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PubMed2005

Late postnatal maturation of excitatory synaptic transmission permits adult-like expression of hippocampal-dependent behaviors.

Sensorimotor systems in altricial animals mature incrementally during early postnatal development, with complex cognitive abilities developing late. Of prominence are cognitive processes that depend on an intact hippocampus, such as contextual-configural learning, allocentric and idiocentric navigation, and certain forms of trace conditioning. The mechanisms that regulate the delayed maturation of the hippocampus are not well understood. However, there is support for the idea that these behaviors come "on line" with the final maturation of excitatory synaptic transmission. First, by providing a timeline for the first behavioral expression of various forms of learning and memory, this study illustrates the late maturation of hippocampal-dependent cognitive abilities. Then, functional development of the hippocampus is reviewed to establish the temporal relationship between maturation of excitatory synaptic transmission and the behavioral evidence of adult-like hippocampal processing. These data suggest that, in rats, mechanisms necessary for the expression of adult-like synaptic plasticity become available at around 2 postnatal weeks of age. However, presynaptic plasticity mechanisms, likely necessary for refinement of the hippocampal network, predominate and impede information processing until the third postnatal week.

Animals↗
PubMed2004

RT-PCR permits simultaneous genotyping of thiopurine S-methyltransferase allelic variants by multiplex induced heteroduplex analysis.

Thiopurine-based drugs are a widely prescribed group of medications. Their tolerance and effectiveness is dependent on an individual's ability to metabolize these compounds. An essential enzyme for the metabolism of these drugs is thiopurine S-methyltransferase (TPMT), whose activity is subject to genetic variation. Genotyping of the most frequent allelic variants in TPMT affords an extremely accurate prediction of the three clinical phenotypes: high, intermediate, and low enzyme activity. One constraint of most genotyping methods is the inability to demonstrate physical linkage between two sequence variants that occur in different exons, e.g., c.460G>A and c.719A>G, which give rise to TPMT*3, the most common defective allele in Caucasian populations. Using mRNA/cDNA as a template enables analysis of both sequence variants in a single assay. This approach could be applicable to other genes where allelic variation (in-cis and in-trans) is due to alterations in different exons. Induced heteroduplex generator analysis has previously been shown to discriminate in-cis and has also been suitable for multiplexing. In this method we have exploited both these features and for the first time have applied them to a RT-PCR analysis. The primary reagent developed allows unequivocal resolution of TPMT*3A and the alleles carrying the c.719A>G allelic variant, TPMT*3C, as well as the silent alteration c.474T>C. The TPMT*3B variant has not been observed. A secondary reagent, which can be multiplexed, identifies the TPMT*2 allele.

Alleles↗
PubMed1981

Cyclosporin a permits the distinction between specific T and NK activity generated in a human MLC.

Human lymphocytes, activated in mixed lymphocyte culture (MLC), express cytolytic activity against a range of target cell types. Cyclosporin A (CyA) efficiently prevents the development of cytotoxic T cells directed against target cells expressing the phenotype of the stimulator cells whilst not preventing an increase in natural killing (NK) activity measured against tumor targets. The drug was further shown not to influence the short-term induction of increased NK activity by interferon. Already established T killer cells are not sensitive to CyA. The drug thus seems to be selectively involved in the induction of immune responses requiring T-cell proliferation.

Cyclosporins↗
PubMed1990

General primer-mediated polymerase chain reaction permits the detection of sequenced and still unsequenced human papillomavirus genotypes in cervical scrapes and carcinomas.

A newly developed general primer-mediated polymerase chain reaction (GP-PCR) was used for the detection of a broad spectrum of Human Papilloma-virus (HPV) genotypes, including unsequenced types, in cytologically normal and abnormal cervical smears and in biopsies of cervical carcinomas. This PCR method used different general primer sets, located in strongly conserved EI and LI regions of the HPV genome. Comparison between results of GP-PCR and HPV-type-specific PCR (TS-PCR) revealed an increase in overall HPV prevalence to 25%, 80% and 88% in scrapes with normally, slightly and severely dysplastic cells, respectively. Unsequenced HPV types were detected in 11% of cytologically normal swabs and in up to 30% of scrapes with dysplastic cells. Further characterization showed that unsequenced types concern HPV 13, 30, 31, 45, 51 and some other, possibly unknown HPV types. More than 90% of carcinomas in situ and invasive cervical carcinomas contained HPV. In the latter, only HPV16 and HPV18 were present. HPV16 was most frequently found in both normal and dysplastic cells, the rate being highest in neoplastic tissue. These results indicate that GP-PCR is a powerful approach for detecting as yet uncharacterized HPV types associated with neoplastic transformation of cervical squamous cell epithelium.

Adolescent↗
PubMed1995

Gene transfer of human TNF alpha into glioblastoma cells permits modulation of mdr1 expression and potentiation of chemosensitivity.

Despite substantial advances in the surgery, radiotherapy and chemotherapy of gliomas, the prognosis of patients with glioblastomas has still not improved. Disappointing results in chemotherapy of glioblastomas resulting from multi-drug resistance (MDR) prompted us to investigate the influence of cytokine gene transfer in glioblastoma cells on the expression of P-glycoprotein and on chemosensitivity of transduced cells. Several investigations have shown that malignant gliomas express P-glycoprotein at high levels. The P-glycoprotein is a product of the multi-drug resistance gene (mdr1) and functions as an energy-dependent efflux pump which decreases drug accumulation and cytotoxicity. Since tumour necrosis factor alpha (TNF alpha) is a powerful anti-cancer agent used in clinical trials and gene therapy protocols, this cytokine gene was chosen for the present investigations. Transduction of the human TNF alpha (hTNF) gene carrying retroviral vector pN2tk-hTNF into U373MG human glioblastoma cells resulted in expression and secretion of biologically active hTNF. Release of transduced hTNF reduces P-glycoprotein expression and is associated with enhanced rhodamine-123 uptake and potentiation of cytotoxicity of the MDR-relevant drugs vincristine and doxorubicin. Furthermore, the transfected cell clones showed a reduced growth rate compared to the parental cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1982

SV40-transformed human cells fail to grow in zinc concentrations which permit normal human fibroblast proliferation.

Zinc is a metal known to be required for normal growth of both cells, and organisms. When normal and SV40-transformed human tumor cells are plated and grown in medium containing zinc sulfate, a significant fraction of the transformed cells fail to grow at zinc concentrations which are relatively nontoxic to the normal fibroblasts. Although cultured cells respond to certain metallic ions by increasing their metallothionein content, no difference in the ability of normal and transformed cells to produce metallothionein in response to zinc exposure could be detected, thus ruling out this mechanism as a basis for the differing abilities of the cells to grow in zinc in vitro. These results suggest that zinc may be capable of differentially regulating the growth of normal and SV40-transformed human fibroblasts.

Cell Division↗
PubMedDate not supplied

Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo.

BACKGROUND: Gene delivery vectors based on poly(L-lysine) and DNA (pLL/DNA complexes) have limited use for targeted systemic application in vivo since they bind cells and proteins non-specifically. In this study we have attempted to form folate-targeted vectors with extended systemic circulation by surface modification of pLL/DNA complexes with hydrophilic polymers. METHODS: pLL/DNA complexes were stabilised by surface modification with a multivalent reactive polymer based on alternating segments of poly(ethylene glycol) and tripeptides bearing reactive ester groups. Folate moieties were incorporated into the vectors either by direct attachment of folate to the polymer or via intermediate poly(ethylene glycol) spacers of 800 and 3400 Da. RESULTS: Polymer-coated complexes show similar morphology to uncoated complexes, their zeta potential is decreased towards zero, serum protein binding is inhibited and aqueous solubility is substantially increased. Intravenous (i.v.) administration to mice of coated complexes produced extended systemic circulation, with up to 2000-fold more DNA measured in the bloodstream after 30 min compared with simple pLL/DNA complexes. In further contrast to simple pLL/DNA complexes, coated complexes do not bind blood cells in vivo. Folate receptor targeting is shown to mediate targeted association with HeLa cells in vitro, leading to increased transgene expression. We demonstrate for the first time that DNA uptake via the folate receptor is dependent on pEG spacer length, with the transgene expression relatively independent of the level of internalised DNA. CONCLUSIONS: We show increased systemic circulation, decreased blood cell and protein binding, and folate-targeted transgene expression using pLL/DNA complexes surface-modified with a novel multireactive hydrophilic polymer. This work provides the basis for the development of plasma-circulating targeted vectors for in vivo applications.

DNA↗
PubMed1991

Isoprenoid modification permits 2',3'-cyclic nucleotide 3'-phosphodiesterase to bind to membranes.

The myelination-related enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a relatively abundant protein in the CNS possesses the C-terminal isoprenylation consensus domain found in a small family that includes the ras oncoproteins and their relatives, some G-proteins, and nuclear lamins. We found that CNP, like these other proteins, is modified posttranslationally by an isoprenoid derived from mevalonic acid. It appears that only the smaller of the two CNP isoforms (CNP1) is isoprenylated, but similar modification of CNP2 cannot be excluded. Inhibition of isoprenoid synthesis by Lovastatin blocks the binding of newly synthesized CNP to cell membranes; binding is restored upon addition of mevalonate to the culture medium. This shows that isoprenylation is permissive for the well-known avid association of CNP with membranes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1988

Venous drainage of the femur permits passage of 100-micron particles.

Iatrogenic metastasis of bone tumor is reportedly increased if aggregates of tumor cells enter the circulation. The current study used a previously developed animal model to explore the movement of 25-, 50-, and 100-micron-diameter particles from an experimental femur biopsy site into the circulation. The results indicate that cell aggregate-sized particles can enter the circulation from the femur at a rate similar to that of the 15-micron particles used in previous studies. Arterial blood samples collected during and after the injection of particles contained no 50- or 100-micron particles, suggesting that the lung is an effective filter for these larger particles.

Animals↗
PubMed1987

The use of the carbon dioxide laser permits primary closure of contaminated and purulent lesions and wounds.

Excision of contaminated and purulent wounds with the carbon dioxide laser makes it safe to close the wounds primarily when certain steps are observed during the operation. The contaminated surface is sterilized with the defocused beam. The surgeons' and assistants' hands should not touch the contaminated areas, and both should wear two pairs of rubber gloves to protect against accidental contamination. All infected tissue is excised completely, and the wound cleansed by copious irrigation and the defocused laser. The wound may then be safely closed by simple sutures, skin graft, or by the rotation of a vascularized musculocutaneous flap. A series of cases are presented in order to illustrate these points.

Abscess↗
PubMed2005

Response to an experimental HBV vaccine permits withdrawal of HBIg prophylaxis in fulminant and selected chronic HBV-infected liver graft recipients.

Strategies using lamivudine and hepatitis B immunoglobulins (HBIg) for prevention of hepatitis B virus (HBV) reinfection after liver transplantation (LT) are expensive since life-long treatment is needed. We evaluated the possibility to obtain protective hepatitis B surface antigen (HBsAg) antibody (anti-HBs) titers after LT and to discontinue HBIg prophylaxis after a reinforced course of vaccination against HBV using an experimental adjuvant HBsAg / AS04 vaccine (GlaxoSmithKline Biologicals [GSK], Rixensart, Belgium) in patients transplanted for hepatitis B. Fifteen LT patients on stable low-level immunosuppression were vaccinated with a double dose of the vaccine at 0, 1, 2, 6, and 12 months: 5 patients were transplanted for nonviral diseases and 10 patients were transplanted for HBV on HBIg monotherapy. HBIg were continued during baseline vaccination (0, 1, and 2 months) and when anti-HBs titers determined every 6 weeks dropped below 150 IU/L. Overall follow-up was 18 months. Sustained long-term response to vaccination was defined as anti-HBs titers >500 IU/L without further need for HBIg administration during a follow-up period of at least 12 months. Overall sustained response to vaccination was 53% (8 / 15 patients); 80% (4 / 5 patients) in the nonviral disease group and 40% (4 / 10 patients) in the HBV group (2 /2 fulminant and 2/8 chronically infected patients) developed a sustained long-term response and were completely free of HBIg at the end of the 18-month follow-up. No HBV recurrence, rejection episodes, or side effects occurred during the follow-up. In conclusion, protective anti-HBs titers were obtained in a substantial number of LT patients following a reinforced course of HBV vaccination with vaccines containing new immunostimulating adjuvants. Vaccination seems well tolerated and safe and allows long-term discontinuation of HBIg.

Adult↗
PubMed1987

Silencing normal input permits regenerating foreign afferents to innervate an identified crayfish sensory interneuron.

Interneuron A, an identified first-order sensory interneuron that is innervated by mechanoreceptors on one side of the crayfish tailfan, normally resists extra innervation by regenerating contralateral mechanoreceptor axons. However, if its normal innervation is silenced by covering mechanosensory hairs with a surgical glue, it accepts contralateral innervation. This finding on an arthropod provides evidence for the generality and antiquity of the principle, critical in development of the vertebrate nervous system, that activity of one set of afferents can control whether other afferents form synapses on a target.

Adhesives↗
PubMed1994

Non-enzymatic retrieval of antigen permits staining of follicle centre cells by the rabbit polyclonal antibody to protein gene product 9.5.

The rabbit polyclonal antibody to protein gene product 9.5 (PGP9.5) will detect the L1 isoenzyme of ubiquitin carboxy-terminal hydrolase (UCH), which is a marker for neurones and neuroendocrine tissue. We re-evaluated this antibody using the technique of non-enzymatic antigen retrieval (boiling sections in citrate buffer, heated by microwave oven) followed by streptavidin-biotin-peroxidase staining. Due to the fortuitous choice of appendix as positive control material containing small nerves, we found strong, repeatable cytoplasmic and nuclear staining of lymphoid follicle centre cells in addition to neural tissue. This effect could be repeated on other lymphoid tissues and was not dependent on microwave heating, but did require boiling in an ionic buffer solution. Staining was also observed with a fresh batch of antibody and with four of the five different batches of antibody which were supplied to us. This pattern was not obtained in fresh tissue, in fixed material following trypsinization, or by increasing the primary antibody concentration. We suggest that the boiling of sections in citrate buffer is exposing an epitope for the anti-PGP9.5 antibody which is inaccessible in the native or fixed state and therefore we would recommend retesting of antibody specificity following non-enzymatic retrieval of antigen.

Animals↗
PubMed2004

Congenital diaphragmatic hernia: prenatal diagnosis permits immediate intensive care with high survival rate in isolated cases. A population-based study.

OBJECTIVES: To estimate the prognosis of prenatally diagnosed isolated congenital diaphragmatic hernia (PDICDH) treated with 'immediate planned care' (IPC) between 1999 and 2003 in Eastern Brittany. METHODS: The prognosis of PDICDH was compared with the prognosis of the other live-born CDH, either prenatally undiagnosed or not having had IPC. IPC consisted in prenatal lung maturation with corticosteroids, elective caesarean section at 37 weeks, immediate intubation, surfactant, high- frequency ventilation or oscillation, nitric oxide, intravenous prostacyclin, anaesthesia and haemodynamic support. Surgical repair was performed in the NICU 34 h after birth. RESULTS: The incidence of CDH was 0.8 per thousand with a prenatal diagnosis rate of 27/30 (90%), leading to a termination of pregnancy in nine cases. Ten CDH were associated with other malformations. IPC in PDICDH was performed in 12 cases. The survival rate of PDICDH with IPC was 11/12 versus 1/9 in CDH with no IPC or no prenatal diagnosis (p < 0.01). Logistic regression analysis showed that IPC was determinant for survival (p < 0.01). CONCLUSION: Prenatal diagnosis of isolated CDH treated with immediate planned care is associated with a high survival rate. This suggests that prenatal diagnosis associated with specifically adapted postnatal procedure may improve the prognosis of isolated CDH.

Adrenal Cortex Hormones↗
PubMed1983

Development of an in vitro clonogenic assay for the R3327 rat prostatic adenocarcinoma permits comparison of the proliferative potential of the R3327, R3327A, and R3327AT tumors.

The R3327 class of rat prostate tumors consists of both the androgen-dependent R3327 adenocarcinoma and androgen-independent sublines, the R3327At spindle cell tumor, and the R3327A, a squamous cell carcinoma. We have developed in vitro clonogenic cell assays to measure and compare systematically the proliferative potential of these tumors following various monodispersion techniques. Linear relationships between the number of monodispersed tumor cells cultured at low cell density and the number of colonies formed 10 days later establish these assays as the first quantitative cellular approach to those proliferative subpopulations ultimately responsible for the growth of these tumors. We have chosen the name colony forming cell-prostatic adenocarcinoma (CFC-PA) to refer to the members of the proliferative subpopulation of the R3327 tumor. An ultrastructural comparison of R3327 adenocarcinoma tissue sections with the cells produced during culture provides evidence that the cells of the proliferative subpopulation may be derived from the acinar epithelium of the tumor.

Adenocarcinoma↗
PubMed2003

A consensus-binding structure for adenine at the atomic level permits searching for the ligand site in a wide spectrum of adenine-containing complexes.

Attempts to derive structural features of ligand-binding sites have traditionally involved seeking commonalities at the residue level. Recently, structural studies have turned to atomic interactions of small molecular fragments to extract common binding-site properties. Here, we explore the use of larger ligand elements to derive a consensus binding structure for the ligand as a whole. We superimposed multiple molecular structures from a nonredundant set of adenosine-5'-triphosphate (ATP) protein complexes, using the adenine moiety as template. Clustered binding-site atoms of compatible atomic classes forming attractive contacts with the adenine probe were extracted. A set of atomic clusters characterizing the adenine binding pocket was then derived. Among the clusters are three vertices representing the interactions of adenine atom N6 with its protein-binding niche. These vertices, together with atom C6 of the purine ring system, complete the set of four vertices for the pyramid-like structure of the N6 anchor atom. Also, the sequence relationship for the adenine-binding loop interacting with the C2-N6 end of the conjugated ring system is expanded to include a third hydrophilic cluster interacting with atom N1. A search procedure involving interatomic distances between cluster centers was formulated and applied to seek putative binding sites in test cases. The results show that a consensus network of clusters, based on an adenine probe and an ATP-complexed training set of proteins, is sufficient to recognize the experimental cavity for adenine in a wide spectrum of ligand-protein complexes.

Adenine↗
PubMed1980

Palate development in the mouse: a quantitative method that permits the estimation of time and rate of palate closure.

A quantitative technique is used to express the morphogenetic stages of palate development in the mouse in terms of gestational age and the normal probability distribution. The qualitative palate rating scale for mouse embryos was modified and the numbers of embryos that had reached the different palate stages at specific gestational ages were fitted to a cumulative multinomial frequency distribution by normal probability regression on gestational age. The median time for each stage can be estimated and the standard deviation of this time (slope of the probability regression) provides a measure of the rate of development. A/J and A.B6 F1 embryos (A/J maternal environment) differed significantly in median time but not in rate of development during a restricted examination time.

Animals↗
PubMed1993

Isolation and characterization of flavoredoxin, a new flavoprotein that permits in vitro reconstitution of an electron transfer chain from molecular hydrogen to sulfite reduction in the bacterium Desulfovibrio gigas.

A new FMN-containing flavoprotein isolated from Desulfovibrio gigas provided maximum coupling efficiency for the reduction of bisulfite from molecular H2. This protein, which is distinct from flavodoxin and for which the name flavoredoxin is proposed, is required for reconstitution of an electron transfer chain between hydrogenase and bisulfite reductase. A Ca(2+)-binding protein functions as a modulator in the presence of Ca2+ in the process. The finding of a membrane-bound cytochrome c with a molecular weight of 104,000 Da that is also active in this electron transfer chain provides an explanation for the energetic linkage between periplasmic and cytoplasmic proteins in this sulfate-reducing bacterium.

Amino Acid Sequence↗