The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization

Dendritic cells (DC) are instrumental in orchestrating an appropriately polarized Th cell response to pathogens. DC exhibit considerable phenotypic and functional plasticity, influenced by lineage, Ag engagement, and the environment in which they develop and mature. In this study, we identify the human cationic peptide LL-37, found in abundance at sites of inflammation, as a potent modifier of DC differentiation, bridging innate and adaptive immune responses. LL-37-derived DC displayed significantly up-regulated endocytic capacity, modified phagocytic receptor expression and function, up-regulated costimulatory molecule expression, enhanced secretion of Th-1 inducing cytokines, and promoted Th1 responses in vitro. LL-37 may be an attractive therapeutic candidate for manipulating T cell polarization by DC.     

C-peptide improves adenosine-induced myocardial vasodilation in type 1 diabetes patients

Patients with type 1 (insulin-dependent) diabetes show reduced skeletal muscle blood flow and coronary vasodilatory function despite intensive insulin therapy and good metabolic control. Administration of proinsulin C-peptide increases skeletal muscle blood flow in these patients, but a possible influence of C-peptide on myocardial vasodilatory function in type 1 diabetes has not been investigated. Ten otherwise healthy young male type 1 diabetic patients (Hb A1c 6.6%, range 5.7-7.9%) were studied on two consecutive days during normoinsulinemia and euglycemia in a double-blind, randomized, crossover design, receiving intravenous infusion of C-peptide (5 pmol.kg-1.min-1) for 120 min on one day and saline infusion on the other day. Myocardial blood flow (MBF) was measured at rest and during adenosine administration (140 microg.kg-1.min-1) both before and during the C-peptide or saline infusions by use of positron emission tomography and [15O]H2O administration. Basal MBF was not significantly different in the patients compared with an age-matched control group, but adenosine-induced myocardial vasodilation was 30% lower (P < 0.05) in the patients. During C-peptide administration, adenosine-stimulated MBF increased on average 35% more than during saline infusion (P < 0.02) and reached values similar to those for the healthy controls. Moreover, as evaluated from transthoracal echocardiographic measurements, C-peptide infusion resulted in significant increases in both left ventricular ejection fraction (+5%, P < 0.05) and stroke volume (+7%, P < 0.05). It is concluded that short-term C-peptide infusion in physiological amounts increases the hyperemic MBF and left-ventricular function in type 1 diabetic patients.     

New equations for redox and nano-signal transduction

Cells maintain redox potentials (Eh) in intracellular compartments, sometimes referred to as redox environments. These potentials are often very reducing, for example in the cytoplasm, but throughout the cell different potentials are maintained, commensurate with the functionality of that particular part of the cell. Furthermore, within a simple cellular compartment, "hot-spots" of redox poise may be maintained. However, despite this complexity, the quantification of such redox potentials has been attempted, and there is indeed a need to accurately assess such potentials, and to monitor how they might change with time. Changes in intracellular potentials may control the oxidation or reduction of protein residues, such as cysteine, which would alter the conformation of those proteins and so modulate their function. Although there are several methods for estimating the intracellular redox potential, the most accessible technique is the measurement of intracellular concentrations of GSH and GSSG, and the calculation of Eh using the Nernst equation. However, using this equation shows that the Eh imposed by the glutathione couple is dependent on the total concentration of glutathione present, and therefore values of Eh obtained may be erroneous. Here, we suggest new equations that can be used to calculate the redox environments of cells.     

Recombinant respiratory syncytial viruses lacking the C-terminal third of the attachment (G) protein are immunogenic and attenuated in vivo and in vitro

The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpDeltaG118, rA2cpDeltaG174, rA2cpDeltaG193, and rA2cpDeltaG213. All rA2cpDeltaG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37 degrees C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpDeltaG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpDeltaG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpDeltaG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.     

Stimulation of vascular smooth muscle cell proliferation and migration by apolipoprotein(a) is dependent on inhibition of transforming growth factor-beta activation and on the presence of kringle IV type 9

Elevated plasma concentrations of lipoprotein(a) are a risk factor for the development of a variety of atherosclerotic disorders. Despite intensive study, the mechanisms by which lipoprotein(a) promotes these disorders remain to be unequivocally defined. It has been demonstrated that lipoprotein(a), through its unique constituent apolipoprotein(a) (apo(a)), stimulates vascular smooth muscle cell (SMC) migration and proliferation. These effects arise from the ability of apo(a) to inhibit the formation of active transforming growth factor beta (TGF-beta) from its latent precursor, which in turn is caused by the ability of apo(a) to decrease the formation of plasmin from its precursor plasminogen. We utilized a battery of recombinant apo(a) variants that represent systematic deletions of the various domains in the molecule to further probe the mechanism underlying the effect of apo(a) on SMC responses. All recombinant apo(a) variants that contained kringle IV type 9 were able to stimulate SMC proliferation and migration and to decrease the formation of active TGF-beta; conversely all recombinant apo(a) variants lacking kringle IV type 9 had no effect on these parameters. The kringle IV type 9-dependent effects of apo(a) on SMC proliferation required the presence of plasminogen, suggesting for the first time that this kringle mediates the ability of apo(a) to inhibit pericellular plasmin formation.     

The two motor domains of KIF3A/B coordinate for processive motility and move at different speeds

KIF3A/B, a kinesin involved in intraflagellar transport and Golgi trafficking, is distinctive because it contains two nonidentical motor domains. Our hypothesis is that the two heads have distinct functional properties, which are tuned to maximize the performance of the wild-type heterodimer. To test this, we investigated the motility of wild-type KIF3A/B heterodimer and chimaeric KIF3A/A and KIF3B/B homodimers made by splicing the head of one subunit to the rod and tail of the other. The first result is that KIF3A/B is processive, consistent with its transport function in cells. Secondly, the KIF3B/B homodimer moves at twice the speed of the wild-type motor but has reduced processivity, suggesting a trade-off between speed and processivity. Third, the KIF3A/A homodimer moves fivefold slower than wild-type, demonstrating distinct functional differences between the two heads. The heterodimer speed cannot be accounted for by a sequential head model in which the two heads alternate along the microtubule with identical speeds as in the homodimers. Instead, the data are consistent with a coordinated head model in which detachment of the slow KIF3A head from the microtubule is accelerated roughly threefold by the KIF3B head.     

Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane.