Global warming-related response after bacterial challenge in Astroides calycularis, a Mediterranean thermophilic coral.

A worldwide increase in the prevalence of coral diseases and mortality has been linked to ocean warming due to changes in coral-associated bacterial communities, pathogen virulence, and immune system function. In the Mediterranean basin, the worrying upward temperature trend has already caused recurrent mass mortality events in recent decades. To evaluate how elevated seawater temperatures affect the immune response of a thermophilic coral species, colonies of Astroides calycularis were exposed to environmental (23 °C) or elevated (28 °C) temperatures, and subsequently challenged with bacterial lipopolysaccharides (LPS). Using immunolabeling with specific antibodies, we detected the production of Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-kB), molecules involved in coral immune responses, and heat shock protein 70 (HSP70) activity, involved in general responses to thermal stress. A histological approach allowed us to characterize the tissue sites of activation (epithelium and/or gastroderm) under different experimental conditions. The activity patterns of the examined markers after 6 h of LPS stimulation revealed an up-modulation at environmental temperature. Under warmer conditions plus LPS-challenge, TLR4-NF-kB activation was almost completely suppressed, while constituent elevated values were recorded under thermal stress only. An HSP70 up-regulation appeared in both treatments at elevated temperature, with a significantly higher activation in LPS-challenge colonies. Such an approach is useful for further understanding the molecular pathogen-defense mechanisms in corals in order to disentangle the complex interactive effects on the health of these ecologically relevant organisms related to global climate change.

Sea anemones, methylmercury, and bacterial infection: A closer look at multiple stressors.

Specimens of the Mediterranean sea anemone Anemonia viridis were exposed to methylmercury (MeHg) and bacterial infection to study their immune responses to a well-known toxic pollutant. Anemones were housed in laboratory conditions and divided into five experimental groups: 1. control (no microinjection); 2. filtered seawater + buffer injection; 3. filtered seawater + Escherichia coli injection; 4. MeHg + buffer injection; 5. MeHg + E. coli injection. Data showed an increase in antioxidant enzyme production compared to the constitutive condition, while methylmercury inhibited lysozyme production. The buffer inoculation had no statistically significant effects on the animals. In addition, electrophoretic and protease analyses revealed differences in the type of proteins produced, as well as a modulation of proteases depending on the treatment. The study demonstrated the immunomodulatory effect of the organic pollutant on A. viridis, validating its use as a model organism for marine coastal biomonitoring programmes and multiple stress studies.

Comparison of bone mass and quality determinants in adolescents and young adults with juvenile systemic lupus erythematosus (JSLE) and juvenile idiopathic arthritis (JIA).

BACKGROUND: Few prospective data have been published on the comparison of bone density and quality in homogeneous groups of patients with juvenile systemic lupus erythematosus (JSLE) and juvenile idiopathic arthritis (JIA). OBJECTIVE AND HYPOTHESIS: The objective of this study is to perform a longitudinal evaluation of the prevalence and the characteristics of bone mass and quality and to evaluate the differences on the bone parameters, using DXA, pQCT and QUS. POPULATION AND/OR METHODS: Forty-three JSLE patients (35 females, 8 males, median age 18.8, range 14.0-34.1 years) have been studied with DXA, pQCT and QUS scans and compared with 138 JIA patients (112 females, 26 males, median age 18.9, range 13.4-33.2 years), and 79 controls (59 females, 20 males; median age 19.3, range 13.5-36.5 years). Of these, 39 patients (32 females and 7 males, median age 20.3, range 16.6-36.8 years) with JSLE were followed longitudinally and compared with 131 patients (108 females, 23 males median age 20.7, range 15.8-37.1 years) with JIA and 63 controls (48 females, 15 males; median age 21.9, range 15.5-38.3 years). RESULTS: JSLE patients have a higher bone cortical density (CrtBMD) than controls and JIA patients (p < 0.005). However, JSLE and JIA patients have a significantly reduced bone trabecular density (TrbBMD) compared to controls (p < 0.0001), with no differences between JSLE and JIA. In addition, JIA patients show a significantly reduced muscle area (MuscleCSA) compared to JSLE and controls (p < 0.001). Conversely, fat area (FatCSA) is significantly increased both in JIA and JSLE patients when compared to controls (p < 0.001), with no differences between the JSLE and JIA groups. Analogous results are observed in the polar resistance to stress (SSIp). On longitudinal evaluation, contrary to CrtBMD, the difference between BMAD SDS, TrbBMD, MuscleCSA and FatCSA remains unchanged; in JSLE patients, SSIp is stable in comparison to JIA and controls without any difference between the two groups. CONCLUSIONS: The evaluation of bone density and structure parameters in JSLE patients highlights significant differences compared with JIA patients and controls. These data might indicate a different pathogenesis of bone damage in the two entities, and suggest a different diagnostic and therapeutic approach to improve the peak bone mass.

Vitamin D levels in children, adolescents, and young adults with juvenile-onset systemic lupus erythematosus: a cross-sectional study.

BACKGROUND: SS and LC contributed equally to this manuscript. Hypovitaminosis D is common in the general population. Although many studies on 25-hydroxyvitamin D (25(OH)D) are available on systemic lupus erythematosus (SLE), few data are reported in juvenile-onset SLE (JSLE) patients. DESIGN: This study aimed to assess serum 25(OH)D levels in JSLE patients and to identify risk factors for vitamin D deficiency in this population. METHODS: Forty-five Caucasian JSLE patients (36 females, nine males; mean age 18.9±6.3 years) and 109 age- and sex-matched healthy controls entered the study. Dual-energy X-ray absorptiometry (DXA) scans of the lumbar spine, serum calcium and phosphate, bone-specific alkaline phosphatase (BSAP), parathyroid hormone (PTH), and 25(OH)D were assessed. The data were compared with an age- and sex-matched control group including 109 Caucasian healthy subjects. RESULTS: JSLE patients exhibited lower 25(OH)D levels than controls (p<0.005), with the lower values observed in patients with active vs. inactive disease (p<0.05). JSLE patients exhibited reduced total calcium levels (p<0.001) and higher phosphate levels (p<0.001), BSAP (p<0.001) and PTH (p<0.001) than controls. In addition, JSLE patients exhibited lower spine bone mineral apparent density (BMAD) SDS values than controls (p<0.001), with higher values in patients with 25(OH)D sufficiency and insufficiency than in those with 25(OH)D deficiency (p<0.001). CONCLUSIONS: Patients with JSLE have significantly lower 25(OH)D levels than controls. Therefore, vitamin D supplementation may be useful to normalize bone mass and quality in subjects with JSLE.

Cross-sectional and longitudinal evaluation of bone mass and quality in children and young adults with juvenile onset systemic lupus erythematosus (JSLE): role of bone mass determinants analyzed by DXA, PQCT and QUS.

INTRODUCTION: There are few prospective data on bone mass and quality in patients with juvenile onset systemic lupus erythematosus (JSLE). There are also few studies analyzing bone mass and quality determinants by using at the same time dual-energy X-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT) and quantitative ultrasound (QUS). OBJECTIVE: The objective of this paper is to evaluate cross-sectionally and longitudinally bone mass and quality determinants in adolescents and young adults with JSLE, and to identify the main predictors of reduced bone mineral density (BMD) and bone quality using these techniques. METHODS: Fifty-six patients with JSLE (mean age 18.5 ± 5.7 years) entered the study. In all subjects DXA scan at the lumbar spine, radius pQCT and phalangeal QUS were performed the same day. Of these, 46 patients (mean age 23.1 ± 6.2 years) were revaluated with a second DXA, pQCT and QUS. The data obtained were compared with 72 and 80 age- and sex- matched healthy controls. RESULTS: At the first evaluation, JSLE patients had a reduced spine BMAD SDS (p < 0.001), and significantly lower levels of TrabBMD (p < 0.0001), SSIp (p < 0.05), AD-SoS and QUS z-score (p < 0.005) but not reduced muscle CSA and CBA values. CortBMD and FatCSA were significantly increased (p < 0.0001). These data were confirmed at longitudinal evaluation regarding spine BMAD SDS (p < 0.001), TrabBMD (p < 0.0001), FatCSA (p < 0.005), AD-SoS (p < 0.001), and QUS z-score (p < 0.005) but not muscle CSA (p ≤ 0.05) and CBA (p < 0.0001). SSIp and CortBMD longitudinal evaluation showed that JSLE patients did not present significant differences in comparison to controls. CONCLUSIONS: Patients with JSLE have a low bone mass without catch-up growth over time, causing a reduction of peak bone mass with high risk of osteoporosis in early adulthood. To reduce the risk, close monitoring of BMD, better control of disease activity, physical activity and dietary intake of calcium and vitamin D are advocated to ameliorate the loss of bone mass. In patients with proved osteoporosis therapeutic approaches including bisphosphonates should be considered.

Push-out strength of translucent fibre posts cemented using a dual-curing technique or a light-curing self-adhering material.

AIM: To compare by means of a push-out test the interfacial strength of a dual-curing resin cement and a light-curing self-adhering resin composite when used in translucent fibre post cementation. METHODOLOGY: Thirty-four extracted human premolars with single canals were selected and root filled. Translucent fibre posts (RelyX Fiber Post) were luted into the root canal using two resinous luting systems (n = 17). Dual-Curing Technique (DC): the specimens were treated with Excite DSC and RelyX ARC, which were light-cured simultaneously through the post for 60 s. Light-Curing Self-Adhering Technique (LCSA): the specimens were treated with Vertise Flow, which was light-cured through the post for 60 s. The specimens were sectioned transversally into six slices to perform the push-out test at the coronal, middle and apical regions of the root canals. Data were analysed by two-way anova. All specimens were analysed by stereomicroscopy and SEM to determine fracture patterns. RESULTS: There were no significant differences between the DC and LCSA techniques (P = 0.703) in any of the canal regions. Root region was not a significant factor for push-out values (P = 0.255) and group-region interactions were not significant (P = 0.740). For the DC technique, the majority of the fracture patterns (73.3%) were adhesive at the interface between dual-curing resin cement and adhesive. For the LCSA technique, the majority of the fracture patterns (71.7%) were adhesive at the interface between light-curing self-adhering resin composite and dentine. CONCLUSIONS: The interfacial strength between light-curing self-adhering resin composite and root canal walls is equivalent to the interfacial strength between dual-curing cement and root canal walls.

A rational use of dental materials in posterior direct resin restorations in order to control polymerization shrinkage stress.

One of the main problems when using resin-based composites is the resulting polymerization shrinkage stress. Composite strain is hindered every time the composite is bonded to the tooth's walls. In the pre-gel phase the shrinkage stress is reduced by the composite flow from the free to the bonded surface areas. Therefore, no stress develops at the dentine-composite interface. When a gel point is reached, the composite flow no longer compensates for the volumetric shrinkage. The generated stress may cause adhesive failure and several other adverse clinical consequences such as enamel fracture, cracked cusps, cuspal movement, microcracking of the restorative material and gaps between the resin and cavity walls which may cause secondary caries and postoperative sensitivity. A sensible use of materials in direct restorations may contribute to a reduced rate of shrinkage stress. To this aim glass-ionomer cement as well as flowable, light-curing and self-curing composites were examined. The aim of this study was to provide some useful information for a sensible choice of restoration materials in order to control shrinkage stress and its negative consequences in direct posterior restorations.

5 '-Amino-4-imidazolecarboxamide riboside induces apoptosis in human neuroblastoma cells via the mitochondrial pathway.

5'-Amino-4-imidazolecarboxamide (AICA) riboside induces apoptosis in neuronal cell models. In order to exert its effect, AICA riboside must enter the cell and be phosphorylated to the ribotide. In the present work, we have further studied the mechanism of apoptosis induced by AICA riboside. The results demonstrate that AICA riboside activates AMP-dependent protein kinase (AMPK), induces release of cytochrome c from mitochondria and activation of caspase 9. The role of AMPK in determining cell fate is controversial. In fact, AICA riboside has been reported to be neuroprotective or to induce apoptosis depending on its concentration, cell type or apoptotic stimuli used. In order to clarify whether the activation of AMPK is related to apoptosis in our model, we have used another AMPK stimulator, metformin, and we have analysed its effects on cell viability, nuclear morphology and AMPK activity. Five mM metformin increased AMPK activity, inhibited viability, and increased the number of apoptotic nuclei. AICA riboside, which can be generated from the ribotide (an intermediate of the purine de novo synthesis) by the action of the ubiquitous cytosolic 5'-nucleotidase (cN-II), may accumulate in those individuals in which an inborn error of purine metabolism causes both a building up of intermediates and/or an increase of the rate of de novo synthesis, and/or an overexpression of cN-II. Therefore, our results suggest that the toxic effect of AICA riboside on some types of neurons may participate in the neurological manifestations of syndromes related to purine dismetabolisms.

The extension of resin tags in etched dentin: a misinterpretation?

AIM: The morphological characteristics of the filament structure, universally recognized as resin tags, in samples prepared using a new SEM methodology, are analyzed . METHODS: Ten non-carious, human third molars were cut transversally to obtain 10 dentinal surfaces. They were filled using an adhesive restorative technique. Subsequently, the samples were cut lengthwise to be SEM observed, and then randomly divided into 6 main groups according to the preparation modality of the section surface: Group A: EDTA; Group B: H(3)PO(4); Group C1: H(3)PO(4)+NaOCl; Group C2 H(3)PO(4)+NaOCl+H(2)O(2); Group D1: HCl+NaOCl; Group D2: HCl+NaOCl+H(2)O(2). RESULTS: The resin tags which originate from resin penetration and polymerization within the dentinal tubules are small conic-trunk shaped plugs that are a few microns long. The thinner extension of the resin tags is probably due to the persistence of the lamina limitans. CONCLUSIONS: A new method of specimen preparation for SEM visualization is proposed in order to effectively evaluate the penetration capacity of the adhesive in the dentinal substratum. In addition to the use of strong acids and bases, an agent capable of degrading the glycosaminoglycans was employed to completely remove the dentinal organic component.

[Use of light-curing composite and adhesive systems for the cementation of tranlucent fiber posts. SEM analysis and pull-out test]. / Utilizzo di adesivi e compositi fotopolimerizzanti nella cementazione di perni translucenti: analisi al SEM e Pull-out test.

AIM: The purpose of this study was to compare 2 different techniques for the cementation of translucent fiber posts. The comparison was carried out by means of tensile bond strength testing and SEM evaluation of the interfaces morphology post/cement and cement/dentin. METHODS: Twenty upper central extracted incisors were first endodontically treated and then randomly divided into 2 groups of 10 samples each. Twenty translucent Endo Light-posts (RTD) were cemented following 2 different METHODS: In Group 1 Excite (Ivoclar Vivadent) and Tetric Flow (Ivoclar Vivadent) were applied and light-cured. In Group 2: All Bond 2 (Bisco) and RelyX ARC (3M) were applied, light-cured and then self-cured. A tensile bond strength test was carried out on 14 roots, 7 from each group. Subsequently, 2 roots were cut lengthwise and observed using a SEM. The remaining 6 roots (3 from each group) were cut into 5 transversal sections and observed using a SEM. The tensile force required to dislodge the cemented posts from the 7 roots of each group was recorded by means of a testing machine. RESULTS: Group 1 revealed a bonding strength which was slightly inferior to the bonding strength registered in Group 2. SEM evaluation showed a good quality of bond. Without taking into account the curing method used, the polymerization carried out by means of light curing seems to result in a better adaptation of cement. CONCLUSIONS: Self curing cement still represents the most reliable alternative, even if photo-curing cement might guarantee a most efficient stress distribution along the walls of the canal.

[Bilateral adrenal hemorrhage with adrenal insufficiency in the framework of primary antiphospholipid antibody syndrome]. / Bilaterale Nebenniereninfarkte mit adrenaler Insuffizienz im Rahmen eines primären Antiphospholipid-Antikörpersyndroms.

We report a case of acute adrenal insufficiency in a context of probable bilateral adrenal haemorrhage, as revealed by CT-scan in a 52-year-old woman with a history of spontaneous abortion and repeated ischaemic stroke without symptoms or signs of collagen vascular disease. The symptoms began after the patient had successfully been treated for pneumonia. She had persistently high titres of IgG anticardiolipin antibodies, antibodies against beta 2-glycoprotein I and a lupus anticoagulant. The diagnosis of primary antiphospholipid syndrome with adrenal insufficiency was postulated.

High-affinity sites for beta-D-galactosidase on membrane-bound vesicles isolated from rat epididymal fluid.

Glycosidases in rat epididymal fluid are secreted under androgen stimulation and possess receptors on the sperm surface. One of these enzymes, beta-D-galactosidase (gal), was found in the epididymal fluid as a soluble enzyme and also in a heterogeneous population of membrane bound vesicles (mbv). beta-D-Galactosidase was specifically localized to a subpopulation of larger, electron-dense mbv. The aim of this study was to analyze the high-affinity sites for gal on the membrane of mbv using two different methods: classical fluorometric assay (used in previous papers) and colloidal gold (20 nm) conjugated to gal as a marker in ultrastructural studies. beta-D-Galactosidase bound to mbv with high-affinity (Kd in a nanomolar range) are in a saturable form. Furthermore, 25 mM fructose-1,6-diphosphate (f-1,6-dip), a sugar that competes for the binding site, showed 50% inhibition of the binding. The gold conjugates were mostly observed on the surface of the large, electron-dense mbv but not on the small, electron-lucent mbv. Gold particles were also observed on the larger vesicles, but less frequently in the presence of f-1.6-dip. Larger mbv possesses high-affinity sites for gal on their membrane.

Purification of proteins from rat sperm membranes that interact with ligands other than phosphomannosyl residues.

In this study proteins were purified from rat sperm membranes which might be the high affinity sites for ligands of epididymal fluid other than the mannose-6-phosphate receptors. The sperm membrane proteins were solubilized and passed over an affinity column containing epididymal fluid proteins coupled to a matrix. Two bands in the range of 45-55 kDa were eluted from the column with fructose-6-phosphate but not with mannose-6-phosphate. Although the molecular weight of these proteins are similar to those of the cation-dependent phosphomannosyl receptors they are not related. These two proteins may correspond either to two different receptors or to forms of the same receptor that recognize ligands from rat epididymal fluid. Sequencing and identification of these proteins will be the aim of future studies.

Arthroscopic meniscectomy for discoid lateral meniscus in children and adolescents: 10-year follow-up.

Seventeen adolescents underwent arthroscopic lateral meniscectomy for discoid lateral meniscus. The average age at surgery was 13.6 years (range: 5-18 years). The main preoperative symptom was pain in 16 knees and extension loss in 1 knee. At arthroscopy, 10 menisci were complete, 4 were incomplete, and 3 were Wrisberg type. Arthroscopic total meniscectomy was performed in the 3 Wrisberg types, 2 complete types, and 1 incomplete type. The remaining menisci underwent partial meniscectomy. The average follow-up was 10 years (range: 5-15 years). According to the Ikeuchi rating system, 12 knees were rated as excellent (no symptoms and full range of motion), 4 were rated as good (occasional pain), and 1 was rated as fair (patellofemoral pain in an obese patient). Radiographic evaluation showed development of minor osteophytes in the lateral compartment of 8 knees and <50% narrowing of the lateral joint space in 11 knees. No correlation was found between meniscal type, type of meniscectomy (partial or total), and clinical and radiographic results. Arthroscopic lateral meniscectomy for discoid lateral meniscus in adolescents was effective in relieving symptoms during a 10-year follow-up period. Longer follow-up is needed to ascertain the significance of the radiographic changes seen in this study.

alpha-Mannosidase from rat epididymal fluid is a ligand for phosphomannosyl receptors on the sperm surface.

This study demonstrates that alpha-mannosidase from rat epididymal fluid is a ligand for phosphomannosyl receptors on the sperm surface. This enzyme was bound to intact epididymal spermatozoa with high affinity and in saturable form, and the binding was inhibited by mannose-6-phosphate but not by phosphorylated derivatives of fructose. Treatment of the enzyme with sodium periodate inhibited the binding of alpha-mannosidase, confirming that a carbohydrate residue is involved in the interaction with spermatozoa. Evidence is also presented that the cation-independent phosphomannosyl receptors are responsible for the interaction with alpha-mannosidase. These findings suggest a new role for extracellular transport mediated by the mannose-6-phosphate receptor.

Is an ATPase involved in uncoating of plasma membrane adaptor complex AP-2?

The coat of clathrin-coated vesicles mostly consists of clathrin and adaptor complexes AP-1 or AP-2. Clathrin is released from the vesicles in an ATP-dependent fashion prior to their fusion with endosomes. In the present study we found that ATP strongly inhibits in vitro binding of cytosolic AP-2 to membranes of stripped vesicles, and promotes the release of endogenous AP-2 from clathrin-deprived coated vesicles. Both effects required hydrolysis of ATP. In contrast, binding of AP-1 to stripped vesicles was not affected by ATP, but was enhanced by GTP-gamma-S. These results point to an ATPase that promotes the release of AP-2 from clathrin-coated vesicles.

Affinity sites for beta-glucuronidase on the surface of human spermatozoa.

Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct. They are believed to play a role in mammalian fertilization. In the present report, we demonstrate that beta-glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner. The binding is Ca(2+)-independent, inhibited by either mannose-6-phosphate, phosphomannan fragments from the yeast Hansenula holstii and alpha-mannosidase from the Dictyostelium discoideum, suggesting that phosphomannosyl receptors are involved in the recognition of the enzyme. The catalytic site of the enzyme is not involved in the binding. The localization of the beta-glucuronidase binding-sites is restricted to the surface of the sperm head. These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.

Electrophoretic analysis of protein selectively extracted with chaotropic agents from membranes of rat sperm.

Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. The 100.000 x g fraction contained only small membranous vesicles and less than 0.5% of the total enzymatic activity. This fraction was considered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetate buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-100, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2% SDS. The extracts and the solubilized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtained with the first three chemicals contained 11,9, and 25% of total proteins respectively. The bicarbonate buffer solubilized 45%, and the detergents 55% and 65% respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbonate buffer, extracted a great number of bands over a wide range of molecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had similar effects: both solubilized four new bands. In residual sediments there were no new proteins and the bands corresponded to those extracted with the detergents, but they varied in staining intensity. According to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50% of the mass of membraneous proteins may be peripheric. Proteins partially extracted with the detergents were also found in the residual sediment, and they may constitute the skeleton of sperm membrane.

Trypanosoma cruzi: involvement of intracellular calcium in multiplication and differentiation.

The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.

Purification and characterization of beta-galactosidase from rat epididymal fluid.

Beta-galactosidase from rat epididymal fluid was purified by a combination of chromatographic techniques and precipitation with ammonium sulphate. Specific activity of the enzyme in the final precipitate was 18 times greater than in the original fluid, and it was practically free of N-acetyl-beta-D-glucosaminidase. A single major band was seen when the precipitate was analysed by sodium dodecylsulphate polyacrylamide gelectrophoresis (SDS-PAGE). The activity of the purified enzyme has an optimum at pH 4.5, and the temperature optimum is around 45 degrees C. The activity was inhibited by p-chloromercuribenzoic acid and ions such as Cd(II), Co(II), Cu(II) and Ag(I). Lactose does not appear to be a substrate for this enzyme.