Shoulder function following autologous latissimus dorsi breast reconstruction. A prospective three year observational study comparing quilting and non-quilting donor site techniques.

Latissimus dorsi harvest and axillary surgery can affect shoulder function. The effect of autologous latissimus dorsi flap (ALD) breast reconstruction and donor site quilting have been inadequately studied. A cohort of ALD flap breast reconstruction patients were assessed pre-operatively and at eight post-operative time-points (up to 3 years after reconstruction) using the self-administered Disabilities of the Arm, Shoulder and Hand (DASH) outcome measure, for which validated normative data is available. Patients with incidental shoulder conditions and bilateral reconstructions were excluded. This was a prospective, observational study with blinded data interpretation: 58 patients, 22 of whom had donor site quilting, were assessed. Groups were compatible demographically, in breast care management and in pre-operative DASH score (quilted 6.5, non-quilted 6.4; P=0.98). Scores were significantly increased at initial post-operative clinic review (mean 49, SD19; P<0.001), 6 week (29, SD20; P<0.001), and 3 month (19, SD19; P<0.01), thereafter remaining at a plateau value of approximately 15 (P>0.05). Seroma incidence was reduced in the quilted group (5% vs 70%). A strong, significant correlation was found between 3 month DASH score and long term function (r=0.66, P<0.0003); patients with DASH >20 fare significantly worse in the longterm (mean 20 point increase, SD5.0, P<0.001). Higher post-operative DASH scores correlated significantly with pre-operative DASH (r=0.58) and BMI (r=0.36). Adjuvant therapy had no effect on shoulder function. Axillary dissection had a weak correlation with a higher DASH score, but only at the 3-month post-operative time-point (r=0.32, P=0.03). ALD flap breast reconstruction generally results in a functionally insignificant increase (6.5 points) in longterm DASH score, although a small subset of patients do develop longterm impairment, and quilting does not appear to inhibit shoulder function.

Physical analysis of the complex rye (Secale cereale L.) Alt4 aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco.

Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library's suitability for investigating Al tolerance genes. The library provides 6 x genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus.

Bilateral internal mammary artery grafts in reoperative and primary coronary bypass surgery.

Bilateral internal mammary artery grafting is recognized as a preferred method of myocardial revascularization. However, its efficacy in coronary bypass reoperation has not been clearly established. From January 1982 through June 1989, 88 patients underwent coronary bypass reoperation with bilateral internal mammary artery grafts. Results were compared with those for a subset of 88 patients receiving primary revascularization with bilateral internal mammary artery grafts who were computer matched for sex, age, left ventricular function, anginal classification, and left main coronary artery disease. In each group, 62.5% (55 patients) had unstable angina, 43.2% (38 patients) had reduced ejection fraction, and 21.6% (19 patients) in the reoperation group and 20.5% (18 patients) in the reference group had left main coronary artery disease. Hospital mortality for the reoperation group was 6.8% (6 patients) and for the reference group, 3.4% (3 patients). No significant difference was found in the incidence of reoperation for bleeding, sternal infection, or stroke in the two groups. The incidence of respiratory insufficiency in the reoperation group was 13.6% (12 patients) and in the reference group, 3.4% (3 patients) (p less than 0.015). Recurrent angina occurred in 13.7% (10 patients) of patients in the reoperation group and 13.3% (10 patients) in the reference group. The long-term survival at 5 years for the reoperation group was 85.3% +/- 5.6% (+/- standard error of the mean) and for the reference group, 91.6% +/- 3.1%. No significant difference was found in the equality of survival distribution for the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of photosynthesis gene transcripts in response to sodium chloride by salt-tolerant alfalfa cells.

A cell line from diploid alfalfa (Medicago sativa L.), HG2-N1, which is stably tolerant to 1% (0.171 M) NaCl in the medium and noticeably more green than the parent line (HG2), was investigated to determine if increased chlorophyll accumulation was related to chloroplast development toward greater photosynthetic activity and increases in chloroplast-gene expression in this cell line. The ctDNA (chloroplast-DNA) copy number in HG2-N1 was found to be increased by 50% from that of the salt-sensitive parent HG2 line. RNA accumulation, as detected by hybridization to a nuclear actin gene, appeared comparable in HG2 and HG2-N1. In contrast, mRNA levels of the plastid-encoded psbA gene were increased in the salt-tolerant HG2-N1 grown on normal medium. The mRNA levels of HG2-N1 were further increased in response to added NaCl in the medium. The mRNA levels from other chloroplast genes necessary for photosynthesis (psbD, psaB, atpB, rbcL), as well as from several nuclear genes (pCab4, pCab1, rbcS) encoding polypeptides participating in photosynthesis, also increased in the salt-tolerant HG2-N1 when it was grown in the presence of NaCl whereas actin-mRNA and chloroplast-rRNA levels remained comparable under growth conditions ± NaCl. Several independently isolated salt-tolerant cell lines showed a similar response to salt, as monitored by accumulation of rbcL mRNA, indicating that transcript accumulation for photosynthesis genes is correlated with the salt response in these salt-tolerant alfalfa cell lines.

Seventeen-year experience with bilateral internal mammary artery grafts.

The internal mammary artery (IMA) is being recognized as the conduit of choice for myocardial revascularization. From January 1972 through June 1988, 1,087 patients received bilateral IMA and supplemental vein grafts. There were 917 men (84.4%) and 170 women (15.6%) with a mean age of 62.4 years (range, 29 to 84 years). Three hundred ninety-four patients (36.2%) had unstable angina, and 194 (17.8%) had left main coronary artery stenosis greater than 50%. In all, 3,741 coronary grafts were performed, with a mean of 3.4 per patient. Hospital mortality was 2.7% (29 patients). Hospital complications included reoperation for bleeding, 19 patients (1.7%); sternal infection, 16 patients (1.5%); respiratory failure, 35 patients (3.2%); perioperative myocardial infarction, 22 patients (2.0%); and stroke, 20 patients (1.8%). Post-operative arteriography in 53 patients (mean postoperative time, 53.0 months) showed that 92.1% (58/63) of the left IMA and 84.9% (45/53) of the right IMA grafts were patent. Follow-up was completed on 1,058 hospital survivors. There were 82 late deaths (7.8%). The actuarial survival for patients discharged from the hospital was 80.0 +/- 3.2% (plus or minus standard error of the mean) at 10 years and 60.0% +/- 5.0% at 15 years. At follow-up, 866 patients (90.3%) were asymptomatic and in New York Heart Association class I and 68 (7.1%) were in class II. This longitudinal analysis demonstrates that bilateral IMA grafting has a low operative risk and provides excellent long-term functional improvement and survival.

Sodium cholate extraction of rat liver nuclear xenobiotic-metabolizing enzymes.

DNA is the purported target of several carcinogenic and mutagenic agents. Nuclear enzymes which could generate or detoxify reactive metabolites are of major concern. Several such enzymes have been identified within nuclei, but obtaining samples with enriched content or activity is difficult, time-consuming, and uses harsh isolation techniques. Extraction of rat liver nuclear suspensions with cholate-containing buffer results in solubilization of 25-30% of the protein. Linear extraction was obtained for total protein and cytochromes P-450 and b5, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, DT-diaphorase, and microsomal-like epoxide hydrolase with specific activities comparable to values reported for isolated nuclear membrane, while the yield was five to ten times greater. Detergent extracts of rat liver nuclei were employed to study the comparative response of microsomal and nuclear enzymes to chemical treatment. While the responses to acute inductive (phenobarbital and 3-methylcholanthrene) and toxic (carbon tetrachloride and dibromochloropropane) treatments were qualitatively similar, an initiation-promotion protocol (diethylnitrosamine with phenobarbital promotion) resulted in divergent responses between the enzymes in the two subcellular fractions. Detergent extracts of nuclei offer an efficient means of recovering xenobiotic-metabolizing enzymes from rat liver nuclei, and have been utilized to demonstrate a differential response of nuclear enzymes during preneoplastic development.

Hepatocarcinogen-induced alterations in nuclear RNA compartmentation.

Prelabeled rat liver nuclei were purified, detergent-rinsed to remove cytoplasmic RNA contaminants and incubated in two in vitro RNA transport assays. Appropriate compartmentation of nuclear RNA sequences was maintained in an assay containing polyvinylpyrrolidone (PVP), which prevents nuclear swelling in aqueous media. Under these conditions, poly(A)+ RNA released in the presence of PVP (and ATP) was significantly more active in directing in vitro protein synthesis than poly(A)+ RNA released in the absence of either additive. Further, the excised fifth intron of alpha 1-acid glycoprotein remained nucleus-restricted, while release of poly(A)+ processing catabolites occurred in the absence of PVP. The appropriate compartmentation of nuclear sequences was lost following treatment of rats with the hepatocarcinogen thioacetamide, paralleling the in vivo situation. This assay should prove useful for subsequent studies on nucleocytoplasmic RNA transport in vitro, in particular for investigations relating to the altered RNA transport associated with the initiation of carcinogenesis.

Photoaffinity labelling of a nuclear matrix nucleoside triphosphatase and its modulation in the acute-phase response.

Photoaffinity labelling has been used to identify the major nuclear matrix nucleoside triphosphatase (NTPase) as a 46 kD polypeptide, which appears to represent the same polypeptide photolabelled in nuclear envelope. Nuclear matrix NTPase and its photolabelling were selectively decreased in the acute phase response of rat liver, which also encompasses decreases (30%) in RNA transport in vitro and nuclear envelope NTPase. These results, and quantitative considerations suggest that the NTPase correlatively linked to RNA transport is not solubilized by detergents; it appears to represent a nuclear matrix component.

Modulation of RNA transport by polyvinylpyrrolidone.

A number of studies have documented substantial loss of nuclear protein during aqueous nuclear isolation procedures. This loss can, to some extent, be counteracted by addition of impermeable macromolecules like polyvinylpyrrolidone, which prevent nuclear swelling. While nucleic acids appear to be much less susceptible to leakage during isolation, the effects of these additives on RNA release during in vitro incubation have not been examined. Here we show that addition of polyvinylpyrrolidone results in significant decreases in RNA transport; inhibition becomes maximal at 50-75 microM addition.

Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope.

We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time, and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport.

Dichlorobenzimidazole-riboside inhibition of nuclear RNA accumulation initiated with gamma-thio analogues of ATP and GTP.

The gamma-thio analogues of ATP and GTP, ATP[S] and GTP[S], have been used as affinity probes to measure RNA synthesis initiated in vitro in L cell nuclei. 5,6-Dichlororibofuranosylbenzimidazole was found to inhibit total RNA synthesis in vitro and the amount bound by mercury-affinity chromatography. Initiation in vitro was also shown by [35S]ATP[S] and [35S]GTP[S] incorporation in RNA molecules with a larger size distribution. Although 66% of RNA molecules labeled with [35S]GTP[S] and 74% of those labeled with [35S]ATP[S] were 3-12 S n size, the remainder of the label was recovered in RNA molecules larger than 12 S and a significant portion in RNA larger tha 18 S. The specificity of the initiation process seems to be indicated by the finding of molecules of the size of pre-tRNA on gel electrophoresis which were labeled with [35S]GTP[S] but not [35S]ATP[S] under our experimental conditions. 5,6-Dichlororibofuranosylbenzimidazole severely inhibited the incorporation of [35S]GTP[S] and [35S]AT[[S] by all size classes, indicating that it can decrease accumulation of RNA initiated in vitro in the L cell nuclear system.

Nuclear ligation of RNA 5'-OH kinase products in tRNA.

Mouse L-cell nuclei incorporate gamma-32P from ATP in vitro predominantly in 5'-monophosphoryl termini and internal phosphodiester bonds with a nonrandom nearest-neighbor distribution. In the presence of 1 microgram of alpha-amanitin per ml the gamma-32P showed a time-dependent appearance in RNA bands which migrated with mature tRNA species but not with pre-tRNA and 5S RNA. The gamma-32P was found in internal phosphodiester bonds as shown by alkaline phosphatase resistance and was identified in 3'-monophosphates after RNase T2, T1, and A digestion. The specificity of this incorporation was indicated by a limited number of labeled oligonucleotides from a T1 digest and identification of 70 to 80% of the 32P label as Cp on complete digestion of the eluted tRNA band. We also observed transiently [gamma-32P]ATP-labeled RNA bands (in 5'-monophosphate positions) that were 32 to 45 nucleotides long. The results presented suggest splicing of several mouse L-cell tRNA species in isolated nuclei which involve the RNA 5'-OH kinase products as intermediates.