High-performance liquid chromatography and micellar electrokinetic chromatography of taxol and related taxanes from bark and needle extracts of Taxus species

High-performance liquid chromatography (HPLC) and micellar electrokinetic chromatography (MEKC) were applied for the separation of taxol, cephalomannine, and baccatin III in crude extracts from the needle and bark of Taxus species. The chromatogram of the bark extract was cleaner than that of the needle allowing a more reliable detection of taxol and cephalomannine in the bark extract. However, HPLC quantitation of taxol in the needle extract would be difficult due to coeluting taxinines. Nevertheless, this was not a problem in the MEKC experiment. In comparison to HPLC, MEKC offered baseline resolution of taxol from taxinines in the needle extract, less solvent waste, a smaller sample requirement, and the simultaneous detection of taxol, cephalomannine and baccatin III in a relatively simpler electrophoretic run.     

Uptake of amino acids by the aquatic resurrection plant Chamaegigas intrepidus and its implication for N nutrition

Chamaegigas intrepidus is a poikilohydric aquatic plant that lives in rock pools on granitic outcrops in Central Namibia. The pools are filled intermittently during the summer rains, and the plants may pass through up 20 rehydration/dehydration cycles during a single wet season. Rehydrated plants also have to cope with substantial diurnal fluctuations in the pH and extreme nutrient deficiency. Ammonium concentrations are normally around 30 μM. Additional nitrogen sources are amino acids. Total free amino acids are up to 15 μM with glycine and serine as the predominant amino acids. Experiments on uptake of radiolabelled amino acids into roots of C. intrepidus showed high␣affinity (K _M= 16 μM) and low-affinity (K _M= 159 μM) uptake systems. The K _M of the high-affinity system is well in accordance with the free amino acid concentration found in the water of the pools. We conclude that amino acids, predominantly glycine and serine, can be utilised by C. intrepidus in its natural habitat. Since glycine uptake showed a strong reduction at pH 10, nitrogen uptake from glycine or serine should occur mainly in the morning when the pH of the pool water is slightly acid. Further experiments with ¹⁵N-labelled ammonium in combination with non-labelled glycine demonstrated high ¹⁵N values in plant tissues. Under experimental conditions C. intrepidus preferred ammonium as a nitrogen source. The implication of amino acids for nitrogen nutrition of C. intrepidus may depend on the relation of inorganic and organic nitrogen available in the pool water and the preferential utilisation of one or the other nitrogen source may change during the day corresponding with pH changes in the water. Received: 28 January 1998 / Accepted: 24 July 1998     

Internalization of V2-vasopressin receptors in LLC-PK1-cells: evidence for receptor-mediated endocytosis.

The mechanism of internalization of the vasopressin-receptor (V2-subtype) of LLC-PK1-cells, a pig renal tubular cell line, is unknown. We studied internalization utilizing a novel, highly specific vasopressin analogue ((125I)-[8-p(OH)-phenylpropionyl]-LVP, 2000 Ci/mmol). Scatchard analysis performed with membranes of LLC-PK1-cells revealed a Kd of 0.8 +/- 0.2 nM and a Bmax of 366 +/- 41 fmol/mg of protein. Degradation of the ligand was excluded by RP-HPLC-analysis. Internalization was proven by the acid-wash technique, quantitative light-microscopic autoradiography and electron microscopy. The ligand was internalized in a time- and temperature-dependent manner. At 4 degrees C, no uptake was found; at 22 degrees C, after 30 min of incubation, more than 50% of the radioligand was found inside the cell. Electron microscopy demonstrated that plasma-membrane bound vasopressin receptors are internalized by receptor-mediated endocytosis via coated pits.     

Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes.

The E6 proteins of the high-risk human papillomaviruses (HPVs) have been shown to form a complex with and induce the degradation of human p53 in vitro. To determine whether p53 is degraded more rapidly in cells expressing E6 in vivo, the half-life of p53 was determined by pulse-chase analysis in early-passage normal human keratinocytes and fibroblasts, human keratinocytes immortalized with HPV type 16 (HPV16) E6 plus E7, and nonimmortal keratinocytes transfected with E6. The results of these experiments indicate that (i) the half-life of newly synthesized p53 is relatively long (4 h) in early-passage human keratinocytes and fibroblasts but short in keratinocytes expressing E6 (15 to 30 min), (ii) a similar increased rate of p53 degradation was measured in lines immortalized with HPV16 E6 plus E7 and senescent cells expressing E6, indicating that this increase is not simply the result of selection in the immortalized lines, and (iii) very low levels of expression of E6 result in a greatly decreased half-life of p53, suggesting that E6 acts in a catalytic manner.     

Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns

A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.     

Cytotoxin production by a merineLyngbya strain (cyanobacterium) in a large-scale laboratory bioreactor

A cytotoxic compound was produced by the marine cyanobacteriumLyngbya sp. Pearl strain in large laboratory-scale batch cultures. Adsorption and fractionation of methanol extracts with reverse phase (C-18) cartridges provided a rapid method for removal of bioassay interference from salts, biopolymers and pigments and concentration of the cytotoxic principles. Cytotoxicity to the murine leukemia cell line P-388 was produced in two cycles coinciding with the initiation of exponential growth and again during the late exponential growth phase. Antiviral activity against influenza virus PR8 was found in extracts prepared from early exponential growth phase cells but antiviral activity was not detected in extracts of mid-log or late-log growth phase cells. These differences in bioactivity suggests that the cytotoxic principles produced during early and late exponential growth may be different compounds. Cytotoxicity assays using murine P-388 leukemia indicates that the semi-pure compound has an IC₅₀ of < 0.25 μg ml⁻¹ to this cell line. P-388 cytotoxicity in cell extracts increased during the late exponential growth phase and the specific yield was estimated at approximately 0.14 mg g⁻¹ (dry cells).     

Papillomavirus polypeptides E6 and E7 are zinc-binding proteins.

Papillomavirus proteins E6 and E7 have Cys-X-X-Cys repeats which have been suggested to mediate zinc binding. We have developed a modification of an assay that detects zinc binding to proteins immobilized on filters. Using well-characterized metalloproteins, we show that, under reducing conditions, this assay distinguishes proteins that coordinate zinc through cysteine residues from those that bind the metal through other amino acids. Under these conditions, E6 and E7 polypeptides of human papillomavirus type 18 and bovine papillomavirus type 1 exhibited high-affinity zinc binding. Our results suggest that E6 and E7 are metalloproteins and may coordinate the metal ions through cysteine residues.     

Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow’s udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.