Biomarkers of Selenium Status

The essential trace element, selenium (Se), has multiple biological activities, which depend on the level of Se intake. Relatively low Se intakes determine the expression of selenoenzymes in which it serves as an essential constituent. Higher intakes have been shown to have anti-tumorigenic potential; and very high Se intakes can produce adverse effects. This hierarchy of biological activities calls for biomarkers informative at different levels of Se exposure. Some Se-biomarkers, such as the selenoproteins and particularly GPX3 and SEPP1, provide information about function directly and are of value in identifying nutritional Se deficiency and tracking responses of deficient individuals to Se-treatment. They are useful under conditions of Se intake within the range of regulated selenoprotein expression, e.g., for humans <55 μg/day and for animals <20 μg/kg diet. Other Se-biomarkers provide information indirectly through inferences based on Se levels of foods, tissues, urine or feces. They can indicate the likelihood of deficiency or adverse effects, but they do not provide direct evidence of either condition. Their value is in providing information about Se status over a wide range of Se intake, particularly from food forms. There is need for additional Se biomarkers particularly for assessing Se status in non-deficient individuals for whom the prospects of cancer risk reduction and adverse effects risk are the primary health considerations. This would include determining whether supranutritional intakes of Se may be required for maximal selenoprotein expression in immune surveillance cells. It would also include developing methods to determine low molecular weight Se-metabolites, i.e., selenoamino acids and methylated Se-metabolites, which to date have not been detectable in biological specimens. Recent analytical advances using tandem liquid chromatography-mass spectrometry suggest prospects for detecting these metabolites.     

Synthesis of stable isotope‐labeled epothilone D using a degradation–reconstruction approach

The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule‐stabilizing drug that has been investigated by Bristol‐Myers Squibb. An important step in the development process was the synthesis of a stable isotope‐labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. A novel synthetic route to stable isotope‐labeled epothilone D is described. The synthetic route was based on a strategy to degrade epothilone B and then use that key intermediate to reconstruct stable isotope‐labeled epothilone D. Epothilone B was treated with potassium osmate and sodium periodate. The thiazole moiety in epothilone B was efficiently cleaved to give (1S,3S,7S,10R,11S,12S,16R)‐3‐acetyl‐7,11‐dihydroxy‐8,8,10,12,16‐pentamethyl‐4,17‐dioxabicyclo[14.1.0]heptadecane‐5,9‐dione. The epoxide in the macrocyclic ring of that intermediate was cleanly removed by treatment with tungsten hexachloride and n‐butyllithium to give the corresponding olefin (4S,7R,8S,9S,16S,Z)‐16‐acetyl‐4,8‐dihydroxy‐5,5,7,9,13‐pentamethyloxacyclohexadec‐13‐ene‐2,6‐dione. Bis(triethylsilyl) protection produced (4S,7R,8S,9S,16S,Z)‐16‐acetyl‐5,5,7,9,13‐pentamethyl‐4,8‐bis(triethylsilyloxy)‐oxacyclohexadec‐13‐ene‐2,6‐dione. This intermediate was coupled to a stable isotope‐labeled thiazole using a Wittig reaction as the key step to provide ¹³C₅, ¹⁵N‐labeled epothilone D. In summary, the synthesis was completed in nine total steps, only six of which involved isotopically labeled reagents. A total of 168 mg of ¹³C₅, ¹⁵N‐labeled epothilone D was prepared in an 8% overall yield from ¹³C₂, ¹⁵N‐labeled thioacetamide and ¹³C₃‐labeled ethyl bromopyruvate.     

Integrin β3 Haploinsufficiency Modulates Serotonin Transport and Antidepressant-Sensitive Behavior in Mice

Converging lines of evidence have identified genetic interactions between the serotonin transporter (SERT) gene and ITGB3, which encodes the β3 subunit that forms the αIIbβ3 and αvβ3 integrin receptor complexes. Here we examine the consequences of haploinsufficiency in the mouse integrin β3 subunit gene (Itgb3) on SERT function and selective 5-hydroxytryptamine (5-HT) reuptake inhibitor (SSRI) effectiveness in vivo. Biochemical fractionation studies and immunofluorescent staining of murine brain slices reveal that αvβ3 receptors and SERTs are enriched in presynaptic membranes from several brain regions and that αvβ3 colocalizes with a subpopulation of SERT-containing synapses in raphe nuclei. Notably, we establish that loss of a single allele of Itgb3 in murine neurons is sufficient to decrease 5-HT uptake by SERT in midbrain synaptosomes. Pharmacological assays to elucidate the αvβ3-mediated mechanism of reduced SERT function indicate that decreased integrin β3 subunit expression scales down the population size of active SERT molecules and, as a consequence, lowers the effective dose of SSRIs. These data are consistent with the existence of a subpopulation of SERTs that are tightly modulated by integrin αvβ3 and significantly contribute to global SERT function at 5-HT synapses in the midbrain. Importantly, our screen of a normal human population for single nucleotide polymorphisms in human ITGB3 identified a variant associated with reductions in integrin β3 expression levels that parallel our mouse findings. Thus, polymorphisms in human ITGB3 may contribute to the differential responsiveness of select patients to SSRIs.     

Electrocoagulation of rectal cancer

Electrocoagulation is an effective treatment modality for localized cancer of the distal rectum. Proper selection remains the key to successful treatment. Of potentially curable patients with cancer of the rectum followed up for a median of five years, 69 per cent had no evidence of cancer at the end of the study period. Gross tumor morphology defined two distinct groups with regard to outcome after electrocoagulation. Ninety-two per cent of patients with polypoid/exophytic tumors as compared to 33 per cent of patients with ulcerative lesions had successful treatment. Based on these results, the authors believe that lesions that are exophytic represent early cancers with a low incidence of nodal spread and, as such, can be treated by electrocoagulation with confidence. As a palliative measure, the authors found electrocoagulation to yield equivocal results. Read at the meeting of the American Society of Colon and Rectal Surgeons, Colorado Springs, Colorado, June 7 to 11, 1981.     

Calpain is a mediator of preservation-reperfusion injury in rat liver transplantation

Proteases as well as alterations in intracellular calcium have important roles in hepatic preservation-reperfusion injury, and increased calpain activity recently has been demonstrated in liver allografts. Experiments were designed to evaluate (i) hepatic cytosolic calpain activity during different periods of cold ischemia (CI), rewarming, or reperfusion, and (ii) effects of inhibition of calpain on liver graft function using the isolated perfused rat liver and arterialized orthotopic liver transplantation models. Calpain activity was assayed using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (AMC) and expressed as mean ± SD pmol AMC released/min per mg of cytosolic protein. Calpain activity rose significantly after 24 hr of CI in University of Wisconsin solution and further increased with longer preservation. Activity also increased within 30 min of rewarming, peaking at 120 min. Increased durations of CI preceding rewarming resulted in significantly higher activity (P < 0.01). Calpain activity increased rapidly upon reperfusion and was significantly enhanced by previous CI (P < 0.01). Calpain inhibition with Cbz-Val-Phe methyl ester significantly decreased aspartate aminotransferase released in the isolated perfused rat liver perfusate (P < 0.05). Duration of survival after orthotopic liver transplantation using livers cold-preserved for 40 hr was also significantly increased (P < 0.05) with calpain inhibitor. In conclusion, calpain proteases are activated during each phase of transplantation and are likely to play an important role in the mechanisms of preservation-reperfusion injury.