A cytochemical method for the demonstration of 5′-nucleotidase in mouse peritoneal macrophages,with cerium ions used as trapping agent

Summary A method was developed for the demonstration of 5-nucleotidase in murine peritoneal resident macrophages. The cells are incubated cytochemically without agitation and cerium chloride is used as a trapping agent. Under these conditions, the great majority of the macrophages in the unstimulated peritoneal cavity show enzyme activity in the plasma membrane. In the presence of AMP-S (an AMP analogue inhibiting 5-nucleotidase, as shown biochemically) there was a decrease in both the number of positive macrophages and the amount of reaction product on the plasma membranes. This indicates that the enzyme activity detected by our cytochemical procedure is attributable to 5-nucleotidase.     

Lipid peroxidation as the mechanism of modification of brain 5′-nucleotidase activity in vitro

The effect of lipid peroxidation on the Mg²⁺-independent and Mg²⁺-dependent activity of brain cell membrane 5-nucleotidase was determined and the affinity of the active sites of Mg²⁺-dependent enzyme for 5-AMP (substrate) and Mg²⁺ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 M ascorbate and 25 M FeCl₂ (resultant) for 10 min. The activity of 5-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg²⁺-independent 5-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 mol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg²⁺, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg²⁺-dependent 5-nucleotidase for 5-AMP tripled, as indicated by a significant decrease inK _m (K _m=95±2 M AMP for control;K _m=32±2 MAMP for peroxidized).V _max was significantly reduced from 3.35±0.16 in control to 1.70±.09 moles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg²⁺ significantly increased (K _m=6.17±0.37 mM Mg²⁺ for control;K _m=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg²⁺-dependent 5-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5-nucleotidase activity and the loss of Mg²⁺-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5-nucleotidase during hypoxia.     

Cytochemical markers of bladder carcinogenesis

Summary Enzyme cytochemical and immunocytochemical techniques at the light and electron microscope levels were used to study the distribution of potential markers of chemical transformation in rodent bladders. In rat tumours induced byin vivo treatment with methylnitrosourea, alkaline phosphatase localization was normal on the external surface of the plasma membranes of some cells but abnormal in others where reaction product was seen only on intracellular membranes. 5-Nucleotidase localization was abnormal in all cells, being seen on endoplasmic reticulum and nuclear membranes only, while in normal bladders only ectoenzyme localization was seen. Heterogeneity of alkaline phosphatase and 5-nucleotidase localization was seen on the plasma membranes of these tumours after 15 days in organ culture. Some cells produced enzyme and others did not; in other cells only parts of the membrane reacted heavily, while other regions were negative.In transformed cell cultures and tumours of mouse bladder derived byin vitro treatment of explants with dimethylbenz (a) anthracene, a bimodal pattern of alkaline phosphatase localization was seen. Cells had either normal ectoenzyme reaction product or abnormal intracellular membrane reaction product. 5-Nucleotidase and ADPase were lost after transformation while cAMP-phosphodiesterase was retained as an ectoenzyme. Mg.ATPase and a cAMP-independent, calcium-insensitive protein phosphatase were induced in transformed cell cultures. An epithelial antigen was detected in the cytoplasm of both normal and transformed cells associated with reticular cytoplasmic ground substance, plasma membrane vesicles and cytoskeletal elements.     

Alpha-thalassemia in Papua New Guinea

Summary A study of the distribution of -thalassemia in Papua New Guinea (PNG) was carried out by DNA analysis. A total of 664 DNA samples were screened for -thalassemia 2 and -thalassemia 1 caused respectively by either deletion of one or both of the duplicated -globin genes. -Thalassemia 2 was detected in high frequencies in coastal and lowland regions where malaria has been holo- to hyperendemic but in low frequencies in non-malarious highland regions. The highest frequency was observed in the north coast of PNG. The distribution of -thalassemia 2 seems to be in accordance with other conditions such as ovalocytosis and G6PD deficiency which are also prevalent in this population, suggesting that they may interact in protection against malaria. However, it appears to be negatively correlated with -thalassemia and -thalassemia 1, the latter being extremely rare in this population. Analysis of the types and subtypes of the single -globin gene deletion revealed a predominance of the –^4.2 type in general, except in some regions in the south where the –^3.7 type is prevalent. The –^3.7 I subtype is the common form of the –^3.7 deletion in the PNG mainland. The –^3.7 III subtype, previously reported to be unique in Melanesians and Polynesians, was detected in an offshore island of PNG. However, this subtype is very rare in Melanesians from the PNG mainland.     

Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom

A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of -mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 59-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5–9.5 and the optimum temperature was 60°C, with activity being rapidly lost within 1 min at 70°C. The K_m of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and -mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.     

Time course of catalase and superoxide dismutase during acclimatization and growth of micropropagated Calathea and Spathiphyllum plants

Catalase and superoxide dismutase (SOD) activities were followed in leaves during ex vitro acclimatization and growth of micropropagated Spathiphyllum floribundum Schott Petite and Calathea louisae Gagnep Maui Queen. During acclimatization of both plants catalase activity increased, reaching a maximum 4 weeks after transplantation, while total superoxide dismutase activity increased with plant growth reaching a maximum in the 24th week. Variations in the pattern of catalase and SOD isoforms were observed; a second Mn-SOD band appeared in Spathiphyllum 12 weeks after transfer from in vitro, while in Calathea plants an additional Mn-SOD form was present from the second until the fourth week after transplantation. The observed changes reflect the plants' capacity to develop antioxidant mechanisms during acclimatization. These findings indicate that the adaptation of micropropagated plants to ex vitro conditions is more extended in time than generally accepted.     

Orientation of the porphyrin ring in artificial chlorophyll membranes

Summary A sensitive photometric method is described by which the dichroism of lipid bilayer membranes in aqueous phase can be measured. The method is applied to black films with incorporated chlorophylla andb. With chlorophylla a relatively large dichroism is found in the Soret band and a much weaker dichroism in the red band. From the experimental data, the angles _B and _R between the blue and red transition moments and the membrane can be obtained. _B and _R are then used to calculate the angle of the porphyrin ring with respect to the membrane surface. For chlorophylla and three different lipids, values of between 44 and 49° are found.     

Geographie variation of α-amylase, β-amylase, β-glucanase,pullulanase and chitinase activity in germinating Hordeum spontaneum barley from Israel and Jordan

The enzyme activities of -amylase, -amylase, -glucanase, pullulanase and chitinase were determined in extracts of wild barley (Hordeum vulgare ssp. spontaneum) germinated for five days under axenic standard conditions. The material comprises 257 accessions, for 242 of which the botanical territory of origin in Israel or Jordan is known. The enzyme activities based on soluble protein in the extracts showed significant differences (P<0.001) among the eleven territories. The territorial moisture parameters mostly correlate with the enzyme activities. As determined by one gene or oligogenes, the significant territorial differences and the correlation with moisture are thought to reflect natural selection of genes responsible for favourable activity, or of genes linked to the enzyme coding loci, or in a coadaptive manner, of physiologically allied genes. Genes for high -glucanase activity at germination are probably coadaptive with genes for high -glucan content of the grain. The generally low starch content of wild barley grains probably makes any high -amylase activity unnecessary at the germination stage. An inverse relationship appears between -glucanase and chitinase activity; these two enzymes are also pathogenesis related proteins.     

Pitfalls in ecto-5''-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages

Summary We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killedEscherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5-nucleotidase onto the adhering particle was fact or artifact, we localized 5-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.     

Influence of fixation and buffer treatment on the release of enzymes from the plasma membrane

Summary 1. Pretreatment of frozen cryostat sections with formaldehyde or calcium ions inhibits diffusion of the plasma membrane enzymes 5-nucleotidase, ATP-ase and alkaline phosphatase during incubation. 2. Treatment of fixed sections with different kinds of buffer at 37°C induces diffusion of enzyme activity from the plasma membrane to other sites of the section and into the incubation medium. This buffer influence depends on temperature: at 4°C only a slight diffusion occurs. Addition of phospholipase C, digitonin or taurocholate to the buffer opposes the buffer effect. 3. Pretreatment of frozen cryostat sections with a mixture of equal parts of chloroform and acetone gives a good fixation of the plasma membrane enzymes 5-nucleotidase, ATP-ase, alkaline phosphatase and leucyl--naphthylamidase. During this treatment the different kinds of lipids present in the membrane are extracted equally. After this fixation buffer treatment does not cause a visible diffusion of enzyme activity in the section. Only a slight diffusion (1 till 7 percent) into the buffer solution takes place. 4. The mentioned treatments open up possibilities to get insight into the membrane anchorage of plasma membrane enzymes.     

Comparison of the carbohydrate moieties of recombinant soluble Fcε receptor (sFcε RII/sCD23) expressed inSaccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFcRII/sCD23) was produced inSaccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFcRII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(2–3)Gal(1–3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only -mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.     

Thermal stability of soluble mitochondrial H+-ATPase

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the -helix of H⁺-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20–40 C. Above 40 C, the pattern of thermal decomposition of the three-dimensional structure of H⁺-ATPase is independent of the nature of nucleotides present. Highly stable -helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

An explication of ‘wind illness’ in Northern Thailand

Wind illness is a very common complaint among the Northern Thai, yet is rarely recognized by Thai physicians trained in biomedicine. Persons most susceptible to wind illness are adult women who have ever borne a child. Consequently, data were obtained from 415 everparous women, 43% of whom reported ever having had wind illness and 57%, never having had it. In addition, 20 individuals who had ever had the syndrome were followed for case study, and 13 indigenous healers who traditionally treat clients suffering wind illness were interviewed. Their perceptions of the etiology, symptomatology and treatment of wind illness are reported in Part I. Part II is an attempt to define wind illness in terms of biomedicine and as a consequence of fertility. Part III synthesizes the emic and etic accounts with explanations for the perdurance of wind illness despite the advances of biomedicine and the recent fertility decline in Northern Thailand.     

Localization of adenylate cyclase and 5′-nucleotidase activities in human thyroid follicular cells

Summary The localization of adenylate cyclase and 5-nucleotidase activities in the follicular cells of adenomatous goiter and normal thyroid was studied by light and electron microscopy. Simultanous biochemical measurement for both activities was carried out to confirm the histochemical findings. Adenylyl-imidodiphosphate (AMP-PNP) was used as an effective substrate for adenylate cyclase. The specificity of the adenylate cyclase reaction was also examined by adding oxalacetic acid or PCMB as an adenylate cyclase inhibitor, and by adding sodium fluoride or TSH as an adenylate cyclase stimulator to the reaction mixture. In the case of tissue from adenomatous goiter, a large amount of the reaction product of the adenylate cyclase activity was found uniformly in the apical and lateral plasma membrane and not in the basal plasma membrane. In the cases of normal thyroid, a small amount of the reaction product of adenylate cyclase activity was demonstrated, and only in the lateral plasma membrane of the follicular cells. On the other hand, the histochemical localization of 5-nucleotidase activity was the same in adenomatous goiter and normal thyroid. The reaction product of 5-nucleotidase activity was found predominantly in the apical plasma membrane of the follicular cells. The biochemical findings indicated that the activity of adenylate cyclase per gram tissue was approximately 2 times higher in the case of adenomatous goiter than that in the case of normal thyroid, while the 5-nucleotidase activity in adenomatous goiter was in slightly higher level than in normal thyroid. Thus the histochemically demonstrable amount of adenylate cyclase and 5-nucleotidase reflected the activity levels measured biochemically. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that this structure may not play any important role in TSH reception.     

Steady state changes in mitochondrial electrical potential and proton gradient in perfused liver from rats fed a high fat diet

In this work the protonmotive force (p), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non acqueous solvents.Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar p to that found in rats fed a low fat diet, but when we consider the two components of p, we find a significant decrease in mitochondrial/cytosolic pH difference (pH_m) and a significant increase in mitochondrial membrane potential (_m) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in pH_m and _m, which represent the respective driving force for their transport.The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.     

High levels of soluble IFN γ receptor α chain in the plasma of rheumatoid arthritis patients

Soluble receptors for hormones and cytokines have beendescribed. They can serve as natural blockers of theirrespective ligands. The natural soluble interferongamma receptor (sIFNR) has been isolated andcharacterized only in urine. Chromatography of human(hu) plasma from rheumatoid arthritis (RA) patientsand controls on immobilized hu IFN orantibodies against IFN R chainpermitted us to isolate the sIFNR. Thereceptor isolated from one control is a protein witha molecular weight between 60-67 kDa depending on thepresence of reducing agents. We detected asignificantly higher level of plasma sIFNR inpatients with rheumatoid arthritis than in apparentlyhealthy subjects.     

Heritable variation in the phaseolin protein of nondomesticated common bean,Phaseolus vulgaris L.

Summary Crude protein extracts from single seeds of nondomesticated Mexican bean accessions were analysed by SDS polyacrylamide gel electrophoresis for variability in phaseolin protein. Six new phaseolin types; M1, M2, M3, M4, M5, M6, which contained polypeptides within the same range of molecular weights (51,000 to 45,000 daltons) as occur in the S, T and C phaseolin types of cultivated beans were identified. No T and C types were found among the non-domesticated Mexican accessions, and the S type occurred in less than 7% of the seeds screened. Genetic analyses of F₂ progenies from crosses between Sanilac (S), and five of the M types showed that each M phaseolin phenotype was allelic to the S type and expressed codominantly.     

Export and activity of hybrid FhuA''-''Iut receptor proteins and of truncated FhuA'' proteins of the outer membrane of Escherichia coli

Summary The FhuA protein in the outer membrane of Escherichia coli serves as a multifunctional receptor for the phages T5, T1, 80, for colicin M, for ferrichrome (Fe³⁺-siderophore) and for the structurally related antibiotic, albomycin. To determine structural domains required for these receptor functions and for export, a fusion protein between FhuA and Iut (receptor for Fe³⁺-aerobactin and cloacin DF13) was constructed. In the FhuA-Iut hybrid protein, 24 amino acids of FhuA were replaced by 19 amino acids, 18 of which were from Iut. The number of plaque forming units of phage T5 and T1 on cells expressing FhuA-Iut was nearly as high as on cells expressing plasmid-encoded wild-type FhuA. However, 10⁷-fold higher concentrations of phage 80 and 10³ times more colicin M were required to obtain a zone of growth inhibition. Truncated FhuA proteins in which the last 24 amino acids at the carboxy-terminus were replaced by 16 (FhuA2) or 3 (FhuAT) amino acids could hardly be detected on polyacrylamide electrophoretograms of outer membrane proteins, due to proteolytic degradation. Sensitivity of cells expressing FhuA2 to phage T5 and T1 was reduced by several orders of magnitude and sensitivity to phage 80 and colicin M was totally abolished. In contrast, cells expressing FhuAT were nearly as sensitive to phage T5, T1, and 80 and to colicin M as cells containing FhuA-Iut. None of the constructs could grow on ferrichrome as sole iron source and none was sensitive to albomycin. Ferrichrome did not inhibit binding of T5 to TonB^– cells expressing FhuA-Iut (as it did in FhuA⁺ cells) due to the lack of ferrichrome binding. It is concluded that very small deletions (relative to the size of FhuA with 714 amino acids) at the C-terminal end render FhuA susceptible to proteolytic cleavage. The C-terminal alterations affect sensitivity to FhuA-specific agents very differently. Apparently, the C-terminus is a very important part of FhuA regarding stability and activity. Expression of active FhuA and partially inactive FhuA derivatives in the same cell revealed no negative complementation, suggesting a FhuA monomer as functional unit.     

Conservation status of parrot populations in an Atlantic rainforest area of southeastern Brazil

A census of four species of syntopic parrots was carried out using distance sampling methods on São Sebastião island, SE Brazil. Most of the 33593 ha island is covered by mature and secondary Atlantic rainforest. Almost 80% of these forests are within the Ilhabela Park. Although the species counted have marked differences in size and weight, density (individuals/km²) and estimated population size in 23500 ha of well-preserved forests were similar: Amazona farinosa (13.82±5.94; 3247±1395), Pionus maximiliani (15.79±7.04; 3712±1654), Brotogeris tirica (15.05±4.87; 3537±1143) and Pyrrhura frontalis (13.06±5.53; 3068±1298). Encounter rates of Forpus crassirostris and Pionopsitta pileata were very low, which suggests that there is only a small population of these species on the island. The São Sebastião forests still support healthy populations of parrots. Although woodpecker population estimates on the island are large enough to provide nesting sites for parrots, competition for holes with other secondary cavity nesters such as toucans, flycatchers and tytiras, and the selective cutting of dead trees for canoe construction, which is a common practice on the island, may limit hole availability for parrots.     

Electron microscopic localization of 5''-nucleotidase in the stratum intermedium and ameloblasts

Synopsis 5-nucleotidase was demonstrated at the fine structural level in the stratum intermedium and ameloblasts of the first mandibular molars of CD-1 mice. The enzyme was localized with the Wachstein & Meisel (1957) method along the plasma membranes of the cells of the stratum intermedium and ameloblasts. While 5-nucleotidase was present throughout the stratum intermedium, only the proximal region of the plasma membranes of ameloblasts was demonstrably active for this enzyme. 5-Nucleotidase has been implicated in transport of metabolites across cell membranes, and its localization in the present study supports this implication as well as the transport functions of the stratum intermedium and the stratum intermedium-ameloblastic interface.