Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGF-activated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass (approximately 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/polyacrylamide gel electrophoresis revealed further that the NGF- or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of approximately 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2-3 h.     

A new approach to determine the stereospecificity in lipase catalysed hydrolysis using circular dichroism (CD): lipases produce optically active diglycerides from achiral triglycerides

We describe a sensitive CD method for determining the stereospecificity in lipase (E.C.3.1.1.3) catalysed hydrolysis of triacyl glycerols into diacyl glycerols. The diglycerols were converted to chiral tert-butyldimethylsilylated 1,2- or 2,3-di-O-benzoyl-sn-glycerol (5 or 5'), and their CD was measured. This approach showed for the first time that lipases produce optically active diacyl glycerides from achiral tripalmitin and tribenzoyl glyceride with a variable extent of enantioselectivity depending on the acyl groups and the enzymes.     

Induction of angiogenesis in chick yolk-sac membrane by polyamines and its inhibition by tissue inhibitors of metalloproteinases (TIMP and TIMP-2).

Treatment of yolk-sac membranes of 4-day-old chick embryos with spermine or spermidine resulted in angiogenesis in the membranes. The angiogenic activity of spermine was stronger than that of spermidine. Putrescine, polylysine and histamine did not induce angiogenesis in the membranes. Administration of putrescine, spermidine and spermine increased their respective levels in yolk-sac membranes, but no interconversion of these amines was observed. The increases in spermidine and spermine levels in yolk-sac membranes preceded induction of angiogenesis. The angiogenesis induced by spermine was inhibited by tissue inhibitors of metalloproteinases, that is, TIMP and TIMP-2. These findings suggest that spermine and spermidine are angiogenesis factors in yolk-sac membranes of chick embryos and that matrix metalloproteinases represented by collagenase are involved in their action.     

Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells

We have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of [32P]Pi-labeled and [3H]leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fractionation studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin.     

Long terminal repeat-like elements flank a human immunoglobulin epsilon pseudogene that lacks introns

There are at least three immunoglobulin epsilon genes (C epsilon 1, C epsilon 2, and C epsilon 3) in the human genome. The nucleotide sequences of the expressed epsilon gene (C epsilon 1) and one (C epsilon 3) of the two epsilon pseudogenes were compared. The results show that the C epsilon 3 gene lacks the three intervening sequences entirely and has a 31-base A-rich sequence 16 bases 3' to the putative poly(A) addition signal, indicating that the C epsilon 3 gene is a processed gene. The C epsilon 3 gene sequence is homologous to the five separate DNA segments of the C epsilon 1 gene; namely, a segment in the 5'-flanking region (100 bases) and four exons, which are interrupted by a spacer region or intervening sequences. Long terminal repeat (LTR)-like sequences which contain TATAAA and AATAAA sequences as well as terminal inverted repeats are present in both 5'- and 3'-flanking regions. The 5' and 3' LTR-like sequences do not, however, constitute a direct repeat, unlike transposable elements of eukaryotes and retroviruses. The 3' LTR-like sequence is repetitive in the human genome, but is not homologous to the Alu family DNA. Models for the evolutionary origin of the processed gene flanked by the LTR-like sequences are discussed. The C epsilon 3 gene has a new open frame which codes potentially for an unknown protein of 292 amino acid residues.     

Carbohydrate fermentation by Clostridium difficile

Biochemical properties of Clostridium difficile were reinvestigated for the practical identification of the organism in clinical laboratories. Bacterial growth in 2% proteose peptone medium supplemented with 0.01% L-cysteine.HCl and 0.1% agar supported sufficient growth to read the fermentation results just as well as did pre-reduced anaerobically sterilized medium. Incubation for 2 days was long enough for determining the ability to ferment fructose, glucose, mannitol, mannose, melezitose, and sorbitol. All of the 82 strains liquefied 2% but not 10% gelatin. The significance of mannitol fermentation and gelatin liquefaction is stressed since C. difficile is the only species fermenting mannitol among the gelatin-liquefying species of clostridia having subterminal spores.     

Studies on the constituents of the seeds of Cassia obtusifolia: The structures of two new lactones,isotoralactone and cassialactone

Torosachrysone and two new naphthalenic lactones, isotoralactone and cassialactone, were isolated from the seeds of Cassia obtusifolia. Their structures were established as 9,10-dihydroxy-7-methoxy-3-methylene-¹H-naphtho(2,3-c)dihydropyrone-1-one and 8-methoxy-4-methyl-1-oxo-4,10,11-trihydroxy-naphtho(2,3-c)oxepin, respectively.     

Carbon Isotope Ratios of Epidermal and Mesophyll Tissues from Leaves of C3 and CAM Plants

The ¹³C values for epidermal and mesophyll tissues of two C₃plants, Commelina communis and Tulipa gesneriana, and a CAMplant, Kalancho daigremontiana, were measured. The values forthe tissues of both C3 plants were similar. In young leavesof Kalancho, the epidermis and the mesophyll showed S13C valueswhich were nearly identical, and similar to those found in C3plants. However, markedly more negative values for epidermalcompared to mesophyll tissue, were obtained in the mature Kalancholeaf. This is consistent with the facts that the epidermis ina CAM leaf is formed when leaves engage in C₃ photosynthesisand that subsequent dark CO₂ fixation in guard cells or mesophyllcells makes only a small contribution to total epidermal carbon. (Received January 27, 1981; Accepted May 14, 1981)     

Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.     

Collection of rice phloem sap from stylets of homopterous insects severed by YAG laser

Pure phloem sap from rice plant was collected by a method similarto the aphid technique using leafhoppers and planthoppers instead.A Yttrium-Aluminium Garnet laser was used to sever the insectstylets. Sap exuded from the plant through the stylet for upto 3 hr at a rate of 0.2 µl/hr. Analysis of the sap forsugars revealed that the only carbohydrate present was sucrose;its content was estimated to be 17% and remained at this levelfor 3 hr. (Received April 16, 1980; )