Standardization of H-reflex analyses.

Variability in the H-reflex can make it difficult to identify significant changes using traditional pooled analysis techniques. This study was undertaken to introduce a normalisation approach to calculate both the relative size and the relative stimulus intensity required to elicit the H-reflex response so that comparisons can be made not only with results obtained during different experimental session but also between different subjects. This normalisation process fits the size of the measured M-responses and H-reflexes over the entire stimulus range with model curves to better facilitate the calculation of important parameters. This approach allows normalisation of not only the size of the response but also the relative stimulus intensity required to elicit the response. This eases the comparison of the reflex responses under various situations, and is capable of bringing out any genuine differences in the reflex in a reliable manner not previously possible. This study illustrates that comparison of the reflex between days is problematic, even in the same subject, as both the reflex size and the relative stimulus intensity required to obtain this reflex changed in all subjects. We suggest that H-reflex studies need to use normalisation not only for size of the reflex but also for the stimulus intensity, and also that all experiments for a single subject should be performed in the same session or during the same day using some level of background muscle activity in the muscle concerned as the variability of the muscle at rest was found to be larger.

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants.

BACKGROUND: The vast majority of BRCA1 missense sequence variants remain uncharacterized for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. OBJECTIVE: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. METHODS AND RESULTS: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. CONCLUSIONS: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

Ontogenic characteristics of lactate dehydrogenase turnover in the mouse.

1. In order to investigate the ontogenic and turnover characteristics of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the foetal mouse, activities of this enzyme and isoenzyme distributions have been measured during development, and the time sequence of incorporation of radioactive amino acids have been determined during the last weeks of gestation. 2. In the early foetal stages, the enzyme was present with low specific activity and in the form of lactate dehydrogenase-5 in all regions, but a marked increase in specific activity became evident about the seventeenth day of gestation, and was accompanied by the formation of tissue specific patterns of isoenzyme distribution. 3. Measurement of turnover parameters was undertaken by both double-label ([3H] and [14C]leucine) and pulse-chase ([3H]leucine) techniques. Both procedures provided indications that appreciable degradation may occur during embryogenesis, with general proteolysis proceeding at a faster rate than with lactate dehydrogenase, and with higher rates of degradation occurring in foetuses which were smaller than average. 4. This data has been discussed in relation to the regional variation in these characteristics and the significance to studies of growth and differentiation.

The turnover of lactate dehydrogenase in skeletal muscle of the mouse. Influence of fibre type and exercise regimen.

1. The influence of fibre-type and an exercise regimen on the turnover characteristics of proteins in mouse skeletal muscle has been studied. 2. Exercise induced a weight increase in most individual muscles and a decrease in lactate dehydrogenase activity which was more marked in white muscle than red. The coincident decrease in M-subunit activity was more noticeable in fast-twitch muscles than in slow-twitch types. 3. Exercise caused a considerable and consistent increase in the rate of degradation of total soluble proteins in individual muscles, with turnover being higher in muscles with a high red fibre content. 4. The response of lactate dehydrogenase to exercise varied considerably between individual muscles as indicated by the comparative rate constants for synthesis and degradation. In general, degradation was greater in muscle with a high slow-twitch or red fibre content. 5. Overall, a considerable redistribution of protein resources was observed during this physiological perturbation (exercise) with the main emphasis away from the pool of total soluble protein towards the myofibrillar constituents. Highly individualistic responses in relation to rates of synthesis and degradation were observed for the specific protein, lactate dehydrogenase, in the separate muscle of this animal.

Hydroxyquinones are competitive non-peptide inhibitors of HIV-1 proteinase.

Quinones with one, two and three aromatic rings are a new class of micromolar non-peptidic inhibitors of HIV-1 proteinase, an enzyme essential for replication of Human Immunodeficiency Virus and an important drug target for AIDS. Substituted anthraquinones bearing hydroxyl substituents on one of their three rings were the most potent of these inhibitors. Comparisons with other small non-peptidic inhibitors that are now emerging, together with enzyme kinetic data indicating that alizarin is a competitive inhibitor, suggest that anthraquinones bind in the active-site groove of HIV-1 proteinase.

Structural analysis of the catalytic site of AcCP-1, a cysteine proteinase secreted by the hookworm Ancylostoma caninum.

Previously, we have reported that hookworms secrete cysteine proteinase activity that is capable of cleaving the cathepsin L-specific substrate Z-Phe-Arg-AMC. We have also reported the gene sequences of novel cathepsin B-like proteinases from hookworms, but have been unable to locate cathepsin L-like genes that could account for the presence of the cathepsin L activity in these parasites. Here we present an homology model for the secreted hookworm cysteine proteinase AcCP-1 based upon the crystal structure co-ordinates of human cathepsin B. The model predicts that substrate binding and specificity differs between AcCP-1 and cathepsin B, and demonstrates that AcCP-1 would preferentially cleave Phe-Arg over Arg-Arg. This thereby provides an explanation for our previous observations that the hookworm proteinase, while structurally cathepsin B-like, displays a cathepsin L-like substrate specificity.

Phylogenetic relationships and theoretical model of human cathepsin W (lymphopain), a cysteine proteinase from cytotoxic T lymphocytes.

The recently described cysteine proteinase cathepsin W, also known as lymphopain, which is expressed specifically by CD8+ T lymphocytes, is phylogenetically related to the cruzipain-like group of the C1 family of peptidases. We have constructed sequence alignments and a theoretical three dimensional homology model of cathepsin W. These have allowed the characterization of signature features of cathepsin W in particular and the cruzipain lineage in general. The signature features are (1) an extended loop structure, Gly 170-Trp 200, in the second or beta-sheet domain; (2) an additional disulfide bond, Cys 25/Cys 60; (3) an additional "orphan" cysteine, Cys 5; (4) an additional residue. Ala 11, inserted after the first beta-sheet sheet; and (5) an S2 pocket lined with Phe 68 and Phe 230 which explains the preference for substrates containing Leu at P2. Further, the model suggested that cathepsin W could exist as a dimer with the Cys 5 of each monomer forming a disulfide bond and the Arg 40 Phe 46 loop (RISFWDF) forming part of the dimeric interface. By comparing cathepsin W with other members of the cruzipain group and with other C1 peptidases, six conserved residues were identified which appear in general to be characteristic of the cruzipain group, and which differentiate cruzipain group members from other C1 peptidases including those of the related cathepsin L lineage. The signature residues of the cruzipain lineage are (cruzipain numbering) Asn 33, Trp 38, Ala 124, Leu 127, Leu 164, and Pro 174.

Effect of pregnancy on the turnover of lactate dehydrogenase and its isoenzymes in mouse tissues.

The turnover characteristics of total protein and of lactate dehydrogenase and its isoenzymes have been determined in several tissues of the mouse during the later stages of pregnancy. Whereas most tissues showed markedly decreased rates of both synthesis and degradation of proteins, the incorporation rate in uterus increased. Significant differences in the extent of isotope distribution between total protein and lactate dehydrogenase and its isozymes were also noted. Overall the concept emerges of a pool of body protein which is responsive to the physiological stresses of pregnancy at the level of both tissue and cellular function.

On the localization of lactate dehydrogenase in the ovaries and reproductive tracts of rats and mice.

The localization of lactate dehydrogenase in the ovaries and reproductive tracts of rats and mice has been studied by a methodology which minimizes loss of this soluble enzyme by diffusion, and allows comment on the subunit composition of the cellular enzyme. The results differ significantly from previous data with conventional methodologies. In particular, the major localization of activity in the present study was identified in interstitial cells, and not the corpora lutea or granulosa cells; and it was noticeable that neither species exhibited massively greater expression of lactate dehydrogenase activity in the oocytes than in adjacent cell types of the reproductive tract. The goblet cells of the Fallopian tube stained intensively for activity of this enzyme. These results have been discussed in relation to the discordant data in the literature, the important role of lactate dehydrogenase in mammalian development, and the evidence for a masking of the activity of this enzyme in oocytes and pre-implantation ova.

Proteolysis of human hemoglobin by schistosome cathepsin D.

Schistosomes feed on human blood. They employ proteases to degrade hemoglobin from ingested erythrocytes, using the residues released for amino acid metabolism. However, the identity and the role of the participating protease(s) are unclear and controversial. Confocal microscopy localized schistosomal cathepsin D to the parasite gastrodermis, and revealed elevated protease expression in females. At sub-cellular level, cathepsin D was localized to superficial digestive vacuoles of the gut and to cisternae of the gastrodermal rough endoplasmic reticulum. Schistosome cathepsin D, expressed in insect cells, autoactivated at pH 3.6 to a approximately 40 kDa form that cleaved the substrates o-aminobenzoyl-Ile-Glu-Phe-nitroPhe-Arg-leu-NH(2) and hemoglobin. The NH(2)-terminal residues of mature cathepsin D of Schistosoma japonicum and Schistosoma mansoni were Asn1 and Gly1, respectively, revealing that the proregion peptide was comprised of 35 residues. The proteases cleaved hemoglobin at pH 2.5--4.6, releasing numerous fragments. S. Japonicum cathepsin D cleaved at 13 sites, S. mansoni cathepsin D at 15 sites. Early cleavage sites were alpha Phe33-Leu34 and beta Phe41-Phe42, while others included alpha Leu109-Ala-110 and beta Leu14-Trp15, demonstrating a preference for bulky hydrophobic residues at P1 and P1'. Most of the schistosomal cathepsin D cleavage sites were discrete from those of human cathepsin D. The gastrodermal location, elevated expression in females, acidic pH optima, similar substrate preferences in two species, and the discrete substrate preferences compared with human cathepsin D together provide compelling support for the hypothesis that schistosomal cathepsin D plays an integral role in hemoglobin proteolysis, and might be selectively targeted by drugs based on protease inhibition.

Host specificity in blood feeding parasites: a defining contribution by haemoglobin-degrading enzymes?

A hypothesis is presented that proposes that the compatibility between species-specific variants of haemoglobin-degrading proteases of blood-feeding parasites (e.g. hookworms, schistosomes, malarial parasites, etc.), and their natural substrates, i.e. haemoglobins from diverse species of mammals, has influenced to evolution of the host range of these parasites. Support for the hypothesis was drawn from molecular modelling studies of the three dimensional structure of an aspartic protease, Acasp, from the canine hookworm Ancylostoma caninum, and models of canine and human haemoglobins docked with the active site of Acasp. The molecular modelling suggested that Acasp, from a canine-specific hookworm, would have a higher substrate affinity for canine haemoglobin than for human haemoglobin.

A device for investigating neuromuscular control in the human masticatory system.

A new apparatus has been developed to study the control of mastication in humans. The subject places his/her teeth on fixed upper and mobile lower bite plates; the device then enables opening and closing movements of the lower jaw against a controlled resistance. It is also possible to vary the number of teeth in contact with the device during an experiment from the entire dental arcade to a single tooth. The specially designed lower bite plate is dynamic and allows for both rotation and translation of the lower jaw during movement, thus, permitting the natural curvilinear trajectory of the jaw. The lower bite plate can follow chewing initiated by the subject without resisting the movement ('no force' mode) via a dedicated microprocessor controlled compensation mechanism. Another function of the device is to inject a constant predetermined load onto the lower bite plate so that the subject 'chews' against a fixed resistance simulating rapidly yielding food bolus ('fixed force' mode). The device can be programmed to increase or decrease the force during the closing or opening phase of chewing by feeding the position information into the force compensation system so both position and force change in parallel, hence, simulating a bite onto a non-yielding, or sticky, food bolus ('normal chewing' mode). By use of a jaw position compensation mechanism, the device can actively move the lower jaw, following any imposed position pattern ('position controlled' mode). The chewing simulator also has a mode that holds the position at a fixed level and allows the force to change ('position hold' mode). Furthermore, the device can inject additional rapid or slow forces or displacements onto the lower bite plate in order to elicit reflexes so that the response of jaw muscles to such stimuli can be examined at various jaw positions, force levels, phases of motion and velocities. The different modes of the apparatus can be used to study the operation and feedback control of human mastication; in particular whether modulations in jaw muscle activity and reflexes are due to changes in force, velocity, position, chewing cycle phase or a combination of these factors.

The envelope glycoprotein of HIV-1 may have incorporated the CD4 binding site from HLA-DQ beta 1.

An hypothesis is presented which states that the increased binding for CD4 by the envelope glycoprotein (gp120) from HIV-1 compared with that from HIV-2 is due to the env gene from HIV-1 having at some stage incorporated exon 2 of the gene coding for the beta subunit of a class II MHC protein, possibly HLA-DQ, which contains part of the CD4 binding site. Evidence is presented from amino acid sequence analysis and consideration of putative binding residues from gp120 and HLA-DQ.

Central nervous system receptor binding profiles of some 2-amino-4-phenyl quinolines: a novel class of alpha 2-adrenoceptor selective ligands.

The 2-amino-4-phenyl quinoline moiety is a structural motif common to a number of central nervous system active agents. Extensive radio-ligand receptor binding profiles for several derivatives of this common structural feature have been determined. A high base level of central nervous system receptor affinity was observed with a distinct preference for the alpha-, and in particular the alpha 2-, adrenoceptors.

Threshold for detection of incisal forces is increased by jaw movement.

Current knowledge regarding the sensitivity of the teeth to forces is based on psychophysical experiments that measured touch detection thresholds under static jaw conditions. It is not known whether jaw movements alter the perception of forces applied to the teeth, but, based on limb movement studies, it is hypothesized that the perception of mechanoreceptor outputs will be downwardly modulated by jaw movements. We predicted that, compared with static jaw conditions, rhythmic jaw movements would be associated with significantly higher psychophysical thresholds for the detection of incisally applied forces. In eight participants, mechanical pulses were delivered to an incisor during static jaw holding or during cyclic jaw opening and closing. Analogous to findings in human limbs, the psychophysical salience of periodontal mechanoreceptor feedback was downwardly modulated by physiologically relevant movements; detection thresholds for mechanical pulses applied to a central incisor were significantly higher during jaw-closing movements than during static jaw positioning.

CD4 and CD8 may adopt a similar mode of binding to their MHC ligands.

A theoretical scheme is proposed by which the type-specific cell surface receptors of T-lymphocytes, CD8 and CD4, bind class I and II MHC proteins in a similar manner. The scheme has equivalent residues in the C'/C'' loop-C'' strand-C''/D loop region in domain 1 of CD4 and CD8 alpha binding to equivalent residues in the C and D beta-strands and C/D loops in HLA-DR beta 2 (class II) and HLA-A2 alpha 3 (class I) respectively through a series of electrostatic, hydrogen and hydrophobic bonds.

Non-peptidic anti-AIDS agents: inhibition of HIV-1 proteinase by disulfonates.

Based upon an earlier observation that sodium docosanedioate (NaO2C-(CH2)20-CO2NA) weakly inhibits HIV-1 proteinase (IC50 12 microM), we have identified a class of more potent inhibitors (sulfonic acids) of this enzyme which are likewise dianionic at pH 5-6.5. Many of the compounds were moderately strong inhibitors of the enzyme (IC50 40nM-10 microM) and some have previously been shown to have anti-HIV activity in lymphocytes. Proteinase inhibition was dependent on the separation between sulfonate/carboxylate substituents, consistent with the hypothesis that negative charged ends of an inhibitor might form ionic bonds with Arg 8 and Arg 108 located at either end of the substrate-binding groove of the enzyme. The binding mode remains to be established by structure elucidation. Results for enzyme inhibition are presented along with structure-activity relationships and evidence for pH dependent inhibition. The general observations reported here may be useful for developing more potent and selective non-peptidic proteinase inhibitors.

Comparative studies of gastrointestinal tolerance and acceptability of milk chocolate containing either sucrose, isomalt or sorbitol in healthy consumers and type II diabetics.

The objective was to compare reaction of adult consumers of confectionery to milk chocolate made with either isomalt, sucrose or sorbitol. Test chocolate was eaten by subjects at home during 7 days in amounts chosen by them up to a maximum of 100 g per day. In a double-blind crossover trial isomalt chocolate was associated in healthy consumers (n = 58) with increased motion frequency, wind and flatulence compared with sucrose chocolate. However, the intensity of these gastrointestinal effects was predominantly slight and insufficient to affect acceptability. In separate crossover trials, reactions of Type II diabetic consumers to eating isomalt chocolate (n = 53) or sorbitol chocolate (n = 51) were compared to reactions when eating no chocolate. Both isomalt and sorbitol chocolate were associated with higher incidence of wind and flatulence than for no chocolate, but only sorbitol chocolate increased motion frequency. Again intensity of gastrointestinal effects was slight. It is concluded that isomalt has potential use in both regular and diabetic chocolate.

Flavones are inhibitors of HIV-1 proteinase.

Substituted gamma-chromones were found to weakly inhibit HIV-1 proteinase, an important enzyme in the replication and processing of the AIDS virus. Chromones bearing hydroxyl substituents and a phenolic group at the 2-position (flavones) were the most active compounds and structure-activity relationships for a limited series of flavone inhibitors are presented. Dixon plots are reported and a possible mechanism for flavone-induced inhibition is proposed. The results are also compared with those for some structurally related non-peptidic inhibitors of HIV-1 proteinase. Since some flavonoid compounds have already been shown to have antiviral activity against AIDS, the present observations of anti-HIV-1 proteinase activity may be particularly significant.