Assembly of microfibrils in vivo and in vitro from (1»3)-β-d-glucan synthesized by protoplasts of Saccharomyces cerevisiae

Polymer chains of (13)--d-glucan were dissolved with 1 M NaOH at 4° C from native microfibrillar protoplast nets. The chains associated into microfibrils during NaOH neutralization or dialysis. In contrast to the native microfibrils which are of uniform width individually (10 to 20 nm) and arranged in flat bundles, the microfibrils formed in vitro showed no band formation and consisted of fibrous spindle-shaped subunits of variable width or loose elementary fibrils about 1.7 nm wide. X-ray diagrams of native nets indicated a fairly high crystallinity and were different for wet and dry specimens. They corresponded to those of paramylon. Precipitated glucans produced diagrams different from the former and revealing a lower crystallinity especially with the dry samples.The X-ray pattern, combined with other data, allowed the precipitated microfibrils to be identified as aggregates of molecular strands composed each of three intertwined helical glucan chains. Since these triple helical chains are about 1.7 nm wide the elementary fibrils of this width can represent only single triple-helical strands. These helices have 7 glucose residues per turn and therefore a low symmetry which explains the poor crystallizing properties. The 7 membered helix represents a basic difference with the well crystallized native glucan which is built of highly symmetrical triple helices with 6 glucose residues per turn. Since 6₁ helical conformation is not formed in vitro at normal temperatures its generation in vivo must be due to the action of synthesizing enzymes at the protoplast membrane. The intertwining of these helices and crystallization of the strands are determined by their symmetry and physical properties of the chains. This characterizes the native microfibrils as products of self-assembly of enzymegenerated 6₁ helices.     

The partial purification and characterisation of gibberellin 2β-hydroxylases from seeds of Pisum sativum

The gibberellin (GA) 2-hydroxylases in mature and immature seeds of Pisum sativum have been partially purified and characterised. The enzymes are unstable when stored below pH 7.0 or in the absence of a thiol reagent. The optimum assay pH is between 7.4 and 7.8 and activity is dependent upon the presence of -ketoglutarate, Fe²⁺ and ascorbate. The 2-hydroxylase activities for GA₁, GA₄, GA₉ and GA₂₀ are chromatographically inseparable and correspond to a protein of M_r 44000. The rate of GA 2-hydroxylation varies according to substrate and some evidence indicates that the 2-hydroxylase activities for GA₁ and GA₄ and for GA₉ and GA₂₀ may reside in different proteins. During pea seed maturation, the specific activity of the enzyme(s) increases dramatically and reaches a maximum at a time when endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ are also at their greatest concentration. This correlation is not the result of substrate induction of enzyme activity. Since the GA 2-hydroxylases operate at maximal rate at low substrate concentrations they are incapable of rapidly 2-hydroxylating excessive quantities of (exogenously applied) GA₁ or GA₂₀. On the basis of the kinetic parameters of the GA 2-hydroxylase activities, a generalised model is discussed for the control of the steady-state levels of bioactive hormone under normal physiological conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - HSS high-speed supernatant - LSS low-speed supernatant - PMSF phenylmethane sulphonyl fluoride     

Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

Binding of 8-bromoguanylic acid to ribonuclease T1 as studied by absorption and circular dichroism spectroscopy

8-Bromoguanosine 2'- and 3'-phosphates have been shown to bind to RNase T1 with the same affinity as the corresponding guanosine phosphates, inducing difference absorption and circular dichroism spectra similar to those induced by the guanosine phosphates. Since the brominated ligands have reduced electron density on N-7 of the guanine ring and syn-fixed conformation due to a bulky, electron-withdrawing Br substituent on C-8, the difference spectra are not attributable to the protonation on N-7 and to the restriction of the ligand to syn-conformation as proposed previously.     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.     

小麦颖果果皮绿色层内同化物的合成与分配

小麦幼嫩颖果中,果皮内侧发育出由内表皮与亚表皮组成的一薄层绿色组织。显微与亚显微结构观察表明,虽绿色层只接受到自然光照的1/4～1/8,叶绿体仍能正常发育,叶绿素含量与叶绿体数量均高出旗叶。大量胞间连丝联接相邻的绿色细胞,并在一定时期形成开放的胞间通道,显然有利于同化物的快速胞间运输。离体颖果饲喂~14CO_2试验证明,新合成的同化物从绿色细胞输向胚珠。绿色层产生的同化物可能通过合点端的珠心进入胚乳或通过珠孔直接汇聚到胚珠和分化中的原胚。     

Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

Summary We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung ₂-adrenergic receptor (amino acids 16–31 and 174–189, respectively). All antisera directed against peptide 1 (four of four rabbits) as well as two antisera directed against peptide 2 (two of four rabbits) recognized the purified ₂-adrenergic receptor in immunoblot conditions when used at a dilution of 1500. Antisera directed against peptide 1 as well as peptide 2 were able to immunoprecipitate iodinated as well as¹²⁵I-cyanopindolol tabeled ₂-adrenergic receptor. This last result implies that the recognized epitopes do not contain the¹²⁵I-cyanopindolol binding domain of the ₂-adrenergic receptor. Immunoblot experiments performed on membrane fractions from hamster lung tissue showed that immunoreactive bands at 64,000, 57,000, 47,000, 44,000 and 38,000 daltons were specifically detected. When purified ₂-adrenergic receptor was iodinated and submitted to glycolytic and/or tryptic treatments, species with similar molecular weights could be recovered. Then, the immunoreactive bands probably correspond to native ₂-adrenergic receptor and to degradative or nonglycosylated species of this molecule. The antisera were also able to detect immunoreactive molecules in murine and human cell lines, suggesting conservation of the probed sequences between these species. Enzymatic linked immunosorbent assay tests on intact cells and immunofluorescence studies confirmed that the amino-terminus and putative first extracellular loop are extracellularly located. Immunofluorescence studies on mouse brain primary cultures showed that cells expressing ₂-adrenergic receptor-like molecules exhibited a neuronal phenotype.     

Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

Localization of α-amylase isozymes within the endomembrane system of barley aleurone

Summary The localization of -amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts was studied using electron microscope immunocytochemistry. Antibodies were raised against total barley -amylase, i.e., -amylase containing both highisoelectric point (high-pI) and low-pI isoforms, as well as against purified high- and low-pI isoforms. All antibodies localized -amylase to the endoplasmic reticulum (ER) and Golgi apparatus (GApp) of the aleurone cell, and various controls showed that the labeling was specific for -amylase. Labeling of protein bodies and spherosomes, which are the most abundant organelles in this cell, was very low. There was no evidence that -amylase isoforms were differentially distributed within different compartments of the endomembrane system. Rather, both high- and low-pI isoforms showed the same pattern of distribution in ER and in the cis, medial, and transregions of the GApp. We conclude that in the Himalaya cultivar of barley, all isoforms of -amylase are transported to the plasma membrane via the GApp.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - GApp Golgi apparatus - PBS phosphate buffered saline - PCR partially coated reticulum - PM plasma membrane - TBS Tris buffered saline - TGN trans-Golgi network     

The inheritance of β-amylase null in storage roots of sweet potato,Ipomoea batatas (L.) Lam.

Summary Several sweet potato genotypes were found to lack completely or to have only traces of-amylase in their storage roots. Such genotypes do not increase in sweetness during cooking because, without a sufficient amount of-amylase, the normal hydrolysis of starch to maltose does not occur in the cooking process. In order to study the inheritance of this biochemical variant in the genotype, 41 families were generated. The following conclusions were drawn from analyzing these families. (1) This trait is controlled by one recessive allele (designated-amy) (2) It is inherited in a hexasomic or tetradisomic manner, but not disomically or tetrasomically. This conclusion supports previous cytological data that sweet potato is an autohexaploid or has two identical genomes plus one genome which is somewhat different. (3) The-amy allele appears to exist at a high frequency in cultivated germplasm. (4) Breeding sweet potato for low-amylase activity is relatively easy. New types of sweet potato without normal-amylase activity have great potential for processing and as a staple food.