Ultrastructure of interstitial cells of Cajal at the gastro-oesophageal junction of the monkey (Macaca fascicularis)

The ultrastructure of the interstitial cells of Cajal (ICC) in the oesophagus of the monkey resembled that described in the oesophagus of other mammalian species but differed in their paucity and almost lack of smooth endoplasmic reticulum, caveolae and filaments. The plasmalemma of the ICC was in close contact (20- to 30-nm gaps) with that of smooth muscle cells. This may occasionally take the form of a desmosome, but gap junctions have not been observed. Vesiculated axon profiles, containing large granular or agranular vesicles were in close contact (20- to 30-nm gaps) with the plasmalemma of ICC. In a few vesiculated profiles a presynaptic density could be recognized. The intercalation of the ICC between the vesiculated axon profiles and the smooth muscle cells suggest a role in oesophageal motility. Between 3 and 21 days following bilateral vagotomy some ICC showed regressive changes such as increased electron density and shrinkage of the cytoplasm, crowding of the organelles and dissolution of the nuclear chromatin material. Axon profiles in the vicinity of the affected ICC contained glycogen granules suggesting injury. In late stages, the number of ICC and smooth muscle contacts was reduced. The results suggest that the vagus nerves exert a trophic influence on the ICC and that the intercellular relationships between ICC and smooth muscle cells possess a degree of plasticity. It is tentatively suggested that these vagal effects may be mediated via the oesophageal myenteric ganglia.     

Ultrastructure of the epithelial cells of the ventral prostate from the hopping mouse Notomys alexis

The large ventral prostate of the hopping mouse has abundant secretory units whose epithelial cells vary in height and which often have nuclei in the apical region of the cell. TEM observations indicated two epithelial cell types in which some unusual features occurred. Type A cells had granular endoplasmetic reticulum (GER) whose membranes often formed intracytoplasmic confronting cisternae. Type B cells had more fragmented and vesiculated GER with sparse ribosomes and less frequently also intracytoplasmic confronting confronting cisternae. In the latter cells, two types of granules were found, one of which was derived from the Golgi and the other possibly directly from the GER. Type A cells only had one type of granule present. A highly convoluted membrane was also found at the basal region in many of the cells. The significance of these unusual ultrastructural features has yet to be ascertained.     

Enhanced production of laccase in Trametes vesicolor by the addition of ethanol

In a medium containing 40 g ethanol l^–1, laccase production by Trametes versicolor was 2.6 unit per ml of the supernatant, which was over 20 times higher than that without ethanol. Laccase activity with ethanol was quite comparable to that with the well-known inducers such as veratryl alcohol, xylidine and guaiacol. With other white-rot fungi, Coriolus hirsutus and Grifola frondosa, ethanol had a similar stimulatory effect on laccase production.     

Enhancement of cell growth by intracellularly expressed herpes simplex virus thymidine kinase

A recombinant cell line (NIH3T3:pLtkSN) was made by infecting parental cells (NIH3T3) with a recombinant retrovirus (pLtkSN) encoding herpes simplex virus thymidine kinase (HSVtk) gene. The cells expressing HSVtk (NIH3T3:pLtkSN) grew 2.3 times more than the parental cells (NIH3T3) in Dulbecco's Modified Eagles Media containing 10% (v/v) horse serum. The NIH3T3:pLtkSN cells also showed a significant enhancement in the maximal cell concentration and the specific growth rate even at 2.5% serum concentration. The specific O2 uptake rate of NIH3T3 was 2.1 times greater than that of NIH3T3:pLtkSN. Under both O2-limited and O2-unlimited conditions, it appears that HSVtk plays an important role in enhancing the growth characteristics of animal cells.     

Anti-coccidial activity of 2-picoline, 6-amino-4-nitro-, 1-oxide

An investigational drug (2-picoline, 6-amino-4-nitro-, 1-oxide) was evaluated to characterize the anti-coccidial spectrum of the compound. Two concentrations of the drug (125 and 250 ppm) were evaluated for bioactivity; weight gain, survival, dropping, and lesion scores were the response variables utilized to ascertain activity. The activities of the picoline derivative were compared with monensin, maduramicin, and a narasin/nicarbazin (1:1) combination. The investigational drug had significant activity against Eimeria tenella and Eimeria necatrix, and the 250-ppm level was significantly more active than 125 ppm. At 250 ppm, the E. tenella activity of the picoline derivative was comparable to both monensin (120 ppm) and the 50-ppm narasin/nicarbazin combination, significantly less effective than maduramicin (6 ppm), and significantly more efficacious than 30 ppm narasin/nicarbazin. At the same level (250 ppm), the picoline derivative had significantly less E. necatrix activity than monensin (120 ppm), maduramicin (6 ppm), and narasin/nicarbazin (50 ppm), and significantly greater activity than 30 ppm narasin/nicarbazin. At best, only extremely weak Eimeria acervulina, Eimeria brunetti, and Eimeria maxima activities were noted with the investigational drug; higher concentrations of the picoline derivative may achieve greater anti-coccidial activity against these species. The efficacy of narasin/nicarbazin compared favorably with monensin and maduramicin; the 50-ppm level of the combination appeared significantly more efficacious than 30-ppm.     

Monitoring signal transduction in cancer: cDNA microarray for semiquantitative analysis.

This study targeted the development of a novel microarray tool to allow rapid determination of the expression levels of 58 different tyrosine kinase (tk) genes in small tumor samples. The goals were to define a reference probe for multi-sample comparison and to investigate the variability and reproducibility of the image acquisition and RT-PCR procedures. The small number of tk genes on our arrays enabled us to define a reference probe by artificially mixing all genes on the arrays. Such a probe provided contrast reference for comparative hybridization of control and sample DNA and enabled cross-comparison of more than two samples against one another. Comparison of signals generated from multiple scanning eliminated the concern of photo bleaching and scanner intrinsic noise. Tests performed with breast, thyroid, and prostate cancer samples yielded distinctive patterns and suggest the feasibility of our approach. Repeated experiments indicated reproducibility of such arrays. Up- or downregulated genes identified by this rapid screening are now being investigated with techniques such as in situ hybridization.     

Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].     

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.