Role of the Eikenella corrodens pilA locus in pilus function and phase variation.

The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.

Eikenella corrodens phase variation involves a posttranslational event in pilus formation.

The human pathogen Eikenella corrodens synthesizes type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying the molecular basis of this phase variation in the clinical isolate E. corrodens VA1. A genomic fragment encoding the major type IV pilin was cloned from the S-phase variant of strain VA1. Sequence analysis of the fragment revealed four tandemly arranged potential open reading frames (ORFs), designated pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin. The protein predicted by pilB shares sequence identity with the Dichelobacter nodosus FimB fimbrial assembly protein. The protein predicted by hagA resembles a hemagglutinin. The region containing these four ORFs was designated the pilA locus. DNA hybridization and sequence analysis showed that the pilA locus of an L-phase variant of strain VA1 was identical to that of the S-phase variant. An abundant pilA1 transcript initiating upstream of pilA1 and terminating at a predicted hairpin structure between pilA1 and pilA2 was detected by several assays, as was a less abundant read-through transcript encompassing pilA1, pilA2, and pilB. Transcription from the pilA locus was nearly indistinguishable between S- and L-phase variants. Electron microscopy and immunochemical analysis showed that S-phase variants synthesize, export, and assemble pilin into pili. In contrast, L-phase variants synthesize pilin but do not export and assemble it into pili. These data suggest that a posttranslational event, possibly involving an alteration in pilin export and assembly, is responsible for phase variation in E. corrodens.

Will elderly rest home residents wear hip protectors?

BACKGROUND: Hip fracture is a common cause of morbidity and mortality in elderly people, for whom osteoporosis, the risk of falling and direct trauma to the hip during the fall are the major risk factors. External hip protectors have been developed which reduce the risk of hip fracture after a fall. However, compliance with their use is uncertain. We addressed this issue in a sample of elderly Dorset rest home residents over a 3-month period. METHODS: 31 rest homes agreed to participate. Of the 288 female subjects approached, 141 gave their informed consent and 101 were allocated to the intervention arm of the study. Their ages ranged from 64 to 98 years, and 44% reported a fall during the preceding 12 months. Each subject was fitted with three pairs of protector pads (Sahvatex, Denmark) sewn into specially designed undergarments. Randomly timed fortnightly visits were made to each subject to assess compliance for 12 weeks. FINDINGS: 27 subjects were compliant for the whole study period; 54 wore the protector pads for less than a week, largely for reasons of poor fitting or discomfort; the remainder withdrew at varying intervals between 1 and 12 weeks. During the study period, there were nine recorded falls onto the hip, six of which occurred in women wearing protectors. None resulted in hip fracture. CONCLUSION: Approximately 50% of elderly rest home residents who are mentally able would wear hip protectors in order to prevent hip fractures. Long-term compliance drops to about 30%. Compliance could be increased substantially if the pads and undergarments were modified to enhance their fit and to reduce the discomfort associated with their use.

Autosomal dominant polycystic kidney disease complicated by glomerulonephritis.

Two patients with autosomal dominant polycystic kidney disease (ADPKD) and concurrent glomerulonephritis are described. Both developed nephrotic-range proteinuria and one showed a concomitant acceleration in the rate of decline of renal function. Subsequent open renal biopsy revealed membrano-proliferative type-1 and mesangio-proliferative glomerulonephritis, respectively. Nephrotic-range proteinuria in the presence of ADPKD, with or without an accompanying decline in renal function, should prompt further investigation to exclude coexisting glomerular disease.

Cloning, expression, isolation and characterization of the pre-S domains of hepatitis B surface antigen, devoid of the S protein.

The 'pre-S' parts of the envelope protein of hepatitis B virus (HBV) have been proposed to be involved in the infection of hepatocytes by HBV. In order to facilitate the study of these processes, we have developed an expression system to allow the production and purification of large quantities of the pre-S protein. To obtain a protein containing all of the pre-S sequence and only this sequence, mutations were introduced into the HBV(ayw) genome to create an NdeI restriction site at the initial ATG of the large surface protein gene. Also, stop codons and a BglII restriction site were introduced after the last codon of pre-S2. This fragment was then cloned into the high-expression vector pET-3A. A protein of the expected Mr was expressed at a level of up to 10% of the total soluble protein in HMS174 (DE3) cells, as judged by SDS/PAGE. A rapid purification method has been developed for this protein. The protein retains the polyalbumin-binding activity ascribed to the pre-S2 sequence, and is recognized by both polyclonal and monoclonal antibodies directed against pre-S determinants. Gel filtration chromatography demonstrates that the protein is monomeric and globular, and c.d. spectroscopy indicates that beta-sheet is the major periodic structure.

Recurrent and fatal haemoptysis caused by an atheromatous abdominal aortic aneurysm.

A 74 year old woman presented with a two month history of recurrent small hemoptyses and died after a subsequent massive haemoptysis. At postmortem examination the source of bleeding was found to be a leaking saccular, atheromatous abdominal aortic aneurysm, which had ruptured through the diaphragm into the lower lobe of the right lung.

[Metal ions in the insulin-receptor interaction in human erythrocytes]. / Iones metálicos en la interacción insulina-receptor en hematíes humanos.

The kinetics of insulin binding to its receptors on human erythrocytes suggests the existence of two types of receptors exhibiting negative cooperativity upon the binding of the hormone. Solubilized and purified receptors were associated to Zn++ or Cu++. The addition of these ions to erythrocytes or to purified insulin receptors from human erythrocytes resulted in an increase of specific insulin binding. Dialysis of solubilized or purified receptors against chelating agents such as EDTA or 1, 10-phenanthroline resulted in a decrease in specific binding of insulin. With the readdition of Zn++ or Cu++ to the medium an increase in specific binding was observed, and values much higher than those of the original preparations were obtained. Dialysis of purified receptors against chelating agents resulted in a decrease in the content of Zn++ and Cu++. These results suggest the possible involvement of a metal ion associated to the receptor in the formation of the insulin-receptor complex.

Phospholipase A2 hydrolysis of membrane phospholipids causes structural alteration of the nicotinic acetylcholine receptor.

Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the alpha-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of alpha-bungarotoxin-binding sites are shifted to lower temperatures by approx. 7 and 5 C degrees, respectively, upon exposure to phospholipase A2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3(n-3] causes detectable destabilization of the alpha-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A2 hydrolysis products.

Thermal perturbation studies of membrane-bound acetylcholine receptor from Torpedo: effects of cholinergic ligands and membrane perturbants.

Thermal perturbation techniques have been used to probe structural features of the nicotinic acetylcholine receptor (AcChR). The information obtained from differential scanning calorimetry (DSC) of AcChR membranes (M.C. Farach and M. Martinez-Carrion (1983) J. Biol. Chem. 258, 4176) in the absence and in the presence of cholinergic ligands and local anesthetics, is comparable to that obtained from a simpler technique of heat inactivation of the alpha-bungarotoxin (alpha-Bgt) binding sites on the AcChR protein in similar samples. When AcChR membranes are heated at approximately 1 degree C/min, heat inactivation of toxin binding sites has a characteristic T50 value (temperature at which 50% of the initial capacity to bind alpha-Bgt remains) of approximately 60 degrees C. When heated at a constant temperature during increasing periods of time, the rate at which heat inactivation occurs is also characteristic of the temperature chosen for the experiment. The above thermal parameters are also sensitive to perturbation of the AcChR membrane matrix by the presence of subsolubilizing concentrations of detergents. Moreover, elimination of detergents by dialysis allows us to evaluate the reversibility or irreversibility of AcChR thermal destabilization induced by detergents or other membrane perturbants. Under the experimental conditions used, structural destabilization induced by octylglucoside or cholate can be fully reversed by detergent dialysis, while that exerted by deoxycholate cannot. "Thermal gel" analysis of the aggregation of AcChR subunits induced by heat (G. Soler, J. R. Mattingly, and M. Martinez-Carrion (1984) Biochemistry 23, 4630) has also been used to assess the effects of detergent presence on the AcChR protein. When deoxycholate is used as the perturbing agent, there is a particularly effective sulfhydryl-mediated aggregation of the gamma-delta subunit group, which appears to correlate with the irreversible destabilization of alpha-Bgt binding sites induced by that detergent.