Temperature and metal ions-dependent activity of the family I inorganic pyrophosphatase from the swine roundworm Ascaris suum.

Temperature dependence, heat stability and metal ions-dependent activity were examined on the Family I inorganic pyrophosphatase (PPase) recently identified from Ascaris suum. Recombinant A. suum PPase (rAsPPase) showed an optimal activity at 55 degrees C. The rAsPPase was heat stable at 40 degrees C in the absence of added Mg(2+) and at 50 degrees C in its presence. The enzyme required divalent metal ions for its activity. The preferences for the metal ions (5 mM concentration) were in the order: Mg(2+)> Co(2+)> Cu(2+)> Fe(2+)> Zn(2+)> Mn(2+). On the contrary, enzyme activity was inhibited by Ca(2+). These findings suggest that catalytic features of AsPPase are consistent with the Family I PPases reported from a wide range of organisms.

Mice intranasally immunized with a recombinant 16-kilodalton antigen from roundworm Ascaris parasites are protected against larval migration of Ascaris suum.

Protective immunity to the pig roundworm, Ascaris suum, has been demonstrated by immunization of pigs with antigens derived from the parasite's larval stages. We identified a protective antigen commonly expressed in the human and pig Ascaris infections as a 16-kDa protein (As16), which has no similarity at the amino acid level to mammalian proteins but has some similarity to those of the filarial parasites and Caenorhabditis elegans gene product. Localization analysis revealed that the native As16 was highly expressed in the adult worm intestine, hypodermis, and cuticles. In addition, As16 was detected in the parasite excretory and secretory products. Mice intranasally vaccinated with Escherichia coli-expressed recombinant As16 (rAs16), coupled with cholera toxin B subunit, generated a significant increase in the level of rAs16-specific immunoglobulin G (IgG) and IgE in serum. Mucosal IgA levels were also increased. The recombinant protein evoked a mixed (both Th1 and Th2) type of immune response characterized by elevated levels of gamma interferon and interleukin-10 in the culture supernatants of activated spleen cells. An increased level of IgG1 and IgG2a in serum was also observed. The vaccinated mice showed a reduction by 58% in the recovery of challenged larvae compared to a nonvaccinated control. These results suggest the possibility of developing a mucosal vaccine for human and pig ascariasis.

Inorganic pyrophosphatase in the roundworm Ascaris and its role in the development and molting process of the larval stage parasites.

Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho-phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli-expressed recombinant enzyme has a specific activity of 937 micro mol Pi.min-1.mg-1 protein corresponding to a kcat value of 638 s-1 at 55 degrees C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose-dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.

Cloning and characterisation of a highly immunoreactive 37 kDa antigen with multi-immunoglobulin domains from the swine roundworm Ascaris suum.

Antigens from larval stages of Ascaris suum have been shown to induce protection against challenge infection with infective A. suum eggs. We previously identified several antigens that reacted strongly with serum from pigs inoculated with infective eggs containing L3. In this study, we isolated an antigen with a molecular mass of 37 kDa and a pI of 4.8 (As37) from A. suum infective eggs using two-dimensional electrophoresis, and obtained a full-length cDNA by reverse transcription-polymerase chain reaction using primers designed based on the internal amino acid sequence of As37. The cDNA sequence consisted of 1,540 bp coding for a protein of 321 amino acids with a complex domain organisation. Simple modular architecture research tool (SMART) analysis indicated that As37 contains three immunoglobulin domains, indicating that it is a member of immunoglobulin superfamily (IgSF). A homology search of GenBank showed that As37 has significant similarity to Caenorhabditis elegans DIM-1 protein and has low similarity to part of the multi-repeat Ig domain from nematode twitchin and mammalian skeleton muscle titin, and to members of the IgSF at the amino acid sequence level. Localisation analysis revealed that antibodies to Escherichia coli-expressed recombinant As37 (rAs37) bound to muscle cells and the hypodermis. The antibodies identified a 37 kDa native antigen in human and dog roundworms, suggesting that there are As37 homologues in ascarid nematodes. Sera from mice, rabbits and pigs immunised with A. suum infective eggs reacted with rAs37 in immunoblot analyses. The potential use of rAs37 for protection against A. suum infection is discussed.

Lung-stage protein profile and antigenic relationship between Ascaris lumbricoides and Ascaris suum.

The protein profile and antigenic properties of lung-stage larvae of Ascaris lumbricoides and A. suum were studied using 2-dimensional electrophoresis and immunoblot analysis, respectively. The protein profiles of the 2 parasites were identical except for the presence of only 1 major protein spot specific for each. There was a complete cross-reactivity between the 2 parasites at the immunological level, and no specific antigen was recognized using specific antibody raised against the 2 parasites in rabbits.

Species-specific proteins identified in Ascaris lumbricoides and Ascaris suum using two-dimensional electrophoresis.

The protein profile of adult female Ascaris lumbricoides and Ascaris suum originating from humans and pigs, respectively, was studied using two-dimensional polyacrylamide gel electrophoresis. Six different major protein spots specific for A. lumbricoides were identified irrespective of their geographical origin and no major specific spot was encountered in A. suum. No major differences in the protein profiles between the extract by phosphate-buffered saline and urea were encountered for either Ascaris species. It is therefore possible to use 2D-PAGE as a tool for discriminating the closely related Ascaris species from humans and pigs.

[The current status and problems of nursing for outpatients department of oral surgery of dental university hospitals in Japan].

Recently, the need for outpatient nursing services attached to general clinics has increased because of the shortening of hospitalization periods and the promotion of home care. The number of outpatients in the field of oral surgery is expected to continue increasing as the number of elderly patients increases and hospitalization periods decrease; therefore, the quality of outpatient nursing services must be improved. In the present study, a questionnaire was distributed to dental university hospitals, to clarify the present condition of outpatient nursing services in the department of oral surgery. The results of the survey showed that nurses routinely provide treatment at oral surgery outpatient clinics, where they perform pre-hospitalization orientations, consultations, and instruction and guidance for patients. Outpatient nurses must be able to help a wide range of patients, from children to the elderly; the contents of the consultations, instructions and guidance performed by the nurses, are also wide-ranging, including social life issues and oral disability rehabilitation. Thus, the various types of support care performed by nurses play an important role in the efficacy of oral surgery outpatient clinics, by contributing to maintenance and improvement of patients' social life and quality of life. However, the survey also found that outpatient services sometimes suffer from a lack of resources, including personnel and facilities. Furthermore, a fee-charging system for nursing services does not presently exist. Active cooperation between dental professionals and oral surgery clinic nurses, and strong appeals to hospital administrative departments, are thus necessary to resolve these problems.

[A study on the relationship between the actual situation of nursing activities, in the outpatient section of oral surgery, and medical fees].

In our outpatient oral surgery section, nurses have performed such nursing activities as guidance and consultation for outpatients. The present survey was conducted to clarify the actual situation of nursing activities, and that of claims for medical fees during fiscal year 2000, the year the claim for medical fees was started. At that time, the problems involved were examined to further improve nursing activities for outpatients. The nurses gave guidance on alimentation, oral hygiene, and breast feeding, from the nurse's record which were classified and totalled according to each disease, guidance item, and time required (less than 30 minutes or more than 30 minutes). The total numbers of patients were 172 persons and 357 cases. Major guidance items were alimentary guidance (179 cases), oral hygiene guidance (105 cases), and breast feeding guidance (73 cases). The items claimed for medical fees were two: "Athrocytosis function therapeutics" (9 cases) and "the guidance fee for the treatment of specific dental disease" (32 cases). The problems that discouraged claiming a fee for performed chargeable nursing activities are listed as 1. there are no exclusive guidance rooms, 2. insufficiency in securing staff, 3. the instructions and the requests by dentists have not been made exactly and smoothly. Thus, the problems to be considered hereafter would be 1. review and improvement in the guidance environment to an appropriate level, 2. make regulations for securing necessary staff, 3. construction of a system for exact and smooth instruction and requests, and 4. promotion to enrich the content of guidance and to make the results public.