In vitro binding of [14C]2,5-hexanedione to rat neuronal cytoskeletal proteins

2,5-Hexanedione (2,5-HD) induces central-peripheral axonpathy characterized by the accumulation of 10-nm neurofilaments proximal to the nodes of Ranvier and a Wallerian-type degeneration. It has been postulated that neurofilament crosslinking may be involved in the production of this axonopathy. A potential initiating event in this neurotoxic process may be the direct binding of 2,5-HD to neurofilament and microtubule proteins. In this study, the in vitro binding of [¹⁴C]2,5-HD to neurofilament and microtubule proteins was examined. Neurofilament proteins isolated from rat spinal cord or microtubule proteins isolated from rat brain were incubated in the presence of 2,5-HD at concentrations ranging 25 to 500 mM. Quantitative analysis of sodium dodecyl sulfate (SDS) polyacrylamide gels revealed a dose- and time-dependent binding of 2,5-HD to both neurofilament proteins and microtubule proteins. Expressed as pmol 2,5-HD bound per g protein, the observed relative binding was MAP2>NF160>NF200>NF68>tubulin. These data demonstrate the direct binding of 2,5-HD to cytoskeletal proteins including both neurofilaments and microtubules.     

The influence of scald water pH on the death rates of Salmonella typhimurium and other bacteria attached to chicken skin

Factory trials where scald tank water was maintained at pH 9.0 +/- 0.2 showed that compared with the usual system of scalding when the water is at pH 6.0 for much of the working day the bacterial counts on carcases post scalding and plucking were significantly lower. In laboratory experiments, attached Salmonella typhimurium and the naturally occurring skin flora were found to be killed significantly more quickly in water at pH 9.0 +/- 0.2.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Multimodal pressure-flow method to assess dynamics of cerebral autoregulation in stroke and hypertension

Background

This study evaluated the effects of stroke on regulation of cerebral blood flow in response to fluctuations in systemic blood pressure (BP). The autoregulatory dynamics are difficult to assess because of the nonstationarity and nonlinearity of the component signals.

Methods

We studied 15 normotensive, 20 hypertensive and 15 minor stroke subjects (48.0 ± 1.3 years). BP and blood flow velocities (BFV) from middle cerebral arteries (MCA) were measured during the Valsalva maneuver (VM) using transcranial Doppler ultrasound.

Results

A new technique, multimodal pressure-flow analysis (MMPF), was implemented to analyze these short, nonstationary signals. MMPF analysis decomposes complex BP and BFV signals into multiple empirical modes, representing their instantaneous frequency-amplitude modulation. The empirical mode corresponding to the VM BP profile was used to construct the continuous phase diagram and to identify the minimum and maximum values from the residual BP (BP_R) and BFV (BFV_R) signals. The BP-BFV phase shift was calculated as the difference between the phase corresponding to the BP_R and BFV_R minimum (maximum) values. BP-BFV phase shifts were significantly different between groups. In the normotensive group, the BFV_R minimum and maximum preceded the BP_R minimum and maximum, respectively, leading to large positive values of BP-BFV shifts.

Conclusion

In the stroke and hypertensive groups, the resulting BP-BFV phase shift was significantly smaller compared to the normotensive group. A standard autoregulation index did not differentiate the groups. The MMPF method enables evaluation of autoregulatory dynamics based on instantaneous BP-BFV phase analysis. Regulation of BP-BFV dynamics is altered with hypertension and after stroke, rendering blood flow dependent on blood pressure.

     

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2- 8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.      

Efflux pumps expression and its association with porin down-regulation and β-lactamase production among <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> causing bloodstream infections in Brazil

Background  

Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

The Rac1 C-terminal polybasic region regulates the nuclear localization and protein degradation of Rac1

We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA). These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasome-mediated degradative pathways involving the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for degradation.     

Simulations of congenital septal defect closure and reactivity testing in patient-specific models of the pediatric pulmonary vasculature: A 3D numerical study with fluid-structure interaction

Clinical imaging methods are highly effective in the diagnosis of vascular pathologies, but they do not currently provide enough detail to shed light on the cause or progression of such diseases, and would be hard pressed to foresee the outcome of surgical interventions. Greater detail of and prediction capabilities for vascular hemodynamics and arterial mechanics are obtained here through the coupling of clinical imaging methods with computational techniques. Three-dimensional, patient-specific geometric reconstructions of the pediatric proximal pulmonary vasculature were obtained from x-ray angiogram images and meshed for use with commercial computational software. Two such models from hypertensive patients, one with multiple septal defects, the other who underwent vascular reactivity testing, were each completed with two sets of suitable fluid and structural initial and boundary conditions and used to obtain detailed transient simulations of artery wall motion and hemodynamics in both clinically measured and predicted configurations. The simulation of septal defect closure, in which input flow and proximal vascular stiffness were decreased, exhibited substantial decreases in proximal velocity, wall shear stress (WSS), and pressure in the post-op state. The simulation of vascular reactivity, in which distal vascular resistance and proximal vascular stiffness were decreased, displayed negligible changes in velocity and WSS but a significant drop in proximal pressure in the reactive state. This new patient-specific technique provides much greater detail regarding the function of the pulmonary circuit than can be obtained with current medical imaging methods alone, and holds promise for enabling surgical planning.