Anti-Leishmania activity of essential oil of Myracrodruon urundeuva (Engl.) Fr. All.: Composition, cytotoxity and possible mechanisms of action.

Myracrodruon urundeuva (Engl.) Fr. All., commonly known as "aroeira-do-sertão", is a medicinal plant from Anacardiaceae family. In this study, the chemical composition of M. urundeuva essential oil (MuEO) was evaluated by gas chromatography-mass spectrometry (GC-MS), as well as its anti-Leishmania potential, cytotoxicity, and macrophage activation capability as possible antiprotozoal mechanism of action were assessed. Fourteen compounds were identified, which constituted 94.87% of total oil composition. The most abundant components were monoterpenes (80.35%), with ß-myrcene (42.46%), α-myrcene (37.23%), and caryophyllene (4.28%) as the major constituents. The MuEO inhibited the growth of promastigotes (IC50 205 ± 13.4 µg mL-1), axenic amastigotes (IC50 104.5 ± 11.82 µg mL-1) and decreased percentage of macrophage infection and number of amastigotes per macrophage (IC50 of 44.5 ± 4.37 µg⋅mL-1), suggesting significant anti-Leishmania activity. The cytotoxicity of MuEO was assessed by MTT test in Balb/c murine macrophages and by human erythrocytes lysis assay and low cytotoxicity for these cells was observed. The CC50 value against macrophages were 550 ± 29.21 µg mL-1, while cytotoxicity for erythrocytes was around 20% at the highest concentration assessed, with HC50 > 800 µg mL-1. While MuEO-induced anti-Leishmania activity is not mediated by increases in both lysosomal activity and nitric oxide production in macrophages, the results suggest the antiamastigote activity is associated with an immunomodulatory activity of macrophages due to an increase of phagocytic capability induced by MuEO. Thus, MuEO presented significant activity against Leishmania amazonensis, probably modulating the activation of macrophages, with low cytotoxicity to murine macrophages and human erythrocytes.

Push-out strength of root fillings with or without thermomechanical compaction.

AIM: To evaluate the influence of thermomechanical compaction (Tagger's hybrid technique - THT) on the push-out strength of several root filling materials to root dentine. METHODOLOGY: Root canals of eighty roots in human canines were prepared with the ProTaper system and filled with one of the following materials, using either lateral compaction (LC) (n = 40) or THT (n = 40): AH Plus/gutta-percha (GP) (n = 10), Sealer 26/GP (n = 10), Epiphany SE/Resilon (n = 10) and Epiphany SE/GP (n = 10). Three 2-mm-thick dentine slices were obtained from each third of each root. The root filling in the first slice was subjected to a push-out test to evaluate the bond strength of the materials to intraradicular dentine. Data (in MPa) were analysed using anova and post hoc Tukey's test (P < 0.05). Failure mode was determined at × 25 magnification. The other two slices were prepared for scanning electron microscopy (SEM) to examine the surface of the filling materials. RESULTS: Lateral compaction (1.34 ± 1.14 MPa) was associated with a significantly higher bond strength (P < 0.05) than the THT (0.97 ± 0.88 MPa). AH Plus/GP (2.23 ± 0.83 MPa) and Sealer 26/GP (1.86 ± 0.50 MPa) had significantly higher bond strengths than the other materials and differed significantly from each other (P < 0.05). There was a significant difference (P < 0.05) between the coronal (1.36 ± 1.15 MPa), middle (1.14 ± 1.05 MPa) and apical thirds (0.95 ± 0.83 MPa). Considering the technique and root filling material interaction, AH Plus/GP-LC was associated with the highest mean values (2.65 ± 0.66 MPa) (P < 0.05). Sealer 26/GP-LC (2.10 ± 0.46 MPa), AH Plus/GP-THT (1.81 ± 0.78 MPa) and Sealer 26/GP-TH (1.63 ± 0.44 MPa) had intermediate values that were not significantly different from each other (P > 0.05). Epiphany SE was associated with the lowest mean values (3.70 ± 0.86 MPa) (P < 0.05), regardless of the root filling technique and type of solid material (cone). Adhesive failures predominated in the specimens filled with Epiphany SE, whilst mixed and cohesive failures were more frequent in those filled with AH Plus and Sealer 26, regardless of the root filling technique. SEM analysis revealed that LC produced a dense and well-compacted filling whilst the use of a hybrid thermomechanical technique resulted in the solid material (GP or Resilon) intermingled within sealer to form a nonhomogenous mass. CONCLUSION: Lateral compaction was associated with higher bond strengths of the materials to intraradicular dentine than a hybrid technique using thermomechanical compaction. The greatest push-out strengths were obtained when the canals were filled with LC of AH Plus and GP cones.

Characterization of the biocompatible magnetic colloid on the basis of Fe3O4 nanoparticles coated with dextran, used as contrast agent in magnetic resonance imaging.

The magnetic resonance imaging contrast agent, the so-called Endorem colloidal suspension on the basis of superparamagnetic iron oxide nanoparticles (mean diameter of 5.5 nm) coated with dextran, were characterized on the basis of several measurement techniques to determine the parameters of their most important physical and chemical properties. It is assumed that each nanoparticle is consisted of Fe3O4 monodomain and it was observed that its oxidation to gamma-Fe2O3 occurs at 253.1 degrees C. The Mössbauer spectroscopy have shown a superparamagnetic behavior of the magnetic nanoparticles. The Magnetic Resonance results show an increase of the relaxation times T1, T2, and T2* with decreasing concentration of iron oxide nanoparticles. The relaxation effects of SPIONs contrast agents are influenced by their local concentration as well as the applied field strength and the environment in which these agents interact with surrounding protons. The proton relaxation rates presented a linear behavior with concentration. The measured values of thermo-optic coefficient dn/dT, thermal conductivity kappa, optical birefringence delta n0, nonlinear refractive index n2, nonlinear absorption beta' and third-order nonlinear susceptibility |chi(3)| are also reported.

Characterization of superparamagnetic iron oxide coated with silicone used as contrast agent for magnetic resonance image for the gastrointestinal tract.

The present work is a report of the characterization of superparamagnetic iron oxide nanoparticles coated with silicone used as a contrast agent in magnetic resonance imaging of the gastrointestinal tract. The hydrodynamic size of the contrast agent is 281.2 nm, where it was determined by transmission electron microscopy and a Fe3O4 crystalline structure was identified by X-ray diffraction, also confirmed by Mössbauer Spectroscopy. The blocking temperature of 190 K was determined from magnetic measurements based on the Zero Field Cooled and Field Cooled methods. The hysteresis loops were measured at different temperatures below and above the blocking temperature. Ferromagnetic resonance analysis indicated the superparamagnetic nature of the nanoparticles and a strong temperature dependence of the peak-to-peak linewidth deltaH(pp), giromagnetic factor g, number of spins N(s) and relaxation time T2 were observed. This behavior can be attributed to an increase in the superexchange interaction.

Cu and Fe metallic ions-mediated oxidation of low-density lipoproteins studied by NMR, TEM and Z-scan technique.

In this work we report on a study of the morphological changes of LDL induced in vitro by metallic ions (Cu(2+) and Fe(3+)). These modifications were characterized by transmission electron microscopy, nuclear magnetic resonance and the Z-scan technique. The degree of oxidative modification of LDL was determined by the TBARS and lipid hydroperoxides assays. It is shown that distinct pathways for modifying lipoproteins lead to different morphological transformations of the particles characterized by changes in size and/or shape of the resulting particles, and by the tendency to induce aggregation of the particles. There were no evidence of melting of particles promoted by oxidative processes with Cu and Fe.

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133+ stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10(-13) mol iron (9.5 pg) or 7.0 x 10(6) nanoparticles per cell (the measurement was carried out in a volume of 2 muL containing about 6.16 x 10(5) pg iron, equivalent to 4.5 x 10(11) SPIONs).

Introduction of air in the anaerobic culture of Streptococcus pneumoniae serotype 23F induces the release of capsular polysaccharide from bacterial surface into the cultivation medium.

AIM: An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed-batch cultivation in bioreactor. METHODS AND RESULTS: Streptococcus pneumoniae serotype 23F was cultivated in a 5-l bioreactor with nitrogen-sparging and followed by addition of air in the stationary phase. The amount of CPS released in the supernatant progressively increased under air sparging. The profile of cellular viability and optical density was similar in both cultures. Immunoelectron microscopy showed that the amount of tightly cell-bound CPS was higher in bacteria cultivated under nitrogen than under air. CONCLUSIONS: The stress caused by the addition of air at the stationary phase promoted a large increase of free CPS into the medium, as a consequence of the morphologic change in the capsule. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of air in the stationary phase of the culture would greatly simplify the subsequent downstream process, allowing CPS purification from the supernatant. The direct consequence of this process improvement is the reduction of vaccine production costs.

Crotoxin induces actin reorganization and inhibits tyrosine phosphorylation and activity of small GTPases in rat macrophages.

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom. Previous work of our group demonstrated that this toxin or its phospholipase A(2) subunit inhibits macrophage spreading and phagocytosis. The phagocytic activity of macrophages is controlled by the rearrangement of actin cytoskeleton and activity of the small Rho GTPases. The effect of crotoxin and its subunit on actin reorganization and tyrosine phosphorylation in rat peritoneal macrophages, during phagocytosis of opsonized zymosan, was presently investigated. The crude venom was used as positive control. In addition, the effect of crotoxin on the activity of Rho and Rac1 small GTPases was examined. Transmission electron studies showed that the venom or crotoxin decreased the extent of spread cells and increased microprojections often extended from macrophage surface. Immunocytochemical assays demosntrated that the venom or toxins increased F-actin content in the cytoplasm of these cells, but induced a marked decrease of phosphotyrosine. These effects were abolished by treatment with zileuton, a 5-lipoxygenase inhibitor. Furthermore, crotoxin decreased membrane-associated RhoA and Rac1 in translocation assays. The present results indicate that the crotalid venom and crotoxin are able to induce cytoskeleton rearrangement in macrophages. This effect is associated with inhibition of tyrosine phosphorylation and of the activity of proteins involved in intracellular signalling pathways important for the complete phagocytic activity of these cells.

Venom production in long-term primary culture of secretory cells of the Bothrops jararaca venom gland.

There is an increasing interest of obtaining venom by other ways than from extracting it from snakes captured in the wild. A readily available source of this venom will be useful for all pharmacological and biotechnological studies, as well as providing an improved avenue for treatments of snakebites. Here, we show that secretory cells of venom gland can be a good in vitro apparatus to produce venom. We have maintained and morphologically characterized the secretory cells of the Bothrops jararaca venom gland cultured up to 21 days. The isolated cells assemble into acini that growth in size up to 21st day, instead of adhering to the substrate. Bothropasin, a venom metalloprotease, was localized in secretory vesicles by immunoelectron microscopy and venom was also detected in culture medium in a concentration as high as 63 microg/ml. These data show that the acini formed in culture are functionally viable; they can produce and secrete venom.

Cytochemical analysis of acid phosphatase activity in the venom secretory cells of Bothrops jararaca.

A study of the histochemical reaction for acid phosphatase (AcPase) in venom gland secretory cells from Bothrops jararaca was done to investigate the distribution of lysosomes and related structures in stages of high- and low-protein synthesis. From this analysis, it was expected to gain insight into the cellular pathway by which AcPase is secreted into the venom. Two subtypes of AcPase reactivities were detected in the venom gland secretory cells: one was found in lysosomes and related structures and in some trans-Golgi network (TGN) elements and reacts with beta-glycerophosphate (betaGP) as substrate; the other was found in secretory vesicles, apical plasmalemma, lysosomes and related structures, and in some TGN elements, and reacts with cytidine monophosphate (CMP). The results are compatible with the possibility that there is a secretory via for AcPase in the venom gland of B. jararaca and that the elements composing this pathway are noted only when CMP is used as substrate. Large autophagosomes reactive to both betaGP and to CMP were commonly observed in the basal region of the secretory cells, and they were more abundant in the glands during the stage of low activity of protein synthesis.

Characterization of beta-adrenoceptors responsible for venom production in the venom gland of the snake Bothrops jararaca.

We have shown that the stimulation of beta-adrenoceptors is an important step in venom production in the Bothrops jararaca venom gland. In the present study, the pharmacological profile of the beta-adrenoceptor present in Bothrops jararaca venom gland was characterized by radioligand binding assay and by the ability of isoprenaline to promote accumulation of cyclic AMP in dispersed secretory cells. In both cases, the venom glands were obtained from non-extracted snakes (quiescent stage) or from snakes which venom was extracted 4 days before sacrifice (venom production stimulated stage). [125I]-iodocyanopindolol ([125I]-ICYP) bound to extracted gland membranes in a concentration-dependent and saturable manner, but with low affinity. Propranolol, beta1- or beta2-selective adrenoceptors ligands displaced the [125I]-ICYP binding with low affinity, while selective beta3-adrenoceptor ligands did not displace the [125I]-ICYP binding. The displacement of [125I]-ICYP by propranolol was similar in non-extracted and extracted glands, showing the presence of beta-adrenoceptors in both stages. In dispersed secretory cells of non-extracted glands, isoprenaline (1 microM) increased the cyclic AMP production and propranolol (10 microM) was able to block this effect. On the other hand, in extracted glands, isoprenaline had no effect. The results suggest that the beta-adrenoceptors present in the Bothrops jararaca venom glands are different from those (beta1, beta2 or beta3) described in mammals, but are coupled to the Gs protein, like the known beta-adrenoceptor subtypes. Moreover, previous in vivo stimulation of venom production desensitizes the beta-adrenoceptors system and, although the receptors could be detected by binding studies, they are not coupled to the Gs protein, indicating that beta-adrenoceptors stimulation contributes to the initial steps of venom synthesis.

One fate of bloodstream trypomastigote forms of Trypanosoma cruzi after immune clearance: an ultrastructural study.

The fate of bloodstream forms of Trypanosoma cruzi in tissues of mice was studied after immune elimination from circulation. Observations using transmission electron microscopy showed platelet thrombi occluding small vessels in the lung, liver, and spleen, and phagocytosed parasites in different stages of destruction within macrophages, neutrophils, and eosinophils. It is suggested that no particular cell population is a potential effector, but that different cells act in concert to destroy the parasites. The mechanism of this destruction might be related to intra- and extracellular mechanisms with trypanolytic activity.

Neisseria meningitidis serogroup C polysaccharide and serogroup B outer membrane vesicle conjugate as a bivalent meningococcus vaccine candidate.

Neisseria meningitidis serogroup C polysaccharide (PS C) was conjugated to serogroup B outer membrane vesicles (OMV) in order to test the possibility of obtaining a bivalent group B and C meningococcus vaccine. The conjugate and controls were injected intraperitoneally into groups of ten mice with boosters on days 14 and 28 after the primary immunization. The following groups were used as control: (i) PS C; (ii) PS C plus OMV; (iii) OMV; and (iv) saline. The serum collected on days 0, 14, 28 and 42 were tested by enzyme-linked immunosorbent assay (ELISA) for PS C and OMV, and by complement mediated bactericidal assay against serogroups B and C. ELISA for PS C as well as bactericidal titres against serogroup C meningococci of the conjugated vaccine increased eight-fold (ELISA) and 32 fold (bactericidal) after 42 days in comparison with the PS C control group. ELISA for OMV and bactericidal titre against serogroup B meningococci of the conjugate showed no significant difference in comparison with the OMV containing controls. Furthermore, Western Blot assay of the conjugate immune serum did not bind OMV class four protein which is related to the complement dependent antibody suppressor. The results indicate that the PS C-OMV conjugate could be a candidate for a bivalent vaccine toward serogroups B and C meningococci.

One fate of epimastigote forms of Trypanosoma cruzi injected in mice. An ultrastructural study.

Recently, we suggested that epimastigote forms of Trypanosoma cruzi are cleared from circulation of mice by a mechanism independent of lysis and that platelets play an important role in this process. These observations prompted us to look at the fate of epimastigotes in the lung, liver, and spleen of mice injected intravenously with these parasite forms. Using transmission electron microscopy, we observed clumps of epimastigotes and platelets in direct contact with phagocytes in the lumen of capillaries. However, the platelets and parasites were probably separated before phagocytosis because only parasites were found inside the phagocytes. Indeed, most of the phagocytes, although containing epimastigotes in different stages of disintegration, contained no platelets. The removal of parasites from platelets was probably mediated by phagocytes through a mechanism similar to the removal of bacteria from the surface of erythrocytes in humans. These observations suggest that the nonvirulence of T. cruzi epimastigotes in mice is not due to lysis but probably to the inability of these parasite forms to escape destruction by the phagocytes.

Control of venom production and secretion by sympathetic outflow in the snake Bothrops jararaca.

Many studies have examined the morphological and biochemical changes in the secretory epithelium of snake venom glands after a bite or milking. However, the mechanisms of venom production and secretion are not yet well understood. The present study was undertaken to evaluate the role of the sympathetic nervous system in the control of venom production and secretion. Venom glands were obtained from Bothrops jararaca (Viperidae) snakes, either unmilked previously or milked 4, 7 or 15 days before they were killed. Levels of tyrosine-hydroxylase-like immunoreactivity were higher in venom glands collected 4 days after milking, coinciding with the maximal synthetic activity of the secretory cells. The only catecholamine detected by high-performance liquid chromatography was noradrenaline, indicating the presence of noradrenergic fibres in these glands. In reserpine-treated milked snakes, no venom could be collected, and electron microscopic analysis showed narrow rough endoplasmic reticulum cisternae, instead of wide cisternae, and less well-developed Golgi apparatus compared with milked untreated snakes, indicating impairment of protein synthesis and secretion. The administration of isoprenaline or phenylephrine (beta- and alpha-adrenoceptor agonists, respectively) to reserpine-treated milked snakes promoted the widening of the rough endoplasmic reticulum and restored venom production, but only phenylephrine restored the development of the Golgi apparatus and the formation of many secretory vesicles. These results provide the first evidence that the sympathetic nervous system plays an important role in venom production and secretion in the venom glands of Bothrops jararaca. Understanding the importance of noradrenergic stimulation in venom production may provide new insights for research into the treatment of snakebites.

Angular leaf spot of phaseolus beans: relationships between disease, healthy leaf area, and yield.

ABSTRACT Five field experiments were conducted to investigate the relationship between the severity of visible disease (X), area under the disease progress curve (AUDPC), healthy leaf area index on any given day (HLAI), radiation intercepted by healthy leaf area on any given day (HRI), healthy leaf area duration (HAD), total healthy leaf area absorption (HAA), and yield of Phaseolus beans, cultivars Rosinha and Carioca, inoculated with Phaeoisariopsis griseola at several doses. In general, yield was not related to disease severity (X) or AUDPC. In contrast, the highest yields were always related to the highest values of HAD and HAA. The relationship between yield and HAD was linear in each of five trials (29.9 < R(2) < 70.2%, P < 0.001). The relationship between yield and HAA was linear in four of the trials (52.3 < R(2) < 70.3%, P < 0.001) and exponential in one of them (in which the plant canopy was the largest). Singlepoint models using HRI to estimate yield at various times during the crop season were developed. The slope of the yield-HRI relationship proved to be stable (26.8 +/-2.4 g MJ(-1)), regardless of cultivar, locale, planting date, and bean growth stage (from R5 to R8). The yield-HLAI relationship proved to be less consistent. HRI is proposed as a key explanatory variable for a transportable system of disease management; it may be useful in producing precise recommendations at the farm level.

Fecal loss and clearance of alpha-1-antitrypsin in children with persistent diarrhea.

1. Daily fecal loss and daily clearance of alpha-1-antitrypsin were determined in 30 infants without intestinal disorders and in 21 with persistent diarrhea. 2. Stools were collected during a 48-h period and a randomly obtained single sample was also collected. Blood samples were also collected from the infants, and alpha-1-antitrypsin was measured by radial immunodiffusion in both stool and serum. 3. No difference in daily fecal loss (mg/d) of alpha-1-antitrypsin was detected between the control group and the group with persistent diarrhea (11 +/- 9.3 vs 18.5 +/- 20 mg/d). No difference in daily alpha-1-antitrypsin clearance (ml/d) was detected between the control group and the group with persistent diarrhea (4.3 +/- 3.6 vs 5.2 +/- 4.8 ml/d). 4. There was a strong correlation between daily fecal loss and daily clearance of alpha-1-antitrypsin (N = 50). There was a weak correlation between the concentrations of alpha-1-antitrypsin in randomly obtained single samples and daily fecal loss of the antiprotease (N = 25; r = -0.183; P < 0.01). 5. We conclude that: a) there is no increased fecal loss of alpha-1-antitrypsin persistent in diarrhea; b) fecal alpha-1-antitrypsin clearance is not necessary to estimate the enteric loss of the antiprotease; c) the determination of alpha-1-antitrypsin in random samples of feces is not a reliable method.

Fecal loss and clearance of alpha-1-antitrypsin in children with persistent diarrhea

1. Daily fecal loss and daily clearance of alpha-1-antitrypsin were determined in 30 infants without intestinal disorders and in 21 with persistent diarrhea. 2. Stools were collected during a 48-h period and a randomly obtained single sample was also collected. Blood samples were also collected from the infants, and alpha-1-antitrypsin was measured by radial immunodiffusion in both stool and serum. 3. No difference in daily fecal loss (mg/d) of alpha-1-antitrypsin was detected between the control group and the group with persistent diarrhea (11 +/- 9.3 vs 18.5 +/- 20 mg/d). No difference in daily alpha-1-antitrypsin clearance (ml/d) was detected between the control group and the group with persistent diarrhea (4.3 +/- 3.6 vs 5.2 +/- 4.8 ml/d). 4. There was a strong correlation between daily fecal loss and daily clearance of alpha-1-antitrypsin (N = 50). There was a weak correlation between the concentrations of alpha-1-antitrypsin in randomly obtained single samples and daily fecal loss of the antiprotease (N = 25; r = -0.183; P < 0.01). 5. We conclude that: a) there is no increased fecal loss of alpha-1-antitrypsin persistent in diarrhea; b) fecal alpha-1-antitrypsin clearance is not necessary to estimate the enteric loss of the antiprotease; c) the determination of alpha-1-antitrypsin in random samples of feces is not a reliable method.

Occurrence of retrovirus-like particles in various cellular and intercellular compartments of the venom glands from Bothrops jararacussu.

Retrovirus-like particles were detected in venom glands from Bothrops jararacussu during electron microscopy. Type C-like particles were found inter- and intracellularly in gland and vessel lumina and scattered in the connective tissue. They were about 100 nm in diameter, with an electron dense core and bilaminar external membrane. Shapes suggestive of a budding process from the plasma membrane were also observed. Less frequently, type A-like particles, about 80 nm diameter with an electronlucent core, appeared in association with the membranes of the endoplasmic reticulum of the secretory cells.

Morphometric studies on venom secretory cells from Bothrops jararacussu (Jararacuçu) before and after venom extraction.

A comparative morphometrical analysis was carried out on secretory cells from Bothrops jararacussu venom glands, before manual extraction of the venom (milking) and 4 and 8 days after milking. At the 8th day after milking, the cytoplasmic volume increased by 160%. The rough endoplasmic reticulum (RER) volume density increase, up to the 8th day after milking, is mainly due to widening of the intra-scisternal space. The total volume and membrane surface of the RER. Golgi apparatus and subcomponents, secretory vesicles and mitochondria, increased during the experimental period while the volume and surface densities of these organelles, with the exception of the RER, did not vary. The numerical density of Golgi-associated microvesicles per Golgi volume unit also increased. The greatest relative increments in these parameters occurred within the first 4 days. These results are compatible with an increased rate of membrane synthesis and transport in the milked glands and suggest that the membrane biogenesis, degradation and circulation that takes place in the first week after milking is achieved through coordinated cellular mechanisms that maintain the rate between total membrane surface and total cytoplasmic volume unaltered.