Identification of the first nucleated phase in submonolayer Ti deposited on Si(111)-7 x 7 by atomic resolution techniques.

Ti5Si4 has been identified to be the first nucleated phase in submonolayer Ti deposited on the Si(111)-7 x 7 surface by ultrahigh vacuum scanning tunneling microscopy in conjunction with atomic-resolution transmission electron microscopy. The direct observation of the formation of clusters surrounded by the heavily damaged silicon lattice strongly suggested that Si is the dominant diffusing species in forming the silicide.

The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles.

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.

Copper-induced conformational changes in the N-terminal domain of the Wilson disease copper-transporting ATPase.

The Wilson disease copper-transporting ATPase plays a critical role in the intracellular trafficking of copper. Mutations in this protein lead to the accumulation of a toxic level of copper in the liver, kidney, and brain followed by extensive tissue damage and death. The ATPase has a novel amino-terminal domain ( approximately 70 kDa) which contains six repeats of the copper binding motif GMTCXXC. We have expressed and characterized this domain with respect to the copper binding sites and the conformational consequences of copper binding. A detailed analysis of this domain by X-ray absorption spectroscopy (XAS) has revealed that each binding site ligates copper in the +1 oxidation state using two cysteine side chains with distorted linear geometry. Analysis of copper-induced conformational changes in the amino-terminal domain indicates that both secondary and tertiary structure changes take place upon copper binding. These copper-induced conformational changes could play an important role in the function and regulation of the ATPase in vivo. In addition to providing important insights on copper binding to the protein, these results suggest a possible mechanism of copper trafficking by the Wilson disease ATPase.

Schistosoma japonicum: size of egg granulomas in vaccinated and non-vaccinated hosts as observed in bovines.

The present paper reported the results of studies on the size of Schistosoma japonicum egg granulomas in susceptible and poorly susceptible, vaccinated and non-vaccinated, hosts as observed in bovines. It was noted that in both cattle and buffaloes, the mean size of the granuloma was smaller in vaccinated than in the non-vaccinated. The vaccinated bovines will accordingly not only be benefited by the reduction of the survival rate of the challenged schistosomula but also by decreasing the size of the egg granulomas.

Pharmacological characterization of muscarinic receptors in neonatal rat cardiomyocytes.

[N-methyl-3H]scopolamine methylchloride ([3H]NMS) was used to characterize the muscarinic receptors (mAChRs) in the intact cardiomyocytes. The specific binding of [3H]NMS was proportional to cell concentration, saturable with respect to [3H]NMS concentration, and time dependent. Scatchard analysis of binding isotherms showed that [3H]NMS bound to the freshly isolated and cultured cardiomyocytes with dissociation constants of 275 +/- 64 and 207 +/- 20 pM as well as maximum receptor densities of 0.13 +/- 0.09 and 5.36 +/- 0.20 fmol/10(5) cells, respectively. Heterogeneity of mAChRs was demonstrated by competitive binding experiments against [3H]NMS with M2 and M3 antagonists. These receptors (80%) exhibited high affinities for 11-([2-[(diethylamino)methyl]-1-piperidinyl]-acetyl)-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine similar to those of M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors and possessed high affinity for 4-diphenylacetoxyl-N-methylpiperidine (4-DAMP) and hexahydrosiladifenidol. On the basis of biochemical studies, AF-DX-116 blocked adenosine 3',5'-cyclic monophosphate (cAMP) inhibition with high affinity (pKB 7.4), while it antagonized inositol phosphate formation with low affinity (pKB 6.5). 4-DAMP possessed high affinity in blocking inositol phosphate formation (pKB 9.0) and low affinity for antagonism of cAMP inhibition (pKB 7.7). Although no other muscarinic receptor mRNA has been detected in these cells, these data suggest the presence of a second population of mAChRs, which may not be identical to the classical cardiac "M2" receptors.

The Hoeppli phenomenon in schistosomiasis japonica: historical findings and additional investigation in bovines.

The present paper deals with a buried knowledge of the early findings of the Hoeppli phenomenon in cattle infected with Schistosoma japonicum together with a revised list of bovines and other mammalian species in which this phenomenon has been found. It was noted that the percentage of the mature-egg granulomas with positive Hoeppli phenomena varied with the species of bovines, ie, higher positive percentage in the more susceptible cattle than in the less susceptible buffalos. The radiating filaments in fringes of the phenomenon were also stronger in cattle than in buffalos.

Autoradiographic study of the attrition of migrating schistosomula in the skin of mice. I. Mice immunized with highly X-irradiated Schistosoma mansoni cercariae.

The migratory pattern in naive mice of Schistosoma mansoni cercariae X-irradiated with OkR, 12kR, or 48rR has been investigated with compressed organ autoradiography. Three groups of 69 mice each were infected with 75Se-labelled cercariae. The skin at the site of infection, as well as the entire lungs and liver were removed from infected mice daily from 1-21d, on 24d, and on 28d; tissues were then processed for autoradiography. Autoradiographic analysis of migratory patterns showed that the schistosomula derived from nonattenuated cercariae passed quickly from the skin to the lungs and at a moderate rate from the lungs to the liver. Schistosomula derived from 12kR-irradiated cercariae were delayed in passing from the skin to the lungs and from the lungs to the liver, so that many remained in the lungs and died there, while only a few died in the skin or liver. Schistosomula derived from 48kR-irradiated cercariae remained mainly in the skin, rarely reached the lungs and were never found in the liver. This study indicates that immunization with 48kR-irradiated cercariae will cause no significant lesions in the lungs or liver.

Vaccination against schistosomiasis in mice with killed schistosomula without adjuvant.

Attempts to develop a killed vaccine against schistosomiasis have generally resulted in failure. There are two recent reports, but unfortunately, harsh adjuvants were used in conjunction with the antigenic materials. In our laboratory, a killed vaccine was developed by freezing (-196 degrees C) and thawing the schistosomula of S. mansoni. The use of such a preparation without adjuvant was effective in vaccinating mice. A worm reduction of 36.4-41.1% was achieved by one vaccinating injection, a 60.2% worm reduction by 3 injections, and a 63.7-66.0% reduction by 5 injections. The sequence of the development and the expression of the immune reactions were similar to those previously found in hosts immunized with highly X-irradiated schistosome organisms. Delayed hypersensitivity was demonstrated in histological sections of the skin in the challenged mice after one vaccination, showing that an adjuvant was not necessary to initiate the induction of cellular immunity.

Vaccination of bovines against schistosomiasis japonica with highly irradiated schistosomula in China.

Vaccination of Chinese bovines (cattle and buffaloes) against schistosomiasis japonica with 36 kR gamma-irradiated schistosomula was done for laboratory challenge and for field trials in China. Altogether, 61 bovines were used. All experimental animals were vaccinated 2-3 times with 10,000 irradiated schistosomula per time. For the laboratory challenge, all experimental and control cattle were challenged with 500 normal cercariae and each buffalo, with 2,000 cercariae. The laboratory-challenged bovines were killed after 54-57 days of challenge; the bovines for the field trial in the lightly endemic area, after 5 months in the field; and the bovines for the field trial in the heavily endemic area, after 58-63 days. When the animals were killed, the number of mature worms in the vaccinated (experimental) and non-vaccinated (control) animals was recorded and the percentage of worm reduction in each group was calculated. The first group, consisting of three vaccinated and three non-vaccinated cattle, was given a laboratory challenge; the worm reduction was 71.6%. The second group, consisting of two vaccinated and three non-vaccinated buffaloes, was also given a laboratory challenge; the worm reduction was 74.4%. The third group, consisting of seven vaccinated and eight non-vaccinated buffaloes, was utilized in a field trial in a lightly endemic area; the worm reduction was 75.6%. The fourth group, consisting of eight vaccinated and nine non-vaccinated cattle, and the fifth group, consisting of nine vaccinated and nine non-vaccinated buffaloes, were pastured in a heavily endemic area. The worm reduction was 65.1% in the fourth group and 75.7% in the fifth group.(ABSTRACT TRUNCATED AT 250 WORDS)

Vaccination against bovine schistosomiasis japonica with highly X-irradiated schistosomula.

Cercariae of the Chinese mainland strain of Schistosoma japonicum were used. Eighteen cattle, divided into six groups of three each, were immunized with schistosomula transformed from cercariae exposed to three different doses of X-irradiation (24, 36, and 48 kR). The immunization was given either once, twice, or thrice, and the number of immunizing schistosomula was 10,000 or more in each immunization. The immunized cattle were challenged with 500 normal cercariae. Five native cattle were similarly infected with normal cercariae as controls. All cattle were killed 32-33 days after challenge or infection, and the worms were obtained by perfusion. The mean worm reduction in the 18 experimental animals varied from 42.1 to 96.0%. The mean percent worm reduction of the six experimental groups varied from 54.8 to 87.1. The reduction was greater with increasing numbers of immunizations, and was higher in the groups immunized with schistosomula exposed to 36 kR than in those exposed to 24 or 48 kR. Statistical analyses showed that all immunized groups yielded significantly fewer worms than controls. However, the three doses of X-irradiation (24, 36, and 48 kR) had no significant effect for a fixed number of immunizations (1, 2, or 3). The means for both two and three immunizations were significantly different from the mean for one immunization, although they were not significantly different from each other.

Histopathological study of the attrition of challenge cercariae of Schistosoma mansoni in the skin of mice immunized by chronic infection and by use of highly X-irradiated cercariae.

To find direct evidence for the sites of death of newly transformed schistosomula of Schistosoma mansoni in the skin of challenged immunized hosts, a histopathological study of the skin of chronically infected mice and of highly X-irradiated cercaria-immunized mice was made at different post-challenge times. Attrition of infecting organisms in naive mice was used as a control. In general, three main kinds of schistosomular attrition were observed: (1) in the granulocytic exudates in the epidermis, (2) in the granulocytic aggregates in the subcutaneous tissue, and (3) in the granuloma-like foci in the subcutaneous tissue. Schistosomula killed by the epidermal granulocytic exudates and by the subcutaneous granuloma-like foci occurred in all three groups of mice, but the schistosomula killed by the subcutaneous granulocytic aggregates were found only in the highly X-irradiated cercaria-immunized animals. Schistosomula killed by the epidermal granulocytic exudates were encountered occasionally in the challenged naive mice but more frequently in both the challenged chronically infected mice and the challenged highly X-irradiated cercaria-immunized mice. The number of subcutaneous granuloma-like foci was greater in the challenged mice immunized with highly X-irradiated cercariae than in the challenged naive mice or the challenged chronically infected mice. Another differentiating character among the three groups of mice was that in the highly X-irradiated cercaria-immunized group, the subcutaneous granuloma-like foci appeared on day 3 after the challenge, but on day 5 in the naive and chronically infected groups. The results clearly indicate that skin is an important site for the attrition of the challenged cercariae in the highly X-irradiated cercaria-immunized mice, and that immunity in the chronically infected and X-irradiated cercaria-immunized mice occurs by different mechanisms.

Schistosoma mansoni: is acquired immunity induced by highly x-irradiated cercariae dependent on the size of the challenging dose?

A high degree of immunity, as shown by a 91% reduction of the number of worms recovered was found in five groups of mice that were immunized five times with highly X-irradiated cercariae and then challenged with 10, 20, 50, 100, or 500 normal Schistosoma mansoni cercariae. The results indicated that there were no significant differences in worm reduction in immunized mice challenged with different numbers of cercariae; consequently the immunity induced by this immunization method did not appear to be challenge-dose-dependent. However, the results also showed that when immunized mice were challenged with 500, 100, 50, 20, and 10 cercariae, 0, 13, 26, 56, and 68%, respectively, of the experimental animals were free of worms. Thus, the percentage of worm-negative cases increased as the number of challenge cercariae decreased. When viewed in this manner, the acquired immunity may be considered challenge-dose-dependent as well. If this method of vaccination is used for schistosomiasis control, we may anticipate that in both hypo- and hyperendemic areas, the intensity of infection and the severity of the disease will be reduced owing to a reduction in worms burdens, and in hypoendemic areas, there will be a number of worm-free cases.

Immunoglobulins and complement in the skin of rhesus monkeys immunized with x-irradiated cercariae of Schistosoma japonicum.

Skin sections of rhesus monkeys immunized with X-irradiated Schistosoma japonicum cercariae were stained by an unlabeled antibody enzyme method for the detection of IgG, IgM, IgA, IgE, and C3. While all of them could be found in the skin lesions, their distribution patterns varied with time and reactions. In whealing reaginic reactions, IgE appeared prominent, having an affinity to the mast cells. In Arthus-like reactions, IgG was predominant. IgG complexes were found on the schistosomula, on the walls of blood vessels, and in granulocytes near the schistosomula. In the late stage of Arthus-like reactions and in delayed hypersensitive reactions, IgA was predominant in granulocytes, mononuclear cells, and macrophages. Characteristics of each immunoglobulin pattern seemed to reflect its function in the effector mechanism. It may be speculated that these immunoglobulins and C3, together with effector cells, synergistically and sequentially destroy schistosomula in the skin.

Eosinophils as effector cells in the destruction of Schistosoma mansoni eggs in granulomas.

Histopathological sections of wedge-biopsied liver and surgically removed gallbladder of a schistosomiasis mansoni patient were studied for the eosinophil-mediated destruction of eggs in granulomas. Apparent degranulation of eosinophils on the eggs in the granulomas and concomittant deterioration of miracidia were observed. It was construed that granule-associated enzymes may be released upon the degranulation of eosinophils, pass through the micropores of the egg shell and cause death of the miracidium inside the egg.

Evaluation of lung recovery assay for schistosomula in mice immunized with x-irradiated cercariae of Schistosoma mansoni.

The lung recovery assay for schistosomula has recently been used as a rapid method for measuring the state of immunity of a challenged animal. Its merit rests on the fact that the peak day of schistosomular recovery in the lungs of both normal and immune hosts falls on the same day and the degree of immunity can be measured by the percent of schistosomular recovery on this particular day. An evaluation of this method for mice immunized with highly X-irradiated cercariae of Schistosoma mansoni was made. The results indicate that in the immunized mice the peak day for the schistosomular recovery was delayed and the number of schistosomula recovered on the peak day was decreased. The delay of the peak day was increased by the degree of host immunity, so its identification for a host of an unknown immunologic status was not a simple matter. In addition, this method measured only the immune effect which took place in the skin and on prepeak days in the lungs. It was not as competent as the conventional liver perfusion method which measures the total result of the immune effect of the skin, lungs, and liver. Our conclusion is that for measuring the immunizing effect of irradiated cercariae, the lung recovery method is not suitable for the determination of the state of immunity induced.