Glycation and phosphorylation of alpha-lactalbumin by dry heating: effect on protein structure and physiological functions.

Alpha-lactalbumin (alpha-LA) was glycated with maltopentaose (MP) through the Maillard reaction (MP-alpha-LA) and subsequently phosphorylated by dry heating in the presence of pyrophosphate to investigate its structure and physiological functions. Glycation occurred effectively, and the sugar content of alpha-LA increased by approximately 22.3% through the Maillard reaction. The phosphorylation of MP-alpha-LA was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorous content of MP-alpha-LA increased by approximately 1.01% by dry heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of alpha-LA increased with an increase in the phosphorylation level. The circular dichroism spectra showed that the change in the secondary structure of the alpha-LA molecule by glycation and subsequent phosphorylation was slight. However, the Trp fluorescence intensity was increased by phosphorylation after glycation. In addition, the differential scanning calorimetry thermograms of alpha-LA showed that the denaturation temperature of MP-alpha-LA was decreased by phosphorylation. These results indicated that molten (partially unfolded) conformations of alpha-LA were formed by dry heating in the presence of pyrophosphate after glycation. The anti-alpha-LA antibody response was significantly reduced by glycation and subsequent phosphorylation. The suppressive effect of alpha-LA on the production of proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha from THP-1 cells after stimulation with lipopolysaccharide was significantly enhanced by glycation with MP and was further enhanced by phosphorylation after glycation. The Ca phosphate-solubilizing ability of alpha-LA was enhanced by phosphorylation. The apoptotic activity of alpha-LA was reduced by glycation and subsequent phosphorylation. These results suggest that phosphorylation by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction is a useful method for improvement of the physiological functions of alpha-LA.

3D morphological assessment of occlusal treatment by measuring dental casts with a micro-focus X-ray CT.

The analysis of occlusal relationship is important for the success of dental treatment. Three-dimensional (3D) computer models of upper and lower dental casts can play a significant role. In this study, we proposed and applied a new method in actual clinical assessment to measure dental casts with occlusal relationship by using a micro-focus X-ray CT system. We examined the modelling accuracy by comparing multiple 3D images taken by shifting the dental cast position. Modelling accuracy was confirmed as 0.03 mm. One occlusal treatment in clinical practice was selected as a case example. The dental casts and bite impression, taken before treatment, were scanned and the occlusal contacts and distance distribution between the upper and lower casts were visualized by a coloured map and overlaid on the computer models. Distances between the upper and lower casts of selected points were compared before and after the treatment. Initially, the subject had early contact on the anterior teeth, where distance was measured as 0.04 mm, and only one area measured less than 0.15 mm. After treatment, five areas measured less than 0.15 mm. Also, by comparing the dental cast models taken before and after occlusal adjustment of the tooth, the position and amount of adjustment were visualized. We successfully demonstrated the quantitative clinical assessment of occlusal treatment.

Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate.

Bovine serum albumin (BSA) was phosphorylated by 2 methods. One is dry-heating in the presence of pyrophosphate, and the other is conjugation with maltopentaose through the Maillard reaction and subsequent dry-heating in the presence of pyrophosphate. The phosphorus content of BSA was increased to approximately 0.45% by dry-heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate, and approximately 0.91% by glycation and subsequent phosphorylation. The circular dichroism spectra showed that the change of secondary structure in the BSA molecule by phosphorylation was mild. However, tryptophan fluorescence intensity of BSA decreased by phosphorylation. The differential scanning calorimetry thermograms of BSA showed a disappearing of the 1st peak and a lowering of the 2nd peak denaturation temperature by phosphorylation. These results indicated molten (partially unfolded) conformations of BSA formed by both phosphorylation methods. The functional properties of BSA such as heat stability and calcium phosphate solubilizing ability were improved by phosphorylation alone and further by phosphorylation after glycation. Transparent gels of BSA with relatively high water-holding capacity were obtained by phosphorylation alone, and the immunogenicity of BSA was reduced significantly by glycation and phosphorylation, respectively.

Improvement of functional properties of whey protein isolate through glycation and phosphorylation by dry heating.

Whey protein isolate (WPI) was glycated with maltopentaose (MP) through the Maillard reaction, and the MP-conjugated WPI (MP-WPI) was then phosphorylated by dry heating in the presence of pyrophosphate. Glycation occurred efficiently, and the sugar content of WPI increased approximately 19.9% through the Maillard reaction. The phosphorylation of MP-WPI was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorus content of WPI increased approximately 1.05% by dry heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of WPI increased with an increase in the phosphorylation level. The stability of WPI against heat-induced insolubility at pH 7.0 was improved by conjugation with MP alone, and further improved by phosphorylation. Although the emulsifying activity of WPI was barely affected by glycation and phosphorylation, the emulsifying stability of phosphorylated MP-WPI (5 d), was 2.2 times higher than that of MP-WPI. Gelling properties such as hardness, resiliency, and water-holding capacity of heat-induced WPI gel were markedly improved, and the gel was rendered transparent by phosphorylation. The calcium phosphate-solubilizing ability of WPI was enhanced by phosphorylation. These results suggested that phosphorylation by dry heating in the presence of pyrophosphate after conjugation with MP is a useful method for improving the functional properties of WPI.

In-vitro uptake of vitamin A by Plasmodium falciparum.

The vitamin-A uptake of Plasmodium falciparum was investigated by culturing a standard isolate of the parasite (FCR-3) with (3)H-labelled vitamin A, at concentrations of the vitamin corresponding to those normally present in human serum. The (3)H-labelled vitamin A accumulated in the parasites from each culture in a parasitaemia-dependent manner. The radioactivity detected in the parasites increased with parasite maturation from the ring to the late-trophozoite stage. In addition, most of the radioactivity incorporated into the parasite cells was in the cytoplasm. The accumulation of vitamin A in the cytoplasm of late trophozoites indicates that P. falciparum may use vitamin A, from its human host, as an antioxidant, to protect itself from oxidative stress while intra-erythrocytic. The amount of the vitamin taken up by the parasite in vitro is small compared with the deficit that sometimes causes severe hypovitaminosis A in malaria cases. Consumption of vitamin A by the parasites together with the systemic decreases in non-enzymatic antioxidants that are seen in malaria may together cause this characteristic hypovitaminosis.

Effect of calcium antagonists, calcium channel blockers and calmodulin inhibitors on the growth and encystation of Entamoeba histolytica and E. invadens.

The effects of calcium antagonists, calcium channel blockers, and calmodulin inhibitors on the growth of Entamoeba histolytica and the growth and encystation of Entamoeba invadens were examined. Calcium chelators ethyleneglycol bis (beta-aminoethyl ether)-N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA) inhibited the growth of both Entamoeba and also the encystation of E. invadens in a dose-dependent manner, with EDTA being more effective than EGTA. A putative antagonist of intracellular calcium flux, 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited both growth and encystation, with the E. histolytica being more sensitive than E. invadens, and with the growth of E. invadens being more sensitive than encystation. The slow Na+-Ca2+ channel blockers bepridil and verapamil inhibited both growth and encystation. Bepridil was more effective than verapamil. The calmodulin (CaM) inhibitors, W-7 (N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide) and trifluoperazine (TFP), were also inhibitory for both the growth and encystation; TFP was more effective than W-7, and encystation was more sensitive than growth in E. invadens. These results indicate that extracellular calcium ions, amebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the growth and encystation of Entamoeba.

Entamoeba invadens: enhancement of excystation and metacystic development by cytochalasin D.

Effects of three actin-modifying drugs, cytochalasin D, latrunculin A, and jasplakinolide, on the excystation and metacystic development in vitro of Entamoeba invadens were examined by transfer of the cysts to growth medium with the drugs. Cytochalasin D unexpectedly increased the number of metacystic amoebae of E. invadens strain IP-1 during incubation. Metacystic development, which was determined by the number of nuclei of metacystic amoebae, was faster in the culture with cytochalasin D than in the culture without the drug. These results suggest that cytochalasin D enhances the excystation and metacystic development. In contrast, latrunculin A and jasplakinolide inhibited these process. No excystation occurred in encystation medium even in the presence of cytochalasin D, suggesting that growth medium is essential for excystation. Excystation was further enhanced when the cysts were incubated with cytochalasin D before culture in growth medium with the drug. The enhancing effect of cytochalasin D on the excystation and metacystic development was abrogated by jasplakinolide. Thus, the results indicate that cytochalasin D, unlike latrunculin A and jasplakinolide, caused enhancement of the excystation and metacystic development of this parasite.

How does heme axial ligand deletion affect the structure and the function of cytochrome b(562)?

We have recently generated a new mutant of cytochrome b(562) (cytb(562)) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb(562)). The M7A cytb(562) can bind heme and the UV-visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the lack of Met7 ligation on the structural integrity of cytb(562), thermal transition analyses of M7A cytb(562) were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb(562) is comparable to the apoprotein despite the presence of heme. On the other hand, exogenous ligands such as cyanide and azide ions are readily bound to the heme iron, indicating that the axial coordination site is available for substrate binding. The peroxidase activity of this mutant is thus examined to evaluate new enzymatic function at this site and M7A cytb(562) was found to catalyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the heme iron is crucial for the stable folding of cytb(562), whereas the functional conversion of cytb(562) is successfully achieved by the loose folding together with the open coordination site.

Inhibition of encystation of Entamoeba invadens by wortmannin.

Using an axenic encystation system in vitro, we examined the effect of wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), which is a signaling molecule responsible for numerous cellular responses, on the encystation of Entamoeba invadens. Wortmannin inhibited both encystation and growth of E. invadens strain IP-1 in a dose-dependent manner, the former being more resistant to the drug than the latter. There was little decrease in the number of trophozoites after 3 days of culture in encystation medium containing wortmannin; and the cells remained motile, suggesting that the inhibitory effect of the drug on encystation was not due to its toxic effect on trophozoites. The addition of wortmannin after the induction of encystation was also inhibitory for encystation. Trophozoites incubated for 1 day in encystation medium with wortmannin did not encyst after removal of the drug, suggesting that the drug effect was not reversible in encystation medium. In contrast, trophozoites cultured in growth medium with wortmannin did encyst after their transfer to encystation medium without the drug. Encystation with wortmannin was more strongly inhibited among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. The process of cyst maturation was slightly affected by wortmannin. These results suggest a possible role for PI 3-kinase in the signaling involved in the encystation of E. invadens.

Modifications of Kohn's chlorazol black E staining and Wheatley's trichrome staining for temporary wet mount and permanent preparation of Entamoeba histolytica.

Preparation of stained smears of Entamoeba histolytica has several drawbacks. We therefore tried to simplify the staining procedures by modifing Kohn's chlorazol black E staining and Wheatley's trichrome staining techniques. Trophozoites and cysts of axenically cultured E. histolytica and Entamoeba invadens, respectively, and trophozoites and cysts of E. histolytica in stools of patients were used. Karyosomes and peripheral chromatin of nuclei and chromatoid bodies became distinctly visible after amoebae were suspended in the basic solution of Kohn's stain. Amoebae fixed in suspension with either basic solution or Bouin's fixative were clearly stained with Kohn's and trichrome preparations, both as wet mounts directly and as permanent slides after processing for mounting. These procedures were easier when the basic solution was used as a fixative and trichrome stain was employed. Erythrocytes ingested by trophozoites, however, were not stained with either of these preparations after fixation in the basic solution but were clearly stained when Bouin's fixative was used. Cysts of E. histolytica in stools concentrated using basic solution (instead of formalin) and ether were also stained with these stains. Consequently, without employing highly toxic mercuric chloride, wet mounts and permanent smears can be prepared with permanent stains, and preserved cysts can be stained after concentration.

Effect of jasplakinolide on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens.

The effect of jasplakinolide. an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 microM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.

[Studies on current trend of imported malaria in Japan--pediatric cases in recent 20 years].

Imported malaria has been increasing according to the recent globalization of Japan. There are about 120 clinical cases of malaria which include a few pediatric cases (approximately 1%) every year. Generally, pediatric cases often have an atypical onset and course compared to adult cases, and also develop serious and fatal effects in a short time. In this study, we examined imported malaria cases in subjects under 15 years old from 1980 to 1999 conducted by Research group on clinical evaluation against orphan drugs in the treatment of imported tropical diseases and parasitic diseases. During the 20 years we found 44 clinical cases in children. Of these 70% were foreign cases. Among the species of parasites, there were 21 cases of Vivax malaria and 17 cases of Falciparum malaria and a few cases of Malariae and Ovale malaria were also found, which is rare even in adults. Concerning the drugs chosen in Japan for chemotherapy to treat malaria, chloroquine and primaquine seemed to be employed most frequently before 1990, however mefloquine or artesunate seemed to be more common after 1990. Also, most pediatric cases were former residents or refugees from tropical countries, however some cases were in Japanese children who had recently visited those areas with their families. There have been no fatalities in pediatric cases of malaria, however tropical diseases, including malaria, must be rule out, when examining pyretic children, considering the number of travelers going abroad has been increasing.

Effect of the antitubulin drug oryzalin on the encystation of Entamoeba invadens.

We have recently demonstrated that the antimicrotubule drug oryzalin inhibits the growth of Entamoeba invadens as well as E. histolytica, the former being more resistant to the drug than the latter, and that effective doses of oryzalin are higher for Entamoeba than for the other parasitic protozoa examined thus far. The aim of the present study was to examine the effect of oryzalin on the encystation of E. invadens using an axenic encystation system in vitro. Oryzalin inhibited the encystation of E. invadens strain IP-1 in a dose-dependent manner. The addition of oryzalin after the induction of encystation was also inhibitory for encystation and cyst maturation. Trophozoites incubated for 1 day in encystation medium with oryzalin did not encyst after removal of the drug. Although trophozoites grown in the presence of 300 microM oryzalin for 2 days did not encyst after their transfer to encystation medium containing the same concentration of drug, a number of trophozoites survived for at least 3 days. In contrast, trophozoites grown in the absence of oryzalin neither survived nor encysted after their transfer to encystation medium supplemented with the drug, which suggests that pretreatment of trophozoites with oryzalin contributes to their continued survival as trophozoites, i.e., without their transforming into cysts, in encystation medium. Trophozoites grown with oryzalin did encyst after their transfer to encystation medium without the drug. Accumulation of trophozoites in the mitotic phase was observed after culture with oryzalin. When cysts prepared at day 1 of encystation, most of which were mononucleate, were reincubated in the presence of oryzalin for an additional 2 days, inhibition of their maturation was observed. Thus, oryzalin is a potent mitotic-phase inhibitor of E. invadens and may become a useful tool for studies on the relationship between the cell cycle and encystation of this parasite.

Effect of cytochalasin D on the growth, encystation, and multinucleation of Entamoeba invadens.

The effect of cytochalasin D, a specific inhibitor of microfilaments, on the growth, encystation, and multinucleation of Entamoeba invadens was examined. Cytochalasin D blocked the growth of axenic E. invadens strain IP-1 in a dose-dependent manner, which suggests that the drug is effective against this species of Entamoeba as well as against E. histolytica strain HM1: IMSS as previously demonstrated. Encystation of E. invadens as induced in vitro was also inhibited by cytochalasin D. This is the first evidence of the participation of microfilaments in the encystation process. Concentrations of cytochalasin D effective for the inhibition of encystation were lower than those effective for the inhibition of growth. Trophozoites grown with cytochalasin D became multinucleate; more than three nuclei per cell were observed in 71% of trophozoites grown in the presence of the drug as opposed to only 5% of those grown in the absence of the drug. Also, trophozoites grown with cytochalasin D produced multinucleate cysts following their transfer to encystation medium. Encystation with cytochalasin D was more strongly inhibited among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. Also, encystation without cytochalasin D was less frequently observed among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. Thus, the multinucleation of trophozoites induced by cytochalasin D had an inhibitory effect on their encystation.

Effect of dinitroaniline herbicides on the growth of Entamoeba histolytica.

The effect of the dinitroaniline herbicides oryzalin and trifluralin on the growth of Entamoeba histolytica was examined. Oryzalin inhibited the growth of E. histolytica strain HM-1:IMSS. Trifluralin was less effective than oryzalin for this parasite. Entamoeba histolytica was more resistant to these dinitroanilines than other parasitic protozoa examined so far, including Leishmania spp., Trypanosoma brucei, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum. Colchicine, a potent microtubule inhibitor of animal cells, was much less effective for E. histolytica, even at very high concentrations. A reptilian parasite, Entamoeba invadens strain IP-1, examined for comparison, was more resistant to these dinitroanilines than E. histolytica. Accumulation of E. histolytica trophozoites in mitosis was observed after culture in 100 microM oryzalin. The inhibitory effect of oryzalin on the growth of E. histolytica trophozoites was abrogated by removal of the drug after exposure to 100 microM for 2 days. In parallel to the recovery of growth after removal of the drug, the percentage of trophozoites in mitosis was reduced to a normal level. The results indicate that treatment of trophozoites with oryzalin arrests mitosis and that its effect is reversible. Therefore, oryzalin is a useful tool for studies relating to the cell cycle of this parasite.

Appearance of a stage-specific immunodominant glycoprotein in encysting Entamoeba invadens.

The appearance of cyst-specific proteins in encysting Entamoeba invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an axenic encystation system in vitro. A rabbit antiserum against trophozoites of E. invadens reacted with a number of proteins of cysts after 1-4 days of incubation. Thus, a number of cyst proteins remained antigenically unchanged as common antigens of the two forms after transformation from trophozoites to cysts. A rabbit antiserum against cysts also reacted with the trophozoite proteins as well as the cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific. The reactivity of the 88-kDa protein of cysts with the absorbed anticyst serum decreased as encystation proceeded. When soluble and particulate fractions prepared from cysts after 1 day of incubation were examined by electrophoresis and immunoblotting, the 88-kDa protein that had reacted with the absorbed anticyst serum was found to be present in the particulate fraction, which was rich in cell-wall fragments, and stained with periodic acid-Schiff's reagent, indicating that it is a glycoprotein. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall.