Comparison of growth properties of carrot hairy root in various bioreactors

Summary Growth properties of carrot hairy root cells in various bioreactors were investigated. A turbine-blade reactor and an immobilized rotating drum reactor were found to be advantageous for the hairy root culture because of a high oxygen transfer coefficient (k in L a). After 30 days of culture, 10 g/l of dry hairy root cells were obtained in both bioreactors and maximum growth rates (V _m ) were found to be 0.63 and 0.61 g/l per day for the turbine-blade reactor and immobilized rotating drum reactor, respectively. Specific growth rates () at various cultivation times were observed to be linearly proportional to X/k _l a for both bioreactor configurations where X is the cell concentration. The estimated specific oxygen uptake rate of 0.34 mmol O₂/g dry cells per hour compares fairly well with an experimental value of 0.3.     

In vitro phosphorylation of the tumor suppressor gene RB protein by mitosis-specific histone H1 kinase

The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed. We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase. Highly purified enzyme was used for this purpose. HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E. coli cells were shown to be phosphorylated by this kinase in vitro. Synthetic peptides for the six expected sites were also phosphorylated. These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase.     

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.     

A re-evaluation of the cytology of cat Pacinian corpuscles

Summary The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.     

Inhibin activity and secretion of gonadotropin during the period of follicular maturation

To evaluate the relative contributions of the ovarian inhibin and estradiol-17 beta (E) on the regulation of FSH secretion, inhibin and E in ovarian venous plasma (OVP) and FSH and LH in peripheral plasma were simultaneously measured using superovulating rats with special reference to follicular maturation. By the transplantation of a pituitary gland from adult male rats under the kidney capsule between 1100 and 1200 hr on diestrus-1 in cyclic rats, superovulation was successfully induced on the morning of the next estrus without any additional treatment with human chorionic gonadotropin (hCG). The number of maturing follicles capable of ovulating in response to hCG significantly increased at 12 hours after the grafting as compared with sham-operated controls and further increases occurred until the afternoon of proestrus. In the superovulating rat, first and second surges of FSH were completely blocked and an LH surge was also partially suppressed during the periovulatory period when surges of FSH and LH were normally observed in controls. Contents of FSH as well as LH in the animal's own pituitary gland were suppressed significantly after the grafting as compared with controls. A marked increase in inhibin activity in OVP of rats with a pituitary transplant occurred concomitantly with an increase in the number of follicles capable of ovulating whereas E levels in OVP did not so. Inhibin activity in OVP at each point was much higher in the pituitary grafted rats than in controls but this was not true for E levels. These results suggest that ovarian inhibin derived from the maturing follicles rather than E may be a primary factor for regulation of FSH secretion, and high levels of endogenous inhibin can suppress synthesis of LH as well as FSH in the pituitary gland of the female rat.     

Relationship between characteristics of immunoreactive LH/FSH cells and the levels of gonadotropin in the female rat

Morphological and functional changes of pituitary LH/FSH cells in the female rat were investigated using the parameters on the radioimmunoassay, immunohistochemistry and ultrastructure. Changes in immunostainability, populations of intensely immunostained LH and FSH cells and total volume of secretory granules were correlated with the changes in pituitary LH and FSH contents during the estrous cycle. The immunohistochemical feature of gonadotropin release is the transformation of intensely immunostained gonadotrophs into the weakly stained ones. Secretory granules of small diameter (less than 150 nm) were numerous just before LH and FSH surges then sharply declined along with LH and FSH surges. The number of secretory granules of large diameter (larger than 150 nm) also decreased when LH and FSH surges took place. Then the number increased progressively until 17.00 h on the day of diestrus, corresponding to the increase in pituitary LH and FSH contents. It is suggested that small secretory granules are a release pool while large ones are a reserve pool.     

Calcium binding to the subunit c ofE. coli ATP-synthase and possible functional implications in energy coupling

The 8-kDa subunit c of theE. coli F₀ ATP-synthase proton channel was tested for Ca⁺⁺ binding activity using a⁴⁵Ca⁺⁺ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds⁴⁵Ca⁺⁺ strongly with Ca⁺⁺ binding properties very similar to those of the 8-kDa CF₀ subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca⁺⁺ binding capacity, shown by loss of Ca⁺⁺ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca⁺⁺ binding, shown by Ca⁺⁺ binding being retained in twoE. coli mutants, Asp61Asn and Asp61Gly. The Ca⁺⁺ binding is pH dependent in both theE. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca⁺⁺ binding affinity to its F₀ binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca⁺⁺ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca⁺⁺ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80–100 nM was obtained using an EGTA-Ca⁺⁺ buffer system to control free Ca⁺⁺ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca⁺⁺ bound to the periplasimic side of theE. coli F₀ proton channel can block H⁺ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca⁺⁺ binding site is on the lumen side of the thylakoid, where Ca⁺⁺ binding can modulate acid-base jump ATP formation. The Ca⁺⁺ binding to the F₀ and CF₀ complexes is consistent with a pH-dependent gating mechanism for control of H⁺ ion flux across the opening of the H⁺ channel.This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia.     

Inhibition of the proliferation of cultured immortalized schwann cells by forskolin with a decreased basal level of diacylglycerol

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 M of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 M forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.     

Superovulation of Holstein heifers by a single subcutaneous injection of FSH dissolved in polyvinylpyrrolidone

This study was undertaken to determine whether a single injection of porcine FSH (pFSH) would induce a superovulatory response in cattle. Holstein heifers were given a single injection of pFSH (30 mg, s.c.) dissolved in saline (Group 1, n = 5); 50% polyvinylpyrrolidone (PVP; Group 2, n = 5); or 25% PVP (Group 3, n = 4). Group-4 heifers (n = 5) were given multiple intramuscular injections of pFSH every 12 h for 3 d at decreasing doses, for a total of 30 mg. All animals received a single injection of 750 microg PGF2 alpha 48 h after the initiation of pFSH treatment. Animals exhibiting estrus were artificially inseminated twice throughout estrus. Ova and embryos were recovered nonsurgically. Ovaries were examined by transrectal ultrasonography or by palpation per rectum on Day 7 or 8 of estrus. Plasma concentrations of pFSH, bovine FSH progesterone, estradiol-17 beta and inhibin were determined by specific radioimmunoassays. The number of corpora lutea (CL) and the numbers of total and transferable embryos which were detected and recovered in Groups 2 and 3 were equivalent to the numbers detected and recovered in Group 4. In Group 1, however, only 1 of 5 animals ovulated even a single oocyte. The present study demonstrated that only a single injection of pFSH dissolved in PVP was capable of inducing a superovulatory response by maintaining a high plasma FSH concentration to allow for the recovery of a sufficient number of embryos for transplantation.