Early growth determines longevity in male rats and may be related to telomere shortening in the kidney

Maternal protein undernutrition can influence the growth and longevity of male offspring in the rat. We tested the hypothesis that these differences in longevity were associated with changes in the rate of telomere shortening. We found age-related shortening of telomeres in the liver and kidney but not in the brain of male rats. Growth retardation in postnatal life was associated with significantly longer kidney telomeres and an increased longevity. Conversely, growth retardation during the foetal life followed by postnatal catch-up growth was associated with a shorter life span and shorter kidney telomeres. These findings may provide a mechanistic basis for epidemiological studies linking early growth retardation to adult degenerative diseases.     

From inhibition to excitation: functional effects of interaction between opioid receptors

Opioids have excitatory effects in multiple regions of the nervous system. Excitation by opioids is generally attributed to inhibition of inhibitory pathways (disinhibition). However, recent studies indicate that opioids can directly excite individual cells. These effects may occur when opioid receptors interact with other G protein coupled receptors, when different subtypes of opioid receptors interact, or when opioids transactivate other receptors such as receptor tyrosine kinases. Changes in the relative level of expression of different receptors in an individual cell may therefore determine its functional response to a given ligand. This phenomenon could represent an adaptive mechanism involved in tolerance, dependence and subsequent withdrawal.     

The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 cooperate to restrict proliferative life span in differentiating ovarian cells

The timing of cellular exit from the cell cycle during differentiation is specific for each cell type or lineage. Granulosa cells in the ovary establish quiescence within several hours after the ovulation-inducing luteinizing hormone surge, whereas they undergo differentiation into corpora lutea. The expression of Cdk inhibitors p21(Cip1/Waf1) and p27(Kip1) is up-regulated during this process, suggesting that these cell cycle inhibitors are involved in restricting proliferative capacity of differentiating granulosa cells. Here we demonstrate that the lack of p27(Kip1) and p21(Cip1) synergistically renders granulosa cells extended an proliferative life span. Immunohistochemical analyses demonstrated that corpora lutea of p27(Kip1), p21(Cip1) double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27(Kip1) single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21(Cip1)-deficient mice ceased proliferation similarly to those in wild-type mice. Interestingly, granulosa cells isolated from p27(Kip1), p21(Cip1) double-null mice exhibited markedly prolonged proliferative life span in culture, unlike cells with other genotypes. Cultured p27(Kip1), p21(Cip1) double-null granulosa cells maintained expression of steroidogenic enzymes and gonadotropin receptors through 8-10 passages and could undergo further differentiation in responses to cAMP accumulation. Thus, the cooperation of p27(Kip1) and p21(Cip1) is critical for withdrawal of granulosa cells from the cell cycle, in concert with luteal differentiation and possibly culture-induced senescence.     

Buthionine sulfoximine embryotoxicity is associated with prolonged AP-1 activation

BACKGROUND: Many teratogens induce oxidative stress, altering redox status and redox signaling; this has led to the suggestion that developmental toxicants act by disturbing redox status. The goal of these studies was to determine the consequences of altering glutathione homeostasis during organogenesis on embryo development, total DNA methylation, and activator protein-1 (AP-1) DNA binding activity and gene expression. METHODS: Gestational day 10.5 rat embryos were cultured in vitro for up to 44 hour in the presence of L-buthionine-S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamyl-cysteine synthetase, the rate limiting step in glutathione biosynthesis. Effects of BSO on total, oxidized and reduced glutathione, embryo development, DNA methylation, AP-1 DNA binding activity and gene expression were investigated. RESULTS: Significant depletion of glutathione by BSO was first noted at 6 hr in the embryo and at 3 hr in the yolk sac; total glutathione in the conceptus was depleted to the same extent after treatment with either 0.1 or 1.0 mM BSO. Exposure to 0.1 mM BSO did not cause a significant increase in embryotoxicity, although some impairment of growth and development was observed. In contrast, exposure to 1.0 mM BSO severely inhibited growth and development, significantly increasing the incidence of swollen hindbrains and of blebs in the forebrain, limb and maxillary regions. No significant treatment-related differences in total DNA methylation were observed. Interestingly, AP-1 DNA binding activity was similar in control and 0.1 mM BSO-treated conceptuses; however, exposure to 1.0 mM BSO increased AP-1 DNA binding at 6, 24, and 44 hr. The expression of several AP-1 family genes and of gamma-glutamylcysteine synthetase was induced in embryos cultured with 1.0 mM BSO. CONCLUSION: Exposure of embryos in vitro to BSO at a concentration that was embryotoxic induced prolonged AP-1 DNA binding activity and altered gene expression. These data suggest that AP-1 induction may serve as a biomarker of embryo stress.     

Variation in Flagellin Genes and Proteins of Burkholderia cepacia

The majority of isolates of Burkholderia cepacia, an important opportunistic pathogen associated with cystic fibrosis, can be classified into two types on the basis of flagellin protein size. Electron microscopic analysis indicates that the flagella of strains with the larger flagellin type (type I) are wider in diameter. Flagellin genes representative of both types were cloned and sequenced to design oligonucleotide primers for PCR amplification of the central variable domain of B. cepacia flagellin genes. PCR-restriction fragment length polymorphism analysis of amplified B. cepacia flagellin gene products from 16 strains enabled flagellin type classification on the basis of product size and revealed considerable differences in sequence, indicating that the flagellin gene is a useful biomarker for epidemiological and phylogenetic studies of this organism.Burkholderia cepacia (formerly Pseudomonas cepacia; a member of the rRNA group II pseudomonads) has emerged as an increasingly important opportunistic pathogen, particularly in relation to patients suffering from cystic fibrosis (CF) (15). Acquisition of B. cepacia, often occurring after lengthy colonization with Pseudomonas aeruginosa, can lead to the rapid deterioration or death of CF patients, and this organism appears to be transmissible between patients (14). There is considerable evidence that some strains of B. cepacia are more virulent than others and that the outcome of colonization by a particular strain can vary from rapidly fatal septicemia to maintenance of stable respiratory function (16). A number of factors have been implicated in the greater virulence of some strains. These include adhesion to respiratory mucin (31, 32) and the presence of cable pili (33).Motility in B. cepacia is by means of polar flagella. Flagella, each consisting of a flagellin filament, hook, and basal body, have been implicated as invasive virulence factors for a number of bacteria (28), including P. aeruginosa (11). Unlike P. aeruginosa, which appears to sit in microcolonies in the viscid mucus, leading to progressive lung damage with episodes of acute debilitating exacerbation, some strains of B. cepacia cause rapidly fatal pneumonia in CF patients (15), suggesting that they may have the ability to move through the mucus. Because of their location on the outside of bacterial cells, flagellins have been targeted in vaccine design. Brett et al. (4) demonstrated that flagellin-specific antisera were capable of protecting diabetic rats from challenge with strains of Burkholderia pseudomallei (another member of rRNA group II). In a recent study, an O-polysaccharide moiety of B. pseudomallei was covalently linked to the flagellin protein from the same strain. O-polysaccharide–flagellin conjugates elicited a high-level immunoglobulin G response capable of protecting diabetic rats from challenge with a heterologous strain of B. pseudomallei (5).Two distinct flagellin protein molecular mass groups in B. cepacia have been reported by Montie and Stover (23). In this previous study, type I flagellins were reported as having a molecular mass of 31 kDa while the molecular mass of type II flagellins was reported as 44 to 46 kDa. This early study, using a limited number of isolates, suggested that with regard to flagellin, B. cepacia is analogous to another CF pathogen, P. aeruginosa, in which two flagellin antigenic types distinguishable by protein or gene size are found (43). Several representatives of the heterologous a-type and homologous b-type fliC loci of P. aeruginosa (encoding flagellins) have been sequenced (37). In addition, PCR amplification of flagellin genes coupled with restriction fragment length polymorphism (RFLP) analysis can be used as a method for differentiating between clinical isolates of P. aeruginosa (7, 43). In this paper we report the development of a similar approach to the study of populations of B. cepacia and discuss the divergence of a highly variable gene, the flagellin gene (fliC), within populations of B. cepacia.     

Posters. P3 North west non‐gynaecological cytology EQA pilot CD ROM scheme 2002–2003

The 8th circulation of this scheme was preceded by a CD ROM circulation of selected digital images and presented as individual JPEG file images in a single folder on the CD ROM. The number of images per case ranged from three to eight. The delegates will have an opportunity to assess these themselves prior to uncovering the consensus diagnosis provided by the participants. Of the 76, 29 participants of the scheme provided a completed answer sheet for the image‐based circulation. To date, 19 have now also supplied answers on the actual slide circulation. The same diagnosis on image and slide circulation varied from 5/19 to 19/19 with a mode of 14/19. The case that provided the poorest correlation was because of a generalized undercall of suspicious in a case which achieved almost complete consensus on the slide circulation. It had been the most difficult to photograph. The case that provided the next poorest correlation 11/19 was the case that had the poorest consensus diagnosis on the slide circulation. The two cases that provided the best correlation 18/19 and 19/19 were two cases that provided 100% consensus diagnosis on the slide‐based circulation. Comments were received from 20 participants about the image circulation ranging from ‘great’ to ‘awful’ with the majority of 12 participants not happy for their diagnostic capability to be assessed on such images alone. Two stated that the CD was easier to use on their home computer than their NHS one. In conclusion an image circulation overcomes many of the inherent problems with a slide circulation for EQA purposes and can provide an overall 70% correlation with a slide circulation but a significant number of pathologists do not find this an acceptable method for EQA.     

P‐15COMPARISON OF RESULTS FOR A NON‐GYNAECOLOGICAL CYTOLOGY EQA SCHEME

The problems encountered with a glass slide circulation are legion but timely circulation is a major problem and is an inherent deficiency of our Non‐gynaecological EQA scheme. This applies not only to consultants but also to specialist registrars (SpRs) and technical staff that are not formally included in the circulation list. In 2005 only 7 technical staff and 4 out of 47 SpRs took part on a formal basis, their participation being dependant on access to slides during their cytology attachment. The results for the 2005 circulation have been analysed and despite the small numbers of participating technical staff and SpRs their answers concur with the consultant body. To address the issues of timeliness and circulation problems a pilot teaching set has been developed by SlidePath into a virtual microscope web based circulation and sent to all SpRs in our region. They have recorded their answers and been given immediate access to the consensus consultant opinion with illustrations of follow up histology. A questionnaire was completed to evaluate the scheme. The facility of immediate feedback of consultant consensus is particularly pertinent to the educational element of the scheme and use of virtual microscopy addresses the issue of timely circulation. If further funding was made available technical staff could also be given the opportunity to try this web‐based circulation.     

Different responses to temperature in three closely-related sympatric cereal aphids

Cotesia glomerata L. (Hymenoptera: Braconidae) is a parasitoid of early instar larvae of Pieris brassicae L. (Lepidoptera: Pieridae). Late instars of P. brassicae can more often overcome parasitization by hemocytic encapsulation of C. glomerata eggs. Short-term hemocyte responses to parasitization were examined in third and fourth instar larvae of P. brassicae. Total and differential hemocyte counts did not differ between parasitized and unparasitized host larvae. A rapid, but temporary decrease of total hemocyte as well as plasmatocyte numbers was observed immediately after oviposition. Numbers of hemocytes adhering to tissues were shown to be the same in untreated, wounded and parasitized P. brassicae larvae by tracing hemocytes with monoclonal antibodies as markers. The in vitro spreading ability of hemocytes from unparasitized third and fourth instar larvae was lower than that of the last instar's; parasitization, however, had no influence on hemocyte spreading. We therefore suggest that the higher parasitization success of C. glomerata in earlier instars of P. brassicae is mainly due to the low spreading ability of the hemocytes. Abbreviations: ACS – anticoagulant saline; BSA – bovine serum albumin; DABCO – 1,4-diazabicyclo-[2,2,2]-octane; DHC – differential hemocyte count; FITC – fluorescein isothiocyanate; GR – granular cells; LPS – lipopolysaccharide; mAb – monoclonal antibody; OE – oenocytoids; PL – plasmatocytes; PRO – prohemocytes; PS – Pieris saline; PVP – polyvinylpyrrolidone; TBS – tris-buffered saline; THC – total hemocyte count.     

The effects of pyridine nucleotides on the activity of a calcium-activated nonselective cation channel in the rat insulinoma cell line,CRI-G1

The activity of a calcium-activated nonselective (Ca-NS⁺) channel in a rat insulinoma cell line (CRI-G1) is inhibited by pyridine nucleotides in excised patches. The effects of all four pyridine nucleotides tested, -NAD⁺, -NADH, -NADP⁺ and -NADPH were very similar when tested at 0.1 mm, and at 1 mm the phosphorylated forms, -NADP⁺ and -NADPH, appeared to be slightly more potent than -NAD⁺ and -NADH. All the pyridine nucleotides tested reduced both the open state probability of the channel and the number of functional channels observed in a single patch.The application of -NAD⁺, but not of the other nucleotides tested, to the cytoplasmic surface of isolated inside-out patches from CRI-G1 cells opened a novel nonselective cation channel (the -NAD⁺-NS⁺ channel). The activity of this new channel is calcium sensitive and may also be inhibited by AMP.