A new quinoline-based chemical probe inhibits the autophagy-related cysteine protease ATG4B.

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4-28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.

Fumonisin B1 causes multiple lesions in common carp (Cyprinus carpio).

Year-1 carp were fed ratios containing 100mg/kg and 10 mg/kg of added fumonisin B1 for 42 days. The experimental and control fish were examined clinically during the experiment and at the end all fish were necropsied and histological changes recorded. Blood vessels, liver, exocrine and endocrine pancreas, excretory and haematopoietic kidney, heart and brain were sensitive both to 100 and 10mg/kg of FB1 in the diet and the rodlet cell (RC) frequency was considerably increased in and around damaged tissues. Many damaged blood vessels contained stacks of RCs above the endothelium. Other changes subsequent to fumonisin exposure that have not been previously reported include scattered lesions in the exocrine and endocrine pancreas, and interrenal tissue, probably due to ischemia and/or increased endothelial permeability. Presented findings indicate the need for more intensive studies of fumonisin-induced toxicity in cultured fish.

Evaluation of functional response of cerebral arteries by a new morphometric technique.

The contractile response of the rabbit basilar artery under four conditions was determined: (1) response in a resting condition without exclusion of the sympathetic nervous system (control group I); (2) response in a resting condition with alpha-adrenoceptor blockade (group II); (3) response to subarachnoid haemorrhage (SAH) (group III); and (4) response to SAH with alpha-adrenoceptor blockade (group IV). It was also ascertained whether it was possible to measure contractile response using a new morphometric method. Vessels were prepared by intracardial perfusion fixation, stained by haematoxylin and eosin, and the length of the intimal corrugations were measured by computer image analysis. Two procedures were followed in order to express the intensity of intimal corrugation, indicating the contractibility of the basilar arteries: (1) the corrugation coefficient (CC) of the basilar artery intima was estimated by dividing the precisely measured length of the intimal corrugations by the length of the measured vessel wall section of the vessel cross-sections (obtained histologically); (2) the lumen reduction coefficient (LRC) of the basilar artery was determined by dividing the "ideal" luminal area (calculated from the total length of the intimal circumference) by the real luminal cross-section area. The results of CC measurements revealed the smoothest intima (mean CC = 1.146, P = 0.00) and the least reduction of lumen (mean LRC = 0.26, P = 0.000) in group II (rabbits without SAH but with alpha-blocker phenoxybenzamine), and in group IV (SAH group of rabbits with alpha-blocker phenoxybenzamine) where the mean CC was 1.141 (P = 0.001) and the mean LRC was 0.33 (P = 0.002) in comparison with the SAH-only group III, pointing out the effectiveness of alpha-blockade even against SAH vasospastic stimuli. Control group I (without SAH and without treatment) showed a greater degree of corrugation in the intima and an increased reduction in the lumina than in groups II and IV, but still significantly less than in group III (mean CC = 1.197, P = 0.001, and mean LRC = 0.40, P = 0.028), thus demonstrating a certain resting tone of the basilar arteries (in an ideal situation, without any tone at all, the CC and LRC would be equal one). The highest degree of intimal corrugation and the greatest lumina reduction were discovered in the SAH-only group III (mean CC = 1.374 and mean LRC = 0.60). The differences among groups I, II and IV were insignificant. The results of this study suggest four conclusions: (i) the possibility of evaluating the functional response of rabbit cerebral arteries using this new morphometric technique; (ii) the adrenergic influence on resting tone of these arteries; (iii) the likely preventive role of an alpha-blockade on post-SAH vasospasm of basilar arteries in rabbits; and (iv) good comparability of the results of CC and LRC measurements with the angiographically estimated vessel diameters of other similar studies.

Sympathetic nervous system exclusion following experimental subarachnoid haemorrhage prevents vasospasm in rabbits.

BACKGROUND: The objective of this study was to establish whether the exclusion of the effect of the sympathetic nervous system prevents vasospasm of cerebral arteries after experimental subarachnoid haemorrhage in rabbits. METHODS: The effect of sympathetic exclusion on vasospasm was studied in 29 New Zealand rabbits under conditions similar to human subarachnoid haemorrhage: 1. The activity of the sympathetic nervous system was excluded only after subarachnoid haemorrhage. 2. The effect of this exclusion was evaluated on the eighth day after subarachnoid haemorrhage. 3. The single haemorrhage model of experimental subarachnoid haemorrhage was chosen. Four groups of rabbits were investigated. The control group A comprised rabbits without subarachnoid haemorrhage; group B consisted of those with subarachnoid haemorrhage (1 ml autologous blood/kg BW suboccipitally into the cisterna magna); group C included those with subarachnoid haemorrhage and pharmacological sympathetic exclusion by the alpha blocker phenoxybenzamine, and group D was composed of those with subarachnoid haemorrhage and operative sympathetic exclusion by cervical gangliectomy. Changes in the basilar arteries of rabbits were evaluated by computer image analysis, using histologic specimens of vessel walls. A new measuring procedure was developed to assess the intensity of vasospasm; the method has a corrugation coefficient that expresses changes in intimal corrugation. RESULTS: Comparison of control group A and group B in regard of vessel intima corrugation showed significantly less corrugated intima in group A (P = 0.0042). In comparison with group B, corrugation of the vessel intima in group C was less intense after sympathetic exclusion by phenoxybenzamine following subarachnoid haemorrhage (P = 0.00012). In comparison with group B, a reduced corrugation was also found in group D after sympathetic exclusion by upper cervical gangliectomy following subarachnoid haemorrhage (P = 0.0026). CONCLUSIONS: The results of the study suggest that exclusion of sympathetic nervous system activity in rabbits prevents vasospasm in circumstances similar to subarachnoid haemorrhage in man. Thus, the sympathetic nervous system could play a critical role in the development of vasospasm in subarachnoid haemorrhage.

Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth.

The initial interaction between B cells and follicular dendritic cells (FDCs) appears to be essential for germinal center (GC) formation. To identify molecules regulating this interaction, we generated FDC-staining monoclonal antibodies (mAbs) and screened them for their ability to block FDC-mediated costimulation of growth and differentiation of CD40-stimulated B cells. Using one of the inhibitory mAbs, 8D6, we expression cloned the cDNA encoding the 8D6 antigen (Ag) from a human FDC line, HK. The 8D6 Ag is a novel protein of 282 amino acids that is expressed abundantly on FDCs. Monolayers of COS cells transiently transfected with the 8D6 Ag cDNA stimulate B cell growth. The mAb 8D6 blocks the costimulatory function completely. The inhibitory activity of the mAb 8D6 was demonstrated to be due to an inhibition of cell cycle progression of CD40 ligand-stimulated GC B cells. In addition, the mAb 8D6 inhibits the growth of a lymphoma of GC origin, L3055, which depends on FDCs or HK cells for its growth. These findings suggest that the primary function of FDCs in the GC is to stimulate B cell growth. An FDC signal molecule, 8D6 Ag, may be an important molecule to mediate this function.

Automated segmentation of muscle fiber images using active contour models.

The cross-sectional area of different fiber types is an important anatomic feature in studying the structure and function of healthy and diseased human skeletal muscles. However, such studies are hampered by the thousands of fibers involved when manual segmentation has to be used. We have developed a semiautomatic segmentation method that uses computational geometry and recent computer vision techniques to significantly reduce the time required to accurately segment the fibers in a sample. The segmentation is achieved by simply pointing to the approximate centroid of each fiber. The set of centroids is then used to automatically construct the Voronoi polygons, which correspond to individual fibers. Each Voronoi polygon represents the initial shape of one active contour model, called a snake. In the energy minimization process, which is executed in several stages, different external forces and problem-specific knowledge are used to guide the snakes to converge to fiber boundaries. Our results indicate that this approach for segmenting muscle fiber images is fast, accurate, and reproducible compared with manual segmentation performed by experts.

Characterization of murine Flt4 ligand/VEGF-C.

Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based affinity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murine flt4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptide-like sequence that is removed to generate the mature N-terminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21000 Mr polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.

Molecular cloning and characterization of murine interleukin-11.

Human interleukin-11 (IL-11) has been shown to have pleiotropic action on hematopoietic, hepatic, stromal, epithelial, neural, and osteoclast cells. In the present work, the murine IL-11 cDNA has been isolated from a fetal thymic cell line, and its structure and function compared with human IL-11. The murine protein was demonstrated to have identical actions on the proliferation of a murine plasmacytoma cell line, murine primitive bone marrow progenitor cells, and megakaryocyte precursors. The murine IL-11 protein was synthesized as a soluble thioredoxin-IL-11 fusion in Escherichia coli and the expression of murine IL-11 was examined by pulse-chase radiolabeling in COS cells. The chromosomal location of the murine IL-11 gene was assigned to the proximal arm of chromosome 7.

A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm.

We have developed a versatile Escherichia coli expression system based on the use of E. coli thioredoxin (trxA) as a gene fusion partner. The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins. Although many of these cytokines previously have been produced in E. coli as insoluble aggregates or "inclusion bodies", we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active. In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels. Two additional properties of E. coli thioredoxin, its ability to be specifically released from the E. coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps. We also find that the active-site loop of E. coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E. coli cytoplasm.

[Mercury concentration in blood and urine--before and after the placement of dental amalgam fillings]. / Quecksilberkonzentration im Blut und Urin--Vor und nach dem Legen von Amalgamfüllungen.

70 patients of dentist's surgery were given MOD amalgam fillings (non-gamma-2 amalgam) for molars. They were allocated for comparison to four groups defined by their treatment, i.e. the number of old and new restorations and whether a rubber dam was used. Blood and urine samples were collected at regular intervals before and during a 14-day period after treatment and tested for mercury concentration (Hg). Over the observation period the groups with the highest exposure (1-2 old restorations replaced by new ones) showed a tendency, in contrast to the less exposed groups (1 new filling with or without dam), towards increases (p less than 0.10) in group average Hg values of approx. 0.2 microgram/L (blood) and 0.3 microgram/g creatinine (urine) as acute treatment effects. The highest values recorded before and after the treatment, 3.3 micrograms/L (blood) and 16.5 micrograms/L (urine) are higher than normal but do not indicate any increase in the risk to health especially if they are not persistent.

Evaluation of elastic matching system for anatomic (CT, MR) and functional (PET) cerebral images.

To evaluate the performance of our elastic matching system, we have created a digitized atlas from the brain of a normal young man, using 135 myelin-stained sections at 700 microns spacing. Software was written to enter and edit regional anatomic contours, which were stacked and aligned to create a three-dimensional atlas. We then evaluated the matching system by comparing computer generated contours with expert defined contours for several subcortical structures, based on CT scans from six neurologically normal patients. The error in positioning, as defined by the distance between the centers of gravity, averaged 4.2 mm for the computer and 1.7 mm for the worst expert's reading, with the computer drawn region frequently inscribed within that of the expert. Comparison was also made for each structure by determining the volume of overlap and the volumes not overlapping. On average, the computer's agreement with the experts was approximately 20% less than the agreement among the experts. This was a preliminary test of the system using only subcortical structures. The results are promising, and techniques are being implemented to overcome the current deficiencies.

The human genes for GM-CSF and IL 3 are closely linked in tandem on chromosome 5.

As demonstrated by long-range mapping of restriction endonuclease recognition sequences and genomic cloning, we found that the human genes encoding interleukin 3 (IL 3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) are tandemly arrayed on the long arm of chromosome 5, separated by 9 kilobases (kb) of DNA. This close physical linkage of genes with similar structure and biologic function suggests that these cytokines may have evolved from a common ancestral gene. This linkage in evolution of two relatively divergent genes further implies that some of the other lymphokine and cytokine genes that appear to share as much or more sequence similarity than do IL 3 and GM-CSF may be distantly related members of a cytokine gene family.

Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3.

A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene, which was readily identified because of its high degree of homology to the gibbon sequence, also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells, proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3.

Biotype determinations for monitoring of EK1 host strains used in recombinant DNA research.

The biotypes of eight Escherichia coli K-12 strains were determined. Eight different biotypes were observed. Based upon the API biotype computer file, the appearance frequency for these K-12 biotypes ranged from 1/127 (infrequent) to 1/18,046 (rare).

Experiences with total hip replacement arthroplasty ad modum ring.

The results of total replacement of the hip joint ad modum Ring are presented. The review includes 63 hips (from among the total number of cases operated by the various methods) followed up for periods ranging from 6 months to 5 years. The assessment has shown excellent results in 33.3 per cent, good results in 50.8 per cent, fair in 9.5 per cent and poor results in 6.4 per cent. Advantages and disadvantages of the method are discussed.