Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme.

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.     

β-glucuronidase as a marker for clonal analysis of tomato lateral roots

In higher plants, the root-shoot axis established during embryogenesis is extended and modified by the development of primary and lateral apical meristems. While the structure of several shoot apical meristems has been deduced by combining histological studies with clonal analysis, the application of this approach to root apical meristems has been limited by a lack of visible genetic markers. We have tested the feasibility of using a synthetic gene consisting of the maize transposable elementActivator (Ac) inserted between a 35S CaMV promoter and the coding region of a -glucuronidase (GUS) reporter gene as a means of marking cell lineages in roots. The GUS gene was activated in individual cells byAc excision, and the resulting sectors of GUS-expressing cells were detected with the histochemical stain X-Gluc. Sectors in lateral roots originated from bothAc excision in meristematic cells and from parent root sectors that bisect the founder cell population for the lateral root initial. Analysis of root tip sectors confirmed that the root cap, and root proper have separate initials. Large sectors in the body of the lateral root encompassed both cortex and vascular tissues. The number of primary initial cells predicted from the size and arrangement of the sectors observed ranged from two to four and appeared to vary between roots. We conclude that transposon-based clonal analysis using GUS expression as a genetic marker is an effective approach for deducing the functional organization of root apical meristems.     

互叶白千层油化学成分的研究

采用水蒸汽蒸馏收集互叶白千层芳香油,对该油进行ＧＣ／ＭＳ／ＤＳ定性分析及其总离于流图的面积归一化定量分析,鉴定出２５种化合物,其中含氧化合物６种,碳氢化合物１９种。直接影响该油商品价值的化学成分４－萜品醇含量约为１７％,桧樟脑含量约为２．４％。     

牛胰多肽与CL／PC脂质体作用后二级结构的变化

用傅立叶变换红外光谱研究了ＢＰＰ与膜作用后其二级结构的变化,通过对红外光谱中酰胺Ｉ谱带进行解卷积、微分及曲线拟合等处理,结果表明,ＢＰＰ与脂膜作用后,其二级结构中α－螺旋成分增多,无规卷曲成分减少。     

Influence of number of gilts per pen on estrous traits in confinement-reared gilts

One hundred forty-four Yorkshire (Y) x Landrace (L) and Chester White (CW) x Large White (LW) reciprocal cross gilts (Trial I) and 147 CW:LW x Y:L reciprocal cross gilts (Trial II), 4.5 to 5 months old, were penned in groups of 3, 9, 17 or 27 (1.1 m(2)/gilt). Estrus was checked daily from seven to nine months of age. Reproductive tracts of all noncyclic gilts were examined at nine months of age. Between seven and nine months of age, 12% to 16% fewer (P<0.05) gilts in pens of three had regular estrous cycles, and the percentage of gilts with regular estrous cycles did not differ with more (9, 17 or 27) gilts/pen. At nine months of age, 56.9% of the gilts penned in groups of three were cyclic as compared to 78%, 80.4% and 80.7% of the gilts penned in groups of 9, 17 and 27, respectively. In Trial I, more (P<0.05) CW x LW reciprocal cross gilts were cyclic compared to Y x L reciprocal cross gilts at nine months of age. The CW x LW group had fewer behaviorally anestrous gilts and more cyclic gilts, regardless of the number of gilts/pen. The social cues involved in the process of sexual development of gilts remain undefined, but extremes in the number of gilts/pen should be avoided.     

Secretion of neurophysins by the ovary in sheep

Measurements of venoarterial concentration differences across the ovary in anesthetized sheep have demonstrated that the ovary secretes ovine neurophysin I/II (oNP I/II) and that this process is stimulated by the prostaglandin F2 alpha analogue, cloprostenol. A parallel increase in the secretion of oxytocin (OT) was observed in response to cloprostenol, and the mean molar ratio of oNP I/II to OT secreted was 1.2. There was no detectable ovarian secretion of oNP III. Secretion of oNP I/II and OT was absent after hysterectomy. The data support other evidence indicating that the corpus luteum synthesizes OT, and confirm that the neurophysin associated with OT in the sheep is oNP I/II.     

PHI--a new brain-gut peptide

The detection of the C-terminal amide structure in porcine intestinal extracts has led to the discovery of a 27 amino acid residue peptide designated PHI (PHI-27, peptide HI). The peptide was found to have structural homologies to vasoactive intestinal peptide (VIP) and growth hormone-releasing factor (GRF). Subsequent studies have revealed that PHI exhibits a variety of biological activities which resemble those of VIP. Moreover, it was found that the peptide is able to inhibit the binding of VIP to its receptors, and to stimulate cyclic AMP production. PHI is present in both brain and gut in high concentrations and probably acts as a neurotransmitter or neuromodulator rather than a hormone. A comparison of the amino acid sequences of porcine, human and bovine PHI indicated that human PHI differs from the porcine peptide in two positions (12 and 27), and bovine PHI differs in one position (10). The amino acid sequence (deduced from the cDNA sequence) of the VIP precursor recently obtained from human neuroblastoma cells also contains an identical sequence to the newly-isolated human PHI from human colonic extracts. PHI has thus been shown to be co-synthesized with VIP in the same precursor molecule.     

On disturbance of homeostasis in organ-cultured tissue

Summary The intact membranous rat mesentery was cultured in Eagle's minimum essential medium containing no serum or only low concentrations of serum. The procedure is in some important respects superior to previous organ culture techniques. To estimate the extent of disturbance of homeostasis of the tissue in culture, the spontaneous mast-cell histamine release was quantitated after preculture preparation of the specimens and after different intervals in culture. Also, the proliferation of fibroblasts and mesothelial cells that predominate in the mesentery was assessed at 48 h by cytofluorometric quantitation of DNA in single-tissue cells. Spontaneous histamine release was time dependent during cultivation, amounting to ca. 50% at 48 h, and was affected by the medium used for moistening the tissue before cultivation. Culturing also brought about great spontaneous increase in the proliferation of fibroblasts and mesothelial cells, the rate being related to the concentration of serum. Addition of the mast-cell secretagogues 48/80 or polymyxin B at 1 h caused rapid release of 50 to 60% of the histamine and was followed by augmented proliferation in the serum-containing media. The spontaneous increase of cell proliferation in tissue culture may be causally related to mast-cell secretion. Further studies are needed to define factors influencing the spontaneous mast-cell secretion and the mast-cell-dependent mitogenesis in normal tissue cells Supported by grants from the Swedish Medical Research Council (Project 5942) and State Board for Animal Experiments.     

Excitation-energy distribution in green algae. The existence of two independent light-driven control mechanisms

Three distinct states can be identified for cells of the green alga Chlorella vulgaris; State 1 and State 2 obtained by preillumination in far-red and red light, respectively, and the dark state obtained by dark-adaptation. Addition of the inhibitor DCMU to algal cells leads to an initial rapid increase in chlorophyll-a fluorescence reflecting the closure of Photosystem II traps. This, in the case of dark and state-2-adapted algae is followed by a slow light-dependent increase to a fluorescence yield typical of State-1-adapted cells. Measurements of low temperature (77 K) emission spectra indicate that the low fluorescence yields of dark and State-2-adapted algae reflect similar balances in excitation-energy distribution between the two photosystems. In both cases, the balance favours PS I and the slow fluorescence increase seen in the poisoned algae reflects a redressing of this balance in favour of PS II. The low fluorescence yield of State-2-adapted algae is thought to be associated with the phosphorylation of chlorophyll a/b light-harvesting protein (Biochim. Biophys. Acta (1983) 724, 94–103). Measurements of the uncoupler and ATPase sensitivity of the light-dependent increases seen in DCMU-poisoned cells indicate that the low fluorescence yield of dark-adapted algae is of different origin. Evidence is presented showing that the light-driven changes in excitation-energy distribution seen in green algae involve two distinct processes; a low-intensity, wavelenght-independent change reflecting simple light/dark changes and a higher intensity, wavelength-dependent change reflecting State 1/State 2 adaptation. The former changes appear to be associated with changes in the local ionic environment within the algal chloroplast, whilst the latter appear to reflect changes in the phosphorylation state of chlorophyll a/b light-harvesting protein.