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.     

Effects of cytochalasin D on the distribution of actin and ACTH-induced steroidogenesis in cultured embryonic adrenal gland cells from the Pekin duck (Anas platyrhynchos)

Cultured steroidogenic cells derived from the adrenal glands of duck embryos were used to study changes in the distribution of actin associated with the corticotropic responsiveness. Actin-containing components were identified by rhodamine-phalloidin staining. The actin in most of the unstimulated cells occurred as stress fibers that either ran parallel throughout the cell or were present as domains of parallel fibers at angles to one another. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH, the cells released approximately equal amounts of corticosterone and aldosterone. Incubation of the cells in buffer containing cytochalasin D caused the cells to lose their stress fibers, and the actin became distributed at the periphery in what appeared to be fragments of stress fibers and clumps of fibrous material in the central cytoplasm. Although cytochalasin D did not affect the basal output of corticosterone and aldosterone, the 1–24 ACTH-induced rates of both hormones were suppressed significantly. After the cells had been washed in unadulterated buffer, the normal distribution of actin stress fibers was restored and the cells responded normally when incubated in buffer containing 1–24 ACTH. These results suggest that the actin components of the cytoskeleton are important determinants of corticotropin-induced steroidogenic responsiveness.     

Mutations of the tyrosinase gene in patients with oculocutaneous albinism from various ethnic groups in Israel.

We have analyzed the tyrosinase (TYR) gene in 38 unrelated patients with oculocutaneous albinism (OCA), derived from several different ethnic groups of the diverse population of Israel. We detected TYR gene mutations in 23 of the 34 patients with apparent type I (i.e., tyrosinase-deficient) OCA and in none of the patients with other clinical forms of albinism. Among Moroccan Jews with type IA (i.e., tyrosinase-negative) OCA, we detected a highly predominant mutant allele containing a missense substitution, Gly47Asp (G47D). This mutation occurs on the same haplotype as in patients from the Canary Islands and Puerto Rico, suggesting that the G47D mutation in these ethnically distinct populations may stem from a common origin.     

A new ATP-binding fold in actin, hexokinase and Hsc70

One of a cell biologist's favourite occupations is to discover the proteins that perform newly described functions in the cell. Very often lately, this has resulted in the identification of protein families whose related amino acid sequences reflect similar functions, but can proteins with totally unrelated sequences have similar structures and functions? In this review, Ken Holmes, Chris Sander and Alfonso Valencia describe the structural similarities between three well-known proteins that have no readily detectable primary sequence similarities but for which X-ray crystallography has revealed very similar structures. A comparison of their structures provides insights into their common mechanisms of action and into protein evolution, and has been used to detect related proteins in sequence data bases.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.