The membrane affinities of the aliphatic amino acid side chains in an alpha-helical context are independent of membrane immersion depth

Understanding, predicting, and designing the binding of peptides and proteins to bilayers require quantifying the intrinsic propensities of individual amino acid residues to bind membranes as a function of structural context and bilayer depth. A host-guest study was performed using the peptide host named helix5 in order to determine the membrane affinities of the aliphatic side chains both in an alpha-helical context and as a function of bilayer depth. Use of the alpha-helical host with a constrained geometry allowed the placement of guest sites at three different depths in bilayers and minimized secondary structural changes due to guest substitutions. Circular dichroism and electron paramagnetic resonance (EPR) were used to characterize the aqueous and bilayer-bound structures of the peptide variants. EPR was also used to measure the bilayer-water partition constants of the peptide variants, and the Delta DeltaGtr values (relative to Gly) of the aliphatic amino acid side chains were subsequently calculated. Surprisingly, the DeltaDeltaGtr values did not significantly vary as a function of the guest site depth in bilayers. In addition, the Delta DeltaGtr values determined in an alpha-helical context are reduced to approximately two-thirds of Delta DeltaGtr values determined in other studies for the bilayer-water and octanol-water partitioning of amino acid side chains in extended and unstructured hosts. Both the relative reduction in Delta DeltaGtr values in the context of an alpha-helical host and the invariance of Delta DeltaGtr values with respect to bilayer depth are consistent with the membrane affinities of the aliphatic residues being largely determined by the classical hydrophobic effect.     

Fetal islet xenotransplantation in rodents and primates

(E)-2'-deoxy-2'-(fluoromethylene) cytidine (FMdC), a novel inhibitor of ribonucleotide-diphosphate reductase, has been shown to have anti-tumor activity against solid tumors and sensitize tumor cells to ionizing radiation. Pentoxifylline (PTX) can potentiate the cell killing induced by DNA-damaging agents through abrogation of DNA-damage-dependent G2 checkpoint. We investigated the cytotoxic, radiosensitizing and cell-cycle effects of FMdC and PTX in a human colon-cancer cell line WiDr. PTX at 0.25-1.0 mM enhanced the cytotoxicity of FMdC and lowered the IC50 of FMdC from 79 +/- 0.1 to 31.2 +/- 2.1 nM, as determined by MTT assay. Using clonogenic assay, pre-irradiation exposure of exponentially growing WiDr cells to 30 nM FMdC for 48 hr or post-irradiation to 0.5 to 1.0 mM PTX alone resulted in an increase in radiation-induced cytotoxicity. Moreover, there was a significant change of the radiosensitization if both drugs were combined as compared with the effect of either drug alone. Cell-cycle analysis showed that treatment with nanomolar FMdC resulted in S-phase accumulation and that such an S-phase arrest can be abrogated by PTX. Treatment with FMdC prior to radiation increased post-irradiation-induced G2 arrest, and such G2 accumulation was also abrogated by PTX. These results suggest that pharmacological abrogation of S and G2 checkpoints by PTX may provide an effective strategy for enhancing the cytotoxic and radiosensitizing effects of FMdC.     

The alloimmune thrombocytopenic syndromes

We have summarized five thrombocytopenic syndromes caused by platelet-reactive alloantibodies. Increased awareness of these five syndromes, together with the greater availability of highly-specialized laboratory methods to detect and to characterize platelet-reactive alloantibodies, will lead to their more frequent diagnosis. It is important for the clinician to consider unusual alloimmune thrombocytopenic disorders in clinical settings in which alloantigens that could be limited to just one family could cause important disease (ie, NAT caused by a private alloantigen; theoretically, PAT or TAT related to directed donations of blood or bone marrow). These considerations underscore the need for serological investigations to involve the family members rather than to rely on standard platelet typing donor pools.     

Brain abscess in solid organ transplant recipients receiving cyclosporine-based immunosuppression

There is currently a stark therapeutic void in the treatment of evolving stroke. Although P-selectin is rapidly expressed by hypoxic endothelial cells in vitro, the functional significance of P-selectin expression in stroke remains unexplored. In order to identify the pathophysiological consequences of P-selectin expression and to identify P-selectin blockade as a potential new approach for the treatment of stroke, experiments were performed using a murine model of focal cerebral ischemia and reperfusion. Early P-selectin expression in the postischemic cerebral cortex was demonstrated by the specific accumulation of radiolabeled anti-murine P-selectin IgG, with the increased P-selectin expression localized to the ipsilateral cerebral microvascular endothelial cells by immunohistochemistry. In experiments designed to test the functional significance of increased P-selectin expression in stroke, neutrophil accumulation in the ischemic cortex of mice expressing the P-selectin gene (PS +/+) was demonstrated to be significantly greater than that in homozygous P-selectin-null mice (PS -/-). Reduced neutrophil influx was accompanied by greater postischemic cerebral reflow (measured by laser Doppler) in the PS -/- mice. In addition, PS -/- mice demonstrated smaller infarct volumes (5-fold reduction, P<.05) and improved survival compared with PS +/+ mice (88% versus 44%, P<.05). Functional blockade of P-selectin in PS +/+ mice using a monoclonal antibody directed against murine P-selectin also improved early reflow and stroke outcome compared with control mice, with reduced cerebral infarction volumes noted even when the blocking antibody was administered after occlusion of the middle cerebral artery. These data are the first to demonstrate a pathophysiological role for P-selectin in stroke and suggest that P-selectin blockade may represent a new therapeutic target in the treatment of stroke.     

Time-dependent effects of repeated amphetamine treatment on norepinephrine in the hypothalamus and hippocampus assessed with in vivo microdialysis

The effects of repeated amphetamine (AMPH) pretreatment on norepinephrine (NE) neurotransmission in the hypothalamus and hippocampus were assessed using in vivo microdialysis. Rats were pretreated with either saline or an escalating-dose AMPH regimen (1-->10 mg/kg) over 10 consecutive days, and then were withdrawn from AMPH for either 1 day or 30 days, at which time the animals underwent two consecutive days of testing. As expected, repeated treatment with AMPH resulted in time-dependent changes in both spontaneous locomotor activity and in the psychomotor response to a subsequent challenge injection of AMPH. In addition, repeated exposure to AMPH resulted in time-dependent and regionally-specific changes in the basal concentrations of NE in dialysate, and in the NE response to an AMPH challenge. For example, AMPH pretreatment produced a persistent (at least one month) increase in the basal concentration of NE in the hippocampus, but not the hypothalamus, although the response to an AMPH challenge was altered in both structures. It is suggested that AMPH treatment produces adaptations in NE systems that far outlast the acute effects of the drug, and that these may contribute to both transient and more persistent behavioral sequelae associated with the discontinuation of chronic AMPH use.