Capacity building through focus group training in community-based participatory research.

BACKGROUND: Community-based participatory research (CBPR) emphasizes collaborative efforts among communities and academics where all members are equitable contributors. Capacity building through training in research methodology is a potentially important outcome for CBPR partnerships. OBJECTIVES: To describe the logistics and lessons learned from building community research capacity for focus group moderation in the context of a CBPR partnership. METHODS: After orientation to CBPR principles, members of a US suburban community underwent twelve hours of interactive learning in focus group moderation by a national focus group expert. An additional eight-hour workshop promoted advanced proficiency and built on identified strengths and weaknesses. Ten focus groups were conducted at an adult education center addressing a health concern previously identified by the center's largely immigrant and refugee population. Program evaluation was achieved through multiple observations by community and academic-based observers. RESULTS: Twenty-seven community and academic members were recruited through established relationships for training in focus group moderation, note-taking, and report compilation. Focus group training led to increased trust among community and research partners while empowering individual community members and increasing research capacity for CBPR. CONCLUSIONS: Community members were trained in focus group moderation and successfully applied these skills to a CBPR project addressing a health concern in the community. This approach of equipping community members with skills in a qualitative research method promoted capacity building within a socio-culturally diverse community, while strengthening community-academic partnership. In this setting, capacity building efforts may help to ensure the success and sustainability for continued health interventions through CBPR.

Saccharomyces cerevisiae Rbg1 protein and its binding partner Gir2 interact on Polyribosomes with Gcn1.

Rbg1 is a previously uncharacterized protein of Saccharomyces cerevisiae belonging to the Obg/CgtA subfamily of GTP-binding proteins whose members are involved in ribosome function in both prokaryotes and eukaryotes. We show here that Rbg1 specifically associates with translating ribosomes. In addition, in this study proteins were identified that interact with Rbg1 by yeast two-hybrid screening and include Tma46, Ygr250c, Yap1, and Gir2. Gir2 contains a GI (Gcn2 and Impact) domain similar to that of Gcn2, an essential factor of the general amino acid control pathway required for overcoming amino acid shortage. Interestingly, we found that Gir2, like Gcn2, interacts with Gcn1 through its GI domain, and overexpression of Gir2, under conditions mimicking amino acid starvation, resulted in inhibition of growth that could be reversed by Gcn2 co-overexpression. Moreover, we found that Gir2 also cofractionated with polyribosomes, and this fractionation pattern was partially dependent on the presence of Gcn1. Based on these findings, we conclude that Rbg1 and its interacting partner Gir2 associate with ribosomes, and their possible biological roles are discussed.

Alloplastic total temporomandibular joint replacement in skeletally immature patients: a pilot survey.

The aim of this study was to survey an international group of temporomandibular joint surgeons regarding their outcomes with alloplastic total joint replacement in skeletally immature patients and to review the literature linked to autogenous reconstruction and alloplastic replacement of the temporomandibular joint (TMJR) in this population. A total of 24 custom/patient-specific TMJ Concepts devices were implanted into 14 patients (eight male and six female). Their mean age was 14 years (range 7-17 years). Nine (64.3%) had bilateral devices and five (35.7%) had unilateral devices. The most prevalent diagnosis was idiopathic condylar resorption (33.3%), followed by ankylosis (16.7%). Concurrent orthognathic surgery was performed in four patients (28.6%). The TMJR was completed as a one-stage procedure in 11 patients (78.6%) and in two stages in three patients (21.4%). All surgeons reported improvements in maximum incisal opening with good function. The respondents reported no asymmetric mandibular growth or retrognathia after either bilateral or unilateral TMJR implantation. This pilot study indicates that the use of TMJR in the growing patient may be a useful modality in select cases. The encouraging results of experienced surgeons demonstrate and support the need for further studies on the utilization of TMJR in this patient population.

Stream-floodplain connectivity and fish assemblage diversity in the Champlain Valley, Vermont, U.S.A.

To evaluate the influence of main channel-floodplain connectivity on fish assemblage diversity in floodplains associated with streams and small rivers, fish assemblages and habitat characteristics were surveyed at 24 stream reaches in the Champlain Valley of Vermont, U.S.A. Fish assemblages differed markedly between the main channel and the floodplain. Fish assemblage diversity was greatest at reaches that exhibited high floodplain connectivity. Whereas certain species inhabited only main channels or floodplains, others utilized both main channel and floodplain habitats. Both floodplain fish alpha-diversity and gamma-diversity of the entire stream corridor were positively correlated with connectivity between the main channel and its floodplain. Consistent with these results, species turnover (as measured by beta-diversity) was negatively correlated with floodplain connectivity. Floodplains with waterbodies characterized by a wide range of water depths and turbidity levels exhibited high fish diversity. The results suggest that by separating rivers from their floodplains, incision and subsequent channel widening will have detrimental effects on multiple aspects of fish assemblage diversity across the stream-floodplain ecosystem.

Bacterial sporulation: pole-to-pole protein oscillation.

Sporulating bacteria need to temporally coordinate DNA replication, chromosome partitioning and sporulation initiation. Recent work has shown that one aspect of this coordination lies with the interdependent subcellular localization of two proteins, Spo0J and Soj, and in the Spo0J-dependent spatial oscillation of Soj.

Bacterial division: Finding the dividing line.

Division of a cell - whether eukaryotic or prokaryotic - requires accurate spatial coordination. Recent work on the bacterium Escherichia coli has shown that correct placement of the cell division site at the midcell position occurs by a combination of selection against potential polar sites and selection of the midcell site.

GLAST1b, the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 is a sensitive marker of neuronal dysfunction in the hypoxic brain.

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.

DA-complex assembly activity required for VP16C transcriptional activation.

One class of transcriptional activation domains stimulates the concerted binding of TFIIA and TFIID to promoter DNA. To test whether this DA-complex assembly activity contributes significantly to the overall mechanism of activation in vivo, we analyzed mutants of the 38-amino-acid residue VP16C activation subdomain from herpes simplex virus. An excellent correlation was observed between the in vivo activation function of these mutants and their in vitro DA-complex assembly activity. Mutants severely defective for in vivo activation also showed reduced in vitro binding to native TFIIA. No significant correlation between in vivo activation function and in vitro binding to human TATA binding protein, human TFIIB, or Drosophila melanogaster TAFII40 was observed for this set of VP16C mutants. These results argue that the ability of VP16C to increase the rate and extent of DA-complex assembly makes a significant contribution to the overall mechanism of transcriptional activation in vivo.

Efficient encapsulation of DNA plasmids in small neutral liposomes induced by ethanol and calcium.

Efficient encapsulation of DNA plasmids inside small, neutral liposomes composed of 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), DOPC/DOPE (1,2-dioleoyl-sn-phosphatidylethanolamine) (1:1) and DOPC/DOPE/cholesterol (1:1:1) was achieved by the addition of ethanol and calcium chloride to an aqueous mixture of small unilamellar vesicles (SUVs) and plasmid. Following dialysis against low-salt buffer, the neutral lipid complexes (NLCs) had average effective diameters less than 200 nm and encapsulated up to 80% of the DNA. Optimum Ca(2+) and ethanol concentrations for each lipid mixture were determined by statistically designed experiments and mathematical modeling of trapping efficiency. NLCs are unilamellar, have neutral surface potentials, and retain entrapped DNA at pH 4.0 and in serum at 37 degrees C. The circulation and clearance properties of the complexes following intravenous administration in mice are similar to empty neutral liposomes, and the toxicity of NLCs are expected to be significantly reduced compared to other non-viral gene-delivery systems. The NLC encapsulation method, if it can be combined with effective targeting and endosome-release technologies to achieve efficient and tissue-specific transfection, may represent an important alternative to current systemic gene therapy approaches.

Preparation and characterization of heat-sensitive immunoliposomes.

Immunoliposomes able to bind specifically to target cells and to release their encapsulated contents upon brief heating were prepared. Monoclonal anti-H2Kk was covalently derivatized with palmitic acid by the method of Huang, A. et al. (Huang, A., Tsao, Y.S., Kennel, S.J. and Huang, L. (1982) Biochim. Biophys. Acta 716, 140-150). The palmitoyl antibody was injected at a controlled rate into a suspension of fused unilamellar dipalmitoylphosphatidylcholine liposomes maintained at a constant temperature. The final protein-to-lipid ratio of the resultant liposomes with incorporated antibody (immunoliposomes) was dependent upon the rate of antibody injection and the lipid concentration. Injection of palmitoyl antibody into a liposome suspension containing 50 mM carboxyfluorescein at 41 degrees C resulted in simultaneous antibody incorporation and entrapment of dye. Immunoliposomes were able to release the entrapped carboxyfluorescein upon heating. The release of dye at temperatures between the pre- and main-transition temperatures of DPPC was abolished by the addition of calf serum (5%). Furthermore, the presence of serum resulted in an increase in the temperature of the maximal release rate and also in the rate of release at that temperature. Retention of antigen-binding capacity was demonstrated by the ability of the immunoliposomes to bind specifically to the target cells. Rapid release of entrapped carboxyfluorescein from immunoliposomes bound to target cells at 4 degrees C was achieved upon brief exposure (less than 3 min) at 41 degrees C. These heat-sensitive immunoliposomes may be useful in enhancing drug delivery to target cells.

Effect of cholesterol on the uptake and intracellular degradation of liposomes by liver and spleen; a combined biochemical and gamma-ray perturbed angular correlation study.

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).

Development of ribozymes for gene therapy.

Ribozymes are a class of RNA molecules that can perform catalytically in the absence of protein. Specifically, they can hybridize to and cleave target RNA molecules independent of cellular proteins. The cleaved target RNA can not be translated thereby preventing synthesis of a specific protein. The therapeutic application is to target the ribozyme to the mRNA of a key protein or proteins involved in maintaining a disease state resulting in a cure. The ribozymes can be chemically synthesized and delivered to cells or they can be expressed from an expression vector following either permanent or transient transfection. Therapeutic applications of ribozymes have been in the areas of AIDS and cancer. The following article describes the ribozymes in more detail with respect to optimizing the design to obtain the maximal cleavage rate, identifying cleavage sites within the target RNA and delivering the ribozymes to cells of interest both in vitro and in vivo.

Cationic lipid-based delivery system for systemic cancer gene therapy.

A cationic lipid-based gene delivery system composed of N-[(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride and cholesterol, at a 4:1 molar ratio, was developed for systemic administration. Plasmid biodistribution and expression were characterized in syngeneic mouse tumor model squamous cell carcinoma VII cells. A reporter gene expression plasmid was used for biodistribution of plasmid and expression. The results showed that lungs and primary tumors were transfected. Fluorescence microscopy showed that fluorescent-labeled transfection complexes were passively targeted to the tumor vasculature and that the endothelial cells internalized the plasmid. Transgene expression was characterized based on duration of expression and dosing schedule. In vivo gene transfer with an interleukin-12 expression plasmid yielded protein levels in blood, lungs, and primary tumor after intravenous administration. Efficacy studies showed that 15 microg of interleukin-12 plasmid was sufficient to produce a gene-specific inhibition of primary tumor growth. These results characterize the vascularity of the tumor model, characterize the in vivo gene transfer properties of the plasmid-based gene delivery system, and show that the transgene expression level was sufficient to elicit a biological response by inhibiting tumor growth.

Non-viral cancer gene therapy: an industrial perspective.

Non-viral gene therapy refers to plasmid-based gene delivery systems for the delivery of genes encoding therapeutic proteins to diseased tissue. The genes are encoded on DNA plasmids that are propagated in bacteria. The purified plasmids can be administered by themselves for certain applications or require formulation with synthetic gene delivery systems to increase the level of cellular uptake and ultimately, gene expression. The resultant transfection complex must yield therapeutic levels of the gene product; it must be scaleable for GMP approved pharmaceutical manufacture; it must be safe with regard to toxicity, immunogenicity and recombination; it must have strong intellectual property protection; and manufacture of the product must be cost effective. The present clinical applications of plasmid-based gene therapy technology fulfill most of these requirements for treatment of cancer and cardiovascular disease, and genetic vaccines for infectious diseases. The product candidates are in phase I or phase I/II clinical trials. Results from phase II clinical trials should be available by or shortly into the new millennium. Success with any of these indications will advance the field of plasmid-based gene therapy from a proof-of-concept into a series of pharmaceutical products.

Electrophysiologic evaluation of pirmenol for sustained ventricular tachycardia secondary to coronary artery disease.

The efficacy and electrophysiologic effects of pirmenol were evaluated in 21 patients with a history of sustained ventricular tachycardia (VT) and coronary artery disease. Intravenous pirmenol (0.7- to 1.1-mg/kg bolus, followed by a 35- to 40-micrograms/kg/min infusion) significantly prolonged the PR, QRS, QT and corrected QT intervals, HV interval and right ventricular effective refractory period, and shortened the sinus cycle length and atrioventricular nodal block cycle length. All 21 patients had inducible VT (20 sustained, 1 nonsustained) during programmed stimulation in the control state. After intravenous pirmenol, 5 patients (24%) no longer had inducible VT. In those in whom VT was still inducible, the VT cycle length was prolonged significantly. The 5 patients who responded to intravenous pirmenol were given oral pirmenol (200 to 250 mg every 8 hours) for 1 to 3 days and retested with programmed stimulation. In 4 of these 5, VT could not be induced with oral pirmenol administration; in 1 patient sustained VT was induced and pirmenol therapy was discontinued. Oral pirmenol suppressed recurrent VT during a follow-up of 315 +/- 133 days in 4 patients. However, pirmenol therapy was discontinued in 2 patients because of possible deleterious effects (worsened heart failure in 1 patient and elevated liver function test results in 1). Thus, pirmenol, a type IA antiarrhythmic drug, had an overall efficacy of approximately 19% in patients with sustained VT secondary to coronary artery disease.

Targeted gene delivery: a two-pronged approach.

The success of gene therapy relies on the ability of gene delivery systems to selectively deliver therapeutic genes to a sufficient number of target cells yielding expression levels that impact the diseased state. The gene delivery systems can be divided into two classes: viral and nonviral (or plasmid DNA-based). The present gene delivery technology being used in clinics today can be considered first generation, in that they possess the ability to transfect or infect target cells through their inherent chemical, biochemical, and molecular biological properties. Relying on these sole properties, however, limits therapeutic applications. Expansion of therapeutic applications or increased effectiveness of current therapies can be achieved by increasing the number of cells and cell types susceptible to gene transfer. This can be achieved through physical targeting and molecular biological targeting. Physical targeting relies on the attachment to the delivery vehicle of ligands that bind to cell surface receptors unique to the target cells. Molecular biological targeting refers to selective expression of the therapeutic gene by the target cell through the use of selective promoters. Selective expression can be further achieved by the use of expression systems controlled by extrinsic induction molecules. This review will describe in detail the advances that have been made in each of these areas of gene targeting.

Purification and characterisation of acetolactate decarboxylase from Leuconostoc lactis NCW1.

A two-step strategy involving DEAE-cellulose and POROS PI anion exchange chromatography has been developed for rapid purification of acetolactate decarboxylase (ALD) from Leuconostoc lactis NCW1. This results in 5333-fold purification with a yield of 30%. Purified ALD is a dimer of 49-kDa subunits, has a pH optimum of 6.0, a pI of 4.2 and its activity is independent of metals or branched chain amino acids. At the optimum pH, the K(m) for 2-acetolactate (ALA) was found to be 1.3 mM and the turnover number was 4000 min(-1). N-terminal sequence comparison with other ALDs showed little sequence conservation in this region. Purified ALD does not catalyse direct production of diacetyl from ALA, unlike the crude extract.

Peptide-mediated gene transfer of cationic lipid/plasmid DNA complexes to endothelial cells.

The purpose of this research is to develop ligand-targeted plasmid based gene delivery systems for gene transfer to tumor endothelium. Cell adhesion assays were used to test the peptide inhibition of human endothelial cell adsorption to vitronectin-treated tissue culture plates. A series of RGD containing peptides were tested in linear form and with one and two disulfide bonds. The linear and two disulfide bond peptides yielded similar IC50 (approximately 1 x 10(-7) M). Substitution of two methionines for cysteines yielded a single disulfide bond that increased the IC50 by 10-fold. The single and double disulfide peptides were derivatized to N-succinyl-dioleoylphopsphatidylethanolamine and incorporated into 100 nm liposomes radiolabeled with H-cholesterylhexadecylether. Liposome uptake by human umbilical vein endothelial cells was tested as a function of lipopeptide surface density. Increase in membrane surface density from 5 to 20mol% increased human umbilical derived endothelial cell (HUVEC) uptake of the liposomes for both the single and double disulfide peptides. Liposome uptake by HUVECs was 3-fold greater for the double disulfide compared to the single disulfide. The single and double disulfide lipopeptides were then tested for gene transfer to HUVECs using DOTMA:Cholesterol cationic liposomes. The polyplexes were formed by rapidly mixing plasmid DNA with DOTMA:CHOL liposomes at a 3:1 charge ratio in 2% ethanol, 10% lactose. The ethanol was removed by lyophilization and upon rehydration, the lipoplexes had a mean diameter of approximately 100nm. HUVEC transfection studies showed that increasing the mol% of the single disulfide RGD lipopeptide to 20mol% increased gene transfer by 10-fold. This increase in transfection could be reduced to that obtained in the absence of lipopeptide by co-incubating the HUVECs with a 100-fold excess of the single disulfide RGD peptide, thus demonstrating lipopeptide mediated gene transfer to endothelial cells.

Obturation of a retained primary mandibular second molar using mineral trioxide aggregate: a case report.

This case report demonstrates Mineral Trioxide Aggregate obturation of the root canal system of a retained primary mandibular second molar where no succedaneous permanent tooth was present. The technique seemed to provide a biocompatible seal of the root canal system in this case. It is not recommended for obturation of primary teeth that are expected to exfoliate since it is anticipated that Mineral Trioxide Aggregate would be absorbed slowly, if at all.

Regeneration of peri-implant infrabony defects using PerioGlas: a pilot study in rabbits.

PerioGlas is a silicate-based synthetic bone augmentation material that has been used to fill periodontal defects with bonding and integration to both soft tissue and bone. The purpose of this research was to determine the PerioGlas interface with titanium dental implants and bone. Seven live rabbits were used; however, one rabbit was euthanized at 3 days as a result of a tibial fracture through the implant placement site. Each rabbit received four 3.3 x 8 mm Imtec titanium plasma-sprayed dental implants, two in each proximal tibia. One implant in each rabbit was placed in the standard fashion. Two implants in each rabbit had a surgically created defect adjacent to one side of the coronal aspect of the implant. The defect was subsequently filled with PerioGlas. One implant in each rabbit had a surgically created defect that was not filled with PerioGlas. The rabbits were sacrificed at 1, 2, 3, 6, 12, and 24 weeks. Each specimen was prepared for histologic viewing, yielding a nondecalcified specimen demonstrating the interface of bone, implant, and PerioGlas. The results demonstrate peripheral formation of osteoid, followed by bone deposition within the defect from host (surgical margin) bone, toward the implant. The new osteoid and bone form around the PerioGlas particles. Newly formed trabeculae connect these areas of osteoid and new bone around the PerioGlas, interconnecting the PerioGlas particles. The new bone eventually reaches the implant, and osseointegration occurs with incorporation of the PerioGlas particles.