In bacterial meningitis cortical brain damage is associated with changes in parenchymal MMP-9/TIMP-1 ratio and increased collagen type IV degradation

Adverse outcome in bacterial meningitis is associated with the breakdown of the blood-brain barrier (BBB). Matrix-metalloproteinases (MMPs) facilitate this process by degradation of components of the BBB. This in turn results in acute complications of bacterial meningitis including edema formation, increased intracranial pressure and subsequent ischemia. We determined the parenchymal balance of MMP-9 and TIMP-1 (tissue inhibitor of MMP) and the structural integrity of the BBB in relation to cortical damage in an infant rat model of pneumococcal meningitis. The data demonstrate that the extent of cortical damage is significantly associated with parenchymal gelatinolytic activity and collagen type IV degradation. The increased gelatinolysis was found to be associated with a brain parenchymal imbalance of MMP-9/TIMP-1. These findings provide support to the concept that MMPs mediated disruption of the BBB contributes to the pathogenesis of bacterial meningitis and that protection of the vascular unit may have neuroprotective potential.

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Quantitative magnetic resonance imaging of enzymatically induced degradation of the nucleus pulposus of intervertebral discs

STUDY DESIGN: The structural integrity of the nucleus pulposus (NP) of intervertebral discs was targeted by enzyme-specific degradations to correlate their effects to the magnetic resonance (MR) signal. OBJECTIVE: To develop quantitative MR imaging as an accurate and noninvasive diagnostic tool to better understand and treat disc degeneration. SUMMARY OF BACKGROUND DATA: Quantitative MR analysis has been previously shown to reflect not only the disc matrix composition, but also the structural integrity of the disc matrix. Further work is required to identify the contribution of the structural integrity versus the matrix composition to the MR signal. METHODS: The bovine coccygeal NPs were injected with either enzyme or buffer, incubated at 37 degrees C as static, unloaded and closed 3-disc segments, and analyzed by a 1.5-Tesla MR scanner to measure MR parameters. RESULTS: Collagenase degradation of the NP significantly decreased the relaxation times, slightly decreased the magnetization transfer ratio, and slightly increased the apparent diffusion coefficient. Targeting the proteoglycan and/or hyaluronan integrity by trypsin and hyaluronidase did not significantly affect the MR parameters, except for an increase in the apparent diffusion coefficient of the disc after trypsin treatment. CONCLUSIONS: Our results demonstrate that changes in the structural integrity of matrix proteins can be assessed by quantitative MR.

Transcriptional dysregulation during myeloid transformation in AML

The current paradigm on leukemogenesis indicates that leukemias are propagated by leukemic stem cells. The genomic events and pathways involved in the transformation of hematopoietic precursors into leukemic stem cells are increasingly understood. This concept is based on genomic mutations or functional dysregulation of transcription factors in malignant cells of patients with acute myeloid leukemia (AML). Loss of the CCAAT/enhancer binding protein-alpha (CEBPA) function in myeloid cells in vitro and in vivo leads to a differentiation block, similar to that observed in blasts from AML patients. CEBPA alterations in specific subgroups of AML comprise genomic mutations leading to dominant-negative mutant proteins, transcriptional suppression by leukemic fusion proteins, translational inhibition by activated RNA-binding proteins, and functional inhibition by phosphorylation or increased proteasomal-dependent degradation. The PU.1 gene can be mutated or its expression or function can be blocked by leukemogenic fusion proteins in AML. Point mutations in the RUNX1/AML1 gene are also observed in specific subtypes of AML, in addition to RUNX1 being the most frequent target for chromosomal translocation in AML. These data are persuasive evidence that impaired function of particular transcription factors contributes directly to the development of human AML, and restoring their function represents a promising target for novel therapeutic strategies in AML.

Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant)

Action of human small intestinal brush border carbohydrate digesting enzymes is thought to involve only final hydrolysis reactions of oligosaccharides to monosaccharides. In vitro starch digestibility assays use fungal amyloglucosidase to provide this function. In this study, recombinant N-terminal subunit enzyme of human small intestinal maltase-glucoamylase (rhMGAM-N) was used to explore digestion of native starches from different botanical sources. The susceptibilities to enzyme hydrolysis varied among the starches. The rate and extent of hydrolysis of amylomaize-5 and amylomaize-7 into glucose were greater than for other starches. Such was not observed with fungal amyloglucosidase or pancreatic alpha-amylase. The degradation of native starch granules showed a surface furrowed pattern in random, radial, or tree-like arrangements that differed substantially from the erosion patterns of amyloglucosidase or alpha-amylase. The evidence of raw starch granule degradation with rhMGAM-N indicates that pancreatic alpha-amylase hydrolysis is not a requirement for native starch digestion in the human small intestine.

Ligand binding induces Cbl-dependent EphB1 receptor degradation through the lysosomal pathway

Eph receptor tyrosine kinases play a critical role in embryonic patterning and angiogenesis. In the adult, they are involved in carcinogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1 and then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1 as kinase-dead EphB1-K652R was resistant to Cbl. Glutathione S-transferase binding experiments showed that Cbl bound to EphB1 through its tyrosine kinase-binding domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.

Secretion and degradation of glucagon by HIT cells

HIT cells have been widely used to study synthesis and secretion of insulin. It has been assumed that this cell line secretes no other islet hormones. To ascertain whether HIT cells synthesize, secrete, and degrade glucagon, we examined cell extracts for this peptide and compared secretion and degradation of glucagon and insulin during stimulation of the cells by arginine. Glucagon levels in acid extracts of HIT cells were found to be 0.72 +/- 0.15 pmol/mg protein. Both glucagon and insulin were maximally stimulated in a glucagon/insulin molar ratio of 0.029 by arginine concentrations of 25-50 nM, and the concentration of arginine that provided half-maximum responses for both hormones was approximately 3 mM. Diminution of arginine-induced glucagon secretion was caused by somatostatin, a physiological inhibitor of pancreatic islet alpha-cell function. HPLC was used to authenticate the glucagon levels stimulated by arginine for 60 min and measured by RIA. Thirty-six percent of immunoreactive glucagon was found in the fractions representing authentic glucagon, whereas the remaining 64% eluted earlier. Experiments examining the fate of radiolabeled glucagon exposed to HIT cells revealed time-dependent degradation of the radioisotope to earlier eluting forms, which accounted for approximately 50% of the radioactivity by 60 min and was complete by 18 h, indicating that the early peak detected by RIA represented a metabolite of glucagon. Radioisotopic insulin was degraded more slowly with an apparent half-life of approximately 36 h. We conclude that HIT cells are not only able to synthesize, secrete, and degrade insulin, but also much smaller amounts of glucagon.

Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20-23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed 'antagomirs'. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.

Appraising and selecting conservation measures to mitigate desertification and land degradation based on stakeholder participation and global best practices

Most desertification research focuses on degradation assessments without putting sufficient emphasis on prevention and mitigation strategies, although the concept of Sustainable Land Management (SLM) is increasingly being acknowledged. A variety of already applied conservation measures exist at the local level, but they are not adequately recognised, evaluated and shared, either by land users, technicians, researchers, or policy makers. Likewise, collaboration between research and implementation is often insufficient. The aim of this paper is to present a new methodology for a participatory process of appraising and selecting desertification mitigation strategies, and to present first experiences from its application in the EU-funded DESIRE project. The methodology combines a collective learning and decision approach with the use of evaluated global best practices. In three parts, it moves through a concise process, starting with identifying land degradation and locally applied solutions in a stakeholder workshop, leading to assessing local solutions with a standardised evaluation tool, and ending with jointly selecting promising strategies for implementation with the help of a decision support tool. The methodology is currently being applied in 16 study sites. Preliminary analysis from the application of the first part of the methodology shows that the initial stakeholder workshop results in a good basis for stakeholder cooperation, and in promising land conservation practices for further assessment. Study site research teams appreciated the valuable results, as burning issues and promising options emerged from joint reflection. The methodology is suitable to initiate mutual learning among different stakeholder groups and to integrate local and scientific knowledge.