Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.     

Oxidative damage increases with age in a canine model of human brain aging

We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) activity and both oxidized and reduced glutathione to study the link between oxidative damage, aging and beta-amyloid (Abeta) in the canine brain. The aged canine brain, a model of human brain aging, naturally develops extensive diffuse deposits of human-type Abeta. Abeta was measured in immunostained prefrontal cortex from 19 beagle dogs (4-15 years). Increased malondialdehyde (MDA), which indicates increased lipid peroxidation, was observed in the prefrontal cortex and serum but not in cerebrospinal fluid (CSF). Oxidative damage to proteins (carbonyl formation) also increased in brain. An age-dependent decline in GS activity, an enzyme vulnerable to oxidative damage, and in the level of glutathione (GSH) was observed in the prefrontal cortex. MDA level in serum correlated with MDA accumulation in the prefrontal cortex. Although 11/19 animals exhibited Abeta, the extent of deposition did not correlate with any of the oxidative damage measures, suggesting that each form of neuropathology accumulates in parallel with age. This evidence of widespread oxidative damage and Abeta deposition is further justification for using the canine model for studying human brain aging and neurodegenerative diseases.     

Implications of axillary sentinel lymph node biopsy in immediate autologous breast reconstruction

For patients with invasive breast cancer, if the results of an axillary sentinel node biopsy are determined to be positive after permanent pathologic examination, the current recommendation is to perform a complete axillary node dissection. Subsequent axillary surgery may compromise the blood supply to an immediate autologous breast reconstruction. The purpose of this study was to determine which clinicopathologic factors in clinically node-negative breast cancer patients may be associated with an increased risk of positive axillary nodes. Identification of these factors will allow surgeons to modify their approach to immediate autologous breast reconstruction in these high-risk patients. The relationship between presenting clinicopathologic characteristics and the incidence of axillary metastases was analyzed by chi-square test and multivariate analysis in 167 patients with invasive breast cancer and a clinically negative axilla who underwent modified radical mastectomy with an immediate free transverse rectus abdominis musculocutaneous (TRAM) flap reconstruction. Axillary nodal metastases were found in 35 percent of clinically node-negative breast cancer patients. Multivariate analysis showed that patient age of 50 years or younger (p = 0.019), T2 tumor stage or greater (p = 0.031), and presence of lymphovascular invasion on the initial biopsy specimen (p < 0.001) were independent predictors of axillary metastases in clinically node-negative patients. Based on these results, the authors propose an algorithm for decision making in clinically node-negative breast cancer patients who desire autologous breast reconstruction and sentinel lymph node biopsy. Options for immediate autologous breast reconstruction in patients undergoing mastectomy and axillary sentinel lymph node biopsy that may minimize the risk of vascular damage on reoperation include the use of the internal mammary artery and vein as recipient vessels for a free TRAM flap or a pedicled TRAM flap. If an axillary-based blood supply is used, the authors are considering the use of cadaveric dermis to isolate the pedicle of the flap away from the remaining axillary contents. New developments in breast cancer diagnosis and treatment necessitate a team approach, with increased communication between the breast surgeon and the plastic surgeon in planning surgery for these patients.     

Comparison of lipids in selected tissues of the Florida manatee (Order Sirenia) and bottlenose dolphin (Order Cetacea; Suborder Odontoceti)

The position, porosity and oil-filled nature of the zygomatic process of the squamosal bone (ZPSB) of the Florida manatee, Trichechus manatus latirostris, suggest that it may have a similar sound conduction function to that of the intramandibular fat body (IMFB) of the bottlenose dolphin, Tursiops truncatus, and other odontocetes. To examine this possibility we determined the lipid composition of the ZPSB and adipose tissue from the dorsal part of the head region of the Florida manatee, and compared it to that of the dolphin IMFB and melon (another fatty area implicated in sound conduction in odontocetes). Lipids from manatee ZPSB and from adipose tissue were composed almost entirely of triacylglycerols. The most abundant fatty acids of the ZPSB were 18:1, 16:0, 14:0 and 16:1. The major fatty acids of the adipose tissue in the head were the four mentioned above, along with 12:0 and 18:0. Manatee samples did not contain isovaleric acid (iso-5:0), which was found in the bottlenose dolphin IMFB and melon, and has been related to sound conduction in dolphins and some other odontocetes. Thus, if manatee tissues are capable of sound conduction, and this process does occur through the ZPSB, a somewhat different suite of lipid components must support this function.     

Patterns of mortality in southern sea otters (Enhydra lutris nereis) from 1998-2001

Detailed postmortem examination of southern sea otters (Enhydra lutris nereis) found along the California (USA) coast has provided an exceptional opportunity to understand factors influencing survival in this threatened marine mammal species. In order to evaluate recent trends in causes of mortality, the demographic and geographic distribution of causes of death in freshly deceased beachcast sea otters necropsied from 1998-2001 were evaluated. Protozoal encephalitis, acanthocephalan-related disease, shark attack, and cardiac disease were identified as common causes of death in sea otters examined. While infection with acanthocephalan parasites was more likely to cause death in juvenile otters, Toxoplasma gondii encephalitis, shark attack, and cardiac disease were more common in prime-aged adult otters. Cardiac disease is a newly recognized cause of mortality in sea otters and T. gondii encephalitis was significantly associated with this condition. Otters with fatal shark bites were over three times more likely to have pre-existing T. gondii encephalitis suggesting that shark attack, which is a long-recognized source of mortality in otters, may be coupled with a recently recognized disease in otters. Spatial clusters of cause-specific mortality were detected for T. gondii encephalitis (in Estero Bay), acanthocephalan peritonitis (in southern Monterey Bay), and shark attack (from Santa Cruz to Point A?o Nuevo). Diseases caused by parasites, bacteria, or fungi and diseases without a specified etiology were the primary cause of death in 63.8% of otters examined. Parasitic disease alone caused death in 38.1% of otters examined. This pattern of mortality, observed predominantly in juvenile and prime-aged adult southern sea otters, has negative implications for the overall health and recovery of this population.     

Effect of ammonia on the growth of Bacillus species and some other bacteria

The tolerance of 26 Bacillus species isolated from alkaline fermented foods, five other bacilli and nine non spore-forming bacteria to alkaline pH and ammonia was determined. All grew at pH 7, 8 and 9 in the presence of 930 mmol l-1 NH4 + at pH 7.0, and in the presence of NH3 concentrations up to 5 mmol l-1 at pH 7.0 and 8.0. At higher NH3 concentrations, growth of some of the bacteria was inhibited and at 500 mmol l-1 only B. pasteurii and B. pumilus grew. Bacteria from alkaline food fermentations included strains relatively sensitive to NH3 (inhibited by 50 mmol l-1) and relatively tolerant strains (grew in the presence of 300 mmol l-1) and there was no evidence that they were more tolerant to NH3 than bacteria not associated with these fermentations.     

Superoxide regulation of endothelin-converting enzyme

Reactive oxygen species (ROS) act as signaling molecules in the cardiovascular system, regulating cellular proliferation and migration. However, an excess of ROS can damage cells and alter endothelial cell function. We hypothesized that endogenous mechanisms protect the vasculature from excess levels of ROS. We now show that superoxide can inhibit endothelin-converting enzyme activity (ECE) and decrease endothelin-1 synthesis. Superoxide inhibits ECE but hydrogen peroxide and nitric oxide do not. Superoxide inhibits ECE by ejecting zinc from the enzyme, and the addition of exogenous zinc restores enzymatic activity. Superoxide may inhibit other zinc metalloproteinases by a similar mechanism and may thus play an important role in regulating the biology of blood vessels.     

Differential Isotope Labeling Strategy for Determining the Structure of Myristoylated Recoverin by NMR Spectroscopy

The three-dimensional solution structure of recombinant bovine myristoylated recoverin in the Ca2+-free state has been refined using an array of isotope-assisted multidimensional heteronuclear NMR techniques. In some experiments, the myristoyl group covalently attached to the protein N-terminus was labeled with 13C and the protein was unlabeled or vice versa; in others, both were 13C-labeled. This differential labeling strategy was essential for structural refinement and can be applied to other acylated proteins. Stereospecific assignments of 41 pairs of -methylene protons and 48 methyl groups of valine and leucine were included in the structure refinement. The refined structure was constructed using a total of 3679 experimental NMR restraints, comprising 3242 approximate interproton distance restraints (including 153 between the myristoyl group and the polypeptide), 140 distance restraints for 70 backbone hydrogen bonds, and 297 torsion angle restraints. The atomic rms deviations about the averaged minimized coordinate positions for the secondary structure region of the N-terminal and C-terminal domains are 0.44 ± 0.07 and 0.55 ± 0.18 Å for backbone atoms, and 1.09 ± 0.07 and 1.10 ± 0.15 Å for all heavy atoms, respectively. The refined structure allows for a detailed analysis of the myristoyl binding pocket. The myristoyl group is in a slightly bent conformation: the average distance between C1 and C14 atoms of the myristoyl group is 14.6 Å. Hydrophobic residues Leu28, Trp31, and Tyr32 form a cluster that interacts with the front end of the myristoyl group (C1-C8), whereas residues Phe49, Phe56, Tyr86, Val87, and Leu90 interact with the tail end (C9-C14). The relatively deep hydrophobic pocket that binds the myristoyl group (C14:0) could also accommodate other naturally occurring acyl groups such as C12:0, C14:1, and C14:2 chains.     

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1). From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.