人粒细胞集落刺激因子（hG－CSF）cDNA3′－UTR对其表达的影响

利用人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）ｃＤＮＡ３′端非翻译区（３′－ＵＴＲ）中存在的ＤｒａⅠ酶切位点,通过部分酶切与完全酶切,删除３′－ＵＴＲ不同长度,构建了四种ｈＧ－ＣＳＦｃＤＮＡ瞬时重组表达质粒。转染ＣＯＳ－７细胞后,生物活性测定结果提示,ｈＧ－ＣＳＦｃＤＮＡ３′－ＵＴＲ对其表达起负调控作用,其关键性序列位于紧接终止密码子ＴＧＡ下游的６５ｂｐ范围内,３′－ＵＴＲ对ｈＧ－ＣＳＦｃＤＮＡ表达的影响与转录水平的差别有一定关系。     

汉防己甲素及克矽平对矽肺大鼠Ⅰ型和Ⅲ型胶原mRNA的影响

汉防己甲素（汉甲）及克矽平（Ｐｏｌｙｖｉｎｙｌｐｙｒｉｄｉｎｅ－Ｎ－Ｏｘｉｄｅ,ＰＶＮＯ）是目前较为有效的抑制矽肺纤维化的药物。本文研究了其对胶原ｍＲＮＡ水平的影响．斑点杂交实验表明大鼠接尘６０天和１２０天后α１（Ⅰ）及α１（Ⅲ）ｍＲＮＡ水平明显上升,经汉甲或克矽平治疗１个月或３个月后,胶原ｍＲＮＡ水平明显下降。原位杂交结果表明胶原ｍＲ－ＮＡ银颗粒与细胞性结节和增厚的肺泡壁的成纤维细胞分布重合。汉甲或克矽平治疗后银颗粒数下降。提示汉甲及克矽平对矽肺进程中的胶原基因表达增强有抑制作用。     

兼具SOD和GPX活力的双功能酶的制备及性质研究

用苯甲基磺酰氟（ＰＭＳＦ）和Ｈ_２Ｓｅ相继处理铜锌超氧化物岐化酶（Ｃｕ,Ｚｎ－ＳＯＤ）,将酶分子中的丝氨酸（Ｓｅｒ）转化为硒代半胱氨酸（ＳｅＣｙｓ）,从而引入了谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团,使其在ＳＯＤ酶活性大部分保留的情况下,具有ＧＰＸ活性,其ＧＰＸ活力是ＰＺ５１活力的３０倍。研究了双功能酶的最佳制备条件,包括ＰＭＳＦ的剂量、反应最适温度及Ｈ_２Ｓｅ处理时间等,并用电子能谱、ＤＴＮＢ等方法测定了双功能酶的硒含量;测定了双功能酶对不同底物的米氏常数及双功能酶的荧光光谱、紫外吸收光谱及稳定性。     

Hypervariable region IV of Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing factor for functional flagella.

To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated fliCd genes with deletions and amino acid alterations in hypervariable region IV and in region of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T.M. Joys and F. Schödel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into functional flagella and conferred motility on flagellin-deficient hosts. Serological analysis of these flagella with different anti-d antibodies revealed that the peptide sequence centered at amino acids 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their flanking sequence by a tripeptide specified by a linker sequence eliminated most reactivity with antisera against wild-type d flagella as tested by enzyme-linked immunosorbent assay or by Western immunoblot. Functional analysis of the mutated flagellin genes with or without an insert suggested that amino acids 180 to 214 in the 5' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins formed by insertion of peptide sequence pre-S1 12-47 of hepatitis B virus surface antigen into the deleted flagellins assembled into functional flagella, and antibody to the pre-S1 sequence was detected after immunization of mice with the hybrid protein. This suggests that such mutant flagellins containing heterologous epitopes have potential as vaccines.     

Intracerebroventricular Administration of AT1 Receptor Antisense Oligonucleotides Inhibits the Behavioral Actions of Angiotensin II

Abstract: Antisense Oligonucleotides were developed to study the expression and function of angiotensin type 1 (AT₁) receptors in cultured cells and brain. In both liver epithelial WB and neuro-blastoma N1E-115 cells AT₁ antisense oligomers substantially decreased AT₁ receptor density, whereas angiotensin type 2 (AT₂) receptors remained unchanged. Similarly, repeated intracerebroventricular injections of AT₁ antisense oligomers in rats decreased AT₁ receptor density in hypothalamic-thalamic-septal tissue, and AT₂ receptors were unaffected. Intracerebroventricular antisense oligomers also attenuated drinking elicited by intra-cerebroventricular angiotensin II but not the cholinomimetic carbachol. Collectively, these results demonstrate that antisense Oligonucleotides attenuate angiotensin receptor expression and function in behaving animals.     

Removal of Adsorbed Toxin Fragments That Modify Bacillus thuringiensis CryIC delta-Endotoxin Iodination and Binding by Sodium Dodecyl Sulfate Treatment and Renaturation

We report that 10- and 25-kDa toxin fragments adhere to CryIC prepared from Bacillus thuringiensis insecticidal crystals, block iodination, and alter membrane binding. There is no apparent affect on CryIC toxicity against Spodoptera exigua. Associated peptides remained bound to CryIC in the presence of 50 mM dithiothreitol or 6 M urea. A novel detergent-renaturation procedure was developed for the purification of B. thuringiensis CryIC toxin. Sodium dodecyl sulfate (SDS) treatment followed by gel filtration chromatography yielded a homogeneous 62-kDa CryIC toxin. After removal of SDS and renaturation, the purified CryIC toxin was fully insecticidal to S. exigua larvae. I-labeled CryIC bound with high affinity to brush border membrane vesicles from S. exigua larvae.     

Reconstitution of iron-sulfur center B of Photosystem I damaged by mercuric chloride

Incubation of thylakoid membranes from spinach with low concentrations of mercuric chloride induces the loss of one of the iron-sulfur centers, F_B, in Photosystem I (PS I) and inhibits the electron transfer from PS I to the soluble electron carrier, ferredoxin. Reconstitution of this damaged iron-sulfur center has been carried out by incubating treated thylakoid membranes with exogenous FeCl₃ and Na₂S in the presence of-mercaptoethanol under anaerobic conditions. Low temperature EPR measurements indicate that center F_B is largely restored. Kinetic experiments show that the restored F_B can be photoreduced from P700. However, these reconstituted thylakoid membranes are still incompetent in the photoreduction of ferredoxin and NADP⁺, even though ferredoxin binding to the modified membranes was not impaired, indicating additional changes in the structure of the PS I complex must have occurred.     

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.)

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.     

Characterization of proteins by means of their buffer capacity, measured with an ISFET-based coulometric sensor—actuator system

Proteins form the specific selector in many biochemical sensors. A change in one of the properties of such a protein has to be detected by an appropriate transducer, which completes the biochemical sensor. One of these properties is the buffer capacity of a protein. If the binding of a substance to a protein can significantly change the proton binding, which accounts for the buffer capacity of proteins, the detection of this changed buffer capacity enables the construction of a new type of biosensor.

It will be shown that the buffer capacity can be measured with an ISFET-based sensor—actuator device. The alternating generation of protons and hydroxyl ions by alternating current coulometry at a porous noble metal actuator electrode causes an associated small pH perturbation, which is detected by the underlying pH-sensitive ISFET. The amplitude of the measured signal is a function of the buffer capacity of the solute, in which proteins can be present (or these proteins can be adsorbed in the porous actuator electrode of the device). A model describing the transfer function from the electrical input signal of the actuator to the resulting chemical output, which is subsequently detected by the ISFET pH sensor, is presented. Preliminary results of the measured buffer capacity of ribonuclease and lysozyme are presented.