AMPK Activation Restores Salivary Function Following Radiation Treatment.

Head and neck cancers represent a significant portion of cancer diagnoses, with head and neck cancer incidence increasing in some parts of the world. Typical treatment of early-stage head and neck cancers includes either surgery or radiotherapy; however, advanced cases often require surgery followed by radiation and chemotherapy. Salivary gland damage following radiotherapy leads to severe and chronic hypofunction with decreased salivary output, xerostomia, impaired ability to chew and swallow, increased risk of developing oral mucositis, and malnutrition. There is currently no standard of care for radiation-induced salivary gland dysfunction, and treatment is often limited to palliative treatment that provides only temporary relief. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme that activates catabolic processes and has been shown to influence the cell cycle, proliferation, and autophagy. In the present study, we found that radiation (IR) treatment decreases tissue levels of phosphorylated AMPK following radiation and decreases intracellular NAD+ and AMP while increasing intracellular adenosine triphosphate. Furthermore, expression of sirtuin 1 (SIRT1) and nicotinamide phosphoribosyl transferase (NAMPT) was lower 5 d following IR. Treatment with AMPK activators, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, attenuated compensatory proliferation (days 6, 7, and 30) following IR and reversed chronic (day 30) salivary gland dysfunction post-IR. In addition, treatment with metformin or AICAR increased markers of apical/basolateral polarity (phosphorylated aPKCζT560-positive area) and differentiation (amylase-positive area) within irradiated parotid glands to levels similar to untreated controls. Taken together, these data suggest that AMPK may be a novel therapeutic target for treatment of radiation-induced salivary damage.

Bundling of actin filaments by alpha-actinin depends on its molecular length.

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

Hair color, molt, and testis size in male, short-tailed weasels treated with melatonin.

Melatonin in beeswax was implanted in male weasels (Mustela erminea). Brown weasels and white animals undergoing the spring change to the brown pelage and reproductive activity molted, grew a new white coat, and became reproductively quiescent after treatment. Controls retained or acquired the brown coat and developed or maintained enlarged testes. Treated weasels with pituitary autografts under the kidney capsule grew brown hair after hair growth was initiated by plucking. It is suggested that the pineal gland product, melatonin, initiates changes in the central nervous system and endocrines which result in molting, growth of the white winter pelage, and reproductive quiescence in the weasel.

Vinculin-lipid monolayer interactions: a model for focal contact formation.

Vinculin-lipid interactions were investigated in a modified Langmuir trough. Provided proper conditions, vinculin had the potential to penetrate into phospholipid monolayers and to form rigid, cohesive protein films even at phospholipid monolayer pressures similar to those assumed to exist in living cell membranes. The equilibrium constant for this reaction was estimated to be on the order of 2.5 X 10(-9) to 2.2 X 10(-7) mol/liter (for pressures between 25 and 35 mN/m). Penetration velocity depended on lipid composition: it was high with acidic phospholipids, intermediate with mixtures of acidic and neutral phospholipids, and low with neutral phospholipids. Electron microscopy of freeze-dried/metal-shadowed vinculin films, recovered from the phospholipid monolayer surface, revealed relatively tightly packed globular particles, 13 to 18 nm in diameter, on average significantly larger than the particles seen in glycerol-sprayed and rotary metal-shadowed preparations of soluble vinculin. The lipid monolayer penetration ability of vinculin appears to depend on its conformation. Acid treatment or low salt buffers induced reversible changes in vinculin conformation such that it abolished its lipid penetration potential. These conformational changes could be documented by both circular dichroism and fluorescence spectroscopy. These results indicate that in the focal contact area vinculin may act like a "glue" and link, in a reversible way, stress fibers of cultured cells via their anchor proteins to the extracellular matrix.

Corpus luteum and fetoplacental functions in monkeys hypophysectomized during late pregnancy.

The hormonal regulation of corpus luteum (CL) function during late pregnancy was studied in hypophysectomized monkeys. Between days 149-154 of gestation, 9 days after hypophysectomy, progesterone in the uteroovarian vein (UOV), uterine vein (UV) and peripheral circulation (P) averaged 179.7 ng/ml, 38.9 ng/ml and 5.5 ng/ml, respectively. Amniotic fluid prolactin ranged from 2150-6700 ng/ml and monkey chorionic somatomammotropin (mCS) in mothers carrying live fetuses ranged from 11.4-30.8 micrograms/ml in the UV and P. Prolactin and monkey chorionic gonadotropin in the UV and P were low or nondetectable as was mCS in 2 mothers carrying dead fetuses. CL function was further studied 7 and 39 days after removal of the fetus alone or both the fetus and placenta. Placental delivery was extremely variable, ranging from 2-greater than 63 days post-fetectomy. Although progesterone was not detectable in the P7 days after cesarean section in those animals in which both fetus and placenta were absent, surprisingly, progesterone was measurable in the UOV (range 1.6-48.2 ng/ml). At 39 days, progesterone was either nondetectable or very low. We have interpreted these data to mean: 1) neither the maternal pituitary gland nor a live fetus is necessary for placental or corpus luteum production of progesterone during late pregnancy, 2) the presence of high levels of circulating prolactin and mCS are apparently not necessary for continued secretion of progesterone from the CL during late pregnancy, 3) the fetoplacental unit may be the source of the luteotropic stimulus of late pregnancy since progesterone in the UOV decreases markedly in the absence of the fetoplacental unit or disruption of the unit brought about by fetectomy, and 4) regression of the CL following cesarean section in hypophysectomized monkeys is exceedingly slow when compared to the precipitous regression characteristic of the CL of the nonfertile menstrual cycle.

Chorionic gonadotropin, chorionic somatomammotropin, and prolactin in the uterine vein and peripheral plasma of pregnant rhesus monkeys.

Seven adult female rhesus monkeys were laparotomized at days 22, 42, and 157 of pregnancy and blood was collected from the uterine vein and peripheral circulation. Plasma samples were analyzed for monkey chorionic gonadotropin (mCG), monkey chorionic somatomammotropin (mCS), and prolactin by radioimmunoassay. Levels of mCG at day 22 of pregnancy were approximately 250 ng/ml; however, during the later stages of gestation mCG was either nondetectable or less than 0.7 ng/ml. There was no statistical difference in prolactin concentrations between days 22 and 42 of pregnancy, mean levels being between 176-424 ng/ml, but by day 157 prolactin levels of greater than 2,000 ng/ml were recorded. No statistical difference existed between peripheral and uterine vein concentrations of either mCG or prolactin at any of the stages of gestation examined. At day 22, mCS was not detectable; however, at day 42 of gestation mCS titers averaged 1.5 micrograms/ml and 2.3 micrograms/ml in the peripheral and uterine vein plasma, respectively. A statistically significant mCS increase occurred by day 157, levels in the periphery and uterine vein averaging 11.0 micrograms/ml and 16.3 micrograms/ml, respectively. Uterine vein titers of mCS were significantly higher than peripheral titers at both days 42 and 157. Thus, the highest levels of mCG were present during early pregnancy, whereas the highest levels of mCS and prolactin were present during late pregnancy.

Nidation in weanling rats treated with PMS and gonadal steroids.

PIP: Implantation was studied in 22-day-old female rats to determine the precise amounts of ovarian hormones needed for implantation of blastocysts. 3 mg of medroxyprogesterone was given to increase the percentage of animals with delayed implantation. .1 mcg estradiol-17beta given on Day 4 produced about 50% blastocyst implantation. 105 or .025 mcg estradiol-17beta given on Day 5 of pregnancy induced nidation in 8 out of 17 and 6 out of 8 rats, respectively. There was no significant difference between control and treated groups with regard to the number of animals with delayed blastocyst implantation and the average number of blastocysts recovered. Daily injection of progesterone helped to cause nidation. High doses of progesterone provide the ratio of progesterone to estrogen needed for implantation and also support the idea that nidation may be triggered by the rise in progesterone level in the presence of a constant amount of estrogen.^ieng

H3-thymidine incorporation by delayed blastocysts from rats treated with subimplantation doses of estrogen.

PIP: Tritiated thymidine incorporation by delayed blastocysts from rats t reated with subimplantation doses of estrogen was studied with autoradio graphic techniques. Gonadotropin-treated prepuberal rats were mated with fertile males and ovariectomized on Day 3 of pregnancy. They also received 2 mg progesterone/day for 6-12 days beginning on Day 3. In addition to the progesterone, .025-.15 mcg estrone had no effect on the percentage of nuclei incorporating thymidine or on the number of nuclei per blastocyst. However, the percentages of nuclei labeled/blastocysts in the rats who received .075 or .1 mcg estrone were significantly lower than in those who received .05 mcg (p less than .05). On Day 12 of pregnancy, a significant increase in thymidine incorporation activity was observed 27 hours postinjection of 1 mcg estrone (p less than .05 as compared with controls). However, the total number of nuclei/blastocyst was decreased after 29-30 hours (p less than .01). Additional studies may establish the extent the uterus must be stimulated before it can depress the synthetic activity of the blastocyst.^ieng