Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Two different dihydroorotate dehydrogenases in Lactococcus lactis.

The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms. The general pathway consists of six enzymatic steps. In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated. The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined. One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis. It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors. We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes. Only the double mutant is pyrimidine auxotrophic.     

Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior.

LamB, a sugar-specific channel of Escherichia coli outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated using fast Fourier transformation. The current noise through the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz. The spectral density of the noise of the open LamB channels was a quadratic function of the applied voltage. Its magnitude was not correlated to the number of channels in the lipid bilayer membrane. Upon addition of sugars to the aqueous phase the current decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the sugars with the binding site inside the channel. The frequency dependence of the spectral density was of Lorentzian type, although the power of its frequency dependence was not identical to -2. Analysis of the power density spectra using a previously proposed simple model (Benz, R., A. Schmid, and G. H. Vos-Scheperkeuter. 1987. J. Membr. Biol. 100: 12-29), allowed the evaluation of the on- and the off-rate constants for the maltopentaose binding to the binding site inside the LamB channels. This means also that the maltopentaose flux through the LamB channel could be estimated by assuming a simple one-site, two-barrier model for the sugar transport from the results of the noise analysis.     

Termination of endothelin signaling: Role of nitric oxide

Cellular mechanisms responsible for the termination of ET-1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET- 1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ET_A receptor cDNA (CHO-ET) were studied. CHO-ET responded to ET-1 with robust [Ca²⁺], transients and developed a long-lasting homologous desensitization. Donors of nitric oxide (NO), 3-morpholino-sydnonimine HCl(SIN-1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca²⁺], recovery, and counteracted the development of homologous desensitization by a cyclic GMP-independent mechanism, suggesting an alternative mode for NO modulation of ET-1 responses. Stimulation of CHO-ET cells with mastoparan, a wasp venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO-induced modulation of [Ca²⁺] mobilization. Using a lys⁹-biotinylated ET-1 (ET-1 [BtK⁹]), binding sites were “mapped” in CHO-ET cells. Receptor-ligand complexes did not exhibit spontaneous dissociation during 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN-1 caused a rapid, concentration-dependent, and reversible dissociation of biotinylated ET- 1 from ET_A receptor (EC₅₀ = 75 μM and 6 μM, respectively), an effect that was not mimicked by 8-bromo-cyclic GMP. “Sandwich” co-culture of endothelial cells with CHO-ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET-1 [BtK⁹] from ET_A receptors. We hypothesize that NO plays a role in physiological termination of ET-1 signalling by dual mechanisms: (1) displacement of bound ET-1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca²⁺] mobilization, hence inhibiting end-responses to ET-1. © 1994 Wiley-Liss, Inc.     

Diurnal variations of amino acids and organic acids in xylem fluid from Lagerstroemia indica: an endogenous circadian rhythm

Diurnal variations in the concentrations of major organic compounds occurred in xylem fluid extracted from Lagerstroemia indica L. The concentration of amino acids and the N/C ratio was at a maximum and that of organic acids was at a minimum between 1230 and 2030 h. Since the concentrations of total organic nitrogen, total amino acids and most individual amino acids (but not organic acids or sugars) were also proportional to xylem tension two experiments were performed to discern whether variations in chemistry were a consequence of diurnal changes in moisture stress. In the first experiment, L. indica , exposed to variable levels of moisture stress during midday, manifested an increase in organic acids and a reduction in the N/C ratio. In the second experiment, chemical profiles of xylem fluid were collected and compared for plants exposed to a natural photoperiod, constant darkness or continuous light at noon and midnight. After 1 day amino acids increased in concentration during midday for all treatments; the variation was greatest (10-fold) for plants in constant darkness where xylem tension varied from 0.20 to 0.25 MPa. Only plants exposed to continuous light lost a diurnal rhythm after 3 days. Thus, the circadian rhythm was endogenous, terminated in continuous light and was not mediated by changes in moisture stress. Glutamine accounted for most of the diurnal variation in total amino acids, organic nitrogen and the N/C ratio in xylem fluid.     

Cumulus cells secrete a meiosi-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed. The GVBD of unstimulated DO also increased significantly when cocultured with cumulus cells from primed CEO. The percentage of GVBD in unprimed DO and in DO isolated from primed CEO was the same. We suggest that within 1–2 hr, forskolin and cAMP stimulate cumulus cells to produce a diffusible meiosis-inducing substance which overcomes HX-inhibition and induces oocyte maturation, including both GVBD and PB formation. The CEO must be primed for more than 2 hr before the resumption of meiosis in DO isolated from such CEO is induced. Oocyte-cumulus connections are crucial as far as initiating the production of a meiosis-inducing substance is concerned. Oocyte-cumulus connections are not needed for transferring this substance to the oocyte. © 1994 Wiley-Liss, Inc.     

Neutrophil priming mechanisms of sulfolipid-I and n-formyl-methionyl-leucyl-phenylalanine

The principal sulfatide of virulentMycobacterium tuberculosis, sulfolipid-I (SL-I), both directly stimulates neutrophil superoxide (O ₂ ^– ) release and, at substimulatory concentrations, primes these cells for markedly enhanced oxidative responsiveness to other stimuli. The present study was undertaken to clarify the priming mechanisms by comparing cellular events following priming doses of SL-I with those following priming with N-formyl-methionyl-leucyl-phenylalanine (FMLP). We compared the involvement of the calcium cation (Ca²⁺), as well as membrane protein kinase C (PKC) activity and the translocation of NADPH oxidase-cytosolic cofactor effected by priming levels of the two agonists. The investigation led to two important conclusions. First, we clearly demonstrate that priming by both SL-I and FMLP results from activation of cellular processes that are not involved in direct oxidative activation. For example, whereas direct induction of O ₂ ^– generation by FMLP and SL-I required increases in intracellular Ca²⁺, an increase in intracellular calcium concentration ([Ca²⁺]_i) above basal levels was not required for priming. Second, we identified key differences in the cellular responses to priming doses of SL-I and FMLP. Whereas increased membrane PKC activity caused by priming doses of FMLP was only partially blocked by chelation of intracellular Ca²⁺, Ca²⁺ chelation completely inhibited the increase in membrane PKC activity caused by SL-I. NADPH oxidase-cytosolic factor translocation to plasma membranes was completely blocked by pertussis toxin when priming doses of SL-I were used. This guanine-nucleotide-binding protein inhibitor had no effect on FMLP-dependent translocation of the oxidase cofactors. The comparative approach introduced in this report provides a valuable and novel method to discern the complex interactions of various cellular processes that regulate the state of activation of stimulated cells.     

Diversity in nucleotide acquisition by antigenically similar Chlamydia psittaci of avian origin

Abstract Two different nucleic acid precursor utilization patterns were obtained for five avian isolates of Chlamydia psittaci . Three of the isolates bahaved in a manner similar to that previously described, showing total dependency on the host cell for ribonucleoside triphosphates and being unable to utilize medium-supplied thymidine. In contrast, the other two isolates were incapable of taking pyrimidine ribonucleotides from the host cell and they could efficiently utilize medium-supplied thymidine. These unusual isolates were resistant to 5-fluorouridine while the other three isolates were sensitive. Of the five isolates only 6BC was sensitive to sulfonamides. The five isolates were divided into two groups by comparing the Alu I restriction endonuclease patterns obtained following digestion of the major outer membrane protein (OMP1) gene, amplified by the polymerase chain reaction. The OMP1 genotyping results were confirmed by serotyping.     

Carbohydrate distribution and cellular injuries in acid rain and cold-treated spruce needles

Summary The cellular structures of acid rain-irrigated needles of several provenances of Norway spruce (Picea abies L. Karst) seedlings were studied after winter experimental freezing. Frost injuries and recovery were characterized by visual damage scoring and classification of mesophyll cell alterations, also using histochemical methods for carbohydrate fluorescent staining. The treatment with-30° C during the late dormancy period was sufficient to cause significant injuries and intracellular degradation in the tissues of the green needles. The most affected seedlings in terms of visual injury scoring were found among those treated with clean water or at pH 3, while freezing injury, defined as an occlusion of phenolic substances in the central vacuole of the mesophyll cells, was most abundant in the needles from spruces irrigated either with clean water or at pH 4 or pH 3. Electron microscopy revealed the details of the injury, e. g. thinning out of the cytoplasm and chloroplast stroma, darkening of the chloroplasts and eventually swelling of the chloroplasts and protoplast. PAS and ConA reactions in the needle tissue revealed intense starch accumulation in the mesophyll and transfusion tissues as early as in March, with a tendency to increase, especially in the untreated needles during the recovery period. Plasma membrane disturbances were indicated by histochemical identification of callose deposits in the mesophyll cell walls, these being most abundant in the acid rain-treated needles. All these findings suggest that freezing at –30° C was more deleterious to the seedlings pretreated with acid or clean water than to those not given additional irrigation.