An automated differential thermal and potentiometric titration apparatus for binding studies

A differential pH-termal titration apparatus is described which can detect pH differences with a sensitivity of ±0.0001 pH units and a thermal sensitivity of ±0.00002°C at a time constant of 0.1 s. With a reaction which yields 1 kcal mol⁻¹, the current system can detect concentrations as low as 4×10⁻⁶ M or, in a 2 ml volume, a total amount of 40 nmol. With a time constant of 0.1 s, the sensitivity is 20±4 μ°C. The experimental protocol is specified by a microprocessor and three modes of operation are possible: titration at constant rate of reagent addition, titration at variable rates of addition so that the contents of both cells are at either constant pH or at a constant temperature and variable rate when a rate of change is specified. Experimental data are collected in files, corrected for heat loss, initial baseline drift, and changes in volume. The final corrected from the standardized run of 0.01338 M HCl in 0.2 M KCl at 25°C calibrate the pH scale yielded the calorimetric conversion constants and pK_w which are calculated and stored for subsequent corrections for the titration of an unknown acid or the measurement of bindin constants and heats.     

Measurements of intra-and extracellular pH in the liverwort Conocephalum conicum during action potentials

During action potentials triggered by electricity and light, measurements of intra- and extracellular pH in the liverwort Conocephalum conicum L. were carried out by the use of antimony-filled H⁺-sensitive microelectrodes. Intracellular pH increased transiently by about 0.05 unit in the course of an action potential, while extracellular pH, measured at the surface of the thallus, remained unchanged. Switching the light off caused a transient increase in intracellular pH by less than 0.1 unit. Turning the light on produced a fast pH decrease by about 0.15 unit followed by a slow increase. When the light was intensive enough, the action potential thus triggered caused a slight increase in intracellular pH superimposed on the phase of a slow pH increment.
The magnitude and time course of the intracellular pH changes seem insufficient for a role as messenger between action potential and the up to 100% increase in the rate of respiration that has been registered in Conocephalum conicum as a consequence of excitation.     

The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light

The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent K_m = 4 μM and saturation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is approx. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.     

The glands of Tamarix aphylla: a system for salt recretion or for carbon concentration?

During periods of high atmospheric humidity, twigs of Tamarix aphylla (L.) Karst. become covered by an alkaline solution. The pH of that solution fluctuates between 8.0 – 8.5 in the dark and 10.5 during the light hours. Such a solution, produced by the glands, constitutes an efficient trap for atmospheric CO₂. Upon the periodic drop in pH, much of the preabsorbed carbon may gradually be released from the solution. This enriches the immediate surroundings of the twigs with CO₂ for prolonged periods of time. The expected concentrations of CO₂, at the boundary layer between the atmosphere and the surfaces of the twigs, are over 1 000 ppm. As net photosynthesis of T. aphylla reaches maximal rates only at CO₂ concentrations of above 500 ppm, the plants may benefit from this extra source of carbon and may exploit it for maximal assimilation during the early morning hours. Thus, the "salt glands'of Tamarix , which are liable for the production of the alkaline recretum, may serve a triple purpose: (a) removal of excess salts out of the twigs, (b) provision of a cover of hygroscopic solutes that moistens the twigs and shortens the duration of transpiration, and (c) providing the plants with an environment enriched in CO₂.     

紫晶香蘑栽培生物学研究

紫晶香蘑栽培生物学研究卢成英，李鹄鸣，钟以举（湖南省吉首大学生态研究所４１６０００）ＢｉｏｌｏｇｉｃａｌＲｅｓｅａｒｃｈｏｎＬｅｐｉｓｔａｓｏｒｄｉｄａＣｕｌｔｉｖａｔｉｏｎ．￥Ｌｕｃｈｅｎｇｙｉｎｇ；ＬｉＨｕｍｉｎｇ；ＺｈｏｎｇＹｉｊｕ（Ｉｎ－ｓｔ...     

Dissecting the signaling pathways involved in the function of sperm flagellum

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.     

Effects of larval age and prolonged simulated acid rain on the susceptibility of European pine sawfly to virus infection

We studied the effect of simulated acid rain treatment of host trees on the susceptibility of the European pine sawfly larvae to virus, and possible differences when larvae of two different ages were infected. Older larvae were less susceptible to virus. Most larvae treated with virus 2 days after they started feeding on experimental foliage (group A) died rapidly within 10 days after the virus treatment, and survival to the end of the larval period was only 8–25%. Larvae treated with virus 1 week later (group B) were less affected by the virus and 36–49% survived. In group A the larval survival in the pH 3 treatment was higher than in other treatments; at the end of the larval period the difference was twofold. In group B there were no clear effects of acid rain on the susceptibility of larvae to virus. The study yielded the following new information: (1) the effect of prolonged acid rain treatment on reducing the efficacy of virus on young larvae was more distinct than in a previous study with shorter exposure to acid rain, and the difference was maintained to the end of larval period; (2) the susceptibility of older larvae to virus was not affected by acid rain treatments; (3) pH inside the needles did not explain the larval mortality caused by virus.     

Growth,radicle and root hair development ofDeschampsia flexuosa (L.) Trin. seedlings in relation to soil acidity

Deschampsia flexuosa (L.) Trin. is an abundant grass species in the ground flora of acidic beech forests in southern Sweden. Generally, the species is restricted to a limited soil pH range (pH 4–5). The main objective was to study the influence of different soil acidities on germination, initial root development and on the growth of the species. The experiments were carried out under controlled conditions and designed to simulate the physico-chemical conditions present in the field. By using forest soils within the pH range 4.0 to 8.3 and artificial variation in pH (3.2 to 7.6) of soil-water extracts, it was possible to evaluate the influence of soil reaction and the H⁺ per se. In all experiments seeds have been used. Germination was significantly delayed in the very acid soil (pH 4.0) in comparison to the germination in soils within the pH range (4.4 to 6.4). Soil substances, other than the H⁺, might be responsible for this delay in germination, whereas development of the radicle was markedly affected by increasing H⁺ concentrations. Especially the development of root hairs was sensitive to H⁺ and was significantly reduced at a pH<-3.8. By increasing soil acidity the injury symptoms, including curling and discolouring, became more intense and at the highest acidity (pH 3.2) the radicles appeared brown, stunted and the root hairs were lacking. Most favourable growth was obtained at pH 4.4 and 5.0. Soil pH levels above and below this range limited both shoot and root growth. The results showed very good correspondence with observations made in Beech forest soils in southern Sweden, where the species was growing in soils within the pH range 3.9 to 5.1 with a peak growth at pH 4.3. This study shows that in soils at pH≤3.8, the poor development of the radicle may be crucial in the establishment ofDeschampsia flexuosa. Root hair development was more sensitive to soil acidity than radicle elongation. Germination was delayed in very acid Beech forest soils but other factors than the H-ion per se may be responsible for this delay.     

Influence of osmolarity and pH increase to achieve a reduction of monoclonal antibodies aggregates in a production process

Anti PSA monoclonal antibodies for diagnostic use were produced in an in vitro system. After purification using Protein G affinity chromatography a percentage of about 10% of antibody aggregates remained. The use of monoclonal antibodies containing aggregates as a capture antibody in a diagnostic kit reduces the performance of the test making it often unacceptable. The aggregates could be eliminated using gel filtration chromatography but, in that way, the final recovery of the whole production process was only about 50%. Aggregation is favoured when the working pH is near to the isoelectric point of the antibody. We varied the culture medium composition, modifying pH and osmolarity. We tested different values of pH and osmolarity: 7.1, 7.5, 8.0, 8.5 for pH, and 300, 340, 367, 395 mOsm/kg H2O for osmolarity. By modification of the cell culture medium we obtained a significant decrease of monoclonal antibody aggregates in the production cycle. In this way we achieved higher recovery rate and could avoid gel filtration polishing step. The experiments were performed in two stages: first in culture flasks changing one parameter in each experiment, and then in spinner bottle using the best conditions obtained in the first stage. During scale up we used the modifications achieved from the experiment showed in this paper in our production by hollow fibre bioreactor with positive results.     

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH

Domain 4 of the anthrax protective antigen (PA) plays a key role in cellular receptor recognition as well as in pH-dependent pore formation. We present here the 1.95 Å crystal structure of domain 4, which adopts a fold that is identical to that observed in the full-length protein. We have also investigated the structural properties of the isolated domain 4 as a function of pH, as well as the pH-dependence on binding to the von Willebrand factor A domain of capillary morphogenesis protein 2 (CMG2). Our results provide evidence that the isolated domain 4 maintains structure and interactions with CMG2 at pH 5, a pH that is known to cause release of the receptor on conversion of the heptameric prepore (PA₆₃)₇ to a membrane-spanning pore. Our results suggest that receptor release is not driven solely by a pH-induced unfolding of domain 4.