Functional conservation of the active sites of human and Drosophila angiotensin I-converting enzyme

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two homologous protein domains, resulting from a tandem gene duplication. It has been proposed that the N- and C-terminal active sites can have specific in vivo roles. In Drosophila melanogaster, Ance and Acercode for two ACE-like single-domain proteins, also predicted to have distinct physiological roles. We have investigated the relationship of Ance and Acer to the N- and C-domains of human sACE by genomic sequence analysis and by using domain-selective inhibitors, including RXP 407, a selective inhibitor of the human N-domain. These phosphinic peptides were potent inhibitors of Acer, but not of Ance. We conclude that the active sites of the N-domain and of Acer share structural features that permit the binding of the unusual RXP407 inhibitor and the hydrolysis of a broader range of peptide structures. In comparison, Ance, like the human C-domain of ACE, displays greater inhibitor selectivity. From the analysis of the published sequence of the Adh region of Drosophila chromosome 2, which carries Ance, Acer, and four additional ACE-like genes, we also suggest that this functional conservation is reflected in an ancestral gene structure identifiable in both protostome and deuterostome lineages and that the duplication seen in vertebrate genomes predates the divergence of these lineages. The conservation of ACE enzymes with distinct active sites in the evolution of both vertebrate and invertebrate species provides further evidence that these two kinds of active sites have different physiological functions.     

The calcium sensing receptor and its alternatively spliced form in murine epidermal differentiation

We have recently reported that human keratinocytes express both the full-length calcium sensing receptor (CaR) and an alternatively spliced form lacking exon 5, which were suggested to be involved in calcium induced keratinocyte differentiation. To understand further the role of these CaRs, we analyzed the structure of mouse CaRs, and investigated their role using a mouse model in which only the full-length CaR was disrupted. Our results show that both the full-length and the alternatively spliced variant lacking exon 5 encoding 77 amino acids of the extracellular domain were expressed in mouse epidermis. The deletion of the full-length CaR increased the production of the alternatively spliced form of CaR in mutant mice. The keratinocytes derived from these mutant mice did not respond to extracellular calcium, suggesting that the full-length CaR is required to mediate calcium signaling in the keratinocytes. The loss of the full-length CaR altered the morphologic appearance of the epidermis and resulted in a reduction of the mRNA and protein levels of the keratinocyte differentiation marker, loricrin. These results indicate that CaR is important in epidermal differentiation, and that the alternatively spliced form does not fully compensate for loss of the full-length CaR.     

A novel conformation of the herpes simplex virus origin of DNA replication recognized by the origin binding protein

The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replication oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop. Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5' side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex. We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.     

Epidermal sphingomyelins are precursors for selected stratum corneum ceramides

Epidermal ceramides (Cer) comprise a heterogeneous family of seven species, including two unique omega-hydroxylated Cer, that are key components of the stratum corneum (SC) intercellular lamellar membranes responsible for the epidermal permeability barrier. Although both glucosylceramide (GlcCer) and the phospho-sphingolipid sphingomyelin (SM) are potential precursors of SC Cer, based on reported chemical structures of epidermal GlcCer and SC Cer, it is assumed that all major subfractions of SC Cer are generated from lamellar body-derived GlcCer. Yet, we and others have shown that SM-derived Cer are required for normal barrier homeostasis. Moreover, two pools of SM, one from plasma membrane, the other from lamellar body-derived contents, are potentially available for Cer production. To clarify the role of SM as a potential precursor of bulk or specific SC Cer, we compared Cer moieties in epidermal SM, Cer generated from epidermal SM by sphingomyelinase treatment, Cer within SC, and Cer that persist in Gaucher SC, where GlcCer cannot generate Cer due to an absence of beta-glucocerebrosidase. Using gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance for Cer characterization, epidermal SM comprise three major subfractions with distinctive amide-linked (N-acyl) fatty acid (FA) compositions: that is, either long-chain FA (SM-1; C(22;-26)), short-chain FA (SM-2; primarily C(16)), and short-chain alpha-hydroxy FA (SM-3; C(16;-18)). In contrast, only trace quantities of omega-hydroxy FA were present. For each SM subfraction, the sphingoid base was either sphingosine or sphinganine, but phytosphingosine was not detected. Comparison of these SM with corresponding sphingomyelinase-generated epidermal Cer and SC Cer revealed that the Cer moieties of SM-1 and SM-3 are equivalent to Cer 2 (NS) and Cer 5 (AS), respectively. Moreover, both Cer 2 and Cer 5 occurred in Gaucher SC, whereas other Cer subfractions did not occur.These results indicate that two epidermal SM, that is, SM-1 and SM-3, are important precursors of two corresponding Cer in mammalian SC, that is, Cer 2 and Cer 5, but other Cer species, including the omega-hydroxy Cer species, do not derive from SM.     

Distribution and characterization of recrystallization inhibitor activity in plant and lichen species from the UK and maritime Antarctic

Extracts from a range of evolutionarily diverse plant and lichen species from the UK and maritime Antarctic have been assayed for inhibition of ice recrystallization. Approximately 25% of overwintering UK species and all Antarctic species exhibited antifreeze activity when exposed to low temperature. Preliminary characterization of the active extracts has demonstrated that the molecules co-opted to antifreeze activity by different species are biochemically diverse.     

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.     

The Incidence of Hepatitis C Virus Positive Serological Test Results Among Cornea Donors

Purpose. We have noticed that the incidence of positive serological tests of hepatitis C virus (HCV) among cornea donors in our eye bank is higher than expected. The purpose of this study was to determine the incidence of these positive findings and identify the contributory risk factors. Methods. All corneas procured between June 1993 and June 1997 were included in this retrospective study. In all cases a routine work-up, including serological testing of donors' sera and evaluation of the procured corneas, was performed prior to corneal transplantation. Donors found to be positive for HCV antibodies were compared to our general population of corneal donors, with respect to the demographic, serological and ophthalmological data obtained during the transplantation work-up. Results. All 851 corneas procured from 438 donors were included. Antibodies to HCV were found in 29 donors (6.6%). Following positive test results, 57 corneas (6.7%) were discarded. The time from donors' death to cornea procurement was significantly longer among HCV-positive patients than in the general donor population (12.3 vs. 9.3h, p<0.0003). No other differences were detected between the two groups. Conclusion. Delay in harvesting of donated corneas may give rise to false positive HCV-antibodies test results, which may be partially responsible for the high rate of positive findings among cornea donors, with consequent tissue wastage.     

Mitral tetrahedron as a geometrical surrogate for chronic ischemic mitral regurgitation

The present study tests the hypothesis that a mitral tetrahedron (MT) is a useful geometrical surrogate for assessment of chronic ischemic mitral regurgitation (CIMR). Fifty-eight subjects were divided into three groups on the basis of left ventricular ejection fraction (LVEF) and the presence or absence of CIMR: LVEF > or =0.5 and negative CIMR (group 1, n = 28), LVEF <0.5 and negative CIMR (group 2, n = 12), and LVEF <0.5 and positive CIMR (group 3, n = 18). MT was defined by its four vertices at the anterior annulus, posterior annulus, and medial and lateral papillary muscle roots, determined by MRI at peak systole. The results showed no clear cutoff values of MT parameters between groups 2 and 1. In contrast, all MT indexes were significantly different between groups 3 and 2 (P < 0.05), and significant cutoff values differentiated the two groups. A scoring system employing parameters of the whole MT confirmed the absence of CIMR with total edge length index <268 mm/BSA(1/3), total surface area index <2,528 mm(2)/BSA(2/3), and volume index <5,089 mm(3)/BSA (where BSA is body surface area). The sensitivity, specificity, and positive and negative predictive values were 1.00. This preliminary study demonstrates that MT might serve as a good geometrical surrogate for assessing CIMR. The derived geometrical criteria of MT may be useful in surgical correction of CIMR.