Melatonin: Translation of Ongoing Studies Into Possible Therapeutic Applications Outside Sleep Disorders.

PURPOSE: Melatonin, a natural hormone mainly synthesized by the pineal gland, is regulated by circadian rhythm. Synthetic melatonin is not approved by the US Food and Drug Administration for any indication. However, melatonin receptor agonists such as ramelteon and tasimelteon are US Food and Drug Administration approved and are considered by the American Academy of Family Physicians for the treatment of insomnia. Due to the availability of over-the-counter products in some countries and the increasing use of melatonin, it is interesting to highlight knowledge regarding the potential benefits of melatonin outside sleep disorders. METHODS: This narrative review included published reports in EMBASE and MEDLINE databases between 1975 and 2021 relating to the therapeutic applications of melatonin. FINDINGS: Based on the quality of the evidence published to date, the most promising non-insomnia indications are for treating ischemia/reperfusion injury, primary headache disorders, fibromyalgia, glucose control, and blood pressure control. IMPLICATIONS: Most of the studies were preclinical and in in vivo and in vitro phases. More clinical trials are needed before recommending melatonin as a treatment in clinical practice.

Roadmap for metal nanoparticles in radiation therapy: current status, translational challenges, and future directions.

This roadmap outlines the potential roles of metallic nanoparticles (MNPs) in the field of radiation therapy. MNPs made up of a wide range of materials (from Titanium, Z = 22, to Bismuth, Z = 83) and a similarly wide spectrum of potential clinical applications, including diagnostic, therapeutic (radiation dose enhancers, hyperthermia inducers, drug delivery vehicles, vaccine adjuvants, photosensitizers, enhancers of immunotherapy) and theranostic (combining both diagnostic and therapeutic), are being fabricated and evaluated. This roadmap covers contributions from experts in these topics summarizing their view of the current status and challenges, as well as expected advancements in technology to address these challenges.

Photothermal therapy in a murine colon cancer model using near-infrared absorbing gold nanorods.

The photothermal ablation of solid tumors using exogenous, near-infrared (NIR)-absorbing nanoparticles has been previously investigated using various preclinical models and is currently being evaluated in the clinic. Here, we evaluate the circulation kinetics, preliminary toxicity, and efficacy of photothermal ablation of solid tumors using gold nanorods systemically delivered and passively accumulated in a murine subcutaneous colon cancer model. Tumored animals were infused with nanorods followed by the percutaneous illumination of the tumor with an 808-nm laser. Control groups consisted of laser-only, nanorod-only, and untreated tumored animals. The survival of the treated and control groups were monitored for 60 days post-treatment. The survival of the photothermally treated group was statistically longer than the control groups, with approximately 44% tumor free through the evaluation period. Histopathology of the major organs of animals infused with nanorods did not indicate any significant toxicity at 60 days post-treatment. Particle biodistribution was evaluated by elemental analysis of the major organs of untumored mice at 1, 7, and 30 days after infusion with nanorods. Elemental analysis indicates nanorod clearance from the blood and retention by the reticuloendothelial system. This study indicates that gold nanorods are promising agents for photothermal ablation of solid tumors.

Awareness and consumption of folate-fortified foods by women of childbearing age in Western Australia.

OBJECTIVES: The introduction of voluntary fortification of some foods with folic acid in Australia has been implemented since evidence of the prevention of neural tube defects with periconceptional folic acid was published. Our objectives were to determine how many women were aware of folate and when they became aware, what was the awareness of labels on foods that mentioned folate, and how much folate-fortified food women ate. METHODS: To address these objectives we collected data by self-administered questionnaire from a random sample of 578 recently pregnant women in Western Australia between September 1997 and March 2000. RESULTS: Overall, 89% of women had heard, seen or read anything about the link between folate and birth defects such as spina bifida, 62% first became aware of the folate message before their recent pregnancy and 42% of women noticed any labels on foods that mention folate before or during their recent pregnancy. Overall, 53% of women were aware of foods that have folate added to them and 33% usually or always read the labels on food packaging. The folate-fortified foods most often consumed by women were cereals (69%), breads (34%) and milk (15%). Of the women who consumed folate-fortified foods (78%), the earlier they became aware of the folate message and noticed labels on food, the more fortified foods they consumed. CONCLUSIONS: These results indicate that staple foods fortified with folate are consumed by almost 80% of women in the population. Therefore, mandatory fortification of staple foods may reach most women, providing improved opportunity for the prevention of neural tube defects in Australia.

Semicarbazone-based inhibitors of cathepsin K, are they prodrugs for aldehyde inhibitors?

Starting from potent aldehyde inhibitors with poor drug properties, derivatization to semicarbazones led to the identification of a series of semicarbazone-based cathepsin K inhibitors with greater solubility and better pharmacokinetic profiles than their parent aldehydes. Furthermore, a representative semicarbazone inhibitor attenuated bone resorption in an ex vivo rat calvarial bone resorption model. However, based on enzyme inhibition comparisons at neutral pH, semicarbazone hydrolysis rates, and 13C NMR experiments, these semicarbazones probably function as prodrugs of aldehydes.

Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles.

The following study examines the feasibility of nanoshell-assisted photo-thermal therapy (NAPT). This technique takes advantage of the strong near infrared (NIR) absorption of nanoshells, a new class of gold nanoparticles with tunable optical absorptivities that can undergo passive extravasation from the abnormal tumor vasculature due to their nanoscale size. Tumors were grown in immune-competent mice by subcutaneous injection of murine colon carcinoma cells (CT26.WT). Polyethylene glycol (PEG) coated nanoshells (approximately 130 nm diameter) with peak optical absorption in the NIR were intravenously injected and allowed to circulate for 6 h. Tumors were then illuminated with a diode laser (808 nm, 4 W/cm2, 3 min). All such treated tumors abated and treated mice appeared healthy and tumor free >90 days later. Control animals and additional sham-treatment animals (laser treatment without nanoshell injection) were euthanized when tumors grew to a predetermined size, which occurred 6-19 days post-treatment. This simple, non-invasive procedure shows great promise as a technique for selective photo-thermal tumor ablation.

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons.

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.

Influence of estrogen and osteopenia/osteoporosis on clinical periodontitis in postmenopausal women.

BACKGROUND: In Western societies, more than one-third of the female population above age 65 suffers from signs and symptoms of osteoporosis, a disorder characterized by low bone mass. Estrogen deficiency is the dominant pathogenic factor for osteoporosis in women. The impact of estrogen deficiency and osteopenia/osteoporosis on periodontitis is unclear, partially due to the lack of longitudinal studies evaluating clinical signs of gingival inflammation and periodontitis progression. The purpose of this investigation was to analyze prospectively the influence of serum estradiol levels and osteopenia/osteoporosis on common clinical measurements of periodontal disease over a 2-year period. METHODS: Fifty-nine moderate/advanced adult periodontitis patients and 16 non-periodontitis subjects, all within 5 years after menopause at baseline, completed the study. Serum estradiol levels (E2) were measured yearly by 125I radioimmunoassay, and osteopenia/osteoporosis was determined by dual energy x-ray absorptiometry of the lumbar spine. Posterior interproximal clinical measurements were obtained every 6 months for the periodontitis patients, including explorer-detectable supragingival plaque, bleeding on probing (BOP) and relative clinical attachment level (RCAL). Baseline probing depths, smoking history, and demographic data also were collected. RESULTS: Data indicated that baseline demographic measurements and bone mineral density (BMD) of the lumbar spine were not different between E2-deficient and E2-sufficient subjects. Smoking activity (packs smoked/day, years smoked) was higher in periodontitis patients (P=0.0001). E2-sufficient periodontitis subjects had a higher frequency of supragingival plaque without increasing gingival inflammation. E2 status did not influence the percentage of sites losing RCAL for either periodontitis or non-periodontitis groups, but when non-smoking osteopenic/osteoporotic periodontitis patients were evaluated, E2-deficient subjects had more BOP (43.8% versus 24.4%, P<0.04) and a trend toward a higher frequency of > or =2.0 mm RCAL loss (3.8% versus 1.2%, P<0.1) than E2-sufficient subjects. CONCLUSIONS: These data suggest that E2 supplementation (serum E2>40 pg/ml) is associated with reduced gingival inflammation and a reduced frequency of clinical attachment loss in osteopenic/osteoporotic women in early menopause.

Substrate binding mutants of the higher plant ADP-glucose pyrophosphorylase.

To explore the structure-function relationships of the heterotetrameric higher plant ADP-glucose pyrophosphorylase, composed of a pair of large and small subunits, the small subunit cDNA was subjected to chemical mutagenesis and then co-expressed with the wild-type large subunit cDNA. Mutants were selected for their inability to complement a defective bacterial ADP-glucose pyrophosphorylase gene and, in turn, to accumulate glycogen as viewed by iodine staining of the cells. Based on these initial analyses, we subsequently identified four distinct classes of mutations which were glycogen-deficient but exhibited enzyme activity levels comparable to the normal recombinant enzyme under saturating reaction conditions. Three classes, each a product of single amino acid substitution, showed altered kinetic constants for substrates. Substitution of Asp252 to Asn conferred the enzyme lower affinity for glucose-1-phosphate, replacement of Asp121 to Asn resulted in an enzyme less responsive to both glucose-1-phosphate and ATP, while the Ala106 to Thr substituted enzyme contains altered sensitivity primarily to ATP. The fourth class, a Pro43 to Ser substitution, resulted in an enzyme with decreased sensitivity (8-fold) to the activator 3-PGA. Overall, the results of this study suggests that the two subunit types do not have identical roles in enzyme function and that the small subunit plays a more dominant role in catalysis than the large subunit.

Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family.

We report the cloning, sequence analysis, tissue distribution, and functional expression of the K-Cl cotransport protein, KCC1. KCC1 was identified by searching the human expressed sequence tag data base, based on the expectation that it would be distantly related to the Na-K-Cl cotransporter. Rabbit KCC1 (rbKCC1) and rat KCC1 (rtKCC1) were cloned by screening rabbit kidney and rat brain cDNA libraries using homologous cDNA probes. Human KCC1 (hKCC1) was obtained from I.M.A.G.E. clones and in part by reverse transcription-polymerase chain reaction; it exhibits 97% identity with rbKCC1. KCC1 encodes a 1085-residue polypeptide with substantial sequence homology (24-25% identity) to the bumetanide-sensitive Na-K-Cl cotransporter (NKCC or BSC) and the thiazide-sensitive Na-Cl cotransporter (NCC or TSC). Hydropathy analysis of KCC1 indicates structural homology to NKCC, including 12 transmembrane domains, a large extracellular loop with potential N-linked glycosylation sites, and cytoplasmic N- and C-terminal regions. Northern blot analysis revealed a ubiquitously expressed 3. 8-kilobase transcript. Much of the genomic sequence of hKCC1 is in the data base, and the gene has been previously localized to 16q22.1 (Larsen, F., Solhein, J., Kristensen, T., Kolsto, A. B., and Prydz, H.(1993) Hum. Mol. Genet. 2, 1589-1595). Epitope-tagged rbKCC1 was stably expressed in human embryonic kidney (HEK 293) cells, resulting in production of a approximately150-kDa glycoprotein. The initial rate of 86Rb efflux from cells expressing rbKCC1 was more than 7 times greater than efflux from control cells and was inhibited by 2 mM furosemide; 86Rb efflux was stimulated by cell swelling. Uptake of 86Rb into rbKCC1 cells after a 15-min pretreatment with 1 mM N-ethylmaleimide was dependent on external chloride but not on external sodium, and was inhibited by furosemide with a Ki of approximately 40 microM and by bumetanide with a Ki of approximately 60 microM. These data demonstrate that the KCC1 cDNAs encode a widely expressed K-Cl cotransporter with the characteristics of the K-Cl transporter that has been characterized in red cells.

Effect of smokeless tobacco extract on human gingival keratinocyte levels of prostaglandin E2 and interleukin-1.

Gingival recession and white mucosal lesions frequently occur at sites of smokeless tobacco (ST) placement. The etiology of these alterations is presumably related to the irritating effects of tobacco components. The purpose of this study was to examine the effect of an aqueous ST extract (STE) on gingival keratinocyte production of prostaglandin E2 (PGE2) and interleukin-1 (IL-1), mediators involved in periodontal destruction and keratinocyte proliferation. Keratinocyte cultures were established from healthy tissues discarded from 8 subjects undergoing crown lengthening procedures. Cells (passage 2-3) were seeded at 2.5 x 10(4) cells/well into 48 well tissue culture plates and maintained in serum-free media at 37 degrees C. On day 4 or 5, the wells were divided into 4 groups receiving either 10%, 5%, 2.5%, or 0% STE for time periods ranging from 30 to 240 minutes. PGE2 levels (pg/10(4) cells), as measured by enzyme immunoassay, were significantly (P < 0.05) increased in the 10% (215.66 +/- 34.58) and 5% STE (151.82 +/- 27.97) treated cultures compared to untreated cells (46.16 +/- 9.58). IL-1 alpha and IL-1 beta proteins were elevated (P < 0.05) in cell lysates (299.45 +/- 38.69 and 28.45 +/- 5.18, respectively) from 5% STE exposed cultures compared to control wells. At 10% STE, secreted IL-1 alpha was decreased (P < 0.05) relative to 2.5% STE. This may reflect a toxic effect, as 10% STE significantly (P < 0.05) depressed cell numbers and viability. Lower tobacco concentrations did not affect cell numbers or viability, but significantly (P < 0.05) increased PGE2 and IL-1 levels. Tobacco-induced synthesis of these mediators may play a role in the development of tobacco-related oral disease.

Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.

A full-length cDNA encoding the murine renal Na-K-Cl cotransporter (NKCC2) was cloned using library screening and anchored polymerase chain reaction. The deduced protein sequence contained 1,095 amino acids and was 93.5% identical to rabbit NKCC2 and 97.6% identical to rat BSC1. Two potential sites of phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase and seven potential sites of phosphorylation by protein kinase C, which were previously identified in the rabbit and rat sequences, were phylogenetically conserved in the mouse. The expression of NKCC2 in the mouse was examined with Northern blot analysis and in situ hybridization. Expression of NKCC2 was kidney specific in both adult and embryonic mice. In the developing metanephros, NKCC2 was induced at 14.5 days post coitus and was expressed in distal limbs of immature loops of Henle but was absent from the ureteric bud, S-shaped bodies, and earlier nephrogenic structures. Similar to the rabbit, isoforms of NKCC2 that differed in the sequence of a 96-bp segment were identified in the mouse. In situ hybridization revealed that the isoforms exhibited different patterns of expression in the mature thick ascending limb of the loop of Henle as follows: isoform F was most highly expressed in the inner stripe of outer medulla, isoform A was most highly expressed in the outer stripe of the outer medulla, and isoform B was most highly expressed in the cortical thick ascending limb. To verify that the isoforms were generated by alternative splicing of mutually exclusive cassette exons, genomic clones encoding murine NKCC2 were characterized. Cassette exons were identified that corresponded to each of the three isoforms and were flanked by consensus splice donor and acceptor sequences.

Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon.

By moving chloride into epithelial cells, the Na-K-Cl cotransporter aids transcellular movement of chloride across both secretory and absorptive epithelia. Using cDNA probes from the recently identified elasmobranch secretory Na-K-Cl cotransporter (sNKCC1) (Xu, J. C., Lytle, C. Zhu, T. T., Payne, J. A., Benz, E., and Forbush, B., III (1994) Proc. Natl. Acad. Sci. 91, 2201-2205), we have identified the human homologue. By screening cDNA libraries of a human colonic carcinoma line, T84 cell, we identified a sequence of 4115 bases from overlapping clones. The deduced protein is 1212 amino acids in length, and analysis of the primary structure indicates 12 transmembrane segments. The primary structure is 74% identical to sNKCC1, 91% identical to a mouse Na-K-Cl cotransporter (mNKCC1), 58% identical to rabbit and rat renal Na-K-Cl cotransporters (NKCC2), and 43% identical to the thiazide-sensitive Na-Cl cotransporters from flounder urinary bladder and rat kidney. Similar to sNKCC1 and mNKCC1, the 5'-end of the human colonic cotransporter is rich in G + C content. Interestingly, a triple repeat (GCG)7 occurs within the 5'-coding region and contributes to a large alanine repeat (Ala15). Two sites for N-linked glycosylation are predicted on an extracellular loop between putative transmembrane segments 7 and 8. A single potential site for phosphorylation by protein kinase A is present in the predicted cytoplasmic C-terminal domain. Northern blot analysis revealed a 7.4-7.5-kilobase transcript in T84 cells and shark rectal gland and a approximately 7.2-kilobase transcript in mammalian colon, kidney, lung, and stomach. Metaphase spreads from lymphocytes were probed with biotin-labeled cDNA and avidin fluorescein (the cotransporter gene was localized to human chromosome 5 at position 5q23.3). Human embryonic kidney cells stably transfected with the full-length cDNA expressed a approximately 170-kDa protein recognized by anti-cotransporter antibodies. Following treatment with N-glycosidase F, the molecular mass of the expressed protein was similar to that predicted for the core protein from the cDNA sequence (132-kDa) and identical to that of deglycosylated T84 cotransporter (approximately 135-kDa). The stably transfected cells exhibited a approximately 15-fold greater bumetanide-sensitive 86Rb influx than control cells, and this flux required external sodium and chloride. Flux kinetics were consistent with an electroneutral cotransport of 1Na:1K:2Cl. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [Cl]-dependent regulatory mechanism.

Bioaccumulation of aromatic hydrocarbons from sediments: a dose-response study with flounder (Pseudopleuronectes americanus).

Pseudopleuronectes americanus were chronically exposed to Hibernia crude oil in sediments, for 4 months. Oil was added to sediments at five concentrations between 0.09 and 4.5 mg/g (dry weight) and was 0.10-0.90 mg/g, at the termination of the exposure. Bioaccumulation measured in terms of fluorescence or in terms of the concentration of specific aromatic targets, increased with dosage. Accumulation of hydrocarbons was observed in muscle tissue (0.22 microgram/g, dry weight), when concentration of the sum of 27 polycyclic aromatic compounds (PAC) in sediments was of 0.65 microgram/g (E-50), at the end of the 4-month period. Of the 27 parental and alkylated polycyclic aromatic compounds analyzed, alkylated naphthalenes predominated in muscle (90-100%) and in sediments (30-60%). Bioaccumulation factors were derived for 13 compounds detected in muscle, at the three higher exposures. Liver concentrations (fluorescence) were higher than in muscle, but did not display a noticeable dose-response. Several alkylbenzenes, a C-2 biphenyl and C-4 acenaphthene were also detected in muscle extracts. The development of dose-response relationships for polycyclic aromatic hydrocarbons (PAH) present in sediment, in relation to bioaccumulation in flatfish, is of major interest for evaluating the environmental effects of oil contamination.

Smokeless tobacco effects on monocyte secretion of PGE2 and IL-1 beta.

The use of smokeless tobacco (ST) products is associated with mucosal lesions, gingival recession, and attachment loss at the site of tobacco placement. Monocytes/macrophages are primary producers of PGE2 and IL-1 beta, inflammatory mediators which are thought to play a role in the destruction of the periodontium. The purpose of this study was to determine the effect of ST alone and in combination with a major stimulator of inflammation, bacterial lipopolysaccharide (LPS), on monocyte secretion of these mediators. Peripheral blood monocytes (PBM) were isolated by counterflow centrifugal elutriation from 15 healthy donors who were non-ST users. PBM were incubated for 24 hours in RPMI 1640 containing various concentrations of ST (0%, 0.005%, 0.01%, 1%) with or without 10 micrograms/ml LPS (Porphyromonas gingivalis LPS or Escherichia coli LPS). Of the ST preparations, only 1% ST resulted in PBM mediator secretion (7.7 +/- 2.0 ng/ml for PGE2 and 1.3 +/- 0.2 ng/ml for IL-1 beta) above that of control (unstimulated) cultures. Furthermore, the combination of 1% ST and LPS resulted in a potentiation of PGE2 release (5-fold for E. coli LPS + 1% ST and 10-fold for P. gingivalis LPS + 1% ST; P < 0.0001, one-way ANOVA) relative to the LPS preparations alone. In contrast, PBM IL-1 beta release decreased more than 2-fold upon E. coli LPS and 1% ST exposure, relative to treatment with E. coli LPS alone (P < 0.0001, one-way ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)