Thomas B. Kardos

Surface-Active Fungicidal d-Peptide Inhibitors of the Plasma Membrane Proton Pump That Block Azole Resistance

ABSTRACT A 1.8-million-member d-octapeptide combinatorial library was constructed in which each member comprised a diversity-containing N-terminal pentapeptide and a C-terminal amidated triarginine motif. The C-terminal motif concentrated the library members at the fungal cell surface. A primary screen for inhibitors of Saccharomyces cerevisiae and Candida albicans growth, together with an in vitro secondary screen with the S. cerevisiae plasma membrane ATPase (Pma1p) as a target, identified the antifungal d-octapeptide BM0 (d-NH2-RFWWFRRR-CONH2). Optimization of BM0 led to the construction of BM2 (d-NH2-RRRFWWFRRR-CONH2), which had broad-spectrum fungicidal activity against S. cerevisiae, Candida species, and Cryptococcus neoformans; bound strongly to the surfaces of fungal cells; inhibited the physiological activity of Pma1p; and appeared to target Pma1p, with 50% inhibitory concentrations in the range of 0.5 to 2.5 μM. At sub-MICs (<5 μM), BM2 chemosensitized to fluconazole (FLC) S. cerevisiae strains functionally hyperexpressing fungal lanosterol 14α-demethylase and resistance-conferring transporters of azole drugs. BM2 chemosensitized to FLC some FLC-resistant clinical isolates of C. albicans and C. dubliniensis and chemosensitized to itraconazole clinical isolates of C. krusei that are intrinsically resistant to FLC. The growth-inhibitory concentrations of BM2 did not cause fungal cell permeabilization, significant hemolysis of red blood cells, or the death of cultured HEp-2 epithelial cells. BM2 represents a novel class of broad-spectrum, surface-active, Pma1p-targeting fungicides which increases the potencies of azole drugs and circumvents azole resistance.

The effect of transforming growth factor beta one (TGF-β1) on wound healing, with or without barrier membranes, in a Class II furcation defect in sheep

The purpose of this study was to analyse the effect of TFG-beta 1 on wound healing in standardized Class II furcation defects of 48 mandibular second premolar teeth in 24 sheep. The experimental design included a control group (carrier only, 25% pluronic F-127), and 2 experimental groups: group A (80 micrograms/ml TGF-beta 1 + carrier) and group B (80 micrograms/ml TGF-beta 1 + carrier covered with a barrier membrane). Sheep were killed either 2 wk or 6 wk after surgery. Mesiodistal sections of the decalcified specimens were quantified histologically using stereology. Percentage volumes of regenerated bone, fibrous connective tissue and cementum were calculated for each furcation defect. Mean values were analysed using multiple ANOVA; p values were calculated using paired and unpaired Students t-tests. After 2 wk there was more bone in group B than either of the other 2 groups, but this was not statistically significant. By 6 wk more bone was present in group A than in the control group (p < 0.02) and also in group B when compared with both group A and the control group (p < 0.02 and p < 0.44), respectively. In the 4 wk between sampling significantly more bone had formed (group A < 0.05 and group B p < 0.003, respectively). A negative correlation existed between volumes of bone and fibrous connective tissue and no significant differences between the volumes of cementum were evident between any of the groups. This study demonstrated that TGF-beta 1 encouraged bone regeneration in Class II furcation defects in sheep, an effect enhanced by the presence of a barrier membrane. This is the first report on the use of TGF-beta 1 in conjunction with GTR in periodontal defects.

Calmodulin-like activity in a mineralising tissue: The rat molar tooth germ

SummaryCalmodulin, a calcium binding protein, has been implicated in the regulation of many calcium-dependent biological processes. Since calcium has an important role in hard tissue genesis, both at intra- and extracellular levels, we anticipate that calcium binding proteins may modulate this process. The present study investigated a mineralising tissue, the rat molar tooth germ, to determine the presence of calmodulin-like activity. A heat-treated cell-free extract of tooth germs provided enhancement of Ca2+-dependent Mg2+-ATPase and 3′:5′-nucleotide phosphodiesterase activity. No enhancement occurred in the absence of calcium or in the presence of trifluoperazine. SDS-polyacrylamide gel electrophoresis of this extract revealed a protein band of approximately 18,000 mol. wt. These findings indicate the presence of calmodulin-like activity in rat molar tooth germs and support the proposal that calcium and calcium binding proteins, in particular calmodulin, have a major regulatory role in the biology of mineralising tissues.

Regulation and pH-dependent expression of a bilaterally truncated yeast plasma membrane H+-ATPase

Constitutive, chromosomal expression of yeast pma1 deletion alleles in Saccharomyces cerevisiae yielded functional, truncated forms of the plasma membrane H+-ATPase which were independently capable of supporting wild type yeast growth rates. Deletion of 27 amino-terminal residues affected neither the enzymes activity nor its responsiveness to changes in glucose metabolism. By contrast, removal of 18 carboxy-terminal amino acids produced an enzyme with a Vmax that was relatively insensitive to glucose-dependent metabolic status and with a Km that was significantly lower than that of the wild type enzyme. These effects were exaggerated when the amino- and carboxy-terminal deletions were combined in a bilaterally truncated H+-ATPase, suggesting that the amino terminus may have a subtle role in modulating ATPase activity. In pma1DeltaDelta cells cultured at pH 6, plasma membrane H+-ATPase levels were much lower than those in cells expressing a wild type ATPase. Increased expression levels could be achieved by growing the pma1DeltaDelta mutant at pH 3, a result that was at least partially due to a sustained, elevated transcription of pma1DeltaDelta mRNA. Our observations suggest that intracellular proton balance can be maintained by regulation of the activity and/or quantity of H+-ATPase in the plasma membrane.

The development of an ePortfolio for life-long reflective learning and auditable professional certification

Recent legislative changes, that affect all healthcare practitioners in New Zealand, have resulted in mandatory audits of practitioners who are now required to provide evidence of competence and continued professional development in the form of a professional portfolio. These changes were the motivation for our development of an electronic portfolio (ePortfolio) suitable for both undergraduate and life-long learning. Bachelor of Oral Health (BOH) students, studying to qualify as Dental Hygienists and Dental Therapists, and BOH teaching staff (who held registrations in Dental Hygiene, Dental Therapy and Dentistry) trialled the use of a personal ePortfolio for advancing their academic and professional development. The ePortfolio enables BOH students to collect evidence of their achievements and personal reflections throughout their 3 years of undergraduate study, culminating in registration and the award of an Annual Practising Certificate (APC). The ePortfolio was designed to allow users to store information and then select appropriate material to be displayed or published, thus assisting health practitioners to present high-quality evidence of their participation and achievements, and to meet the professional requirements for their APC.

A new periodontal membrane biology based upon thixotropic concepts

The biology of the periodontal membrane is not completely understood, and tissue changes associated with orthodontic tooth movement are assumed to be the result of a recoverable pathologic process. The response of this tissue to dynamic loading may, however, be explained by changes in viscosity of the collagenous matrix.

FUNCTIONAL COMPLEMENTATION BETWEEN TRANSMEMBRANE LOOPS OF SACCHAROMYCES CEREVISIAE AND CANDIDA ALBICANS PLASMA MEMBRANE H+-ATPASES

Saccharomyces cerevisiae PMA1 sequences encoding a putative antifungal target site comprising transmembrane loops 1 + 2 and/or 3 + 4 were replaced with the homologous sequences from Candida albicans PMA1 by using PCR-mediated domain transfer. The chimeric pma1 mutants and an isogenic wild type S. cerevisiae strain had similar growth rates, growth yields, glucose-dependent proton pumping rates, acid-activated omeprazole sensitivities, salt tolerances and antifungal sensitivities. The yields and kinetic properties of H(+)-ATPases in plasma membranes of mutant and wild type strains were comparable. Single heterologous transmembrane loops caused deleterious phenotypes at low pH and elevated temperature. Inclusion of both heterologous transmembrane loops fully suppressed the temperature sensitivity caused by heterologous transmembrane loop 1 + 2, partially suppressed the pH sensitivity and gave Candida-like in vitro sensitivity to vanadate, suggesting that the loops operate as a domain. The fully functional chimeric H(+)-ATPase containing C. albicans transmembrane loops 1 + 2 and 3 + 4 demonstrates this domains complementarity to the equivalent region of the S. cerevisiae enzyme and validates the wild type S. cerevisiae H(+)-ATPase as an antifungal screening target.

Rapid dissection of rodent molar-tooth germs

A rapid rate of isolation of molar-tooth germs from rodents aged from 19 embryonic days to 7 days postnatal can be achieved. The procedure is of direct application to biochemical studies of odontogenesis where relatively large amounts of tissue are often required. The quality of dissection, assessed by morphological and organ culture criteria, extends the application of this procedure to general use in the isolation of molar-tooth germs from rodents.

Scanning Electron Microscopy of Trypsin-Treated Enamel from Fluorosed Rat Molars

Fluoride-induced pitting and porosity of teeth have long been observed, but little progress has been made in determining their origin. We have observed, in the trypsin-treated surfaces of enamel, pits that disappear on completion of maturation, following the removal of the protein matrix and full mineralization. Since these pits were considered to be similar to those seen in fluorotic teeth, this scanning electron microscope (SEM) study was undertaken to determine the effect of fluoride on these transient developmental pits during enamel matrix maturation. A group of 20 eight-day-old rats was given daily intraperitoneal injections of NaF (20 mg/kg [9 mg F-/kg] body weight) for five days. Twenty control animals received intraperitoneal injections of isotonic saline. Maxillary and mandibular molars were dissected from the 13-day-old animals, washed in HEPES buffered (Ca2+/Mg2+) free basal medium, Eagles (BME), incubated in 3% trypsin/BME for 5-10 min at room temperature, then indirectly sonicated in BME for 2-4 min. Clean crowns were fixed in 2.5% glutaraldehyde for three hr, dehydrated, critical-point-dried, and sputter-coated for SEM. Pits in the surfaces of developing enamel were observed in all groups. In control teeth, the pitting was restricted to the cervical margin, whereas in teeth from the fluoride-treated animals, pits were observed on some cuspal surfaces in addition to the cervical margin. These results confirmed that pits in trypsin-treated surfaces of developing enamel are a transient developmental event and showed that, in the presence of a high dose of fluoride, the maturation of enamel is modified with retention of the pits.