Anticancer actions of carnosine in cellular models of prostate cancer.

Treatments for organ-confined prostate cancer include external beam radiation therapy, radical prostatectomy, radiotherapy/brachytherapy, cryoablation and high-intensity focused ultrasound. None of these are cancer-specific and are commonly accompanied by side effects, including urinary incontinence and erectile dysfunction. Moreover, subsequent surgical treatments following biochemical recurrence after these interventions are either limited or affected by the scarring present in the surrounding tissue. Carnosine (ß-alanyl-L-histidine) is a histidine-containing naturally occurring dipeptide which has been shown to have an anti-tumorigenic role without any detrimental effect on healthy cells; however, its effect on prostate cancer cells has never been investigated. In this study, we investigated the effect of carnosine on cell proliferation and metabolism in both a primary cultured androgen-resistant human prostate cancer cell line, PC346Flu1 and murine TRAMP-C1 cells. Our results show that carnosine has a significant dose-dependent inhibitory effect in vitro on the proliferation of both human (PC346Flu1) and murine (TRAMP-C1) prostate cancer cells, which was confirmed in 3D-models of the same cells. Carnosine was also shown to decrease adenosine triphosphate content and reactive species which might have been caused in part by the increase in SIRT3 also shown after carnosine treatment. These encouraging results support the need for further human in vivo work to determine the potential use of carnosine, either alone or, most likely, as an adjunct therapy to surgical or other conventional treatments.

Longitudinal Changes in Cerebral Perfusion, Perivascular Space Volume, and Ventricular Volume in a Healthy Cohort Undergoing a Spaceflight Analog.

BACKGROUND AND PURPOSE: A global decrease in brain perfusion has recently been reported during exposure to a ground-based spaceflight analog. Considering that CSF and glymphatic flow are hypothesized to be propelled by arterial pulsations, it is unknown whether a change in perfusion would impact these CSF compartments. The aim of the current study was to evaluate the relationship among changes in cerebral perfusion, ventricular volume, and perivascular space volume before, during, and after a spaceflight analog. MATERIALS AND METHODS: Eleven healthy participants underwent 30 days of bed rest at 6° head-down tilt with 0.5% atmospheric CO2 as a spaceflight analog. For each participant, 6 MR imaging brain scans, including perfusion and anatomic-weighted T1 sequences, were obtained before, during, and after the analog period. Global perfusion, ventricular volume, and perivascular space volume time courses were constructed and evaluated with repeated measures ANOVAs. RESULTS: Global perfusion followed a divergent time trajectory from ventricular and perivascular space volume, with perfusion decreasing during the analog, whereas ventricular and perivascular space volume increased (P < .001). These patterns subsequently reversed during the 2-week recovery period. CONCLUSIONS: The patterns of change in brain physiology observed in healthy participants suggest a relationship between cerebral perfusion and CSF homeostasis. Further study is warranted to determine whether a causal relationship exists and whether similar neurophysiologic responses occur during spaceflight.

The Effect of a Combination Saliva Substitute for the Management of Xerostomia and Hyposalivation.

OBJECTIVE: The objective of this study was to evaluate the difference between the combination agent of xylitol, beatine and olive oil in a chewable capsule versus the control agent of a sorbitol tablet in subjects with hyposalivation and xerostomia. MATERIALS AND METHODS: The subjects had xerostomia over 3 months and a measured hyposalivation. The study was 3 weeks in duration, with 2 treatment phases of 1 week and a 7 day wash out period in between. At the end of each treatment phase, subjects returned for a follow up evaluation. At this visit they were given the subjective sensation questionnaire, as well as their unstimulated whole salivary flow and stimulated whole salivary flow were measured. RESULTS: There was a greater increase in the unstimulated and stimulated whole salivary flow rate, although the results were not statistically significant. The subjective evaluation as measured by the questionnaire showed that both agents reduced the mean score as compared to the baseline, although only the findings in the active agent was statistically significant (p = 0.0015). CONCLUSION: The significant conclusions found in this study were that the active agent provided a significant subjective improvement in speech, swallowing, and decreased subjective xerostomia as compared to the control tablet. CLINICAL RELEVANCE: This combination agent has a significant effect on patients with subjective xerostomia but does not have a significant effect on objective hyposalivation.

Circular dichroism in biological photonic crystals and cubic chiral nets.

Nature provides impressive examples of chiral photonic crystals, with the notable example of the cubic so-called srs network (the label for the chiral degree-three network modeled on SrSi2) or gyroid structure realized in wing scales of several butterfly species. By a circular polarization analysis of the band structure of such networks, we demonstrate strong circular dichroism effects: The butterfly srs microstructure, of cubic I4(1)32 symmetry, shows significant circular dichroism for blue to ultraviolet light, that warrants a search for biological receptors sensitive to circular polarization. A derived synthetic structure based on four like-handed silicon srs nets exhibits a large circular polarization stop band of a width exceeding 30%. These findings offer design principles for chiral photonic devices.

Groundtruthing and potential for predicting acid deposition impacts in headwater streams using bedrock geology, GIS, angling, and stream chemistry.

Atmospheric acid deposition is of environmental concern worldwide, and the determination of impacts in remote areas can be problematic. Rainwater in central Pennsylvania, USA, has a mean pH of approximately 4.4. Bedrock varies dramatically in its ability to neutralize acidity. A GIS database simplified reconnaissance of non-carbonate bedrock streams in the Valley and Ridge Province and identified potentially chronically impacted headwater streams, which were sampled for chemistry and brook trout. Stream sites (n=26) that originate in and flow through the Tuscarora had a median pH of 5.0 that was significantly different from other formations. Shawangunk streams (n=6) and non-Tuscarora streams (n=20) had a median pH of 6.0 and 6.3, respectively. Mean alkalinity for non-Tuscarora streams (2.6 mg/L CaCO(3)) was higher than the mean for Tuscarora streams (0.5 mg/L). Lower pH and alkalinity suggest that the buffering capability of the Tuscarora is inferior to that of adjacent sandstones. Dissolved aluminum concentrations were much higher for Tuscarora streams (0.2 mg/L; approximately the lethal limit for brook trout) than for non-Tuscarora streams (0.03 mg/L) or Shawangunk streams (0.02 mg/L). Hook-and-line methods determined the presence/absence of brook trout in 47 stream reaches with suitable habitat. Brook trout were observed in 21 of 22 non-Tuscarora streams, all 6 Shawangunk streams, and only 9 of 28 Tuscarora stream sites. Carefully-designed hook-and-line sampling can determine the presence or absence of brook trout and help confirm biological impacts of acid deposition. 15% of 334 km of Tuscarora stream lengths are listed as "impaired" due to atmospheric deposition by the Pennsylvania Department of Environmental Protection. 65% of the 101 km of Tuscarora stream lengths examined in this study were impaired.

A novel TNFRSF1A splice mutation associated with increased nuclear factor kappaB (NF-kappaB) transcription factor activation in patients with tumour necrosis factor receptor associated periodic syndrome (TRAPS).

OBJECTIVE: To characterise and investigate the functional consequences of a novel TNFRSF1A splice site mutation causing tumour necrosis factor receptor associated periodic syndrome (TRAPS) in a 16-year-old male patient and his mother. METHODS: Mutational DNA screening was performed in the patient and his mother. Western blotting was used to analyse protein expression levels of TNFR1. A multiplex bead immunoassay was used to quantify serum levels of range of cytokines, and an ELISA-based transcription factor assay to measure nuclear factor (NF)-kappaB transactivation. Serum levels of soluble TNFR1 (sTNFR1) were measured by ELISA and fluorescence-activated cell sorting (FACS) analysis used to measure monocyte TNFR1 cell surface expression. RESULTS: A novel mutation, c.472+1G>A (C158delinsYERSSPEAKPSPHPRG), involving a splice site in intron 4 of TNFRSF1A, was found in the proband and affected mother leading to a 45 nucleotide insertion of intronic DNA into the mRNA, resulting in an in-frame insertion of 15 amino acids in the mature TNFR1 protein and a deletion of a cysteine residue C129 (158) in cysteine rich domain (CRD)3. The patients had reduced serum sTNFR1 and surface expression levels of TNFR1, with marked increases in pro- and anti-inflammatory cytokine. Their peripheral blood mononuclear cells (PBMC) had increased basal NF-kappaB activation compared with healthy controls and also had increased p50 nuclear expression following tumour necrosis factor (TNF) stimulation compared with PBMC from healthy controls, as well as T50M (T79M) and C88R (C117R) patients with TRAPS and patients with rheumatoid arthritis (RA). CONCLUSION: A novel, TRAPS causing, TNFRSF1A splice site mutation is associated with decreased sTNFR1 levels, cell surface and whole cell extract expression and increased NF-kappaB transcription factor activation.

Protein traffic from the secretory pathway to the endosomal system in pancreatic beta-cells.

Constitutive-like secretion involves vesicular trafficking corresponding kinetically and biochemically with a post-trans-Golgi network (TGN) origin. In pancreatic beta-cells, the budding of AP-1/clathrin-coated vesicles, a portion of which is derived from immature secretory granules, has been hypothesized to initiate constitutive-like trafficking. However, approximately 30 min after release of a 20 degrees C intracellular transport block in pancreatic beta-cells (to synchronize protein egress from the TGN), addition of brefeldin A (BFA) (which inhibits AP-1 recruitment) was reported not to block subsequent constitutive-like secretion. To further explore post-TGN trafficking in pancreatic beta-cell lines, we have followed the fate of pulse-labeled procathepsin B (ProB, a lysosomal proenyzme) after postpulse wortmannin treatment or the BFA treatment described above. We find that continuous wortmannin treatment allows ProB to reach immature secretory granules but inhibits its egress from maturing granules. Remarkably, BFA treatment causes augmented unstimulated secretion of newly synthesized ProB that is not paralleled by insulin. This effect requires a delay of 25-35 min after release from the 20 degrees C block. Further, when ProB delivery to endosomes is inhibited, its BFA-augmented secretion is eliminated. We hypothesize that the constitutive-like pathway involves an endosomal intermediate.

The effects of nicotine on learning and memory: a neuropsychological assessment in young and senescent Fischer 344 rats.

The effects of chronic nicotine on the behavioral performance of young (4 month) and old (24 month) Fischer-344 rats were assessed on four behavioral tasks: activity chamber. rotating rod, serial pattern learning, and Morris water maze paradigm. Old and young nicotine-treated rats received an intraperitoneal injection of nicotine (0.20 mg/kg) 15 min prior to all behavioral testing, and old and young saline-treated rats received saline injections 15 min prior to all behavioral testing. Nicotine improved motor coordination and increased the general activity levels of the old rats compared to old saline-treated rats. There were no significant differences in the behaviors of the young rats in these behavioral evaluations. In young rats, nicotine improved the acquisition of a serial pattern, suggesting an improvement in working memory or related processes. Nicotine was found to increase swim speed in a Morris water maze paradigm with a hidden platform; however, no beneficial effects of nicotine in reference memory were obtained for either age group. These results suggests that nicotine may not be as beneficial in attenuating age-related learning and memory deficits as once proposed.

A Rab GTPase is required for homotypic assembly of the endoplasmic reticulum.

To define the requirements for the homotypic fusion of mammalian endoplasmic reticulum (ER) membranes, we have developed a quantitative in vitro enzyme-linked immunosorbent assay. This assay measures the formation of IgG (H2L2) following the fusion of ER microsomes containing either the heavy or light chain subunits. Guanine nucleotide dissociation inhibitor (GDI), a protein that extracts Rab GTPases in the GDP-bound form from membranes, potently inhibits fusion. Inhibition was not observed using GDI mutants defective in Rab binding. Kinetic analysis of the inhibitory effects of GDI revealed that Rab activation is required immediately preceding or coincident with fusion and that this step is preceded by a priming event requiring a member of the AAA ATPase family. Our results suggest that homotypic fusion of ER membranes requires Rab and that Rab activation is a transient event necessary for the formation of a fusion pore leading to the mixing of luminal contents of ER microsomes.

A rabbit tooth-pulp assay to quantify efficacy and duration of antinociception by local anesthetics infiltrated into maxillary tissues.

The rabbit tooth-pulp assay is well established as a method for measuring the efficacy and potency of parenteral analgesic drugs. We describe a method for administration of local anesthetic drugs into the maxillary arch and subsequent measurements of antinociceptive action. It was possible to use two different methods of ED50 estimation and to provide measures of the potency, efficacy, and duration of local anesthetic drugs. These measurements corresponded with in vitro estimates of potency and duration and with intrinsic observations of the clinical actions.

Vectorial secretion by constitutive and regulated secretory pathways in mammary epithelial cells.

Lactating mammary epithelial cells synthesize large quantities of milk proteins, which they secrete vectorially at the apical membrane into the alveolar lumen of the gland. Recent work suggested that mammary protein secretion is not wholly constitutive, but may also occur in part through a regulated secretory pathway. This study used mouse mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix to compare the proportions of basally and apically-directed proteins secreted constitutively or in a regulated manner. On EHS matrix, mammary cells formed mammospheres, multicellular structures enshrouded in matrix material, within which they became polarised, formed tight intercellular junctions, and secreted milk proteins vectorially. Protein secreted basolaterally was collected in culture medium, whereas apically-secreted milk proteins accumulated in a closed lumen within the mammosphere, and were recovered by EGTA treatment of the cells in situ. Protein secretion was measured by following the release of radiolabelled protein after pulse-labelling with [35S]-methionine. Basolateral and apical secretion of [35S]-protein appeared complete within 1 h of pulse-labelling, consistent with immediate secretion through constitutive secretory pathways. However, addition of the calcium ionophore ionomycin induced a second wave of secretion in both directions. Ca(2+)-stimulated secretion occurred within 15 min of ionomycin addition, doubled the extent of basolateral and apical secretion, but did not change the populations of proteins secreted. Ionomycin treatment did not affect mammosphere morphology or mammary cell ultrastructure. The results suggest that lactating mammary epithelial cells secrete proteins apically and basolaterally by two pathways, one a Ca(2+)-independent constitutive pathway, the other a regulated pathway stimulated by elevation of intracellular Ca2+.

Fetal methylmercury study in a Peruvian fish-eating population.

Maternal consumption during pregnancy of methylmercury (MeHg)-contaminated fish in Japan and of MeHg-contaminated bread in Iraq caused psychomotor retardation in the offspring. Studies in Iraq suggested adverse fetal effects when maternal hair mercury concentrations were as low as 20 ppm. This prospective study involved 131 infant-mother pairs in Mancora, Peru with peak maternal hair MeHg levels during pregnancy from 1.2 ppm to 30.0 ppm, geometric mean 8.3. The MeHg was believed to be derived from marine fish in the diet. There was no increase in the frequency of neurodevelopmental abnormalities in early childhood. The possible role of selenium or other protective mechanisms in marine fish is discussed. This previously unpublished study was conducted between 1981 and 1984. Our report of August 1985 to the funding agencies has been circulated, and the data were presented at the Twelfth International Neurotoxicology Conference in Hot Spring, Arkansas, October 30 to November 2, 1994. The current account has not been modified or updated since 1985. For reference to interim publications on fetal MeHg studies in Iraq and New Zealand see Marsh et al., 1995.

The kinetics of intravenously administered methyl mercury in man.

We describe a compartmental pharmacokinetic model for methyl mercury and its metabolite mercuric mercury in humans. A tracer dose of 203Hg-labeled methyl mercury was administered iv to seven healthy young adult male volunteers. Blood samples were obtained periodically and urine and feces were collected throughout the 70 days of the study. The blood contained predominantly methyl mercury, while the excreta contained principally inorganic mercury. The behavior of both methyl mercury and inorganic mercury in the body was modeled with the simplest compartmental model which fit the data. This five-compartment model shows that inorganic mercury accumulates in the body and at longer times is the predominant form of mercury present. The biological half-life of methyl mercury in the body is 44 days and 1.6% of the body burden is lost each day by both metabolism and excretion. This rate of loss is 60% greater than that currently accepted (1.0% per day). Thus, the risk associated with dietary methyl mercury may have been overestimated.

Differential effect of brefeldin A on phosphorylation of the caseins in lactating mouse mammary epithelial cells.

The major milk proteins, the caseins, contain multiple phosphorylation sites. Phosphorylation of the caseins is necessary to allow Ca2+ binding and aggregation of the caseins to form micelles. We have followed the phosphorylation of the caseins in isolated acini from lactating mouse mammary gland. Incubation of mammary cells with [32P]orthophosphate revealed that phosphorylation of newly synthesised caseins was complete within 20 minutes of synthesis. Extensive secretion of alpha-, beta- and gamma-caseins occurred over a 2 hour period. Activation of the regulated secretory pathway using ionomycin over the last hour resulted in a preferential increase in secretion of alpha- and gamma-caseins. Brefeldin A (BFA) inhibited protein secretion and synthesis in mammary cells in prolonged incubations. An examination of short-term treatments with BFA on 32P incorporation into the caseins revealed a differential effect of BFA in which the drug inhibited phosphorylation of beta- and gamma- but not alpha-caseins. These results suggest that phosphorylation of alpha-casein normally occurs in Golgi cisternae whereas that of beta- and gamma-caseins occurs in the trans-Golgi network. Phosphorylation of specific secretory proteins may, therefore, occur in different Golgi compartments.

Inhibition of constitutive protein secretion from lactating mouse mammary epithelial cells by FIL (feedback inhibitor of lactation), a secreted milk protein.

The effect of a protein feedback inhibitor of lactation (FIL) on casein synthesis and secretion was examined using isolated acini from lactating mouse mammary gland. As previously found, FIL partially inhibited protein synthesis but produced an additional inhibition of constitutive casein secretion. The inhibition of synthesis and secretion showed similar dose-dependency and the inhibition was fully reversible. Constitutive secretion of pre-formed protein was inhibited by FIL in a pulse-chase protocol, indicating that the inhibitor regulated protein secretion by reducing protein movement through the secretory pathway independently of any initial inhibition of synthesis. Regulated exocytosis was not inhibited since casein release due to elevation of cytosolic Ca2+ concentration by the ionophore ionomycin was unaffected. Brefeldin A, which is known to block ER-to-Golgi transport, also inhibited both protein synthesis and secretion in mammary cells. The action of FIL on synthesis and secretion and previously described actions on casein degradation would be consistent with a block at an early stage in the secretory pathway. In support of this idea FIL treatment was found to result in vesiculation and swelling of the endoplasmic reticulum. These data provide evidence for a novel control of a constitutive secretory pathway by a physiological extracellular regulatory protein.

Exocytosis from permeabilized lactating mouse mammary epithelial cells. Stimulation by Ca2+ and phorbol ester, but inhibition of regulated exocytosis by guanosine 5'-[gamma-thio]triphosphate.

Lactating mouse mammary epithelial cells secrete large amounts of milk protein via constitutive or regulated exocytotic pathways. Secretion through both pathways was quantified by assaying the release of [35S]methionine-labelled trichloroacetic acid-precipitable proteins from digitonin-permeabilized secretory acini isolated from mammary glands of 10-day-post-partum lactating mice. Protein secretion from the isolated permeabilized cells was either Ca(2+)-dependent (regulated) or Ca(2+)-independent (constitutive). In both cases there was a requirement for ATP. Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) caused a marked increase in the percentage protein secretion from the cells in a Ca(2+)-independent manner. However, the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused a partial inhibition of Ca(2+)-dependent exocytosis, while having no significant effect on Ca(2+)-independent exocytosis. Thus the GTP[S] is exerting its effect on the regulated pathway at a site subsequent to protein sorting and packaging into secretory vesicles at the trans-Golgi network.

Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells.

Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.

Electrophoretic studies of human upper gastrointestinal mucosal acid proteinases.

The pattern of acid proteinase zymogens, seven pepsinogens (Pg) and slow moving protease (SMP), in normal human gastric mucosa has been reported. No significant differences were found in appearance of individual pepsinogens in oxyntic mucosa in the two sexes, but in pyloric mucosa, Pg 3 occurred significantly more often in men. Rapidly migrating pepsinogens (constituents of Group I pepsinogens) were seen in pyloric mucosa as well as in oxyntic mucosa. The duodenal mucosa contained small amounts of proteinases, the activity being largely confined to the slower moving proteinases (constituents of Group II pepsinogens).

Experimental small bowel transplantation utilizing cyclosporine.

Cyclosporine prolonged the survival of ectopic small bowel allografts in a canine model. A 9-fold increase in mean survival as compared with controls was obtained. Addition of prednisone to cyclosporine did not result in further graft prolongation, but improved the gross and histological appearance of the allograft. Monitoring the motility and histology of the allograft appears to be useful in predicting approaching rejection. Xylose absorption was not helpful in this regard. A new technique for measuring fat absorption in the transplant is described. Following allografting, fat absorption is resumed by 14 days posttransplantation.