Continuous glucose production using encapsulated mycelial-associated β-glucosidase from Trichoderma E58

Summary Cellobiose and salicin were continuously hydrolysed in a packed bed reactor containing Trichoderma sp. E-58, encapsulated in calcium alginate beads. Continuous -glucosidase activities were measured using inlet substrate concentrations of 10 mM and 35 mM for cellobiose and salicin respectively. Maximal activities achieved using the described immobilization procedure were between 70 and 90 moles substrate reacted per minute per liter of bead volume. Immobilized mycelial-associated -glucosidase activity was shown to have a half-life of greater than 1000 hours when operating continuously at 50°C.     

Lateral mobility of lipid analogues and GPI-anchored proteins in supported bilayers determined by fluorescent bead tracking

Lipid analogues and glycosylphosphati-dylinositol (GPI)-anchored proteins incorporated in glass-supported phospholipid bilayers (SBL) were coupled to small (30 nm diameter) fluorescent beads whose motion in the liquid phase was tracked by intensified fluorescence video microscopy. Streptavidin (St), covalently attached to the carboxyl modified surface of the polystyrene bead, bound either the biotinylated membrane component, or a biotinylated monoclonal antibody (mAb) directed against a specific membrane constituent. The positions of the beads tethered to randomly diffusing membrane molecules were recorded at 0.2 sec intervals for about l min. The mean square displacement () of the beads was found to be a linear function of diffusion time t, and the diffusion coefficient, D, was derived from the relation, (t) = 4Dt. The values of D for biotinylated phosphatidylethanolamine (Bi-PE) dispersed in an egg lecithin: cholesterol (80:20%) bilayer obtained by this methodology range from 0.05 to 0.6 m²/sec with an average of D = 0.26 m²/sec, similar to the value of D = 0.24 m²/sec for fluorescein-conjugated phosphati-dylethanolamine (Fl-PE) linked to St-coupled beads by the anti-fluorescein mAb 4-4-20 or its Fab fragment. These values of D are comparable to those reported for Fl-PE linked to 30 nm gold particles but are several times lower than that of Fl-PE in the same planar bilayer as measured by fluorescence photobleaching recovery, D = 1.3 m²sec. The mobilities of two GPI-anchored proteins in similar SBL were also determined by use of the appropriate biotinylated mAb and were found to be D = 0.25 and 0.56 m²/sec for the decay accelerating factor (DAF, CD55) and the human FcRIIIB (CD16) receptors, respectively. The methodology described here is suitable for tracking any accessible membrane component.This work was supported by National Institutes of Health grants 1R24 RR05272 and AI-24322.     

Osmotic measurements on stomatal cells ofCommelina communis L.

Summary Observations of aperture changes as sucrose is added to the solution bathing epidermal strips ofCommelina communis L. allow calculation of the osmotic changes required to open or close the stomatal pore, for comparison with changes in potassium content. With isolated guard cells, in strips in which all cells other than guard cells have been killed, the internal osmotic changes required are 83 mosmol kg^–1 m^–1 below 10m aperture, 129 mosmol kg^–1 m^–1 in the range 10–15 m, and 180 mosmol kg^–1 m^–1 above 15 m. For opening against subsidiary cell turgor in addition to guard cell turgor, in intact strips with live subsidiary and epidermal cells, these figures should each be increased by about 33 mosmol kg^–1 m^–1. A change in subsidiary cell turgor is magnified in its effects on the water relations of the guard cell by a factor greater than 3.7 for equal changes in the water potential of the two cells, or greater than 4.7 at constant volume of the guard cell.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Influence of nitrogen on the behaviour of nickel in wheat

A glasshouse experiment was conducted to study the effect of Ni on the growth and nutrients concentration in wheat (Triticum aestivum Cv. WH 291) in the presence and absence of applied N as urea. Responses to N application were observed up to 120 g N g^–1 soil. No response to Ni was observed in the dry matter yield of wheat tops (leaves + stem) in the absence of applied N while in the presence of applied N, significant yield increases were obtained at 12.5g Ni g^–1 soil. Nickel was not toxic to wheat up to 50g Ni g^–1 soil in the presence of 120g N g^–1 soil. Nitrogen and Ni concentration in wheat tops and roots increased with increasing levels of applied N and Ni, respectively. Applied Ni had an antagonistic effect on N concentration. Similarly, N reduced the Ni concentration in the wheat tissues. Positive growth responses to Ni were associated with 22 and 15g Ni g^–1 in wheat tops, in the presence of applied N at 60 and 120g N g^–1 soil, while Ni toxicity was associated with 63, 92.5 and 112.5g Ni g^–1 in wheat tops, in the absence and presence of applied N at 60 and 120g N g^–1 soil, respectively.     

Effect of culture medium ions on chromate reduction by resting cells of Agrobacterium radiobacter

The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe²⁺ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg²⁺, 20 m Ca²⁺ and 3.6 m Mn²⁺. Chromate reduction was not affected by the addition of MgCl₂, CdCl₂, ZnCl₂, MnCl₂, Na₂SO₄ (1000 m), and Na₂MoO₄ (100 m) to the activity assays. However, activity was inhibited by the presence of Na₂SO₄ (10 mm), Na₂MoO₄ (200 m) and ferric citrate.     

The Sertoli cell of the water buffalo (Bubalus bubalis) during the spermatogenic cycle

Summary Ultrastructural features and morphometric evaluations of buffalo Sertoli cells are reported for the six phases of the spermatogenic cycle. The phases of the tubular seminiferous epithelium are identified according to characteristic cellular associations with completed spermiation as demarcation between two cycles. Average tubular diameter (245 m) and epithelial height (61 m) do not vary significantly during the cycle. The relative Sertoli cell volume in the seminiferous epithelium varies between 30% (phase 4) and 39% (phase 8). The calculated volume of a single Sertoli cell increases from a nadir of 7118 m³ in phase 3 abruptly to a maximum of 8968 m³ in phase 4 and is then gradually reduced during the following phases. The Sertoli cell surface area shows a similar trend: it amounts to 11105 m² in phase 3 and to 14260 m² in phase 4. The contact area of the Sertoli cell with adjacent cells and structures is subject to characteristic changes; from the expansion of basal Sertoli-Sertoli contacts it is concluded that the blood-testis barrier in the buffalo is particularly tight during phases 8, 1 and 2. The irregularly contoured nucleus contains a vesicular nucleolus, has a calculated volume from 465 m³ to 543 m³ and occupies 5 to 7% of the cell. Volume percentages of mitochondria (4%), Golgi apparatus and lysosomal bodies are rather constant during the cycle. Whorls and orderly arranged aggregates of the smooth endoplasmic reticulum occur in basal location as well as in close association with elongating spermatids. Smooth ER is the organelle that exhibits the most prominent changes during the Sertoli cell cycle: it occupies 5.79% in phase 3 and 20.9% in phase 4 of the total cellular volume. Phagocytosis of residual bodies is insignificant in this species and a lipid cycle is absent in buffalo Sertoli cells.     

Effect of 2-hydroxybenzoate on the rate of naphthalene mineralization in soil

The effect of 2-hydroxybenzoate (2-OHB, salicylate) on the mineralization rate of [¹⁴C]naphthalene, the population density of naphthalene-degrading bacteria, and the concentration of genes encoding for naphthalene dioxygenase in a soil bacterial community was investigated. Six different concentrations of 2-OHB (10, 20, 50, 100, 150 and 200 g g^–1 soil) were tested in 100-g portions of soil. The addition of 10, 20 or 50 g 2-OHB g^–1 soil produced a general increase in total soil bacterial population density, whereas the addition of 100 g or 200 g 2-OHB g^–1 soil specifically increased the proportion of naphthalene degraders relative to the total population. The addition of 50 g 2-OHB g^–1 soil produced a fourfold increase (the maximum observed) in the rate of naphthalene mineralization relative to the rate in unamended soil. The concentration of 2-OHB ( 100 g/g) added to soil correlated with the population density of naphthalene degraders (r=0.961). Addition of up to 200 g 2-OHB g^–1 correlated with the abundance of DNA sequences homologous to known naphthalene dioxygenase genes (nahAB) (r=0.958). However, mineralization of [¹⁴C]naphthalene was stimulated significantly only by the addition of 50 g 2-OHB g^–1 soil. Results of the mineralization experiments were supported by the detection of nahAB mRNA extracted directly from soil. The specificity of the effect of 2-OHB on naphthalene biodegradation was confirmed in a control experiment using equivalent concentrations of 4-OHB which repressed naphthalene mineralization by about 50%. Addition of ammonium nitrate to the soil also increased the rate of naphthalene mineralization. Ammonium nitrate added together with 2-OHB reduced the mineralization enhancement effect of either compound alone. The study confirmed that specific induction of biodegradative genes can enhance chemical pollutant removal in situ. Correspondence to: O. A. Ogunseitan     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Influence of sarmesin on some dopamine-related types of behaviour

Summary The present study was undertaken to investigate the effects of sarmesin, an analogue of [Sar¹] angiotensin II (ANG II) where the tyrosine hydroxyl group in position 4 is methylated, on dopamine (DA)-related paradigms: locomotor and exploratory behaviour as well as apomorphine (3 mg/kg, ip)-induced stereotypy in rats. Sarmesin (0.5 and 1 g, icv) significantly decreased ambulation and rearing movements, and blocked the inhibitory effect of ANG II (0.1 g) on both types of activity. Sarmesin induced biphasic effects on apomorphine-induced stereotypy depending on the dose increase (0.5 and 5 g, icv) and decrease (10 g). Moreover, sarmesin (5 g) blocked the inhibitory effect of ANG II (2 g, icv) on apomorphine stereotypy. Taken together, these results suggest that sarmesin might interact with AT₁ and AT₂ receptor subtypes. The results further confirm the statement for ANG II-DA interaction in brain structures involved in these types of behaviour.     

5′-AMP hydrolysis by suspensions and homogenates of pancreatic islet cells from normal and cortisone-treated rats

Summary Suspensions of endocrine pancreas cells were prepared by shaking collagenase-isolated rat islets of Langerhans in calcium-free buffer. When incubated with 1.0 mM substrate at pH 7.4, the cells split,P _i from 5-AMP at a rate of 87 nmol/h per g DNA, and from-glycerophosphate at a rate of 25 nmol/h per g DNAK _m for 5 AMP was about 54 M. Adenosine or theophylline inhibited the 5-AMP hydrolysis. Homogenization of the cells increased the activity toward 5-AMP by 23% and that toward-glycerophosphate by 115%. Injecting rats with cortisone had no effect on the 5-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular activity toward 5-AMP was more than doubled by the cortisone treatment. Staining whole islet cells for 5-AMP-splitting activity resulted in a demarcation of the cell periphery in control rats. Cells from cortisone-treated rats showed heavier deposits of reaction product, and their cell periphery did not stand out as clearly. It is suggested that 5-nucleotidase is largely an ectoenzyme in normal rat islet cells. The cells also contain an as yet unidentified intracellular phosphatase that seems to be solely responsible for the increased hydrolysis of 5-AMP in cortisone-treated rats.     

Ultrastructural immunocytochemical localization of l-glutamate decarboxylase and GABA in rat pancreatic zymogen granules

Summary The ultrastructural immunohistochemical localization of gamma aminobutyric acid (GABA) and its regulating enzymes, l-glutamate decarboxylase (GAD) and gamma aminobutyrate--ketoglutarate transaminase, was determined utilizing an immunogold post-embedding protocol in pancreatic exocrine tissue. Within the acinar cell, GABA and its biosynthetic enzyme, GAD, were localized in zymogen granules. Quantitative analysis of the GABA immunoreactivity in the acinar cell revealed 1.7±0.5 gold particles/m² over the cytoplasm, 36.6±14.1 gold particles/ m² over the zymogen granules, and 2.9±2.1 gold particles /m² over the mitochondria. Quantitative analysis of the distribution of colloidal gold particles, representing glutamate decarboxylase immunoreactivity in the acinar cells, revealed 38.4±2.5 gold particles/m² over the zymogen granules, 4.7±1.1 gold particles/m² over the mitochondria and 6.3±0.5 gold particles/m² over the remainder of the cytoplasm. Substitution of normal sheep serum for the sheep anti-glutamate decarboxylase serum revealed a significant (p< 0.001) decrease of the colloidal gold particle distribution over the zymogen granules and cytoplasmic compartments of the acini. Gamma aminobutyrate --ketoglutarate transaminase, the catabolic enzyme for GABA, was not detected in the mitochondria, zymogen granules, and cytoplasm of the acinar cell, suggesting that GABA is not catabolized within the acinar cell. Preabsorption and substitution controls resulted in an absence of labeling. These results suggest that GABA may act extracellularly and/or have a role within the zymogen granule in the exocrine pancreas.     

Three-dimensional organization of smooth muscle cells in blood vessels of laboratory rodents

Summary Three-dimensional aspects of smooth muscle cells of the microvas-culature were studied ultrastructurally in laboratory rodents by means of serial thin sections and reconstruction of muscle cell models. It was demonstrated that a muscle cell of an arteriole (luminal diameter (LD) 17 m) in hamster striated muscle was spindle-shaped, 70 m long, and wound twice round the vessel axis. The volume of the cell was calculated as 750 m³ and its surface area as 1330 m². A muscle cell in an arteriole (LD 6 m) in the rat retina was irregular in shape, about 22 m long, and had branched processes. The cell volume was calculated as 139 m³ and its surface area as 298 m².     

Male reproductive effect of arsenic in mice

Arsenic, a known human carcinogen, was given to mice via drinking water as sodium arsenite at a dose 53.39, 133.47, 266.95 and 533.90 mol l for 35 days. A decrease in the activity of 17 HSD along with increase in LDH, GT activity were observed at 533.90 mol l. The observed sperm count, motility and morphological abnormalities in sperm were similar to control at lower dose levels. However at 533.90 mol l a significant decrease in sperm count and motility along with increase in abnormal sperm were noticed. Significant accumulation of arsenic in testes and accessory sex organs may be attributed to the arsenic binding to the tissues or greater cellular uptake. No effects were observed on indices studied for reproductive effects at 53.39 mol l arsenic close to which human being are exposed through drinking water under the present set of experimental conditions.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Stereological model system for free cells and base-line data for human peripheral blood-derived small t-lymphocytes

Summary T-lymphocytes derived from human peripheral blood and passed through a nylon-wool column, were employed to develop and test a new Stereological model system for free spherical cells, allowing a quantitative characterization of the cell and its components at the ultrastructural level. Electron micrographs were recorded in a hierarchical manner at three different levels of magnification and subjected to point counting procedures. The resulting parameters were expressed in relation to various reference compartments, both absolute and relative. Results indicated that the average volume of a small, non-activated T-lymphocyte was 103.8 m³, the nuclear volume 47.5 m³ and the cytoplasmic volume 55.9 m³. On the average, the cytoplasm contained 30 mitochondria, 0.7 m³ RER-cisternae, 0.2 m³ cisternae and vesicles of the Golgi apparatus and about 231,000 free ribosomes (most of them single). The ratio of eu- to heterochromatin volume was 0.5. The design and application of the Stereological model system are discussed with regard to dynamic studies of a variety of free cells, such as macrophages, neutrophilic granulocytes and various lymphocytes.

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Zusammenfassung Menschliche, in Nylonwolle gereinigte T-Lymphozyten aus dem peripheren Blut dienten als repräsentatives Untersuchungsobjekt zur Schaffung eines neuen stereologischen Modellsystems für freie, sphärische Zellen. Dieses System erlaubt, die Zelle und die darin enthaltenen Strukturkomponenten auf ultrastruktureller Ebene quantitativ zu charakterisieren.

     

Phase-I study of intravenous modified lipid A

Summary Endotoxin and the lipid-A portion of the molecule have a variety of biological effects, including the induction of necrosis and regression of malignancy. To date extensive clinical trials of endotoxin as a potential therapeutic agent have been shunned due to the toxicity of the material.Several lipid-A analogues have been described which have reduced toxicity and retain antitumor activity. We have investigated in a phase-I trial the clinical toxicity and immunological effects of monophosphoryl lipid A prepared from Salmonella typhimurium and Salmonella minnesota.Patients entered on the study received IV monophosphoryl lipid A twice weekly for a total of 4 weeks. At least three patients were entered sequentially at each of the dose levels of 10, 25, 50, 100, and 250 g/m² body surface area. One patient was treated at the dose level of 500 g/m². The major clinical toxicity was fever, chills, and rigor, which occurred in over 50% of the treatments at doses of 250 g/m². Two instances of bronchospasm occurred in one patient who received 250 g/m². One patient received 500 g/m² and became hypotensive.Sequential clinical data showed no evidence of renal or hepatic toxicity. A transient decrease in the WBC and platelets occurred during the first 24 h after therapy. Immune function testing measured T cells, monocyte cytostasis, monocyte suppressor cell activity, and NK activity. These data suggested a shift in monocyte populations with activated cells moving into the tissue.Direct objective antitumor activity or necrosis was not observed in this group of patients. We conclude that monophosphoryl lipid A can be given to patients in a dose of up to 100 g/m² with acceptable toxicity. Its clinical activity as a single agent in combination with other immunomodulators remains to be demonstrated.     

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca²⁺ (Mg²⁺)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent ⁴⁵Ca²⁺ uptake into IP₃-sensitive Ca²⁺ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca²⁺ uptake was inhibited by TG up to 10 nm (apparent K_i4.2 nm, Ca²⁺ pool I). An additional increase of inhibition up to 85–90% of total Ca²⁺ uptake could be achieved at 15 to 20 nm of TG (apparent K_i12.1 nm, Ca²⁺ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent K_i10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca²⁺ uptake. About 30–40% of total Ca²⁺ uptake was inhibited by 100 m of vanadate (apparent K_i18 m, Ca²⁺ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent K_i300 m) or by TG up to 10 nm (Ca²⁺ pool I). The amount of IP₃-induced Ca²⁺ release was constant at 25% over a wide range of Ca²⁺ filling. About 10–20% remained unreleasable by IP₃. Reduction of IP₃ releasable Ca²⁺ in the presence of inhibitors showed similar dose-response curves as Ca²⁺ uptake (apparent K_i 3.0 nm for IP₃-induced Ca²⁺ release as compared to 4.2 nm for Ca²⁺ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca²⁺ pump fills the IP₃-sensitive Ca²⁺ pool I. At TG concentrations >10 nm which blocked Ca²⁺ pool II the apparent K_i values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent K_i values were 18 m for Ca²⁺ uptake and 7 m for Ca²⁺ release (Ca²⁺ pool II). At vanadate concentrations up to 1 mm the apparent K_i values were 300 and 200 m, respectively (Ca²⁺ pool I). Both Ca²⁺ pools I and II also showed different sensitivities to IP₃. Dose-response curves for IP₃ in the absence of inhibitors (control) showed an apparent K_m value for IP₃ at 0.6 m. In the presence of TG (inhibition of Ca²⁺ pool I) the curve was shifted to the left with an apparent K_m for IP₃ at 0.08 m. In the presence of vanadate (inhibition of Ca²⁺ pool II), the apparent K_m for IP₃ was 2.1 m. These data allow the conclusion that there are at least three different Ca²⁺ uptake mechanisms present in pancreatic acinar cells: TG- and IP₃ insensitive but highly vanadate-sensitive Ca²⁺ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca²⁺ pools with different TG-, vanadate- and IP₃-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help.     

The nutritional status of the apical meristem of Lactuca sativa as affected by NaCl salinization: An electron-probe microanalytic study

A volume of tissue of lettuce (Lactuca sativa L.) plants extending 2 mm basipetally from the apical meristem and including leaf primordia and young expanding leaves was surveyed using electron-probe microanalysis (EPMA) on both frozen-hydrated and freeze-dried samples. This analysis was carried out either 2 or 5 d following NaCl salinization of the medium from the 10 mol · m^–-3 control level up to 80 mol · m^–-3. The objective was the investigation of possible changes in the nutritional status of the apical meristem that might account for some aspects of salt-induced growth inhibition. Sodium and chloride increased significantly in tissues basal to the apical meristem, while both phosphorus and potassium decreased in the same region. These changes were evident in specimens collected just 2 d after the commencement of salinization (20 h after completion of the salinization) and were not exacerbated by an additional 3 d of treatment; they were present in tissue as close as 100 m to the meristem and extending down to 500 m. The apical 10–50 m were relatively protected from both the increase in sodium and chloride and the decrease in phosphorus and potassium that occurred in more basal regions. Young leaves (up to 1.5 mm in length) appear to control their own mineral nutrient levels when challenged by salinization of the medium, presumably because of altered growth. A decrease in the concentration of total Ca as a result of salinization was significant in cells 500 m basal to the meristem, but was evident as a tendency in the data even within the first 50 m. Using an improved automatic method for the analysis of calcium by EPMA, it was found that total Ca was reduced by salinization, especially in basal regions (500 m below the apex) and also in young leaves (1–1.5 mm in length). We suggest that the nutrition of the shoot apical meristem may be disturbed soon after salinization and that the shoot meristem might be the source of a signal to expanding leaves, as well as exerting its own direct influence over leaf emergence.Abbreviation EPMA electron-probe microanalysis This work was supported by U.S. Department of Agriculture grant 87-CRCR-1-2462.     

Structural and enzymatic studies on the plasma membrane domains and sodium pump enzymes of absorptive epithelial cells in the avian lower intestine

Summary The coprodaeum of the domestic hen maintained on a low-NaCl diet adapts by enhanced sodium transport. This study examines the adaptive response at the single cell and whole organ levels. Surface areas of apical (microvillous) and basolateral plasma membranes of columnar absorptive epithelial cells were estimated by use of ultrastructural stereology. The activities of succinic dehydrogenase (a mitochondrial enzyme) and ouabain-sensitive, potassium-dependent paranitrophenyl phosphatase (a sodium pump enzyme) were determined in tissue homogenates. Sodium, potassium-ATPase (pump enzyme) activity in cell membranes was localized by ultrastructural cytochemistry. Apical and basolateral membranes responded differently. In high-NaCl hens, the membrane signature of the average cell was 32 m² (apical), 932 m² (lateral) and 17 m² (basal). Cells from low-NaCl hens had more apical membrane (49 m² per cell) but essentially the same area of basolateral membrane. However, total surfaces per organ were greater for all membranes. Sodium pump enzymes were localized in basolateral membranes. Enzyme activities per unit mitochondrial volume and per unit basolateral membrane surface were higher in low-NaCl birds. These findings are discussed in the context of known mechanisms of transcellular sodium transport via apical ion channels and basolateral pumps.