Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.     

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Regulatory volume decrease in small intestinal crypts is inhibited by K+ and Cl- channel blockers.

Total crypt volume has been estimated by analysis of photographic images of intact viable crypts isolated from guinea-pig small intestine. Exposing these crypts to a hypotonic medium, led to transient swelling followed by regulatory volume decrease (RVD) in 12-20 min. RVD was blocked by inhibitors of K+ and Cl- conductance, suggesting that it occurs by activation of K+ and Cl- permeability pathways and loss of these ions.     

Effect of stobadine on indomethacin- and ethanol-induced stomach lesions and gastric secretion.

Stobadine was found to inhibit the ulcerogenic activity of indomethacin in relation to the dose but was ineffective against the direct necrotizing action of ethanol. It also inhibited gastric acid secretion when administered intraduodenally. Although stobadine is considered to be a scavenger of free radicals, our results indicate that, under the given experimental conditions, it is rather the inhibition of gastric acid secretion that is responsible for its antiulcerogenic effect. The preliminary results do not allow the exclusion of other mechanisms for explaining its antiulcerogenic effect.     

Reactor design for the enzymatic isomerization of glucose to fructose

A comprehensive methodology is presented for the design of reactors using immobilized enzymes as catalysts. The design is based on material balances and rate equations for enzyme action and decay and considers the effect of mass transfer limitations on the expression of enzyme activity. The enzymatic isomerization of glucose into fructose with a commercial immobilized glucose isomerase was selected as a case study. Results obtained are consistent with data obtained from existing high-fructose syrup plants. The methodology may be extended to other cases, provided sound expressions for enzyme action and decay are available and a simple flow pattern within the reactor might be assumed.List of Symbols C kat/kg specific activity of the catalyst - D m²/s substrate diffusivity within the catalyst particle - Dr m reactor diameter - d d operating time of each reactor - E kat initial enzyme activity - E _i kat initial enzyme activity in each reactor - F m³/s process flowrate - F _i m³/s reactor feed flowrate at a given time - F ₀ m³/s initial feed flowrate to each reactor - H number of enzyme half-lives used in the reactors - K mole/m³ equilibrium constant - K _S mole/m³ Michaelis constant for substrate - K _P mole/m³ Michaelis constant for product - K _m mole/m³ apparent Michaelis constant f(K, K _s, K_p, s₀) - k mole/s · kat reaction rate constant - k _d d^–1 first-order thermal inactivation rate constant - L m reactor height - L _r m height of catalyst bed - N _R number of reactors - P _i kg catalyst weight in each reactor - p mole/m³ product concentration - R m particle radius - R _P ratio of minimum to maximum process flowrate - r m distance to the center of the spherical particle - s mole/m³ substrate concentration - s _0i mole/m³ substrate concentration at reactor inlet - s ₀ mole/m³ bulk substrate concentration - s mole/m³ apparent substrate concentration - T K temperature - t d time - t _i d operating time for reactor i - t _s d time elapsed between two successive charges of each reactor - V m³ reactor volumen - V _m mole/m³ s maximum apparent reaction rate - V _p mole/m³ s maximum reaction rate for product - V _R m³ actual volume of catalyst bed - V _r m³ calculated volume of catalyst bed - V _S mol/m³ s maximum reaction rate for substrate - v mol/m³ s initial reaction rate - v _i m/s linear velocity - v _m mol/m³ s apparent initial reaction rate f(K_m, s,V_m) - X substrate conversion - X _eq substrate conversion at equilibrium - =s/K dimensionless substrate concentration - ₀=s₀/K bulk dimensionless substrate concentration - _eq=s_eq/K dimensionless substrate concentration at equilibrium - local effectiveness factor - mean integrated effectiveness factor - Thiéle modulus - =r/R dimensionless radius - _s kg/m³ hydrated support density - substrate protection factor - s residence time     

Ecological aspects of Sargassum muticum (Fucales,Phaeophyta) in Baja California,Mexico: reproductive phenology and epiphytes

The reproductive phenology and epiphytic macroalgae of Sargassum muticum were studied through an annual cycle (September 1987 to November 1988) at two sites on the northwestern coast of Baja California, Mexico, which were subjected to different degrees of wave exposure. Sargassum muticum is a brown alga of Japanese origin, now considered a permanent member of the marine flora of Baja California. A similar reproductive development was observed at both sites, with a maximum percentage of reproductive plants from May to July (spring–summer) and minimum from December to March (winter). Reproductive plants were found throughout the year. A total of 48 species of epiphytes were identified and seasonal variation in their diversity was observed. The greatest diversity was found at the more protected site.     

In situ nick translation of human chromosomes using Alu I: unmasking of recognition sites by proteinase K pretreatment

Human chromosomes prepared according to routine methods were treated with the restriction endonuclease Alu I followed by staining with Giemsa solution or fluorescent dyes. This procedure results in a C-band-like appearance of the chromosomes due to removal of DNA from euchromatic chromosomal regions. The resistance of heterochromatic regions against cleavage by the enzyme has mainly been interpreted by the absence or rareness of recognition sites for this particular enzyme in these regions. Proteinase K pretreatment followed by a nick translation procedure with Alu I was combined to check this hypothesis. The results show that heterochromatic chromosomal regions can also be labelled. Thus, they are not characterized by a lack of recognition sites. Gradual deproteinisation of chromosomes changes the labelling pattern from a reverse C-banding pattern to a C-band-like appearance. The resistance of heterochromatic chromosomal parts revealed by the technique is mainly due to local chromatin configuration rather than to the underlying DNA sequence itself.     

The Roberts syndrome

Summary Four siblings with the autosomal recessive Roberts syndrome are reported, and we discuss the phenotypic overlap of this syndrome with other similar radial, aplasia syndromes.