2-Hydroxypropyl-beta-cyclodextrin enhances phorbol ester effects on glucose transport and/or protein kinase C-beta translocation to the plasma membrane in rat adipocytes and soleus muscles

In rat adipocytes and soleus muscles, 2-hydroxypropyl-beta-cyclodextrin (CD) was found to have a relatively small or no effect on basal or insulin-stimulated hexose uptake, but markedly enhanced hexose uptake effects of phorbol esters and/or diacylglycerol. In rat adipocytes, the CD-induced enhancement of hexose uptake during concurrent phorbol ester treatment was not associated with an increase in GLUT4 glucose transporter translocation to the plasma membrane, which was stimulated comparably by insulin and phorbol esters. Moreover, CD appeared to activate or facilitate the activation of glucose transporters subsequent to their translocation to the plasma membrane during ongoing phorbol ester treatment. In rat adipocytes, CD also enhanced the translocation of protein kinase C (PKC)-beta to the plasma membrane during the action of phorbol esters, which alone had little or no effect on this specific PKC translocation. Although it is uncertain how CD alters the function of plasma membranes to enhance the translocation of PKC-beta to, and the activation of glucose transporters within, this subcellular fraction during phorbol ester treatment, our findings provide direct support for a two-step model in the activation of glucose transport. In addition, it seems clear that, at least in some cell types, simple phorbol ester treatment does not necessarily serve as a ubiquitous activator of all activable PKC pools and all potential PKC-mediated responses.     

Plant genetics: fast flowering

Bacillus cereus hemolysin III activity was tested in crude extracts, from Escherichia coli carrying the hly-III gene. It was concluded that hemolysin III is a pore-forming hemolysin with functional pore diameter of about 3-3.5 nm. Hemolysis occurs in at least three steps: (i) the temperature-dependent binding of the Hly-III monomers to the erythrocyte membrane; (ii) the temperature-dependent formation of the transmembrane oligomeric pore; (iii) the temperature-independent erythrocyte lysis.     

Mass spectrometric analysis of 2-deoxyribonucleoside and 2'-deoxyribonucleotide adducts with aldehydes derived from lipid peroxidation

An important emerging issue in chemical carcinogenesis is the role that products of endogenous metabolism play in formation of covalently modified DNA. One example is the formation of alpha, beta-unsaturated aldehydes as a result of endogenous and drug-stimulated lipid peroxidation. Malondialdehyde (MDA), crotonaldehyde (CR), 2-hexenal (HX), and 4-hydroxy-2-nonenal (HNE) react covalently with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) residues on DNA to form promutagenic cyclic adducts that may be important in the etiology of cancer in humans and animals. The accurate quantification of such adducts provides a powerful tool in molecular epidemiology for assessing carcinogenic risks from various lifestyle choices (e.g. diet, drug use) in humans. 32P-Postlabeling is recognized as one of the most sensitive methods available for detection of DNA adducts in human tissues, but without adequate validation such methodology can yield inaccurate quantitative measurements. We have used LC separations in conjunction with electrospray ionization MS and tandem MS (triple quadrupole and hybrid quadrupole-orthogonal acceleration time of flight analyzers) to characterize MDA-, CR-, HX- and HNE-modified dG and nucleotide (3'- and 5'-monophosphate; 3',5'-bisphosphate) adducts. These data have been used to validate 32P-postlabeling methods for quantification of low level MDA-dG adducts formed in DNA of human and animal tissues. Availability of reliable methods for quantification of endogenous DNA damage in humans and animals is essential for determining unknown etiologies of cancer and for the assessment of cancer risks in humans.     

Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.     

Capillary tube resistive thermal cycling

A system that performs rapid thermal cycling of microliter and smaller liquid volumes inside glass capillary tubes that have an optically transparent thin film of indium-tin oxide (ITO) covering the exterior is described. The ITO film acts as both a heater and a temperature sensor, while cooling is accelerated with forced air. Unlike existing batch-mode thermal cycling systems, this system allows control over each sample's temperature profile. Temperature transition rates of 44 degrees Celsius per second during heating and 15 degrees Celsius per second during cooling have been achieved, allowing successful polymerase chain reaction (PCR) experiments to be performed in 20 min. Capillary external temperature can be regulated typically to within +/- 0.25 degrees Celsius, and peak temperatures more than 800 degrees Celsius have been demonstrated. Capillary internal (sample) temperatures at present are controllable typically to within 2 degrees Celsius. The resistive film can be used as a temperature sensor, and the optical transparency of the thin-film coating could permit fluorescent monitoring of the sample during thermal cycling, making this method well suited for real-time quantitative PCRs.     

Pharmacological activities of trimetoquinol and 1-benzyl halogen-substituted analogues on rat beta-adrenoceptor subtypes

The beta-adrenoceptor activity profile of trimetoquinol and its 1-benzyl halogen-substituted analogues was studied in rat tissues containing primarily beta 1 (atria)-, beta 2 (trachea)- and atypical beta/beta 3 (distal colon and brown adipose tissue)-adrenoceptors. Functional biological activity resided in the (-)-isomer of trimetoquinol which was 112-, 275-, 372- and 513-fold more potent than (+)-trimetoquinol in trachea, right atria, distal colon and brown adipose tissue, respectively. (+/-)-Trimetoquinol was equally or slightly less active than (-)-trimetoquinol. The 1-benzyl halogen-substituted analogues of trimetoquinol exhibited differential activation of beta-adrenoceptor subtypes. In functional assays, 3'-iodotrimetoquinol was a potent activator of all beta-adrenoceptor subtypes. 3',5'-Diiodotrimetoquinol was 10-fold more potent as an agonist in tissues containing atypical beta/beta 3-adrenoceptors than those tissues containing beta 1- and beta 2-adrenoceptor sites. Furthermore, this drug was a partial agonist as compared to (+/-)-trimetoquinol and 3'-iodotrimetoquinol on beta 1-adrenoceptors. Pharmacological properties of the compounds on rat beta 3-adrenoceptors expressed in Chinese hamster ovary (CHO) cells were consistent with results observed in functional assays. 3',5'-Diiodotrimetoquinol possessed the greatest potency for activation of adenylyl cyclase. Rank order of affinity for rat beta 3-adrenoceptor was 3'-iodotrimetoquinol = 3',5'-diiodotrimetoquinol > (+/-)-trimetoquinol > (-)-isoprenaline. These results suggest that 3',5'-diiodotrimetoquinol is a promising drug for further chemical modification in the development of selective beta 3-adrenoceptor ligands.