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The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.

Objective and Design: Gross cystic disease (GCD) of the breast is reported to occur in 7% of women in the developed world and, although not premalignant, is thought to be associated with an increased risk of breast cancer. Hormone and growth factor concentration levels were measured in breast cyst fluid (BCF) to correlate them with their mitogenic activity in tumour (MCF-7) or nontransformed (MCF-10A) cells., Results: Oestradiol (E2), oestrone (E1), E2-sulfate (E2-S), E1-sulfate (E1-S) and epidermal growth factor (EGF) concentrations were, as expected, significantly higher in type I than in type II cysts, while transforming growth factor-beta 2 (TGF-beta2) showed higher levels in type II cysts. Fifty per cent of the BCF samples stimulated [3H]-thymidine incorporation into MCF-7 cells while 34.5% inhibited this parameter. In MCF-10A cells, most BCF samples were stimulatory (85%). E2, E1 and EGF concentrations in BCF samples correlated significantly and positively with cell proliferation in MCF-7 cells, whereas a significant negative correlation was found for TGF-beta2. In MCF-10A cells, only E2-S and E1-S exhibited significant positive correlation, whereas a significant negative correlation was found for TGF-beta2. Progesterone (Pg), E2 and EGF incubated under the same conditions had a stimulatory effect on [3H]-thymidine incorporation into MCF-7 cells, whereas TGF-beta2 inhibited this parameter. Pg, E2, E1 and EGF significantly stimulated this parameter in MCF-10A cells., Conclusions: The stimulatory action of BCF on cell proliferation in a model of human breast epithelial cells could partly explain the increased incidence of breast cancer in cyst-bearing women.

The proximal mechanisms leading to monandry have been little studied in most insect orders, including Hymenoptera. In the parasitoid wasp Spalangia endius, mated females are less attractive (less often mounted) than virgins and are unreceptive (unlikely to allow copulation). Which aspects of mating are responsible was tested by observing male responses toward females whose mating had been interrupted at various stages. All females were allowed to receive precopulatory courtship and to open their genital aperture to copulate. Then some were interrupted before copulation, some after copulation but before postcopulatory courtship, and some were allowed to complete postcopulatory courtship. Females that had copulated were not less attractive than females that had not. In contrast, females that had received postcopulatory courtship were clearly both less attractive and less receptive. Thus, postcopulatory courtship functions as extended mate guarding, by making the female less attractive and less receptive to subsequent males even after the original male is no longer present. The effect of postcopulatory courtship on female attractiveness was persistent but imperfect: when males were presented sequentially to mated females, most but not all males retreated without mounting, and a female could repulse more than twenty males in succession.

For the development of phage therapy, systematic understanding mechanisms of bacteriophage resistance will be required. We describe a new strain of Escherichia coli O157:H7, named Mu(L), which stably co-exists with the O157:H7-specific lytic bacteriophage PP01. Chemostat cultures of E. coli O157:H7 infected with PP01 showed unchanging cell concentration, but phage concentrations which increased by approximately 10(8) PFU mL(-1). However, the latent period, burst size, and growth rate of Mu(L) were the same as in a PP01-susceptible strain. The binding rate of PP01 to the cell surface was diminished 8.5-fold in Mu(L). By observation of the binding of fluorescently labeled O157:H7-specific phage to individual Mu(L) cells, we found that clonal Mu(L) cultures were heterogeneous in their ability to bind bacteriophage. 15% of the Mu(L) population was completely resistant to PP01 infection. Mu(L) also co-existed with bacteriophages unrelated to PP01. Broad-range phage resistance by clonal heterogeneity represents a new class of bacteria-phage interactions.

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h(-1) and by 3 orders of magnitude at a lower dilution rate (0.327 h(-1)). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h(-1) and persisted until the end of the experiment (approximately 200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.

The previously isolated T-even type coliphage PP01, specifically infective to Escherichia coli O157:H7, uses the outer membrane protein OmpC as a receptor. The characterization of a spontaneous PP01-resistant strain indicated that it had lost ompC due to the deletion of a 14-kbp region upstream of and partially including ompC. Two host range mutants, able to infect an ompC null mutant, were isolated. Sequencing of gene 38, which codes for the receptor recognition protein Gp38, indicated three mutations in one mutant and two in the other. Both mutant proteins had a Gly208Arg, a Gly161Arg or Gly101His replacement, respectively, and the one mutant phage in addition a Trp189Arg replacement. These alterations suggest that the host range was mediated by a more positively charged Gp38.

Background: Homograft aortic valve replacement has been performed in 107 patients during the past 7 years. Two primary methods of implantation were used (intraaortic and root replacement). Results of both methods are presented., Methods: Intraaortic implantation (subcoronary or cylinder technique) was performed in 36 patients (mean age, 54 years) for aortic stenosis or regurgitation (31 patients) and endocarditis (5 patients). Aortic root replacement was performed in 71 patients (mean age, 62 years). The majority (58 patients) had complex root pathologies such as ascending aneurysm, dissection, or prosthetic endocarditis with annular destruction. Early results were assessed with intraoperative or predischarge echocardiography; annual echocardiograms provided long-term follow-up. Left ventricular mass was calculated in patients with long-standing pathology for whom preoperative and postoperative data were available., Results: Early valvular insufficiency was documented in 16 of the 36 intraaortic implants (44%); 9 of these have had progression of the insufficiency. Of the 20 patients who had trivial or no early insufficiency, significant insufficiency has developed in 7 and mild insufficiency has developed in 5. Calculation of left ventricular mass revealed a mean reduction of 11% at 1 year. There has been no mortality, endocarditis, or homograft-related reoperation in the intraaortic group with a mean follow-up of 50 months. The root replacement group had a hospital mortality of 17%. The cardiac pathology was limited to the aortic valve in 12 patients; mortality in this subset was zero. There has been no significant early or late postoperative valvular insufficiency in the 59 surviving patients. More rapid left ventricular mass reduction was seen in this group with a 26% mean reduction within 1 year. A mean follow-up of 32 months in the root replacement group has seen no homograft-related reoperations., Conclusions: Although the lack of early mortality in the intraaortic group makes this technique appealing, the high incidence of early insufficiency with the realistic expectation of progression has led to our abandonment of the intraaortic technique. Homograft aortic root replacement confers a higher mortality based on the severity of aortic pathology, but offers excellent long-term hemodynamics in any patient. We have expanded our indication for homograft root replacement to include patients with isolated valvular disease rather than reserving it for those patients with extensive root pathology.

Background: Complex aortic valve and root pathology presents a serious operative challenge., Methods: During the last 45 months 21 cryopreserved homograft root replacements have been performed in 19 patients. The ages of the patients ranged from 28 to 77 years; there were 15 men and four women. Six patients had endocarditis, four prosthetic and two native valve endocarditis. One patient underwent homograft aortic root replacement twice; one underwent concomitant homograft right ventricular outflow tract reconstruction for pulmonic endocarditis. Causes of aortic pathology in the other 13 patients included ascending aortic aneurysm with valvular regurgitation in seven, Marfan syndrome in two, and four aortic dissections with aortic regurgitation. Ten of the nineteen patients were undergoing reoperative procedures., Results: There were three hospital deaths and one death at home of unknown cause. Two of the three patients who died in hospital had undergone redo cardiac procedures. Dramatic functional class improvement was seen in all survivors., Conclusions: Follow-up from 2 to 45 months in the fifteen survivors revealed no clinical or significant echocardiographic evidence of valvular regurgitation or recurrent infection. We believe homograft root replacement provides an excellent method of reconstruction with several advantages unique to human valved conduits, including ease of handling, lack of synthetic nidus for recurrent infection, and absence of thrombogenic materials. Homografts should be considered the approach of choice in complex valvular and root infectious processes.

Three open heart surgery patients developed noncardiogenic pulmonary edema after administration of protamine following cardiopulmonary bypass. A catastrophic series of events are characteristic of this reaction: 1) sudden onset; 2) severe bronchoconstriction with early extreme difficulty in ventilation; 3) hyperinflation of the lungs; 4) pulmonary hypertension with normal pulmonary wedge or left atrial pressures; 5) progression to fulminant noncardiogenic pulmonary edema; 6) significant mortality; and 7) ventilatory perfusion abnormalities in survivors. Review of the literature reveals three types of reactions to protamine injection of varying severity: 1) brief hypotension; 2) anaphylactoid generalized reaction; and 3) high protein noncardiogenic pulmonary edema with cardiopulmonary collapse. The severity of the reaction had no relation to the dose of protamine. Previous protamine exposure was documented in 14 of 35 cases. Awareness of this reaction is essential for prompt treatment if fulminant pulmonary edema occurs. Administration of epinephrine, steroids, vasopressors, and potassium replacement may be required. Needless use of protamine sulfate should be discouraged.

The objective of this study was to evaluate the controlled microscopic development and fate of the neointima of Gore-Tex graft pretreated with multiple regular perforations, compared to nonperforated controls. Gore-Tex grafts (each 7 cm x 8 mm), perforated at regular 2 mm intervals with a special needle (0.8 mm internal diameter) for one half the length, with the remaining half kept intact, were implanted in 37 canine abdominal aortas in an end-to-end fashion. Standard untreated Gore-Tex grafts, of identical size, were similarly implanted in ten as controls. The perforated portion of the artificially treated Gore-Tex grafts demonstrated early neointimization at one week, the presence of fibroblasts and developing vasovasorum at 4 weeks, and complete endothelialization at 12 weeks. In contrast, the unperforated portion of implanted grafts demonstrated delayed endothelialization, requiring 24 weeks for complete development. Untreated controls demonstrated incomplete healing of the central luminal portion of the graft, with reddish thrombi still present at 24 weeks. Our study demonstrated that the poor development of neointima persists in the central portion of standard Gore-Tex controls in the last postoperative period, while pretreatment with multiple small perforations significantly hastens the development of a smooth neointima.

To reassess the role and timing of operative intervention for spontaneous pneumothorax, 119 patients were retrospectively reviewed to compare recurrences, complications, and hospital stay between a nonoperative group (Group 1) and an operative group (Group 2). Total hospital days were greater in Group 2, but excluding the length of preoperative stay, the number of hospital days were similar in both groups. Group 1 patients more than 40 years old had a longer postoperative hospitalization, but not a higher rate of complication. Overall, morbidity was not different between the two groups, and there were no immediate or perioperative deaths in either group. There were no recurrences in Group 2. At least 11 of the 49 patients in Group 1 had a recurrence (p = .012). Considering the excellent results achieved with operative pleurodesis and the total hospital days accrued with nonoperative therapy, operative pleurodesis should be considered if an active leak persists more than three days after the initial episode of spontaneous pneumothorax or at the time of the first recurrence in the hospitalized patient.