Mandibular shape analysis in fossil hominins: Fourier descriptors in norma lateralis.

Biological shape can be defined as the boundary of a form in 2-space (R(2)). An earlier study (Lestrel et al., 2010, HOMO-J. Comp. Hum. Biol.) of the cranial vault found that there were statistically significant differences between each of the three groups: H. erectus, H. heidelbergensis, and H. neanderthalensis compared with H. sapiens. In contrast, there was no statistically significant difference among the first three groups. These results suggest that these three groups may have formed single evolving lineage while H. sapiens represents a separate evolutionary development. The purpose of the current research was to discern if the mandible reflected a similar pattern as the cranial vault data. This study used lateral jpeg images of the mandible. Five fossil samples were used: A. robustus (n=7), H. erectus (n=12), H. heidelbergensis (n=4), H. neanderthalensis (n=22) and H. sapiens (n=61). Each mandible image was pre-processed with Photoshop Elements. Each image was then submitted to a specially written routine that digitized the 84 points along the mandible boundary. Each mandible was fitted with elliptical Fourier functions (EFFs). Procrustes superimposition was imposed to insure minimum shape differences. The mandible results largely mirrored the earlier cranial vault study with one exception. Statistically significant results were obtained for the mandible between the H. erectus and H. neanderthalensis samples in contrast to the earlier cranial vault data. F-tests disclosed that the statistical significance was limited to the anterior symphysis of the mandible. This mosaic pattern may be explained by the reduction in prognathism with the concomitant if rudimentary development of the chin as seen in H. neanderthalensis compared to H. erectus.

Cranial vault shape in fossil hominids: Fourier descriptors in norma lateralis.

Two major views of human evolution have elicited considerable controversy. These are: [1] the "out of Africa" hypothesis and [2] the "multiregional" hypothesis. This paper is an attempt to try to reconcile these two scenarios using hominid cranial vault data. Elliptical Fourier functions (EFFs) were used to describe, in visual and numerical terms, the shape of the human cranial vault in norma lateralis. Using jpeg images, contours of the cranial vault of a large sample of hominid specimens were pre-processed in Photoshop CS and rotated in 2D space (positional-orientation) so that a line drawn from nasion to porion was horizontal. The cranial vault image was then digitized with 72 closely-spaced points and submitted to a specially written routine that computed EFFs normalized by scaling (size-standardization). This ensured that the representation was invariant with respect to starting point, size and orientation. Statistically significant differences were found between the H. sapiens sample and both the H. erectus and H. neanderthalensis samples. In contrast, there were no statistically significant differences between the H. erectus and H. neanderthalensis groups, leading to three conclusions: [1] the similarity in cranial vault shape between H. erectus and H. neanderthalensis suggests a single gradually evolving lineage; [2] The taxon H. heidelbergensis can be embedded into the H. erectus→H. neanderthalensis line; and [3] H. sapiens seems to be a separate evolutionary development and is considered here either as a separate species or as a possible example of an allopatric semispecies (Grant, 1977). The results here suggest that human evolution over the last 2 Ma may turn out to be neither totally multiregional or simply out of Africa but rather represents a considerably more complicated picture.

Single oral doses of 13C forms of pteroylmonoglutamic acid and 5-formyltetrahydrofolic acid elicit differences in short-term kinetics of labelled and unlabelled folates in plasma: potential problems in interpretation of folate bioavailability studies.

Single (13)C6-labelled doses of pteroylmonoglutamic acid (PteGlu; 634 nmol) or 5-formyltetrahydrofolic acid (431-569 nmol) were given to fasted adult volunteers, and the rise in total and (13)C-labelled plasma 5-methyltetrahydrofolic acid metabolite monitored over 8 h by HPLC and liquid chromatography-MS. The dose-adjusted area under the curve (AUC) for total (labelled plus unlabelled) plasma 5-methyltetrahydrofolic acid following a 5-formyltetrahydrofolic acid test dose was 155 % that obtained following a PteGlu test dose. Surprisingly, an average 60 and 40 % of the total plasma 5-methyltetrahydrofolic acid response to [(13)C6]PteGlu and [(13)C6]5-formyltetrahydrofolic acid, respectively, was unlabelled; an observation never before reported. Short-term kinetics of plasma [(13)C6]5-methyltetrahydrofolic acid showed a slower initial rate of increase in plasma concentration and longer time to peak following an oral dose of [(13)C6]PteGlu compared with that for an oral dose of [(13)C6]5-formyltetrahydrofolic acid, while the [(13)C6]5-methyltetrahydrofolic acid AUC for [(13)C6]5-formyltetrahydrofolic acid was 221 % that for [(13)C6]PteGlu. These data indicate that PteGlu and 5-formyltetrahydrofolic acid, which are thought to be well absorbed (about 90 %) at physiological doses, exhibit dramatically different rates and patterns of plasma response. A limitation in the rate of reduction of PteGlu before methylation could result in slower mucosal transfer of [(13)C6]5-methyltetrahydrofolic acid derived from [(13)C6]PteGlu into the plasma. This, when coupled with an observed similar plasma clearance rate for [(13)C6]5-methyltetrahydrofolic acid metabolite derived from either folate test dose, would yield a comparatively smaller AUC. These findings suggest potential problems in interpretation of absorption studies using unlabelled or labelled folates where the rate of increase, the maximum increase, or the AUC, of plasma folate is employed for test foods (mainly reduced folates) v. a 'reference dose' of PteGlu.

Lipid dynamics in the plasma membrane of ram and bull spermatozoa after washing and exposure to macromolecules BSA and PVP.

Seminal plasma proteins and macromolecules in the external medium have a major influence on the functionality of sperm plasma membranes. In this investigation we have examined their effects on lipid diffusion in the surface membrane of ram and bull spermatozoa as measured by fluorescence recovery after photobleaching (FRAP). Results show that progressive removal of seminal plasma from ram spermatozoa by repeated centrifugation and resuspension in media +/- 4% bovine serum albumin (BSA) or 0.4% polyvinlypyrrolidone (PVP) causes a reduction in lipid diffusion in all regions of the membrane. By contrast, bull sperm membranes respond with an increase in diffusion in all regions. Repeated washing of bull spermatozoa whose membranes were previously immobile (i.e., showed no recovery after FRAP) restored lipid diffusion suggesting an inhibitory effect of seminal plasma proteins. Further analysis by atomic force microscopy revealed a close association between BSA and the plasma membrane. It is concluded that diffusion of lipids in the plasma membrane of ejaculated ram and bull spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium. Mol. Reprod. Dev. 59:306-313, 2001.

Membrane interactions of the putative fusion peptide (MF alpha P) from fertilin-alpha, the mouse sperm protein complex involved in fertilization.

Mammalian fertilization depends upon successful binding and fusion between the membranes of the spermatozoon and the oocyte. These processes are thought to be mediated by a series of protein-protein interactions in which sperm antigens known as fertilins are thought to play a key role. Using a recently developed fluorescence technique, the interactions of the oligopeptide sequence corresponding to the fusogenic domain of mouse fertilin-alpha (MF alpha P) and phospholipid vesicles have been investigated. Following stopped-flow mixing, MF alpha P bound rapidly to phospholipid membranes in a co-operative manner with a Hill coefficient of 2.4 and binding rate constants in excess of 1000 s-1. The co-operative nature of the binding process is suggested to represent evidence of a structural mechanism to prevent egg fertilization by immature spermatozoa. The subsequent membrane insertion was found to take place over a longer time period (with rate constants of up to 6.3 s-1), and was linear with respect to peptide concentration. Comparison of these processes with similar time-resolved circular dichroism measurements revealed that changes in peptide secondary structure were very rapid. Fourier transform infrared spectroscopy measurements confirmed changes in the secondary structure of MF alpha P during interaction with PC phospholipid membranes, indicating that the peptide is mainly present in a beta-structure with a small proportion of alpha-helix. These results are consistent with the hypothesis that fertilin-alpha is the fusogenic species with an important role in fertilization.

Lipid dynamics in the plasma membrane of fresh and cryopreserved human spermatozoa.

Preserving the integrity of the plasma membrane of spermatozoa is crucial for retention of their fertilizing capacity, especially after stressful procedures such as freezing and storage. In this investigation we have measured lipid diffusion in different regions of the plasma membrane of fresh and cryopreserved human spermatozoa using a sensitive, high resolution fluorescence photobleaching technique (FRAP) with 5-(N-octadecanyl)aminofluorescein as reporter probe. Results show that diffusion was significantly faster on the plasma membrane overlying the acrosome and decreased progressively in the postacrosome, midpiece and principal piece. The midpiece plasma contains a higher proportion of immobile lipids than other regions. In cryopreserved spermatozoa, lipid diffusion in the plasma membrane was significantly reduced on the acrosome, postacrosome and midpiece relative to fresh spermatozoa. Diffusion, however, could be restored to normal levels by washing spermatozoa in a medium containing 0.4% polyvinylpyrrolidine but not in medium alone or in medium containing 0.4% albumin. These results suggest that (i) lipid dynamics in the plasma membrane of human spermatozoa varies significantly between surface regions; (ii) in-plane diffusion is adversely affected by cryopreservation; and (iii) washing frozen spermatozoa in 0.4% polyvinylpyrrolidine restores membrane lipid fluidity to normal levels. The latter finding has important implications for improving the fertility of human spermatozoa following cryopreservation.

Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching.

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5-(octa-decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity.

Regionalized lipid diffusion in the plasma membrane of mammalian spermatozoa.

The plasma membrane of mammalian spermatozoa shows pronounced lateral asymmetry with many glycoproteins restricted to specific domains. Some of these antigens are freely diffusing throughout the membrane whereas others appear static in position. It is not clear whether these concepts also apply to membrane lipids. In this investigation we have used fluorescence recovery after photobleaching (FRAP) techniques to spatially resolve lipid dynamics in various surface domains of 5 species of mammalian spermatozoa (bull, boar, ram, mouse, and guinea pig). Sperm plasma membranes were loaded with 5-(N-octadecanoyl)aminofluorescein (ODAF) reporter probe, and its diffusion was measured in various domains by FRAP analysis. Results showed that in live bull, boar, ram, and mouse spermatozoa, diffusion coefficients (D) were significantly higher over the acrosome and postacrosome than on the midpiece and principal piece of the tail. In dead or permeabilized cells, on the other hand, large immobile phases developed, particularly on the sperm tail, that severely reduced D values. ODAF diffusion was also sensitive to temperature and cross-linking of protein components within the membrane with paraformaldehyde. Guinea pig spermatozoa were different in almost all respects from those of the other species tested. It is concluded that lipid diffusion in the plasma membrane of live spermatozoa varies significantly between surface domains, because of either compositional heterogeneity, or differences in bilayer disposition, or the presence of intramembranous barriers that impede free exchange between domains. This study emphasizes the important role of membrane lipids in regulating polarized migration of sperm surface antigens during developmental processes such as maturation and capacitation.

Contribution of basic residues of the D and H helices in heparin binding to protein C inhibitor.

Protein C inhibitor (PCI) is a heparin-binding serine protease inhibitor (serpin) that regulates hemostatic proteases such as activated protein C (APC) and thrombin. The work described here provides further evidence that the PCI H helix, but not the D helix, has a major role in heparin-accelerated inhibition of APC and thrombin. We previously identified Arg-269 and Lys-270 of the H helix [R269A/K270A "H1" recombinant PCI (rPCI)] as important residues both for heparin-accelerated inhibition of thrombin and APC and for heparin-Sepharose binding (Shirk, R. A., Elisen, M. G. L. M., Meijers, J. C. M., and Church, F. C. (1994) J. Biol. Chem. 269, 28690-28695). H1 rPCI was used as a template for Ala-scanning mutagenesis of other H helix basic residues (H1-K266A, H1-K273A, and H1-K266A/K273A) and of the D helix basic residues (H1-K82A, H1-K86A, H1-R90A, and H1-K82A/K86A/R90A). Compared to wild-type rPCI/heparin (k2 = 2.2 x 10(7) M-1 min-1 for thrombin), heparin-accelerated thrombin inhibition was decreased 2.4-fold by H1 rPCI, 4.4-fold by H1-K266A rPCI, and 8-fold by H1-K273A rPCI. H1-K266A/K273A rPCI thrombin inhibition was essentially not accelerated by heparin. A similar trend was found for APC-heparin inhibition using these H helix rPCI mutants. In contrast, the D helix rPCI mutants did not have further reduced heparin-stimulated thrombin or APC inhibition compared to H1 rPCI. Interestingly, all of the H and D helix rPCI mutants had reduced heparin-Sepharose binding activity (ranging from 180 to 360 mM NaCl) compared to wild-type rPCI and H1 rPCI, which eluted at 650 and 430 mM NaCl, respectively. These data suggest that all four basic residues (Lys-266, Arg-269, Lys-270, Lys-273) in the H helix of PCI form a heparin binding site. Our results also imply that while the D helix basic residues (Lys-80, Lys-86, and Arg-90) contribute to overall heparin binding, they are not necessary for heparin-accelerated activity. We conclude that the primary heparin binding site of PCI is the H helix and not the D helix as found in other homologous heparin-binding serpins such as antithrombin III, heparin cofactor II, and protease nexin 1.

Development of a kinetic model to describe the effective rate of antibody oxidation by periodate.

The oxidation of antibody carbohydrate residues by periodate is a common approach used for site-specific antibody modification and immobilization. This study sought to develop a general kinetic model that could be used to describe the effective rate of this oxidation for process control. A detailed analysis of previous data collected for rabbit immunoglobulin G in the presence of excess periodate indicated that the reaction followed a pseudo-first-order mechanism in which two general classes of sites were being oxidized. The first class of sites was oxidized fairly rapidly (i.e., within 15-30 min), while the second class of sites reacted over the course of several hours. From these results, an equation was developed that gave a good fit under a variety of reaction conditions to the production of oxidized sites available for coupling with a hydrazide label. On the basis of this equation, data obtained at several periodate concentrations under the same pH and temperature conditions were used to estimate the apparent rate and equilibrium constants for the oxidation of each class of sites. The values obtained by using this approach could be used not only to predict the effective rate of oxidation at other periodate concentrations but also to provide information on the individual steps involved in the oxidation process.

Studies on the rate and control of antibody oxidation by periodate.

The oxidation of antibody carbohydrate residues by periodate is a common approach for the site-specific immobilization or modification of antibodies for use in various bioanalytical methods. This study examined the time dependence of this oxidation process under a variety of pH, temperature, and concentration conditions. Polyclonal rabbit immunoglobulin G (IgG)was used as the model system for these studies. Flow-injection analysis and a hydrazide label (Lucifer yellow CH) were used to monitor the progress of the oxidation reaction. It was found that the number of oxidized sites that were available for labeling could be varied between one and eight groups per antibody by adjusting the time, pH, periodate concentration, or reaction temperature. In each case, most of these groups were produced during the first 30-60 min of the reaction. A comparison was made between these results and those of previous studies that have examined the effects of periodate treatment on amino acid residues and antibody activity. From this work, general guidelines were developed for the control and optimization of antibody oxidation for use with assays that require either high or low levels of antibody modification.

Automated determination of antibody oxidation using flow injection analysis.

The oxidation of antibody carbohydrate residues is a common approach used for site-specific antibody immobilization or modification. In this study a flow injection analysis system (FIA) was developed for monitoring antibody oxidation. Antibodies were oxidized with periodate and the resulting aldehyde groups were labeled with Lucifer yellow CH (LyCH). The labeled antibodies were then injected onto an FIA system where the amount of LyCH label was determined by absorbance measurements at 428 nm and the amount of antibody was determined using an on-line bicinchoninic acid protein assay. The analysis time was 2 min per 20 microliters sample injection. The limits of detection for rabbit immunoglobulin G (IgG) and LyCH were 1 x 10(-8) and 4 x 10(-7) M, respectively. The dynamic ranges for IgG and LyCH extended to 2 x 10(-5) and 7 x 10(-3) M. The within-run precision was +/- 5% or less for both analytes. Studies with known LyCH/antibody mixtures indicated that the FIA system had greater accuracy than manual methods at high LyCH levels. One specific application studied for this system was its use in monitoring the time course of periodate-antibody oxidation.

Optimization of oxidized antibody labeling with lucifer yellow CH.

The oxidation of antibody carbohydrate residues is a common approach used for the site-specific immobilization or modification of antibodies. One way of following this oxidation process is to label the resulting aldehyde groups with a dye such as Lucifer yellow CH (LyCH). This study examined the optimum conditions for preparing and purifying antibody-LyCH conjugates. A 250-fold excess of LyCH reacted with antibody at pH 6.5 for two or more hours gave maximum labeling. Nonreacted LyCH could be effectively removed by passing the labeled antibody through a size exclusion column, followed by one or two dialysis cycles. The LyCH antibody conjugates were found to be stable for at least three weeks when stored in pH 7.4 phosphate buffer.

An unusual cause of massive pleural effusion in pregnancy.

A 40 year old woman at 30 weeks of her eighth pregnancy presented with acute onset of dyspnoea and a large left pleural effusion after the onset of premature labour. A barium enema showed diaphragmatic rupture with intestinal contents in the thorax. Repair was accomplished through simultaneous left subcostal and thoracic incisions.

Preparation of guidelines for the avoidance and treatment of extravasation due to antineoplastic drugs.

The preparation of guidelines that address antineoplastic drug administration is described. Vesicant drugs are identified, and specific techniques to avoid extravasation and tissue injury are included as are specific treatment recommendations for extravasation injury. The guidelines were developed by a multidisciplinary ad hoc task force that focused on a major objective of permitting particular treatments to be initiated immediately after extravasation is noted.