Oligomerization of expanded-polyglutamine domain fluorescent fusion proteins in cultured mammalian cells

Six inherited neurologic diseases, including Huntington's disease, result from the expansion of a CAG domain of the disease genes to produce a domain of more than 40 glutamines in the expressed protein. The mechanism by which expansion of this polyglutamine domain causes disease is unknown. Recent studies demonstrated oligomerization of polyglutamine-domain proteins in mammalian neurons. To study oligomerization of polyglutamine proteins and to identify heterologous protein interactions, varying length polyglutamine-green fluorescent protein fusion proteins were expressed in cultured COS-7 cells. The 19- and 35-glutamine fusion proteins (non-pathologic length) distributed diffusely throughout the cytoplasm. In contrast, 56- and 80-glutamine fusion proteins (pathologic length) formed fibrillar arrays resembling those previously observed in neurons in Huntington's disease and in a transgenic mouse model. These aggregates were intranuclear and intracytoplasmic. Intracytoplasmic aggregates were surrounded by collapsed intermediate filaments. The intermediate filament protein vimentin co-immunoisolated with expanded polyglutamine fusion proteins. This cellular model will expedite investigations into oligomerization of polyglutamine proteins and their interactions with other proteins.     

Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.     

A protective epitope of Moraxella catarrhalis is encoded by two different genes

The high-molecular-weight UspA protein of Moraxella catarrhalis has been described as being both present on the surface of all M. catarrhalis disease isolates examined to date and a target for a monoclonal antibody (MAb 17C7) which enhanced pulmonary clearance of this organism in a mouse model system (M. E. Helminen et al., J. Infect. Dis. 170:867-872, 1994). A recombinant bacteriophage that formed plaques which bound MAb 17C7 was shown to contain a M. catarrhalis gene, designated uspA1, that encoded a protein with a calculated molecular weight of 88,271. Characterization of an isogenic uspA1 mutant revealed that elimination of expression of UspA1 did not eliminate the reactivity of M. catarrhalis with MAb 17C7. In addition, N-terminal amino acid analysis of internal peptides derived from native UspA protein and Southern blot analysis of M. catarrhalis chromosomal DNA suggested the existence of a second UspA-like protein. A combination of epitope mapping and ligation-based PCR methods identified a second M. catarrhalis gene, designated uspA2, which also encoded the MAb 17C7-reactive epitope. The UspA2 protein had a calculated molecular weight of 62,483. Both the isogenic uspA1 mutant and an isogenic uspA2 mutant possessed the ability to express a very-high-molecular-weight antigen that bound MAb 17C7. Southern blot analysis indicated that disease isolates of M. catarrhalis likely possess both uspA1 and uspA2 genes. Both UspA1 and UspA2 most closely resembled adhesins produced by other bacterial pathogens.     

Regional proliferative patterns in the colon of patients at risk for hereditary nonpolyposis colorectal cancer

Between 1989 and 1993, 20 intercondylar fractures of the distal humerus were treated by open reduction, internal fixation and early postoperative mobilisation. One patient died on the third postoperative day as a result of multiple injuries, leaving 19 patients for evaluation. The mean follow-up period was 4.1 years (range 2.0 to 6.7 years). According to the Muller system, there were 7 type C1 and 12 type C2 fractures. Using the Jupiter criteria, 6 elbows were rated as excellent, 9 good and 4 fair. Complications included late ulnar neuritis in one patient and wound infection in another patient.     

Cyclosporine A inhibits lymphocyte migration into ovine peripheral nerve allografts

Lymphocyte migration into nerve allografts was measured to estimate the cyclosporine A (CsA) dose required to suppress rejection. Twelve outbred sheep received daily subcutaneous CsA at 0, 5, 10, or 15 mg/kg/day for 2 weeks prior to implantation of multiple heterotopic subcutaneous nerve grafts. Lymphocyte migration was determined after 7 days by an intravenous pulse of autologous 111indium-labeled lymphocytes and subsequent quantitation of gamma radioactivity in nerve tissue (CPM/g, mean +/- SEM). Measurement by radioimmunoassay revealed a dose-dependent increase in blood cyclosporine levels. Lymphocyte migration into autografts (404+/-44) was significantly less than migration into allografts (16,554+/-2,049), in control animals (P < 0.01). A dose-dependent inhibition of lymphocyte migration into nerve allografts was observed with counts of 7,662+/-1,692, 4,083+/-1,112, and 1,561+/-232 in sheep receiving 5, 10, or 15 mg/kg/day of CsA, respectively. Daily CsA administration produced effective blood levels and immunosuppression sufficient to inhibit lymphocyte migration into nerve allografts.     

Decompression illness in sports divers detected with technetium-99m-HMPAO SPECT and texture analysis

Diving for sport and recreation has increased in recent years, resulting in more incidences of diving illness. Therefore, we studied potential use of regional cerebral blood flow SPECT imaging with 99mTc-HMPAO in the management of divers who have experienced decompression illness (DCI). METHODS: A group of ten sports divers who had no experience of DCI were compared with ten sports divers who had experienced at least one episode of DCI. Transaxial SPECT images were first compared objectively using a first-order texture measure and then subjectively using a receiver operator characteristic (ROC) experiment. Experienced observers were asked to rate images subjectively in terms of the images' textural appearance. RESULTS: Both these techniques showed that there is a statistically significant difference between the two groups and the images produced by the DCI divers were generally more coarsely patchy when compared to the non DCI divers. The quantitative texture technique proved significantly better in identifying divers with DCI than the visual analysis by observers using ROC curves. CONCLUSION: Differences between the cerebral blood flow patterns of sports divers who have experienced DCI and sports divers who have no experience of DCI can be detected using 99mTc-HMPAO SPECT and a texture analysis technique.     

Maturation alters the contractile role of calcium in ovine basilar arteries

OBJECTIVE: To investigate the distribution patterns of primary and permanent teeth in the cleft area and the numerical variation in teeth in unilateral complete cleft lip and palate (UCLP) patients. DESIGN: A survey of the dentition in UCLP patients. SETTING: Craniofacial Center, Chang Gung Memorial Hospital, Taipei, Taiwan. PATIENTS: 137 UCLP patients who met the following criteria: (1) have had at least one panoramic film taken, (2) the first panoramic film illustrates either primary or early mixed dentition. Evaluation of both permanent and primary dentition was available in 91 cases. MAIN OUTCOME MEASURES: Two evaluators performed independent evaluations of number and distribution of teeth in UCLP patients. The hypothesis that there are two odontogenic origins for maxillary lateral incisors was proposed to explain the occurrence of distribution patterns of dentition in the cleft area and to explain differences between primary and permanent dentition in UCLP patients. RESULTS: Four distribution patterns in the cleft area were identified in both the primary and the permanent dentition. In the primary dentition, placement of the lateral incisor distal to the alveolar cleft was the predominant pattern (pattern y, 82.4%), followed by absence of the cleft side maxillary lateral incisor (pattern ab, 9.9%), presence of one tooth on each side of the alveolar cleft (pattern xy, 5.5%), and placement of the lateral incisor mesial to the alveolar cleft (pattern x, 2.2%). In the permanent dentition, the most common pattern was the absence of the maxillary lateral incisor on the cleft side (pattern AB, 51.8%), followed by lateral incisor placement distal to the alveolar cleft (pattern Y, 46%), lateral incisor placement mesial to the alveolar cleft (pattern X, 1.5%) and the presence of one tooth on each side of the alveolar cleft (pattern XY, 0.7%). The discrepancy between the distribution patterns of primary dentition and permanent dentition successors is 57.1%. Variations in tooth number in both primary and permanent dentition of UCLP patients occurred most often in the cleft area. Abnormalities in the number of teeth (hypodontia or hyperdontia) outside the cleft area were more common in the permanent dentition than in the primary dentition (24.1% versus 4.4%). CONCLUSIONS: Four distribution patterns in the cleft area were identified in both sets of dentition. Our findings of distribution patterns in UCLP patients support the hypothesis that there may be two odontogenic origins for maxillary lateral incisors. Clinicians involved in managing the dentition of UCLP patients should consider the high frequency of numerical variation both in and outside the cleft area before starting dental treatment.     

Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.     

Roentgen stereophotogrammetric analysis using computer-based image-analysis

The two-dimensional position of markers in radiographs for Roentgen Stereophotogrammetric Analysis (RSA) is usually determined using a measuring table. The purpose of this study was to evaluate the reproducibility and the accuracy of a new RSA system using digitized radiographs and image-processing algorithms to determine the marker position in the radiographs. Four double-RSA examinations of a phantom and 18 RSA examinations from six patients included in different RSA-studies of knee prostheses were used to test the reproducibility and the accuracy of the system. The radiographs were scanned at 600 dpi resolution and 256 gray levels. The center of each of the tantalum-markers in the radiographs was calculated by the computer program from the contour of the marker with the use of an edge-detection software algorithm after the marker was identified on a PC monitor. The study showed that computer-based image analysis can be used in RSA-examinations. The advantages of using image-processing software in RSA are that the marker positions are determined in an objective manner, and that there is no need for a systematic manual identification of all the markers on the radiograph before the actual measurement.