Biomedical subjects

B Andersson

Publications and source records attributed to B Andersson.

At least 163 records · Page 9Linked to original sources

PubMed1997

Large-scale concatenation cDNA sequencing.

A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

DNA Transposable Elements↗

PubMed1997

In situ hybridisation mapping of genomic clones for five human respiratory chain complex I genes.

Using human and bovine short cDNA sequences as probes we screened human cosmid and P1 libraries for components of the complex I multi-subunit enzyme of oxidative phosphorylation. We isolated genomic recombinants encoding cI-B8 (gene NDUFA2), cI-B14 (gene NDUFA6), cI-B14.5a (gene NDUFA7), cI-ASHI (gene NDUFB8) and cI-23kD (gene NDUFS8). Genomic versions of these genes have not been previously cloned in the human although they are represented as anonymous entries in public cDNA databases. By using the derived genomic clones for in situ hybridisation studies we determined the following chromosome locations: NDUFA2, 5q31; NDUFA6, 21q22; NDUFA7, 20p13; NDUFB8, 12q21; NDUFS8, 3q28.

Animals↗

PubMed1997

Association of serum antibodies to heat-shock protein 65 with borderline hypertension.

Heat-shock proteins protect cells from damage but are also often the target of immune responses in inflammation and may therefore both induce and perpetuate the chronic inflammation characterizing atherosclerosis. Hypertension is a well-established risk factor for atherosclerosis, and recently, borderline hypertension also has been related to atherosclerosis. The present study investigated the possible role of heat-shock proteins in borderline hypertension and their relation to atherosclerosis by investigating antibody titers against the 65-kD heat-shock protein (HSP65). Sixty-six men with borderline hypertension and 67 age-matched normotensive men (diastolic pressure, 85 to 94 and < 80 mm Hg, respectively) were recruited from a population screening program. Titers of antibodies to HSP65 were determined by enzyme-linked immunosorbent assay. The presence of carotid atherosclerosis was determined by B-mode ultrasonography. Twenty-seven individuals had atherosclerotic plaques: 48 were smokers (more than one to two cigarettes per day). Borderline hypertensive men had higher anti-HSP65 reactivity than normotensive control subjects (P = .034). Smokers with atherosclerosis had low levels of antibodies to HSP65 compared with nonsmokers with atherosclerosis (P = .002). Furthermore, when high-risk individuals (borderline hypertension plus plaque, n = 15) were compared with matched low-risk individuals (normotensive with no plaque, n = 15), the high-risk men had significantly enhanced antibody titers to HSP65 (P = .041). In conclusion, we demonstrate that serum antibody titers to HSP65 are enhanced in individuals with borderline hypertension, which may indicate an ongoing immune reaction in the artery wall.

Adult↗

PubMed1997

Phase I trial of cyclosporine-induced autologous graft-versus-host disease in patients with multiple myeloma undergoing high-dose chemotherapy with autologous stem-cell rescue.

PURPOSE: To determine the feasibility and toxicity of inducing autologous graft-versus-host disease (GVHD) with cyclosporine in patients with multiple myeloma undergoing autologous stem-cell transplantation. PATIENTS AND METHODS: Fourteen multiple myeloma patients with a median age of 50 years (range, 41 to 63) were enrolled. The median time from diagnosis to transplant was 651 days (range, 229 to 3,353). Ten patients had primary refractory disease, two were in first remission, and two were responsive to salvage therapy. The preparative regimen consisted of thiotepa, busulfan, and cyclophosphamide. Cyclosporine was administered daily for 28 days after the stem-cell infusion, and the dose was adjusted to maintain whole-blood cyclosporine levels between 50 and 150 ng/dL in the first seven patients (low-level group) and between 150 and 300 ng/dL in the other seven patients (high-level group). RESULTS: All patients achieved neutrophil engraftment a median of 11 days after transplant. Four patients developed > or = grade 2 hepatic toxicity, six developed > or = grade 2 nephrotoxicity, and four developed reversible cardiac toxicity. Only one treatment-related death occurred. Cyclosporine was withheld in seven patients for a median of 6 days because of renal and/or liver dysfunction. One patient developed clinical skin GVHD, which responded to corticosteroid therapy. Six patients developed histologic evidence of GVHD without clinical signs of GVHD (subclinical GVHD). The incidence of clinical and subclinical GVHD was similar in both cyclosporine groups. Three of 11 patients assessable for response achieved remissions. Three patients experienced disease progression 80, 160, and 354 days after transplant. Ten patients are alive without progression between 56 and 444 days after transplant. CONCLUSION: Induction of autologous GVHD by posttransplant cyclosporine is feasible and well tolerated in patients with multiple myeloma.

Adult↗

PubMed1997

Estrogen replacement therapy decreases hyperandrogenicity and improves glucose homeostasis and plasma lipids in postmenopausal women with noninsulin-dependent diabetes mellitus.

Hyperandrogenicity in women is closely associated with insulin resistance and a risk factor for cardiovascular disease and noninsulin-dependent diabetes mellitus (NIDDM). Therefore, 25 postmenopausal women with NIDDM and sex hormone-binding globulin values less than 60 nmol/L, as an indicator of a moderate hyperandrogenicity, were treated with 2 mg 17-beta-estradiol orally for 3 months in a double-blind, cross-over, placebo-controlled trial. During the last 16 days of active treatment, 1 mg norethisterone acetate was added for 10 days for endometrial protection. Blood glucose, glycosylated hemoglobin, insulin, c-peptide, lipoprotein profile, sex steroid hormones, GH, and insulin-like growth factor I (IGF-I) were measured, and insulin sensitivity was determined by the euglycemic hyperinsulinemic clamp method. All metabolic measurements were taken at baseline and after 68 days of active or placebo treatment. Estradiol treatment, compared with the placebo period, was followed by a marked increase of sex hormone-binding globulin and a decrease of free testosterone. Blood glucose, glycosylated hemoglobin, c-peptide, total cholesterol, low-density lipoprotein cholesterol, and IGF-I decreased significantly (P < 0.01-P < 0.001), whereas high-density lipoprotein cholesterol rose (P < 0.001). In conclusion, estrogen replacement therapy in postmenopausal women with NIDDM ameliorated hyperandrogenicity, and this was accompanied by marked improvements in glucose homeostasis and lipoprotein profile.

Androgens↗

PubMed1997

Simultaneous shotgun sequencing of multiple cDNA clones.

This study describes an efficient method for the rapid sequencing of DNA fragments in the size range 1-5 kb. Individual fragments, here cDNA inserts, are purified by restriction enzyme digestion and gel purification, pooled and concatenated by ligation. The concatamers are sheared and cloned randomly into M13, followed by random sequencing. The sequences of the individual cDNA inserts are obtained at the assembly stage using restriction enzyme sites as "tags' for the ends of each fragment. In this study the sequencing of two libraries containing 7 and 16 cDNA inserts with an average length of 2.5 kb is described. The results show that of the shotgun sequencing of large fragments, and that the method compares favorably to alternative strategies, such as primer walking.

Animals↗

PubMed1997

Cerebrospinal fluid interleukin-1 receptor antagonist and tumor necrosis factor-alpha following subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) causes an inflammatory reaction and may lead to ischemic brain damage. Experimental ischemia has been shown to be connected with the alarm-reaction cytokines interleukin-1 receptor antagonist (IL-1Ra) and tumor necrosis factor-alpha (TNF alpha). Increased levels of these cytokines, however, have not been detected thus far in patients following an SAH event. For this reason daily cerebrospinal fluid (CSF) samples were collected from 22 consecutively enrolled patients with SAH and from 10 non-SAH patients (controls). The CSF samples were studied using immunoassays for IL-1Ra and TNF alpha to investigate whether an SAH caused increased cytokine levels. The mean IL-1Ra levels were significantly higher in patients with SAH who were in poor clinical condition on admission than in those who were in good condition (318 pg/ml vs. 82 pg/ml, p < 0.02). The IL-1Ra levels increased during delayed ischemic episodes and after surgery in patients who were in poor clinical condition. Significant increases in IL-1Ra and TNF alpha were detected during Days 4 through 10 in patients suffering from SAH who eventually had a poor outcome (p < 0.05). Patients with good outcomes and control patients had low levels of these cytokines. The levels of IL-1Ra increased after surgery in patients with Hunt and Hess Grades III through V, but not in those with Grade I or II. This finding indicates that patients in poor clinical condition have a labile biochemical state in the brain that is reflected in increased cytokine levels following the surgical trauma. Both IL-1Ra and TNF alpha are known to induce fever, malaise, leukocytosis, and nitric oxide synthesis and to mediate ischemic and traumatic brain injuries. The present study shows that levels of these cytokines increase after SAH occurs and that high cytokine levels correlate with brain damage. It is therefore likely that fever, leukocytosis, and nitric oxide synthesis are also mediated by IL-1 in patients suffering from SAH and it is probable that the inflammatory mediators contribute to brain damage.

Adult↗

PubMed1997

Clinical evaluation of ceramic veneered titanium restorations according to the Procera technique.

The aim of the present study was to present the design and one-year results of a study of crowns and fixed partial dentures (FPDs) made with the Procera system. All patients at one clinic needing fixed prosthodontic restorations during a two-year period were invited. All 260 patients accepted to participate and they received 333 ceramic veneered Procera restorations-242 single crowns and 91 FPDs. The loss of patients during the one-year follow-up was only 5%. The restorations were evaluated according to a modified version of the California Dental Association rating system. Practically all Procera restorations were judged as satisfactory both at base line and at the one-year follow-up examination. Two crowns and one FPD showed fractures of the ceramic veneering. Another FPD had a fracture of a welded joint. Three single crowns came loose but could be recemented. In conclusion, the ceramic veneered Procera titanium restorations were well accepted by the patients and the success rate was high during the one-year follow-up period.

Adult↗

PubMed1997

Electrophysiological characterization of the prokinetic agents cisapride and mosapride in vivo and in vitro: implications for proarrhythmic potential?

In the present study the electrophysiological characteristics and the proarrhythmic potential of cisapride and a structurally related drug, mosapride, were compared. In the anesthetized guinea pig, cisapride and d-sotalol (0.01-10 micromol/kg i.v., n = 6) dose-dependently prolonged the duration of the monophasic action potential recorded from the left ventricle. The maximal lengthening was 18 +/- 3.2% at 1.0 micromol/kg (mean +/- S.E.M., P < .01 vs. base line) and 19 +/- 2.5% at 10 micromol/kg (P < .001) for cisapride and d-sotalol, respectively. In contrast, mosapride did not increase this variable. In a rabbit model of the acquired long QT syndrome, infusion of cisapride (0.3 micromol/kg/min for 10 min maximum, n = 6), but not mosapride or vehicle, was associated with a significant lengthening of the QTU interval (43 +/- 3.8 ms, P < .01). Furthermore, torsades de pointes appeared in two of the six rabbits given cisapride. In isolated rabbit Purkinje fibers (PF), cisapride increased the action potential duration (48 +/- 5.6% at 0.1 micromol/l, P < .01 vs. control, n = 4). Mosapride did not significantly influence the action potential duration (3 +/- 2.0% increase at 1.0 micromol/l, n = 6). However, after mosapride was washed out, the addition of cisapride (0.1 micromol/l) caused a 46 +/- 3.2% lengthening of the action potential duration (P < .01 vs. 1.0 micromol/l mosapride). Early afterdepolarizations and triggered activity appeared in four of eight cisapride-superfused PF stimulated at a very low frequency (0.1 Hz). In isolated rabbit cardiomyocytes, cisapride concentration-dependently blocked (IC50 = 9 nmol/l) the rapid component of the delayed rectifying K+ current (I(Kr)). Mosapride was approximately 1000-fold less potent in blocking I(Kr) (IC50 = 4 micromol/l). It is concluded that the electrophysiological characteristics of cisapride may explain the recently reported propensity to prolong the QT interval and to induce torsades de pointes in susceptible patients, although a structurally related benzamide, mosapride, did not appear to have electrophysiological features of relevance for induction of torsades de pointes in common with cisapride.

Action Potentials↗

PubMed1997

The impact of T-cell depletion on the effects of HLA DR beta 1 and DQ beta allele matching in HLA serologically identical unrelated donor bone marrow transplantation.

Unrelated donor bone marrow transplants have been associated with relatively high rates of acute graft-vs.-host disease and treatment-related mortality. These complications reflect histo-incompatibility between donor and recipient. Molecular technology has recently been applied to HLA typing to identify alleles not distinguishable with serologic typing techniques. We report results in 92 unrelated marrow transplant recipients who were HLA seroidentical with donor HLA-A, -B, and -DR antigens and assess the effect of DR beta 1 and DQ beta compatibility using sequence specific oligonucleotide primers. Forty-eight patients received T-cell depleted marrow grafts, and 44 received unmodified grafts. Among recipients of unmodified marrow grafts, matching for both DR beta 1 and DQ beta reduced the rate of grade 3-4 acute graft-vs.-host disease to 38 +/- 20% vs. 73 +/- 20% among recipients mismatched for either allele (p = 0.02). This difference was not observed in recipients of T-cell depleted marrow grafts. Multivariate analysis confirmed matching for both DR beta 1 and DQ beta loci (p = 0.015), and receiving a T-cell depleted graft (p = 0.008) independently predicted for reduced risk of grade 3-4 acute graft-vs.-host disease. In conclusion, both DR beta 1 and DQ beta appear biologically important for development of acute graft-vs.-host disease in patients receiving unmanipulated marrow grafts for unrelated donor transplant.

Adult↗

PubMed1997

Allogeneic transplantation for recurrent or refractory non-Hodgkin's lymphoma with poor prognostic features after conditioning with thiotepa, busulfan, and cyclophosphamide: experience in 44 consecutive patients.

We report the outcomes of 44 consecutive patients with non-Hodgkin's lymphoma (NHL) who participated in prospective studies of allogeneic transplantation after conditioning with thiotepa, busulfan and cyclophosphamide. Within a range of 27-57 years, the median age was 37. Of the 44 patients, 12 (27.2%) had high-grade lymphomas, 27 (61.4%) had intermediate-grade lymphomas, and five (11.3%) had low-grade lymphomas. Twenty-eight (63.6%) patients had chemotherapy refractory disease. Thirty (68.2%) patients had stage IV disease at the time of transplantation, involving the bone marrow in 19 (43.2%). Eight (18.1%) patients had undergone previous transplantation, and 13 (29.5%) patients had received high-dose CVP as induction within 2 months prior to transplantation. Thirty-eight (86.3%) patients had an HLA-identical donor, and 6 (13.6%) had a one-antigen mismatched related donor. Twenty (45.4%) patients received bone marrow and 24 (54.6%) received granulocyte colony-stimulating factor (G-CSF) mobilized stem cells. The graft-versus-host disease (GVHD) prophylaxis contained cyclosporine or tacrolimus in combination with either methylprednisolone in 32 (72.7%) patients or with methotrexate in 12 (27.2%) patients. The actuarial probability of disease-free survival at 2 years is 23% (95% CI 13%-40%). Donor stem cell use was associated with a significantly decreased risk of treatment-related toxicity (p < 0.001), but with an increased risk for GVHD and delayed fungal and viral infections. These infections are linked not only to the use of donor-stem cells, but also to the methylprednisolone in the GVHD prophylaxis regimen. Improvements in the outcome of patients with advanced NHL and undergoing allogeneic transplantation will depend on the development of effective and non-toxic regimens for conditioning, GVHD prophylaxis, and opportunistic infections prophylaxis.

Adult↗

PubMed1996

Allogeneic transplantation for advanced leukemia: improved short-term outcome with blood stem cell grafts and tacrolimus.

We have evaluated the use of blood stem cell grafts for rapid hematopoietic recovery and tacrolimus (FK506) as GVHD prophylaxis to reduce early mortality after allogeneic transplantation. Eighty-five adults with advanced leukemia received high-dose thiotepa, busulfan, and cyclophosphamide as a preparative regimen in a prospective Phase II study. All donors were HLA-matched and related. Marrow (BMT) was used for 44 patients and filgrastim-mobilized blood stem cells (SCT) for 41 patients. GVHD prophylaxis consisted of cyclosporine (CsA) or FK506 with methotrexate (MTX) or methylprednisolone (MP). The median time to neutrophil recovery was earlier after SCT than after BMT (day 10 vs. 17, P<0.001), but this was due to the selective use of MTX only in the BMT patients. The risk of grades 2-4 GVHD was lower with FK506 than with CsA (16% vs. 45%, P=0.02) and was the same for SCT recipients as for BMT recipients (33% vs. 34%). Regimen-related toxicity was significantly lower after SCT than after BMT but did not differ between the FK506 and CsA patients. In comparison with those receiving the standard transplant (BMT with CsA and MTX), only the SCT recipients using FK506 and MP had a significantly higher survival at day 180 posttransplant (84% vs. 53%, P=0.014). In multivariate analyses, use of FK506 was associated with a lower risk of treatment-related mortality and a higher survival at day 180, while the diagnosis of acute lymphoblastic leukemia was associated with a higher risk of treatment-related mortality. These data suggest that the use of blood stem cell grafts and FK506 can reduce the early mortality after allogeneic transplantation for advanced leukemia.

Adolescent↗

PubMed1996

Tacrolimus and minidose methotrexate for prevention of acute graft-versus-host disease after matched unrelated donor marrow transplantation.

Thirty adults with leukemia or lymphoma undergoing marrow transplantation from HLA-compatible unrelated donors received tacrolimus (FK506), a new immunosuppressive macrolide lactone, and minidose methotrexate to prevent acute graft-versus-host disease (GVHD). The group had a median age of 36 years (range 21 to 49 years). Twenty-four patients had advanced disease, and 11 were resistant to conventional therapy. Tacrolimus was administered at 0.03 mg/kg/d intravenously (i.v.) by continuous infusion from day -2, converted to oral at four times the i.v. dose following engraftment, and continued through day 180 posttransplant. Methotrexate 5 mg/m2 was given i.v. on days 1, 3, 6, and 11. All patients engrafted. Grades 2-4 GVHD occurred in 34% (95% CI, 17% to 52%), and grades 3-4 GVHD in 17% (95% CI, 3% to 31%). Mild renal toxicity was common before day 100; 63% of patients had a doubling of creatinine, and 52% had a peak creatinine greater than 2 mg/dL, but only one patient was dialyzed. The median last i.v. dose of tacrolimus was 53% of the scheduled dose, and the median oral dose on day 100 was 41% of that scheduled. Overall survival at 1 year was 47% (95% CI, 27% to 66%). We conclude that tacrolimus can be combined safely with minidose methotrexate, and the combination has substantial activity in preventing acute GVHD after unrelated donor marrow transplantation.

Actuarial Analysis↗

PubMed1996

Identification, characterization, and molecular cloning of a homologue of the bacterial FtsH protease in chloroplasts of higher plants.

In an attempt to identify and characterize chloroplast proteases, we performed an immunological analysis of chloroplasts using an antibody against Escherichia coli FtsH protease, which is an ATP-dependent metalloprotease bound to the cytoplasmic membrane. A cross-reacting protein of 78 kDa was found in the thylakoid membrane of spinach, but not in the soluble stromal fraction. Alkali and high salt washes, as well as trypsin treatment of thylakoid membranes, suggest that the chloroplastic FtsH protein is integral to the membrane, with its hydrophilic portion exposed to the stroma. The protein is not bound to any photosynthetic complex and is exclusively located in the stromally exposed regions of the thylakoid membrane. Its expression is dependent on light, as it is present in green pea seedlings, but absent from etiolated ones. An Arabidopsis cDNA was isolated, and the deduced amino acid sequence demonstrated high similarity to the E. coli FtsH protein, especially in the central region of the protein, containing the ATP- and zinc-binding sites. The product of this clone was capable of import into isolated pea chloroplasts, where it was processed to its mature form and targeted to the thylakoid membrane. The trans-bilayer orientation and lateral location of the FtsH protein in the thylakoid membrane suggest its involvement in the degradation of both soluble stromal proteins and newly inserted or turning-over thylakoid proteins.

ATP-Dependent Proteases↗

PubMed1996

Changes in early and late diastolic filling patterns induced by long-term adrenergic beta-blockade in patients with idiopathic dilated cardiomyopathy.

BACKGROUND: beta-Blockers have been used in patients with idiopathic dilated cardiomyopathy to improve cardiac performance and theoretically would be beneficial to diastolic function. However, there are few reports on changes in diastolic function during chronic pharmacological treatment of congestive heart failure. METHODS AND RESULTS: The present study was a substudy in the international Metoprolol in Dilated Cardiomyopathy Trial. Transmitral Doppler echocardiography was used to evaluate diastolic function in 77 patients randomly assigned to placebo (n = 37) or metoprolol (n = 40). The patients were treated for 12 months. Changes in Doppler flow variables in the metoprolol group implied a less restrictive filling pattern, expressed as an increase in E-wave deceleration time (placebo, 185 +/- 126 to 181 +/- 64 ms; metoprolol, 152 +/- 63 to 216 +/- 78 ms; P = .01, placebo versus metoprolol). Maximal increase in deceleration time had occurred by 3 months, whereas systolic recovery was achieved gradually and maximal effect was seen by 12 months of treatment. Although deceleration time was correlated to heart rate at baseline, changes in deceleration time were not significantly correlated to changes in heart rate during treatment. CONCLUSIONS: During the first 3 months of treatment, maximal effects on diastolic variables were reached, whereas the most prominent effect on systolic function was seen late in the study. It is suggested that effects on diastolic filling account for subsequent later myocardial systolic recovery. The E-wave deceleration time, which in recent studies has been shown to be a powerful predictor of survival, was significantly improved in the metoprolol-treated patients.

Adrenergic beta-Antagonists↗

PubMed1996

Kinetic resolution of the incorporation of the D1 protein into photosystem II and localization of assembly intermediates in thylakoid membranes of spinach chloroplasts.

The chloroplast-encoded D1 protein of photosystem II (PSII) has a much higher turnover rate than the other subunits of the PSII complex as a consequence of photodamage and subsequent repair of its reaction center. The replacement of the D1 protein in existing PSII complexes was followed in two in vitro translation systems consisting of isolated chloroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed translation elongation, release of proteins from the ribosomes, and subsequent incorporation of newly synthesized products into PSII (sub)complexes. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gradient centrifugation. Immediately after termination of translation, the D1 protein was found both unassembled in the membrane as well as already incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII reaction center components. Later steps in the reassembly of PSII were clearly post-translational and sequential. Different rate-limiting steps in the assembly process were found to be related to the depletion of nuclear encoded and stromal components as well as the lateral migration of subcomplexes within the heterogeneous thylakoid membrane. The slow processing of precursor D1 in the thylakoid translation system revealed that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precursor could take place. The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was clearly post-translational in both translation systems. Radiolabeled assembly intermediates smaller than the PSII core complex were found to be located in the stroma-exposed thylakoid membranes, the site of protein synthesis. Larger PSII assembly intermediates were almost exclusively located in the appressed regions of the membranes.

Centrifugation, Density Gradient↗

PubMed1996

A "double adaptor" method for improved shotgun library construction.

The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

Base Sequence↗

PubMed1996

Isolation of hydrophobic membrane proteins by perfusion chromatography--purification of photosystem II reaction centers from spinach chloroplasts.

Ion exchange perfusion chromatography has been introduced for the isolation of hydrophobic membrane protein complexes from thylakoid membranes of spinach chloroplasts. By using this chromatographic technique, previously shown to be useful for the rapid isolation of soluble proteins (Regnier, F. E. (1991) Nature 350, 634-635), we have been able to isolate oxygen evolving photosystem II core complexes and photosystem II reaction center particles. Pure reaction centers could be isolated from photosystem II core complexes after a chromatographic step requiring only 6.5 mm, which is a substantial improvement in comparisons with previous procedures. The entire preparation of photosystem II core complexes and reaction center II particles could be completed in less than 2 h. The use of perfusion chromatography, as a versatile method for the isolation of hydrophobic membrane proteins from photosynthetic membranes, as well as for other biological membranes, will be discussed.

Chloroplasts↗