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Abstract Chronic inflammation provides a favorable microenvironment for tumorigenesis, which opens opportunities for targeting cancer development and progression. Piplartine (PL) is a biologically active alkaloid from long peppers that exhibits anti-inflammatory and antitumor activity. In the present study, we investigated the physical and chemical interactions of PL with anti-inflammatory compounds and their effects on cell proliferation and migration and on the gene expression of inflammatory mediators. Molecular docking data and physicochemical analysis suggested that PL shows potential interactions with a peptide of annexin A1 (ANXA1), an endogenous anti-inflammatory mediator with therapeutic potential in cancer. Treatment of neoplastic cells with PL alone or with annexin A1 mimic peptide reduced cell proliferation and viability and modulated the expression of MCP-1 chemokine, IL-8 cytokine and genes involved in inflammatory processes. The results also suggested an inhibitory effect of PL on tubulin expression. In addition, PL apparently had no influence on cell migration and invasion at the concentration tested. Considering the role of inflammation in the context of promoting tumor initiation, the present study shows the potential of piplartine as a therapeutic immunomodulator for cancer prevention and progression.

Background: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. Results: The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related betaalpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. Conclusions: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis. [ABSTRACT FROM AUTHOR]

Chronic food restriction (FR) of rats and mice results in moderate hyperadrenocorticism, which may play a role in activating cellular mechanisms that retard aging. Previously, we reported that the FR-induced hyperadrenocorticism is not due to an activated hypothalamo-pituitary unit. Therefore, we investigated in a series of experiments whether adrenal responsiveness to adrenocorticotropic hormone (ACTH), in vitro and in vivo, is enhanced by FR. Three mo-old male Fischer 344 rats were either given free access to food (AL rats) or restricted to 60% of food consumed by AL rats (FR rats) from 6 wk of age. They were killed by decapitation in the morning (AM) and afternoon (PM) when endogenously circulating corticosterone levels are at their nadir and peak, respectively. In vitro, adrenal glands from FR rats (1.5 mo FR) produced more corticosterone per mg at all doses of ACTH than those from AL rats in both the AM and PM (diet main effect, P < 0.001). FR (1.5 to 2.5 mo) also enhanced adrenal responsiveness to physiologic (diet main effect, P < 0.05) and superphysiologic (diet main effect, P < 0.001) levels of ACTH administered in vivo to dexamethasone-treated rats. ACTH-receptor (ACTH-R) mRNA, normalized to adrenal mass or to total RNA, was not influenced by FR (1.5 mo). However, adrenal ACTH-R mRNA, as well as adrenal mass, per unit body weight was greater in FR than in AL rats (diet main effect, P < 0.001). These results indicate that enhanced adrenocortical responsiveness to ACTH plays a major role in the hyperadrenocortical state of chronically FR rats. [ABSTRACT FROM AUTHOR]

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes., Methods: Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries., Results: Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues., Conclusion: To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor.

Because neuroendocrine mechanisms may contribute to the antiaging effects of food restriction (FR), we measured the effect of FR on mRNAs encoding anterior pituitary (AP) tropic hormones. Slot blots or RNase protection assays were done on AP RNA from 3-, 6-, 12-, 18- and 24-mo-old male F344 rats consuming food ad libitum (AL) or food restricted (FR; to 60% of AL food intake) from 6 wk. Both AL and FR rats gained body weight during the study (P < 0.05), but FR rats weighed approximately 40% less (P < 0.0001). Messenger RNA levels were expressed in two ways, i.e., per total AP and per microgram total AP RNA. Proopiomelanocortin (POMC) mRNA/microg RNA was higher (P < 0.0005) in FR than in AL rats at all ages. Thyroid-stimulating hormone (TSH) beta mRNA declined with age (P < 0.05) in AL but not FR rats and was reduced by FR up to 12 mo (P < 0.01). Growth hormone (GH) mRNA/microg RNA declined with age (P < 0.05) in AL but not FR rats, and total GH mRNA in the AP was reduced by FR at early ages (P < 0.05). FR reduced prolactin (PRL) mRNA and its age-related increase (P < 0.0005). Levels of luteinizing hormone (LH) beta and follicle-stimulating hormone (FSH) beta mRNAs did not differ between AL and FR rats until 12 mo, but thereafter rose in FR (LH beta mRNA; P < 0.01, FSH beta mRNA; P < 0.05). Many of these changes in gene expression corroborate previously reported hormonal changes in FR rodents and mutant mice with extended life spans, and thus provide further support for the hypothesis that an altered hormonal milieu contributes to the antiaging effects of food restriction.

The life-prolonging effects of calorie restriction (CR) may be due to reduced damage from cumulative oxidative stress. Our goal was to determine the long-term effects of moderate dietary CR on the myocardial response to reperfusion after a single episode of sublethal ischemia. Male Fisher 344 rats were fed either an ad libitum (AL) or CR (40% less calories) diet. At age 12 mo the animals were anaesthetized and subjected to thoracotomy and a 15-min left-anterior descending coronary artery occlusion. The hearts were reperfused for various periods. GSH and GSSG levels, nuclear factor-kappaB (NF-kappaB) DNA binding activity, cytokine, and antioxidant enzyme expression were assessed in the ischemic zones. Sham-operated animals served as controls. Compared with the AL diet, chronic CR limited oxidative stress as seen by rapid recovery in GSH levels in previously ischemic myocardium. CR reduced DNA binding activity of NF-kappaB. The kappaB-responsive cytokines interleukin-1beta and tumor necrosis factor-alpha were transiently expressed in the CR group but persisted longer in the AL group. Furthermore, expression of manganese superoxide dismutase, a key antioxidant enzyme, was significantly delayed in the AL group. Collectively these data indicate that CR significantly attenuates myocardial oxidative stress and the postischemic inflammatory response.

Objectives: To quantitate and compare granulosa cell alpha-inhibin messenger RNA (mRNA) levels in IVF-ET poor and good responders and thereby learn how alpha-inhibin mRNA levels change in states of diminished ovarian responsiveness., Design: Ribonucleic acid analysis of stored luteinized granulosa cell samples., Setting: Academic tertiary care institution., Patients: Fifty-three women undergoing follicle aspiration for IVF-ET were studied. Patients were classified as poor responders (n = 16) or good responders (n = 37) according to their E2 concentration on the day of hCG; the E2 of poor responders was < 1,000 pg/mL (3,671 pmol/L) and that of good responders was > or = 1,000 pg/mL (3,671 pmol/L)., Main Outcome Measures: Messenger RNA levels were measured using dot blot RNA analysis. The following parameters were determined or derived: total mRNA levels, total alpha-inhibin mRNA levels, alpha-inhibin mRNA per follicle, and proportional alpha-inhibin mRNA as the ratio of alpha-inhibin mRNA:total mRNA., Results: Proportional alpha-inhibin mRNA and alpha-inhibin mRNA per follicle were not significantly different between poor responders and good responders. Total mRNA and total alpha-inhibin mRNA levels, however, were diminished significantly in poor responders., Conclusions: The observations that proportional alpha-inhibin mRNA and alpha-inhibin mRNA per follicle do not significantly change in poor responders, whereas total alpha-inhibin mRNA does, indicate that the decrease in total alpha-inhibin mRNA in poor responders reflects a decreased pool of total mRNA, likely because of a reduction in follicle number. These findings are in contrast to other recent reports that describe a change in granulosa cell function accompanying states of decreased ovarian responsiveness.

Despite expanding knowledge on the kinetic aspects of folliculogenesis, the question of what initiates follicle growth remains unanswered. Efforts to solve this problem have been thwarted by the absence of sensitive markers to identify the onset of follicular growth. In this study, we determined whether increased proliferating cell nuclear antigen (PCNA) correlates with initiation of follicle growth and might therefore be useful for studying early events in this process. Paraffin sections of ovaries from cycling adult rats, prepubertal eCG+hCG-primed rats, and prepubertal control rats were processed for immunocytochemistry by use of a PCNA primary antibody. In primordial follicles, neither granulosa cells nor oocytes stained for PCNA. PCNA immunoreactivity coincided with the earliest sign of follicle growth, appearing in pregranulosa cells of early primary follicles just beginning to grow. In all primary follicles, some granulosa cells were PCNA-positive. PCNA immunoreactivity in oocytes first appeared in primary follicles, preceding oocyte enlargement. In preantral and antral follicles, granulosa cell PCNA staining was uniform throughout the granulosa cell layers. Oocytes were positive for PCNA in both preantral and antral follicles. PCNA expression diminished in atretic follicles. In CL, granulosa cell PCNA expression was also decreased. Theca cell PCNA expression was first evident in the transitional follicle (1-2-layer granulosa cells) and was present in all stages thereafter. The pattern of PCNA expression did not differ among adult cycling, prepubertal eCG+hCG-stimulated, and control rat ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies in C57BL/6J, DBA/2J and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1) 2-5 months old revealed marked genotypic differences among inbred strains. C57 mice had three times as many regular (3-6 days) cycles as DBA and C3H mice, due largely to fewer pseudopregnant-like (7-14 day) cycles. C57 had longer regular cycles than DBA and C3H mice. Although the frequencies of regular cycles of DBA and C3H mice were similar, the cycles of C3H mice were shorter than those of DBA mice. The results indicated that the genetic determinants of the frequency of regular cycles differ from those specifying cycle length. Frequency of regular cycles of F1 hybrids was either intermediate between the parent strains (B6D2F1) or similar to the C57 strain (B6C3HF1), suggesting that regular cycle frequency shows additive genetic variation in the former crosses, but mostly dominant variance in the latter background. Regular cycles were either shorter than in both parent strains (B6D2F1) or similar to one of them (B6C3HF1), indicating heterosis and dominance for genes specifying short cycles. Although the lack of reciprocal crosses meant that maternal effects and possible genomic imprinting effects could not be assessed, these results reveal marked genetic influences on cycle length and frequency and suggest that some of the genes specifying these two traits differ.

Long-term exposure to ovarian hormones contributes to age-related changes in estrous cyclicity in rodents. Estrogens are implicated in this process, but the concentration of estrogen required to exert these effects is not well established. Also, although estrogens are presumed to alter vaginal cyclicity by affecting the hypothalamic-pituitary axis, they may also impair the ability of the vaginal epithelium to cornify. To address these issues, young and middle-aged ovariectomized (ovx) C57BL/6J mice were exposed for 7-10 wk to plasma levels of estradiol (E2) at one of three ranges (30-40, 50-80, or 120-160 pg/ml). Ovaries from young mice were then transplanted under the renal capsule, and vaginal cyclicity was monitored for 4 mo. Mice exposed to the lowest level of E2 not only failed to stop cycling, but had a higher monthly frequency of estrous cycles than did controls (nearly 1 extra cycle/mo). Mice exposed to the intermediate level of E2 showed no impairment in cyclicity. Although mice exposed to the highest concentrations of E2 showed no vaginal cyclicity, they continued to ovulate as evidenced by fresh, albeit reduced, numbers of corpora lutea. These results indicate that, in ovx mice, (1) chronic exposure to relatively low concentrations of E2 potentiates cyclicity, (2) very high levels of E2 are required to induce acyclicity, and (3) this acyclicity reflects vaginal as well as neuroendocrine alterations. The results also indicate that vaginal acylicity may be a poor indicator of ovulatory acyclicity in mice that have been chronically exposed to E2.

Genetic influences on the timing of three pubertal events--vaginal opening, first vaginal cornification, and onset of cyclicity--were studied in C57BL/6J, DBA/2J, and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1). Marked genotypic differences were found. Among inbred strains, differences in the onset of vaginal opening and first vaginal cornification (C3H less than DBA less than C57) did not parallel those for the onset of cyclicity (C3H much greater than DBA = C57). Compared to parental strains, F1 hybrid strains were intermediate for times of vaginal opening and first vaginal cornification, consistent with the model in which the genetic effects on the timing of these events are additive. By contrast, onset of cyclicity occurred significantly earlier in the F1 hybrids than in their parent strains, indicating heterosis for one or more genes specifying this event. Body weights also differed among the genotypes from weaning onward, but these differences were only partially correlated with the differences in the timing of the pubertal events. Thus, genetic influences other than those affecting body weight contribute to the differential timing of pubertal events in these mouse strains. These results reveal marked genetic variation in the timing of puberty, and indicate that the set of genes specifying the timing of vaginal opening and first vaginal cornification differs from those specifying the onset of cyclicity.

Cycle frequency, length, and vaginal cytology were measured longitudinally in three cohorts of singly housed virgin mice staggered across a 3-year interval. The age profiles of these parameters were qualitatively similar, but quantitatively different, among cohorts. Cycle frequency was initially low (Phase I), due to prolonged cycles and late-starting cycles, and did not peak (Phase II) until mice were 3-5 months old. Phase II lasted for 7-10 months, depending on the cohort. Thereafter cycle frequency declined steadily (Phase III). The average age of cessation of cyclicity varied among cohorts, occurring between 13 and 16 months of age. Age changes in cycle length paralleled those of cycle frequency. During Phase II, median cycle length was less than 5 days and variance was lowest. During Phases I and III, variance was about twofold greater and median cycle length was greater than 5 days. Although median cycle length remained stable for several months during Phase II, the peak period of 4-day cycles was much shorter. In all cohorts, 4-day cycles did not peak until 7-8 months of age and began to decline by 9 months. The decrease in 4-day cycles was associated with a progressive lengthening of cycles-first from 4 to 5 days, then to longer cycles. The fraction of cycles with extended cornification (greater than 2 days) increased with advancing age from less than 0.35 during the initial period of cycle lengthening to a maximum of 0.60. The observation that the initial phase o cycle prolongation was not usually associated with extended cornification is consistent with earlier evidence that this period is characterized by a delayed, rather than prolonged, preovulatory rise of estradiol. However, the increased fraction of prolonged cycles with extended cornification at later ages suggests that the preovulatory elevation of estradiol may ultimately be prolonged.

The of onset of acyclicity and the duration of persistent vaginal cornification (PVC), the predominant postcyclic anovulatory condition, were measured in five cohorts of virgin mice staggered across a 6-yr interval. The average age of cessation of cyclicity varied significantly among cohorts, ranging from 11 to 16 mo of age. Seventy-five percent of the mice entered PVC after ceasing to cycle, the remainder entered directly into a persistent diestrous condition. The duration of PVC, which averaged between 2 and 4 mo, varied markedly among individuals and was inversely related to the age of onset of acyclicity. Consequently, the variance associated with the cessation of cyclicity was about 4-fold greater than that associated with the cessation of PVC. These differing variances suggest that the factors governing ovulatory failure and PVC cessation may differ.

Long-term exposure of adult female rodents to estrogen has many deleterious effects on reproductive neuro-endocrine structure and function, but its effects on peripheral target tissues are not well known. This study was designed to determine whether chronic exposure of young mice to estradiol (E2) alters the response of the vagina to E2, and if so, whether aging potentiates this alteration. Eight-week-old mice were ovariectomized (ovx) and given subcutaneous Silastic or polyethylene (PE) implants containing E2. Silastic implants produced supra-physiologic E2 levels, while E2 levels in PE-implanted mice were within the physiologic range. Initially all E2-exposed mice showed vaginal cornification (CORN). However, CORN soon began to decline and was virtually absent 3-5 mo after implantation, despite evidence of continued, albeit reduced, release of E2 from the implants. Mice were reimplanted with new E2 implants to determine whether the loss of CORN resulted from an altered response to E2 or from a decreased release of E2 from the implants. Vaginas of mice previously exposed to either Silastic (high E2) or PE (low E2) implants failed to cornify in response to new E2 implants, whereas vaginas of mice that had been initially exposed to implants without E2 cornified in response to identical E2 implants. When old (23 mo) acutely ovx mice were given E2-containing Silastic implants, the peak level and duration of CORN were only one-third and one-fifth, respectively, of that seen in young mice. Non-cornifying epithelia from both young and old chronically E2-exposed mice were as hyperplastic and active mitotically as cornifying epithelia, indicating that the loss of CORN was not a result of decreased epithelial proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative contributions of ovarian failure and hypothalamic-pituitary dysfunction to the prolongation and cessation of estrous cycles were assessed by measuring the ability of acutely ovariectomized (OVX) middle-aged (12 mo) mice to cycle after receiving grafts (under the renal capsule) of ovaries from young (2 mo) mice. The potentially disruptive effect of the acyclic state on the cycling response to grafted, young ovaries was avoided restricting grafting to middle-aged hosts that were still cycling. The effect of chronic exposure to ovarian secretions before the cessation of cyclicity on age-related hypothalamic-pituitary dysfunction was also assessed. The cycling ability of long-term OVX middle-aged mice (i.e., OVX at 3 mo) bearing grafts of young ovaries was compared to that of age-matched acutely OVX controls. Grafted young ovaries extended the cycling lifespan of acutely OVX middle-aged hosts by 60%. The length of this extended cycling lifespan, however, was only 80% of that achieved by young hosts bearing grafts of young ovaries. Young ovaries in middle-aged mice markedly lowered the incidence of long cycles (greater than 5 days), shifting the modal cycle length to 5 days. However, young ovaries in middle-aged mice failed to increase the incidence of 4-day cycles, the modal cycle of young controls. Middle-aged ovaries grafted into young hosts lengthened their cycles and shortened their cycling lifespan to middle-aged values. Long-term ovariectomy failed to increase the cycling lifespan of middle-aged hosts bearing grafts of young ovaries beyond that achieved in acutely OVX mice. Long-term ovariectomy did shorten the modal cycle length of middle-aged mice to 4 days, although the duration of 4-day cycling was only one-third (2 mo) that of young controls. These results indicate that the relative contributions of ovarian and neuroendocrine factors to three major events of reproductive aging vary with each event. Whereas the hypothalamic-pituitary unit appears to play an important role in the initial shift from 4- to 5-day cycles, the aging ovary plays the major role in the subsequent shift to longer cycles and in the ultimate cessation of cyclicity. Although chronic exposure to ovarian secretions during the period of cyclicity does not play a major role in the cessation of cyclicity, it appears to contribute to the hypothalamic-pituitary changes responsible for the initial shift from 4- to 5-day cycles.

Restricting the food intake of female mice by alternating days of feeding and fasting delayed the age-related loss of estrous cycling potential and retarded the rate of follicular depletion, as determined after reinstatement of ad libitum (AL) feeding. During the period of food restriction (FR; 3.5-10.5 mo), food intake and body weight were about 80% of AL values. Mice were acyclic and predominantly in a state of diestrus during FR, but after reinstatement of an AL diet at 10.5 mo all FR mice resumed cycling regularly. By contrast, 80% of AL controls had become acyclic by this age, and the cycles of the remaining mice were significantly longer than those of the reinstated FR mice. Follicular reserves of 12.5-mo-old FR mice were twice those of age-matched AL controls. Cycling performance of reinstated FR mice, measured by cycle length and the proportion of mice still cycling, was equivalent to that of AL mice when the latter were 2-5 mo younger. Ovarian age, measured by the size of the follicular reserve, was similarly retarded in FR mice. Based on these data and previous evidence that follicular depletion plays a major role in the cessation of cyclicity in this strain, we hypothesize that the delayed loss of estrous cyclicity in aging FR mice is mediated at least in part by the retarding effect of dietary restriction on the rate of follicular depletion.