Expression of human IL-1 beta in Salmonella typhimurium. A model system for the delivery of recombinant therapeutic proteins in vivo.

The feasibility of using Salmonella typhimurium aroA mutant (SL3261) to deliver protein therapeutic agents was investigated in a murine model system. We have constructed an Escherichia coli expression plasmid designed to express the human protein IL-1 beta. This plasmid expresses IL-1 beta to high levels (greater than 30% total cell protein) in E. coli. In Salmonella the IL-1 beta is expressed constitutively to about 10% total cell protein, as verified by Western blotting analysis using polyclonal rabbit anti-IL-1 beta antibody. The protein is produced in a soluble and biologically active form. BALB/c mice administered orally or i.v. with S. typhimurium aroA mutants carrying the plasmid produced highly significant antibody responses against human IL-1 beta as determined by a solid-phase RIA. Furthermore, mice injected with the construct were significantly protected against lethal gamma-irradiation (850 rad). This study therefore demonstrates that the vaccine strain of Salmonella mutants can also be used effectively to deliver therapeutic proteins in vivo.     

Subgroups of Tcr α chains and correlation with T-cell function

T-cell receptor (Tcr) chains are classified into four subgroups (I, II, III, and miscellaneous) based on the amino acid residues at positions 61 and 62. Subgroup I has Gly Phe at these positions, subgroup II has Arg Phe, subgroup III has Arg Leu, and subgroup miscellaneous has several other combinations. Variability plots for subgroups I, II, and III sequences show higher values around positions 93–103, 105, 108, 111, 113, and 115, suggesting that these positions may interact with the processed antigen molecules. Smaller peaks are present at various other regions which may bind the major histocompatibility complex class I or II molecules. The patterns of variability within one subgroup are similar for all species, for human alone, and for mouse alone. These subgroup patterns appear much less complicated than patterns for sequences in all subgroups taken together, implying that subgroups may be related to Tcr functions. Among 83 mouse chains, 15 are from cytotoxic cells and 40 from helper cells. Of the 15 from cytotoxic cells, 11, 2, 0, and 2 are in subgroups I, II, III, and miscellaneous; and of the 40 from helper cells, 9, 16, 12, ans 3 are in subgroups I, II, III, and miscellaneous, respectively. Thus, a correlation between sequence and function of Tcr chains seems possible. Address correspondence and offprint requests to: M. Schiffer.     

Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and [35S]methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.     

Cytochalasin B interferes with conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding.

To gain insight into the mechanism of facilitated sugar transport and possible mechanisms by which glucose transporter intrinsic activity might be altered, we have investigated conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding and by the transporter inhibitor, cytochalasin B. Changes in the ability of thermolysin to digest glucose transporters present in erythrocyte ghosts were used to monitor conformational changes of the glucose transporter. The degree of protease digestion was determined by the amount of undigested glucose transporter remaining after the protease treatment, as assessed in Western blots using the glucose transporter specific monoclonal antibody 7F7.5. D-Glucose, the physiological substrate of the transporter, increased the transporter's susceptibility to cleavage by thermolysin. Nontransportable glucose analogues which bind specifically to either an internal or external glucose transporter sugar binding site also altered susceptibility of the transporter to thermolysin. Both methyl and propyl glucoside, which preferentially bind the internal sugar site, increased thermolysin susceptibility of the glucose transporter in a manner similar to that of D-glucose. In contrast, 4,6-O-ethylideneglucose, which preferentially binds the external sugar site, protected the transporter from thermolysin digestion. These results suggest that sugar binding to internal and external sugar sites induces distinct conformational changes and that the observed D-glucose effect on the susceptibility of the glucose transporter to thermolysin is due to D-glucose at equilibrium predominantly forming a complex with the internal sugar site. The protection from cleavage by thermolysin caused by external sugar binding is attenuated by the addition of an internally binding sugar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.)

We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4-dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and embryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 M dicamba, addition of 2.25 to 9 M BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 M dicamba and 2.25 M BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse.Abbreviations MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - dicamba 3, 6-dichloro-o-anisic acid     

稻瘟病菌株的DNA指纹分析及小种鉴别

基于对稻瘟病菌(Pyricularia oryzae)基因文库的分析,我们找到了一套含重复顺序的克隆。其中POR6和POR7被证实具有高度的多态性并随机散布于稻瘟病菌生理小种的致病性时,可以获得可分辨的基因组特异的杂交带型。我们还分析了致病性与8个稻瘟病菌株DNA指纹图谱之间的关系,结果表明各个小种组合间的百分相似率S_xy,值与该小种组合间共同侵染的鉴别品种数目有正相关性。     

The conventional short-circuiting technique under-short-circuits most epithelia

Summary The relationships among ion current, membrane potential difference, and resistance of an epithelium are studied. The short-circuit technique introduced by Ussing and Zerahn does not completely short circuit the epithelium if the series resistance parallel to the cell layer between the voltage electrodes is not properly compensated. The residual potential difference across the epithelial cell layer in the short-circuit state is proportional to both the measured short-circuit current and the resistance of the diffusion barriers not compensated. In the conventionally short-circuited small intestinal mucosa the villus and crypt areas are hypo-polarized to different degrees rather than simultaneously hyper- and hypo-polarized. Short-circuiting the whole tissue reduces but does not abolish the passive net ion movement across the tissue. Measurements of the electrical properties of the whole and denuded rat distal small intestine in HCO₃-Ringer solution containing 10mm glucose reveal that the measured short-circuit current has under-estimated approximately 33% of the true short-circuit current and that the passive net Na flux from serosa to mucosa and Cl flux from mucosa to serosa are not negligible in the short-circuit state.     

Release of arachidonate from diglyceride in human platelets requires the sequential action of a diglyceride lipase and a monoglyceride lipase

Release of arachidonate from 2-arachidonyl diglyceride by human platelet microsomes was investigated. Diglycerides labeled with ¹⁴C-stearate at sn-1 and with ³H-arachidonate at sn-2 were used as a substrate for microsomal diglyceride lipase. Diglyceride was deacylated first at sn-1 as evidenced by the accumulation of 2-arachidonyl monoglyceride but not of 1-stearoyl monoglyceride. Subsequent release of arachidonate from monoglyceride required the action of a monoglyceride lipase. Studies on substrate specificity indicated that diglyceride lipase utilized 2-arachidonyl diglyceride as the best substrate.     

Equine follicle-stimulating hormone. Purification, acid dissociation, and binding to equine testicular tissue

A simple method of purification of equine follicle-stimulating hormone is described by which two forms of the hormone are obtained. The acid dissociation of the most active preparation was studied and a pKa of 5.8 was determined at 37 degrees C. This value is 2 pH units higher than that observed for pregnant mare serum gonadotropin suggesting that the binding areas between subunits are not identical in the two hormones. We also describe an homologous radioreceptor assay of equine follicle-stimulating hormone which is highly specific for this hormone in contrast to the heterologous systems described so far. The analysis of the properties of equine gonadotropins in homologous and heterologous radioreceptor assays suggests that all glycoprotein hormones share a common high affinity binding site and that specificity of binding is due to binding prohibition sites preventing fixation of each hormone to the receptors of the other glycoprotein hormones. This specific hindering is defined as "negative specificity."     

Triphasic concentration effects of gentamicin on activity and misreading in protein synthesis.

Gentamicin is shown to exert a triphasic concentration effect on peptide synthesis in vitro with natural messengers. Low concentrations (up to 2 micron) caused slowing and a decrease in total synthesis, but little misreading (assayed with extracts lacking Glu-tRNA); the inhibition was greater with an initiating system (with phage RNA as messenger) than with pure chain elongation on purified endogenous polysomes of Escherichia coli. Moderate concentrations (up to 100 micron) slowed synthesis less, markedly increased its duration in the noninitiating system, and strongly stimulated misreading; at optimal concentrations total synthesis was even greater than normal. Moreover, with phage RNA these concentrations increased the synthesis of large polypeptides. We conclude that binding of gentamicin to its first site causes inhibition but little misreading; binding to additional site(s) partly reverses the inhibition by first-site binding and markedly stimulates misreading, and the misreading appears to favor "readthrough" of termination codons. In the third phase (greater than 100 micron) synthesis is slowed again but the pattern of misreading does not appear to be altered; this effect need not involve a specific further action on the ribosome.