Cellular stages in cartilage formation as revealed by morphometry, radioautography and type II collagen immunostaining of the mandibular condyle from weanling rats

The role played by cell addition, cell enlargement, and matrix deposition in the endochondral growth of the condyle was assessed in weanling rats by four approaches making use of the light microscope: morphometry, 3H-thymidine radioautography, 3H-proline radioautography, and immunostaining for the cartilage-specific type II collagen. From the articular surface down, the condyle may be divided into five layers made up of cells embedded in a matrix: 1) the articular layer composed of static cells in a matrix rich in fibers presumed to be of type I collagen, 2) the polymorphic cell layer including the progenitor cells from which arise the cells undergoing endochondral changes, 3) the flattened cell layer in which cells produce a precartilagenous matrix devoid of type II collagen while undergoing differentiation in two stages: a "chondroblast" stage and a short "flattened chondrocyte" stage when intracellular type II collagen elaboration begins, 4) the upper hypertrophic cell layer, in which cells are "typical chondrocytes" that enlarge at a rapid rate, actively produce type II collagen, and deposit it into a cartilagenous matrix, and 5) the lower hypertrophic cell layer, composed of chondrocytes at a stage of terminal enlargement while the cartilagenous matrix is adapting for mineralization. 3H-thymidine radioautographic results indicate that the turnover time of progenitor cells in the polymorphic cell layer is about 2.9 days. The time spent by cells at each stage of development is estimated to be 1.4 days as chondroblasts, 0.5 days as flattened chondrocytes, 2.3 days as the chondrocytes of the upper hypertrophic cell layer, and 1.1 days as those of the lower hypertrophic cell layer. Calculations referring to a 1 x 1-mm square-sided column extending from the articular surface to the zone of vascular invasion provide the daily rate of cell addition (0.0077 mm3), extracellular matrix deposition (0.0127 mm3), and cell enlargement (0.0302 mm3). Hence the respective contribution of the three factors to condyle growth is in a ratio of about 1:1.6:4. This result emphasizes the role played by cell enlargement in the overall growth of the condyle.     

Studies on the effect of stigmatellin derivatives on cytochrome b and the Rieske iron-sulfur cluster of cytochrome c reductase from bovine heart mitochondria

Stigmatellin and its derivatives represent a third class of Qo site inhibitors besides the hydroxyquinone derivatives and the E-beta-methoxyacrylate (MOA) inhibitors [von Jagow and Link (1986) Methods Enzymol. 126, 253-271]. The stigmatellins consist of a chromone ring system connected to an substituted alkenyl side chain. Alterations in the side chain, i.e. saturation of the C = C double bonds, shift of a methoxy group or loss of the methyl groups, specifically affect the binding characteristics. Besides changing the red shift spectrum of reduced cytochrome b566 and the EPR spectrum of the Rieske iron-sulfur cluster, the side chain alterations diminish the binding affinity and the extent of the midpoint potential shift of the iron-sulfur protein. Thus, the side chain of the molecule makes an essential contribution to the binding energy and is not necessary solely for partitioning the molecule into the hydrophobic phase, as assumed so far.     

Filtration rates of mosquito larvae in suspensions of latex microspheres and yeast cells

Filtration rates of fourth instars of Aedes aegypti L., Anopheles albimanus Wiedemann, Anopheles quadrimaculatus Say and Culex quinquefasciatus Say (Diptera: Culicidae) were determined by quantifying removal rates of suspended latex microspheres or yeast cells. Average filtration rates were 33–34 l/larva/h (An. quadrimaculatus), 49–55 (An. albimanus), 490–590 (C. quinquefasciatus) or 590–690 l/larva/h (Ae. aegypti) for larvae exposed to latex beads suspended in phagostimulant yeast extract solutions. In suspensions of yeast cells, filtration rates of Ae. aegypti and C. quinquefasciatus were not significantly different from filtration rates in latex bead suspensions. Larval density, ranging from 0.3 to 2.4 individuals/ml in tests with Ae. aegypti and C. quinquefasciatus and up to 4.8 larvae/ml in tests with Anopheles, did not influence filtration rates.

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Zusammenfassung Die Filtrierraten von Viertlarven der Stechmückenarten Aedes aegypti, Anopheles albimanus, Anopheles quadrimaculatus und Culex quinquefasciatus wurden in Laborversuchen bestimmt. Dabei wurde die Filtrierrate definiert als ein Wasservolumen, welches pro Stunde von einer Larve von den Testpartikeln (Hefezellen oder Latexkugeln mit einem Durchmesser von 2 m) befreit wurde. Nach Exposition der Larven in Dichten zwischen 0.15 und 2.4 Larven/ml (Ae. aegypti und C. quinquefasciatus), oder zwischen 0.6 und 4.8 Larven/ml (Anopheles) wurde der Partikelgehalt der Suspensionen in einem elektronischen Partikelzähler bestimmt. Die Filtrierraten wurden über die Verringerung der Partikeldichte mit zunehmender Larvendichte entsprechend einer in der Literatur angegebenen Formel berechnet.Suspensionen von Hefezellen wurden von Ae. aegypti Larven mit einer Leistung von 680±220 l/Larve/h gefiltert. Bei Larven von C. quinquefasciatus wurden Filtrierraten von 600±120 l/Larve/h gemessen. Die Filtrierraten beider Arten waren unabhängig von der Larvendichte. In Suspensionen von Latexpartikeln (zur Phagostimulation der Larven wurden diese Partikel in Lösungen von Hefeextrakt angeboten) wurden die folgenden Filtrierraten festgestellt: An quadrimaculatus: 33–34, An. albimanus: 49–55, C. quinquefasciatus: 490–590, und Ae. aegypti: 560–690 l/Larve/h. Die Larvendichte hatte auch hier keinen Einfluss auf die Filtrierrate. Hefezellen und Latexpartikel wurden von Ae. aegypti und C. quinquefasciatus mit statistisch nicht signifikant verschiedenen Filtrierraten aufgenommen. Die Filtrierraten der Anopheles Larven waren um mehr als eine Zehnerpotenz kleiner als die Filtrierraten von Culex und Aedes, und auch untereinander signifikant verschieden. Der Einfluss der Filtrierraktivität von Stechmückenlarven auf das Seston der Brutgewässer wird diskutiert.

     

Aberrant DNase I digestion kinetics of nucleosomal core particles from sea urchin sperm

The pancreatic deoxyribonuclease (DNase I) digestion rates at the susceptible sites on nucleosomal core particles from blastula, gastrula and sperm cells of the sea urchin, Parechinus angulosus, have been determined. Although there are differences in their isohistone composition, the rates of digestion are similar for both embryonic stages. The rates of digestion for sperm core particles are 3-5 times lower than for embryo core particles at the more, and up to 2.5 times lower at the less susceptible sites. An explanation for these differences could be sought in the sperm isohistones H2B which are characterized by N-terminal extensions of 20-25 amino acid residues.     

Isolation and characterization of three protein proteinase isoinhibitors from the granular fraction of horse neutrophilic granulocytes

Three cathodically migrating protein protease isoinhibitors were isolated from the granule-rich fraction of equine neutrophilic granulocytes by means of FPLC chromatography, in addition to two previously described anodically migrating inhibitors. The three isoinhibitors had an identical enzyme specificity which was equal to the two previously described isoinhibitors; they inhibited exclusively proteinase K and subtilisin. The inhibitors retained their activity between pH 1 and 12. They also were heat stable at 100 degrees C for 20 min. Neither the biological function of isoinhibitors nor the fundamental role of granular protease inhibitors of such narrow and peculiar enzyme specificity are known.     

Structure elucidation of the core regions from Citrobacter O4 and O36 lipopolysaccharides by chemical and enzymatic methods, gas chromatography/mass spectrometry, and NMR spectroscopy at 500 MHz

Novel enterobacterial core oligosaccharides were isolated from Citrobacter O4 and O36 lipopolysaccharides, and their structures were determined by methylation analysis, Smith degradation and enzymatic degradations, gas chromatography/mass spectrometry, and two-dimensional phase-sensitive correlated, relayed coherence transfer, double-quantum, triple-quantum-filtered, and nuclear Overhauser effect (NOE) 1H NMR spectroscopy at 500 MHz. In the formulas, all hexose residues are D-hexopyranoses, and heptoses are L-glycero-D-manno-heptopyranoses; the alternative locations of the side-chain heptose and pyrophosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3-deoxy-D-manno-octulosonic acid. (formula; see text) Along with these complete cores, incomplete ones, lacking the hexosamine trisaccharides, occur in the lipopolysaccharides of both types. Qualitative NOE data were in good agreement with the minimum energy conformation of the above O36 oligosaccharide, calculated with the aid of the SUGAR program [Sundin, A., Carter, R. E., & Liljefors, T. (1988) J. Mol. Graphics (in press)].     

Isolation and characterization of two new low-molecular-weight protein proteinase inhibitors from the granule-rich fraction of equine neutrophilic granulocytes

A new species of protein proteinase inhibitors was detected in the granule-rich fraction of equine neutrophilic granulocytes. Five isoinhibitors were identified with a narrow enzyme specificity towards two microbial proteinases, e.g., proteinase K and subtilisin. Two isoinhibitors were purified and partially characterized. They had an Mr of 11,300 and 7400, respectively, and were resistant to perchloric acid and heat treatment at 100 degrees C for 20 min. The inhibitors retained their activity over a broad range of pH (1-9 and 1-12, respectively). The possible biological function of this species of protein proteinase inhibitors as defensins (= endogenous antibiotics) is tentatively discussed.     

Transcending the impenetrable: how proteins come to terms with membranes

In the living cell, proteins are efficiently sorted to a whole range of subcellular compartments. In many cases, sorting specificity is mediated by short 'sorting signals' attached either permanently or transiently to the protein. At long last, a fairly coherent picture of the design and function of many such sorting signals is beginning to emerge.     

Treatment with natural human interferon alpha of a CML-patient with antibodies to recombinant interferon alpha-2b

A patient with Philadelphia-chromosome positive chronic myelogenous leukemia developed interferon antibodies on treatment with recombinant interferon alpha-2b. Clinically this event corresponded with progressive disease. No cross-reactivity of antibodies with human leukocyte interferon was found by Western blot. Treatment was switched to human leukocyte interferon with an obvious clinical effect: WBC was reduced and platelet count stabilized, but the effect was transient and no hematologic remission was achieved. Human leukocyte interferon may be an alternative in CML-patients with neutralizing antibodies to recombinant interferon alpha.