A case of nonscarring inflammatory epidermolysis bullosa acquisita: characterization of IgG autoantibodies by immunofluorescence, immunoblotting and immunogold electron microscopy

We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.     

Determination of copy number of rRNA genes in Pneumocystis carinii f. sp. hominis

Differential PCR was performed to determine the copy number of rRNA genes in Pneumocystis carinii f. sp. hominis. Two different reference genes, thymidylate synthase (TS) and beta-tubulin (BTU) genes, were used. Primers for the internal transcribed spacer (ITS) region of nuclear rRNA genes and either the TS or BTU gene were mixed together to perform PCR on seven different bronchoalveolar lavage specimens from patients with P. carinii pneumonia. The radioactivity derived from the incorporated radioactive nucleotides of each PCR product band was then used to calculate the copy number of the ITS relative to that of the TS or BTU gene. The copy number ratio between the ITS and the TS gene was determined to be 0.8, and that between the ITS and the BTU gene was also 0.8. These results suggest that the ITS has the same copy number as the TS or BTU gene. Since the copy number of the TS or BTU gene is presumed to be 1, the results also suggest that P. carinii f. sp. hominis has only one copy of the ITS and thus one copy of the nuclear rRNA genes. Therefore, two types of ITS sequences derived from a specimen would indicate that the patient is infected by two types of P. carinii f. sp. hominis.     

Identification of common and distinct residues involved in the interaction of alphai2 and alphas with adenylyl cyclase

The G protein alpha subunits, alphas and alphai2, have stimulatory and inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent conformational change that involves three alpha subunit regions (Switches I-III). alphas residues in three adjacent loops, including Switch II, specify activation of adenylyl cyclase. The adenylyl cyclase-specifying region of alphai2 is located within a 78-residue segment that includes two of these loops but none of the conformational switch regions. We have used an alanine-scanning mutagenesis approach within Switches I-III and the 78-residue segment of alphai2 to identify residues required for inhibition of adenylyl cyclase. We found a cluster of conserved residues in Switch II in which substitutions cause major losses in the abilities of both alphai2 and alphas to modulate adenylyl cyclase activity but do not affect alpha subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of alphai2 in which substitutions reduce the ability of alphai2 to inhibit adenylyl cyclase, one of which corresponds to an effector-activating region of alphas. Thus, both alphai2 and alphas interact with adenylyl cyclase using: 1) conserved Switch II residues that communicate the conformational state of the alpha subunit and 2) divergent residues that specify particular effectors and the nature of their modulation.     

Computer-assisted frontal sinusotomy

This study investigated the effects of bilateral, selective lesions of subfield CA3, produced by intrahippocampal administration of kainic acid, on the generation of hippocampal type 2 RSA. Within 4 weeks of lesioning, animals were anesthetized with urethane and microelectrode depth profiles were performed throughout the dorsal-ventral extent of the hippocampus. In control animals, spontaneous and stimulation-induced RSA was present at the amplitude maxima in stratum oriens of the CA1 and at the level of the hippocampal fissure. Animals that received intrahippocampal microinfusions of kainic acid showed a significant reduction of RSA amplitude at both the stratum oriens and fissure regions. These results suggest that the CA3 subfield may play an important role in the production of type 2 RSA.     

Laparoscopic cholecystectomy. Do preoperative factors predict the need to convert to open?

We reviewed our experience with the last 587 laparoscopic cholecystectomies performed between May 1990 and January 1993 to correlate preoperative findings that may predict the conversion of a laparoscopic cholecystectomy to that of an open procedure. The prediction of a need to convert to an open cholecystectomy would allow the surgeon to discuss the higher risk of conversion with the patient and also allow for an earlier intraoperative decision to convert if difficulty was encountered. In addition to routine demographic data, ultrasound reports were available for 526 patients and the following information was recorded: presence of stones, thickened gallbladder wall, common bile duct dilatation, gallbladder sludge, and cystic duct impaction. Overall, a two times higher rate of conversion was found for male patients and patients with a body mass index > 27.2 kg/m2. Additionally, a thickened gallbladder wall on preoperative ultrasound was correlated with a six times higher conversion rate to open cholecystectomy. As expected, the positive intraoperative cholangiogram was associated with a higher incidence of conversion. Additionally, finding a dilated common bile duct on ultrasound was found to be associated with a nearly seven times higher rate of positive intraoperative cholangiogram. No statistical significance was found between conversion and age, previous abdominal operations, the presence of stones, common bile duct dilatation, gallbladder sludge, cystic duct impaction, or a distended gallbladder. Thus, these predictive findings allow the surgeon to preoperatively discuss the higher risk of conversion and allow for an earlier judgment decision to convert if intraoperative difficulty is encountered.     

Long term clinical evaluation of three luting materials

A last critical step in the completion of a cast restoration is permanent cementation. With the development of new luting agents the practitioner has to be aware of the physical and biological properties as well as the long term clinical performance of these materials. In this study three commonly used luting agents were evaluated over a four year period. The three cements, a zinc phosphate, a glass ionomer and a resin cement were tested for their reaction to periodontal tissues, pulpal response and other clinical parameters. In twenty six patients, 61 crowns, onlays or bridge abutments were cemented. Evaluations were made at 0 hour, after 7 days, 6 and 12 months and 4 years. In general the cements performed satisfactorily. Health of periodontal tissues adjacent to the crowns improved considerably after 7 days. Postcementation hypersensitivity appeared to be a negligible problem, which was attributed to the time span between preparation and cementation, which exceeded in most instances four weeks. The choice of a final luting agent should be based on anticipated postoperative pulpal reactions, rather than exclusive reliance on physical properties.     

Clinical applications of the camptothecins

In this paper, the pattern of induction of heat shock proteins (hsps) was studied in cultured Reuber H35 rat hepatoma cells by sequential application of different stressors. We analyzed whether a specific stress condition is able to induce an enhanced sensitivity to a subsequent application of a low dose of either the same or another stressor (self-sensitization and cross-sensitization, respectively). As a measure of sensitization, the stimulation of hsp induction was employed. Three different stressor conditions (heat shock, sodium arsenite and cadmium chloride) were used in doses which exerted a similar impact on overall protein synthesis. A synergistic effect in induction of the synthesis of various hsps was observed when a high stressor dose was followed by an 8-h incubation in a lower stressor dose in both self- and cross-sensitization experiments. The low-dose conditions used as second treatments did not induce any responses in non-pretreated cells. Studies in cultured cells have demonstrated stressor-specific hsp induction patterns. In this study we analyzed whether the pattern of hsps induced by the low-dose condition is characteristic for the first sensitizing stressor or for the secondary stressor applied in a low dose. The pattern of hsps which was induced above the level of the high-dose effect, due to the incubation with the secondary applied low-dose condition, was found to be characteristic for the secondary stressor and not for the sensitizing primary treatment. These results are of importance for an improved understanding of the regulation of heat shock protein synthesis in conditions of self- and cross-sensitization, as well as for a proper use of hsps as biomarkers of exposure to environmental stress.     

Two new coccidian parasites from the grand anglehead lizard, Gonocephalus grandis from Peninsular Malaysia

During 3 collecting expeditions between October 1996 and December 1996, fecal samples were obtained from 43 adult Gonocephalus grandis from Tanah Rata and the Cameron Highlands in Peninsular Malaysia. Two species of coccidia (Isospora gonocephali n. sp. [9/43, 23%] and Eimeria cameronensis n. sp. [3/43, 7%]) were discovered. Sporulated oocysts of I. gonocephali are subspherical to ovoidal, 22.3 x 18.7 (19-25 x 17-23) microm with a bilayered wall composed of a thin inner wall and a striated outer wall with a pitted surface; oocyst residuum absent; 1 polar granule present; sporocysts are almond-shaped, 13.5 x 9.2 (12-15 x 8.5-10) microm, Stieda body broad, domelike, substieda body fanlike, sporocyst residuum consisting of coarse, nonuniform granules in an amorphous cluster; sporozoites sausage-shaped with 1 large terminal, refractile body and lay randomly in the sporocyst. Sporulated oocysts of E. cameronensis are bilayered, smooth-walled, ellipsoidal, 26.5 x 12.4 (25-28 x 12-13) microm; with 1, small, polar granule composed of 2-3 splinter-like structures fused together; oocyst residuum absent; sporocysts ovoidal, almost rectangular-shaped 8.8 x 6.6 (8-9 x 5-7) microm, with no Stieda or substieda bodies, containing scattered residuum and 2 sausage-shaped sporozoites with 1 terminal, ovoidal refractile body. No individual lizard was host to both coccidian species.     

Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.