Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

Long term urinary platinum, palladium, and gold excretion of patients after insertion of noble metal dental alloys

The aim of this study was to investigate to which extent noble-metal dental alloys contribute to the total platinum (Pt), palladium (Pd), and gold (Au) body burden of the general population. The urinary Pt, Pd, and Au excretion was determined in three non-occupationally exposed volunteers before and up to 3 months after insertion of a highgold dentalalloy. The in-vitro release of Pt, Pd, and Au from four different types of dental alloys into either artificial saliva or 1% lactic acid solution was additionally investigated. The Pt, Pd, and Au concentrations were determined by sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). Before insertion of the high-gold dental alloy, the Pt excretion of the patients ranged between 1.0 and 7.4 ng l-1 (0.6-3.3 ng g-1 creatinine). In the immediate post-insertion phase the Pt excretion rose to 10.5-59.6 ng l-1 (14.5-33.2 ng g-1 creatinine). This is a mean increase by a factor of 12 compared with the average Pt excretion before insertion. Three months after insertion, the Pt excretion was still elevated by a factor of 7. Contrary to Pt, the Au and Pd excretion in urine was not significantly increased after insertion of this type of high-gold dental alloy. Our in-vitro investigations confirm the assumption that Pt, Pd, and Au are released from noble metal containing dental alloys by corrosion. Under the applied conditions, the release was in the lower ng cm-2 range. It can be concluded that the Pt release from dental alloys can predominantly contribute to the Pt exposure of non-occupationally exposed persons. It can exceed the exposure from all other environmental sources including the Pt release from automobile exhaust catalysts.     

In-vitro processing of yeast α-factor leader fusion proteins using a soluble yscF (Kex2) variant

Summary The Saccharomyces cerevisiae KEX2 gene encodes the membrane-bound endoprotease yscF, which is responsible for the site-specific endoproteolytic cleavages at pairs of basic amino acid residues in the -factor precursor. In order to obtain soluble yscF activity, a mutant KEX2 gene lacking 600 bp coding for the C-terminal 200 amino acids was constructed. Expression of the truncated KEX2 gene in yeast led to the secretion of an active soluble yscF protein (yscF_s). The soluble yscF protein is able to efficiently cleave heterologous protein precursors in-vitro, as demonstrated for -factor leader-hIGF1 and -factor leader-hirudin fusion proteins. Offprint requests to: P. G. Seeboth     

Acetylene inhibition technique underestimates in situ denitrification rates in intact cores of freshwater sediment

Abstract Denitrification in intact sediment cores was measured by the acetylene inhibition technique and compared with the nitrate flux between water and sediment. Less than half of the nitrate-N consumed by the sediment could be recovered as nitrous oxide-N. The low recovery rate of nitrous oxide from intact sediment cores indicated losses of nitrous oxide by diffusion down to nitrate-free sediment layers, where reduction of nitrous oxide may take place. In sediment slurries 100% of nitrate-N could be recovered as nitrous oxide-N as long as the nitrate concentration in the liquid phase was above 10 μM. Nitrous oxide added to nitrate-free sediment slurries was reduced regardless of whether acetylene was present or not. Therefore denitrification may be significantly underestimated by this method.     

Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.     

2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.     

Development of Grossensee (Holstein,Germany): variations in trophic status from the analysis of subfossil microfauna

Microfossil analysis was carried out on 90 samples of a sediment core from Grossensee. The diversity of subfossil Cladocera, Chironomidae, Chaoborus, and Ostracoda reflects at least five different stages of development. Three extended cycles correspond to different climatic periods in the first 10 000 years of the lake's history, while during the last 2 000 years, two shorter cycles can be identified which correlate with proofs or even historical events for human activity in the catchment area. During this time the profundal fauna changed from stenoxibiontic to euryoxibiontic species. In the planktonic fauna a succession of three Eubosmina types, B. longispina, B. coregoni kessleri, and B. coregoni coregoni, was observed. The chydorid and ostracod assembly of the littoral showed no clear change.     

13C NMR Analysis of some simple tetrahydroisoquinolines

¹³C NMR resonances of 15 simple tetrahydroisoquinolines have been assigned on the basis of chemical shift theory, ¹³C-¹H coupling constants     

The Receptor for Urokinase-Type Plasminogen Activator of a Human Keratinocyte Line (HaCaT)

It is assumed that plasmin participates in pericellular proteolysis in the epidermis. Plasmin is generated by keratinocyte-associated plasminogen activators from the proenzyme plasminogen; plasminogen activation can proceed at the keratinocyte surface. The resultant plasmin interferes with cell to matrix adhesion and does possibly contribute to keratinocyte migration during reepithelialization. Here we describe the receptor for urokinase-type plasminogen activator (uPA-R) in the human keratinocyte cell line HaCaT, which serves to direct plasminogen activation to the cell surface; we relate the receptor to the uPA-R previously described in human myclo-/monocytes. Binding of uPA to the receptor accelerated plasminogen activation by a factor of ≈10, compared to uPA in solution. Receptor-bound uPA was susceptible to inhibition by the plasminogen activator inhibitors 1 and 2. uPA and uPA-R antigen, as well as uPA activity, were localized to the leading front of expanding sheets of HaCaT cells. Exposure of HaCaT cells to plasminogen was followed by detachment of the cells. Detachment was prevented by an anti-catalytic anti-uPA antibody, by the plasmin-specific inhibitor aprotinin, and by the lysine analogue tranexamic acid, the latter of which prevents plasmin(ogen) binding to the cell surface. Our findings support the hypothesis that uPA-mediated plasminogen activation is characteristic of mobile rather than sessile keratinocytes. Moreover, the uPA-R seems to focalize plasminogen activation to the surface of cells at the site of keratinocyte migration.