Is methyl-branching in alpha-tocopherol's "tail" important for its in vivo activity? Rat curative myopathy bioassay measurements of the vitamin E activity of three 2RS-n-alkyl-2,5,7,8-tetramethyl-6-hydroxychromans

The vitamin E activity of the acetates of three 2RS-n-alkyl-2,5,7,8-tetramethyl-6-hydroxychroman analogs of alpha-tocopherol have been measured and compared directly with all-rac-alpha-tocopheryl acetate, or indirectly via 2R,4'R,8'R-alpha-tocopheryl acetate, using the rat curative myopathy, plasma pyruvate kinase assay. The analogs with alkyl chain lengths of 11 and 13 carbons have activities which not only do not differ significantly (p greater than 0.05) from each other but also do not differ from that of all-rac-alpha-tocopheryl acetate. This finding indicates that methyl branching in the phytyl tail at the 4', 8', and 12' positions has little if any influence upon vitamin E activity. Thus physical interactions involving the methyl branches of the phytyl tail and the polyunsaturated moieties of membrane phospholipids are unimportant in vivo, insofar as this bioassay is concerned. However, the length of the hydrocarbon tail is important. This is indicated by the result obtained with the acetate of the analog with an alkyl chain length of 15 carbon atoms which had only 15% of the activity of 2R,4'R,8'R-alpha-tocopheryl acetate, i.e., 22% of the activity of all-rac-alpha-tocopheryl acetate since this form is 1.47 times less active than 2R,4'R,8'R-alpha-tocopheryl acetate in the curative myopathy bioassay (Weiser, Vecchi, & Schlachter, Internat. J. Vit. Nutr. Res. 55:149-158, 1985).     

Free fatty acids inhibit cytotoxic T lymphocyte-mediated lysis of allogeneic target cells

Short term exposure of murine CTL clones to long chain cis unsaturated free fatty acid (FFA) inhibits alloantigen specific lysis of cognate target cells, whereas long-chain saturated FFA have no effect. Inhibition of lysis occurs when cis FFA is added before or within 10 min after CTL-target cell conjugate formation and thus appears to interfere with lethal hit delivery. Our previous studies have shown that similar treatment with cis FFA inhibits, in CTL, the Ag stimulated increase in intracellular calcium and degranulation, suggesting that inhibition of lysis probably results from perturbation of the CTL signaling pathway. However, inhibition of lysis is probably not due to the inhibition of the rise in intracellular calcium or degranulation, because lysis can occur under conditions in which FFA inhibit degranulation and because cis FFA inhibit calcium-independent killing. Inhibition of lysis is detectable at unbound FFA concentrations less than 1 microM and is generally complete at concentrations less than 5 microM. Although these levels of FFA are somewhat higher than reported for normal physiologic conditions, plasma FFA levels can be elevated into this range in states of stress and disease, suggesting that FFA modulation of the immune response has important physiologic consequences.     

The Effects of Brown Rust and Phosphate on Cold Acclimation in Winter Barley

The green leaf area of winter barley, cv. Sonja, sampled fromthe field at different times during winter was always greatestin plants grown at high soil phosphate and smallest in plantsgrown at low soil phosphate, and at each fertilizer level wasgreater in healthy plants than in plants infected by rust (Pucciniahordei). In leaves that survived the coldest period of winter,the percentage area that was damaged was increased by rust infectionwhich prevented the ameliorating effects of high soil P. Rustand low P interacted to reduce the increases in leaf area andshoot d. wt that occurred when higher temperatures prevailedin spring. Under controlled conditions in the laboratory, phosphate reducedthe injury suffered when plants not acclimated to low temperatureswere exposed to freezing conditions, but this effect was removedby rust infection. After rust infection, freezing temperatureswere damaging even to acclimated plants, particularly if grownwith low soil P. Evidence of visible symptoms, and quantitativemeasurements of electrolyte efflux from intact leaves, chlorophyllfluorescence in vivo, and ethane and ethylene evolution fromcold-acclimated plants, showed that infection raised the minimumtemperature at which tissues could survive without injury. Infectedleaves were more sensitive to low temperature post-sporulationthan presporulation. Measurements of electrolyte efflux andchlorophyll fluorescence on plants growing under cold conditionsshowed that infection inhibited the processes of acclimationto low temperatures. Winter barley, Puccinia hordei, injury, low temperature, acclimation     

Neurophysins as markers of vasopressin and oxytocin release. A study in carcinoma of the lung

Vasopressin-neurophysin (hNpI), oxytocin-neurophysin (hNpII) and blood osmolality were assayed before any treatment in basal conditions in 35 patients suffering from lung carcinoma (20 oat cell, 6 undifferentiated and 9 well-differentiated epidermoid cell carcinomas). Plasma vasopressin (antidiuretic hormone, ADH) was also assayed in 7 of the 20 patients suffering from oat cell carcinoma. We found a close correlation (r = 0.98) between plasma ADH and hNpI levels in the 7 patients. Further, hNpI was elevated in 13 out of the 20 oat cell carcinoma patients and in none of the epidermoid-cell carcinoma group; however, searching for an abnormality of ADH secretion as reflected by a detectable plasma hNpI level together with subnormal plasma osmolality revealed 2 additional positive results in the oat cell carcinoma group, and 2 out of the 6 in the undifferentiated-cell carcinoma group. hNpII was increased together with an increase in hNpI in 6 oat cell carcinoma patients; it was specifically increased without hNpI increment in 2 additional oat cell carcinoma patients and in 2 patients of the undifferentiated-cell carcinoma group (different from the 2 positive for the hNpI-osmolality ratio). hNpI and hNpII were normal in the majority of undifferentiated and all of the differentiated epidermoid-cell carcinoma group. Hence, our results show that simultaneous measurements of hNpI, hNpII, and blood osmolality could detect abnormalities in 17 out of 20 oat cell carcinoma patients, in 4 of the 9 undifferentiated-cell carcinoma patients, but in none of the differentiated epidermoid-cell carcinoma patients, suggesting that the neurophysin assay can be used for the early detection of oat cell- and possibly other neuroendocrine-derived carcinomas.     

Electrostatic forces at helix-coil boundaries in DNA

The Tm of internal loop-forming (dA.dT)N domains in pBR322 DNA has been measured over a tenfold range of [Na+]. The slopes SN = dTm/d log [Na+] are linear and decrease in magnitude with decreasing loop size N, signaling a reduction in Na+ released during the transition of these domains to the coil state. Values of SN decrease linearly with increasing N-1 in accordance with the expectation of a simple model for the occurrence of a gradient of long-range electrostatic forces at helix-coil boundaries, and extrapolate almost precisely to the value of S infinity observed for (dA.dT) infinity. These results indicate (1) less counterion is released per phosphate residue from the finite loop than from the infinite-sized loop, and (2) the difference in binding is constant for each boundary formed and independent of the size of the loop within the range examined: approximately 350 base pair (bp) greater than N greater than 71 bp. The slope of the dependence of SN on N-1 indicates the region of higher charge density at the boundary extends at least 18 A into the coil and probably 40-50 A before dropping to a value characteristic of the unperturbed coil. The free energy for excess counterion binding at boundaries can be expressed by -delta G/RT = 10.47 log[Na+] + 5.234 When the loop entropy function in a statistical mechanical algorithm for the dissociation of DNA is weighted by this quantity, calculated Tm are seen to vary by only +/- 0.09 degrees C from observed.     

Measurement of active-site homology between potato and rabbit muscle alpha-glucan phosphorylases through use of a linear free energy relationship

The Michaelis-Menten parameters (Vmax and Km) for turnover of an extensive series of deoxy and deoxyfluoro derivatives of alpha-D-glucopyranosyl phosphate by the alpha-glucan phosphorylase from potato tuber have been determined. Very large rate reductions are observed as a consequence of each substitution, primarily due to losses in specific binding interactions, most likely hydrogen bonding, at the enzymic transition state. Comparison of the Vmax/Km values so determined with those measured for rabbit muscle alpha-glucan phosphorylase [Street et al. (1989) Biochemistry 28, 1581] reveals an astonishingly similar specificity, especially in light of the phylogenetic separation of their host organisms. This indicates that very similar hydrogen-bonding interactions between the enzyme and the substrate must be present at the transition states for the two enzymic reactions; therefore, they have very similar active sites. Quantitation of this similarity is achieved by plotting the logarithm of the Vmax/Km value for each substrate analogue with the potato enzyme against the same parameter for the muscle enzyme, yielding straight lines (p = 0.998 and 0.999) of slope 1.0 and 1.2 for the deoxy and deoxyfluoro substrates, respectively. Since the correlation coefficient of such plots is a direct measure of the similarity of the two transition-state complexes, thus of the enzyme active sites, it can be used as a measure of active-site homology between the two enzymes. The extremely high homology observed in this case is consistent with the observed sequence homology at the active site.     

Inactivation of pyruvate decarboxylase by 3-hydroxypyruvate.

Pyruvate decarboxylase from Zymomonas mobilis is inhibited by 3-hydroxypyruvate, which can also act as a poor substrate. While catalysing the decarboxylation of this alternative substrate, the enzyme undergoes a progressive but partial inactivation over several hours. The extent of inactivation depends upon the pH and upon the concentration of 3-hydroxypyruvate. After partial inactivation and removal of unchanged 3-hydroxypyruvate, enzymic activity recovers slowly. We suggest that inactivation results from accumulation of enzyme-bound glycollaldehyde, which is relatively stable, possibly because it is dehydrated to form an acetyl group.     

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine. An effective, mild procedure based on N alpha-fluorenylmethyloxycarbonyl (Fmoc) protection and side-chain anchoring to a tris(alkoxy)benzylamide (PAL) handle

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N alpha-Fmoc-C alpha-tert.-butyl aspartate or glutamate via the free omega-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF3COOH--CH2Cl2, 90 min at 25 degrees, releases the desired peptides in greater than 95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.     

Effect of mastectomy versus mastectomy plus adjuvant ovariectomy on prolactin response to TRH in breast cancer

The endocrine effects of ovariectomy need to be further investigated. The present study was carried out to evaluate the influence of the adjuvant ovariectomy on the mastectomy-induced changes in PRL response to TRH in breast cancer. The study included 34 patients with locally limited breast carcinoma, 18 of whom were treated with radical mastectomy, whereas the other 16 underwent mastectomy plus adjuvant ovariectomy. PRL secretion in response to TRH (200 mcg I.V. as bolus) was evaluated one day before and 7 days after surgery. In patients treated with mastectomy only, PRL increase after TRH was significantly higher after surgery than before. On the contrary, no difference was seen in patients treated with mastectomy plus ovariectomy. This study shows that the adjuvant ovariectomy may block the increase in PRL response to TRH induced by mastectomy in breast cancer.