Hemoglobins are ancient O(2)-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum x C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full-length globins with the classical 8 helix Mb-like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant non-symbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the class 2 globins and show no hexacoordination, very high rates of O(2) binding ( approximately 250 microM(-1)s(-1)), moderately high rates of O(2) dissociation ( approximately 5-15 s(-1)), and high oxygen affinity (P(50) approximately 50 nM). These properties both facilitate O(2) diffusion to respiring N(2) fixing bacteria and reduce O(2) tension in the root nodules of legumes. The class 1 plant Hbs show weak hexacoordination (K(HisE7) approximately 2), moderate rates of O(2) binding ( approximately 25 microM(-1)s(-1)), very small rates of O(2) dissociation ( approximately 0.16 s(-1)), and remarkably high O(2) affinities (K(d) or P(50) approximately 2 nM), suggesting a function involving O(2) and nitric oxide (NO) scavenging. The class 2 Hbs exhibit strong hexacoordination (K(HisE7) approximately 100), low rates of O(2) binding ( approximately 1 microM(-1)s(-1)), moderately low O(2) dissociation rate constants ( approximately 1 s(-1)), and moderate, Mb-like O(2) affinities (K(d) or P(50) approximately 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.(c) 2009 Wiley Periodicals, Inc. Biopolymers, 2009.

Nitrosyl hydride, HNO or nitroxyl, is the one-electron reduced and protonated form of nitric oxide. HNO is isoelectronic to singlet O(2), and we have previously reported that deoxymyoglobin traps free HNO to form a stable adduct. In this report, we demonstrate that oxygen-binding hemoglobins from human, soy, and clam also trap HNO to form adducts which are stable over a period of weeks. The same species can be formed in higher yields by careful reduction of the ferrous nitrosyl adducts of the proteins. Like the analogous O(2)-Fe(II) adducts, the HNO adducts are diamagnetic, but with a characteristic HNO resonance in (1)H NMR at ca. 15 ppm that splits into doublets for H(15)NO adducts. The (1)H and (15)N NMR resonances, obtained by HSQC experiments, are shown to differentiate subunits and isoforms of proteins within mixtures. An apparent difference in the reduction rates of the NO adducts of the two subunits of human hemoglobin allows assignment of two distinct nitrosyl hydride peaks by a combination of UV-vis, NMR, and EPR analysis. The two peaks of the HNO-hHb adduct have a persistent 3:1 ratio during trapping reactions, demonstrating a kinetic difference between HNO binding at the two subunits. These results show NMR characterization of ferrous HNO adducts as a unique tool sensitive to structural changes within the oxygen-binding cavity, which may be of use in defining modes of oxygen binding in other heme proteins and enzymes.

Errors in blood flow delivery due to shunting have been reported to reduce flow by, potentially, up to 40-83% during cardiopulmonary bypass. The standard roller-pump measures revolutions per minute and a calibration factor for different tubing sizes calculates and displays flow accordingly. We compared displayed roller-pump flow with ultrasonically measured flow to ascertain if measured flow correlated with the heart-lung pump flow reading. Comparison of flows was measured under varying conditions of pump run duration, temperature, viscosity, varying arterial/venous loops, occlusiveness, outlet pressure, use of silicone or polyvinyl chloride (PVC) in the roller race, different tubing diameters, and use of a venous vacuum-drainage device.

OBJECTIVE: To determine the distribution of 2 intracellular oxygen-carrying molecules, neuroglobin (NGB) and cytoglobin (CYGB), in specific retinal cell types of human retinas. METHODS: Specific antibodies against NGB and CYGB were used in immunohistochemical studies to examine their distribution patterns in human retinal sections. Double-labeling studies were performed with the anti-NGB and anti-CYGB antibodies along with antibodies against neuronal (microtubule-associated protein 2, class III beta-tubulin [TUJ1], protein kinase C alpha, calretinin) and glial (vimentin, glial fibrillary acid protein) markers. Confocal microscopy was used to examine the retinal sections. RESULTS: Immunohistochemical analysis of human retinal tissue showed NGB and CYGB immunoreactivity in the ganglion cell layer, inner nuclear layer, inner and outer plexiform layers, and retinal pigment epithelium. Neuroglobin immunoreactivity was also present in the outer nuclear layer and photoreceptor inner segments. Neuroglobin and CYGB were coexpressed in the neurons in the ganglion cell layer and inner nuclear layer but not within glial cells. CONCLUSION: Neuroglobin and CYGB are colocalized within human retinal neurons and retinal pigment epithelium but not within glial cells. Clinical Relevance Our results suggest that NGB and CYGB may serve a neuroprotective role as scavengers of reactive oxygen species and therefore should be considered when developing therapeutic strategies for treatment of hypoxia-related ocular diseases.

This study provides a detailed description of immunolocalization of two oxygen-binding proteins, neuroglobin (Ngb)()and cytoglobin (Cygb), in the anterior segment of healthy human and canine eyes. Specific antibodies against Ngb and Cygb were used to examine their distribution patterns in anterior segment structures including the cornea, iris, trabecular meshwork, canal of Schlemm, ciliary body and lens. Patterns of immunoreactivity (IR) were imaged with confocal scanning laser and conventional microscopy. Analysis of sectioned human and canine eyes revealed Ngb- and Cygb-IR in the corneal epithelium and endothelium. In the iris, Ngb and Cygb-IR was localized to the anterior border and the stroma, iridal sphincter and dilator muscle. In the iridocorneal angle, Ngb and Cygb were detected in endothelial cells of the trabecular meshwork and canal of Schlemm in human. In the ciliary body, Ngb- and Cygb-IR was localized to the nonpigmented ciliary epithelium of the pars plana and pars plicata, and in ciliary body musculature. Ngb and Cygb distribution was similar and co-localized within the same structures of healthy human and canine anterior eye segments. Based on their immunolocalization and previously reported biochemical features, we hypothesize that Ngb and Cygb may function as scavengers of reactive oxygen species. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

We have previously demonstrated the role of univentricular pacing modalities in influencing coronary conduit flow in the immediate post-operative period in the cardiac surgery patient. We wanted to determine the mechanism of this improved coronary conduit and in addition to explore the possible benefits with biventricular pacing. Sixteen patients undergoing first time elective coronary artery bypass grafting who required pacing following surgery were recruited. Comparison of cardiac output and coronary conduit flow was performed between VVI and DDD pacing with a single right ventricular lead and biventricular pacing lead placement. Cardiac output was measured using arterial pulse waveform analysis while conduit flow was measured using ultrasonic transit time methodology. Cardiac output was greatest with DDD pacing using right ventricular lead placement only [DDD-univentricular 5.42 l (0.7), DDD-biventricular 5.33 l (0.8), VVI-univentricular 4.71 l (0.8), VVI-biventricular 4.68 l (0.6)]. DDD-univentricular pacing was significantly better then VVI-univentricular (P=0.023) and VVI-biventricular pacing (P=0.001) but there was no significant advantage to DDD-biventricular pacing (P=0.45). In relation to coronary conduit flow DDD pacing again had the highest flow [DDD-univentricular 55 ml/min (24), DDD-biventricular 52 ml/min (25), VVI-univentricular 47 ml/min (23), VVI-biventricular 50 ml/min (26)]. DDD-univentricular pacing was significantly better then VVI-univentricular (P=0.006) pacing but not significantly different to VVI-biventricular pacing (P=0.109) or DDD-biventricular pacing (P=0.171). Pacing with a DDD modality offers the optimal coronary conduit flow by maximising cardiac output. Biventricular lead placement offered no significant benefit to coronary conduit flow or cardiac output. Keywords: Pacing; Biventricular; Cardiac output; Coronary conduit flow; DDD; VVI.

Genomics has produced hundreds of new hemoglobin sequences with examples in nearly every living organism. Structural and biochemical characterizations of many recombinant proteins reveal reactions, like oxygen binding and NO dioxygenation, that appear general to the hemoglobin superfamily regardless of whether they are related to physiological function. Despite considerable attention to "hexacoordinate" hemoglobins, which are found in nearly every plant and animal, no clear physiological role(s) has been assigned to them in any species. One popular and relevant hypothesis for their function is protection against NO. Here we have tested a comprehensive representation of hexacoordinate hemoglobins from plants (rice hemoglobin), animals (neuroglobin and cytoglobin), and bacteria (Synechocystis hemoglobin) for their abilities to scavenge NO compared to myoglobin. Our experiments include in vitro comparisons of NO dioxygenation, ferric NO binding, NO-induced reduction, NO scavenging with an artificial reduction system, and the ability to substitute for a known NO scavenger (flavohemoglobin) in E. coli. We conclude that none of these tests reveal any distinguishing predisposition toward a role in NO scavenging for the hxHbs, but that any hemoglobin could likely serve this role in the presence of a mechanism for heme iron re-reduction. Hence, future research to test the role of Hbs in NO scavenging would benefit more from the identification of cognate reductases than from in vitro analysis of NO and O(2) binding.