Human hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase: evidence for the regulation of enzymic activity by a bicyclic phosphorylation cascade

Microsomal human liver HMG-CoA reductase has been shown to exist in active (dephosphorylated) and inactive (phosphorylated) forms. Microsomal HMG-CoA reductase was inactivated in vitro by ATP-Mg in a time dependent manner; this inactivation was mediated by reductase kinase. Incubation of inactivated enzyme with phosphatase resulted in a time dependent reactivation (dephosphorylation). Polyacrylamide gel electrophoresis of purified HMG-CoA reductase incubated with reductase kinase and radiolabeled ATP revealed that the 32P radioactivity and HMG-CoA reductase enzymic activity were localized in a single electrophoretic position. Partial dephosphorylation of the phosphorylated enzyme was associated with loss of 32P and increase in HMG-CoA reductase activity. Human reductase kinase also exists in active and inactive forms. The active (phosphorylated) form of reductase kinase can be inactivated by incubation with phosphatase. Phosphorylation of inactive reductase kinase with ATP-Mg and a second kinase, reductase kinase kinase, was associated with a parallel increase in the enzymic activity of reductase kinase and the ability to inactivate HMG-CoA reductase. The combined results present initial evidence for the presence of human HMG-CoA reductase and reductase kinase in active and inactive forms, and the in vitro modulation of its enzymic activity by a bicyclic phosphorylation cascade. This bicyclic cascade system may provide a mechanism for short-term regulation of the pathway for cholesterol biosynthesis in man.     

Phosphoregulation of STIM1 Leads to Exclusion of the Endoplasmic Reticulum from the Mitotic Spindle

The endoplasmic reticulum (ER) undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown [1]. Stromal interaction molecule 1 (STIM1) is an ER Ca(2+) sensor that activates store-operated Ca(2+) entry (SOCE) [2, 3] and also functions in ER morphogenesis through its interaction with the microtubule?+TIP protein end binding 1 (EB1) [4]. We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE [5]. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM1(10A)) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM1(10A), demonstrating that aberrant association of STIM1(10A) with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired?+TIP tracking in interphase but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca(2+) signaling during mitosis require multimodal STIM1 regulation by phosphorylation.     

Stimulation of the largest subordinate follicle by intrafollicular treatment with insulin-like growth factor 1 is associated with inhibition of the dominant follicle in heifers

The effect of intrafollicular treatment of the second-largest follicle (F2) with insulin-like growth factor (IGF) 1 on the largest follicle (F1) and F2 was studied in heifers. Treatment of F2 was done when F1 reached ≥8.2 mm (expected beginning of follicle deviation; Day 0 or Hour 0). In each of two experiments, three groups (n = 6 or 7 heifers/group) were used: controls, F2 treated with vehicle and F2 treated with IGF1. The IGF1 treatment consisted of 200 μg of recombinant human IGF1 (pharmacological dose) in 20 μL of vehicle. In Experiment 1, the hypothesis that treatment of F2 with IGF1 has a stimulatory effect on F2 was supported by a greater (P < 0.05) incidence of F2 dominance (≥10 mm) in the IGF1 group (71%) than in the other two groups (8%), and a greater (P < 0.02) growth rate of F2 on Days 0-2. Unexpectedly, treatment of F2 with IGF1 had an inhibitory effect on F1, as indicated by a reduced (P < 0.03) growth rate of F1 during Days 0-1 and Days 0-4 and a lesser (P < 0.05) maximum diameter of F1 in the IGF1 group. In Experiment 2, the hypothesis of an inhibitory effect on F1 when F2 was treated with IGF1 was supported by a lesser (P < 0.04) increase in diameter of F1 and a lesser (P < 0.04) percentage of follicle wall with power-Doppler signals of blood flow between Hours 0 and 14 in the IGF1 group. Circulating concentrations of FSH and LH were not altered significantly in either experiment. In conclusion, treatment of F2 with IGF1 at the expected beginning of deviation had a stimulatory effect on F2, but an inhibitory effect on F1.     

Disruption of the periovulatory LH surge by a transient increase in circulating 17β-estradiol at the time of ovulation in mares

The mechanism for a reported temporal association between ovulation and a transient disruption in the periovulatory increase in LH concentrations was studied in nine mares treated with human chorionic gonadotropin when the preovulatory follicle was ≥32 mm. Examinations for ovulation detection and blood collection were done at 2-h intervals and the results were retrospectively centralized to ovulation (Hour 0). Concentrations of LH began to increase (P < 0.03) rapidly at Hour ?18, decreased (P < 0.04) between Hours 0 and 6, and again increased (P < 0.0001) after Hour 12. A progressive decrease (P < 0.0001) in estradiol between Hours ?30 and 24 was interrupted temporarily by an increase (P < 0.01) between Hours ?2 and 0 and a decrease (P < 0.007) between Hours 0 and 2. Results indicated that a disruption and depression in the periovulatory LH surge occurred at the time of detected ovulation in mares and was temporally associated with a transient increase in estradiol during an overall progressive decline. The disruption in LH concentrations is attributable to the estradiol increase, based on a reported negative effect of estradiol on LH. The source of the circulating estradiol was likely from the discharge of estradiol-laden follicular fluid into the abdomen during ovulation and rapid absorption of estradiol into the circulation.     

In vivo effects of an intrafollicular injection of insulin-like growth factor 1 on the mechanism of follicle deviation in heifers and mares

In cattle and mares, free insulin-like growth factor 1 (IGF-1) is higher in the future dominant follicle (F1) than in the future largest subordinate follicle (F2) before deviation in diameter or selection is manifested between the two follicles. The effect of IGF-1 on other follicular-fluid factors and on the destiny of F2 were studied in two experiments in each species, using a total of 40 heifers and 42 mares. An injection of IGF-1 was made into F2 at the expected beginning of deviation (heifers, F1 >or= 8.5 mm; mares, F1 >or= 20.0 mm; Hour 0). In heifers, follicular fluid was taken from F2 at Hours 3, 6, 12, or 24; each heifer was sampled only once. In mares, sequential F2 samples were taken from each mare at Hours 0, 6, and 24 or at Hours 12 and 24. Transvaginal ultrasound guidance was used for treatment and sample collection. In heifers, IGF-1 treatment of F2 stimulated the secretion of estradiol (P < 0.05) between Hours 3 and 6 and androstenedione (P < 0.05) between Hours 3 and 12. In F2 of control heifers, estradiol decreased (P < 0.05) and androstenedione did not change significantly. In mares, IGF-1 treatment of F2 did not affect the concentrations of estradiol during the 24-h posttreatment period; androstenedione decreased (P < 0.04) in the IGF-1 group and increased (P < 0.006) in the controls. Compared with control mares, the IGF-1 group had higher (P < 0.04) activin-A at Hours 12 and 24 and higher (P < 0.0006) inhibin-A at Hour 24. After ablating F1 at Hour 24 in mares, F2 became dominant and ovulated in more mares (P < 0.0002) in the IGF-1 group (12/14) than in the control group (2/14). These results are consistent with reported temporal relationships among follicular factors during deviation in both species and indicate that IGF-1 plays a key role in controlling the temporal relationships; however, no indication was found that IGF-1 stimulated estradiol production in mares during the 24 h after treatment.     

Associated and independent comparisons between the two largest follicles preceding follicle deviation in cattle

Follicle diameters and concentrations of follicular fluid factors were studied in the two largest follicles (F1 and F2) using F1 diameters in increments of 0.2 mm (equivalent to 4 h intervals) and extending from 7.4 to 8.4 mm (12 heifers in each of 6 groups). Changes were compared between follicles using the F2 associated with each F1-diameter group. Diameter deviation began in the 8.2-mm group as indicated by a greater (P < 0.05) diameter difference between F1 and F2 in the 8.4-mm group than in the 8.2-mm group. In the 8.0-mm group, estradiol concentrations began to increase (P < 0.05) differentially in F1 versus F2, and free insulin-like growth factor-1 (IGF-1) began to decrease differentially in F2 (P < 0.06). Combined for F1 and the associated F2, activin-A concentrations increased (P < 0.05) between the 7.6- and 8.2-mm groups and then decreased (P < 0.05). Results supported the hypothesis that estradiol and free IGF-1 concentrations simultaneously become higher in F1 than in the associated F2 by the beginning of diameter deviation. Results did not support the hypothesis that a transient elevation in activin-A is present in F1 but not in the associated F2 at the beginning of the estradiol and IGF-1 changes; instead, a mean transient elevation in activin-A occurred at this time only when data for the two follicles were combined. Comparisons between F1 and F2 also were made by independently grouping F2 and using diameter groups at 0.2-mm increments for F2 as well as for F1. In the diameter groups common to F1 and F2 (7.4, 7.6, 7.8, and 8.0 mm) there was a group effect (P < 0.003) for estradiol involving an increase (P < 0.05) beginning at the 7.6-mm group averaged over F1 and F2. For free IGF-1 concentrations, a fluctuation (a significant increase followed by a significant decrease) occurred independently in F1 between the 7.4- to 7.8-mm groups and independently in F2 between the 7.0- to 7.4-mm groups.     

Regulation of developing B cell survival by RelA-containing NF-kappa B complexes

Mice deficient in the RelA (p65) subunit of NF-kappaB die during embryonic development. Fetal liver (FL) hemopoietic precursors from these mice were used to generate RelA-deficient lymphocytes by adoptive transfer into lethally irradiated mature lymphocyte-deficient recombination-activating gene-1(-/-) mice. Strikingly, RelA(-/-) lymphocyte generation was greatly diminished compared with that of RelA(+/+) lymphocytes. The most dramatic reduction was noticed in the numbers of developing B cells, which were considerably increased when RelA(-/-) FL cells that were also TNFR1 deficient were used. The role of RelA was further investigated in FL-derived developing B cells in vitro. Our results show that RelA is a major component of constitutive and TNF-alpha-induced kappaB site-binding activity in developing B cells, and provide evidence for a direct role of TNF-alpha in killing RelA(-/-) B cells. The absence of RelA significantly reduced mRNA expression of the antiapoptotic genes cellular FLICE-inhibitory protein and Bcl-2. Retroviral transduction of RelA(-/-) B cells with either cFLIP or Bcl-2 significantly reduced TNF-alpha killing. Together, these results indicate that RelA plays a crucial role in regulating developing B cell survival by inhibiting TNF-alpha cytotoxicity.     

An overview on fermentation,downstream processing and properties of microbial alkaline proteases

Microbial alkaline proteases dominate the worldwide enzyme market, accounting for a two-thirds share of the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus sp. dominate the industrial sector. To develop an efficient enzyme-based process for the industry, prior knowledge of various fermentation parameters, purification strategies and properties of the biocatalyst is of utmost importance. Besides these, the method of measurement of proteolytic potential, the selection of the substrate and the assay protocol depends upon the ultimate industrial application. A large array of assay protocols are available in the literature; however, with the predominance of molecular approaches for the generation of better biocatalysts, the search for newer substrates and assay protocols that can be conducted at micro/nano-scale are becoming important. Fermentation of proteases is regulated by varying the C/N ratio and can be scaled-up using fed-batch, continuous or chemostat approaches by prolonging the stationary phase of the culture. The conventional purification strategy employed, involving e.g., concentration, chromatographic steps, or aqueous two-phase systems, depends on the properties of the protease in question. Alkaline proteases useful for detergent applications are mostly active in the pH range 8-12 and at temperatures between 50 and 70 degrees C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80-90 degrees C. Alkaline proteases mostly have their isoelectric points near to their pH optimum in the range of 8-11. Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and three-dimensional structures.