Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6-Hydroxydopamine

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 m M 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 m M 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Drugs derived from phage display: From candidate identification to clinical practice

Phage display, one of today’s fundamental drug discovery technologies, allows identification of a broad range of biological drugs, including peptides, antibodies and other proteins, with the ability to tailor critical characteristics such as potency, specificity and cross-species binding. Further, unlike in vivo technologies, generating phage display-derived antibodies is not restricted by immunological tolerance. Although more than 20 phage display-derived antibody and peptides are currently in late-stage clinical trials or approved, there is little literature addressing the specific challenges and successes in the clinical development of phage-derived drugs. This review uses case studies, from candidate identification through clinical development, to illustrate the utility of phage display as a drug discovery tool, and offers a perspective for future developments of phage display technology.     

Eight new microsatellite markers in Atlantic cod (Gadus morhua L.) derived from an enriched genomic library

One hundred and fifty random clones from an enriched genomic library of Atlantic cod were sequenced. Primer pairs were designed for 15 microsatellites containing perfect di‐, tri‐, tetra‐ and hexanucleotide repeats and characterized in 96 unrelated fish. Eight markers were successfully amplified, with the number of alleles ranging from two to nine per locus and observed heterozygosity ranging from 0.341 to 0.977. Loci Gmo‐G13 and Gmo‐G14 had a significant excess of homozygotes. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.     

Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction

Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.     

Novel peptide inhibitors of angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2), a recently identified human homolog of ACE, is a novel metallocarboxypeptidase with specificity, tissue distribution, and function distinct from those of ACE. ACE2 may play a unique role in the renin-angiotensin system and mediate cardiovascular and renal function. Here we report the discovery of ACE2 peptide inhibitors through selection of constrained peptide libraries displayed on phage. Six constrained peptide libraries were constructed and selected against FLAG-tagged ACE2 target. ACE2 peptide binders were identified and classified into five groups, based on their effects on ACE2 activity. Peptides from the first three classes exhibited none, weak, or moderate inhibition on ACE2. Peptides from the fourth class exhibited strong inhibition, with equilibrium inhibition constants (K(i) values) from 0.38 to 1.7 microm. Peptides from the fifth class exhibited very strong inhibition, with K(i) values < 0.14 microm. The most potent inhibitor, DX600, had a K(i) of 2.8 nm. Steady-state enzyme kinetic analysis showed that these potent ACE2 inhibitors exhibited a mixed competitive and non-competitive type of inhibition. They were not hydrolyzed by ACE2. Furthermore, they did not inhibit ACE activity, and thus were specific to ACE2. Finally, they also inhibited ACE2 activity toward its natural substrate angiotensin I, suggesting that they would be functional in vivo. As novel ACE2-specific peptide inhibitors, they should be useful in elucidation of ACE2 in vivo function, thus contributing to our better understanding of the biology of cardiovascular regulation. Our results also demonstrate that library selection by phage display technology can be a rapid and efficient way to discover potent and specific protease inhibitors.     

Metastatic sweat gland adenocarcinoma: A clinico-pathological dilemma

Background  

Sweat gland adenocarcinoma is a rare malignancy with high metastatic potential seen more commonly in later years of life. Scalp is the most common site of occurrence and it usually spreads to lymph nodes. Liver, lung and bones are the distant sites of metastasis with fatal results. The differentiation between apocrine and eccrine metastatic sweat gland carcinoma is often difficult. The criteria's are inadequate to be of any practical utility.