Plastic encapsulation of stabilized Escherichia coli and Pseudomonas putida

Escherichia coli and Pseudomonas putida dried in hydroxyectoine or trehalose are shown to be highly resistant to the organic solvents chloroform and acetone, and consequently, they can be encapsulated in a viable form in solid plastic materials. Bacteria are recovered by rehydration after physical disruption of the plastic. P. putida incorporated into a plastic coating of maize seeds was shown to colonize roots efficiently after germination.     

Serum prolactin is associated with apoptosis in men with human immunodeficiency virus infection

We examined the in vivo and in vitro production of prolactin (PRL) in 20 untreated HIV-infected men compared to 14 uninfected men and its association with the cell cycle and apoptosis. Compared to uninfected men, the HIV-infected men had: (i) higher fasting serum bioactive (BIO) PRL; (ii) lower serum immunoreactive (RIA) and BIO-PRL responses to intravenous metoclopramide; (iii) greater BIO-RIA PRL ratio both fasting and during intravenous metoclopramide; (iv) lower percentage of non-stimulated PBMC in the G0/G1 phase, but a higher percentage in the S phase, of the cell cycle with normal response to Concanavalin-A; and (v) higher in vitro production of BIO-PRL by non-stimulated PBMC, which was blocked after Concanavalin-A. Fasting serum BIO-PRL positively correlated with the percent of non-stimulated PBMC in S + G2/M phases. The percentage of apoptotic PBMC negatively correlated with CD4+ T lymphocytes and with the area under the serum RIA-PRL curve, but positively correlated with the area under the curve for the BIO/RIA ratio. These results suggest that in these HIV-infected men: (i) a diminished dopaminergic tone may exist, as an adaptive mechanism attempting to survive; and (ii) BIO-PRL may participate as a cofactor in the stimulation of T-cell proliferation.     

Upregulation of indoleamine 2,3-dioxygenase in hepatitis C virus infection

Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Long-term treadmill exercise improves spatial memory of male APPswe/PS1dE9 mice by regulation of BDNF expression and microglia activation

Increasing evidence suggests that physical activity could delay or attenuate the symptoms of Alzheimer''s disease (AD). But the underlying mechanisms are still not fully understood. To investigate the effect of long-term treadmill exercise on the spatial memory of AD mice and the possible role of β-amyloid, brain-derived neurotrophic factor (BDNF) and microglia in the effect, male APPswe/PS1dE9 AD mice aged 4 months were subjected to treadmill exercise for 5 months with 6 sessions per week and gradually increased load. A Morris water maze was used to evaluate the spatial memory. Expression levels of β-amyloid, BDNF and Iba-1 (a microglia marker) in brain tissue were detected by immunohistochemistry. Sedentary AD mice and wildtype C57BL/6J mice served as controls. The results showed that 5-month treadmill exercise significantly decreased the escape latencies (P < 0.01 on the 4th day) and improved the spatial memory of the AD mice in the water maze test. Meanwhile, treadmill exercise significantly increased the number of BDNF-positive cells and decreased the ratios of activated microglia in both the cerebral cortex and the hippocampus. However, treadmill exercise did not significantly alleviate the accumulation of β-amyloid in either the cerebral cortex or the hippocampus of the AD mice (P > 0.05). The study suggested that long-term treadmill exercise could improve the spatial memory of the male APPswe/PS1dE9 AD mice. The increase in BDNF-positive cells and decrease in activated microglia might underpin the beneficial effect.     

Functional Dissection of the Conjugative Coupling Protein TrwB

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwBΔN70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.Bacterial conjugation can be viewed mechanistically as a rolling-circle replication system linked to a type IV secretion process. The two processes come into contact through the activity of a protein that couples the plasmid replication machinery to the export system in the membrane, allowing horizontal dissemination of the replicating DNA molecule (35). This key protein is called “coupling protein” (here “T4CP” for “type IV CP”). It is present in all conjugative systems as well as in many type IV secretion systems (T4SS) involved in bacterial virulence (16). The secreted substrate in bacterial conjugation is the relaxase or pilot protein, attached to the DNA strand. The shoot-and-pump model for bacterial conjugation proposes that, after secretion of the protein through the T4SS, the T4CP works as a motor for export of the rest of the DNA molecule (36). In addition to its presumed role as a DNA transporter, TrwB is also required for transport of relaxase TrwC in the absence of DNA transfer (15).In accordance with its proposed coupling activity, early genetic experiments made patent that the function of conjugative T4CPs depended on interactions with both the cytoplasmic substrate complex (the relaxosome) and the T4SS (6, 7). Thus, T4CP interactions with other conjugation proteins are a key aspect of their function. There have been several reports of interactions between T4CPs from conjugative plasmids and either relaxosomal components—such as F-TraD with TraM (14, 38), RP4-TraG with TraI (49), and pCF10-PcfC with PcfF and PcfG (11)—or T4SS components such as R27-TraG with TrhB (17). T4CP-T4SS interactions have also been reported for the VirB/D4 T4SS involved in DNA transfer from Agrobacterium tumefaciens to plant cells (1, 9). Both sets of interactions have only been concurrently shown for TrwB, the T4CP of plasmid R388. TrwB interacts with proteins TrwA and TrwC, which form the R388 relaxosome, and with the R388 T4SS component TrwE (37). While the interaction with the relaxosome is highly specific for its cognate system (24, 37, 48), the interaction between the T4CP and the T4SS is less specific: a single T4CP can interact functionally with several conjugative T4SS. Interestingly, a correlation was observed between the strength of the T4CP-TrwE-like interaction and the efficiency of DNA transfer (37). T4CPs also interact with TrwE-like components of T4SS involved in virulence (13). In the case of the highly related Trw T4SS systems of plasmid R388 and the human pathogen Bartonella, it was further demonstrated that R388 TrwE could be functionally replaced by the Bartonella tribocorum TrwE homolog, TrwE_Bt (13).T4CPs are integral membrane proteins anchored to the inner membrane by an N-terminal transmembrane domain (TMD). The soluble cytoplasmic domain of TrwB (TrwBΔN70), lacking this TMD, has been biochemically and structurally analyzed in detail. It retains the ability to bind NTPs and to unspecifically bind DNA (42). The characterization of its DNA-dependent ATPase activity (53) strengthened the possibility that T4CPs work as DNA motors. This activity is also stimulated by the oriT-binding protein TrwA (52).The determination of the three-dimensional (3D) structure of TrwBΔN70 indicated a quaternary structure consisting of hexamers that form an almost spherical, orange-shaped structure with a 20-Å inner channel (ICH) (18, 19). Each monomer is composed of two main structural domains: the nucleotide-binding domain (NBD) and the all-alpha domain (AAD). The NBD has α/β topology and is reminiscent of RecA and DNA ring helicases. The AAD is facing the cytoplasmic side and bears significant structural similarity to the N-terminal domain of site-specific recombinase XerD and also to a 40-residue segment of the DNA binding domain of protein TraM, the component of the relaxosome of F-like plasmids that interacts with its cognate T4CP, TraD. The structure of the hexamer as a whole resembles that of the F₁-ATPase, raising interesting perspectives into the possible way of action of coupling proteins as molecular motors in conjugation (5).There have been several attempts to functionally dissect T4CPs. In F-TraD, it was determined that its C terminus is essential for relaxosomal specificity, probably through an interaction with TraM (4, 39, 48). The cytoplasmic domain of the related TraD protein of plasmid R1 stimulates both transesterase and helicase activities of its cognate relaxase, TraI (41, 51). A series of random mutations were shown to affect TraD oligomerization (23). In VirD4, the T4CP of the VirB T4SS of A. tumefaciens, both the periplasmic domain plus key residues of the NBD are required for its location at the cell poles (31); its interaction with the T4SS protein substrate VirE2 does not require the N-terminal TMD (2). Mutational analysis of R27 TraG showed that the periplasmic residues are essential for interaction with the T4SS (22). An N-terminal deletion variant of PcfC, the T4CP of the Enterococcus plasmid pCF10, loses its membrane localization but retains its ability to bind relaxosomal components (11). Biochemical analysis of full-length R388 TrwB showed that the N-terminal TMD stabilizes the protein, aids oligomerization, and affects nucleotide selection (25-27). This region is essential for T4SS interaction, but TrwBΔN70 retains the ability to interact with the relaxosomal components TrwA and TrwC (37). Taken together, these analyses suggested that the N-terminal TMD of the T4CPs is necessary for T4SS interaction, oligomerization, and cellular location and that the C-terminal cytoplasmic domain is necessary for relaxosomal interactions and ATPase activity associated with DNA transport.In this study, we set up different assays to search for mutants affecting TrwB function in DNA and protein transfer. We constructed a series of TrwB point mutants based on the 3D structure of TrwBΔN70. Most selected residues were essential for TrwB function in conjugation, especially under conditions where TrwB was in limiting quantities. We analyzed the in vivo properties of selected mutants with a battery of in vivo assays to map functional domains. Also, random mutants in the TMD were screened for improved interactions with the T4SS, which allowed mapping of the TrwB-TrwE interaction domain.