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Biomedical subjects

R M Green

Publications and source records attributed to R M Green.

At least 19 recordsLinked to original sources
PubMed2000

Molecular cloning and characterization of the murine bile salt export pump.

Hepatic bile salt secretion and bile formation are essential functions of the mammalian liver, and the rate-limiting step of hepatocellular secretion of bile salts is canalicular secretion. Recently, the rat sister-of-p-glycoprotein/bile salt export pump (spgp/BSEP) was demonstrated to encode for the rat ATP-dependent canalicular bile salt export protein, and mutations of human BSEP were identified as the cause of PFIC 2. Since mouse models are vital for studies in hepatocellular transport and metabolism, cloning and characterization of the murine gene are essential. In this study, we have cloned a full-length, functional cDNA for the mBsep. The deduced amino acid sequence encodes for a 1321-amino-acid protein and is 94% similar to rat and 89% similar to human bsep. Western immunoblotting using an antibody directed against a carboxy-terminal peptide of mbsep protein reveals a 160kDa protein, which is highly enriched in mouse canalicular membranes. Transfection of mBSEP into Sf-9 insect cells or mammalian Balb-3T3 cells confers functional transport of the bile salt taurocholate. The mBsep mRNA is expressed in murine liver, but not in other tissues. Hepatic mBsep levels appear highly regulated, being markedly diminished in both LPS and estrogen models of cholestasis. These data are important for further murine studies of hepatocellular transport physiology and metabolism.

3T3 Cells↗
PubMed2000

Direct graft puncture with use of a crossed catheter technique for thrombolysis of peripheral bypass grafts.

PURPOSE: To determine the efficacy and safety of direct graft puncture of peripheral arterial bypass grafts with placement of retrograde and antegrade catheters within the graft for thrombolytic therapy. This study also evaluated potential clinical benefit to patients. MATERIALS AND METHODS: A retrospective study was performed on 19 patients with 24 peripheral bypass grafts and lower extremity ischemia of less than 1 month duration. Thrombolysis was performed with a continuous high-dose infusion of urokinase. Successful lysis was defined as greater than 95% clot dissolution with antegrade flow within the graft. RESULTS: Technical success was achieved in 17 of 19 patients (89%). The complexity of operative intervention was diminished in 12 of 19 patients (63%). The major complication rate (16%) was significantly higher and, therefore, this technique has a role for patients in whom traditional access is not optimal, such as in those in whom access cannot be achieved or in those with long bypass grafts. CONCLUSION: Direct graft puncture with placement of catheters across the proximal and distal anastomoses of bypass grafts is a safe method of access, with a major complication rate similar to conventional access techniques. This mode of graft access demonstrates efficacious thrombolysis and acts as a conduit for ancillary procedures.

Aged↗
PubMed2000

Irradiation for intimal hyperplasia: implications for peripheral arterial bypass.

Irradiation has been shown to inhibit postangioplasty intimal hyperplasia ("restenosis") in unbranched tubes. It seems likely that irradiation will similarly be able to inhibit intimal hyperplasia after a surgical anastomosis at a biochemical and cellular level, but whether it will produce a clinically relevant or even clinically detectable difference is unproved. One possibility is that no clinical effect may occur; the search for a "cure" for intimal hyperplasia has been long and, as yet, unsuccessful. On the other hand, if a strong effect without insurmountable logistical problems could be produced, one major cause of bypass graft failure would be preventable. Not only would the incidence of late graft occlusion, need for reoperation, and limb loss be reduced, but, if patency of prosthetics could be sufficiently improved, the initial operation could be made much easier, faster, and perhaps safer.

Angioplasty, Balloon↗
PubMed2000

The anatomy of deep venous thrombosis of the lower extremity.

BACKGROUND: The diagnosis, treatment, and long-term sequelae of lower extremity deep venous thrombosis (DVT) depend on the anatomic location and extent of the process, yet a lack of such fundamental knowledge has limited the development of effective protocols for managing patients with DVT. METHODS: Venograms with evidence of acute DVT were evaluated, and the extent of the thrombotic process was recorded and correlated with the clinical presentation. Thrombi were classified according to the venous segments involved and to the thrombus' isolation to one segment or multiple segments. The left-to-right ratio of the DVT was assessed for various etiologic subgroups. RESULTS: Among 2762 venograms performed in 2541 patients over a 10-year period, there were 885 cases (34.8%) of DVT documented. Of these cases, 344 cases (39%) were idiopathic, 307 cases (35%) were postoperative, 84 cases (10%) occurred in the setting of malignancy, and 70 cases (8%) occurred as the result of trauma. Distal thrombi were more common than proximal thrombi, with calf involvement in 734 patients (83%), femoropopliteal involvement in 470 patients (53%), and iliac involvement in 75 patients (9%). The most common site of thrombus was the peroneal vein, which was involved in 595 patients (67%). The ratio of left-to-right-sided DVT was 1.32:1 overall but was greater for proximal thrombi, with a ratio of 2.4:1 for iliac DVT versus 1.3:1 for infrainguinal DVT. The preponderance of left-sided DVT appeared to be related to the high-frequency, left common iliac vein involvement; the left-to-right ratio was much closer to equality (1.09:1) for isolated infrainguinal DVT. The anatomic configuration of the DVT was correlated with the etiologic subgroup; postoperative DVTs were more often distal, whereas DVT developing in the setting of malignancy was more frequently proximal and often right sided. Proximal, left-sided DVTs were common in the idiopathic subgroup, presumably as a result of undiagnosed left iliac vein webs. CONCLUSIONS: The frequency of distal vein involvement greatly exceeds that of proximal involvement in patients with DVTs. Proximal DVTs are more frequently left sided, whereas distal DVTs occur with a more equal left-to-right distribution. The anatomic extent of DVTs appears to depend on the etiology of the process. These observations may shed light on the pathophysiology of venous thrombosis. The findings are of value in planning therapeutic interventions directed at venous recanalization.

Female↗
PubMed2000

A peptide permease mutant of Mycobacterium bovis BCG resistant to the toxic peptides glutathione and S-nitrosoglutathione.

Oligopeptides play important roles in bacterial nutrition and signaling. Using sequences from the available genome database for Mycobacterium tuberculosis H37Rv, the oligopeptide permease operon (oppBCDA) of Mycobacterium bovis BCG was cloned from a cosmid library. An opp mutant strain was constructed by homologous recombination with an allele of oppD interrupted by kanamycin and streptomycin resistance markers. The deletion was complemented with a wild-type copy of the opp operon. Two approaches were taken to characterize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide variety of peptides as sole source of carbon and/or nitrogen. Among 25 peptides ranging from two to six amino acids in length, none was capable of supporting measurable growth as the sole carbon source in either wild-type or mutant strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (gamma-glutamyl-L-cyteinylglycine [GSH]) is toxic to wild-type BCG and was used successfully to characterize peptide uptake in the opp mutant. In 2 mM GSH, growth of the wild-type strain is inhibited, whereas the opp mutant is resistant to concentrations as high as 10 mM. Similar results were found with the tripeptide S-nitrosoglutathione (GSNO), thought to be a donor of NO in mammalian cells. Using incorporation of [(3)H]uracil to monitor the effects of GSH and GSNO on macromolecular synthesis in growing cells, it was demonstrated that the opp mutant is resistant, whereas the wild type and the mutant complemented with a wild-type copy of the operon are sensitive to both tripeptides. In uptake measurements, incorporation of [(3)H]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three strains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.

Cloning, Molecular↗
PubMed2000

An endoluminal method of hemorrhage control and repair of ruptured abdominal aortic aneurysms.

PURPOSE: To report our initial experience with endovascular grafting to treat ruptured abdominal aortic aneurysms (AAAs). METHODS: Three consecutive patients with severe comorbid illnesses and symptoms of aneurysm rupture and hemodynamic instability were treated with aortomonoiliac grafts. The Z-stent-based devices were implanted with the assistance of an occlusion balloon placed in the distal descending thoracic aorta. RESULTS: All patients survived the procedure with successfully excluded AAAs. Two patients had relatively short hospital stays (4 and 14 days), while the third required prolonged treatment for pre-existing conditions. All patients required blood transfusions; 2 developed significant coagulopathies. Definitive management was delayed significantly by imaging protocols and graft construction. CONCLUSIONS: Endovascular repair of ruptured aortic aneurysms is feasible. Proximal aortic control is readily attainable with the use of an aortic occlusion balloon placed through the left axillary artery. The absence of a laparotomy, extensive retroperitoneal dissection, and aortic cross-clamping likely contributes to patient survival; however, the delay in operative therapy to obtain adequate imaging and construct an endograft could be a hindrance to the ultimate success of this approach. The concepts of alternative aortic imaging techniques and endograft design, construction, and storage must be addressed.

Aged↗
PubMed2000

Prosthetic above-knee femoropopliteal bypass grafting: five-year results of a randomized trial.

PURPOSE: This trial was designed to identify factors affecting patency rates of primary prosthetic above-knee femoropopliteal bypass grafts at 5 years. METHODS: A multi-institutional, prospective trial randomized 240 patients to compare patency rates of Gore-tex and Hemashield above-knee femoropopliteal bypass grafts at 5 years. Univariate comparisons of patency between levels of each prognostic variable were made with the Kaplan-Meier method. Variables that had a univariate P value less than.25 or those known to be important were submitted to a Cox regression analysis. RESULTS: The patient survival rate at 5 years was 59.4%. There were no differences in primary or secondary patency rates at 5 years between the two graft materials (primary, 45% vs 43% and secondary, 68% vs 68%). The risk for graft occlusion was significantly increased for patients younger than 65 years (2.1; P =.001) and for grafts with a diameter less than 7 mm (1.65; P =.0219). Variables with no apparent independent effect on patency rates were smoking status, runoff, diabetes mellitus, sex, presenting symptoms, and postoperative treatment with aspirin or Coumadin. Noninvasive test results were not predictive of subsequent graft function. CONCLUSION: Although the type of prosthetic used for above-knee femoropopliteal bypass grafts does not affect 5-year patency rates, age and graft size do influence results. These factors should be considered before a prosthetic bypass grafting procedure. Furthermore, these data should serve as a contemporary standard, with which evolving and conventional procedures can be compared.

Aged↗
PubMed1999

Cloning, tissue-specific expression, gene structure and chromosomal localization of human phosphatidylcholine transfer protein.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein that catalyzes intermembrane transfer of phosphatidylcholines in vitro. We have cloned a cDNA encoding the human ortholog of PC-TP and have determined its tissue-specific expression as well as genomic organization. Radiation hybrid mapping localized the human gene, PCTP, to chromosome 17q21-22 and PCR-based single strand conformation polymorphism analysis of an interspecific backcross assigned mouse Pctp to the region of syntenic conservation on chromosome 11.

Amino Acid Sequence↗
PubMed1999

The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis.

A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock remains unknown. Here we show that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortened the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduced phytochrome induction of an Lhcb gene. Finally, we found that the photoperiodic flowering response, which is influenced by circadian rhythms, was diminished in the transgenic lines, and that the plants flowered earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis.

Amino Acid Sequence↗
PubMed1999

Loss of the circadian clock-associated protein 1 in Arabidopsis results in altered clock-regulated gene expression.

Little is known about plant circadian oscillators, in spite of how important they are to sessile plants, which require accurate timekeepers that enable the plants to respond to their environment. Previously, we identified a circadian clock-associated (CCA1) gene that encodes an Myb-related protein that is associated with phytochrome control and circadian regulation in plants. To understand the role CCA1 plays in phytochrome and circadian regulation, we have isolated an Arabidopsis line with a T DNA insertion that results in the loss of CCA1 RNA, of CCA1 protein, and of an Lhcb-promoter binding activity. This mutation affects the circadian expression of all four clock-controlled genes that we examined. The results show that, despite their similarity, CCA1 and LHY are only partially redundant. The lack of CCA1 also affects the phytochrome regulation of gene expression, suggesting that CCA1 has an additional role in a signal transduction pathway from light, possibly acting at the point of integration between phytochrome and the clock. Our results indicate that CCA1 is an important clock-associated protein involved in circadian regulation of gene expression.

Arabidopsis↗
PubMed1999

A novel putative transporter maps to the osteosclerosis (oc) mutation and is not expressed in the oc mutant mouse.

The phenotype of mice homozygous for the osteosclerosis (oc) mutation includes osteopetrosis, and a variety of studies demonstrate that osteoclasts in these mice are present but nonfunctional. We have identified a novel gene that has homology to a family of 12-transmembrane domain proteins with transport functions and maps to proximal mouse chromosome 19, in a region to which the oc mutation has been previously assigned. The putative transporter is abundant in normal kidney, but its expression is markedly reduced in kidneys from oc/oc mice when tested using Northern and Western analyses. Southern analysis of this gene, which we call Roct (reduced in oc transporter), demonstrates that it is intact and unrearranged in oc/oc mice. In situ studies show that Roct is expressed in developing bone. We propose that the absence of Roct expression results in an osteopetrosis phenotype in mice.

Amino Acid Sequence↗
PubMed1999

Cloning and functional expression of a mouse liver organic cation transporter.

Hepatic uptake of organic cations is essential for the metabolism and secretion of numerous endobiotics and drugs. Several hepatic organic cation transporters have been kinetically defined, yet have not been isolated or cloned. We have isolated a complementary DNA (cDNA) from both murine liver and kidney cDNA libraries (mOct1/Slc22a1), and have functionally expressed it in Xenopus laevis oocytes. Although mOct1/Slc22a1 is homologous to previously cloned rat and human organic cation transporters, organic cation transport kinetics differed markedly. mOct1/Slc22a1-RNA injection of oocytes resulted in the saturable, time- and temperature-dependent uptake of the quaternary organic cation [14C]-tetraethylammonium ([14C]-TEA), with a Km of 38 micromol/L. TEA uptake was inhibited by several other organic cation drugs, but was not inhibited by the organic cation n-methyl-nicotinamide (NMN), being instead stimulated by it (fourfold). [14C]-TEA uptake was also stimulated by an inside-outside proton gradient. mOct1/Slc22a1-injected oocytes transported the organic cations [3H]-1-methyl-4-phenylpyridium and [3H]-choline chloride, but did not transport other classes of organic compounds. mOct1/Slc22a1 encodes for a hepatic and renal organic cation transporter which may be important for the uptake and secretion of cationic drugs and endobiotics.

1-Methyl-4-phenylpyridinium↗
PubMed1999

A volumetric index for the quantification of deep venous thrombosis.

PURPOSE: The evaluation of treatment strategies for deep venous thrombosis (DVT) is assessed through the use of a reliable method of quantifying the extent of the thrombotic process. Previous indices of thrombus burden have suffered from various limitations, including lack of clinical relevance, poor correlation with actual thrombus mass, and failure to include important venous segments in the methodology. The use of a novel scheme of quantifying venous thrombus was evaluated as an alternative method that would avoid some of the drawbacks of existing indices. METHODS: The volumes of 14 venous segments (infrarenal inferior vena cava, common iliac, hypogastric, external iliac, common femoral, profunda, superficial femoral, and popliteal and six tibial veins) were calculated from computed tomography (pelvic vein diameter), duplex ultrasound scan (infrainguinal vein diameter), and contrast venography (length of all segments) measurements. A venous volumetric index (VVI) was assigned with the normalization of the values to the volume of a single calf vein. The VVI was validated with the assessment of the ability to discriminate between asymptomatic and symptomatic DVT and between those DVT that were associated with pulmonary emboli and those that were not. RESULTS: With the imaging data, the VVI ranged from 1 for a single calf vein thrombus to 26 for the infrarenal inferior vena cava. Each VVI unit represented 2.3 mL of thrombus, with a maximum possible score of 63 representing a thrombus burden of 145 mL for a single extremity, including the infrarenal inferior vena cava. In 885 patients with DVT, the VVI ranged from 1 to 56, averaging 3.9 +/- 0.2 in patients who were asymptomatic and 8.7 +/- 0.3 in patients who were symptomatic (P <.001). The VVI was similar in the patients with pulmonary emboli as compared with those without (9.6 +/- 1.2 vs 7.7 +/- 0.2, respectively). In comparison with the three existing methods of quantifying venous thrombus burden, the receiver operating characteristic curve analysis results suggested that the VVI and the Venous Registry index were better than the other two indices in the discrimination of patients with symptomatic and asymptomatic DVT (P <.001). CONCLUSION: A novel index of venous thrombus burden, on the basis of actual venous volume measurements, was more accurate than present indices in the differentiation between clinical categories of patients with DVT. As such, it offers an acceptable alternative to current scoring systems.

Diagnostic Imaging↗
PubMed1999

Photoplethysmography and calf muscle pump function after subfascial endoscopic perforator ligation.

OBJECTIVE: Subfascial endoscopic perforator surgery (SEPS) results in acceptable healing and recurrence rates. The role of hemodynamic venous testing in this situation, however, is poorly understood and inconsistently used. Our ongoing experience was reviewed to explore how SEPS affects the photoplethysmographic assessment of the leg. METHODS: Preoperative and postoperative venous refill times (VRTs) were measured with photoplethysmography in 30 limbs in 28 patients who underwent SEPS and superficial ablation, when indicated, with complete clearing of the anterolateral surface of the tibia, thus opening the deep posterior compartment from mid calf to close to the malleolus. Postoperative healing and duplex scanning were used to assess clinical and anatomic success, respectively. The VRTs were classified as "interpretable" if the leg emptied or "uninterpretable" if the calf could not empty. The "interpretable" study results were further classified as "normal" if the refill took 20 seconds or more or "abnormal" if less. RESULTS: Before the patients underwent SEPS, six study results (20%) showed inability of the calf to empty and thus were judged uninterpretable. After the patients underwent SEPS, 12 study results (40%) were uninterpretable (NS; P =.09 with the chi(2) test). Of the 24 preoperative interpretable study results, two (8%) were normal, and of the 18 postoperative interpretable study results, seven (39%) were normal (P <.03). With the consideration of only interpretable study results, the mean VRT increased slightly from 12.0 +/- 5.1 seconds (mean +/- standard deviation) to 14.3 +/- 8.1 seconds (NS). Seventeen of 19 ulcers (89%) had healed at a mean follow-up period of 8.6 +/- 4.8 months. CONCLUSION: Although VRT is unpredictably affected by SEPS, the most consistent finding is the inability of the calf to empty, which invalidates the remainder of the test. In addition, most ulcers heal, even with uninterpretable or abnormal postoperative VRTs. This suggests that photoplethysmography is a poor method of assessment of venous reflux after SEPS.

Adult↗
PubMed1999

Intracerebral hemorrhage after carotid endarterectomy: incidence, contribution to neurologic morbidity, and predictive factors.

PURPOSE: With a diminishing rate of cardiac and neurologic events after carotid endarterectomy, intracerebral hemorrhage is gaining increasing importance as a cause of perioperative morbidity and mortality. To date, information has been largely anecdotal, and there has been no comparison with a control group of patients. METHODS: The records of all patients experiencing symptomatic intracerebral hemorrhage after carotid endarterectomy were reviewed and compared with data from 50 randomly selected patients who did not experience intracranial bleeding. Univariate analyses were performed, using the Fisher exact test for dichotomous data and the Student t test for continuous data. RESULTS: During a 6-year period, symptomatic intracranial hemorrhage developed in 11 (0.75%) of 1471 patients undergoing carotid endarterectomy, accounting for 35% of the 31 total perioperative neurologic events. Hemorrhage occurred a median of 3 days postoperatively (range, 0 to 18 days). Signs and symptoms included hypertension in all 11 patients, headache in 7 conscious patients (64%), and bradycardia in 6 patients (55%). Massive hemorrhage with herniation and death occurred in 4 patients (36%). Moderate hemorrhage developed in 5 patients (45%); 3 of these patients had partial recovery, and 2 had complete recovery. Petechial hemorrhage occurred in the remaining 2 patients (18%), 1 with partial and 1 with complete recovery. In comparison with the control group, there were no differences in respect to sex, indication for operation, smoking or diabetic history, and antiplatelet therapy or perioperative heparin management. Patients with intracranial hemorrhage were, however, younger, more frequently hypertensive, had a higher degree of ipsilateral and contralateral carotid stenosis, and had a higher rate of contralateral carotid occlusion. CONCLUSION: Intracranial hemorrhage occurs with notable frequency after carotid endarterectomy and accounts for a significant proportion of neurologic morbidity and mortality. Younger patients, hypertensive patients, and patients with severe cerebrovascular occlusive disease appear to be at greatest risk for the complication.

Age Factors↗
PubMed1999

Azoxymethane-induced fulminant hepatic failure in C57BL/6J mice: characterization of a new animal model.

Without transplantation, approximately 50-90% of all patients with fulminant hepatic failure (FHF) die. This poor outcome is due in part to the absence of an appropriate animal model, which would allow for a greater understanding of the pathophysiology of this syndrome. Given the reports of liver injury in humans and livestock fed cycad palm nuts on the island of Guam, we hypothesized that the active ingredient azoxymethane (AOM) could cause FHF. We therefore evaluated AOM in C57BL/6J mice. Histologically, we observed microvesicular steatosis 2 h, sinusoidal dilatation 4 h, and centrilobular necrosis 20 h after AOM administration, and transmission electron microscopy showed that this agent causes mitochondrial injury. FHF was associated with all four stages of encephalopathy, as well as by a prodromal period of decreased eating and drinking lasting approximately 15 h before the development of stage I encephalopathy (i.e., loss of scatter reflex). Late encephalopathy was associated with increased arterial ammonia, decreased serum glucose, and evidence of brain edema (astrocyte swelling). We show that AOM-induced FHF is highly reproducible, without evidence of lot-to-lot variability, and is dose dependent. These findings therefore suggest that AOM is an excellent agent for the study of FHF, as well as indicate that Guamanian FHF may be due to AOM found in unwashed cycad palm nuts.

Animals↗
PubMed1999

Prosthetic above-knee femoropopliteal bypass.

One of the classic and as yet unresolved arguments in vascular surgery is whether using prosthetic for a first-time above-knee femoropopliteal bypass, to "save the vein" for a later, more distal bypass, is of net benefit. Most of the arguments supporting use of prosthetic for a first bypass in this situation are, in fact, not supported by the literature, whereas most of the arguments in favor of using vein first are. In addition, decision analysis shows that overall amputation-free survival and number of operative procedures required are clearly better if vein is used first under essentially all conditions--the overwhelmingly strongest determinant of outcome is patency of the first bypass, and the superior patency of initial bypass with vein mathematically outweighs the ability of the "preserved" vein to salvage failures after an initial bypass with prosthetic. Theoretical, empirical, and mathematical arguments all strongly favor preferential use of vein for a first bypass to the above-knee popliteal artery.

Blood Vessel Prosthesis Implantation↗