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Hazardous area classification is well established for dust and vapours, however this is not the case for high flashpoint liquid fuels. This study highlights the limitations of current guidance in relation to flammable mists, through demonstration of flammability of a representative high flashpoint fuel for releases in the range of representative industrial operating pressure, complemented by a phenomenological analysis and semi-quantification of the results observed.\ud \ud Flammability results are presented from low-pressure practical releases ( 61 °C), through a plain orifice, at temperatures well below its flashpoint. Based on a proposed two-phase flow-regime diagram, a semi-quantitate analysis of the results observed is offered via a simple 1-D phenomenological model, accommodating jet breakup length, spray quality, air entrainment and droplet dynamics.\ud \ud The complex scenario of liquid releases impinging onto an unheated flat surface is also considered. An impingement model is utilised to show the relative increase in volume of fine secondary spray induced post-impingement relative to the unobstructed case, resulting in a significant volume of flammable mist. This is demonstrated experimentally by showing flammability of a 5 barg release post impingement whereas the unobstructed 10 barg case would not ignite.

Background:Electroencephalography (EEG) is playing an increasingly important role in the management of comatose patients in the intensive care unit.Methods:The techniques of EEG monitoring are reviewed. Initially, standard, discontinuous recordings were performed in intensive care units (ICUs). Later, continuous displays of “raw EEG” (CEEG) were used. More recently, the addition of quantitative techniques allowed for more effective reading.Results and Conclusions:Applications of continuous EEG to clinical problems are discussed. The most useful role of CEEG appears to be the detection and management of nonconvulsive seizures. There is a need for controlled studies to assess the role for CEEG in neuro-ICUs and general ICUs.

The gene encoding the proteinase from equine infectious anaemia virus (EIAV) was cloned and expressed in Escherichia coli. The recombinant EIAV proteinase was purified to homogeneity and shown to have the ability to process polyprotein and synthetic peptide substrates of human immunodeficiency virus (HIV) origin with an efficiency that can approach that exhibited by HIV proteinase. EIAV proteinase, however, was not susceptible to inhibition by a wide variety of inhibitors of HIV-1 proteinase, including those which have been licenced as anti-AIDS drugs. In this respect, EIAV proteinase behaves like an extreme case of a drug-resistant mutant of HIV-1 proteinase that has arisen under selective drug pressure. Only one potent inhibitor (HBY-793) of HIV-1 proteinase showed comparable efficiency against the EIAV enzyme; the compounds A-77003 and A-76889, which differ only in their stereochemistry and which are otherwise structurally identical to HBY-793 from residues P2 to P2' [nomenclature of Schechter, I.Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162], were not effective inhibitors of EIAV proteinase. Mutant forms of EIAV proteinase (Thr30--Asp and Ile54--Gly) were generated and their ability to interact with substrates and inhibitors was characterised. HBY-793 inhibited [Gly54]proteinase as effectively as the wild-type proteinase but was tenfold less potent against [Asp30]proteinase. Data interpretations are presented, based on the structure solved for the complex between HBY-793 and EIAV [Gly54]proteinase [Gustchina A., Kervinen, J., Powell, D. J., Zdanov, A., Kay, J.Wlodawer, A. (1996) Protein Sci. 5, 1453-1465].

After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus. Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site.

Genes were constructed to encode single-chain tethered human immunodeficiency virus HIV-1/HIV-1 and HIV-2/HIV-2 homodimeric proteinases and two HIV-1/HIV-2 heterodimers which differed in the nature of the interface strands. All four constructs under the control of a heat-inducible promoter were expressed in E. coli and the resultant proteinases were purified therefrom. Kinetic parameters (Km, kcat and kcat/Km) were derived for the interaction of the tethered homo and heterodimeric proteinases with two distinct substrates at a variety of pH values. All four enzymes were comparably active toward one substrate. With the second substrate at pH 4.7, the kcat/Km value was best for the HIV-1/1 tethered homodimer, 15-fold lower for the two heterodimeric proteinases, and was reduced by an additional 6-fold for the HIV-2/2 homodimer. From the Ki values determined for the interactions of the four tethered dimer proteinases with a systematic series of synthetic inhibitors, a parallel trend was observed. Whereas several inhibitors were equipotent against all four enzymes, two were discriminatory in that they inhibited strongly the HIV-1/1 homodimer and the two heterodimeric proteinases but had little effect on the HIV -2/2 tethered homodimer (or its untethered wild-type counterpart from HIV-2). The significance of these findings for active site interaction with HIV-proteinases is considered.

Long-term demonstration tests of advanced sorbent enhancement additive (SEA) technologies have been completed at five coal-fired power plants. The targeted removal rate was 90% from baseline conditions at all five stations. The plants included Hawthorn Unit 5, Mill Creek Unit 4, San Miguel Unit 1, Centralia Unit 2, and Hoot Lake Unit 2. The materials tested included powdered activated carbon, treated carbon, scrubber additives, and SEAs. In only one case (San Miguel) was >90% removal not attainable. The reemission of mercury from the scrubber at this facility prevented >90% capture.

Metallothioneins (MTs) are ubiquitous low-molecular-weight proteins with a high affinity for heavy metal ions such as zinc, copper and cadmium. MT over-expression has been associated with resistance against anticancer drugs. In the present study we investigated 86 cases (45 cases of tumour category pT1 and 41 of category pT2) of routinely fixed and paraffin-embedded primary breast carcinomas immunohistochemically with a monoclonal antibody to an epitope of MT shared by its I and II isoforms. Immunohistochemically demonstrated MT over-expression was found in the invasive components of 7 of 32 pT1 and 17 of 28 pT2 invasive ductal carcinomas, whereas all 26 invasive lobular carcinomas gave weak or negative results. Fourteen of 17 pT2 and 2 of 7 pT1 invasive ductal carcinomas with MT over-expression developed metastases during follow-up with poor prognostic outcome. In contrast only 3 of 11 pT2 and none of the 25 pT1 cases without MT over-expression had a poor clinical course (P < 0.001). It is concluded that MT over-expression is associated with significantly poor prognosis particularly in pT2 invasive ductal breast carcinomas.

Antisera were raised against synthetic peptides from the prosegment of human prorenin. The use of each of these for detection of the appropriate prosegment region of prorenin was validated by development of an ELISA protocol standardised with recombinant prorenin present in culture medium conditioned by myeloma cells transfected with a prorenin expression plasmid. Detection of the respective epitopes in the prosegment required prior exposure of the prorenin in the medium to acid pH in order to partially unfold the prorenin molecule by dislodging the prosegment from the main body of the protein. By these ELISA protocols, the form of latent renin present in representative samples from ovarian cyst and follicular fluids was analysed; one follicular cyst fluid was found to contain full-length prorenin whereas the fluid from a benign cyst and ovarian follicular fluid samples contained the precursor in truncated form.

The wild-type -Phe*Pro- bond located at the N-terminus of the mature aspartic proteinase of HIV-1 was replaced by -Ile-Pro- or -Val-Pro-. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV-proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31-8959, was assessed. A hitherto unobserved cleavage junction (at approximately Ala-Phe*Leu-Gln approximately) in the frame-shift region of the gag-pol viral genome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self-processing at neural pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31-8959.

A family of synthetic genes was constructed encoding a rainbow trout metallothionein (MT), a human MT, and two chimeric molecules which contained respectively (i) the N-terminal (or head) domain of human MT followed by the C-terminal (or tail) domain of a fish MT (termed mermaid MT) and (ii) the head domain of fish MT fused with the tail domain of human MT (denoted fishman MT). These were expressed in Escherichia coli and the four recombinant proteins were purified to homogeneity therefrom. All four were found to bind 7 g atoms of Cd(II) per mol; at pH 7.0, but not at pH 8.6, four Cd(II) ions were sequestered preferentially in the tail (or alpha) domain. Reciprocally, copper was found to bind preferentially in the head (or beta) domain. The human and fishman MTs displayed a stoichiometry of 12 g atoms of Cu(I) per mol, while rainbow trout and mermaid MTs bound only 10. The significance of these findings is discussed in relation to the different positional organization of cysteine residues close to the N and C termini of mammalian and piscine metallothioneins.

In the United States, testing has been under way at electric coal-fired power plants to find viable and economical mercury control strategies to meet pending regulations. San Miguel Electric Cooperative (SMEC) engaged the Energy & Environmental Research Center (EERC) through a request for proposal (RFP) to perform research tests to evaluate sorbent-based technologies at its coal-fired San Miguel Generating Station to identify possible technology options that could be used by SMEC to meet the mercury reduction requirements of future U.S. federal standards. The goal of the testing was to target a mercury removal of {ge}90%. The EERC has successfully field-tested several sorbent-based technologies in previous projects that offer promise and potential to achieve a target removal of {ge}90%. Based on these field test results, yet recognizing that fuel type and plant operating conditions affect mercury capture significantly, the EERC proposed research tests to evaluate potential sorbent-based technologies provided by Norit Americas and the EERC that could potentially meet SMEC's mercury control objectives. Over the period of May through mid-June 2008, the EERC tested injection of both treated and nontreated activated carbon (AC) provided by Norit Americas and sorbent enhancement additives (SEAs) provided by the EERC. Tests were performed at Sanmore » Miguel Unit 1 (450 MW) and included injection at the inlet of the air heater (AH) (temperature of 720 F). The test coal was a Texas lignite fuel with an average moisture content of 31.19%, an ash content of 26.6%, a heating value of 5,094 Btu/lb, a sulfur content of 2.7%, and a mercury concentration of 0.182 ppm, all reported on an as-received basis. Pilot-scale testing results identified DARCO{reg_sign} Hg-LH, SEA2 + DARCO{reg_sign} Hg, and the ChemMod sorbents as technologies with the potential to achieve the target mercury removal of {ge}90% at the full-scale test. Mercury concentrations were tracked with continuous mercury monitors (CMMs) at the electrostatic precipitator (ESP) inlet (ESP In), scrubber inlet, and scrubber outlet of San Miguel Unit 1, and a dry sorbent trap method was used to take samples periodically to measure mercury concentrations at the each of the CMM sampling locations described above. A limited number of Ontario Hydro (OH) measurements were also conducted. Removal efficiencies were calculated from mercury-in-coal values to scrubber out CMM values. Sorbent trap samples taken at the each sampling location outlet were found to be fairly consistent with CMM values. A maximum mercury removal of 78.5% was achieved with the SEA2 + DARCO Hg sorbent combination at injection rates of 50 ppm and 4 lb/Macf, respectively. An injection rate of 4 lb/Macf for DARCO Hg-LH and DARCO Hg resulted in mercury removals of 70.0% and 64.2%, respectively. These mercury reduction values were achieved at full load and at stable plant operating conditions. Scrubber reemission was observed during sorbent injection and had a significant effect on coal to scrubber out mercury removal values. When the sorbents were injected into San Miguel Unit 1 at the AH inlet, no effects on unit operations were observed. ESP performance throughout the test period was fairly steady, with only one minor breakdown. However, it should be noted that test durations were short.« less

Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.

A neutral proteinase has been solubilized from rat intestinal muscle by extraction at low ionic strength. It has an apparent mol. wt. of 33,000 and is stable only around neutral pH. Characterization studies with specific inhibitors and substrates have shown it to be a trypsin-like serine proteinase. The rat proteinase and bovine pancreatic trypsin have equivalent activities as measured with peptide and denatured protein substrates but the rat proteinase is about 300 times more active than an equimolar amount of trypsin towards proteins in their native conformation. It has been shown that the activity of the rat proteinase can be modulated (1) by changing the conformation of the substrate protein(s) and (2) by means of an endogenous inhibitor. The inhibitor has been purified to homogeneity from rat intestinal muscle. It has a mol. wt. of 8,000 and binds only weakly to the rat proteinase (Ki approximately equal to 10(-6) M). It did not inhibit any of the other proteinases tested. The implications for such a proteinase--inhibitor system in the non-lysosomal pathway of intracellular protein degradation are considered.

Kinetic constants (Km,Kcat) are derived for the hydrolysis of a number of chromogenic peptide substrates by the aspartic proteinase from HIV-2. The effect of systematic replacement of the P2 residue on substrate hydrolysis by HIV-1 and HIV-2 proteinases is examined.

1. When rainbow trout were exposed to cadmium by intraperitoneal injection, there was a rapid (within 3hr) and significant (approx. 63%) loss of the metal from the whole bodies of the fish. 2. Of the metal retained in the bodies of the fish (approx. 37% of the injected dose), more than 98% was accounted for collectively among the liver, kidney and gills. 3. Subsequent maintenance of the rainbow trout in fresh water for up to 98 days post-metal administration, indicated that there was no further loss of the cadmium accumulated in the organs studied and that the distribution of the metal among the liver, kidney and gills remained unchanged over that period. 4. During this 98-day period of maintenance of the fish, tissue concentrations of metallothionein-specific mRNA and metallothionein protein were quantified using riboprobe and ELISA systems respectively. Metallothionein-specific mRNA concentrations increased rapidly (within 24 hr) before falling back to levels similar to, or slightly greater than, those found in control animals. The concentration of metallothionein protein also increased significantly (within 3 days) then remained elevated thereafter. 5. Throughout the experimental period, the concentrations of zinc and copper were also monitored in the liver, kidney and gills of the rainbow trout. The concentrations of each ion differed between each of the organs but did not change during the experiment. 6. The induction of metallothionein gene expression by cadmium in the liver, kidney and gill of rainbow trout and the subsequent sequestration of the toxic metal is discussed with regard to the relative levels of these other essential metal ions.

1. 1. Pike were exposed to cadmium dissolved in their aquarium water at separate concentrations of 60, 125 and 225 μg/l and the distribution of the metal accumulated in the major body organs was determined. 2. 2. The fractional retention coefficient ([μg Cd/100 g body wt]/[(μg Cd/1) × weeks]) for cadmium was constant at each concentration and at different periods of exposure to the metal. The values measured were remarkably low. 3. 3. When cadmium was introduced into the fish by intraperitoneal injection, the metal was found associated with metallothionein. 4. 4. The amino acid composition and immunochemical cross-reactivity of pike metallothionein were comparable to those of other piscine metallothioneins. 5. 5. The unexpected tolerance of pike to the toxic effects of cadmium when exposed to the metal in the aquatic environment is discussed.

The two human pronapsin genes which are located immediately adjacent to one another on chromosome 19 were shown, using quantitative RT-PCR, to have distinctly different expression patterns. Transcription of the pronapsin A gene in normal human lung tissue was not appreciably altered in lung carcinomas and comparable pronapsin A mRNA levels were also quantified in lung epithelial cell lines and in tumour cell lines originating from human lung epithelium. Pronapsin A may thus have considerable diagnostic value as a marker for primary lung cancer. In contrast, the pronapsin B gene, which lacks an in-frame stop codon and so may be a transcribed pseudogene, is expressed at comparable levels in normal human spleen, thymus, cytotoxic and helper T-lymphocytes, natural killer (NK) cells and B-lymphocytes. The mRNA levels quantified in B-cells from adults with chronic lymphoblastic leukaemia were not significantly different from those measured in B-cells from healthy individuals. Similarly, comparable levels of expression were quantified in monocytes from healthy individuals and from a patient with acute myeloid leukaemia, whereas in alveolar macrophages, which are terminally differentiated myeloid cells, transcription of the pronapsin B gene was down-regulated by approximately one order of magnitude. Reciprocally, an approximately 20-fold up-regulation in expression of the procathepsin D gene in the macrophages relative to the circulating monocytes was revealed by quantitative measurements made in parallel throughout all of this study for the respective mRNAs encoding the intracellular aspartic proteinases, procathepsin D and procathepsin E. Thus, while there appears to be little difference in expression of the pronapsin A and B genes between healthy and malignant human cells and tissues, the reciprocal alterations in the levels of transcription of the pronapsin B and procathepsin D genes may have significance in the developmental processes associated with myeloid cell differentiation into macrophages.

Swamp fever or equine infectious anaemia is an infectious disease caused by a non-oncogenic retrovirus. The primary mode of transmission between horses is via a blood-sucking insect. Equine infectious anaemia virus (EIAV) is closely related to other lentiviruses of ruminants and primates, such as HIV, SIV and FIV (feline immunodeficiency virus). Just as with these other viruses, EIAV encodes an aspartic proteinase which is essential for processing gag and gag-pol polyproteins in order to generate new infectious viral particles.1 The sequences of EIAV and HIV-1 proteinases have ~30% identity overall. With the large number of HIV-1 PR inhibitors that have been generated,2 including those now licensed by the FDA (Invirase™, Norvir™ and Crixivan™), it was important to establish whether any of these might be effective against EIAV PR. Since no effective treatment currently exists for affected horses, this might be a useful approach for management of this wasting disease.

Malaria is one of the major diseases of the world. Between 2 to 3 million deaths occur each year, mainly children under 5 years of age living in sub-Saharan Africa. Up to 300 million people are infected at any given time and up to 2 billion people (close to half the world population) live in malarious areas and are at risk of infection. The most virulent of the four malaria species which infect humans is Plasmodium falciparum and the spread of resistance by this species to the available drugs, such as chloroquine, has resulted in a critical world health situation with a desperate need to develop new drugs.

Only a few natural inhibitors of aspartic proteinases have been discovered to date. A small number of peptides from bacterial origins have been shown to inhibit members of this protease family.1-2 Larger proteinaceous inhibitors of aspartic proteases have been discovered in the potato3 (cathepsin D inhibitor), in yeast,4 and the intestinal nematode Ascaris.5–7 Similar genes have been discovered in other parasitic nematodes.8 The Ascaris aspartic proteinase inhibitor (PI-3) is a 16.7 kDa protein that specifically inhibits pepsin, gastricsin, and, less strongly, cathepsin E. This inhibitor possesses six cysteine residues which form three disulfide bridges, and it contains helical and strand structural motifs.

Currently, three inhibitors of HIV-I protease are showing promise in clinical use against HIV infection. However, the development of resistance continues to be an important consideration in protease inhibitor therapy. Mutations in protease which lead to resistance have been observed in both inhibitor treated and untreated individuals (Borman et al., 1996; Condra et al., 1995). Variations have also been observed to a higher degree in areas of the gag region including p7, and the cleavage site sequences (Barrie et al., 1996).

Varicella-zoster virus (VZV) genes 33 and 33.5 are predicted to encode the VZV proteinase and its substrate (the assembly protein) respectively. These genes were expressed in insect cells using recombinant baculovirus and it was confirmed that gene 33 encodes a proteinase capable of autoproteolytic processing at two positions. When VZV gene 33.5 was co-expressed with the VZV proteinase, processing of the VZV33.5 gene product was observed. A polyclonal antiserum to the VZV assembly protein domain highlighted a set of proteins in VZV infected HEL cells identical to those identified in insect cells expressing VZV genes 33 and 33.5. To facilitate further characterization of the VZV proteinase the enzyme was purified by affinity chromatography from an E. coli expression system and in vitro activity was observed.

Genes encoding a number of mutants of HIV-1 proteinase were sub-cloned and expressed in E. coli. The proteinases containing mutations of single residues (e.g., G48V, V82F, I84V and L90M) were purified and their catalytic efficiencies relative to that of wild-type proteinase were examined using a polyprotein (recombinant HIV-1 gag) substrate and several series of synthetic peptides based on the -Hydrophobic * Hydrophobic-, -Aromatic * Pro- and pseudo-symmetrical types of cleavage junction. The L90M proteinase showed only small changes, whereas the activity of the other mutant enzymes was compromised more severely, particularly towards substrates of the -Aromatic * Pro- and pseudo-symmetrical types. The susceptibility of the mutants and the wild-type proteinase to inhibition by eleven different compounds was compared. The L90M proteinase again showed only marginal changes in its susceptibility to all except one of the inhibitors examined. The K(i) values determined for one inhibitor (Ro31-8959) showed that its potency towards the V82F, L90M, I84V and G48V mutant proteinases respectively was 2-, 3-, 17- and 27-fold less than against the wild-type proteinase. Several of the other inhibitors examined form a systematic series with Ro31-8959. The inhibition constants derived with these and a number of other inhibitors, including ABT-538 and L-735,524, are used in conjunction with the data on enzymic efficiency to assess whether each mutation in the proteinase confers an advantage for viral replication in the presence of any given inhibitor.

Malaria is endemic throughout much of the tropics, causing in excess of 100 million clinical cases and 1 million child deaths in Africa per year. With increasing resistance of the parasites to currently available anti-malarials, paralleled by the increasing resistance of vector mosquitoes to pesticides, malaria is resurgent and represents the most serious threat to human health in the developing world.

The specific conversion of human endothelin (ET) precursors big ET-1, big ET-2 and big ET-3 into their respective ET by cathepsin E was examined. Comparable pH optima were obtained for ET-1 and ET-2 generation, whereas effective conversion of big ET-3 into ET-3 necessitated a lower pH value. Determination of kinetic parameters (Km, kcat.) for all three conversions indicated that the precursors were efficiently bound by cathepsin E. The significance of the values obtained for the catalytic-centre activities and the effect of a specific inhibitor are discussed.

A metallothionein cDNA was generated from pea (Pisum sativum L.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of approximately 6 g.atoms per mole of protein was estimated. N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

The clinical efficacy of converting enzyme inhibitors1 for reducing blood pressure in a large percentage of hypertensive patients has aroused considerable interest in developing agents that interrupt the renin-angiotensin system at other points, for example by blockade of the angiotensin II receptor2 or by inhibition of the aspartic proteinase, renin. Substrate based design of renin inhibitors in which the scissile P1-P1′ dipeptide is replaced with a non-hydrolyzable group, often a mimetic of a tetrahedral transition state for amide bond hydrolysis, has provided a useful approach for obtaining a variety of inhibitor structure types,3 many with Ki’s of better than 10−9 M. A clinically useful renin inhibitor, however, has not yet emerged due to poor pharmacokinetics (i.e., metabolism or rapid clearance) or poor oral absorption, problems often encountered with peptidic drug targets. An example of this is seen with 1, a “tetrapeptide” inhibitor4 that spans the P4 through P3′ sites of the renin substrate and which utilizes the statine analog, ACHPA5, as a P1-P1′ dipeptide replacement (Figure 1). Although 1 is quite potent in vitro, very low levels of drug are found in the blood after oral administration to the rhesus monkey at 50 mg/kg, and the half life after intravenous administration is short (< 1 h). Rapid biliary excretion of intact drug has been demonstrated for a number of other renin inhibitors.6

The aspartic proteinase encoded by the HIV virus is required for the processing of the structural gag and enzymatic pol precursor proteins1 and is therefore essential for the generation of mature virus particles.2 The proteinase itself is autocatalytically released from the gag-pol fusion precursor to yield an 11 kDa active species.3 This crucial role in the life cycle of the HIV virus has identified this aspartic proteinase as a target for therapeutic intervention to inhibit disease progression.4

The pol open reading frame (ORF) of the human immunodeficiency virus (HIV-1) encodes three distinct enzymes, one of which is an aspartic proteinase (PR). Point mutations introduced at the active site of this enzyme result in an inability to form infectious virions1 thus emphasizing the paramount importance of PR in viral maturation. Proteinase has thus become a strategic target for the development of compounds that might have therapeutic value in the treatment of AIDS.2 By preference, such compounds should be specific for the target, HIV-PR and should not have side effects by interacting with similar enzymes present in the human body, such as have been observed for example with anti-viral agents (e.g. AZT that act against reverse transcriptase RT). The design of specific PR inhibitors is facilitated considerably by a detailed understanding of the molecular topography of the PR active site. To this end, several series of synthetic chromogenic substrates have been used to unravel the subsite preferences of this important enzyme. In some cases, the requirement for certain residues to be present in particular locations in native protein substrates in order to ensure effective hydrolysis by PR has also been examined.

A synthetic gene for rainbow trout metallothionein was constructed and inserted into a dual origin plasmid where expression was induced by a temperature shift in a proteinase-deficient strain of Escherichia coli . The recombinant protein was purified to homogeneity, and a partial amino acid sequence was determined to confirm its identity. Its immunochemical characteristics were similar to those of native metallothionein from rainbow trout. The amounts of recombinant metallothionein produced were quantified in soluble cell extracts by ELISA. Low concentrations were detected when growth was performed either in L-broth or defined (GMM-II) medium. Supplementation of the medium with zinc or copper had no effect on the amount of metallothionein produced. By contrast, when cadmium was included in either L-broth or GMM-II medium, much higher concentrations of the protein within the cells (approx. 13 μg/mg soluble cell protein) were detected. This stabilisation of the protein by metal reconstitution in vivo is considered in relation to the selective uptake / exclusion of metals by the cells and its significance for the scavenging of certain precious or toxic heavy metals is discussed.

The aspartic proteinase zymogen, progastricsin, which occurs in normal gastroduodenal mucosa and prostate, has been localized by the immunogold-silver method in formalin-fixed, paraffin-embedded sections of metastatic adenocarcinoma in lymph nodes and liver, the primary site being known in each case. Progastricsin was demonstrated in 16 of 84 lymph node metastases, including 7 of 17 from stomach, 2 of 2 from prostate, and 7 of 65 from other sites. Progastricsin was also found in 19 of 98 hepatic metastases, including 8 of 22 from stomach, 6 of 24 from pancreas, and 5 of 52 from other sites. The presence of progastricsin in a metastasis correlated well with a primary tumour in the stomach or prostate or, less significantly, pancreas. Immunolocalization of progastricsin in a histological section of metastatic adenocarcinoma may help to locate the primary site.

Rainbow trout were exposed to either cadmium (9 micrograms/l) or zinc (100 micrograms/l) in their aquarium water. They were then transferred to water containing concentrations of cadmium (54 micrograms/l) that would have otherwise proved fatal to the majority of the fish without the pretreatment. Most of the fish survived under both sets of conditions. However, two different mechanisms seem to be involved in the protection of the animals against the toxic manifestations of cadmium. In both cases, more than 99% of the total body load of cadmium was found in the liver, kidney and gills of the animals. Analysis of the metal-binding proteins in these organs was carried out. In the fish exposed to the two concentrations of cadmium, the toxic metal was found only in association with two low mol. wt specific binding proteins despite the presence of zinc- (and copper)-containing isometallothioneins in all three organs. On the other hand, cadmium was distributed between these binding-proteins and metallothioneins in the liver, kidney and gill of the trout pretreated with zinc before their exposure to cadmium.

1. Rainbow trout were exposed to cadmium in their aquarium water for different lengths of time and at different concentrations. 2. More than 99% of the total body loads accumulated under all such conditions of exposure was found in the liver, kidney and gills. 3. Comparison of the metal-binding proteins in these organs indicated that two low mol. wt proteins sequestered the cadmium while zinc was bound to metallothionein. 4. Cadmium was not found in association with metallothionein unless artificially high concentrations were introduced either by intraperitoneal injection of the fish or by dialysis of tissue extracts in vitro .

Summary The 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV), have been demonstrated to be cysteine-type proteinases, involved in the processing of the respective polyproteins expressed by the monocistronic RNA genome. Nucleotide sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome encodes a homologous 3C protein. The HRV-14 3C protein was purified to homogeneity from Escherichia coli expressing the cloned 3C genomic fragment. The enzyme was assayed against peptides corresponding to those residues, predicted (by nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein. The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase. By contrast, peptides corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys→Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV-14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an important role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy.

Inhibitors of the conversion of angiotensinogen to the vasoconstrictor angiotensin II have considerable value as antihypertensive agents. For example, captopril and enalapril are clinically useful as inhibitors of angiotensin-converting enzyme. This has encouraged intense activity in the development of inhibitors of kidney renin, which is a very specific aspartic proteinase catalysing the first and rate limiting step in the conversion of angiotensinogen to angiotensin II. The most effective inhibitors such as H-142 and L-363,564 have used non-hydrolysable analogues of the proposed transition state, and partial sequences of angiotensinogen (Table 1). H-142 is effective in lowering blood pressure in humans but has no significant effect on other aspartic proteinases such as pepsin in the human body (Table 1). At present there are no crystal structures available for human or mouse renins although three-dimensional models demonstrate close structural similarity to other spartic proteinases. We have therefore determined by X-ray analysis the three-dimensional structures of H-142 and L-363,564 complexed with the aspartic proteinase endothiapepsin, which binds these inhibitors with affinities not greatly different from those measured against human renin (Table 1). The structures of these complexes and of that between endothiapepsin and the general aspartic proteinase inhibitor, H-256 (Table 1) define the common hydrogen bonding schemes that allow subtle differences in side-chain orientations and in the positions of the transition state analogues with respect to the active-site aspartates.

The aspartic proteinase cathepsin D was purified from human spleen and localised in various formalin fixed paraffin embedded human tissues using the peroxidase-antiperoxidase (PAP) technique. Cathepsin D was shown not only in macrophages but also in other connective tissue cells, and in epithelium. It was present in spleen (littoral cells and cells within Malpighian bodies), liver (hepatocytes and Kupffer cells), lung (alveolar macrophages and bronchial epithelium), brain (neurones), lymph nodes (histiocytes in germinal centres, sinusoid lining cells) and stomach (parietal and mucous neck cells). Cathepsin D was also found in carcinomas of bronchus, stomach, colon, kidney, breast, ovary, bladder and pancreas, both in neoplastic epithelium and in stromal cells, but was seldom present in connective tissue neoplasms. A group of malignant lymphomas also contained the enzyme within scattered cells. The distribution of cathepsin D seems to be much wider than that of the structurally related aspartic proteinases pepsin, gastricsin, and renin.

SummaryCommercial and pure chicken pepsin preparations were compared with rennet as cheesemaking coagulants. From the milk clotting and caseinolytic activities and enzymic stabilities, predictions were made of the relative activities of the chicken pepsins and rennet during cheese ripening. By this means mixtures of chicken and pig pepsins for cheesemaking trials were formulated. These and mixtures of chicken pepsins with rennet were approximately additive in coagulating activity provided they were used immediately. The amounts of chicken pepsins used in laboratory scale Cheddar cheesemaking trials were minimized by acidification and calcification of the milk. The nature of the coagulant did not affect the cheesemaking conditions, yields or cheese composition. Cheeses made with chicken pepsins alone showed faster proteolysis, more intense flavour, off-flavours and bitterness and were softer than those made with rennet. Cheeses made with chicken pepsin/rennet mixtures were intermediate and those made with chicken pepsin/pig pepsin mixtures were similar to those made with rennet. It was concluded that neither of the chicken pepsin preparations was suitable for Cheddar cheesemaking and that the predictive tests had led to correct assessments.

1. The effect of exogenous lipases on fatty acid synthesis from [14C]malonyl-CoA by the microsomal and soluble fractions from germinating peas was studied. 2. Addition of phospholipase A2 or the lipase from Rhizopus arrhizus had no effect on total fatty acid synthesis by the soluble fraction but caused severe inhibition of that by the microsomal fraction. 3. The addition of enzymes with phospholipase activity particularly inhibited the microsomal stearate elongase. 4. Control studies indicated that the phospholipase-induced inhibition of fatty acid synthesis was due to the location of fatty acid synthetase, palmitate elongase and stearate elongase on the outside of the microsomal vesicles. 5. Experiments with a trypsin-like proteinase showed that approximately half the microsomal fatty acid synthesis was resistant to proteolysis. 6. Although addition of exogenous phospholipases had no effect on total fatty acid synthesis by the soluble fraction, it did increase alpha-hydroxylation of newly-formed palmitate and stearate. 7. The results provide further evidence for differences between the soluble and particulate fatty acid synthetase and palmitate elongase activities of germinating pea.

1. 1. Treatment of glutamate dehydrogenase with chymotrypsin causes a transient increase in activity. 2. 2. The allosteric inhibition of dehydrogenase activity by GTP is not affected, whereas activation by ADP is lost during proteolysis. 3. 3. NADP protects the dehydrogenase from these effects.

Three aspartic proteinases with similar Mr values (approx. 80,000) but from distinct sources (human gastric mucosa, human erythrocyte membranes and rat spleen) were shown to have immunological cross-reactivity and comparable mobilities when subjected to polyacrylamide-gel electrophoresis under non-denaturing conditions. Kinetic parameters (kcat, Km and Ki) were determined for the interactions of the three enzymes with two synthetic chromogenic substrates and five inhibitors (naturally occurring and synthetic). On this basis it would appear that all of the enzymes should be considered equivalent to cathepsin E. pH-activity measurements indicated that the aspartic proteinase that originated from the erythrocyte membranes retained activity at a higher pH value than either of its readily soluble counterparts.

Bovine pepsinogen is converted into pepsin by removal of 45 amino acid residues from the NH2 terminus of the single polypeptide chain. The complete sequence of the activation peptides has been deduced from two overlapping sets of peptides, one set obtained by autoactivation of the zymogen at pH 2 and the second set from a tryptic digest of maleylated pepsinogen. A peptide derived from the 17 residues at the NH2 terminus of bovine pepsinogen has been identified as an inhibitor of the milk-clotting action of pepsin. Extensive sequence homologies exist among the activation peptides of bovine and porcine pepsinogens and bovine prochymosin. In the peptides from the two pepsinogens, 30 of the 44 or 45 residues are identical, and most of the substitutions in the other residues are conservative. The preservation of positive charges in the NH2-terminal portion of the zymogen molecules, lost during activation, agrees with current ideas on the involvement of this segment in stabilizing the physical structure of the zymogens.

Human gastric mucosa contains three immunochemically distinguishable aspartic proteinases, pepsinogen I (pepsinogenA), pepsinogen II (pepsinogen C, progastricsin), and a nonpepsinogen proteinase also termed slow moving proteinase (SMP). The properties of SMP, and in particular its relationship to another aspartic proteinase, cathepsin D, were examined in this study. Slow moving proteinase and cathepsin D were isolated, respectively, from gastric mucosa and human spleen. Antiserum specific to each proteinase was prepared in rabbits. Rabbit anti-SMP did not recognize cathepsin D, and conversely, anticathepsin D did not react with SMP. Immunohistochemical studies localized SMP to surface epithelial cells in both the fundic and pyloric gland areas of the stomach. In contrast, cathepsin D was found mainly in mononuclear cells in the lamina propria and in parietal cells. Slow moving proteinase exhibited considerably lower K m values for its interaction with two chromogenic substrates than did cathepsin D. An even greater distinction between the two enzymes was found with the protein inhibitor from Ascaris lumbricoides; the activity of SMP was inhibited very strongly, whereas that of cathepsin D was not affected. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions, SMP consisted of two subunits with apparent molecular weights of 42,500 and 41,000. The last two properties characterize a less-well-known aspartic proteinase, cathepsin E. We conclude that SMP is not cathepsin D, but that it may be cathepsin E.