High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties

Bovine beta-lactoglobulin (BLG) variant A has been expressed in the methylotropic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the alpha-mating factor prepro-leader peptide from Saccharomyces cerevisiae. P. pastoris Mut+ transformants were obtained by single cross-over integration of the BLG-containing vector into the AOX1 locus. In a fed-batch fermenter, a cell density of approximately 300 mg/ml was achieved by controlled glycerol feeding for a total of 24 h. After 72 h of methanol induction, the secreted BLG reached levels of > 1 g/l. The secreted protein could be purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the secreted BLG revealed that the Glu-Ala spacer repeats inserted between the mature protein and the alpha-factor prepro-leader were still present. The purified protein was characterized by a number of methods, including CD spectroscopy, guanidine-HCl unfolding, crystallization and two-dimensional 1H-NMR spectroscopy. By all of these measures, the physical characteristics of recombinant BLG were indistinguishable from those of the native purified bovine BLG, making it useful as a model for protein folding and other biophysical studies.     

Longitudinal weight changes, length of survival, and energy requirements of long-term care residents with dementia

OBJECTIVE: We hypothesized that institutionalized patients with dementia, who frequently have feeding problems and require supervised and assisted feeding, would lose more weight during their residency than nondemented, independently functioning residents and have compromised survival. To test this hypothesis, we examined the survival and longitudinal changes in weight of two cohorts of institutionalized residents with dementia and compared these cohorts with a cohort of nondemented residents. We also measured the resting energy expenditures of a subset of the subjects with dementia as an indicator of their energy needs. DESIGN: A longitudinal cohort study with retrospective baseline chart review and subsequent follow-up of monthly weights and mortality over 4 years. SETTING: A 725-bed long-term care institution with specified levels of care. SUBJECTS: Two cohorts of residents with dementia, one consisting of subjects who required total care throughout their institutional stay (n = 31) and another group who did not initially require total care (n = 48); these were compared with a cohort with normal mentation who were functionally independent in their daily activities (n = 26). The total number of subjects was 105. MEASUREMENTS: Demographics, medical problems, and medications by chart review; functional and mental status evaluations; longitudinal monthly weights and mortality for the 48-month study period; and resting energy expenditures by indirect calorimetry. MAIN RESULTS: Residents with dementia had lower weights on admission and throughout their stay than nondemented, independently functioning residents, and they were more likely to have a weight loss of 10 lbs or more at some point during the 4-year study period. However, their mean weights did not change during the study period. The mean survival from admission of those demented residents who died was more than 3 years. Resting energy expenditures of women residents with advanced dementia were 12% lower than predicted from the Harris Benedict equations. CONCLUSION: Dementia is not necessarily associated with unremitting weight loss during institutionalization despite the frequent occurrence of feeding difficulties and temporary weight loss. This may be caused partly by the lower than expected resting energy expenditures and, hence, energy needs of affected residents as their dementia progresses. Demented residents weighed significantly less than nondemented, independently functioning residents throughout their institutional stay. Nevertheless, nursing staff are able to maintain weight and survival for extended periods even in very impaired residents.     

Aminoglycosides prevent and dissociate the aggregation of platelets in patients with EDTA-dependent pseudothrombocytopenia

Although EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is of practical importance because failure to recognize this clinical entity may result in misdiagnosis and subsequent mismanagement of the patients, the pathophysiological nature of EDTA-PTCP remains unknown. To develop an effective way to evaluate the platelet counts in patients with EDTA-PTCP, we introduced aminoglycosides-supplemented anticoagulating agents. When kanamycin was pre-supplemented with EDTA for anticoagulating blood samples from EDTA-PTCP patients there was no significant change in the platelet counts and the morphology of blood cells after 150 min of incubation at room temperature. Furthermore, when kanamycin was added to EDTA-anticoagulated blood samples from EDTA-PTCP patients within 30 min after blood withdrawal, rapid dissociation of platelets without apparent morphological changes of blood cells was observed, and complete blood cell counts as well as the histogram patterns were almost the same as those examined immediately after blood sampling. The dissociation of aggregated platelets was also detected when other antibiotics were used, although it was associated with some extent of morphological changes of blood cells. These findings indicate that the supplementation of aminoglycosides either before or after blood sampling is a useful method for the diagnosis EDTA-PTCP and for the evaluation of platelet counts in patients with EDTA-PTCP.     

Life-size, computer-generated skull replica to assist surgery of craniofacial fibrous dysplasia

Craniofacial fibrous dysplasia is a benign pathological condition but it replaces bone and expands gradually, leading to facial distortion. Surgery is the only effective treatment. The authors describe the usefulness of a life-size, computer-generated skull replica which assists surgeons pre- and intraoperatively. Surgery can be safely performed with less invasion and a shorter operation time by referring to the skull replica. Partial bone resection and bone contouring have been carried out, assisted by skull replicas, in 8 patients with craniofacial fibrous dysplasia, resulting in good cosmesis.     

Induction of neutralizing antibody in mice by immunization with recombinant 56 kDa protein of Orientia tsutsugamushi

Anti-oriential antibody inhibits Orientia tsutsugamushi attachment to, and penetration of, host cells. However, O. tsutsugamushi antigens that induce the production of a neutralizing antibody have not been identified. The authors immunized mice and rabbits with the recombinant 56 kDa protein of O. tsutsugamushi fused to the maltose binding protein of Escherichia coli (MBP-Bor56) and analysed their effect on O. tsutsugamushi attachment to or penetration of L929 cells. O. tsutsugamushi attachment and penetration were measured by using an indirect immunofluorescent antibody assay (IFA). O. tsutsugamushi growth in L929 cells was determined by [3H]thymidine uptake assay. By IFA, we observed a 96% reduction of attachment or penetration of O. tsutsugamushi treated with rabbit anti-MBP-Bor56 sera. [3H]thymidine uptake showed that mouse anti-MBP-Bor56 sera caused a 91% reduction in O. tsutsugamushi growth, when compared to mouse anti-MBP sera. These results suggest that the 56 kDa protein of O. tsutsugamushi plays an important role in O. tsutsugamushi attachment to or penetration of cells.     

Efficacy of surgical treatment in traumatic central cord syndrome

BACKGROUND: Controversy surrounds the treatment of traumatic central cord syndrome (TCCS), as there are strong advocates for nonsurgical treatment for most patients. However, conservative treatment has been shown to yield a longer period of discomfort from pain and weakness in certain cases. METHODS: In a retrospective review of 114 patients presenting with acute or chronic TCCS from 1988-94, four different age groups were separately observed under different treatments. Motor and sensory recovery were assessed. RESULTS: Better results were achieved in younger patients, with or without radiographic abnormalities, and in patients with clinically correlated encroaching cord lesions who received early surgical decompression. CONCLUSIONS: Surgical intervention for TCCS must be addressed with careful clinical and radiographic survey. Removal of offending lesions in the subacute period results in significant motor and sensory improvement in short-term and long-term follow-up.     

Retrospective comparison of infusional 5-fluorouracil, doxorubicin, and mitomycin-C (modified FAM) combination chemotherapy versus palliative therapy in treatment of advanced gastric cancer

About one-third of patients with gastric cancer are unresectable at the time of diagnosis. Their median survival is < 6 months, with a grave prognosis. The purpose of this study was to assess the efficacy of a modified FAM (mFAM) regimen in advanced gastric cancer. We retrospectively reviewed the clinical records of 409 advanced gastric cancer patients who had not received curative surgery. Among 409 patients, 202 patients were treated with an mFAM regimen (infusional 5-FU + doxorubocin + mitomycin-C), and 207 patients received no chemotherapy (control group). No differences were found in clinical parameters between the two groups. The 1-year survival rates were 34.1% for the mFAM-treated group and 22.5% for the control group (p = 0.0135). In subset analysis, a higher 1-year survival rate was demonstrated in patients with mFAM and palliative surgery. Of the 154 evaluable patients in the mFAM-treated group, the response rate was 17.5%. In these patients, median response duration was 30 weeks, and progression-free survival was 23 weeks. Overall toxicity of mFAM regimen was relatively tolerable and reversible. In conclusion, FAM combination chemotherapy, which has been used as a standard therapy, prolonged survival after modification of the administration schedule and combination with palliative surgery. A prospective randomized study is warranted to confirm this conclusion from our retrospective study.     

Functional role of high-affinity anandamide transport, as revealed by selective inhibition

Anandamide, an endogenous ligand for central cannabinoid receptors, is released from neurons on depolarization and rapidly inactivated. Anandamide inactivation is not completely understood, but it may occur by transport into cells or by enzymatic hydrolysis. The compound N-(4-hydroxyphenyl)arachidonylamide (AM404) was shown to inhibit high-affinity anandamide accumulation in rat neurons and astrocytes in vitro, an indication that this accumulation resulted from carrier-mediated transport. Although AM404 did not activate cannabinoid receptors or inhibit anandamide hydrolysis, it enhanced receptor-mediated anandamide responses in vitro and in vivo. The data indicate that carrier-mediated transport may be essential for termination of the biological effects of anandamide, and may represent a potential drug target.