Crosstalk of AsA/DHA and GSH/GSSG ratios' role in growth-phase dependent antioxidative defense in euryhaline and freshwater microalgae: explored for the first time.

The cooperative role of vital components of the antioxidative defense pathway in addition to redox couples was studied in a growth-phase dependent manner at 20, 30, and 40 days after subculturing (DAS) in five different euryhaline microalgal strains (EMS) Scenedesmus MKB (B-S), Spirulina subsalsa (B-6), Anabaena sp. (B-7), Chlorella sp. (B-8), and Chlorosarcinopsis eremi (B-18) collected from waterlogged areas of Punjab, India and in two freshwater microalgal strains (FMS). EMS surpasses to maintain a high redox couple's ratio ascorbic acid/dehydroascorbate (AsA/DHA), and reduced glutathione/oxidized glutathione (GSH/GSSG) through a close-knit pattern of antioxidative enzymes including high specific activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and less specific activity of glutathione peroxidase (GPX). While FMS struggled for the same irrespective of near similar total glutathione and higher specific activity of GPX might be answerable for the lesser redox ratio than EMS. However, high specific activity of catalase (CAT) might be sought to compensate for the less increase of APX in FMS. The fact significantly less H2O2, and malondialdehyde (MDA) with DAS in EMS than in FMS and higher redox ratios exquisitely elevate their tolerance ability making EMS a captivating prospect for cultivation in waterlogged areas. Additionally, their abundance of potent antioxidants further highlights the potential of EMS as an excellent source of these beneficial compounds.

Does daily energy and macronutrient intake differ between work and non-workdays in shift workers? A mixed methods study.

Shift workers are at increased risk of obesity and metabolic diseases, but their eating patterns on work and non-workdays are understudied. We aimed to examine whether energy intake and macronutrient intake of day and night shift nurses were different during work and non-workdays. We used a mixed-methods approach to study food intake of shift working nurses from two hospitals during day and night shifts. Participants completed baseline questionnaires about eating behaviour, sleep, chronotype, mood and shift work disorder. Participants then completed a 4-d food diary which included a non-workday prior to the first shift, the first and last shift (either day or night) and the following non-workday. After completion of the food diaries, we used semi-structured interviews to explore the qualitative aspects of eating behaviours. Seventy-nine shift-working nurses participated in the study. Daily energy intake was not significantly different on work and non-workdays in day or night shift workers (p > 0.05). Whilst macronutrient consumption was also not different between day and night shift workers (p > 0.05), sugar intake was higher in day compared to night shift workers (p = 0.02) on the non-workday prior to the first workday. In qualitative interviews, participants reported their eating to be different on day and night shifts as well as work and non-workdays. Eating behaviour in day and night shift workers was highly influenced by food availability, convenience, peers, and family members. Nurses qualitatively report that night and day shifts result in them eating differently despite no statistically discernible difference in energy intake.

Exploring the Potential of Bee-Derived Antioxidants for Maintaining Oral Hygiene and Dental Health: A Comprehensive Review.

Honey bee products comprise various compounds, including honey, propolis, royal jelly, bee pollen, bee wax and bee venom, which have long been recognized for their pharmacological and health-promoting benefits. Scientists have discovered that periodontal disorders stem from dental biofilm, an inflammatory response to bacterial overgrowth produced by dysbiosis in the oral microbiome. The bee products have been investigated for their role in prevention of oral diseases, which are attributed to a myriad of biologically active compounds including flavonoids (pinocembrin, catechin, caffeic acid phenethyl ester (CAPE) and galangin), phenolic acids (hydroxybenzoic acid, hydroxycinnamic acid, p-coumaric, ellagic, caffeic and ferulic acids) and terpenoids. This review aims to update the current understanding of role of selected bee products, namely, honey, propolis and royal jelly, in preventing oral diseases as well as their potential biological activities and mechanism of action in relation to oral health have been discussed. Furthermore, the safety of incorporation of bee products is also critically discussed. To summarize, bee products could potentially serve as a therapy option for people suffering from a variety of oral disorders.

Genotypic Spectrum and its Correlation with Alopecia and Clinical Response in Hereditary Vitamin D Resistant Rickets: Our Experience and Systematic Review.

Alopecia in hereditary vitamin D resistant rickets (HVDRR) has some correlation with severe rickets and poor overall response. However, these observations are based on small series. Hence, we aim to assess the genotypic spectrum of HVDRR and its correlation with alopecia and clinical response. Seven genetically-proven HVDDR patients from five unrelated families and 119 probands from systematic review were analysed retrospectively for phenotypic and genotypic data and overall response to therapy. In our cohort mean age at rickets onset was 12 (± 3.4) months. Alopecia was present in all patients but one. All patients had poor overall response to oral high-dose calcium and calcitriol and most required intravenous calcium. Genetic analyses revealed four novel variants. On systematic review, alopecia was present in majority (81.5%) and preceded the onset of rickets. Patients with alopecia had higher serum calcium (7.6 vs.6.9 mg/dl, p = 0.008), lower 1, 25(OH)2 D (200 vs.320 pg/ml, p = 0.03) and similar overall response to oral therapy (28.7% vs. 35.3%, p = 0.56). Alopecia was present in 51.4% of non-truncating (NT) ligand-binding domain (LBD) variants, whereas it was universal in truncating LBD and all DNA binding-domain (DBD) variants. Overall response to oral therapy was highest in LBD-NT (46.4%) as compared to 7.6% in LBD-truncating and 19% in DBD-NT variants. Among LBD-NT variants, those affecting RXR heterodimerization, but not those affecting ligand affinity, were associated with alopecia. Both alopecia and overall response have genotypic correlation. Age at diagnosis and overall response to oral therapy were similar between patients with and without alopecia in genetically proven HVDRR.

Variation of Mineral Composition in Different Fruit Parts of Bitter Gourd (Momordica charantia L.).

Bitter gourd (Momordica charantia L.), belonging to family Cucurbitaceae, is a good source of carbohydrates, proteins, vitamins, minerals, and bioactive compounds. In the present study, fruits (and its parts-epicarp, mesocarp, endocarp, seed, and whole fruit) of 56 accessions and 4 cultivars of Momordica spp. were assayed and compared for macro-minerals magnesium (Mg), potassium (K), sodium (Na), phosphorus (P), and calcium (Ca), and microminerals iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu). Potassium was the most abundant macro-mineral found in whole fruit ranging from 78.40 to 483.49 mg/100 g dry weight (DW), followed by Mg (13.23-101.70 mg/100 g DW) in epicarp, P (32.22-98.24 mg/100 g DW) in endocarp, Ca (23.41-71.39 mg/100 g DW) in whole fruit, and Na (6.09-18.56 mg/100 g DW) in epicarp. The concentration of microminerals was recorded higher in seeds compared to other fruit parts. Levels of Fe were higher (0.76-6.14 mg/100 g DW), followed by Zn (0.87-2.64 mg/100 g DW), Cu (137.68-525.45 µg/100 g DW), and Mn (46.92-179.05 µg/100 g DW). The analysis depicted bitter gourd to be a potential source of both macro-minerals (K and Mg) and microminerals (Fe and Zn). The consumption of bitter gourd could be a health-promoting strategy to meet daily dietary intake requirements of essential minerals for human health.

Comparison of Mineral Composition in Microgreens and Mature leaves of Celery (Apium graveolens L.).

Celery (Apium graveolens L.), a medicinal crop, occupies a significant position in the human diet possessing several essential macro- and microelements. For proper proximate analysis, an experiment was executed by taking 20 celery genotypes. The genotypes were analyzed for macro- and microminerals which include nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), sodium (Na), sulfur (S), zinc (Zn), iron (Fe), copper (Cu), and manganese (Mn). Results from analysis revealed that the  amount of N, P, Ca, Na, and S was higher in microgreens, whereas a higher value for K was found in mature leaves. Zn, Cu, and Mn contents were found to be higher in mature leaves, while no significant difference was observed for Fe content in microgreens and mature leaves. The inclusion of celery microgreens in our daily diet would fulfill a significant portion of our daily mineral requirement. This is the first report on mineral comparison between microgreens and mature leaves of celery. It opens a new avenue for further enhancement of minerals via biofortification of this medicinal wonder crop through systematic breeding efforts. On the basis of mineral analysis, three genotypes, namely PAU2, PAU4, and PAU16, were found superior in terms of mineral composition in microgreens and mature leaves of celery. Principal component and cluster analyses divide the genotypes into two different clusters on the basis of variability in mineral composition.

Mineral Content Variation in Leaves, Stalks, and Seeds of Celery (Apium graveolens L.) Genotypes.

Celery is an important nutritionally rich crop in the family Apiaceae. It is cultivated worldwide for food as well as for use in pharmaceutics. It is an excellent source of minerals, vitamins, and phytochemicals. Identification of superior genotypes with improved nutritional content is the requirement to develop cultivars for commercial cultivation. For mineral analysis of celery, an experiment was carried out taking 20 diverse genotypes. These genotypes were analysed for macro- and micronutrients which include nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), and sodium (Na). The study revealed high content of K (20.3-26.1 mg/g dry weight (DW)) and Zn (0.09-0.14 mg/g DW) in leaves while the stalks were rich in Ca (41.5-51.3 mg/g DW) and Na (5.2-8.0 mg/g DW). High contents of P (5.2-6.8 mg/g DW), Fe (0.41-0.56 mg/g DW), Cu (0.015-0.026 mg/g DW), and Mn (0.020-0.029 mg/g DW) were observed in seeds. Based on the mineral content, three genotypes, viz., PAU2, PAU4, and PAU7, were found to be superior in terms of mineral composition in leaves, stalks, and seeds. Cluster analysis divided the genotypes into two major groups. These genotypes can be used in crosses as they showed great potential for use in biofortification. This study opens newer avenues for future research, encouraging researchers to enhance the product quality and production efficiency of the leaves, stalks, and seeds valuable for human consumption.

Lipidomics-Assisted GWAS (lGWAS) Approach for Improving High-Temperature Stress Tolerance of Crops.

High-temperature stress (HT) over crop productivity is an important environmental factor demanding more attention as recent global warming trends are alarming and pose a potential threat to crop production. According to the Sixth IPCC report, future years will have longer warm seasons and frequent heat waves. Thus, the need arises to develop HT-tolerant genotypes that can be used to breed high-yielding crops. Several physiological, biochemical, and molecular alterations are orchestrated in providing HT tolerance to a genotype. One mechanism to counter HT is overcoming high-temperature-induced membrane superfluidity and structural disorganizations. Several HT lipidomic studies on different genotypes have indicated the potential involvement of membrane lipid remodelling in providing HT tolerance. Advances in high-throughput analytical techniques such as tandem mass spectrometry have paved the way for large-scale identification and quantification of the enormously diverse lipid molecules in a single run. Physiological trait-based breeding has been employed so far to identify and select HT tolerant genotypes but has several disadvantages, such as the genotype-phenotype gap affecting the efficiency of identifying the underlying genetic association. Tolerant genotypes maintain a high photosynthetic rate, stable membranes, and membrane-associated mechanisms. In this context, studying the HT-induced membrane lipid remodelling, resultant of several up-/down-regulations of genes and post-translational modifications, will aid in identifying potential lipid biomarkers for HT tolerance/susceptibility. The identified lipid biomarkers (LIPIDOTYPE) can thus be considered an intermediate phenotype, bridging the gap between genotype-phenotype (genotype-LIPIDOTYPE-phenotype). Recent works integrating metabolomics with quantitative genetic studies such as GWAS (mGWAS) have provided close associations between genotype, metabolites, and stress-tolerant phenotypes. This review has been sculpted to provide a potential workflow that combines MS-based lipidomics and the robust GWAS (lipidomics assisted GWAS-lGWAS) to identify membrane lipid remodelling related genes and associations which can be used to develop HS tolerant genotypes with enhanced membrane thermostability (MTS) and heat stable photosynthesis (HP).

Early Pulse Glucocorticoid Therapy and Improved Hormonal Outcomes in Primary Hypophysitis.

INTRODUCTION: The role of glucocorticoids in primary autoimmune hypophysitis (PAH) has been fraught with variability in regimens, leading to inconsistent outcomes in terms of anterior pituitary (AP) hormonal recovery. Hence, we aimed to compare the clinical, hormonal, and radiological outcomes of a standardized high-dose glucocorticoid therapy group (GTG) in PAH with a matched clinical observation group (COG). METHODS: Thirty-nine retrospective patients with PAH evaluated and treated at a single center in western India from 1999 to 2019 with a median follow-up duration of 48 months were subdivided into the GTG (n = 18) and COG (n = 21) and compared for the outcomes. RESULTS: Baseline demographic, hormonal, and radiological features matched between the groups, except pituitary height, which was significantly higher in GTG. Cortisol, thyroid, and gonadal axes were affected in 25 (64%), 22 (56%), and 21 (54%) patients, respectively, and central diabetes insipidus was seen in 7 (18%) patients. Panhypophysitis (PH) was the most common radiological subtype (n = 33, 84.6%). Resolution of mass effects was similar in both groups. Overall and complete AP hormonal recovery was significantly higher in the GTG than in the COG (12/14 [85.7%) vs. 6/14 [42.8%], p = 0.02; 10/14 [71.4%] vs. 1/14 [7.7%], p = 0.0007, respectively). Proportion of cases with empty sella were significantly higher in the COG (9/20 [45%] vs 1/17 [5.9%], p = 0.001). Among PH patients in the GTG (n = 17), we found duration from symptoms onset to treatment as the predictor of recovery. CONCLUSION: In a PH subtype-predominant PAH cohort, a standardized high-dose glucocorticoid regimen resulted in higher overall and complete AP hormonal recovery than that in the COG. Initiation of glucocorticoids in the early disease course may have been contributory.

177Lu-DOTATATE therapy in metastatic/inoperable pheochromocytoma-paraganglioma.

INTRODUCTION: 177Lu-DOTATATE-based peptide receptor radionuclide therapy (PRRT) is a promising therapy for metastatic and/or inoperable pheochromocytoma and paraganglioma (PPGL). We aim to evaluate the efficacy and safety of and identify predictors of response to 177Lu-DOTATATE therapy in metastatic and/or inoperable PPGL. METHODS: This retrospective study involved 15 patients of metastatic or unresectable PPGL, who received 177Lu-DOTATATE PRRT therapy. Clinical, biochemical (plasma-free normetanephrine), and radiological (anatomical and functional) responses were compared before and after the last therapy. RESULTS: A total of 15 patients (4 PCC, 4 sPGL, 5 HNPGL, 1 PCC + sPGL, 1 HNPGL + sPGL) were included. The median duration of follow up was 27 (range: 11-62) months from the start of PRRT. Based on the RECIST (1.1) criteria, progressive disease was seen in three (20%), stable disease in eight (53%), partial response in one (7%), and minor response in three (20%) and controlled disease in 12 (80%). On linear regression analysis the presence of PGL (P= 0.044) and baseline SUVmax >21 (P < 0.0001) were significant positive predictors of early response to PRRT. Encouraging safety profiles were noted with no long term nephrotoxicity and hematotoxicity. CONCLUSION: 177Lu-DOTATATE therapy is an effective and safe modality of treatment for patients with metastatic/inoperable PPGL. Although it is not prudent to withhold PRRT in metastatic PPGL with baseline SUVmax < 21, baseline SUVmax >21 can be used to predict early response to PRRT.

A carbohydrate binding module-5 is essential for oxidative cleavage of chitin by a multi-modular lytic polysaccharide monooxygenase from Bacillus thuringiensis serovar kurstaki.

Conversion of crystalline chitin to soluble sugar molecules, using lytic polysaccharide mono-oxygenases (LPMOs) has emerged as a new avenue for the production of biofuels. The present study describes the role of accessory domains in a multi-modular LPMO from Bacillus thuringiensis serovar kurstaki (BtLPMO10A). The full length BtLPMO10A (BtLPMO10A-FL) possesses an N-terminal LPMO of AA10 family (BtAA10) and a C-terminal CBM5 (BtCBM5) connected via two fibronectin (Fn) III domains (aligned as AA10-FnIII-FnIII-CBM5 from N- to C-terminus). To determine the role of individual domains, we generated truncation mutants of BtLPMO10A-FL. Substrate binding and kinetic studies revealed that BtCBM5 was involved in increasing binding efficiency of BtAA10 which otherwise has feeble binding towards ß-chitin and could not bind to α-chitin. Furthermore, binding assays also indicated that the presence of CBM5 increases the binding efficiency of BtLPMO10A-FL under extreme pH conditions. FnIII domains neither bind nor assist BtLPMO10A-FL in chitin binding and serve as linkers in BtLPMO10A-FL. BtLPMO10A-FL and BtAA10 generated oxidized chito-oligosaccharides from the insoluble ß-chitin substrate. It is concluded that BtCBM5 is responsible for increasing binding efficiency of BtLPMO10A-FL, whereas; BtAA10 domain is accountable for oxidative cleavage of recalcitrant chitin.

Testosterone Retention Mechanism in Sertoli Cells: A Biochemical Perspective.

Mechanism(s) involved in regulating Intratesticular Testosterone levels (iT) have assumed importance in recent years, from the point of view of hormonal contraception. Contraceptives using Testosterone (T) in combination with Progestins (P), for more effective suppression of pituitary gonadotropins thereby iT, are not 100% effective in suppressing spermatogenesis in human males, likely due to pesrsistence of Intratesticular Dihydrotestosterone (iD) in poor-responders. Several lacunae pertaining to the mechanism of action of principal male hormone T during spermatogenesis remain to be resolved. Notably, the mechanism through which T brings about the stage-specific differentiation of germ cells lacking Androgen Receptors (AR). Testosterone is a highly anabolic steroid with a rapid tissue clearance rate. T is intratesticular substrate for synthesis of Dihydrotestosterone (DHT) and Estradiol (E2) involved in spermtaogenesis. Therefore, it is important to delineate the mechanism(s) for retention of iT, in order to understand regulation of its bioavailability in testis. In depth studies, pertaining to the role of androgen-binding protein(s) in sequestration, retention and bioavailability of T/DHT are required to understand male fertility regulation. The appropriate approach to overcome this lacuna would be development of mice lacking functional testicular Androgen-Binding Protein (ABPKO), but not deficient T/DHT, Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH), in order to understand its physiological functions. Insights gained about androgen retention mechanism(s) from the ABPKO murine model will be of immense help in improving the efficacy of male hormonal contraceptives and infertility management.

Biotechnological approaches for field applications of chitooligosaccharides (COS) to induce innate immunity in plants.

Plants have evolved mechanisms to recognize a wide range of pathogen-derived molecules and to express induced resistance against pathogen attack. Exploitation of induced resistance, by application of novel bioactive elicitors, is an attractive alternative for crop protection. Chitooligosaccharide (COS) elicitors, released during plant fungal interactions, induce plant defenses upon recognition. Detailed analyses of structure/function relationships of bioactive chitosans as well as recent progress towards understanding the mechanism of COS sensing in plants through the identification and characterization of their cognate receptors have generated fresh impetus for approaches that would induce innate immunity in plants. These progresses combined with the application of chitin/chitosan/COS in disease management are reviewed here. In considering the field application of COS, however, efficient and large-scale production of desired COS is a challenging task. The available methods, including chemical or enzymatic hydrolysis and chemical or biotechnological synthesis to produce COS, are also reviewed.

Bacterial chitin binding proteins show differential substrate binding and synergy with chitinases.

Glycosyl hydrolase (GH) family 18 chitinases (Chi) and family 33 chitin binding proteins (CBPs) from Bacillus thuringiensis serovar kurstaki (BtChi and BtCBP), B. licheniformis DSM13 (BliChi and BliCBP) and Serratia proteamaculans 568 (SpChiB and SpCBP21) were used to study the efficiency and synergistic action of BtChi, BliChi and SpChiB individually with BtCBP, BliCBP or SpCBP21. Chitinase assay revealed that only BtChi and SpChiB showed synergism in hydrolysis of chitin, while there was no increase in products generated by BliChi, in the presence of the three above mentioned CBPs. This suggests that some (specific) CBPs are able to exert a synergistic effect on (specific) chitinases. A mutant of BliChi, designated as BliGH, was constructed by deleting the C-terminal fibronectin III (FnIII) and carbohydrate binding module 5 (CBM5) to assess the contribution of FnIII and CBM5 domains in the synergistic interactions of GH18 chitinases with CBPs. Chitinase assay with BliGH revealed that the accessory domains play a major role in making BliChi an efficient enzyme. We studied binding of BtCBP and BliCBP to α- and ß-chitin. The BtCBP, BliCBP or SpCBP21 did not act synergistically with chitinases in hydrolysis of the chitin, interspersed with other polymers, present in fungal cell walls.

Role of endogenous psychosine accumulation in oligodendrocyte differentiation and survival: implication for Krabbe disease.

Krabbe disease is a lethal, demyelinating condition caused by genetic deficiency of galactocerebrosidase (GALC) and resultant accumulation of its cytotoxic substrate, psychosine (galactosylsphingosine), primarily in oligodendrocytes (OLs). Psychosine is generated by galactosylation of sphingosine by UDP-galactose:ceramide galactosyltransferase (CGT), a galactosylceramide synthesizing enzyme which is primarily expressed in OLs. The expression of CGT and the synthesis of galactosyl-sphingolipids are associated with the terminal differentiation of OL, but little is known about the participation of endogenous psychosine accumulation in OL differentiation under GALC deficient conditions. In this study, we report that accumulation of endogenous psychosine under GALC deficient Krabbe conditions impedes OL differentiation process both by decreasing the expression of myelin lipids and protein and by inducing the cell death of maturating OLs. The psychosine pathology under GALC deficient conditions involves participation of secretory phospholipase A2 (sPLA2) activation and increase in its metabolites, as evidenced by attenuation of psychosine-induced pathology by treatment with pharmacological inhibitor of sPLA2 7,7-dimethyleicosadienoic acid (DEDA). These observations suggest for potential therapeutic efficacy of sPLA2 inhibitor in Krabbe disease.

Putative molecular mechanism underlying sperm chromatin remodelling is regulated by reproductive hormones.

BACKGROUND: The putative regulatory role of the male reproductive hormones in the molecular mechanism underlying chromatin condensation remains poorly understood. In the past decade, we developed two adult male rat models wherein functional deficits of testosterone or FSH, produced after treatments with 20 mg/Kg/d of cyproterone acetate (CPA) per os, for a period of 15 days or 3 mg/Kg/d of fluphenazine decanoate (FD) subcutaneously, for a period of 60 days, respectively, affected the rate of sperm chromatin decondensation in vitro. These rat models have been used in the current study in order to delineate the putative roles of testosterone and FSH in the molecular mechanism underlying remodelling of sperm chromatin. RESULTS: We report that deficits of both testosterone and FSH affected the turnover of polyubiquitylated histones and led to their accumulation in the testis. Functional deficits of testosterone reduced expression of MIWI, the 5-methyl cap binding RNA-binding protein (PIWIlike murine homologue of the Drosophila protein PIWI/P-element induced wimpy testis) containing a PAZ/Piwi-Argonaut-Zwille domain and levels of histone deacetylase1 (HDAC1), ubiquitin ligating enzyme (URE-B1/E3), 20S proteasome α1 concomitant with reduced expression of ubiquitin activating enzyme (ube1), conjugating enzyme (ube2d2), chromodomain Y like protein (cdyl), bromodomain testis specific protein (brdt), hdac6 (histone deacetylase6), androgen-dependent homeobox placentae embryonic protein (pem/RhoX5), histones h2b and th3 (testis-specific h3). Functional deficits of FSH reduced the expression of cdyl and brdt genes in the testis, affected turnover of ubiquitylated histones, stalled the physiological DNA repair mechanism and culminated in spermiation of DNA damaged sperm. CONCLUSIONS: We aver that deficits of both testosterone and FSH differentially affected the process of sperm chromatin remodelling through subtle changes in the 'chromatin condensation transcriptome and proteome', thereby stalling the replacement of 'dynamic' histones with 'inert' protamines, and altering the epigenetic state of condensed sperm chromatin. The inappropriately condensed chromatin affected the sperm chromatin cytoarchitecture, evident from subtle ultrastructural changes in the nuclei of immature caput epididymal sperm of CPA- or FD-treated rats, incubated in vitro with dithiothreitol.

Sperm chromatin protamination: an endocrine perspective.

During spermiogenesis, the elongating rat spermatid chromatin undergoes a gradual process of condensation which is initiated in the round spermatids at "step 7" of cytodifferentation in stage VII and extending to elongated spermatids at "step 19" of cytodifferentiation in stage VIII. The mechanism of chromatin condensation in the elongating spermatids is an elaborate process that encompasses several biochemical and biological aspects culminating in the deposition of protamine in DNA grooves. The protamination of sperm chromatin involves expression and storage of proteins involved in condensation, removal and degradation of nuclear histones and their replacement by transition proteins and protamine 1, transcriptional silencing and DNA repair, reduction of nuclear volume, repackaging of protaminated chromatin in torroids and development of a characteristic head shape and perforatorium. A study was undertaken in my laboratory to delineate the role of follicle stimulating hormone (FSH) and testosterone in the condensation of nuclear chromatin in the elongating spermatids of sexually competent species of rat. Towards this end, sexually competent male Holtzmann rats were treated with 20 mg/Kg/d per os (oral supplementum) of cyproterone acetate and 3 mg/Kg/d i.p (intra peritoneal) of fluphenazine decanoate to induce a functional deficiency in either testosterone or FSH. In both rat models, membrane-impermeable CMA3 fluorescent dye uptake assay for GC-rich prospective sites of DNA protamination, was indicative of insufficiency of protamine 1 in spermatozoa taken from caput epididymides of treated rats whereas a fluorescent TUNEL assay indicated the presence of nicked chromatin strands only in protamine-deficient spermatozoa derived from caput epididymides of fluphenazine-treated rats with functional deficiency of FSH. Western blotting of acid-soluble sperm basic proteins had confirmed the near absence of protamine 1 in treated rat spermatozoa in both models. Electron Microscopic evaluation too revealed fine ultrastructural changes in the nuclear membrane of cyproterone acetate as well as fluphenazine decanoate treated spermatozoa derived from caput epididymides. Electrophoretic analysis of caput sperm nuclear basic proteins substantiated the observations at cellular level and revealed a pattern of abnormal persistence of acid-soluble nuclear basic proteins in both rat models, the levels being more prominent in fluphenazine treated rats. Our studies suggest that adequate levels of both FSH and testosterone could be essential during the stages of spermatidal condensation and led us to hypothesize the existence of an endocrine-regulated molecular mechanism for histone to protamine transition and maintenance of chromatin integrity during chromatin condensation in the testis during spermiogenesis.

Inhibition of rho family functions by lovastatin promotes myelin repair in ameliorating experimental autoimmune encephalomyelitis.

Impaired remyelination is critical to neuroinflammation in multiple sclerosis (MS), which causes chronic and relapsing neurological impairments. Recent studies revealed that immunomodulatory activity of statins in an experimental autoimmune encephalomyelitis (EAE) model of MS are via depletion of isoprenoids (farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate) rather than cholesterol in immune cells. In addition, we previously documented that lovastatin impedes demyelination and promotes myelin repair in treated EAE animals. To this end, we revealed the underlying mechanism of lovastatin-induced myelin repair in EAE using in vitro and in vivo approaches. Survival, proliferation (chondroitin sulfate proteoglycan-NG2(+) and late oligodendrocyte progenitor marker(+)), and terminal-differentiation (myelin basic protein(+)) of OPs was significantly increased in association with induction of a promyelinating milieu by lovastatin in mixed glial cultures stimulated with proinflammatory cytokines. Lovastatin-induced effects were reversed by cotreatment with mevalonolactone or geranylgeranyl-pyrophosphate, but not by farnesyl-pyrophosphate or cholesterol, suggesting that depletion of geranygeranyl-pyrophosphate is more critical than farnesyl-pyrophosphate in glial cells. These effects of lovastatin were mimicked by inhibitors of geranylgeranyl-transferase (geranylgeranyl transferase inhibitor-298) and downstream effectors {i.e., Rho-family functions (C3-exoenzyme) and Rho kinase [Y27632 (N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride)]} but not by an inhibitor of farnesyl-transferase (farnesyl transferase inhibitor-277). Moreover, activities of Rho/Ras family GTPases were reduced by lovastatin in glial cells. Corresponding with these findings, EAE animals exhibiting demyelination (on peak clinical day; clinical scores >/=3.0) when treated with lovastatin and aforementioned agents validated these in vitro findings. Together, these data provide unprecedented evidence that-like immune cells-geranylgeranyl-pyrophosphate depletion and thus inhibition of Rho family functions in glial cells by lovastatin promotes myelin repair in ameliorating EAE.

NADPH oxidase in human lung fibroblasts.

Reactive oxygen species produced by NADPH oxidase appear to play a role in the response of human lung fibroblast cells to rhinovirus infection. The purpose of the following studies was to characterize the NADPH oxidase components in these cells, to examine the effect of rhinovirus challenge on the expression of these proteins, and to confirm previous studies suggesting a role for p47-phox in the oxidant response to rhinovirus challenge. The results revealed that the NADPH oxidase components p47-phox, p67-phox, p22-phox, and NOX4 were expressed in lung fibroblast cells. In contrast, gp91-phox was not expressed in this cell line. Expression of p67-phox was upregulated by rhinovirus challenge. The functional role of NADPH oxidase in the rhinovirus-induced oxidant stress and elaboration of IL-8 was confirmed by detection of significant reductions in oxidant stress and IL-8 elaboration following transfection of the cells with antisense nucleotides to p47-phox. The lack of gp91- phox in cultured lung fibroblast cells, the induction of p67-phox by rhinovirus, and the confirmation of participation of p47-phox in rhinovirus-induced oxidant stress are significant findings of this study and form a basis for future investigations into understanding the mechanisms of the NADPH oxidase response to rhinovirus infection.

Regulation of gene expression associated with acute experimental autoimmune encephalomyelitis by Lovastatin.

The attenuation of experimental autoimmune encephalomyelitis (EAE) by Lovastatin (LOV) has now been well established. The present study was designed to explore the global effect of LOV treatment on expression of immune-related genes in lumbar spinal cord (LSC) during acute EAE by using Affymetrix DNA microarrays. LOV treatment demonstrated the limited infiltration of inflammatory cells into the LSC, and microarray analysis further validated those interpretations by demonstrating relatively less alteration in expression of immune response genes in LOV-treated EAE rats on peak clinical day and recovery vs. untreated EAE counterparts. There was significant change in expression of about 158 immune-related genes (including 127 genes reported earlier) in LOV-treated vs. untreated EAE (>1.5 or <-1.5 fold change; P