Single copy Babesia microti hsp70

The glucose-6-phosphate dehydrogenase-encoding gene (G6PD) belongs to a group with constitutive expression in all tissues. The regulation of these housekeeping genes is poorly understood, as compared to what is known about many genes whose expression is restricted to a particular tissue or stage of development, and which are often regulated by locus control regions (LCR) able to act over wide distances. In order to identify sequences in human G6PD which are necessary for its expression, we have generated transgenic mice carrying a 20-kb G6PD construct, including only 2.5 kb of upstream and 2.0 kb of downstream flanking sequence. All mice which carried the transgene (TG) expressed it, and the levels of expression detected in a range of tissues from three independent lines of mice were comparable to that of the endogenous murine G6PD. The variation in enzyme activity from tissue to tissue was remarkably similar for both the TG and the endogenous gene, and was shown to be due in both cases to variations in the steady-state mRNA levels.     

Site-directed mutagenesis of human leukotriene C4 synthase

The functional characteristics of leukotriene C4 synthase (LTC4S), which specifically conjugates leukotriene A4 with GSH, were assessed by mutagenic analysis. Human LTC4S and the 5-lipoxygenase-activating protein share substantial amino acid identity and predicted secondary structure. The mutation of Arg-51 of LTC4S to Thr or Ile abolishes the enzyme function, whereas the mutation of Arg-51 to His or Lys provides a fully active recombinant protein. The mutations Y59F, Y97F, Y93F, N55A, V49F, and A52S increase the Km of the recombinant microsomal enzyme for GSH. The mutation Y93F also markedly reduces enzyme function and increases the optimum for pH-dependent activity. The deletion of the third hydrophobic domain with the carboxyl terminus abolishes the enzyme activity, and function is restored by the substitution of the third hydrophobic domain and carboxyl terminus of 5-lipoxygenase-activating protein for that of LTC4S. Mutations of C56S and C82V alone or together and the deletion of Lys-2 and Asp-3 of LTC4S do not alter enzyme function. The direct linkage of two LTC4S monomers by a 12-amino acid bridge provides an active dimer, and the same bridging of inactive R51I with a wild-type monomer creates an active pseudo-dimer with function similar to that of the wild-type enzyme. These results suggest that in the catalytic function of LTC4S, Arg-51 probably opens the epoxide ring and Tyr-93 provides the thiolate anion of GSH. Furthermore, the monomer has independent conjugation activity, and dimerization of LTC4S maintains the proper protein structure.     

The rat biliary metabolites of dihydroartemisinin, an antimalarial endoperoxide

[13-14C]Dihydroartemisinin was administered to male rats (35 micromol kg-1, iv). Within 0-1 hr and 0-5 hr of dosing, 34.8 +/- 5. 2% (mean +/- SD, N = 6) and 48.4 +/- 5.9% of the radiolabel, respectively, was recovered in bile. Only 1.1 +/- 1.2% was recovered in bladder urine after 5 hr. The biliary metabolites were identified by LC/MS. The principal metabolite (21.1 +/- 9.3% of dose) was the biologically inactive dihydroartemisinin (DHA) glucuronide. The other metabolites were products of reductive cleavage and rearrangement of the endoperoxide bridge, a process known to generate reactive radical intermediates and abolish antimalarial activity. They were desoxy-DHA (3.3 +/- 2.0%) and its glucuronide (1.1 +/- 1.0%), 3-hydroxydesoxy-DHA glucuronide (2.9 +/- 1.8%), and the glucuronide of a ring-contracted tetrahydrofuran acetate isomer of DHA (6.9 +/- 5.6%).     

Manometry of individual segments of the distal esophageal sphincter. Its relation to functional incompetence

The morphology of the arteries in the uterine wall was studied in three multiparous aged mares that had suffered repeated pregnancy failure. The uterine wall arteries exhibited elastosis of the intima or adventitia, or both, resembling "physiological pregnancy sclerosis". In areas affected by elastosis, degeneration of the pre-existing elastic fibres and increased glycosaminoglycans were frequently observed. Newly formed elastic fibres were not evident. Delayed resorption due to disordered metabolic turnover of the elastin was thought to be an important factor in the pathogenesis of the arterial elastosis in the uterine wall.     

Neurocutaneous melanosis: clinical features of large congenital melanocytic nevi in patients with manifest central nervous system melanosis

BACKGROUND: Patients with a large congenital melanocytic nevus (LCMN) may have associated leptomeningeal melanocytosis with or without central nervous system (CNS) melanomas. These patients are considered to have neurocutaneous melanosis, a disorder that, when symptomatic or otherwise manifest neurologically, carries a poor prognosis even in the absence of malignancy. OBJECTIVE: Our purpose was to identify typical clinical features in patients who have manifest CNS melanosis in association with LCMN. METHODS: The records of 117 patients with LCMN in the New York University Registry of LCMN and the reports of 172 cases of LCMN in the world literature were included for features that might signal a high risk for the development of manifest CNS involvement. RESULTS: Of the 289 patients with LCMN, 33 had manifest CNS melanosis. In all 33 in whom symptomatic neurocutaneous melanosis was diagnosed, the LCMNs were present in a posterior axial location on the head, neck, back, and/or buttocks. "Satellite" nevi were known to be present in 31 of the 33 patients. CONCLUSION: Patients with LCMN in a posterior axial location, especially when associated with "satellite" melanocytic nevi, are at greater risk for the development of manifest neurocutaneous melanosis than patients with LCMN limited to the extremities or those who are lacking satellite nevi.     

Decreased paralysis and better motor coordination with microspinal versus PE10 intrathecal catheters in pain study rats

BACKGROUND: The sentinel lymph node is the first node or nodes to drain a cutaneous melanoma. Sentinel lymph node biopsy is performed to determine whether regional metastases are present. The authors' experience with the new technique of sentinel lymph node biopsy for melanoma of the head and neck is reported. PATIENTS AND METHODS: During the period of January of 1992 to December of 1995, 58 consecutive patients were identified from the melanoma database who had localization of the sentinel lymph node for primary cutaneous melanoma of the head and neck. Techniques for identification of the sentinel node(s) include preoperative lymphoscintigraphy and intraoperative Lymphazurin dye (vital blue dye) and technetium-99m-labeled sulfur colloid injection around the primary tumor site with Neoprobe mapping. RESULTS: Fifty-eight patients (13 female, 45 male), mean age 61 years, with melanoma of the head and neck with a mean Breslow thickness of 2.21 mm. (range, 0.82-6.87 mm.) and no regional lymphadenopathy underwent sentinel node mapping. The sentinel node was successfully identified in 55 patients (95 percent). Blue dye was visualized in 85 of 126 sentinel nodes excised (67 percent), whereas the remainder of the sentinel nodes were localized with the Neoprobe. Forty-nine patients who had successful mapping and sentinel node biopsy had no evidence of metastatic disease in the sentinel node or other nodes in the basin. Six of the fifty-five patients (11 percent) had evidence of micrometastatic disease, and all six had the sentinel node as the only site of metastasis. Five of six patients with micrometastases had a subsequent neck dissection and/or superficial parotidectomy. None of these patients had evidence of "skip metastases" with a negative sentinel node and higher level nodes positive for metastases. In total, 6 of the 18 sentinel nodes (33 percent) identified in these six patients contained micrometastatic disease, whereas none of the 139 other nodes sampled had any evidence of metastases. The exact probability that all six unpaired observations would consist of involvement of only the sentinel nodes is p = 0.0312. CONCLUSIONS: By combining the two mapping techniques in patients with melanoma of the head and neck, the sentinel node(s) can be mapped and identified individually, similar to melanoma in other locations. The sentinel nodes have been shown to contain the first evidence of regional metastatic melanoma. This staging information can be used to plan therapeutic node dissections and adjuvant therapy that may have a survival benefit in patients with stage III melanoma of the head and neck. Lymphatic mapping can be used to make the surgical care of the melanoma patient more conservative, so that only those patients with solid evidence of regional node metastases are subjected to the morbidity and expense of a complete node dissection and the toxicities of adjuvant therapy.     

Isolation of anti-T cell receptor scFv mutants by yeast surface display

Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.