Biomedical subjects
Publications and source records attributed to R S Kaplan.
Recent decisions by the Supreme Court give some indication of how a court would rule in a sexual harassment or disability discrimination suit. Home care providers need to understand the effect of these rulings and ensure that their own policies are clear and clearly disseminated to employees.
A gene encoding the mitochondrial dicarboxylate transport protein (DTP) has been identified for the first time from any organism. Our strategy involved overexpression of putative mitochondrial transporter genes, selected based on analysis of the yeast genome, followed by purification and functional reconstitution of the resulting protein products. The DTP gene from the yeast Saccharomyces cerevisiae encodes a 298-residue basic protein which, in common with other mitochondrial anion transporters of known sequence and function, displays the mitochondrial transporter signature motif, three homologous 100-amino acid sequence domains, and six predicted membrane-spanning regions. The product of this gene has been abundantly expressed in Escherichia coli where it accumulates in inclusion bodies. Upon solubilization of the overexpressed DTP from isolated inclusion bodies with Sarkosyl, 28 mg of DTP was obtained per liter of E. coli culture at a purity of 75%. The purified, overexpressed DTP was then reconstituted in phospholipid vesicles where both its kinetic properties (i.e. Km = 1. 55 mM and Vmax = 3.0 micro;mol/min/mg protein) and its substrate specificity were determined. The intraliposomal substrates malonate, malate, succinate, and phosphate effectively supported [14C]malonate uptake, whereas other anions tested did not. External substrate competition studies revealed a similar specificity profile. Inhibitor studies indicated that the reconstituted transporter was sensitive to inhibition by n-butylmalonate, p-chloromercuribenzoate, mersalyl, and to a lesser extent pyridoxal 5'-phosphate but was insensitive to N-ethylmaleimide and selective inhibitors of other mitochondrial anion transporters. In combination, the above findings indicate that the identified gene encodes a mitochondrial transport protein which upon overexpression and reconstitution displays functional properties that are virtually identical to those of the native mitochondrial dicarboxylate transport system. In conclusion, the present investigation has resulted in identification of a gene encoding the mitochondrial DTP and thus eliminates a major impediment to molecular studies with this metabolically important transporter. Based on both structural and functional considerations, the yeast DTP is assignable to the mitochondrial carrier family. Additionally, the development of a procedure that enables the expression and isolation of large quantities of functional DTP provides the foundation for comprehensive investigations into the structure/function relationships within this transporter via site-directed mutagenesis, as well as for the initiation of crystallization trials.
Thirty-two genes have been identified within the genome of the yeast Saccharomyces cerevisiae which putatively encode mitochondrial transport proteins. We have attempted to overexpress a subset of these genes, namely those which encode mitochondrial transporters of unknown function, and have succeeded in overexpressing 19 of these genes. The overexpressed proteins were then isolated and tested for five well-characterized reconstituted transport activities (i.e., the transport of citrate, dicarboxylates, pyruvate, camitine, and aspartate). Utilizing this approach, we have clearly identified the yeast mitochondrial dicarboxylate transport protein, as well as two additional lower-magnitude transport functions (i.e., tricarboxylate and dicarboxylate transport activities). The implications of these results and the considerations relevant to this approach are discussed.
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The nuclear gene encoding the mitochondrial citrate transport protein (i.e., CTP1) has been deleted from a haploid yeast strain. The stable yeast deletion strain was constructed by homologous recombination of the HIS3 gene at the CTP1 gene locus. Deletion of the CTP was confirmed by PCR. Immunoblot analysis provided the first quantitative estimate of the level of the CTP in wild-type yeast mitochondria and indicated the absence of expressed CTP in mitochondria isolated from the deletion strain. Deletion of CTP1 did not lead to a phenotype on any carbon source tested, indicating that CTP1 is not an essential gene. This suggests that either known alternative pathways are able to produce sufficient acetyl-CoA to support biosynthetic reactions, or there exists a second CTP gene. The ability of the deletion strain to serve as a host for the correct targeting and overexpression of a mutated CTP was then demonstrated. These studies provide a system which permits the use of site-directed mutagenesis to examine both CTP targeting to mitochondria, as well as the molecular basis underlying CTP function. Moreover, this system will not only facilitate the study of the yeast CTP, but also CTPs expressed from the cDNAs of higher eukaryotes.
OBJECTIVE: To determine the effect of application of back supports on back strength in subjects with osteoporosis. DESIGN: In a prospective, randomized, controlled study, we compared compliance and evaluated changes in back strength in 45 women with osteoporosis who were older than 40 years of age and were randomly assigned to one of three groups: (1) postural exercise only, (2) postural exercise and a conventional thoracolumbar support, or (3) postural exercise and a Posture Training Support (PTS) (a weighted kypho-orthosis). MATERIAL AND METHODS: Of the 45 study participants, 15 were assigned to a PTS group, 15 to conventional thoracolumbar support, and 15 to no orthosis. All subjects were instructed in basic body mechanics and postural exercises. Back extensor strength, grip strength, and patient's physical activity were measured at baseline and at subsequent 8-week intervals for a 16-week period. Each patient's compliance during the study period was also recorded. RESULTS: compliance was poor among the thoracolumbar group; only 5 of the 15 subjects completed the study (P<0.001), in comparison with 11 of the 15 patients in the control group and all 15 in the PTS group. Analysis revealed statistically significant mean increases in back strength in the PTS group (23%) and the control group (13%) and a nonsignificant increase in the thoracolumbar group (15%), although poor compliance in the thoracolumbar group yielded insufficient power to detect a significant difference in this group. No statistically significant difference was found between the improvements in the PTS and control groups, possibly because of the small sample size in this pilot study. One patient who wore the PTS for only 4 hours a day rather than 8 hours had the largest percentage increase in back extensor strength of this group (78%). At 16 weeks, decreases in back strength of more than 5% below the initial measurements were noted in 1 of 11 subjects (9%) who completed the control arm of the study, 2 of 14 (14%) who completed the PTS arm, and 2 of 5 (40%) who completed the thoracolumbar arm. CONCLUSION: Compliance with use of the PTS was better than that with the thoracolumbar support. Back extensor strength may increase in patients who comply with the PTS and postural exercise program. We caution, however, that this pilot study requires replication in a larger series to determine the clinical and statistical generalizability of these findings to a wider population.
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The inability to obtain abundant quantities of purified, functional membrane proteins has represented a significant impediment to the goal of obtaining high-resolution structural information. In this review, procedures are described which have been developed in this laboratory and enable the high-level bacterial expression and subsequent purification of functional mitochondrial citrate transport proteins from yeast and rat liver. The data that we have obtained using these procedures and related results from other laboratories are discussed. Additionally, the general applicability of this approach to most mitochondrial transport proteins as well as to other types of membrane proteins is considered. Finally, relevant considerations when contemplating the use of this methodology and the likely value of this approach in future areas of research are explored.
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BACKGROUND: The National Surgical Adjuvant Breast and Bowel Project (NSABP) Protocol B-06, a clinical trial sponsored by the National Cancer Institute (NCI), has provided evidence of the value of lumpectomy and breast irradiation for treating women with breast cancer in an early stage. Publicity generated by the discovery that the study included fraudulent data on patients enrolled by St. Luc Hospital in Montreal aroused concern about the overall accuracy of the data and conclusions. To address this concern, the NCI conducted an audit of other participating institutions. METHODS: In 1994, data on 1554 of the 1809 randomized patients (85.9 percent) enrolled by centers other than St. Luc Hospital were audited at 37 clinical sites in North America. The audit included data on eligibility, survival, disease-free survival, the length of time to a recurrence of cancer in the ipsilateral breast, and documentation of signed informed consent. RESULTS: End points were assessed for all 1554 patients, and eligibility was assessed for 1507 patients; 47 patients were excluded because their forms were not complete or not returned. A total of 1429 patients had their eligibility status verified. Of a total of 7770 data points examined with respect to the number of positive nodes at base line, treatment characteristics, first events (excluding death), recurrence of cancer in the ipsilateral breast, and survival, 7577 (97.5 percent) were verified, 123 (1.6 percent) could not be verified, and 70 (0.9 percent) were discrepant with the NSABP file. Of the 1554 patients, 1340 (86.2 percent) had all audited items (including eligibility) verified, 69 (4.4 percent) had at least one discrepant item, and 113 (7.3 percent) had at least one unverified item (as a result of missing or incomplete data); 32 (2.1 percent) were not assessed for eligibility but had no other discrepant or unverifiable items. Written informed consent was documented for 1098 patients before surgery and 210 after surgery; no date appeared on the signed form for 137. The informed-consent status was not verified for 71 patients and could not be determined for 38. The rates of verification of end-point data and documentation of written informed consent were similar among the total-mastectomy group, the lumpectomy group, and the group treated by lumpectomy and breast irradiation. CONCLUSIONS: The audit confirms the adequacy of the data on which the reanalysis of Protocol B-06 and the results after 12 years of follow-up are based.
The gene encoding the mitochondrial citrate transport protein (CTP) in the yeast Saccharomyces cerevisiae has been identified, and its protein product has been overexpressed in Escherichia coli. The expressed CTP accumulates in inclusion bodies and can be solubilized with sarkosyl. Approximately 25 mg of solubilized CTP at a purity of 75% is obtained per liter of E. coli culture. The function of the solubilized CTP has been reconstituted in a liposomal system where both its kinetic parameters (i.e. Km = 0.36 mM and Vmax = 2.5 mumol/min/mg protein) and its substrate specificity have been determined. Notably, the yeast CTP displays a stricter specificity for tricarboxylates than do CTPs from higher eukaryotic organisms. Dot matrix analysis of the yeast CTP sequence indicates the presence of three homologous sequence domains (each approximately 100 residues in length), which are also related to domains in other CTPs. Thus, the yeast CTP displays the tripartite structure characteristic of other mitochondrial transporters. Alignment of the yeast CTP sequence with CTPs from other sources defines a consensus sequence that displays 89 positions of amino acid identity, as well as the more generalized mitochondrial transporter-associated sequence motif. Based on hydropathy analysis, the yeast CTP contains six putative membrane-spanning alpha-helices. Finally, Southern blot analysis indicates that the yeast genome contains a single gene encoding the mitochondrial CTP. Our data indicate that, based on both its structural and functional properties, the expressed yeast CTP can be assigned membership in the mitochondrial carrier family. The identification of the yeast CTP gene, and the expression and purification of large quantities of its protein product, pave the way for investigations into the roles of specific amino acids in the CTP translocation mechanism, as well as for the initiation of crystallization trials.
The rat liver mitochondrial tricarboxylate transport protein has been overexpressed in E. coli. The expressed transporter, which contains a 21 amino acid N-terminal fusion sequence, accumulates in inclusion bodies. Subsequent extraction of the tricarboxylate transporter from isolated inclusion bodies yields approximately 90 mg of transport protein per liter of E. coli culture at a purity of greater than 90%. Upon incorporation into phospholipid vesicles the purified, overexpressed transporter catalyzes a 1,2,3-benezenetricarboxylate-sensitive citrate/citrate exchange (i.e., the defining reaction of the mitochondrial tricarboxylate transporter). Kinetic characterization of the reconstituted transporter indicates a Km of 0.37 mM and a Vmax of 101 nmol/min/mg protein. The substrate specificity of the reconstituted, expressed transporter is virtually identical to that of the native transporter. These studies represent the first overexpression of the rat liver mitochondrial tricarboxylate transporter. By providing a large amount of highly-purified, functionally competent transporter this system will now enable a variety of structural studies, including site-directed mutagenesis, which heretofore could not be performed.
The effect of pyridoxal 5'-phosphate (PLP), a lysine-selective reagent, on the function of the purified and reconstituted mitochondrial tricarboxylate transport protein has been studied. PLP caused a concentration-dependent inhibition of citrate transport with an IC50 value of 0.09 mM. At 10 mM PLP virtually complete inhibition of transport was observed (i.e. 97%), thereby indicating that modification of a residue(s) whose participation is essential to the translocation mechanism had occurred. Substrate protection studies demonstrated that substrates for the tricarboxylate transporter (i.e., citrate, isocitrate, phosphoenolpyruvate, and malate) effectively protected against PLP inhibition, whereas other organic anions which are not transported by the tricarboxylate carrier (i.e., malonate, alpha-ketoglutarate, phosphate, and succinate) afforded considerably less protection. Kinetic studies in which the transport rate was measured at varying citrate and PLP concentrations indicated that PLP caused an increase in the apparent Km of transport with little change in the Vmax, thereby resulting in a primarily competitive inhibition pattern. In combination, the above findings indicate that PLP interacts with the tricarboxylate transporter at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal alanine residue) that is important in the translocation mechanism and may reside within or near the substrate binding site.
A 29-year-old man presented 45 minutes after sustaining a blow to the right side of his head when his motorcycle slid on gravel and flipped on the side of the road. After walking away from the accident, the patient noted burning pain in his neck. Shortly thereafter, complete quadriplegia with a sensory level at C4 developed. The patient was given a high dose of methylprednisolone and transferred to a tertiary care center. En route, neurological testing demonstrated rapid improvement, and on arrival, all abnormal motor and sensory findings had completely resolved. Radiographic examination and magnetic resonance imaging of the entire spine showed no abnormality. Torg's ratio was > 1 at vertebral level C3 through C6. There was no evidence of developmental stenosis, congenital fusion, cervical instability, or intervertebral disk protrusion.
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From 1978 to 1988, 314 patients with malignant astrocytoma were treated by our neuro-oncology team. Twenty-five patients were excluded from further analysis because of a lack of adequate follow-up, the brain-stem location of the tumor, or an age of less than 18 years. Of the 289 remaining patients in the valid study group, 213 had Grade IV tumors (73.7%) and 76 had Grade III tumors; 167 patients were male (57.8%) and 112 were female, and 89 were less than 40 years of age (30.8%). There were 58 long-term survivors (> 36 mo) in the series (20%). Long-term survivors were much more likely to be less than 40 years of age (x = 41.8; P < 0.005), to have undergone repeated surgery (x = 17.3; P < 0.005), to have received more than 60 Gy of radiation (x = 11.6; P < 0.005), to have Grade III tumors (x = 10.6; P < 0.005), and to have received nitrosoureas (x = 6.09; P < 0.02). Neither sex nor blood type were significantly associated with long-term survival. Patients undergoing repeated surgery were more likely to be less than 40 years of age (x = 5.72; P < 0.02), but neither sex nor histological findings was associated with repeated surgery. For the series as a whole, the observed 5-year survival rate was 6%. We conclude that an aggressive multidisciplinary approach can produce sizable numbers of long-term survivors in malignant astrocytoma patients with favorable prognostic factors.
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