Cardiopulmonary Bypass in a Patient with Factor XII Deficiency.

The performance of cardiopulmonary bypass (CPB) in the factor XII-deficient patient is challenging in that the normal method for monitoring anticoagulation is ineffective as a result of an impaired contact activation system. We report the case of a factor XII-deficient patient who underwent surgical revascularization on CPB. His factor XII level was replenished with fresh-frozen plasma immediately before surgery. This management strategy lowered the baseline activated clotting time (ACT) to near normal, providing a meaningful ACT value for CPB. Factor XII is also a key component in the fibrinolytic system and its deficiency is associated with increased thrombosis. Because the factor XII level quickly returns to baseline postoperatively, perioperative care must include strategies to avoid postoperative thromboembolic events.

Lymphangioma-like Kaposi's sarcoma: etiology and literature review.

Much confusion surrounds a rare and occasionally described variant of Kaposi's sarcoma (KS) known as lymphangioma-like or bullous KS. We describe the typical clinical and histologic features and elucidate the etiologic cell in lymphangioma-like KS. A computer-based review of the English-language literature was performed. Routine histologic and immunoperoxidase techniques were performed on formalin-fixed tissue. Immunoperoxidase staining for anti-CD31, anti-CD34, and anti-factor VIII-related antigen suggests that the etiologic cell of origin is the vascular endothelial cell. Lymphangioma-like KS has been described for more than a century and given various names. Dilated vascular spaces correlate with the clinically bullous lesions, which are veritable KS and not a secondary reaction to it.

Anaesthetic management of a parturient with a colloid cyst of the third ventricle.

A colloid cyst in the third ventricle near the foramen of Monroe can obstruct cerebrospinal fluid (CSF) flow from the lateral ventricles. Any change in the CSF pressure on either side of the cyst can lead to displacement and thus precipitate acute hydrocephalus. Management of the confinement of a patient with a colloid cyst must therefore aim to minimize changes in CSF pressure. We describe our management of a patient with a small colloid cyst who was permitted to labour with the assistance of patient-controlled epidural analgesia. The available alternatives are discussed.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Fibrous hamartoma of infancy.

Fibrous harmartoma of infancy is a benign soft tissue tumor that occurs in the first few years of life. Although the lesion is not distinctive clinically, it has a characteristic microscopic appearance. Only 12 cases of fibrous hamartoma of infancy have been reported in the dermatology literature.

Cytologic-pathologic correlations: fine needle aspiration of three cases of steatocystoma multiplex.

Steatocystoma multiplex is a rare condition characterized by multiple subcutaneous nodules classically located on the anterior chest. Diagnosis usually is made clinically or by biopsy. The lesions may be numerous, and surgical treatment may not be practical or the results may be unsatisfactory. Three patients with multiple subcutaneous nodules underwent fine needle aspiration, which produced smears with acellular debris. A diagnosis was possible in the first patient because of close collaboration with a dermatologist. The lesions in the other patients were recognized clinically and were confirmed cytologically by fine needle aspiration. A syringe holder allows the use of thin needles (22-gauge) to aspirate very small lesions (4 to 5 mm). Fine needle aspiration may provide a useful alternative to surgical methods in the treatment of these lesions because it is inexpensive, well-tolerated by the patients, and has no associated morbidity.

A preliminary study of immunologic and hematologic profiles of peripheral blood from Nebraska farmers who apply pesticides to their fields.

OBJECTIVE: Laboratory studies have documented a wide range of pesticide-induced changes in the hematopoietic and lymphoreticular systems. Some of these are expressed as altered serum values, blood cell counts, and leucocyte functions. The goal of the present study was to determine whether these alterations were evident in peripheral blood of Nebraska farmers who applied pesticides to their fields. METHODS: An invitation to participate was mailed to 100 residents (70 farmers; 30 controls) of Butler County, Nebraska. All respondents (51 farmers and 21 controls) were enrolled and surveyed by written questionnaire for health status and pesticide use. Our analysis included 45 farmers and 18 controls. The farmers were divided into a high (n = 23) and a low (n = 22) pesticide use group. Statistical correlations of ten blood values with both pesticide use and age were evaluated, since pesticide use correlated with age. RESULTS: Four of the ten blood values correlated with pesticide use and age (Spearman Rho). In a multiple regression model, pesticide use (not age) proved to be a predictor of red blood cell count and hematocrit. In the same model, pesticide use was not a predictor of mean red cell volume or candida antigen-induced T-lymphocyte proliferation. Serum complement activity did not correlate with pesticide use among the farmers (n = 45) but was significantly reduced (ANOVA) in the high pesticide use group, compared to controls. CONCLUSIONS: A preliminary study of blood values in a small cohort of Nebraska farmers found no pesticide-associated effects on 1) leucocyte count, 2) antigen- and mitogen-stimulated T-cell proliferation, 3) mitogen-stimulated B-cell proliferation, and 4) concentrations of serum IgG and IgM. The study found small but statistically significant pesticide-associated effects on red blood cells and serum complement.

Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding.

Uracil phosphoribosyltransferase (UPRTase) catalyzes the transfer of a ribosyl phosphate group from alpha-D-5-phosphoribosyl-1-pyrophosphate to the N1 nitrogen of uracil. The UPRTase from the opportunistic pathogen Toxoplasma gondii is a rational target for antiparasitic drug design. To aid in structure-based drug design studies against toxoplasmosis, the crystal structures of the T.gondii apo UPRTase (1.93 A resolution), the UPRTase bound to its substrate, uracil (2.2 A resolution), its product, UMP (2.5 A resolution), and the prodrug, 5-fluorouracil (2.3 A resolution), have been determined. These structures reveal that UPRTase recognizes uracil through polypeptide backbone hydrogen bonds to the uracil exocyclic O2 and endocyclic N3 atoms and a backbone-water-exocyclic O4 oxygen hydrogen bond. This stereochemical arrangement and the architecture of the uracil-binding pocket reveal why cytosine and pyrimidines with exocyclic substituents at ring position 5 larger than fluorine, including thymine, cannot bind to the enzyme. Strikingly, the T. gondii UPRTase contains a 22 residue insertion within the conserved PRTase fold that forms an extended antiparallel beta-arm. Leu92, at the tip of this arm, functions to cap the active site of its dimer mate, thereby inhibiting the escape of the substrate-binding water molecule.

Anabolic steroid use among adolescents in Nebraska schools.

The extent of anabolic steroid use among adolescent sports participants and nonparticipants in Nebraska schools was studied. The Nebraska Secondary School Survey administered in 1991 contained 19 new items designed to determine rates of use of anabolic steroids and to measure participation in school sports. Schools participating in the survey are part of the Toward a Drug Free Nebraska training project; when the 1991 survey was conducted, training did not address ergogenic drug use. The survey was administered on a voluntary and confidential basis to Nebraska students in grades 7 through 12. A total of 4722 students in 62 secondary schools were surveyed; 3183 (67.4%) identified themselves as participants in school-sponsored sports. Of all the respondents, 117 (2.5%) reported having used anabolic steroids in the preceding 30 days. Some 4.5% of all the male respondents were steroid users, versus 0.8% of all the females. Of the students who reported using anabolic steroids, 72.6% were sports participants. Steroid users, whether they participated in sports or not, were more likely to use alcohol, tobacco, and other drugs than were nonusers of steroids. Among sports participants and nonparticipants, anabolic steroid users were more likely than nonusers to report acting violently. Nebraska students (grades 7 through 12) who participated in school-sponsored sports were more likely than non-participants to use anabolic steroids. Steroid-using athletes were more likely to use alcohol, tobacco, and other drugs than athletes who did not use steroids.

Respiratory sensation during bronchial challenge testing with methacholine, sodium metabisulphite, and adenosine monophosphate.

BACKGROUND: There is some evidence that the perception of bronchoconstriction may very according to the nature of the provoking stimulus. The aims of this study were, firstly, to develop a method for measuring dyspnoea during induced bronchoconstriction in patients with asthma and, secondly, to apply this method to testing differences between directly and indirectly acting bronchoconstricting stimuli. METHODS: Descriptive terms suitable for quantifying respiratory discomfort due to bronchoconstriction in patients with asthma were identified in a preliminary investigation. The relation between reduction in forced expiratory volume in one second (FEV1) and respiratory discomfort, measured using a visual analogue scale (VAS), was then studied during challenges with three different inhaled stimuli: methacholine (MCH), sodium metabisulphite (MBS), and adenosine monophosphate (AMP). Three indices were calculated to describe the relation: the VAS value associated with a 20% fall in FEV1 (FEV20 VAS); the ratio of the final VAS value to the final percentage fall in FEV1 (VAS-FEV1 ratio); and the regression coefficient for predicting VAS from the percentage fall in FEV1 within each challenge (beta VAS FEV1). RESULTS: "Difficulty in breathing" and "chest tightness" were selected as suitable terms for quantifying respiratory discomfort. There were no differences between the three agonists in the qualitative aspects of the respiratory sensation. In paired challenges with the same agonist the three indices were all found to be reproducible for both sensations measured. MCH induced less intense difficulty in breathing and chest tightness for a given fall in FEV1 than did AMP. There was a trend in the same direction for the comparison between MCH and MBS. There were no differences between AMP and MBS. FEV20 VAS was less powerful in discriminating between agonists than the two slope indices. CONCLUSIONS: The relation between induced reduction in FEV1 and the intensity of respiratory discomfort can be measured reliably. The indirectly acting bronchoconstricting agonists AMP and MBS induced more intense respiratory discomfort for a given fall in FEV1 than the direct agonist MCH. This may be due to differences in unmeasured mechanical changes in the lungs or to an additional action on airway sensory nerves.

Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma.

Asthma and chronic obstructive pulmonary disease are characterized by chronic airway inflammation. Studies using bronchoalveolar lavage (BAL) have shown an increased proportion of eosinophils in the BAL fluid from asthmatics compared with that from normal subjects, whereas studies of chronic obstructive pulmonary disease (COPD) have shown increased numbers of neutrophils. Induced sputum allows sampling of respiratory tract secretions from patients and control subjects, providing a noninvasive method of studying airway secretions and allowing characterization of cells and measurement of soluble markers. We investigated whether induced sputum was a useful method of studying airway fluid from patients with moderate to severe COPD and whether it could be used to compare inflammation in this condition with that in asthma. An initial reproducibility study was undertaken. Sputum was induced twice in 13 patients with severe COPD at a 14-d interval. Total and differential cell counts were carried out and were found to be reproducible over this period. Sputum was then induced in 14 patients with COPD, 23 patients with asthma, 12 healthy cigarette smokers, and 16 normal nonsmoking control subjects. We found a significant increase in neutrophils and increased concentrations of tumor necrosis factor-alpha (TNF alpha) and interleukin-8 (IL-8) in the patients with COPD compared with the smoking and nonsmoking control subjects. Interleukin-8, but not TNF alpha, was significantly higher in the COPD group than in the asthmatic group. We conclude that the cytokines TNF alpha and IL-8 may be involved in the inflammation in COPD.

A survey of alcohol and other drug use behaviors and risk factors in health profession students.

This survey assessed the alcohol and other drug (AOD) use habits and risk factors of health profession students at a midwestern university health science center. The authors administered a 75-item survey to 1,707 students in selected classrooms: 984 students responded for a return rate of 57.6%. In 1990, they found, alcohol use among the health profession students in the past year was comparable to that of undergraduate college students nationally (86%), although significantly fewer health profession students drank heavily (27% had five or more drinks in the past 2 weeks, compared with 41% of college students). The percentage of health profession students who reported using tobacco or illicit drugs was lower than the percentage of undergraduate students who used these substances. At the time of the study, 16% of the respondents may have had a potential current alcohol problem and 3.5% a potential drug problem. Pharmacy students most often reported negative consequences from their AOD use. Peer pressure influenced the drinking decisions of 55% of the respondents; students in dentistry and pharmacy experienced the least support from peers for their decisions to abstain from drinking. Family histories of alcohol problems were reported by 38% of the respondents, and family histories of drug use by 14.8%. Male health profession students, when compared with the female professional students, drank more and experienced more consequences of their drinking or drug use and were also more influenced by peers.

A new approach to the cloning of genes encoding T-cell epitopes.

The molecular structure of antigens recognized exclusively by T cells, such as minor histocompatibility antigens and some antigens that provoke autoimmune responses, has proved difficult to determine. Recently, several antigens induced on tumor cells by mutagen treatment have been cloned by transfection of genomic DNA libraries into P1.HTR cells, screening for antigen expression using T-cell clones, and subsequent recovery of the integrated DNA by cosmid rescue. We have modified this technique and have stably transfected P1.HTR cell lines with polyoma T antigen, which allows episomal replication of the shuttle vector, pCDM8. Using pCDM8-CAT constructs, we have determined the frequency of transfection and plasmid copies taken up per cell under optimal transfection conditions. Using a pCDM8 construct which expresses the tumor-specific antigen, P91A (pCDM8-tum-), that is recognized by a T-cell clone, we have found that cells transfected with this antigen can be recognized by the T-cell clone when they are present at only 1%-3% of a mixed population. Progeny of a single cell transfected with pCDM8-tum-: pCDM8-CAT at proportions of 1:10, 1:25, and 1:50 are recognized by the T-cell clone. Furthermore, Hirt extracted plasmid DNA from transfectants expressing the tum- antigen can be amplified in bacteria, transfected back into P1.HTR recipients, and recognized by the T-cell clone. This approach should enable reasonably rapid screening of cDNA libraries for even relatively low abundance messages encoding, for example, minor histocompatibility and alloantigens, and allow their subsequent cloning.

Studies on the phenotype and karyotype of immortalized rabbit kidney epithelial cell lines.

Differentiated mammalian cell lines can be isolated by immortalizing primary cells by transfection with DNA from plasmids containing sequences from SV40 early region. These cell lines show cytogenetic abnormalities but the degree of aneuploidy is considerably less than that observed in other established cell lines. No correlation was observed between the degree of differentiation of a clone and the extent of chromosomal damage.

Rabbit urinary tamm-horsfall glycoprotein. Chemical composition and tentative carbohydrate structure.

The urinary Tamm-Horsfall protein (THP) is the major glycoprotein secreted by the mammalian kidney. We recently isolated and immortalized thick ascending limb of Henle cells from rabbit kidney, which produce Tamm-Horsfall protein in cell culture in vitro. In order to further study the yet undefined functional role and biosynthetic pathways of this protein, we first re-examined the chemical composition and the carbohydrate structure of rabbit urinary Tamm-Horsfall protein. Using precipitation with 0.58 mol/l NaCl a protein was isolated from rabbit urine which showed extensive microheterogeneity and had an average molecular mass of 95 kDa. Deglycosylation of the protein led to a loss of microheterogeneity and yielded a molecular mass of 58-60 kDa. Amino-acid analysis of the native and deglycosylated protein revealed a lower cysteine (20 mol/mol THP) and a higher histidine (20 mol/mol THP) content than described previously. Chemical analysis of the carbohydrates showed a high glucosamine (50 mol/mol THP), galactose (43 mol/mol THP), and mannose (24 mol/mol THP) content. The amount of sialic acid was 15 mol/mol THP. Using lectins to identify the structure of the carbohydrate chains it was shown that rabbit Tamm-Horsfall protein possesses complex-type oligosaccharide chains with terminal sialic acid, beta-galactose, and probably alpha-fucose and chains of the mucin type. These results indicate that some of the cysteine residues in the polypeptide chain of THP can be replaced by histidine, suggesting a role of some cysteins in metal binding rather than intramolecular stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of Tamm-Horsfall protein in chloride transporting epithelia: lack of correlation with the Na, K, Cl cotransporter.

In order to test the hypothesis that Tamm-Horsfall protein (THP) and the Na, K, Cl cotransporter in chloride transporting epithelia are functionally related, the presence of THP in various rat epithelia was investigated by indirect immunocytochemical procedures. Positive staining was found in the apical cytoplasmic portion of the renal thick ascending limb, in apical large secretory granules of the pancreatic acinar cells, in the cytoplasm of the serous acinar cells of the salivary gland, and in the apical cytoplasm of mucosal cells in the jejunum. No staining was observed in the tracheal epithelial cells. These studies show that THP, or antigenically related substances, can be found in a variety of extrarenal organs. Only in the renal thick ascending limb the intracellular distribution of THP coincides partly with the distribution of the Na, K, Cl cotransporter. In all other epithelia investigated no correlation between THP staining and presence of the cotransporter was found. Thus, it seems very unlikely that Tamm-Horsfall protein and the Na, K, Cl cotransporter are functionally related.

Differentiation in vitro of primary cultures and transfected cell lines of epithelial cells derived from the thick ascending limb of Henle's loop.

The use of primary cell cultures derived from defined locations of the kidney has enabled the study of certain kidney cell type-specific characteristics under defined environmental conditions. The use of primary cell cultures, however, has a number of inherent disadvantages, many of which may be overcome by the use of differentiated cell lines of defined origin. In this paper I describe in detail an approach to: (a) the isolation and culture of primary cultures derived from the thick ascending limb of Henle's loop (TALH), and (b) the production of differentiated cell lines by the transfection of these primary cell cultures with early region SV40 virus genes. The characteristics of these cultures and other TALH-derived cell lines are described.