Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.     

Synchronization of estrus in early diestral dairy heifers with Prostaglandin F(2)alpha and estradiol benzoate

Following observation of estrus, 134 Holstein heifers were given injections of Prostaglandin F(2)alpha (PGF(2)alpha) between Days 5 and 10 of their cycle (estrus = Day 0). They were then randomly assigned to either a group receiving 400 mug of estradiol benzoate (E(2)B) 40 h or maintained as controls. Heifers observed in estrus within 120 h of PGF(2)alpha administration were inseminated (approximately 12 h after initial observation of estrus). Blood samples for progesterone determination were drawn from the coccygeal vein on Days 15 and 21 after insemination. Pregnancy was confirmed by palpation per rectum between Days 5.0 and 60 post insemination. When control and treated heifers were compared it was found that a higher percentage of heifers treated with E(2)B exhibited estrus after PGF(2)alpha, but there had been no effect on subsequent progesterone concentrations or pregnancy rates.     

A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.     

Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements

Normal human epidermal melanocytes were selectively propagated from mixed (keratinocyte-melanocyte) cultures and primary epidermal cell suspensions in serum-free medium, MCDB 153 containing insulin, bovine pituitary extract (BPE), phorbol-12-myristate-13-acetate (PMA), ethanolamine, phosphoethanolamine, and hydrocortisone. Neonatal foreskin melanocytes (NFMs) replicated more readily than adult melanocytes in culture. Early passage NFMs grown in serum-free medium exhibited a population generation time of 24-48 hours. NFMs assumed a less dendritic appearance and were less pigmented than adult melanocytes. PMA or other protein kinase C-activating phorbol esters significantly enhanced mitogenesis of NFMs; however, cAMP-elevating agents were not required for efficient replication of NFMs. Basic fibroblast growth factor (bFGF) was a potent mitogen for NFMs and replaced the requirement for BPE in the culture medium. NFMs expressed a single class of specific, high-affinity receptors for bFGF, exhibiting a Kd = 3 x 10(-11) M and approximately 76,500 receptors/cell. Neither EGF nor TGF-alpha were mitogenic for NFMs, and TGF-beta reversibly inhibited NFM growth. Rapidly growing, early passage NFMs were shown to have cell cycle times of 19.5, 7.5, and 9 hours for G1, S, and G2/M phases of the cell cycle, respectively. Culture of NFMs to confluence or depletion of growth factors from the culture medium caused reversible, G1 phase-specific, cell cycle growth arrest. Senescence of NFMs was associated with irreversible growth arrest in the G1 phase after 40-45 population doublings in culture. Our data demonstrate that basal medium MCDB 153 can be supplemented with defined factors to cultivate selectively two major constituent cell types of the epidermis, the melanocyte and the keratinocyte.     

The fate of the fusarium mycotoxin zearalenone in maize cell suspension cultures

The fusarium mycotoxin zearalenone was transformed in cell suspension cultures of Zea mays giving α- and β-zearalenol and the β-D-glu cos ides of zearalenone and α- and β-zearalenol. The structure of zearalenone-4-β-D-glucopyranoside was determined by liquid — chromatography-mass spectrometry and specific hydrolysis with β-glucosidase. α- and β-zearalenol and their glucosides were identified by co chromatography using tic and HPLC and glucosidase — treatment Up to 50% of the mycotoxin added was bound to a non extractable or “bound” residue fraction. After treating this residue by a sequential cell wall fractionation procedure, zearalenone was found to be bound mainly to starch, hemicellulose, and lignin fractions.     

Characterization of the L lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition (L kappa----L lambda) at temperatures similar to the L beta'----(P beta')----L alpha transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. The L kappa phase shows typical gel-phase X-ray diffraction patterns. In contrast, the L lambda-phase diffraction patterns indicate disordered hydrocarbon chains. 2H NMR of specifically 2H chain-labeled DPPC confirmed that the acyl chains are disordered in the L lambda phase over their entire lengths. 2H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and 13C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicating very little motion at the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. We postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluidity of the hydrophobic region in the L lambda phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.