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WE report here the discovery of the first ear ossicle, an incus, of a Plio–Pleistocene hominid. It is substantially different from that of modern man, and the dissimilarity exceeds that between the ear bones of Homo sapiens and of the African apes. The new incus is of interest particularly in view of the unique advantages that ear ossicles have for taxonomic and phylogenetic studies. (The only other fossil hominid ear ossicles are from Qafzeh1 and are indistinguishable from those of modern man.)

A brief description of the exposed middle ear from two Swartkrans specimens demonstrates the feasibility of examining the inner auditory structures of existing fossil hominid material. These Swartkrans fossils provide the first clear view of the early hominid middle ear, and their structures appear to be morphologically distinct. The bony anatomy of the external ear of Australopithecus robustus is found to be unique among the South African hominids.

Publisher Summary This chapter provides an overview of the inclusion complexes of cyclomalto-oligosaccharides (cyclodextrins). Inclusion complexes are chemical species consisting of two or more associated molecules in which one of the molecules—the “host”—forms or possesses a cavity into which it can admit a “guest” molecule, resulting in a stable association without formation of any covalent bonds. Secondary forces are alone responsible for the maintenance of the integrity of all inclusion complexes. One of the most important properties of the cyclodextrins is their ability to form complexes with a variety of organic and inorganic compounds. 1 H-Nuclear magnetic resonance (NMR) spectroscopy provided the first direct evidence of inclusion within the cyclodextrin cavity in solution. Using aromatic “guest” molecules, it was found that, on the addition of the guest, the resonances of the hydrogen atoms of the cyclodextrin situated on the inside of the cavity were shifted significantly upfield due to shielding by the aromatic guest. This chapter discusses determination of the structure of the cyclodextrins, and formation of the cyclodextrins from starch. It provides details about formation of inclusion complexes, detection of complex formation, and thermodynamics of complex formation. Kinetics of complex formation is also explained in the chapter.

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol V required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.

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A complete understanding of how biological membranes function can be obtained only by considering how their various components influence membrane properties and their interactions with biomolecules. In membrane charge transport processes, such as ion permeation, or the movements of membrane-active peptides, a better understanding of lipid-dependent membrane electrostatics is needed. We have varied lipid components that influence membrane chemistry, structure, hydration and packing in ways that will alter the membrane's electrostatic (dipole) potential. We have compared simulations with different electrolyte composition to study the electrostatic influences of ions. We have simulated zwitterionic and 1:1 mixtures with charged lipids, to analyse how the head group controls the dipole potential. We varied the glycerol backbone of the lipids, studying ether lipids that have no carbonyl groups, leading to changes in dipole potential by up to 250 mV. We discuss an effect of interfacial water on the dipole potential and explain the origin of the change. We have simulated a 1:1 mixture of mono- and di-myristoylphosphatidylcholine to isolate the contribution from the single carbonyl group. Lipid chain length effects have been studied using saturated and unsaturated bilayers of different thickness, spanning approximately 7 A. Simulations of monounsaturated and polyunsaturated bilayers were compared to evaluate the roles of double bond position and extent of unsaturation. Differences in dipole potential for all systems are in a good agreement with experiment. Important contributions from the different lipid components along with methods to extract membrane electrostatic properties will be discussed.

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.

Measurements of the u.v.–visible, fluorescence and circular dichroic spectra of roccellin (RO) in the presence of α-, β- and γ-cyclodextrin (αCD, βCD and γCD) have been carried out. From equilibrium u.v.–visible spectral measurements, in the case of βCD, a single 1:1 complex was observed (298.2 K): RO +βCD⇌RO·βCD (K1) where K1=(7.20 ± 0.88)× 102 dm3 mol–1.In the case of γCD a single 2:1 complex was observed (298.2 K): 2RO +γCD⇌(RO)2·γCD (K12) where K12=(9.0 ± 1.8)× 1010 dm6 mol–2. No complexation was observed between roccellin and αCD. Measurements of induced circular dichroism and of fluorescence were consistent with the reaction schemes proposed above for both βCD and γCD, but no interaction was observed with αCD.Measurements of the u.v.–visible spectra of aqueous solutions of roccellin allowed characterisation of the dimerisation equilibrium: 2RO⇌(RO)2(Kd) where Kd is (1.64 ± 0.12)× 104 dm3 mol–1. Measurements of the u.v.–visible and fluorescence spectra of roccellin, in increasing concentrations of lithium sulphate up to 0.5 mol dm–3, showed marked changes, consistent with the presence of increasing concentrations of aggregates of dye molecules with increasing electrolyte concentration.

Measurements of the temperature-jump and equilibrium u.v.–visible and induced circular dichroic spectra of tropaeolin 000 No. 2 (TR) in the presence of α-, β- and γ-cyclodextrin (αCD, βCD and γCD) have been carried out. Three complexation steps are detected in the presence of γCD through the temperature-jump data (298.2 K): TR +γCD ⇌ TR·γCD fast (K1), [graphic omitted], γCD +(TR)2·γCD ⇌(TR)2·(γCD)2 fast (K3), where K1, K2 and K3 are (4.18 ± 1.47)× 102, (1.68 ± 0.54)× 106 and (1.77 ± 1.54)× 102 dm3 mol–1, respectively; k2=(2.27 ± 0.61)× 109 dm3 mol–1 s–1 and k–2=(1.35 ± 0.23)× 103 s–1. In the presence of βCD the third complexation was not detected and K1=(7.1 ± 0.7)× 102 and K2=(4 ± 7)× 106 dm3 mol–1; k2=(5 ± 6)× 109 dm3 mol–1 s–1 and k–2=(1.3 ± 1.5)× 103 s–1. No complexation reactions were detected in the presence of αCD. The equilibrium u.v.–visible spectra and the circular and linear dichroic spectra are consistent with these reaction schemes, but suggest different orientation and penetration for the (TR)2 dimer included in (TR)2·βCD compared with (TR)2·γCD.For the equilibrium [graphic omitted] the equilibrium constant Kd=(9.10 ± 4.28)× 102 dm3 mol–1 has been determined by u.v.–visible spectroscopy, and k–d=(2.24 ± 0.40)× 103 s–1 has been estimated from temperature-jump experiments. Hence kd=(2.0 ± 1.0)× 106 dm3 mol–1 s–1. Thus the increase in stability of (TR)2 included in (TR)2·γCD and (TR)2·βCD over that observed in the absence of cyclodextrin is a consequence of k2≈ 103kd.

Exposure of voltage clamped cardiomyocytes to receptor-coupled oxidant signalling has been associated with a decrease in electrogenic Na+, K+ pump current activity vattributed to glutathionylation of the pump's β1 subunit. The mechanism by which glutathionylation, a reversible oxidative modification, affects turnover is not understood. We investigated the effect of β1 subunit glutathionylation on the E1Na3 → E2P forward reaction using the fluorescent probe anthryl-ouabain (AO) that binds to E2P species.We induced glutathionylation in Na+, K+ ATPase enriched pig kidney membrane fragments by exposure to the chemical oxidant peroxynitrite. The Na+, K+ ATPase was stabilised in the E1Na3 poise by incubation in 100 mM NaCl and 20 mM Histidine. Glutathionylation of the β1 subunit was confirmed by immunoblotting techniques. MgATP was added to initiate the E1Na3 → E2P conversion and the E2P species was detected by the binding of AO. Addition of MgATP induced an increase in AO fluorescence indicative of a shift to E2P. The increase in fluorescence was blocked by the exposure to peroxynitrite. When glutathionylation was reversed by exposure to glutaredoxin 1, confirmed by the immublotting technique, the shift to E2P was restored as indicated by AO fluorescence.In an independent series of experiments we utilised baseline glutathionylation of a fraction of the Na+, K+ ATPase that is detectable without exposure to oxidants. We followed the E1Na3 → E2P conversion with RH421 fluorescence. As expected, addition of MgATP markedly increased fluorescence. We added 2.5 mM dithiothreitol (DTT) to rapidly reverse glutathionylation. This further increased RH421 fluorescence indictating that DTT made additional Na+, K+ ATPase available to undergo the E1Na3 → E2P conversion.We conclude that glutathionylation of the β1 subunit blocks the forward E1Na3 → E2P reaction.