Dynamics of cyanide binding to ferrous Scapharca inaequivalvis homodimeric hemoglobin

Flash photolysis experiments have been carried out for the first time on a hemoglobin ferrous cyanide adduct with an 8 ns laser pulse. A 95% nonexponential rebinding process occurs within 2 micros after full photolysis in ferrous cyanide dimeric Scapharca inaequivalvis hemoglobin (HbI), indicating that once photolyzed the cyanide anion is not able to escape from the protein matrix and rebinds to the heme iron. The resonance Raman spectrum of the 10 ns photoproduct is identical to that of the fully relaxed deoxy derivative, indicating that in the ferrous cyanide HbI adduct protein relaxation occurs within 10 ns after photolysis. This behavior is at variance with that of the carbonmonoxy HbI derivative in which very little geminate rebinding is observed and the photoproduct relaxes with a lifetime of 1 micros. The fast relaxation of the cyanide HbI photoproduct can be accounted for by the small perturbation of the heme structure induced by cyanide binding to ferrous HbI. This is consistent with a deoxy-like conformation of the HbI ferrous cyanide adduct and implies that the pathway for relaxation involves only minor local rearrangements of the heme moiety. Photolysis experiments carried out on ferrous cyanide horse myoglobin, which can be saturated only partially, show a qualitatively similar behavior in ligand rebinding, indicating that the geminate process of the cyanide anion is a general phenomenon in hemoproteins.     

The impact of ischemic heart disease on main pulmonary artery blood flow patterns: a comparison between magnetic resonance phase velocity mapping and transesophageal color Doppler

OBJECTIVE: To give a detailed evaluation on main pulmonary artery blood velocity patterns, in patients with ischemic heart disease and to provide recommendations for pulsed Doppler sample volume placement, in order to optimize cardiac output estimation. METHODS: Using magnetic resonance phase and esophageal color Doppler velocity mapping in 12 patients with ischemic heart disease and undergoing coronary artery by-pass grafting, very similar data on pulmonary artery blood velocity patterns were provided for comparison with each other. RESULTS: Peak blood velocities were located in the inferior half of the main pulmonary artery cross-sectional area. Early after peak systole the highest velocities shifted towards the superior/left (major curvature) with a simultaneous decrease in velocities inferiorly. The velocity decrease further evolved into retrograde flow to the inferior/right (minor curvature). This feature was significantly enhanced compared to earlier findings in healthy volunteers. The mean temporal blood velocity profiles were asymmetrically skewed, thereby giving unreliable cardiac output estimates based on single point Doppler blood velocity recordings. The error incurred may amount to more than 100% in extreme cases. According to our data, optimal assessment of cardiac output should be based on multiple sample volumes placed along the inferior/right to superior/left diameter. CONCLUSIONS: MR-phase velocity mapping and multiplane transesophageal color Doppler recordings provided similar blood velocity patterns in patients with ischemic heart disease. The skewness of the mean temporal blood velocity profile is enhanced compared with healthy subjects, resulting in error in the assessment of CO by means of pulsed Doppler echocardiography. By using multiple Doppler sample volumes, the error can be minimized.     

Fast ion-exchange HPLC of proteins using porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths grafted with poly(2-acrylamido-2-methyl-1-propanesulfonic acid)

Porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths with different porous properties grafted with poly(2-acrylamido-2-methyl-1-propanesulfonic acid) chains using cerium(IV) initiated free-radical polymerization have been prepared and used for the separation of proteins in ion-exchange HPLC mode. Because of the presence of the large pores that are typical of monolithic separation media which allow easy flow of all of the mobile phase, the efficiency of the columns does not deteriorate even at high flow velocities as a result of the specific morphology of the monoliths. Optimization of the chromatographic conditions such as the shape of the mobile phase gradient and the flow rate allows for very fast separation of three proteins in less than 1.5 min.     

Vitamin A deficiency and mutations of RXRalpha, RXRbeta and RARalpha lead to early differentiation of embryonic ventricular cardiomyocytes

Knock-out of the mouse RXRalpha gene was previously shown to result in a hypoplastic heart ventricular wall, histologically detectable in 12.5 dpc fetuses. We show here that a precocious differentiation can be detected as early as 8.5 dpc in ventricular cardiomyocytes of RXRalpha(-/-) mutants. This precocious differentiation, which is characterized by the presence of striated myofibrils, sarcoplasmic reticulum and intercalated disks, is found after 9.5 dpc in about 50% of RXRalpha(-/-) subepicardial myocytes. In contrast, wild-type subepicardial myocytes remain morphologically undifferentiated up to at least 16.5 dpc. A similar precocious differentiation was observed in 9.5 dpc subepicardial myocytes of several RXRbeta(-/-) and RARalpha(-/-) mutants, as well as in vitamin A-deficient embryos. The proportion of differentiated subepicardial myocytes almost reached 100% in RXRalpha/RXRbeta double null mutants, indicating a partial functional redundancy between RXRalpha and RXRbeta. This differentiation defect was always paralleled by a decrease in the mitotic index. In addition, subepicardial myocytes of RXRalpha(-/-), RXRalpha(-/-)/RXRbeta(-/-) or vitamin A deficient, but not of RXRbeta(-/-) and RARalpha(-/-) embryos, were often flattened and more loosely connected to one another than those of WT embryos. Thus, retinoids are required at early stages of cardiac development to prevent differentiation, support cell proliferation and control the shape of ventricular myocytes, and both RXRs and RARs participate in the mediation of these functions.     

Comparison of closed-suction drainage and no drainage after primary total knee arthroplasty

One hundred thirty-six primary total knee arthroplasty patients were randomized for the use of closed-suction, nonreinfusable wound drains. Blood loss was identical in the drained and undrained groups. Forty percent of undrained wounds compared with 0% of drained wounds required dressing reinforcement. Sixty-nine percent of undrained wounds compared with 39% of drained wounds developed ecchymosis, measuring 92 cm2 in the undrained group and 28 cm2 in the drained group. This study concludes that a simple wound drain effectively minimizes the undesirable accumulation of blood in the surrounding soft tissues and the postoperative wound dressing after total knee arthroplasty.     

Echocardiographic Doppler study of left ventricular filling in the diagnosis of minimal or moderate rejection in cardiac transplantation

The study of left ventricular filling by Doppler echocardiography may be a non-invasive diagnostic method of detection of acute rejection of cardiac transplants. The aim of this study was to assess the value of the method for diagnosis of minimal to moderate rejection (grades 1 to 3 of the Billingham classification). A total of 466 Doppler echocardiographic studies were performed in 23 cardiac transplantation patients (21 men, mean age 49.3 +/- 10 years) with endomyocardial biopsy as the reference method for the diagnosis of rejection. Over a follow-up period of 18.5 +/- 10 months, 22.7% of biopsies showed minimal or moderate rejection. The Doppler measurements of the isovolumic relaxation period and peak early diastolic (E) velocity with respect to the mitral velocity-time integral were no different in cases of acute rejection. The only difference observed was in the mitral half-pressure time which was much shorter in cases of rejection. However, after drawing a ROC graph, the accuracy of this parameter was insufficient for diagnosing rejection irrespective of the threshold of variation considered (23% sensitivity for a 20% shortening and 36% sensitivity for a 10% shortening). The authors conclude that Doppler echocardiographic study of left ventricular filling is of limited value for the diagnosis of acute minimal or moderate rejection in cardiac transplant patients. The half-pressure time may be a useful complement to endomyocardial biopsy or when biopsy investigations are performed less frequently.     

The RepA protein of plasmid RSF1010 is a replicative DNA helicase

The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication. RepA is one of the smallest known helicases. The protein forms a homohexamer of 29,896-Da subunits. A variety of methods were used to analyze the quaternary structure of RepA. Gel filtration and cross-linking experiments demonstrated the hexameric structure, which was confirmed by electron microscopy and image reconstruction. These results agree with recent data obtained from RepA crystals diffracting at 3.5-A resolution (R?leke, D., Hoier, H., Bartsch, C., Umbach, P., Scherzinger, E., Lurz, R., and Saenger, W. (1997) Acta Crystallogr. Sec. D 53, 213-216). The RepA helicase has 5' --> 3' polarity. As do most true replicative helicases, RepA prefers a tailed substrate with an unpaired 3'-tail mimicking a replication fork. Optimal unwinding activity was achieved at the remarkably low pH of 5.5. In the presence of Mg2+ (Mn2+) ions, the RepA activity is fueled by ATP, dATP, GTP, and dGTP and less efficiently by CTP and dCTP. UTP and dTTP are poor effectors. Nonhydrolyzable ATP analogues, ADP, and pyrophosphate inhibit the helicase activity, whereas inorganic phosphate does not. The presence of Escherichia coli single-stranded DNA-binding protein stimulates unwinding at physiological pH 2-3-fold, whereas the RSF1010 replicon-specific primase, RepB' protein, has no effect, either in the presence or in the absence of single-stranded DNA-binding protein.     

Midwifery education for safe motherhood

A series of new safe motherhood midwifery education modules was evaluated in nursing and midwifery education institutions, regional training centers, acute care hospitals, and community settings in Ethiopia, Fiji, Lesotho, Mozambique, and Nepal in 1995. The series was developed by the World Health Organization's Maternal Health and Safe Motherhood Program. A total of 36 teachers, 82 midwives or nurse-midwives, and 60 post-basic midwifery students were enrolled in a 2-week clinical skills course and an 8-day training in module use. In subsequent questionnaires and focus group discussions, participants indicated the modules were understandable, relevant, easy to use, and of high quality and the guidelines for assessing competence were adequate. Difficulties encountered included insufficient recommended time frames for some of the sessions, a limited availability of clinical cases for teaching the specific skills in the modules, difficulties obtaining data for a community profile, and a lack of resources to support application of skills learned. Participants indicated they would benefit from having copies of the technical material used in the modules for reference after the course. Overall, these findings indicate the modules have the potential to strengthen the education of midwives in developing countries and thereby to make motherhood safer. Weak health system infrastructures, including regulatory measures, represent the major obstacle to successful program application.     

Comparative analysis of antibiotic resistance, immunofluorescent colony staining, and a transgenic marker (bioluminescence) for monitoring the environmental fate of rhizobacterium

Field releases of the wild-type plant growth-promoting rhizobacterium Pseudomonas fluorescens 89B-27, its bioluminescent derivative GEM-8 (89B-27::Tn4431), and a spontaneous rifampin-resistant variant estimating the wild-type population. Seed and root samples were taken 0, 7, 14, 21, or 28, 35 or 42, and 70 days after planting in each year and processed for enumeration by spiral plating or immunofluorescent colony staining (IFC). In both years, the populations of 89B-27, R34, and GEM-8, as measured by IFC, were not significantly different (P > 0.05) from each other at each sampling time. However, the populations of R34 and GEM-8, as measured by spiral plating and differentiation based on their respective phenotypes, were significantly lower (P < 0.05) than the wild-type populations and their IFC-determined populations. These data indicate that traditional marker systems may underestimate populations and hence the survival and colonization of genetically marked bacteria.