Activated platelets rescue apoptotic cells via paracrine activation of EGFR and DNA-dependent protein kinase.

Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK--an endogenous cytoprotective cascade.

Towards third generation matrix metalloproteinase inhibitors for cancer therapy.

The failure of matrix metalloproteinase (MMP) inhibitor drug clinical trials in cancer was partly due to the inadvertent inhibition of MMP antitargets that counterbalanced the benefits of MMP target inhibition. We explore how MMP inhibitor drugs might be developed to achieve potent selectivity for validated MMP targets yet therapeutically spare MMP antitargets that are critical in host protection.

Time-dependent XAS studies of trapped enzyme-substrate complexes of alcohol dehydrogenase from Thermoanaerobacter brockii.

The understanding of structure-function relationships in proteins has been significantly advanced with the advent of the biotechnological revolution. A goal yet to be realized for many metalloenzyme systems is to characterize the dynamic changes in structure that bridge the static endpoints provided by crystallography. We present here a series of edge and EXAFS spectra of the metalloenzyme alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) complexed with its substrate. The enzyme-substrate complexes were trapped by fast freezing at various times, following their enzyme activity. Our edge and EXAFS analyses both reveal the time-dependent changes in the structure of the active site of TbADH.

X-ray absorption studies of human matrix metalloproteinase-2 (MMP-2) bound to a highly selective mechanism-based inhibitor. comparison with the latent and active forms of the enzyme.

Malignant tumors express high levels of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metastasis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been implicated in malignant processes, and thus, they have been a target for drugs designed to block their activity. Therefore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full-length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of MMP-2 in complex with SB-3CT, a recently reported novel mechanism-based synthetic inhibitor that was designed to be highly selective in gelatinases. It is shown that SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme.

Biological Control of the Root-Knot Nematode Meloidogyne javanica by Trichoderma harzianum.

ABSTRACT The fungal biocontrol agent, Trichoderma harzianum, was evaluated for its potential to control the root-knot nematode Meloidogyne javanica. In greenhouse experiments, root galling was reduced and top fresh weight increased in nematode-infected tomatoes following soil pretreatment with Trichoderma peat-bran preparations. The use of a proteinase Prb1-transformed line (P-2) that contains multiple copies of this gene improved biocontrol activity in the greenhouse experiments compared with the nontransformed wild-type strain (WT). All the Trichoderma strains showed the ability to colonize M. javanica-separated eggs and second-stage juveniles (J2) in sterile in vitro assays, whereas P-2 also penetrated the egg masses. This protease-transformed line presented the same nematicidal and overall proteolytic activity as the WT in in vitro tests in which concentrated soil extracts from Trichoderma-treated soils immobilized the infective J2. However, the J2 immobilization and proteolytic activities of both P-2 and the WT were higher than those obtained with strain T-203. Characterization of the activity of all Trichoderma strains soil extracts on J2 showed that it was heat resistant and restricted to the low-molecular-weight fraction (less than 3 kDa). It is suggested that improved proteolytic activity of the antagonist may be important for the biological control of the nematodes.

Structural characterization of the catalytic active site in the latent and active natural gelatinase B from human neutrophils.

Matrix metalloproteinases are endopeptidases that have a leading role in the catabolism of the macromolecular components of the extracellular matrix in a variety of normal and pathological processes. Human gelatinase B is a zinc-dependent proteinase and a member of the matrix metalloproteinase family that is involved in inflammation, tissue remodeling, and cancer. We have conducted x-ray absorption spectroscopy, atomic emission, and quantum mechanics studies of natural and activated human gelatinase B. Our results show that the natural enzyme contains one catalytic zinc ion that is central to catalysis. In addition, upon enzyme activation, the catalytic zinc site exhibits a conformation change that results in the expansion of the bond distances around the zinc ion and the replacement of one sulfur with oxygen. Interestingly, quantum mechanics calculations show that oxygen ligation at the catalytic zinc ion exhibits a greater affinity to the binding of an oxygen from an amino acid residue rather than from an external water molecule. These results suggest that the catalytic zinc ion plays a key role in both substrate binding and catalysis.

Spectroscopic studies of inhibited alcohol dehydrogenase from Thermoanaerobacter brockii: proposed structure for the catalytic intermediate state.

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP(+) as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.

The effects of apolipoprotein E deficiency on brain cholinergic neurons.

Previous studies utilizing apolipoprotein E (apoE)-deficient mice revealed distinct decreases in the levels of cholinergic synaptic markers of projecting basal forebrain cholinergic neurons and no such alterations in other brain cholinergic systems. In order to investigate the mechanisms underlying these neuron-specific cholinergic effects, primary neuronal cultures from apoE-deficient and control mice were prepared and characterized. These include basal forebrain cultures, which are enriched in projecting cholinergic neurons, and cortical cultures, which contain cholinergic interneurons. The levels of cholinergic nerve terminals in these cultures were assessed by ligand binding measurements of the levels of the vesicular acetylcholine transporter (VAChT). This revealed that basal forebrain cultures of apoE-deficient mice contain markedly lower VAChT levels (approximately 50%) than do control cultures, but that VAChT levels of the corresponding cortical cultures of the apoE-deficient and control mice were the same. Time course studies revealed that VAChT levels of the basal forebrain cultures increased with culture age, but that the relative reduction in VAChT levels of the apoE-deficient cholinergic neurons was unaltered and was the same for freshly prepared and for 96 h old cultures. These in vitro observations are in accordance with the in vivo findings and suggest that projecting basal forebrain cholinergic neurons, but not cholinergic interneurons, are markedly dependent on apoE and that similar mechanisms mediate the in vivo and in vitro effects of apoE deficiency on cholinergic function.

[Biometry of the lens with respect to age and cataract morphology. Evaluation of Scheimpflug photos of the anterior segment]. / Biometrie der Linse in Abhängigkeit vom Lebensalter und von der Kataraktmorphologie. Auswertung von Scheimpflug-Photos des vorderen Augenabschnittes.

Thickness of the lens, cortex and nucleus or nucleus with supranuclear layer, respectively, were determined for 58 controls aged 11-69 years (115 lenses), and for 175 patients aged 30-86 years with different types of cataract (247 lenses). Measurements of the densitograms of Scheimpflug photos (taken along the optical axis, magnified 10-fold) were performed, accurate to 0.5 mm (corresponding to 0.05 mm original size). The cataractous lenses were grouped in 5 morphological classes and 3 degrees of opacification (density according to the Scheimpflug photos). As regards normal lenses, the present authors' findings correspond to earlier findings of Niesel et al. (1976). Deviations from the normal pattern are found in typical cortical cataract (water-cleft and spokes cataract, wedge-shaped cataract) where nucleus and nucleus with supranuclear layer are thinner than the normal lenses, while with nuclear cataracts and mixed types the opposite is the case. In eyes with posterior cortical opacities no increase in the thickness of the anterior cortex is found with aging. With the true nuclear cataracts the increase in the thickness of the anterior cortex is negligible. The degree of opacification is closely related to an increase in the thickness of the nucleus and supranuclear layer, which implies a decrease in the thickness of the anterior cortex. Water-clefts and spokes of larger size cause the anterior cortex to become thinner. In eyes with wedge-shaped cataracts the different degrees of opacification did not affect the parameters measured.

[Computer tomography in unilateral exophthalmos caused by an osteomas of the orbital wall (author's transl)]. / Computertomographie bei einem einseitigen Exophthalmus bedingt durch einen Knochentumor der Orbitalwand (Osteom).

A 67-year-old woman patient presented with protrusion of the right globe of unknown origin. Computer tomography revealed a probably benign space-occupying lesion (osteoma) originating in the orbital wing portion of the sphenoidal bone and extending to the orbital roof of the frontal bone.

[Fistulating operations with Walser's punch Modifications without scleral flap and with fixed or loose scleral flap (author's transl)]. / Fistulierende Operationen mit Walser-Stanze Modifikationen ohne Skleradeckel, mit fixiertem und mit nicht fixiertem Skleradeckel.

Long term results of fistulating operations with Walser's punch are reported. A comparison between two methods without and with fixed scleral flap shows that the first method was more often associated with hypotonia, while the latter is more often followed by unsatisfactory pressure regulation necessitating additional drug therapy. That is why a third method of operation was developed. Here the scleral flap is shortened by scissors and not resutured to the sclera. The first clinical results indicate that the method makes use of the advantages of a scleral flap with less risk of an insufficient pressure regulation.

[On malignant melanoma of the cornea and its treatment (author's transl)]. / Uber das maligne Melanom der Hornhaut und seine Therapie

A report is given of an amelanotic malignant malanoma of the cornea without previous changes in the cornea and without any connection with the limbus. Treatment was by simple removal after cauterisation of the nutrient vessels coming from the conjunctiva. Histologically, alongside naevoid cell structures were seen epithelioid to anaplastic cells a striking wealth of plasma cells, which were in places so numerous as the melanoma cells and diffusely or like streets infiltrated them.