Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes

Oocytes induced to undergo meiotic maturation by progesterone possess a cytoplasmic activity that causes germinal vesicle breakdown (GVBD). The cytoplasmic factor postulated to be responsible for this activity is designated as the maturation promoting factor (MPF). The activity of MPF was assayed by injecting cytoplasm into fully-grown oocytes to induce GVBD. It was found that maturing oocyte cytoplasm possesses MPF activity before GVBD begins. Treatment of progesterone stimulated oocytes with cycloheximide, either applied externally or injected, inhibited the appearance of MPF in the cytoplasm as well as GVBD when the inhibitor treatment was initiated before the cytoplasm exhibited MPF activity. In contrast, the same treatment did not inhibit GVBD when it was applied to oocytes after the cytoplasm possessed MPF activity. Furthermore, cycloheximide treatment of recipient oocytes did not inhibit the induction of GVBD by injected cytoplasm containing MPF. Cytoplasm of oocytes injected with MPF subsequently possessed MPF activity as high as that of the original donor cytoplasm in spite of its extensive dilution. This suggests that amplification of MPF took place in the recipient. Cycloheximide treatment did not inhibit the amplification of MPF. It was concluded that cycloheximide inhibits only the initial phase of induction of MPF activity, but neither its amplification nor its action on the nucleus that causes GVBD. From these results, a hypothesis concerning the cytoplasmic mechanism for the induction of GVBD has been proposed.     

Isoforms of NADP-Malic Enzyme from Mesembryanthemum crystallinum L. That are Involved in C3 Photosynthesis and Crassulacean Acid Metabolism

Exposure of the facultative halophyte Mesembryanthemum crystallinumL. to salt stress induces a shift from C₃ photosynthesis toCrassulacean acid metabolism (CAM). During induction of CAM,the activity of NADP-malic enzyme (EC 1.1.1.40 [EC] ) increased asmuch as 12-fold in leaves, while the enzymatic activity in rootsfell to half of the original level. These changes in the activityof the enzyme corresponded to changes in levels of the enzymeprotein. NADP-malic enzymes extracted from leaves in the C₃and CAM modes could be distinguished by differences in electrophoreticmobility during electrophoresis on a non-denaturing polyacrylamidegel. NADP-malic enzyme extracted from roots in the C₃-mode andin the CAM mode migrated as fast as the enzyme extracted fromleaves in the CAM mode on the same gel. Although the patternof peptide fragments from NADP-malic enzyme from CAM-mode leaveswas similar to that from C₃-mode leaves, as indicated by peptidemapping, both immunoprecipitation and an enzyme-linked immunosorbentassay revealed some antigenic differences between the enzymesextracted from leaves in the C₃ and the CAM modes. These resultssuggest the existence of at least two isoforms of NADPmalicenzyme that differ in their levels of expression during inductionof CAM. (Received April 21, 1994; Accepted September 5, 1994)     

Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions.

Rational shift of the optimum pH toward alkalinity and enhancement of thermostability were investigated by using a thermostable extremely alkaline protease (optimum pH, 12 to 13) from the alkaliphilic and thermophilic Bacillus sp. strain B18'. The protease gene (aprM) was cloned, and the sequence analysis revealed an open reading frame of 361 amino acids that was composed of a putative signal sequence (24 amino acids), a prosequence (69 amino acids), and a mature enzyme (268 amino acids) (molecular weight, 27,664). The amino acid sequence of this protease was compared with those of other serine proteases. A direct correlation of higher optimum pH with an increase in the number of arginine residues was observed. An even more thermostable mutant enzyme was created by introducing a point mutation. When the position of the beta-turn, Thr-203, was replaced by Pro, the residual activity of this mutant enzyme at 80 degrees C for 30 min was higher than that of the wild-type enzyme (50% versus 10%). The specific activity of this mutant enzyme at 70 degrees C was 105% of that of the wild-type enzyme under nondenaturation condition. These data suggest that the higher content of Arg residues favors the alkalinity of the serine protease and that introduction of a Pro residue into the beta-turn structure stabilizes the enzyme.     

The amino acid sequence of the 9 kDa polypeptide and partial amino acid sequence of the 20 kDa polypeptide of mitochondrial NADH:ubiquinone oxidoreductase.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated enzyme in the respiratory chain and is composed of at least 26 distinct polypeptides. Two hydrophilic subfractions of bovine heart complex I were systematically resolved into individual polypeptides by chromatography. Three polypeptides (51, 24, and 9 kDa) were isolated from the flavoprotein fraction (FP) of complex I, and the complete amino acid sequence of the 9 kDa polypeptide was determined. The 9 kDa polypeptide is composed of 75 amino acids with a molecular weight of 8,437. This protein exhibits no obvious sequence similarity to other proteins. The iron-sulfur protein fraction (IP) of complex I was separated into eight polypeptides, 75, 49, 30, 20, 18, 15, 13 kDa-A, and 13 kDa-B. The 20 kDa polypeptide was recognized as a novel component of IP for the first time. The N-terminal and several peptide sequences of the 20 kDa polypeptide were determined. Comparison of the sequences revealed significant sequence similarities of the 20 kDa polypeptide to the psbG gene products encoded in the chloroplast genome. The conserved sequence in these proteins was also found in the small subunit of the nickel-containing hydrogenases. These results suggest that complex I is related to other redox enzyme complexes.     

Functional implications of interleukin-1 beta based on the three-dimensional structure.

The molecular structure of interleukin-1 beta, a hormone-like cytokine with roles in several disease processes, has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. The framework of this molecule consists of 12 antiparallel beta-strands exhibiting pseudo-3-fold symmetry. Six of the strands make up a beta-barrel with polar residues concentrated at either end. Analysis of the three-dimensional structure, together with results from site-directed mutagenesis and biochemical and immunological studies, suggest that the core of the beta-barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL-1 interacts with its receptor.     

Development of Microtubule-Dependence of the Chromosome Cycle at the Midblastula Transition in Xenopus laevis Embryos

Animal-cap cells isolated from Xenopus laevis morulae and blastulae are cultured for 2 to 6 hr in medium containing nocodazole, Colcemid or taxol, at concentrations completely inhibiting cell division. At 20°C, cells from each control embryo undergo synchronous cell cycles up to the 12th, with a period of 32 min, of which 60% represents the chromosome condensation (mitotic or M-) phase, and the average mitotic index remains near 50%. Cells treated with nocodazole, Colcemid or taxol before 12th cleavage undergo chromosome cycles with a similar period as controls, albeit without chromosome segregation, and the average mitotic index remains near 50%. From the 12th to 15th cycles, control cycles become asynchronous, their period gradually increases 2 to 3 times, and the mitotic index declines to 10%. In cells treated after 12th cleavage with taxol, the mitotic index declines, similarly to control cells. However, in nocodazole-treated cells, it increases steadily, and exceeds 70% at 2 hr of treatment, but gradually declines to 40% at 6 hr. Therefore, while inhibition of microtubule activities does not significantly alter the timing of chromosome condensation cycles during synchronous cleavage, inhibition of microtubule assembly can prolong M-phase during asynchronous cleavage after the midblastula transition.     

Promyelocytic leukemia cell line, HL-60, produces human hepatocyte growth factor.

The human promyelocytic leukemia cell line, HL-60, stimulated with PMA, produced human HGF-like immunoreactivity (HL-60 HGF), which was detected with human HGF-specific ELISA. The purified HL-60 HGF was indistinguishable from human HGF in the plasma of patients with fulminant hepatic failure by studies of subunit constitution and amino acid composition. The HL-60 HGF mRNA corresponded to 6 kb, which was consistent with previous reported data in rat and human HGF mRNA, was detected in stimulated HL-60, by northern hybridization analysis using human HGF cDNA probe. These findings indicated that HL-60 HGF was identical to, or closely resembled, human plasma HGF. The HL-60 cell is an attractive model for studies of HGF-producing mechanisms, the manner of secretion and the nature of induction signals.     

Crystal structure of Thermus thermophilus HB8 UvrB protein, a key enzyme of nucleotide excision repair

In the nucleotide excision repair system, UvrB plays a central role in damage recognition and DNA incision by interacting with UvrA and UvrC. We have determined the crystal structure of Thermus thermophilus HB8 UvrB at 1.9 A resolution. UvrB comprises four domains, two of which have an alpha/beta structure resembling the core domains of DNA and RNA helicases. Additionally, UvrB has an alpha-helical domain and a domain consisting of antiparallel beta-sheets (beta-domain). The sequence similarity suggests that the beta-domain interacts with UvrA. Based on the distribution of the conserved regions and the structure of the PcrA-DNA complex, a model for the UvrB-DNA complex is proposed.     

Ca and Mg control of cytostatic factors from Rana pipiens oocytes which cause metaphase and cleavage arrest.

A Ca-sensitive cytoplasmic factor is extractable from mature oocytes of Rana pipiens, but not from immature oocytes or fertilized embryos, and is capable of inducing cleavage arrest at metaphase when microinjected into one blastomere of a cleaving embryo. This factor, designated “primary cytostatic factor” (1°CSF), is active in the presence of Mg or EGTA, but is rapidly inactivated by Ca at concentrations as low as 10 μM or by EDTA. 1°CSF is also inactivated in vivo by injection into the cytoplasm of a zygote during the first 45 min following insemination. Another factor, the “secondary cytostatic factor” (2°CSF), develops in vitro during the aging of extracts prepared from immature oocytes, mature oocytes, and fertilized eggs, and is also capable of arresting cleavage at metaphase. 2°CSF is insensitive to exogenous Ca or EDTA, and also remains active when injected into a zygote at any time following insemination. Since the responses of the fertilized egg to 1°CSF and to mature oocyte cytoplasm are quite similar, it appears that 1°CSF is equivalent to the factor which maintains metaphase II arrest in the mature oocyte. The possibility of 1°CSF inactivation by endogenous free Ca at the time of fertilization is discussed in the context of changes in the free Ca concentration.     

Isolation and characterization of cyclic AMP-resistant variants from a functional adrenal cortical cell line.

We have isolated a number of cyclic AMP-resistant cell lines and characterized two of them, 3B4 and 10F2s, from a functional adrenal cortical cell line, Y-1. At seeding densities above $\text{100}\text{cells}\text{10}\text{cm}$ diameter plates, the variant cells are resistant in their morphological change, plating efficiency and growth to the normal effects of dibutyryl cyclic AMP (db-cAMP). At lower seeding densities, 3B4 and 10F2 have retained a slight sensitivity to db-cAMP in their plating efficiency and in their morphology. Studies with various nucleotides and cAMP analogues show that the inhibitory effects of db-cAMP on the growth and morphology of Y-1 cells are not due to degradation products of db-cAMP. There is loss of response to ACTH in the variant cell lines such that there are no effects of ACTH on plating efficiency, growth, morphology, steroidogenesis and cAMP excretion. In addition, the variant cell lines show lowered activities of the cAMP binding receptor and cAMP-dependent protein kinase. Preliminary studies indicate that in Y-1, cAMP markedly reduces protein phosphorylation, and it inhibits phosphate uptake. In the variants, the protein phosphorylation and phosphate uptake are maintained even in the presence of db-cAMP. The maintenance of phosphorylation in the presence of db-cAMP may play an important role in the ability of the cells to survive in high concentrations of db-cAMP. The variant cell lines can be stimulated by db-cAMP to increase steroidogenesis, although the stimulated levels of steroidogenesis in 3B4 and 10F2 are less than those in Y-1. The variant phenotype is stable in vitro and in vivo.