The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.     

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.     

Gamma-tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during drosophila oogenesis

bicoid (bcd) RNA localization requires the activity of exuperantia and swallow at sequential steps of oogenesis and is microtubule dependent. In a genetic screen, we identified two novel genes essential for bcd RNA localization. They encode maternal gamma-Tubulin37C (gammaTub37C) and gamma-tubulin ring complex protein 75 (Dgrip75), both of which are gamma-tubulin ring complex components. Mutations in these genes specifically affect bcd RNA localization, whereas other microtubule-dependent processes during oogenesis are not impaired. This provides direct evidence that a subset of microtubules organized by the gamma-tubulin ring complex is essential for localization of bcd RNA. At stage 10b, we find gammaTub37C and Dgrip75 anteriorly concentrated and propose the formation of a microtubule-organizing center at the anterior pole of the oocyte.     

The mu2 subunit of the clathrin adaptor AP-2 binds to FDNPVY and YppØ sorting signals at distinct sites

The endocytic sorting signal on the low-density lipoprotein receptor for clathrin-mediated internalization is the sequence FDNPVY in the receptor's cytosolic tail. We have used a combination of surface plasmon resonance and crosslinking with a photoactivated peptide probe to demonstrate the interaction between FDNPVY-containing peptides and the μ2 chain of purified AP-2 clathrin adaptors (the complexes responsible for plasma membrane sorting). We show that recognition of the FDNPVY signal is mediated by a binding site in the μ2-subunit that is distinct from the site for the more general YppØ sorting signal, another tyrosine-based sequence also recognized by μ2-adaptin. These results suggest the possibility that low-density lipoprotein receptor uptake may be modulated specifically and independently of other proteins in the clathrin pathway.     

Beach morphology and food web structure: comparison of an eroding and an accreting sandy shore in the North Sea

Food web components and inorganic nutrients were studied on two sandy shores of the adjacent barrier islands of Sylt and R?m? in the North Sea, differing in morphodynamics. Implications of high and low wave energy on the food web structure were assessed. The Sylt shore represents a dynamic intermediate beach type, while the R?m? shore is morphologically stable and dissipative. On the steep-profiled, coarse-grained Sylt shore, strong hydrodynamics resulted in erosion and high fluxes of organic material through the beach, but prevented any storage of food sources. In contrast, the flat-profiled, fine-grained R?m? shore, with low wave energy and accretion, accumulated organic carbon from surf waters. At Sylt, oxic nutrient regeneration prevailed, while anoxic mineralization was more important at R?m?. Macrofauna on the Sylt shore was impoverished compared with the community at R?m?. Correspondingly, abundances of epibenthic predators such as shrimps, crabs, fish, and shorebirds were also lower at Sylt. Meiofauna was abundant on both shores, but differed in taxonomic composition. Several major taxa were represented in fairly equal proportions of individual numbers on the well-oxygenated Sylt shore, while nematodes strongly dominated the assemblage at R?m?. Thus, on cold-temperate, highly dynamic intermediate shores with high wave energy and subject to erosion, the "small food web" dominates. Organisms are agile and quickly exploit fresh organic material. Larger organisms and nematodes abound under stable, dissipative and accreting shore conditions, where some food materials may accumulate and zoomass builds up to support numerous visitors from higher trophic levels. Electronic Publication     

Studies of strontium biokinetics in humans

The radioactive isotopes of strontium, mainly (90)Sr, which are common fission products, may significantly contribute to the internal exposure of the population in case of their accidental release into the environment and transfer to the food chain. For (90)Sr, the internal radiation dose is significantly dependent on the fractional absorption of the ingested activity (f(1)-value). Human data on the absorption of dietary strontium and of soluble forms of the element give values ranging from about 0.15 to 0.45 (up to 1.0) for adults. The International Commission on Radiological Protection (ICRP) has adopted f(1)-values of 0.6 for children of less than 1 year of age, 0.4 for children between 1 and 15 years and 0.3 for adolescents above 15 years of age. This study was aimed at investigating how far these values correspond to the actual uptake of strontium from contaminated foodstuffs. A methodology is presented that has been developed for preparing foodstuffs intrinsically labelled with stable isotopes and that will be used in tracer kinetic investigations. The results show that cress and salad can be adequately labelled, i.e. a strontium concentration of 1.36+/-0.47 g per kg of cress (wet weight) and of 0.29+/-0.04 g per kg of salad (wet weight) may be obtained within 15 days and 24 days, respectively. For the biokinetic investigations on humans, applying stable isotopes of Sr as tracers, about 0.1-1 mg strontium is required per volunteer, i.e. a few grams of the edible parts of the labelled material are sufficient.     

Amino acid replacements in transmembrane domain 1 influence osmosensing but not K<Superscript>+</Superscript> sensing by the sensor kinase KdpD of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Expression of the kdpFABC operon coding for the high affinity K+ -translocating KdpFABC complex of Escherichia coli is induced by K+ limitation or high osmolality. This process is controlled by the sensor kinase/response regulator system KdpD/KdpE. To study the importance of the transmembrane domains of KdpD for stimulus perception, each amino acid residue of the transmembrane domain 1 and Asp-424 of the adjacent periplasmic loop were replaced with Cys in a KdpD derivative devoid of native Cys residues. In vivo analysis of KdpD proteins with a single Cys residue revealed that 14 out of 18 amino acid replacements caused an altered response towards an osmotic upshift imposed by NaCl, whereby only four replacements also altered the response towards changes in the K+ concentration. The in vitro activities of most of the KdpD derivatives were in the range of KdpD devoid of native Cys residues. The results reveal that the osmosensing and K+ -sensing properties of KdpD can be dissected. Furthermore, the data support the hypothesis that osmosensing involves amino acid residues of the transmembrane domains.     

Interleukin-12 but not interleukin-18 is required for immunity to Trypanosoma cruzi in mice

Protective immunity to the parasite Trypanosoma cruzi in mice depends on a pro-inflammatory T cell response involving the production of interferon-gamma (IFN-gamma). In conjunction with interleukin-12 (IL-12), IL-18 promotes the synthesis of IFN-gamma and a T helper type 1 immune response. We investigated the requirements of IL-12 and IL-18 in murine T. cruzi infection by use of C57BL/6 mice genetically deficient in either cytokine. IL-12p40(-/-) mice succumbed to infection at doses of 100 parasites, whereas IL-18(-/-) and wild-type mice resisted infectious doses up to 1000 parasites to the same extent. Levels of parasitemia were comparable between the latter groups, as were tissue parasite burdens according to quantitative real-time PCR. In contrast, IL-12p40(-/-) mice displayed vastly increased levels of parasites both in blood and in tissue. IFN-gamma concentrations in the serum of infected mice and in supernatants of splenocytes stimulated in vitro were decreased in IL-18(-/-) mice, whereas in IL-12p40(-/-) mice, IFN-gamma was undetectable in the serum and drastically reduced in cell supernatants. Levels of IL-12 production were generally comparable between wild-type and IL-18(-/-) mice, as were levels of IL-4, IL-2 and nitric oxide. Thus, the requirement for endogenous pro-inflammatory cytokines for a protective murine immune response against T. cruzi is satisfied by the expression of IL-12, while IL-18 is dispensable.     

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.