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LytB and GcpE, because they are codistributed with other pathway enzymes, have been predicted to catalyze unknown steps in the nonmevalonate pathway for isoprenoid biosynthesis. We constructed a conditional Escherichia coli lytB mutant and found that LytB is essential for survival and that depletion of LytB results in cell lysis, which is consistent with a role for this protein in isoprenoid biosynthesis. Alcohols which can be converted to pathway intermediates beyond the hypothesized LytB step(s) support limited growth of E. coli lytB mutants. An informatic analysis of protein structure suggested that GcpE is a globular protein of the TIM barrel class and that LytB is also a globular protein. Possible biochemical roles for LytB and GcpE are suggested.

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Expression of the prion protein gene (Prnp) and production of the PrP protein are essential requirements for acquisition and spread of transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) in humans. Here we have developed an in situ hybridization method for use on human post-mortem central nervous system (CNS) tissues in order to determine those cell which are transcribing the Prnp gene and thus expressing PrP mRNA. Tissues from 11 adult individuals (age range 21-79 years) were analysed. Similar to previous studies in other animal systems, it was shown that PrP production occurs primarily in neuronal populations throughout the human brain. Neurones of the hippocampus, cortex, thalamus, cerebellum and medulla all synthesize PrP mRNA at readily detectable levels. No age-related differences were observed between the cases studied. It was also found that the ependymal cells produced PrP mRNA; these were the only non-neuronal cell type expressing the Prnp gene in the CNS. It is hoped that the information produced here will be helpful in understanding the pathology associated with CJD and other prion diseases in humans.

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We investigated the role of the basal ganglia (BG) in motor imagery in patients with Huntington's disease (HD). A visually guided pointing task assessed whether patients could predict actual movement time (MT) through motor imagery. Executed and imagined movements were performed when vision was constrained centrally, or was free to move. Participants completed a series of imagined and actual movements, with and without central fixation, between two target circles. Patients with HD and controls' imagined MTs were significantly faster than their executed MTs. In compliance with Fitt's law, both actual and imagined MTs increased as a function of increasing task difficulty. We conclude that motor imagery is relatively preserved in HD.

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Chaperonins participate in the facilitated folding of a variety of proteins in vivo. To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an Escherichia coli strain engineered so that the groE operon is under strict regulatory control. We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES.

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Deletion of ftsK results in the inhibition of cell division, but this inhibition can be reversed by a plasmid carrying only the first approximately 17% of ftsK. The division block can be suppressed in most mutants by deletion of dacA, which codes for the D-alanine:D-alanine carboxypeptidase PBP5, or in all mutants by overexpression of ftsN. Overexpression of ftsK inhibits cell division and the formation of FtsZ rings. This division block is not due to the induction of either the SOS or the heat shock regulons.

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OBJECTIVE: To measure the potential for secondary prevention of coronary disease in the United Kingdom. DESIGN: Cross sectional survey of a representative sample of coronary patients from a retrospective review of hospital medical records and patient interview and examination. SETTING: Stratified random sample of 12 specialist cardiac centres and 12 district general hospitals drawn from 34 specialist cardiac centres and 261 district general hospitals in 12 geographic areas in the United Kingdom. SUBJECTS: 2583 patients < or = 70 yr; 25 consecutive males and 25 consecutive females identified retrospectively in each of four diagnostic categories: coronary artery bypass grafting, percutaneous transluminal coronary angioplasty, acute myocardial infarction, and acute myocardial ischaemia without evidence of infarction. MAIN OUTCOME MEASURES: Risk factor recording and management in medical records; the prevalence and control of risk factors at interview six months after the procedure or event. RESULTS: Recording of coronary risk factors in patient's records was incomplete and this varied by risk factor. Smoking habit and blood pressure were most completely recorded, whereas a history of hyperlipidaemia and blood cholesterol concentrations were least complete. Risk factor records were more likely to be complete in cardiac centres than in district hospitals. At interview 10% to 27% of patients were still smoking cigarettes and 75% remained overweight, females more severely so. Up to a quarter of patients remained hypertensive, males more severely so than females. Over three quarters had a total cholesterol > 5.2 mmol/l. In patients on medication for blood pressure, cholesterol or glucose, risk factor profiles were little better than in those who were not. Only about one patient in three was taking a beta blocker after infarction. Up to a fifth of patients who had had acute myocardial ischaemia were not taking aspirin at follow up. CONCLUSIONS: There is considerable potential to reduce the risk of a further major ischaemic event in patients with established coronary disease. This can be achieved by effective lifestyle intervention, the rigorous management of blood pressure and cholesterol, and the appropriate use of prophylactic drugs.

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Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino-acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns. Pure substrate-free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers. The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence. GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformers. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences.

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The active form of the HSP60 molecular chaperone of Escherichia coli, GroEL, is a pair of seven-membered rings. We have used site-directed mutagenesis to construct forms of the 547-amino-acid monomer truncated at the C-terminus. We show here that forms that are 520 amino acids long or longer are close to being fully functional. Removing one further amino acid, however, results in a protein, GroEL519, which retains little function. This truncated form is metabolically stable but is not recovered from the cell in particle form. When synthesized at high levels, it prevents the normal assembly of GroEL547 present in the same cell. When synthesized at low levels, it can be included, probably at low molar ratios, in particles formed by assembly-competent forms of GroEL. This can be seen as partial complementation of the temperature-sensitive mutant groEL44. We conclude that amino acid 520 is crucial for particle assembly. GroEL516 has in vivo properties similar to those of GroEL516 has in vivo properties similar to those of GroEL519, but the still shorter form, GroEL504, appears to be inactive.

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The universally distributed heat-shock proteins (HSPs) are divided into classes based on molecular weight and sequence conservation. The members of at least two of these classes, the HSP60s and the HSP70s, have chaperone activity. Most HSP60s and many HSP70s feature a striking motif at or near the carboxyl terminus which consists of a string of repeated glycine and methionine residues. We have altered the groEL gene (encoding the essential Escherichia coli HSP60 chaperonin) so that the protein produced lacks its 16 final (including nine gly, and five met) residues. This truncated product behaves like the intact protein in several in vitro tests, the only discernible difference between the two proteins being in the rate at which ATP is hydrolysed. GroELtr can substitute for GroEL in vivo although cells dependent for survival on the truncated protein survive slightly less well during the stationary phase of growth. Elevated levels of the wild-type protein can suppress a number of temperature-sensitive mutations; the truncated protein lacks this ability.

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