. More recently, Zhu et al. (45) have shown that mice lacking NFs due to a targeted disruption of the NF-L gene have diminished axonal calibers and delayed maturation of regenerating myelinated axons.Although these models clearly suggest a role for NFs in establishing axonal diameter, they contribute only limited information concerning the roles of the individual NF subunits. During development, NF-L and NF-M are coexpressed initially whereas NF-H appears later (4). Studies in transgenic mice have found that overexpressing mouse NF-L leads to an increased density of NFs, but no increase in axonal caliber (25). More recently, Xu et al. (43) overexpressed each of the mouse NF subunits either individually or in various combinations. They found that only when NF-L was overexpressed in combination with either NF-M or NF-H was axonal growth significantly increased. Interestingly, when NF-M and NF-H were overexpressed alone or in combination with one another, radial axonal growth was inhibited.It also remains incompletely understood how NF stoichiometries are regulated and the degree to which any one NF subunit is dominant in this regulation. Recently, conflicting data has appeared concerning the role of NF-M in regulating NF stoichiometries. We found that overexpression of human NF-M in transgenic mice increases the levels of endogenous mouse NF-L protein and decreases the extent of phosphorylation of NF-H (39). These results imply that NF-M may play a dominant role in regulating the levels of NF-L protein, the relative stoichiometry of NF subunits, and the phosphorylation status of NF-H. However different results were obtained by Wong et al. (40) who found that overexpression of mouse NF-M in transgenic mice did not effect the levels of axonal NF-L, and although it reduced NF-H, it did not effect its phosphorylation status.To further address these issues we generated mice bearing a null mutation in the mouse NF-M gene. Here we describe the effects of this mutation on nervous system development with particular reference to the role of the NF-M subunit in specifying axonal diameter and its effect on levels of the remaining NF subunits.     

Analysis of the RNA of defective VSV particles

Viral mRNA isolated from infected cells and the virion RNA isolated from two classes of defective interfering particles have been analyzed by RNA-RNA duplex reactions. The results show that the RNA of both defective interfering particles is viral, not host in origin. The RNA isolated from the two defective particles represents homogeneous populations of molecules containing only part of the genetic information present in the whole VSV genome. Annealing competition experiments indicate that if any overlap exists between the two, it is less than 220 nucleotides. We conclude from the data presented that a rudimentary physical map of the VSV and DI particle genomes is

Our results suggest that there is not a single specific site that is required for autointerference.     

Quality and intensity of light affect <Emphasis Type="Italic">Lippia gracilis</Emphasis> Schauer plant growth and volatile compounds in vitro

The aim of this study was to evaluate the effects of different intensities and quality of light and explant type on the growth of and volatile compounds in Lippia gracilis in vitro. The treatments were as follows: light intensities of 26, 51, 69, 94, or 130 µmol m^?2 s^?1 from fluorescent lamps and light-emitting diode (LED) lamps at different wavelengths, namely, white, red, blue, and combinations of red and blue light at ratios of 2.5:1 and 1:2.5, respectively, and two explant types, namely, nodal and apical segments. On the 30th day of culture on half-strength Murashige and Skoog (Physiol Plant 15(3):473–497, 1962) medium, growth, production of photosynthetic pigments, chlorophyll a and b, total chlorophyll, carotenoids, and volatile constituents (using headspace gas chromatography-mass spectrometry) were analyzed. The light quality and intensity significantly influenced the in vitro growth of L. gracilis. The apical segments were superior in all parameters evaluated compared to nodal segments. The number of segments plantlet^?1, root length, and leaf, shoot, root, and total weight were higher with increasing light intensity, especially under the 94 µmol m^?2 s^?1 treatment, for both explant types. The red light showed the highest leaf (32.28 mg plantlet^?1) and total (58.33 mg plantlet^?1) dry weight of all the light qualities. Major constituents, namely, ρ-cymene, γ-terpinene, thymol, carvacrol, and E-caryophyllene, were identified, regardless of light conditions. The amount and composition of volatile compounds varied according to light intensity and quality. Low intensity (26 µmol m^?2 s^?1) increased γ-terpinene content (12.42%) and concomitantly decreased carvacrol (38.52%). Blue LED light showed higher production of carvacrol (48.11%).