The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.     

The role of the renin-angiotensin system in malignant vascular injury affecting the systemic and cerebral circulations

Malignant hypertension is a rare but serious syndrome complicating 1% of essential hypertension and causing neurological, renal and cardiac complications. Despite improved anti-hypertensive medication, the incidence of this condition fails to decline. In the first part of this review, we discuss transgenic rat models of malignant hypertension, generated by over-expressing renin, to illustrate the role of the renin–angiotensin system in the development of systemic hypertensive vascular remodelling and hypertension. In the second part, we focus on the cerebrovascular response to hypertension and discuss new data using a conditional, transgenic model of malignant hypertension, the inducible hypertensive rat (IHR). Cerebral infarction associates strongly with hypertension in man and the mechanisms by which hypertension predisposes to different types of stroke remains poorly understood. Rats have similar cerebrovascular anatomy and structure to humans and as such provide a good experimental tool. To date, such models lack controllability and blood-pressure matched controls. Using the IHR, we have manipulated dietary salt and water intake to generate a novel, controllable stroke phenotype. Hypertensive small-vessel stroke develops over a predictable time period, permitting the study of developing cerebrovascular lesions. Systemic end-organ injury and hypertension are not affected. Dissociation of the systemic and central vascular responses in this way, will allow for comparative study of animals with equivalent hypertension, genetic background and systemic features of hypertension with or without stroke.     

Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I

Maternal factors control development prior to the activation of the embryonic genome. In vertebrates, little is known about the molecular mechanisms by which maternal factors regulate embryonic development. To understand the processes controlled by maternal factors and identify key genes involved, we embarked on a maternal-effect mutant screen in the zebrafish. We identified 68 maternal-effect mutants. Here we describe 15 mutations in genes controlling processes prior to the midblastula transition, including egg development, blastodisc formation, embryonic polarity, initiation of cell cleavage, and cell division. These mutants exhibit phenotypes not previously observed in zygotic mutant screens. This collection of maternal-effect mutants provides the basis for a molecular genetic analysis of the maternal control of embryogenesis in vertebrates.     

Transforming growth factor beta (TGFbeta ) signaling via differential activation of activin receptor-like kinases 2 and 5 during cardiac development. Role in regulating parasympathetic responsiveness

Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor beta (TGFbeta) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Galpha(i2). Here in atrial cells 5 days in ovo, TGFbeta increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Galpha(i2). TGFbeta also stimulated Galpha(i2) mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFbeta receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Galpha(i2) promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Galpha(i2) promoter activity independent of embryonic age. In 5-day atrial cells, TGFbeta stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFbeta stimulated both pVent and p3TP-lux. Thus TGFbeta exerts opposing effects on parasympathetic response and Galpha(i2) expression by activating different type I TGFbeta receptors at distinct stages during cardiac development.     

Genetic heterogeneity in Bacillus sporothermodurans as demonstrated by ribotyping and repetitive extragenic palindromic-PCR fingerprinting

Thirty-eight strains of Bacillus sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. sporothermodurans that can be found in UHT-treated or sterilized milk products.     

Hyphal tip growth in Achlya bisexualis. I. Distribution of 1,3-{beta}-glucans in elongating and non-elongating regions of the wall

We have approached the problem of hyphal tip growth by comparing the cell wall composition of elongating and non-elongating regions of the hyphae of Achlya bisexualis. To ensure that we could distinguish between elongating and non-elongating hyphae, light microscopic observations were used to determine the rates of elongation under growing and non-growing conditions. When elongation was measured in 10 min intervals it was found to consist of fluctuating periods of fast and slow growth rates, in the form of cycles. Even under our growing conditions, however, a very small number of hyphae in a colony are not elongating. SEM analysis revealed that elongating hyphae have tapered apices, whereas non-elongating hyphae have a rounded apex. The major matrix wall components, 1,3-β-glucans, were localized with an indirect immunogold technique specific for these polymers. This method resulted in their localization to all regions of both elongating and non-elongating hyphae, including the apex.     

Effect of linker sequence on the stability of circularly permuted variants of ribonuclease T1

Circularly permuted variants of ribonuclease T1 were constructed with a library of residues covalently linking the original amino and carboxyl terminal ends of the wild-type protein. The library of linking peptides consisted of three amino acids containing any combination of proline, aspartate, asparagine, serine, threonine, tyrosine, alanine, and histidine. Forty two unique linker sequences were isolated and 10 of these mutants were further characterized with regard to catalytic activity and overall thermodynamic stability. The 10 mutants with the different linking sequences (HPD, TPH, DTD, TPD, PYH, PAT, PHP, DSS, SPP, and TPS), in addition to GGG and GPG, were 4.0-6.2 kcal/mol less stable than the wild-type ribonuclease T1. However, these circular permuted variants were only 0.4-2.6 kcal/mol less stable than the direct parent protein that is missing the disulfide bond connecting residues 2 and 10. The most stable linking peptide was HPD.     

Membrane topology of the Mep/Amt family of ammonium transporters

The Mep/Amt proteins constitute a new family of transport proteins that are ubiquitous in nature. Members from bacteria, yeast and plants have been identified experimentally as high-affinity ammonium transporters. We have determined the topology of AmtB, a Mep/Amt protein from Escherichia coli, as a representative protein for the complete family. This was established using a minimal set of AmtB-PhoA fusion proteins with a complementary set of AmtB-LacZ fusions. These data, accompanied by an in silico analysis, indicate that the majority of the Mep/Amt proteins contain 11 membrane-spanning helices, with the N-terminus on the exterior face of the membrane and the C-terminus on the interior. A small subset, including E. coli AmtB, probably have an additional twelfth membrane-spanning region at the N-terminus. Addition of PhoA or LacZ alpha-peptide to the C-terminus of E. coli AmtB resulted in complete loss of transport activity, as judged by measurements of [14C]-methylammonium uptake. This C-terminal region, along with four membrane-spanning helices, contains multiple residues that are conserved within the Mep/Amt protein family. Structural modelling of the E. coli AmtB protein suggests a number of secondary structural features that might contribute to function, including a putative ammonium binding site on the periplasmic face of the membrane at residue Asp-182. The implications of these results are discussed in relation to the structure and function of the related human Rhesus proteins.