HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

The covalent addition of mono-AMP to target proteins (AMPylation) by Fic domain-containing proteins is a poorly understood, yet highly conserved post-translational modification. Here, we describe the generation, evaluation, and application of four HypE-specific nanobodies: three that inhibit HypE-mediated target AMPylation in vitro and one that acts as an activator. All heavy chain-only antibody variable domains bind HypE when expressed as GFP fusions in intact cells. We observed localization of HypE at the nuclear envelope and further identified histones H2–H4, but not H1, as novel in vitro targets of the human Fic protein. Its role in histone modification provides a possible link between AMPylation and regulation of gene expression.     

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.     

Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD

We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G-->T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC.     

Matrix Metalloproteinase-2 (MMP-2) Gene Deletion Enhances MMP-9 Activity,Impairs PARP-1 Degradation,and Exacerbates Hepatic Ischemia and Reperfusion Injury in Mice

Hepatic ischemia and reperfusion injury (IRI) is an inflammatory condition and a significant cause of morbidity and mortality after surgery. Matrix metalloproteinases (MMPs) have been widely implicated in the pathogenesis of inflammatory diseases. Among the different MMPs, gelatinases (MMP-2 and MMP-9) are within the most prominent MMPs detected during liver IRI. While the role of MMP-9 in liver damage has been fairly documented, direct evidence of the role for MMP-2 activity in hepatic IRI remains to be established. Due to the lack of suitable inhibitors to target individual MMPs in vivo, gene manipulation is as an essential tool to assess MMP direct contribution to liver injury. Hence, we used MMP-2^-/- deficient mice and MMP-2^+/+ wild-type littermates to examine the function of MMP-2 activity in hepatic IRI. MMP-2 expression was detected along the sinusoids of wild-type livers before and after surgery and in a small population of leukocytes post-IRI. Compared to MMP-2^+/+ mice, MMP-2 null (MMP-2^-/-) mice showed exacerbated liver damage at 6, 24, and 48 hours post-reperfusion, which was fatal in some cases. MMP-2 deficiency resulted in upregulation of MMP-9 activity, spontaneous leukocyte infiltration in naïve livers, and amplified MMP-9-dependent transmigration of leukocytes in vitro and after hepatic IRI. Moreover, complete loss of MMP-2 activity impaired the degradation of poly (ADP-ribose) polymerase (PARP-1) in extensively damaged livers post-reperfusion. However, the administration of a PARP-1 inhibitor to MMP-2 null mice restored liver preservation to almost comparable levels of MMP-2^+/+ mice post-IRI. Deficient PARP-1 degradation in MMP-2-null sinusoidal endothelial cells correlated with their increased cytotoxicity, evaluated by the measurement of LDH efflux in the medium. In conclusion, our results show for the first time that MMP-2 gene deletion exacerbates liver IRI. Moreover, they offer new insights into the MMP-2 modulation of inflammatory responses, which could be relevant for the design of new pharmacological MMP-targeted agents to treat hepatic IRI.     

An upper limit to the abundance of aquatic organisms

Summary The maximum density achievable by aquatic organisms is an inverse linear function of their body size. As a consequence, the maximum achievable biomass is independent of body size, and is 2 orders of magnitude higher than the biomass in natural populations. The minimum interorganismic terorganismic distance, calculated from the maximum density to allow comparison between aquatic and terrestrial organisms, scales as the 1/3 power of body size in both habitats. The similarities in the interorganismic distance of terrestrial and aquatic plant and animal communities suggest a fundamental regularity in the way organisms use the space.     

Ovarian cycle of southern brown howler monkey (<Emphasis Type="Italic">Alouatta guariba clamitans</Emphasis>) through fecal progestin measurement

The ovarian cycle in howler monkeys (genus Alouatta) has beean investigated through several biological parameters (ranging between 16.3 and 29.5 days); however, no data exist concerning the ovarian activity of the southern brown howler monkey (Alouatta guariba clamitans). This study aimed to describe the ovarian cycle of A. g. clamitans by profiling fecal progestin concentrations. Over 20 weeks, fecal samples of eight captive adult females of A. g. clamitans were collected. The collections were made at dawn, 5 days a week, and the samples were frozen immediately following collection. Next, they were dried, pulverized and hormonal metabolites were extracted to determine progestin concentrations by enzyme immunoassay. Of the 758 samples tested, the mean concentration of fecal progestins was 2866.40 ± 470.03 ng/g of dry feces, while the mean concentration at baseline was 814.47 ± 164.36 ng/g of dry feces. Among the eight females, one showed no ovarian cyclicity and three presented periods of probable absence of cyclicity and low progestin concentrations. A mean duration of 16 ± 0.52 days was observed for the 35 cycles studied. The interluteal phase lasted 4 ± 0.37 days on average, with a mean concentration of fecal progestins of 467.98 ± 29.12 ng/g of dry feces, while the luteal phase lasted 11 ± 0.50 days, with a mean concentration of 4283.27 ± 193.31 ng/g of dry feces. Besides describing the characteristics of the ovarian cycle, possible causes for the low concentrations of fecal progestins and periods of absence of cyclicity are also discussed.     

Random amplified polymorphic DNA analysis of DNA extracted from Trichuris trichiura (Linnaeus, 1771) eggs and its prospective application to paleoparasitological studies

Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing. T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.     

Synthesis and antidepressant-like action of stereoisomers of imidobenzenesulfonylaziridines in mice evaluated in the forced swimming test

The present study describes the chemical synthesis and pharmacological evaluation of a new series of eleven compounds stereoisomers of imidobenzenesulfonylaziridines in the forced-swimming test (FST) in mice. The pharmacological results of these compounds show that six of them, given intraperitoneally, reduced the immobility time of mice evaluated in the FST, an antidepressant-like profile of action similar to imipramine, a well-known standard antidepressant drug used for comparison, without compromising the animals' motor performance. The putative antidepressant-like action demonstrated here indicates their viability for the development of new therapeutic options for the treatment of depression.