Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

The metal directed assembly of a trinuclear macrocyclic copper(II) complex

A trinuclear macrocyclic complex is reported from the metal directed condensation between melamine, formaldehyde and the Cu-II complex of a linear tetraamine.

The first structurally characterized discrete dinuclear mu-cyano hexacyanoferrate complex

Mixed valence complexes containing ferro- and ferricyanide have been known for almost 300 years, but no dinuclear, non-polymeric examples of these complexes have been structurally characterized. Here we report the first such example, comprising ferrocyanide coordinated to a pentaaminecobalt(III) complex. This Fe-II-Co-III complex may be reversibly oxidized to the Fe-III-Co-III analogue.

Regional and total body bone mineral density in elite collegiate male swimmers

Back,ground To examine the role of long-term swimming exercise on regional and total body bone mineral density (BMD) in men. Methods. Experimental design: Cross-sectional. Setting: Musculoskeletal research laboratory at a medical center, Participants:We compared elite collegiate swimmers (n=11) to age-, weight-, and height-matched non-athletic controls (n=11), Measures: BMD (g/cm(2)) of the lumbar spine L2-4, proximal femur (femoral neck, trochanter, Ward's triangle), total body and various subregions of the total body, as well as regional and total body fat and bone mineral-free lean mass (LM) was assessed by dual-energy X-ray absorptiometry (DXA, Hologic QDR 1000/W). Results. Swimmers, who commenced training at 10.7+/-3.7 yrs (mean+/-SD) and trained for 24.7+/-4.2 hrs per week, had a greater amount of LM (p<0.05), lower fat mass (p<0.001) and percent body fat (9.5 vs 16.2 %, p<0.001) than controls. There was no significant difference between groups for regional or total body BRID, In stepwise multiple regression analysis, body weight was a consistent independent predictor of regional and total body BMD, Conclusions. These results suggest that long-term swimming is not an osteogenic mode of training in college-aged males. This supports our previous findings in young female swimmers who displayed no bone mass benefits despite long-standing athletic training.

Crystal and molecular structure of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH) and its iron(III) complex: an iron chelator with anti-tumour activity

Previous studies have demonstrated that 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH) and several other aroylhydrazone chelators possess anti-neoplastic activity due to their ability to bind intracellular iron. In this study we have examined the structure and properties of NIH and its Fe-III complex in order to obtain further insight into its anti-tumour activity. Two tridentate NIH ligands deprotonate upon coordination to Fe-III in a meridional fashion to form a distorted octahedral, high-spin complex. Solution electrochemistry of [Fe(NIH-H)(2)](+) shows that the trivalent oxidation state is dominant over a wide potential range and that the Fe-II analogue is not a stable form of this complex. The fact that [Fe(NIH-H)(2)](+) cannot-cycle between the Fe-II and Fe-III states suggests that the production of toxic free- radical species, e.g. OH. or O2(.-),is not part of this ligand's cytotoxic action. This suggestion is supported by cell culture experiments demonstrating that the addition of Fe-III to NIH prevents its anti-proliferative effect. The chemistry of this chelator and its Fe-III complex are discussed in the context of understanding its anti-tumour activity.

m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Metal-centred versus ligand-centred luminescence quenching of a macrocyclic copper(II) complex

The new macrocyclic ligand trans-6-(9-anthracenylmethylamino)-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecan-13-amine has been synthesized and characterised as its copper(II) complex and the crystal structure of this complex has been determined. Fluorescence of the anthracenyl group of the macrocycle is quenched in its free base form and when complexed with Cu-II. Fluorescence returns when Lewis acids such as H+ and Zn-II are added to solutions of the ligand, indicating that photoinduced electron transfer from the amine lone pairs is responsible for fluorescence quenching in the free base form. By contrast, fluorescence of the complex is quenched by intramolecular electronic energy transfer.

Synthesis of [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)] (PF6)(2), a hydroxo-bridged copper(II) complex of N,N '-dimethyl-1-thia-4,7-diazacyclononane (Me-2[9]aneN(2)S). X-ray structural analysis, magnetic susceptibility, EPR and visible spectroscopy

The bis(mu-hydroxo) complex [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)](PF6)(2) (Me-2[9]aneN(2)S = N,N'-dimethyl-1-thia-4,7-diazacyclononane) results after reaction of [Cu(Me-2[9]aneN(2)S)(MeCN)] (PF6) with dioxygen at -78 degrees C in acetonitrile. The complex has been characterized by X-ray crystallography: orthorhombic, space group Pnma, with a 18.710(3), b 16.758(2), c 9.593(2) Angstrom, and Z = 4. The structure refined to a final R value of 0.051. The complex contains two copper(II) ions bridged by two hydroxo groups with Cu ... Cu 2.866(1) Angstrom. The solid-state magnetic susceptibility study reveals ferromagnetic coupling, the fitting parameters being J = +46+/-5 cm(-1), g = 2.01+/-0.01 and theta = -0.58+/-0.03 K. The frozen-solution e.p.r. spectrum in dimethyl sulfoxide is characteristic of a monomeric copper(II) ion (g(parallel to) 2.300, g(perpendicular to) 2.063; A(parallel to) 156.2 x 10(-4) cm(-1), A(perpendicular to) 9.0 x 10(-4) cm(-1)) with an N2O2 donor set. Thioether coordination to the copper(II) in solution is supported by the presence of an intense absorption assigned to a sigma(S)-->Cu-II LMCT transition at c. 34000 cm(-1). The single-crystal spectrum of [Cu-2(Me-2[9]aneN(2)S)(2)(OH)(2)] (PF6)(2) (273 K) reveals d-->d transitions at 14500 and 18300 cm(-1) and a weak pi(S)-->Cu-II charge-transfer band at approximately 25000 cm(-1).

Life tables of the processionary caterpillar Ochrogaster lunifer Herrich-Schaffer (Lepidoptera : Thaumetopoeidae) at local and regional scales

A major ongoing debate in population ecology has surrounded the causative factors underlying the abundance of phytophagous insects and whether or not these factors limit or regulate herbivore populations. However, it is often difficult to identify mortality agents in census data, and their distribution and relative importance across large spatial scales are rarely understood. Were, we present life tables for egg batches and larval cohorts of the processionary caterpillar Ochrogaster lunifer Herrich-Schaffer, using intensive local sampling combined with extensive regional monitoring to ascertain the relative importance of different mortality factors at different localities. Extinction of entire cohorts (representing the entire reproductive output of one female) at natural localities was high, with 82% of the initial 492 cohorts going extinct. Mortality was highest in the egg and early instar stages due to predation from dermestid beetles, and while different mortality factors (e.g. hatching failure, egg parasitism and failure to establish on the host) were present at many localities, dermestid predation, either directly observed or inferred from indirect evidence, was the dominant mortality factor at 89% of localities surveyed. Predation was significantly higher in plantations than in natural habitats. The second most important mortality factor was resource depletion, with 14 cohorts defoliating their hosts. Egg and larval parasitism were not major mortality agents. A combination of predation and resource depletion consistently accounted for the majority of mortality across localities, suggesting that both factors are important in limiting population abundance. This evidence shows that O. lunifer is not regulated by natural enemies alone, but that resource patches (Acacia trees) ultimately, and frequently, act together to limit population growth.

Regional-scale investigation of the spatial distribution and origin of soluble salts in central north Queensland

A method for regional assessment of the distribution of saline outbreaks is demonstrated for a large area (68 000 km(2)) in north Queensland, Australia. Soil samples were used in conjunction with a digital elevation model and a map of potentially saline discharge zones to examine the landscape distribution of soluble salts in the region. The hypothesis of atmospheric accession of salt was tested for the topographically defined catchment regions feeding into each potentially saline discharge area. Most catchments showed a salt distribution consistent with this hypothesis, i.e. %TSS was large near the discharge areas and decreased rapidly with distance uphill from the discharge areas. In some catchments, however, local saline outbreaks were apparent at significant distances uphill from discharge areas. The possibility of geological sources of this salt was examined by comparing random point distributions with the location of saline points with distance downhill from geological units (excluding points near discharge zones). The distribution of some saline outbreaks was consistent with the occurrence of Cambro-Ordovician metasediments, Devonian limestone, Upper Devonian-Lower Carboniferous volcanics, and Triassic sediments. Copyright (C) 2000 John Wiley & Sons, Ltd.

Identifying regional dust transport pathways: Application of kinematic trajectory modelling to a trans-Tasman case

PI kinematic trajectory model is used to investigate potential pathways of dust transport from Australia to New Zealand. Historically, these have been assumed to follow rather direct west-east trajectories spanning 2 to 3 days, often resulting in red snow events in the Southern Alps of New Zealand. However, results from the present study which examined the route taken by air parcels originating in southern Australia during dust storms on 24 and 25 May 1994, indicate that trans-Tasman dust transport trajectories are more diverse than previously thought, and display considerable variation during single events. These mon divergent pathways tie in more closely with aeolian dust sedimentation patterns identified by ocean coring in the Tasman Sea, and may account for the deposition of Australian dust on sub-Antarctic islands located well south of the Australian continent. Copyright 2000 John Wiley Sons, Ltd.

Glucocorticoid receptors in human preadipocytes: regional and gender differences

Glucocorticoid excess causes visceral obesity and its accompanying insulin resistance, dyslipidemia and hypertension. Glucocorticoids enhance preadipocyte (PA) differentiation and increase their aromatase activity (oestrogen production) and there is regional variability in these PA processes. Therefore, we studied human PAs for the presence of, and any regional or gender differences in, glucocorticoid receptors (GRs). Confluent subcultured human subcutaneous (Sc) and visceral (Vis) PAs from both genders contained GRs as assessed by GR gene expression and specific glucocorticoid (dexamethasone) binding. The dissociation constant was similar to that of other human cells and there was no difference between Sc and Vis sites or between males and females. There was significantly less GR mRNA in Vis PAs compared with Sc PAs in females (P=0.008) but not in males. There was less glucocorticoid binding in Vis compared with Sc PAs in females, measured by maximal binding capacity (P=0.035) or single saturating dose glucocorticoid binding (Bssd) (P=0.019). There was no regional difference in specific glucocorticoid binding in males. There was a gender difference with fewer GRs in Vis PAs in females compared with males measured by Bssd (P=0.006). In summary, GRs are present in human PAs. There is a lower GR density in Vis compared with Sc PAs in females, and females have fewer GRs in Vis PAs compared with males. These differences are likely to affect regional aromatase activity and to contribute to the smaller visceral fat mass in females compared with males.

X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure

Plant performance is, at least partly, linked to the location of roots with respect to soil structure features and the micro-environment surrounding roots. Measurements of root distributions from intact samples, using optical microscopy and field tracings have been partially successful but are imprecise and labour-intensive. Theoretically, X-ray computed micro-tomography represents an ideal solution for non-invasive imaging of plant roots and soil structure. However, before it becomes fast enough and affordable or easily accessible, there is still a need for a diagnostic tool to investigate root/soil interplay. Here, a method for detection of undisturbed plant roots and their immediate physical environment is presented. X-ray absorption and phase contrast imaging are combined to produce projection images of soil sections from which root distributions and soil structure can be analyzed. The clarity of roots on the X-ray film is sufficient to allow manual tracing on an acetate sheet fixed over the film. In its current version, the method suffers limitations mainly related to (i) the degree of subjectivity associated with manual tracing and (ii) the difficulty of separating live and dead roots. The method represents a simple and relatively inexpensive way to detect and quantify roots from intact samples and has scope for further improvements. In this paper, the main steps of the method, sampling, image acquisition and image processing are documented. The potential use of the method in an agronomic perspective is illustrated using surface and sub-surface soil samples from a controlled wheat trial. Quantitative characterization of root attributes, e.g. radius, length density, branching intensity and the complex interplay between roots and soil structure, is presented and discussed.

Glutamate-mediated transmission, alcohol, and alcoholism

Glutamate-mediated neurotransmission may be involved in the range of adaptive changes in brain which occur after ethanol administration in laboratory animals, and in chronic alcoholism in human cases. Excitatory amino acid transmission is modulated by a complex system of receptors and other effecters, the efficacy of which can be profoundly affected by altered gene or protein expression. Local variations in receptor composition may underlie intrinsic regional variations in susceptibility to pathological change. Equally, ethanol use and abuse may bring about alterations in receptor subunit expression as the essence of the adaptive response. Such considerations may underlie the regional localization characteristic of the pathogenesis of alcoholic brain damage, or they may form part of the homeostatic change that constitutes the neural substrate for alcohol dependence. (C) 2000 Elsevier Science Ltd. All rights reserved.

Th structure of the P-II-ATP complex

P-II is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of P-II was solved using a hexagonal crystal form (form I). The more physiologically relevant form of P-II is a complex with small molecule effecters. We describe the structure of P-II with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co-crystallization of P-II with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the P-II/ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that P-II and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.