Breast reconstruction with abdominal flaps after abdominoplasties.

After the reported safe transverse rectus abdominis myocutaneous (TRAM) flap after liposuction of the abdomen, two cases of bipedicled reconstruction with this flap after abdominoplasty were successfully performed. This operation has not previously been considered possible because of the transection of the perforator arteries during the undermining of the abdomen. To examine the possible reperfusion of the perforator arteries, the authors studied the perforator arteries of 10 patients before they underwent abdominoplasty and at 1 week, 3 months, and 6 months after the operation. The arteries were studied with color-duplex scanning and power Doppler, using 10-MHz superficial probes, and their position was marked on a map. A cadaver study of a woman who had had an abdominoplasty 10 years before her death is also presented. In every patient, reperfusion of all perforator arteries was documented, starting from the control at 1 month. In no case was the caliber of the reperfused vessels more than 40 percent of the original diameter (maximum: 0.53 mm). This was also confirmed by the cadaver study. In conclusion, after an abdominoplasty operation, constant reperfusion of the perforator arteries of the rectus muscles occurs. However, the diameter of the arteries may not be enough to provide the necessary blood supply for a TRAM flap, which is therefore strongly discouraged by the authors after abdominoplasty in favor of a vertical rectus abdominis muscle (VRAM) flap. A liposuction, which does not necessarily disrupt the perforators, is not an absolute contraindication for a TRAM flap, provided that an accurate color-duplex scanning study is done.

The latissimus dorsi added fat flap for natural tissue breast reconstruction: report of 15 cases.

The latissimus dorsi added fat flap is an alternative method of natural tissue breast reconstruction. A significant volume of additional subcutaneous back fat is left attached to a traditional latissimus dorsi flap, avoiding the need for an additional implant. The surgical technique and results in 15 patients are discussed.

The cephalic vein: an aid in free TRAM flap breast reconstruction. Report of 12 cases.

The harvesting of the cephalic vein is a simple and effective technique for providing or augmenting venous drainage in free TRAM flap breast reconstruction. It may be divided distally and rotated about the infraclavicular fossa as a cephalic vein transfer or used as a source of free vein grafts. It is easily harvested with minimal morbidity. Its anatomy, surgical technique, indications, and results of use are discussed. In some circumstances, a cephalic vein transfer may allow greater areas of the free TRAM flap to be used more safely. This is discussed.

Ornithine decarboxylase gene of Neurospora crassa: isolation, sequence, and polyamine-mediated regulation of its mRNA.

Ornithine decarboxylase (ODC), which initiates the biosynthesis of the polyamines putrescine, spermidine, and spermine, is encoded by the spe-1 gene of the fungus Neurospora crassa. This gene and its cDNA have been cloned and sequenced. The gene has a single 70-nucleotide intron in the coding sequence. The cDNA, comprising the entire coding region, recognizes a single 2.4-kb mRNA in Northern (RNA) blots. The mRNA transcript, defined by S1 mapping, has an extremely long, 535-base leader without strong secondary-structure features or an upstream reading frame. The translational start of the protein is ambiguous: a Met-Val-Met sequence precedes the Pro known to be the N terminus of the ODC polypeptide. The polypeptide encoded by the N. crassa spe-1 gene (484 amino acids) has 46% amino acid identity with that of Saccharomyces cerevisiae (466 amino acids) and 42% with that of mouse (461 amino acids). Alignment of the longer N. crassa sequence with S. cerevisiae and mouse sequences creates gaps in different sites in the S. cerevisiae and mouse sequences, suggesting that N. crassa ODC is closer to an ancestral form of the enzyme than that of either yeast or mouse ODC. N. crassa ODC, which turns over rapidly in vivo in the presence of polyamines, has two PEST sequences, found in most ODCs and other proteins with rapid turnover. In striking contrast to other eucaryotic organisms, the variation in the rate of ODC synthesis in response to polyamines in N. crassa is largely correlated with proportional changes in the abundance of ODC mRNA. Spermidine is the main effector of repression, while putrescine has a weaker effect. However, putrescine accumulation appears to increase the amount of active ODC that is made from a given amount of ODC mRNA, possibly by improving its translatability. Conversely, prolonged starvation for both putrescine and spermidine leads to the differentially impaired translation of ODC mRNA.

Calcium modulation of polyamine transport is lost in a putrescine-sensitive mutant of Neurospora crassa.

Putrescine transport in Neurospora is saturable and concentrative in dilute buffers, but in the growth medium putrescine simply equilibrates across the cell membrane. We describe a mutant, puu-1, that can concentrate putrescine from the growth medium because the polyamine transport system has lost its normal sensitivity to Ca2+. The wild type closely resembles the mutant if it is washed with citrate and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The mutant phenotype also appears in the wild type after treatment with cycloheximide. The results suggest that putrescine uptake is normally regulated by an unstable Ca(2+)-binding protein that restricts polyamine uptake. This protein is evidently distinct from the polyamine-binding function for uptake, which is normal in mutant and in cycloheximide-treated wild type cells. The puu-1 mutation, stripping of Ca2+, and cycloheximide treatment all cause an impairment of amino acid transport, indicating that other membrane transport functions rely upon the product of the puu-1+ gene. Preliminary evidence suggests that the putrescine carrier is not the Ca(2+)-sensitive, low-affinity K(+)-transport system, but K+ efflux does accompany putrescine uptake.

Measurement of IgM responses to a subunit influenza A vaccine by sucrose-gradient centrifugation and membrane-filtration enzyme immunoassays.

IgM responses to a deoxycholate-split influenza vaccine containing the surface antigens of the H3N2 virus A/Philippines/2/82 were studied in five volunteers, three of whom were seronegative by haemagglutination inhibition (HI) tests. Responses were measured by a sucrose-gradient centrifugation technique, in which IgM-specific HI activity was computed as a proportion of total IgM and IgG-specific HI activity, and by a membrane filtration-enzyme immunoassay (MF-EIA). Responses could be detected in all volunteers when measured by sucrose-gradient centrifugation within 1-2 weeks, and the IgM induced was 5-40% of total HI-specific activity. The response was also observed in the presence of low levels of pre-existing antibody. Levels of IgM antibody, when measured by MF-EIA, could be easily detected in two of the volunteers, while those of two others were very low and there was no response in a fifth. No biphasic virus-specific response to vaccination, involving first IgM and then IgG, could be measured by either technique. From these studies, the sucrose-gradient fractionation technique appears to be the more sensitive procedure.

The "chemical leech": intra-replant subcutaneous heparin as an alternative to venous anastomosis. Report of three cases.

Three successful cases of distal finger replantation are described where suitable veins were unavailable for anastomosis after arterial flow had been re-established. To prevent infarction, calcium heparin was injected subcutaneously into the replants at intervals over 9 days. This allowed the venous blood to escape into the dressings while an adequate microvenous circulation was re-established, thus simulating the effect of a leech. Complete survival of all three distal replantations was observed. No attempted case has been unsuccessful. The procedure is simple and can be administered by the nursing staff. It avoids some of the problems associated with the use of medicinal leeches and systemic heparin. Applications of this technique in other areas of replantation and flap surgery are suggested.

An improved membrane-filtration enzyme immunoassay for the rapid serological diagnosis of viral infections.

A one-step modification of the membrane-filtration enzyme immunoassay (MF EIA) (Barnett et al., J. Clin. Microbiol., 23:385-399, 1987), for estimation of virus-specific antibody is described. The modified MF EIA allowed serum, antigen and enzyme-conjugated anti-globulin to be incubated together in membrane-based 96-well plates to enable the formation of immune complexes in solution at 37 degrees C. The assay required only 45 min for completion and polyethylene glycol was shown to be an essential component in reaction mixtures for IgG assays to enhance immune complex formation. The modified MF EIA was as sensitive as the previous two-step method for monitoring responses to influenza vaccine, and control antigen backgrounds were significantly reduced. The one-step procedure was also shown to be suitable for the rapid serodiagnosis of naturally acquired influenza A and B infections. However, MF EIA detected cross-reactive H1N1 responses in 57.7% of naturally-acquired H3N2 infections, suggesting that responses to common internal antigens were being measured. Cross-reactive responses to influenza A viruses could not be detected in volunteers receiving subunit vaccines.

Putrescine and spermidine control degradation and synthesis of ornithine decarboxylase in Neurospora crassa.

Neurospora crassa mycelia, when starved for polyamines, have 50-70-fold more ornithine decarboxylase activity and enzyme protein than unstarved mycelia. Using isotopic labeling and immunoprecipitation, we determined the half-life and the synthetic rate of the enzyme in mycelia differing in the rates of synthesis of putrescine, the product of ornithine decarboxylase, and spermidine, the main end-product of the polyamine pathway. When the pathway was blocked between putrescine and spermidine, ornithine decarboxylase synthesis rose 4-5-fold, regardless of the accumulation of putrescine. This indicates that spermidine is a specific signal for the repression of enzyme synthesis. When both putrescine and spermidine synthesis were reduced, the half-life of the enzyme rapidly increased 10-fold. The presence of either putrescine or spermidine restored the normal enzyme half-life of 55 min. Tests for an ornithine decarboxylase inhibitory protein ("antizyme") were negative. The regulatory mechanisms activated by putrescine and spermidine account for most or all of the regulatory amplitude of this enzyme in N. crassa.

Membrane filtration enzyme immunoassay, a novel, rapid method for measurement of virus-specific immunoglobulins G and M and detection of viral antigens.

A novel membrane filtration enzyme immunoassay (MF EIA) is described in which virus-antibody complexes formed are trapped onto the surface of membranes with low protein-binding affinity by vacuum filtration. Class-specific immunoglobulin G (IgG) or IgM antibody was measured by adding enzyme-conjugated antiimmunoglobulin and incubating prior to the final wash and addition of enzyme substrate. Influenza A virus-specific IgG antibodies measured by MF EIA showed similar sensitivity for detecting seroconversion in volunteers administered influenza virus subunit vaccines and subtype specificity comparable to that observed by the hemagglutination inhibition technique. Cytomegalovirus-specific IgG antibodies measured by MF EIA with commercially available complement fixation antigens gave results similar to those of conventional enzyme-linked immunosorbent assay and complement fixation tests. The MF EIA method is also suitable for detection of rotavirus antigen in feces.

Purification and properties of ornithine decarboxylase from Physarum polycephalum.

Ornithine decarboxylase (EC 4.1.1.17) has been purified 3,500-fold from the plasmodia of Physarum polycephalum. The purified material exhibited a Km for ornithine of 0.6 mM and Vmax of 20 mumol of CO2 formed per min/mg at 30 degrees C (62 mumol/min/mg at 37 degrees C). It migrated as a single protein and activity species on high pressure liquid chromatography (TSK-3000) in 0.15 M NaCl (Mr = 80,000) and in native gels containing 5, 6.5, 8, and 9.5% acrylamide. A single protein band (Mr = 43,000) was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity was lost upon incubation with alpha-difluoromethyl[5-14C] ornithine, and the inactivated material appeared as a single Mr = 43,000 14C-band on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels. The decarboxylase activity was specific for ornithine and was pyridoxal-P-dependent. The Km for pyridoxal-P (10 microM) was identical with the Kd for pyridoxal-P binding determined from the quenching of protein fluorescence (lambda ex = 282 nm, lambda em = 350 nm, maximal quenching 81%). Using specific antibody obtained from rabbit hyperimmune serum as a probe, an Mr = 43,000 immunoreactive species was detected on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels of plasmodial homogenates and all pooled purification fractions, but no higher molecular weight cross-reactive material was detected.