Early activation of bradykinin B2 receptor aggravates reactive oxygen species generation and renal damage in ischemia/reperfusion injury

The kallikrein/kinin system is beneficial in ischemia/reperfusion injury in heart, controversial in brain, but detrimental in lung, liver, and intestine. We examined the role of the kallikrein/kinin system in acute ischemia/reperfusion renal injury induced by 40 min occlusion of the renal artery followed by reperfusion. Rats were infused with tissue kallikrein protein 5 days before (pretreated group) or after (treated group) ischemia. Two days later, the pretreated group exhibited the worst renal dysfunction, followed by the treated group, then the control group. Kallikrein increased tubular necrosis and inflammatory cell infiltration with generation of more tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Reactive oxygen species (ROS), malondialdehyde, and reduced/oxidized glutathione measurement revealed that the oxidative stress was augmented by kallikrein administration in both ischemic and reperfusion phases. The groups with more ROS generation also had more apoptotic renal cells. The deleterious effects of kallikrein on ischemia/reperfusion injury were reversed by cotreatment with bradykinin B2 receptor (B2R) antagonist, but not B1 receptor antagonist, and were not associated with hemodynamic changes. We conclude that early activation of B2R augmented ROS generation in ischemia/reperfusion renal injury, resulting in subsequent apoptosis, inflammation, and tissue damage. This finding suggests the potential application of B2R antagonists in acute ischemic renal disease associated with bradykinin activation.     

Nonviral gene carriers based on diblock copolymers of poly(2-ethyl-2-oxazoline) and linear polyethylenimine

Diblock copolymers that consist of poly(2-ethyl-2-oxazoline) (PEOz) and linear polyethylenimine (LPEI) were prepared for use as nonviral gene carriers. The PEOz-b-LPEI copolymers were synthesized by coupling PEOz with LPEI in a thiol-disulfide exchange reaction between the sulfhydryl and pyridyl disulfide terminal groups. A polymer/DNA weight ratio (P/D) of over 12 was required to enable PEOz-b-LPEI to condense DNA completely. The DNA-condensing capability of the diblock copolymers was increased with increasing the hydrolytic degrees of the LPEI segment. The PEOz-b-LPEI polyplexes were stable in 150 mM NaCl aqueous solution and had a mean diameter around 190 nm, whereas BPEI and LPEI polyplexes formed large aggregates in the range 300-500 nm. In addition, these polyplexes exhibited the sensitivity to solution pH and were dissociated in the acidic buffers (pH < or = 5.5). The results of in vitro cell viability and luciferase assay indicated that PEOz-b-LPEI showed not only low cytotoxicity but also high transfection efficiency in gene expression.     

Malaria: therapy, genes and vaccines

Malaria kills over 3,000 children each day. Modern molecular and biochemical approaches are being used to help understand and control Plasmodium falciparum, the parasite that causes this deadly disease. New drugs are being invented for both chemoprophylaxis and therapeutic treatments and their use is discussed along side that of the more commonly used treatments. Classical genetic crosses coupled with molecular analysis of gene loci are use to explain the genetics behind the development of specific drug resistances that the parasites have naturally developed. Rapid advances in DNA sequencing techniques have allowed the compete sequencing of the P. falciparum and several other rodent malaria parasite genomes. Proteomics and computational analysis of these vast databanks are being used to model and investigate the three-dimensional structure of many key malaria proteins in an attempt to facilitate drug design. Recombinant protein expression in bacteria and yeast coupled with cGMP purification technologies and conditions have lead to the recent availability of several dozen malaria protein antigens for human-use Phase I and Phase II vaccine trials. Drug companies, private foundations, and key government agencies have contributed to the coordinated efforts needed to test these antigens, adjuvants and delivery methods in an effort to find an effective malaria vaccine that will prevent infection and disease.     

Proapoptotic effects of NARC 1 (= PCSK9), the gene encoding a novel serine proteinase.

BACKGROUND: NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC 1 might be involved in the propagation of apoptotic signaling in neurons. METHODS: CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scanning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC 1 proapoptotic effect. RESULTS: Wild-type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF-sensitive component of cell death. NH(2)-terminal deletion constructs of NARC 1 had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAF. CONCLUSION: The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc 1 active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc 1 protein.     

Contrasting phylogeographical patterns between mainland and island taxa of the Pinus luchuensis complex

Species whose geographical distribution encompasses both mainland and island populations provide an ideal system for examining isolation and genetic divergence. In this study, paternally transmitted chloroplast DNA (cpDNA) and maternally transmitted mitochondrial DNA (mtDNA) were used to estimate population structure and phylogeography of Pinus luchuensis, a species found in eastern China (ssp. hwangshanensis), Taiwan (ssp. taiwanensis), and the Ryukyu Archipelago (ssp. luchuensis). Gene genealogies of both mtDNA and cpDNA reveal two major lineages. Molecular dating indicates that these lineages diverged before the colonization of P. luchuensis subspecies in Taiwan and the Ryukyu Archipelago. Both mtDNA and cpDNA show a lack of correspondence between molecular phylogeny and subspecies designation. Phylogeographical analysis suggests that paraphyly of the subspecies is the result of recent divergence rather than secondary contacts. In spite of the short divergence history of P. luchuensis on islands, the island populations show the same degree of genetic divergence as mainland populations. Low levels of genetic diversity in the mainland ssp. hwangshanensis suggest demographic bottlenecks. In contrast, the high heterogeneity of genetic composition for island populations is likely to be associated with a history of multiple colonization from the mainland. The spatial apportionment of organelle DNA polymorphisms is consistent with a pattern of stepwise colonization on island populations.     

Cutting edge: novel human dendritic cell- and monocyte-attracting chemokine-like protein identified by fold recognition methods

Chemokines play an important role in the immune system by regulating cell trafficking in homeostasis and inflammation. In this study, we report the identification and characterization of a novel cytokine-like protein, DMC (dendritic cell and monocyte chemokine-like protein), which attracts dendritic cells and monocytes. The key to the identification of this putative new chemokine was the application of threading techniques to its uncharacterized sequence. Based on our studies, DMC is predicted to have an IL-8-like chemokine fold and to be structurally and functionally related to CXCL8 and CXCL14. Consistent with our predictions, DMC induces migration of monocytes and immature dendritic cells. Expression studies show that DMC is constitutively expressed in lung, suggesting a potential role for DMC in recruiting monocytes and dendritic cells from blood into lung parenchyma.