Efficacy of Melaleuca alternifolia and chlorhexidine mouth rinses in reducing oral malodor and Solobacterium moorei levels. A 1 week, randomized, double-blind, parallel study.

OBJECTIVES: The objective is to evaluate the association of Solobacterium moorei (S. moorei) to halitosis and to also check for the effects of two different mouth rinses on levels of S. moorei in saliva and tongue coating and its impact on oral halitosis. MATERIALS AND METHODS: This was a placebo-controlled parallel study of 160 individuals who were randomized and the study was performed using double-blinded protocol. Enrolled individuals filled a structured questionnaire regarding demographic data, oral hygiene habits, and dietary habits. Full mouth organoleptic odor scores (OLR), volatile sulfur compounds levels, Miyazaki's tongue coating index, and Plaque scores were recorded before intervention (baseline) and after 1-week post treatment. Microbiological samples obtained from the tongue and saliva was investigated for S. moorei levels using real time polymerase chain reaction. Participants were randomly assigned for two test mouth rinses (Melaleuca alternifolia and Chlorhexidine) and placebo groups. RESULTS: All salivary and tongue coating samples were tested positive for S. moorei in the halitosis group. One week post-treatment S. moorei counts in saliva and tongue coating samples of test group showed a significant reduction at P < 0.001. Paired t-test results showed that Melaleuca alternifolia was comparable with chlorhexidine in reduction of OLR, and VSC scores (P < 0.001). Salivary levels of S. moorei in Melaleuca alternifolia group showed a higher reduction (5.67 log10 copies/mL) than chlorhexidine group (5.1log10 copies/mL). CONCLUSION: S. moorei showed a positive correlation with oral halitosis scores. Both Melaleuca alternifolia and chlorhexidine were equally effective in reducing S. moorei levels and halitosis score.

Microbial contamination and plaque scores of nanogold-coated toothbrush.

OBJECTIVE: The study aimed to evaluate the microbial contamination and plaque scores of nanogold-coated and uncoated toothbrushes. METHODS: This study was designed as a single-centre, parallel, examiner-blinded, randomized, two-group clinical trial. Eighty-four participants were enrolled and randomly assigned to receive either a nanogold or uncoated toothbrush. Basic periodontal therapy was performed for all the recruited subjects, and plaque scores of zero were considered baseline values. All participants were instructed to follow a twice-daily brushing regimen without dentifrice and to refrain from other oral hygiene care during the one-week study period. Plaque levels were assessed after one week using the Turesky modification of the Quigley-Hein Plaque Index (TMQHPI). The bristles were tested for microbial contamination by viable cell counting. The recorded data were statistically analysed, and a P-value of <.05 was accepted as statistically significant. RESULTS: After one week of brushing without using toothpaste, the mean plaque index scores were 0.37 ± 0.07 in the nanogold group and 0.58 ± 0.10 in the uncoated group. A significant difference in the mean plaque scores was observed between the groups (P < .001). The mean colony-forming unit (CFU) was 21 ± 48.8 for the nanogold-coated group and 100 ± 128.4 for the uncoated group. The difference in the mean CFUs observed between the groups was significant (P = .014). CONCLUSION: The use of a nanogold-coated toothbrush demonstrated significantly lower bristle contamination and lower plaque scores after one week compared with uncoated toothbrushes without using dentifrice.

28-Homobrassinolide: a novel oxysterol transactivating LXR gene expression.

Cholesterol is the template for steroid hormone biosynthesis. Cholesterol homeostasis is regulated by Cyt-P450 oxygenated cholesterols acting as ligands on LXR-α and LXR-ß transcription factors that are now emerging as drug targets. Heterodimerization of LXRs with retinoic acid receptor is considered a prerequisite for target gene activation. Dietary plant oxysterol 28-homobrassinolide (28-HB) is a proven antihyperglycemic and a pro-steroidogenic agent in the rat. Whether 28-HB has a role in LXR gene expression was therefore investigated using oral gavage (15 days) of 28-HB (333 µg/kg b w) to normal and diabetic rat. PCR amplified LXR-α and ß mRNA transcripts from treated rat liver and testis exhibited quantitative differences in their expression. Conformational differences in 28-HB docking to LXR-α and ß binding domains were also noted through in silico studies, LXR-ß adopting lesser specificity. We report that 28-HB transactivates LXR genes in the rat tissues.

Gibberellic acid acts as an agonist of steroidogenesis in male rats.

Testicular steroidogenesis has significant implication in male reproductive function. Although the effects of various signalling molecules on testicular functions have been studied earlier, the influence of the plant hormone gibberellic acid (GA3 ) on steroidogenesis has not been investigated. Acute (4 h) and subacute (15 days) studies using this compound through oral administration (150 µg day(-1) ) to groups of normal and diabetic Wistar male rats were therefore carried out. Results indicate that (i) enhanced activity of steroidogenic markers 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), elevated tissue testosterone (T) content, increased steroidogenic acute regulatory protein (StAR) and androgen binding protein (ABP) levels with reduced lipid peroxidation and improved antioxidant defence in this treatment group of normal and diabetic rat testis, and (ii) elevated lipid peroxidation and diminished antioxidant defence, with insignificant change in 3ß-HSD and 17ß-HSD activity and testosterone level in acute treatment group of normal and diabetic rats testis, were noted. The observed increase in the activity of testicular 3ß-HSD and 17ß-HSD along with elevated testosterone content established GA3 as an inducer of steroidogenesis in rat.

A phytooxysterol, 28-homobrassinolide modulates rat testicular steroidogenesis in normal and diabetic rats.

Steroidogenesis in testicular cells depends upon the availability of cholesterol within testicular mitochondria besides the activities of 3ß-hydroxysteroid dehydrogenase (3ß-HSD, 17ß-hydroxysteroid dehydrogenase [17b-HSD]), and the tissue levels of steroidogenic acute regulatory protein (StAR), androgen-binding protein (ABP), and testosterone (T). Cellular cholesterol biosynthesis is regulated by endogenous oxycholesterols acting through nuclear hormone receptors. Plant oxysterols, such as 28-homobrassinolide (28-HB), available to human through diet, was shown to exhibit antihyperglycemic effect in diabetic male rat. Its role in rat testicular steroidogenesis and lipid peroxidation (LPO) was therefore assessed using normal and streptozotocin-induced diabetic male rats. Administration of 28-HB (333 µg/kg body weight) by oral gavage for 15 consecutive days to experimental rats diminished LPO, increased antioxidant enzyme, 3ß-HSD and 17ß-HSD activities, and elevated StAR and ABP expression and T level in rat testis. We report that 28-HB induced steroidogenesis in normal and diabetic rat testis.

DNA barcoding and development of species-specific markers for the identification of tea mosquito bugs (Miridae: Heteroptera) in India.

Rapid, accurate, and timely identification of insects as a group is important and challenging worldwide, as they outnumber all other animals in number and diversity. DNA barcoding is a method for the identification of species in a wide range of animal taxa, which uses the 5' region of the mitochondrial cytochrome c oxidase-I (CO-I). Yet another easy, accurate, and economical method of species discrimination is by developing species-specific markers, which produce specific amplicon for the species in question. The method is handy because it is not limited by life stages, sex, polymorphism, and other factors. Herein, we measured the usefulness of CO-I for the species discrimination of mirids in India viz. Helopeltis antonii Signoret, H. thievora Waterhouse, H. bradyi Waterhouse, and Pachypeltis maesarum Kirkaldy in their various life stages. Furthermore, our study showed the utility of species-specific markers in differentiating H. antonii (295) and H. bradyi (514) regardless of their life stages. Analysis of CO-I gene revealed <1% intraspecific divergence for all four species examined, whereas the interspecific distances ranged from 7 to 13%. This study showed that the DNA barcode and species-specific markers will aid the identification of mirids in India and will stand as a decisive tool in formulating integrated pest management (IPM) strategy, quick identification of invasive and cryptic species, haplotypes, biotypes, and other factors, if any.

Enhanced expression of hexokinase I mRNA in male rat tissues by homobrassinolide.

The subchronic effect of the plant hormone homobrassinolide, a dietary constituent of vegetables and green leaves, was investigated in male albino Wistar strain rats. Blood sugar and serum insulin content, tissue hexokinase enzyme activity, and mRNA expression were studied using homobrassinolide administered orally by gavage at 50 µg (333 µg/kg body weight) for 15 consecutive days. Selected tissue responses were determined at 16 days post administration in control and experimental animals employing established methods. Homobrassinolide reduced the circulating blood sugar and increased the serum insulin level significantly. Hexokinase activity in the brain, heart, liver, kidney, and testis of experimental rats was found elevated. Hexokinase mRNA expression detected employing polymerase chain reaction (PCR) technique was found significantly increased in the brain and liver compared to other tissues. It is suggested that this plant hormone is a transcriptional activator of hexokinase gene, promoting enhanced hexokinase mRNA synthesis in vivo in rat tissues.

Induction of hexokinase I expression in normal and diabetic rats by a brassinosteroid isoform.

This study aimed to investigate the effect of the plant growth regulator 28-homobrassinolide (HB) on the hexokinase I (HK I) enzyme gene expression in the tissues of normal and streptozotocin-induced diabetic rats. Normal and diabetic rats were administered 50 microg of HB for 15 consecutive days. The tissues level of HK I mRNA expression was quantitated by PCR and densitometry analysis, HK I protein expression was quantitated by Western blot and densitometry analysis, localization of HK I was done by immunohistochemistry and HK enzyme activity was determined by coupled enzyme assay. Subchronic treatment of rats with HB enhanced HK I enzyme expression in diabetic rat compared to the control rat. From these experimental evidences, we came to the conclusion that HK I played a vital role in the regulation of blood sugar in streptozotocin-induced diabetic rats. A direct role for hexokinase enzyme activity in the control of diabetes is presented.

Alterations in beta-islets of Langerhans in alloxan-induced diabetic rats by marine Spirulina platensis.

Marine Spirulina platensis may potentially influence the metabolic process in animal cells, and the effect of marine Spirulina platensis in normal and alloxan-induced diabetic rats was therefore investigated. Normal and diabetic rats (albino Wistar strain) were orally administered marine Spirulina platensis for 30 days and their blood levels of glucose and insulin and body weight changes were determined. Pancreatic histopathology was also noted. Treatment with marine Spirulina platensis caused significant alterations in the content of these indicators and therefore in the antidiabetic capacity of the treated animals compared to control rats.

A comparative study on the effect of homobrassinolide and gibberellic acid on lipid peroxidation and antioxidant status in normal and diabetic rats.

Dietary content of phytohormones may potentially influence metabolic processes in animal cells. This study therefore aimed to investigate the effect of two plant growth regulators homobrassinolide (HB) and gibberellic acid (GBA) on the antioxidant defense status and lipid peroxidation level in the tissues of normal and streptozotocin- induced diabetic rats. Normal and diabetic rats (Albino -wistar strain) were administered 50 microg HB and GBA intradermally each day for seven days and their tissue and blood levels of malondialdehyde (MDA), 4-hydroxy-2-nonenol (4-HNE), reduced glutathione (GSH) content and catalase (CAT) activity were determined. Subchronic treatment of rats with HB reduced lipid perioxidation and elevated antioxidant defense whereas GBA caused enhancement of lipid peroxidation and reduction of antioxidant defense in treated animals compared to the control rats.

Toxic effects of carbendazim at low dose levels in male rats.

Carbendazim is a systemic broad-spectrum fungicide controlling a wide range of pathogens. It is also used as a preservative in paint, papermaking and leather industry, and as a preservative of fruits. In the present study, low dose intracellular effect of carbendazim was investigated employing 5, 10, 25 and 50 mM of the compound administered to male rats intradermally. Blood and liver of each animal was collected 6 hrs later to analyze serum and tissue enzyme activities, tissue lipid peroxidation and hematological and biochemical parameters. The experimental results of low dosage carbendazim use indicated augmentation of investigated parameters. However, the higher dosage of carbendazim use resulted in renormalization of investigated parameters to control levels or to values below control, providing a U-shaped hormesis type dose-response profile. Histopathological sections revealed portal vein congestion, mononuclear cell infiltration and hydropic degeneration of the liver tissue. These results indicated that carbendazim even at low dose exhibited toxicity, affected the liver and also caused specific changes in hematological and biochemical parameters in the rat.

Metabolic dysregulation and inhibition of spermatogenesis by gibberellic acid in rat testicular cells.

Enzymatic and histological change in the testicular cells of rats treated orally and intradermally for 45 days with gibberellic acid (GBA) in independent studies is reported. Assay of hexokinase (HK), acid phosphatase (AcP) and alkaline phosphatase (AkP) in rat testicular tissue homogenate preparations yielded results that suggested changes in these enzyme activities relative to their respective controls. Histological studies showed loss of germ cells, derangement of the germinal cells, and reduction in the size of the seminiferous tubules and dystrophy of Leydig cells. More importantly decreased sperm count in the lumen was observed. A dysregulatory role is thus established for GBA in rat testicular cell function. This compound may serve as an inhibitor of testicular cell function.

Effect of gastrointestinal proteases on purified human intrinsic factor-vitamin B12 (IF-B12) complex.

Intrinsic factor (IF) from human gastric juice was purified and complexed with vitamin B12 (IF-B12 complex) on Sepharose-vitamin B12 affinity matrix. By labeling studies, using [(57)Co] vitamin B12 and (125)I, the specific B12 binding activity of IF was found to be 23 microg B12/mg protein, and the molecular size by gel filtration 60 kDa. Proteolysis of the IF-B12 complex by sequential treatment with pepsin, trypsin, alpha-chymotrypsin and carboxypeptidase A, followed by chromatography of proteolysed complex and IF-B12 showed higher mobility of proteolysed fraction. Gel filtration, however, showed same molecular size for both proteolysed and the IF-B12 complex. On SDS-PAGE, purified IF-B12 appeared as a single band of 60 kDa. The proteolysed complex had higher mobility on SDS-PAGE and did not bind to zirconium phosphate gel. Immunodiffusion with rabbit antisera had positive reaction with IF-B12, but there was no reaction with the proteolysed sample.

Long-term marrow cultures: human and murine systems.

The intramedullary control of marrow cell production has been a difficult area to approach experimentally. The introduction by Dr. Dexter and colleagues of long-term stromal dependent culture systems for murine marrow and the adaptation of these systems to human marrow growth have allowed for in-vitro studies of stromal dependent hemopoiesis. Despite some controversy in this area, most studies appear to show that adherent murine or human stromal cells are capable of producing a relatively large number of hemopoietic growth factors including G-CSF, GM-CSF, CSF-1, IL-6 and, at least by PCR analysis, IL-3. Other work indicates that the most primitive hemopoietic cells which appear to be multifactor responsive adhere directly to these stromal cells presumably through mediation of various adherence proteins. An early acting, multilineage factor termed hemolymphopoietic growth factor-1 (HLGF-1) has been isolated from a murine stromal cell line and may be identical to the recently described ligand for the c-kit receptor. This may represent an important early survival/maintenance factor for stem cells in this system. Studies on primitive stem cells, especially the high proliferative potential colony forming cell (HPP-CFC), indicate that they are responsive to varying combinations of growth factors and that with increasing numbers of growth factors, as studied in serum-free systems, decreasing concentrations of the factors may be biologically active. These observations altogether suggest that intramedullary hemopoiesis may be regulated by the positioning of early multifactor responsive stem cells via adherent proteins in juxtaposition to synergistically acting combinations of growth factors attached to stromal cell surfaces or the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Colony-stimulating factor-1 modulates alpha 2-macroglobulin receptor expression in murine bone marrow macrophages.

Primary cultures of murine bone marrow macrophages (BMMs) were prepared from marrow cell suspensions. These cells expressed specific receptors that recognized the transformed conformation of human alpha 2-macroglobulin (alpha 2M) generated by reaction with CH3NH2. alpha 2M receptor expression was regulated by colony-stimulating factor-1 (CSF-1). The BMMs were deprived of CSF-1 for 6 h and then treated with different concentrations of the purified cytokine. After 18 h, binding of 125I-alpha 2M-CH3NH2 was examined at 4 degrees C. Analysis of the saturation isotherms and Scatchard transformations indicated that the KD was not affected by CSF-1 (1.9-2.4 nM), whereas the maximum specific radioligand binding capacity (Bmax) was increased from 5.6 x 10(4) receptors/cell in the absence of CSF-1 to 2.2 x 10(5) and 2.6 x 10(5) receptors/cell for BMMs treated with 1,000 and 10,000 units/ml CSF-1, respectively. The difference in total cellular protein after exposure to different levels of CSF-1 for 18 h was small (1.50-1.92 ng/cell) and not statistically significant. A 6-12-h lag phase was identified between the time of CSF-1 exposure and increased alpha 2M receptor expression. Cycloheximide completely blocked the increase in alpha 2M receptor expression when added simultaneously with the CSF-1; greater than 50% inhibition was observed when the cycloheximide was added up to 8 h later. The RNA synthesis inhibitors, actinomycin D and daunomycin, prevented increased alpha 2M receptor expression when added up to 4 h after the CSF-1, but had no effect at 8 h. At 37 degrees C, uptake and digestion of 125I-alpha 2M-CH3NH2 was increased in BMMs treated with 1,000 units/ml CSF-1 for 18 h compared with untreated cells. These studies demonstrate that CSF-1 increases the expression of alpha 2M receptors in BMMs through a pathway that requires new RNA and protein synthesis. We hypothesize that increased alpha 2M receptor expression may play an important role in cellular growth and differentiation.

The role of selenium-dependent and selenium-independent glutathione peroxidases in the formation of prostaglandin F2 alpha.

In recent years, growing evidence suggests that glutathione peroxidases (GSH-Pxs), both selenium-dependent GSH-Px (Se-GSH-Px) and selenium-independent GSH-Px (non-Se-GSH-Px) play an important role in the biosynthesis of prostaglandins and leukotrienes and in the regulation of key enzymes associated with the arachidonic acid cascade. The precise nature of their involvement in eicosanoid metabolism, however, is not yet completely understood. In the study reported here, we have systematically determined the catalytic efficiencies of Se-GSH-Px and non-Se-GSH-Px toward prostaglandin (PG) G2 (PGG2) and PGH2. Se-GSH-Px exhibited high catalytic activity for the reduction of PGG2 as indicated by Km and Vmax values of 12 microM and 78 mumol/min/mg, respectively, whereas PGH2 was found to be a poor substrate, an indication that Se-GSH-Px reduces the hydroperoxide moiety but not the endoperoxide moiety of PGG2. The kinetic constants of Se-GSH-Px toward PGG2 were comparable to those determined for such classical substrates as H2O2 and cumene hydroperoxide. In contrast to Se-GSH-Px, non-Se-GSH-Px associated with cationic isozyme II of glutathione S-transferases (GSTs) from sheep lung cytosol was very active in the conversion of PGH2 to PGF2 alpha with a Vmax of 960 nmol/min/mg and a Km of 77 microM. This study shows that PGF2 alpha formation by non-Se-GSH-Px occurred in a GSH-dependent reduction of either PGG2 or PGH2. When PGG2 was used as the substrate for non-Se-GSH-Px, a novel intermediate compound appeared and was later identified by several methods of structural analysis as 15-hydroperoxy PGF2 alpha. Thus, the reductive cleavage of the endoperoxide occurs faster than the 15-hydroperoxide reduction allowing 15-hydroperoxy PGF2 alpha to accumulate briefly. A study of GSTs from several different tissues and species indicated that the transformation of PG endoperoxides to PGF2 alpha is catalyzed specifically by GST isozymes, which contain Ya size subunits. This specificity of GST isozymes in PG biosynthesis, coupled with their tissue-specific expression, may be a mechanism by which the body modulates the type of PGs produced in these tissues. Also, these results suggest a possible interaction of Se-GSH-Px and non-Se-GSH-Px in the biosynthesis of PGF2 alpha.

Aggregation and thermo-inactivation of glutamine synthetase from an extreme thermophile, Bacillus caldolyticus.

The extreme thermophile, Bacillus caldolyticus, contains two regulatory isoforms of glutamine synthetase (glutamate-ammonia ligase, EC 6.3.1.2), E-I and E-II, produced as separate gene products. Light scattering and electron microscopy data indicate that these thermophilic enzymes aggregate to higher molecular weight species in two stages: initial polymerization of native dodecamers, followed by 'melting' of the aggregated species to produce amorphous denatured protein. The initial stages of the aggregation occurred at temperatures below those for time-dependent denaturation, especially for E-II. In contrast, mesophilic (B. subtilis) enzyme showed no evidence of temperature-dependent aggregation. Thus, aggregation may be a stabilizing mechanism for the thermophilic systems. Bound metal ions and substrates caused dramatic increases in the temperatures at which aggregation and loss of activity occurred for thermophilic enzymes. Certain combinations of ligands (e.g., MnATP + L-glutamate) acted synergistically, so that these complexes denatured only above 90 degrees C. Various models were considered for heat-driven aggregation followed by denaturation, plus ligand stabilization. Taken together, the data are most consistent with unfolding of subunits within the dodecameric unit, rather than unfolding to monomers prior to aggregation.

Immunological and sequence interrelationships between multiple human liver and rat glutathione S-transferases.

The 13 forms of human liver glutathione S-transferases (GST) (Vander Jagt, D. L., Hunsaker, L. A., Garcia, K. B., and Royer, R. E. (1985) J. Biol. Chem. 260, 11603-11610) are composed of subunits in two electrophoretic mobility groups: Mr = 26,000 (Ha) and Mr = 27,500 (Hb). Preparations purified from the S-hexyl GSH-linked Sepharose 4B affinity column revealed three additional peptides at Mr = 30,800, Mr = 31,200, and Mr = 32,200. Immunoprecipitation of human liver poly(A) RNAs in vitro translation products revealed three classes of GST subunits and related peptides at Mr = 26,000, Mr = 27,500, and Mr = 31,000. The Mr = 26,000 species (Ha) can be precipitated with antisera against a variety of rat liver GSTs containing Ya, Yb, and Yc subunits, whereas the Mr = 27,500 species (Hb) can be immunoprecipitated most efficiently by antiserum against the anionic isozymes as well as a second Yb-containing isozyme (peak V) from the rat liver. The Mr = 31,000 band can be immunoprecipitated by antisera preparations against sheep liver, rat liver, and rat testis isozymes. Human liver GSTs do not have any subunits of the rat liver Yc mobility. Antiserum against the human liver GSTs did not cross-react with the Yc subunits of rat livers or brains in immunoblotting experiments. The human liver GST cDNA clone, pGTH1, selected human liver poly(A) RNAs for the Ha subunit(s) in the hybrid-selected in vitro translation experiments. Southern blot hybridization results revealed cross-hybridization of pGTH1 with the Ya, Yb, and Yc subunit cDNA clones of rat liver GSTs. This sequence homology was substantiated further in that immobilized pGTH1 DNA selected rat liver poly(A) RNAs for the Ya, Yb, and Yc subunits with different efficiency as assayed by in vitro translation and immunoprecipitation. Therefore, we have demonstrated convincingly that sequence homology as well as immunological cross-reactivity exist between GST subunits from several rat tissues and the human liver. Also, the multiple forms of human liver GSTs are most likely encoded by a minimum of three different classes of mRNAs. These results suggest a genetic basis for the subunit heterogeneity of human liver GSTs.