Increased shikonin production inLithospermum erythrorhizon suspension cultures within situ extraction and fungal cell treatment (elicitor)

Summary The effect ofin situ extraction and elicitor treatment on shikonin production was studied with the suspension cultures ofLithospermum erythrorhizon. Shikonin concentration of 60 mg/L was achieved by the use of both techniques which was 24 times higher than that of control culture, and 65 times higher in terms of shikonin productivity. The host-pathogen effect of elicitor treatment andin situ extraction for product removal were effective for shikonin production.     

The influence of various physical parameters on anther culture of broccoli (Brassica oleracea var. italica)

Embryo formation by cultured broccoli (Brassica oleracea L. var. italica) anthers was best in the pH range of 5.5 to 5.8. Manipulation of the initial medium pH showed, however, that embryos could be recovered throughout the entire pH range tested. Experiments designed to test the influence of anther density on embryo production exhibited an apparent population effect. Comparison of anthers cultured with and without filaments showed a significantly lower level of embryo formation with filaments attached. The importance of anther orientation with the adaxial surface up was also demonstrated. Detailed studies of the effect of temperature on anther response showed the importance of 35°C treatments. Other temperatures and a variety of temperature manipulations were either comparatively ineffective or inhibitory. The duration of 35°C exposure required for optimal response varied widely between 18 and 48 h. Wide variation in plant to plant response was observed despite attempts to optimize the manipulation of physical parameters. Individual plants were identified that reliably formed many thousands of embryos, whereas other plants failed to form embryos under all tested conditions.     

Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers.

Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.     

Restriction site mapping for three or more enzymes

Restriction site mapping requires a generator to put forwardpossible maps and a constraint checker to reject false maps.Ideally these combine to give an algorithm which calculatesa sound and complete solution set. Three algorithms for generationare presented and compared. Two decompose a multi-enzyme problem(3) into subproblems. The constraint checker is based on separationtheory. Some insights into the extent of constraint checkinginvolved in and the feasibility of more checking for three ormore enzymes are discussed. The trade-off between computationtime and the soundness of the solution set is examined. Received on July 30, 1989; accepted on April 4, 1990     

Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection.

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.     

Inter- and intraclade neutralization of human immunodeficiency virus type 1: genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes.

We have studied genetic variation among clades A through E of human immunodeficiency virus type 1 (HIV-1) at the levels of antibody binding to gp120 molecules and virus neutralization. We are unable to identify neutralization serotypes that correspond to the genetic clades. Instead, we observe that inter- and intraclade neutralization of primary isolates by HIV-1-positive sera is generally weak and sporadic; some sera show a reasonable degree of neutralization breadth and potency whereas others are relatively sensitive to neutralization, but no consistent pattern was found. However, a few sera were able to neutralize across clades with significant potency, an observation which may have implications for the feasibility of a broadly effective HIV-1 vaccine involving humoral immunity. Serological assays measuring anti-gp120 antibody binding also failed to identify serotypes that correspond precisely to the genetic clades, but some indications of clade-specific binding were observed, notably with sera from clades B and E. A representative protein for each clade (A through E) was selected on the basis of its specificity, defined as high seroreactivity with sera from individuals infected with virus of that clade and lower reactivity with sera from individuals infected with viruses from other clades. The seroreactivity patterns against these five proteins could be used to predict the genotype of the infecting virus with moderate success.     

龙胆属的系统发育分析

本文运用支序分类的原理和方法,对龙胆科龙胆属的属下等级进行了重新归类和系统发育分析。龙胆属是一个单系群,以3项近裔共性为归类依据。性状分析作了性状同源性分析和性状极性分析。性状极化主要以外类群比较、性状相关性及染色体资料为依据,其它方法,如生物重演律原则、地理递进原则以及孢粉形态等也被结合使用。分析结果,双蝴蝶属和蔓龙胆属被选择为外类群,71个性状被选择作为建立数据矩阵的基本资料。使用PAUP程序对矩阵进行了运算,得到4个最简约的谱系分支图,它们均具一致性系数0.637,支序长度为160步,f-比值范围为0.179～0.189,其中具最低f-比值的图被选作为类群归类和讨论亲缘关系的基础。在支序图上龙胆属归为15个组;其中5个组又划分为系,共包括23个系,其余组为单型组,故共有33个属下类群。一个严格的一致性谱系分支图总结了所有的一致点,从而支持了支序分析的结果。     

Synthesis of biodegradable emulsifier using lipase in anhydrous pyridine

Summary Synthesis of a bioemulsifier using a lipase from Pseudomonas sp. with fructose and vinyl laurate was carried out in anhydrous pyridine. The synthetic product was identified as laurylfructose with an emulsifying activity on various hydrocarbons, edible oils and petroleum oils. The compound reduced the surface tension of distilled water from 72 mN/m to 29 mN/m and the interfacial tension of water/n-hexadecane from 50 mN/m to 6 mN/m.     

Somatic mosaicism in a patient with neurofibromatosis type 1.

Using loss of heterozygosity analysis, a method designed to detect moderate to large gene deletions, we have identified a new-mutation neurofibromatosis type 1 (NF1) patient who is somatically mosaic for a large maternally derived deletion in the NF1 gene region. The deletion extends at least from exon 4 near the 5' end of the gene to intron 39 near the 3' end. The gene-coding region is, therefore, mostly or entirely deleted, encompassing a loss of > or = 100 kb. We hypothesize that the deletion occurred at a relatively early developmental timepoint, since signs of NF1 in this patient are not confined to a specific body region, as seen in "segmental" NF, and since both mesodermally and ectodermally derived cells are affected. This report provides the first molecular evidence of somatic mosaicism in NF1 and, taken together with a recent report of germ-line mosaicism in NF1, adds credence to the concept that mosaicism plays an important role in phenotypic and genetic aspects of NF1 and may even be a relatively common phenomenon.     

Chaperonins GroEL and GroES: views from atomic force microscopy.

The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.