A study of methods for in situ X-ray energy dispersive analysis of polyphosphate bodies in Plectonema boryanum

A variety of preparative methods for in situ X-ray energy dispersive analysis were tested to determine their effects on the elemental composition of polyphosphate bodies in P. boryanum. The bodies were found to contain large amounts of P and K and small amounts of Ca and Mg. Air drying, freeze-drying and freeze-drying from a liquid nitrogen slush all gave similar results. Fixation of the cells in glutaraldehyde and/or OsO₄ resulted in loss of the K and enhancement of the Ca peak. Magnesium was lost during embedding in epoxy.     

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.     

Effect of Somatostatin Analog on Postprandial Satiation in Obesity

Objective: Altered satiation may impact postprandial symptoms and potentially change food intake in obesity. Our aim was to compare effects of octreotide and placebo on postprandial symptoms, satiation, and gastric volumes in obesity. Research Methods and Procedures: In a randomized, parallel‐group, double‐blind, placebo‐controlled study, 26 obese but otherwise healthy participants received 100 μg of octreotide or placebo subcutaneously 30 minutes before each study. Studies were performed on 2 separate days and included validated non‐invasive techniques: ^99mTc‐single photon emission computed tomography imaging to measure fasting stomach volume and gastric volume changes after 90 mL of water and 240 mL of Ensure and a standardized nutrient drink test to measure the maximum tolerated volume and postprandial symptoms. Results: Relative to placebo, octreotide increased gastric volume after 90 mL of water; however, fasting and gastric volume change post‐Ensure and maximum tolerated volume of Ensure were not different. Octreotide decreased sensations of fullness (p = 0.035) and bloating (p = 0.05) and tended to reduce aggregate symptoms (p = 0.07) after the fully satiating meal. Discussion: In obese individuals, somatostatin analog significantly reduced postprandial sensations after a satiating meal without altering maximum tolerated meal volume or postnutrient gastric volume, suggesting an effect on upper gut sensation. The role of somatostatin as a permissive factor in the development of obesity by reducing postprandial sensations deserves further study.     

Classification of the insulin-like growth factor binding proteins into three distinct categories according to their binding specificities

Competitive binding experiments with insulin-like growth factor (IGF)-1, IGF-2 and des-(1-3)-IGF-1 have confirmed the interpretation based on limited amino-terminal sequence analysis that at least three types of IGF binding protein occur. In addition to the acid stable subunit of the large serum binding protein which exhibits des-(1-3)-IGF-1 binding only slightly less than IGF-1, the small IGF binding proteins can be separated into two classes based on differences in des-(1-3)-IGF-1 and IGF-2 binding potencies.     

Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.     

Mitochondrial defects and oxidative damage in patients with peripheral arterial disease

Abnormal mitochondrial function is present in patients with peripheral arterial disease and may contribute to its clinical manifestations. However, the specific biochemical mitochondrial defects and their association with increased oxidative stress have not been fully characterized. Gastrocnemius muscle was obtained from peripheral arterial disease patients (n = 25) and age-matched controls (n = 16) and mitochondrial parameters were measured. Complexes I through IV of the electron transport chain were individually evaluated to assess for isolated defects. Muscle was also evaluated for protein and lipid oxidative changes by measuring the levels of protein carbonyls, lipid hydroperoxides, and total 4-hydroxy-2-nonenal binding and for the activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. Mitochondrial electron transport chain complexes I, III, and IV in arterial disease patients demonstrated significant reductions in enzymatic activities and mitochondrial respiration compared to controls. Oxidative stress biomarker analysis demonstrated significantly increased levels of protein carbonyls, lipid hydroperoxides, and 4-hydroxy-2-nonenal compared to control muscle. Antioxidant enzyme activities were altered, with a significant decrease in superoxide dismutase activity and significant increases in catalase and glutathione peroxidase. Peripheral arterial disease is associated with abnormal mitochondrial function and evidence of significant oxidative stress.