A family of brevinin-2 peptides with potent activity against Pseudomonas aeruginosa from the skin of the Hokkaido frog, Rana pirica

Nine peptides displaying varying degrees of antimicrobial activity were extracted from the skin of the Hokkaido frog, Rana pirica. Five structurally related peptides were identified as members of the brevinin-2 family. These peptides were active against reference strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) and Gram-positive (Staphlococcus aureus) bacteria but displayed relatively low hemolytic activity. The most abundant peptide, brevinin-2PRa (680 nmol/g weight of dry skin) showed high potency [minimal inhibitory concentration (MIC) values between 6 and 12 microM] against a range of clinical isolates of P. aeruginosa. In addition, activity was unaffected by NaCl concentrations up to 200 mM. Cladistic analysis based on the primary structures of brevinin-2 peptides supports a close phylogenetic relationship between R. pirica and Japanese mountain brown frog Rana ornativentris. One peptide of the ranatuerin-2 family and one strongly hemolytic peptide of the brevinin-1 family were also isolated from the extract along with two members of the temporin family, temporin-1PRa (ILPILGNLLNGLL.NH(2)) and temporin-1PRb (ILPILGNLLNSLL.NH(2)) that atypically lacked basic amino acid residues and showed only very weak antimicrobial and hemolytic activity.     

A mutation in the <Emphasis Type="Italic">Cap Binding Protein 20</Emphasis> gene confers drought

In a genetic screen for Arabidopsismutants displaying pleiotropic alterations in vegetative development and stress responses we have identified a T-DNA insertion mutation in the Cap Binding Protein 20 (CBP20) gene, that encodes the 20kDa subunit of the nuclear mRNA cap binding complex (nCBC). Plants homozygous for the recessive cbp20 mutation show mild developmental abnormalities, such as serrated rosette leaves, delayed development and slightly reduced stature. Loss of the cbp20 function also confers hypersensitivity to abscisic acid during germination, significant reduction of stomatal conductance and greatly enhanced tolerance to drought. Expression of the wild type cDNA by CaMV35S promoter provides full genetic complementation of the pleiotropic cbp20phenotype. Phenotypic characteristics of the cbp20 mutant are very similar to those of recently described abh1mutant that is defective in the 80kDa subunit of nCBC. Our data thus confirm that both genes are dedicated to the same function. CBP20 provides a new target for breeding efforts that aim at the improvement of drought tolerance in plants. Our results also show that screening for pleiotropic phenotypes in mutant plant populations may be a fruitful strategy to isolate genes for agronomically important traits.     

Distinct temperature-dependent dopamine-releasing effect of drugs of abuse in the olfactory bulb

It was recently shown in the olfactory bulb (OB) that the response to olfactory stimulation might be related to local reinforcement mechanisms involved in discrimination of different odors. Therefore, it seemed interesting to study the effects of several drugs of abuse on the release of dopamine (DA) in the OB. Nicotine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), and cocaine at 37 degrees C increased the release of [3H] DA from olfactory bulb slice preparations of the rats. While nicotine, amphetamine, and MDMA directly evoked DA release, cocaine, by inhibiting the reuptake processes, enhanced the electrical stimulation-evoked release. At low temperature (17 degrees C), a condition in which the transmitter uptake carriers of the plasma membrane in both the normal and reverse mode of operation are inhibited, the nicotine-evoked [3H] DA release was potentiated, whereas those evoked by amphetamine and MDMA were inhibited. At low temperature the field stimulation-evoked [3H] DA release was potentiated, but under this condition cocaine failed to increase the release. Our results show that low temperature (a) increases the concentration of extracellular DA released by Ca(2+)-dependent vesicular exocytosis elicited by nicotine, (b) inhibits the extracellular Ca(2+)-independent amphetamine- and MDMA-induced release of DA that occurs by the reverse operation of membrane carriers transporting DA from the cytoplasm of presynaptic terminals to the extraneuronal space, and (c) does not alter the inhibitory effect of cocaine on DA uptake that increases the concentration of extracellular DA released by field stimulation. The findings that the drugs of abuse tested all enhanced the release of DA in the olfactory bulb suggest that local reinforcing mechanisms may also exist in this brain area. In addition, we also show that lowering the temperature in in vitro experiments is an easy and straightforward method for separating vesicular and cytoplasmic release of transmitters, and is suitable for studying the mechanism of catecholamine release evoked by drugs of abuse. This technique may be applicable in other neurochemical studies that need inhibition of the uptake carriers without the blockade of the ligand-gated ion channels caused by reuptake inhibitor drugs.     

Phosphatidylglycerol is essential for oligomerization of photosystem I reaction center

Our earlier studies with the pgsA mutant of Synechocystis PCC6803 demonstrated the important role of phosphatidylglycerol (PG) in PSII dimer formation and in electron transport between the primary and secondary electron-accepting plastoquinones of PSII. Using a long-term depletion of PG from pgsA mutant cells, we could induce a decrease not only in PSII but also in PSI activity. Simultaneously with the decrease in PSI activity, dramatic structural changes of the PSI complex were detected. A 21-d PG depletion resulted in the degradation of PSI trimers and concomitant accumulation of monomer PSI. The analyses of PSI particles isolated by MonoQ chromatography showed that, following the 21-d depletion, PSI trimers were no longer detectable in the thylakoid membranes. Immunoblot analyses revealed that the PSI monomers accumulating in the PG-depleted mutant cells do not contain PsaL, the protein subunit thought to be responsible for the trimer formation. Nevertheless, the trimeric structure of PSI reaction center could be restored by readdition of PG, even in the presence of the protein synthesis inhibitor lincomycin, indicating that free PsaL was present in thylakoid membranes following the 21-d PG depletion. Our data suggest an indispensable role for PG in the PsaL-mediated assembly of the PSI reaction center.     

The zygomatic flap: a further possibility in reconstructing soft-tissue defects of the nose and upper lip

Through the dissection and localization of the cutaneous zygomatic branch, as previously described by the authors, a vessel is available that plays an important role in reconstructive surgery. The performance of this anatomical study has enabled designing of the so-called zygomatic flap, which can be considered as a further possibility in the reconstruction of soft-tissue defects of the upper lip and nose. This new island axial pattern flap provides a reliable source of skin, with color match for facial resurfacing, and leaves a well-hidden donor site similar to that of the nasolabial flap. The flap must be carefully raised, and when properly designed, it can follow naturally existing contour lines, thus respecting and preserving the normal facial topography and leaving the patient with minimal surgical deformity. In this article, the authors report the clinical application of the zygomatic flap and the outcome of 10 cases. In a follow-up period from 1998 to the end of 2002, there was no flap loss, and in all cases, the aesthetic results were excellent and highly acceptable to the patients. The authors' experience with this new island axial pattern flap has been good, and they recommend this technique.     

Kinetic and crystallographic studies on 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site

In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of beta-D-glucopyranose, 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc-1-P) with K(i) values of 145.2 (+/-11.6), 76 (+/-4.8), and 8.6 (+/-0.7) muM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb-methyl-oxadiazole complex, 1.92 A; GPb-benzothiazole, 2.10 A; GPb-benzimidazole, 1.93 A). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (approximately 32 A) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.     

Iron gathering of opportunistic pathogenic fungi. A mini review

Iron is an essential nutrient for most organisms because it serves as a catalytic cofactor in oxidation-reduction reactions. Iron is rather unavailable because it occurs in its insoluble ferric form in oxides and hydroxides, while in serum of mammalian hosts is highly bound to carrier proteins such as transferrin, so the free iron concentration is extremely low insufficient for microbial growth. Therefore, many organisms have developed different iron-scavenging systems for solubilizing ferric iron and transporting it into cells across the fungal membrane. There are three major mechanisms by which fungi can obtain iron from the host: (a) utilization of a high affinity iron permease to transport iron intracellularly, (b) production and secretion of low molecular weight iron-specific chelators (siderophores), (c) utilization of a hem oxygenase to acquire iron from hemin. Patients with elevated levels of available serum iron treated with iron chelator, deferoxamine to remedy iron overload conditions have an increased susceptibility of invasive zygomycosis. Presumably deferoxamine predisposes patients to Zygomycetes infections by acting as a siderophore]. The frequency of zygomycosis is increasing in recent years and these infections respond very poorly to currently available antifungal agents, so new approaches to develop strategies to prevent and treat zygomycosis are urgently needed. Siderophores and iron-transport proteins have been suggested to function as virulence factors because the acquisition of iron is a crucial pathogenetic event. Biosynthesis and uptake of siderophores represent possible targets for antifungal therapy.     

T-cell recognition of differentially tolerated epitopes of cartilage proteoglycan aggrecan in arthritis

Proteoglycan (PG) aggrecan, a major macromolecular component of cartilage, is highly immunogenic; it induces arthritis in genetically susceptible BALB/c mice. The present study maps the T-cell epitope repertoire of cartilage PG by identifying a total of 27 distinct T-cell epitopes. An epitope hierarchy, accounting for the different effector functions of PG-specific T cells, and determinant spreading, has been found. T-cell responses to four epitopes were associated with arthritis induction. Some of the T-cell epitopes were full T-cell activators, whereas a number of subdominant and cryptic epitopes proved to be partial activators in vitro, inducing either cytokine secretion or T-cell proliferation, but not both. A few T-cell epitopes of the core protein of cartilage PG were clearly recognized by T cells in PG-immunized arthritic animals, but the corresponding peptides did not induce T-cell responses when injected into naive BALB/c mice; thus these T-cell epitopes were designated as "conditionally immunogenic."     

Astrocyte Mitochondrial Mechanisms of Ischemic Brain Injury and Neuroprotection

Research on ischemic brain injury has established a central role of mitochondria in neuron death. Astrocytes are also damaged by ischemia, although the participation of mitochondria in their injury is ill defined. As astrocytes are responsible for neuronal metabolic and trophic support, astrocyte dysfunction will compromise postischemic neuronal survival. Ischemic alterations to astrocyte energy metabolism and the uptake and metabolism of the excitatory amino acid transmitter glutamate may be particularly important. Despite the significance of ischemic astrocyte injury, little is known of the mechanisms responsible for astrocyte death and dysfunction. This review focuses on differences between astrocyte and neuronal metabolism and mitochondrial function, and on neuronal-glial interactions. The potential for astrocyte mitochondria to serve as targets of neuroprotective interventions is also discussed.