Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.      

Immobilized Clostridium acetobutylicum P262 Mutants for Solvent Production

The production of acetone, butanol, and ethanol by two immobilized, sporulation-deficient (spo) Clostridium acetobutylicum P262 mutants which were held in the solventogenic phase was investigated. The spoA2 mutant, which was an early-sporulation mutant and did not form a forespore septum, produced higher solvent yields than did the spoB mutant which was a late-sporulation mutant and was blocked at a stage after forespore septum formation. The spoA2 mutant was also granulose and capsule negative. In a conventional batch fermentation, the wild-type strain produced 15.44 g of solvents per liter after 50 h at a productivity of 7.41 g of solvents per liter per day. The spoA2 mutant produced 15.42 g of solvents per liter at a productivity of 72.4 g of solvents per liter per day, with a retention time of 2.4 h in a continuous immobilized cell system employing a fluidized bed reactor. This represents a major advance, since the immobilization of wild-type cells showed similar increases in productivity but a ca. fivefold reduction in final product concentrations.     

Mutation in mce operons attenuates Mycobacterium tuberculosis virulence

On the Mycobacterium tuberculosis genome there are four mce operons, all of which are similar in sequence and organization, and code for putatively exported proteins. To investigate whether Mce proteins are essential for virulence, we generated knock-out mutants in mce1, mce2 and mce3 operons of M. tuberculosis and evaluated their ability to multiply in a mammalian host. The allelic replacement was confirmed in each mutant strain by Southern blotting. RT-PCR experiments demonstrated the lack of in vitro expression of mutated genes in Deltamce1 and Deltamce2 mutants. On the other hand, no expression of mce3 was detected in either the wild-type or mutant strains. Similar doubling time and growth characteristics in in vitro culture were observed for mutants and parental strains. The intratracheal route was used to infect BALB/c mice with the Deltamce3, Deltamce2 and Deltamce1 mutants. Ten weeks after infection, all mice infected with the Deltamce mutants survived, while those infected with the wild-type strain died. This long survival correlated with very low counts of colony-forming units (CFU) in the lungs. Deltamce1-infected mice developed very few and small granulomas, while animals infected with Deltamce3 or Deltamce2 mutants showed delayed granuloma formation. Mice infected with Deltamce1 did not develop pneumonia, while animals infected with Deltamce3 and Deltamce2 mutants showed small pneumonic patches. In spleens, bacterial counts of mutant strains were less reduced than in lungs, compared with those of wild-type. In contrast, no such attenuation was observed when the intraperitoneal route was used for infection. Moreover, Deltamce1 mutants appear to be more virulent in lungs after intraperitoneal inoculation. In conclusion, mce operons seem to affect the virulence of M. tuberculosis in mice, depending on the route of infection. Hypotheses are discussed to explain this last issue. Thus, mutants in these genes seem to be good candidates for vaccine testing.     

Effects of 5-hydroxytryptamine on the neuronal firing rate of bulbar reticular neurons

The effects of 5-hydroxytryptamine (5-HT) on neuronal firing rate were studied in the reticular gigantocellular nucleus (GRN) and, for a comparison, in the interstitial (IRN), the parvicellular (PRN) and the lateral (LRN) nuclei, sharing some of GRN functional characteristics. Unitary extracellular recordings performed in anesthetized rats demonstrated that microiontophoretic application of 5-HT modulated the background firing rate in 92% of GRN, in 100% of IRN and LRN, and in 77% of PRN tested neurons. In GRN, 5-HT application induced excitatory responses in 49% of the neurons tested and inhibitions in 43% of them. Both types of effects were dose dependent and appeared scattered throughout the nucleus. Enhancements and decreases of firing rate in response to 5-HT application were also recorded in IRN (58% and 42% respectively), LRN (43% and 57%) and PRN (36% and 41%). The 5-HT(1A) receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT) mimicked 5-HT evoked inhibitions in all the nuclei tested and induced weak inhibitory responses also in neurons excited by 5-HT. The 5-HT2A receptor agonist alphamethyl-5-hydroxytryptamine (alpha-me-5-HT) mimicked excitatory as well as inhibitory responses to 5-HT, the former prevailing in GRN and the latter in the remaining reticular nuclei. Both excitatory and inhibitory responses to 5-HT were partially or totally blocked by the application of 5-HT2 receptor antagonist ketanserin. It is concluded that an extended, strong and differentiated control is exerted by 5-HT on the electrical activity of bulbar reticular neurons. Both 5-HT(1A) and 5-HT(2A) receptors mediate these effects, but the involvement of other receptors appears probable.     

Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells

Real-time visualization of specific endogenous mRNA expression in vivo has the potential to revolutionize medical diagnosis, drug discovery, developmental and molecular biology. However, conventional liposome- or dendrimer-based cellular delivery of molecular probes is inefficient, slow, and often detrimental to the probes. Here we demonstrate the rapid and sensitive detection of RNA in living cells using peptide-linked molecular beacons that possess self-delivery, targeting and reporting functions. We conjugated the TAT peptide to molecular beacons using three different linkages and demonstrated that, at relatively low concentrations, these molecular beacon constructs were internalized into living cells within 30 min with nearly 100% efficiency. Further, peptide-based delivery did not interfere with either specific targeting by or hybridization-induced fluorescence of the probes. We could therefore detect human GAPDH and survivin mRNAs in living cells fluorescently, revealing intriguing intracellular localization patterns of mRNA. We clearly demonstrated that cellular delivery of molecular beacons using the peptide-based approach has far better performance compared with conventional transfection methods. The peptide-linked molecular beacons approach promises to open new and exciting opportunities in sensitive gene detection and quantification in vivo.     

Estrogen receptors in rabbit thymus

The estrogen receptors presence or absence in the thymus gland represents the basic element to assert or to exclude any estrogenic action in the thymus. Now we have measured the presence of cytoplasmatic estrogen receptors in the female rabbits thymus, using some methods (sucrose gradient, protamine sulphate, dextran-coated charcoal). Finally, we have investigated, using the sucrose gradient assay, the nuclear ER presence in 1000g thymic pellet. The results exclude high levels of estrogen-binding proteins while don't suggest their total absence in the female rabbit thymus.