The Research Funding Service: a model for expanded library services

Traditionally, libraries have provided a modest amount of information about grants and funding opportunities to researchers in need of research funding. Ten years ago, the University of Washington (UW) Health Sciences Libraries and Information Center joined in a cooperative effort with the School of Medicine to develop a complete, library-based grant and funding service for health sciences researchers called the Research Funding Service. The library provided space, access to the library collection, equipment, and electronic resources, and the School of Medicine funded staff and operations. The range of services now includes individual consultation appointments, an extensive Web site, classes on funding database searching and writing grant applications, a discussion series that frequently hosts guest speakers, a monthly newsletter with funding opportunities of interest to the six health sciences schools, extensive files on funding sources, and referral services.

Public library consumer health information pilot project: results of a National Library of Medicine evaluation

In October 1998, the National Library of Medicine (NLM) launched a pilot project to learn about the role of public libraries in providing health information to the public and to generate information that would assist NLM and the National Network of Libraries of Medicine (NN/LM) in learning how best to work with public libraries in the future. Three regional medical libraries (RMLs), eight resource libraries, and forty-one public libraries or library systems from nine states and the District of Columbia were selected for participation. The pilot project included an evaluation component that was carried out in parallel with project implementation. The evaluation ran through September 1999. The results of the evaluation indicated that participating public librarians were enthusiastic about the training and information materials provided as part of the project and that many public libraries used the materials and conducted their own outreach to local communities and groups. Most libraries applied the modest funds to purchase additional Internet-accessible computers and/or upgrade their health-reference materials. However, few of the participating public libraries had health information centers (although health information was perceived as a top-ten or top-five topic of interest to patrons). Also, the project generated only minimal usage of NLM's consumer health database, known as MEDLINEplus, from the premises of the monitored libraries (patron usage from home or office locations was not tracked). The evaluation results suggested a balanced follow-up by NLM and the NN/LM, with a few carefully selected national activities, complemented by a package of targeted activities that, as of January 2000, are being planned, developed, or implemented. The results also highlighted the importance of building an evaluation component into projects like this one from the outset, to assure that objectives were met and that evaluative information was available on a timely basis, as was the case here.

Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences.

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."

Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice.

Transgenic mice were generated in which the cDNA for the human insulin-like growth factor 1B (IGF-1B) was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene, IGF-1B mRNA was not detectable in the fetal heart at the end of gestation, was present in modest levels at 1 day after birth, and increased progressively with postnatal maturation, reaching a peak at 75 days. Myocytes isolated from transgenic mice secreted 1.15 +/- 0.25 ng of IGF-1 per 10(6) cells per 24 hr versus 0.27 +/- 0.10 ng in myocytes from homozygous wild-type littermates. The plasma level of IGF-1 increased 84% in transgenic mice. Heart weight was comparable in wild-type littermates and transgenic mice up to 45 days of age, but a 42%, 45%, 62%, and 51% increase was found at 75, 135, 210, and 300 days, respectively, after birth. At 45, 75, and 210 days, the number of myocytes in the heart was 21%, 31%, and 55% higher, respectively, in transgenic animals. In contrast, myocyte cell volume was comparable in transgenic and control mice at all ages. In conclusion, overexpression of IGF-1 in myocytes leads to cardiomegaly mediated by an increased number of cells in the heart.

Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with late-phase long-term potentiation in both Schaffer collateral and mossy fiber pathways.

The gene encoding tissue-type plasminogen activator (t-PA) is an immediate response gene, downstream from CREB-1 and other constitutively expressed transcription factors, which is induced in the hippocampus during the late phase of long-term potentiation (L-LTP). Mice in which the t-PA gene has been ablated (t-PA-/-) showed no gross anatomical, electrophysiological, sensory, or motor abnormalities but manifest a selective reduction in L-LTP in hippocampal slices in both the Schaffer collateral-CA1 and mossy fiber-CA3 pathways. t-PA-/- mice also exhibit reduced potentiation by cAMP analogs and D1/D5 agonists. By contrast, hippocampal-dependent learning and memory were not affected in these mice, whereas performance was impaired on two-way active avoidance, a striatum-dependent task. These results provide genetic evidence that t-PA is a downstream effector gene important for L-LTP and show that modest impairment of L-LTP in CA1 and CA3 does not result in hippocampus-dependent behavioral phenotypes.

Interaction of the histone (H3-H4)2 tetramer of the nucleosome with positively supercoiled DNA minicircles: Potential flipping of the protein from a left- to a right-handed superhelical form.

We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two H3s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a left- to a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame, R. W., Wang, B. C., Love, W. E. & Moudrianakis, E. N. (1991) Proc. Natl.Acad. Sci. USA 88, 10148-10152] that this change in DNA topology is mediated by a similar change in the topology of the tetramer itself, which may occur through a rotation (or a localized deformation) of the two H3-H4 dimers about their H3-H3 interface. Potential implications of this model for nucleosome dynamics in vivo are discussed.

The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for Cln G1 cyclin function at Start.

In yeast, commitment to cell division (Start) is catalyzed by activation of the Cdc28 protein kinase in late G1 phase by the Cln1, Cln2, and Cln3 G1 cyclins. The Clns are essential, rate-limiting activators of Start because cells lacking Cln function (referred to as cln-) arrest at Start and because CLN dosage modulates the timing of Start. At or shortly after Start, the development of B-type cyclin Clb-Cdc28 kinase activity and initiation of DNA replication requires the destruction of p40SIC1, a specific inhibitor of the Clb-Cdc28 kinases. I report here that cln cells are rendered viable by deletion of SIC1. Conversely, in cln1 cln2 cells, which have low CLN activity, modest increases in SIC1 gene dosage cause inviability. Deletion of SIC1 does not cause a general bypass of Start since (cln-)sic1 cells remain sensitive to mating pheromone-induced arrest. Far1, a pheromone-activated inhibitor of Cln-Cdc28 kinases, is dispensable for arrest of (cln-)sic1 cells by pheromone, implying the existence of an alternate Far1-independent arrest pathway. These observations define a pheromone-sensitive activity able to catalyze Start only in the absence of p40SIC1. The existence of this activity means that the B-type cyclin inhibitor p40SIC1 imposes the requirement for Cln function at Start.

Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins.

We used novel immunofluorescence strategies to demonstrate that outer surface proteins (Osps) A, B and C of Borrelia burgdorferi have limited surface exposure, finding that contradicts the prevailing viewpoint that these antigens are exclusively surface exposed. Light labeling was observed when antibodies to OspA or OspB were added to motile organisms, whereas intense fluorescence was observed when the same slides were methanol-fixed and reprobed. Modest labeling also was observed when spirochetes encapsulated in agarose beads (gel microdroplets) were incubated with antibodies to these same two antigens. This contrasted with the intense fluorescence observed when encapsulated spirochetes were probed in the presence of 0.06% Triton X-100, which selectively removed outer membranes. Proteinase K (PK) treatment of encapsulated spirochetes abrogated surface labeling. However, PK-treated spirochetes fluoresced intensely after incubation with antibodies to OspA or OspB in the presence of detergent, confirming the existence of large amounts of subsurface Osp antigens. Modest surface labeling once again was detected when PK-treated spirochetes were reprobed after overnight incubation, a result consistent with the existence of a postulated secretory apparatus that shuttles lipoproteins to the borrelial surface. Last, experiments with the OspC-expressing B. burgdorferi strain 297 revealed that this antigen was barely detectable on spirochetal surfaces even though it was a major constituent of isolated outer mem- branes. We propose a model of B. burgdorferi molecular architecture that helps to explain spirochetal persistence during chronic Lyme disease.

Suppression of diet-induced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase.

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. Conflicting results have been reported concerning its role in atherogenesis. To determine the effects of the overexpressed LPL on diet-induced atherosclerosis, we have generated low density lipoprotein receptor (LDLR) knockout mice that overexpressed human LPL transgene (LPL/LDLRKO) and compared their plasma lipoproteins and atherosclerosis with those in nonexpressing LDLR-knockout mice (LDLRKO). On a normal chow diet, LPL/LDLRKO mice showed marked suppression of mean plasma triglyceride levels (32 versus 236 mg/dl) and modest decrease in mean cholesterol levels (300 versus 386 mg/dl) as compared with LDLRKO mice. Larger lipoprotein particles of intermediate density lipoprotein (IDL)/LDL were selectively reduced in LPL/LDLRKO mice. On an atherogenic diet, both mice exhibited severe hypercholesterolemia. But, mean plasma cholesterol levels in LPL/ LDLRKO mice were still suppressed as compared with that in LDLRKO mice (1357 versus 2187 mg/dl). Marked reduction in a larger subfraction of IDL/LDL, which conceivably corresponds to remnant lipoproteins, was observed in the LPL/LDLRKO mice. LDLRKO mice developed severe fatty streak lesions in the aortic sinus after feeding with the atherogenic diet for 8 weeks. In contrast, mean lesion area in the LPL/LDLRKO mice was 18-fold smaller than that in LDLRKO mice. We suggest that the altered lipoprotein profile, in particular the reduced level of remnant lipoproteins, is mainly responsible for the protection by LPL against atherosclerosis.

Hippocampal acetylcholine release during memory testing in rats: augmentation by glucose.

Several lines of evidence indicate that a modest increase in circulating glucose levels enhances memory. One mechanism underlying glucose effects on memory may be an increase in acetylcholine (ACh) release. The present experiment determined whether enhancement of spontaneous alternation performance by systemic glucose treatment is related to an increase in hippocampal ACh output. Samples of extracellular ACh were assessed at 12-min intervals using in vivo microdialysis with HPLC-EC. Twenty-four minutes after an intraperitoneal injection of saline or glucose (100, 250, or 1000 mg/kg), rats were tested in a four-arm cross maze for spontaneous alternation behavior combined with microdialysis collection. Glucose at 250 mg/kg, but not 100 or 1000 mg/kg, produced an increase in spontaneous alternation scores (69.5%) and ACh output (121.5% versus baseline) compared to alternation scores (44.7%) and ACh output (58.9% versus baseline) of saline controls. The glucose-induced increase in alternation scores and ACh output was not secondary to changes in locomotor activity. Saline and glucose (100-1000 mg/kg) treatment had no effect on hippocampal ACh output when rats remained in the holding chamber. These findings suggest that glucose may enhance memory by directly or indirectly increasing the release of ACh. The results also indicate that hippocampal ACh release is increased in rats performing a spatial task. Moreover, because glucose enhanced ACh output only during behavioral testing, circulating glucose may modulate ACh release only under conditions in which cholinergic cells are activated.

Hemodynamic effects of epidermal growth factor in conscious rats and monkeys.

The therapeutic application of growth factors to human disease has become closer to reality with the advent of faster means of synthesizing these molecules and novel drug delivery strategies. Epidermal growth factor (EGF) belongs to a large family of molecules with the ability to modulate growth. Purified extracts of EGF have been used clinically to modulate gastrointestinal secretion of hormones and accelerate healing. EGF is also reported to have both vascular smooth muscle contractile and relaxing activity Cardiovascular studies were performed with the bioactive 48-amino acid fragment of human EGF in rodents and primates to determine the effects of EGF on blood pressure and heart rate in conscious animals. Intravenous infusion of EGF induced an initial pressor response in rats followed by a prolonged decrease in blood pressure. In contrast, in monkeys, EGF had dose-related blood pressure-lowering effects only; significant hypotension was observed at doses ranging from 3 to 300 microg/kg i.v. Hypotension was associated with modest tachycardia in both species. To our knowledge, this is the first report of hemodynamic effects of EGF in primates, and it clearly documents that the mitogenic role of growth factors such as EGF is but one aspect of their physiology.

On the mechanism of the anticlotting action of vitamin E quinone.

Vitamin E in the reduced, alpha-tocopherol form shows very modest anticlotting activity. By contrast, vitamin E quinone is a potent anticoagulant. This observation may have significance for field trials in which vitamin E is observed to exhibit beneficial effects on ischemic heart disease and stroke. Vitamin E quinone is a potent inhibitor of the vitamin K-dependent carboxylase that controls blood clotting. A newly discovered mechanism for the inhibition requires attachment of the active site thiol groups of the carboxylase to one or more methyl groups on vitamin E quinone. The results from a series of model reactions support this interpretation of the anticlotting activity associated with vitamin E.

Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

We and other groups have recently reported the potentiation by ribonucleotide reductase inhibitors such as hydroxyurea of the anti-human immunodeficiency virus type 1 (HIV-1) activity of purine and pyrimidine 2',3'-dideoxynucleosides in both resting and phytohemagglutinin-stimulated peripheral blood mononuclear cells. Little agreement prevails, however, as to the mechanism of the synergistic effects described. We report here that in phytohemagglutinin-stimulated peripheral blood mononuclear cells, two mechanisms exist for the potentiation of the anti-HIV-1 activity by low-dose hydroxyurea of the purine-based dideoxynucleoside 2',3'-dideoxyinosine and the pyrimidine-based dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine. For 2',3'-dideoxyinosine, the enhancement arises from a specific depletion of dATP by hydroxyurea, resulting in a favorable shift of the 2',3'-dideoxyadenosine 5'-triphosphate/dATP ratio. For the pyrimidine dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, the more modest anti-HIV enhancement results from hydroxyurea-induced increases of pyrimidine kinase activities in the salvage pathway and, hence, increased 5'-phosphorylation of these drugs, while depletion of the corresponding deoxynucleoside 5'-triphosphates (dTTP and dCTP) plays no significant role.

Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.

The very low density lipoprotein (VLDL) receptor is a recently cloned member of the low density lipoprotein (LDL) receptor family that mediates the binding and uptake of VLDL when overexpressed in animal cells. Its sequence is 94% identical in humans and rabbits and 84% identical in humans and chickens, implying a conserved function. Its high level expression in muscle and adipose tissue suggests a role in VLDL triacylglycerol delivery. Mutations in the chicken homologue cause female sterility, owing to impaired VLDL and vitellogenin uptake during egg yolk formation. We used homologous recombination in mouse embryonic stem cells to produce homozygous knockout mice that lack immunodetectable VLDL receptors. Homozygous mice of both sexes were viable and normally fertile. Plasma levels of cholesterol, triacylglycerol, and lipoproteins were normal when the mice were fed normal, high-carbohydrate, or high-fat diets. The sole abnormality detected was a modest decrease in body weight, body mass index, and adipose tissue mass as determined by the weights of epididymal fat pads. We conclude that the VLDL receptor is not required for VLDL clearance from plasma or for ovulation in mice.

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.