Age estimation in cadavers, human remains and living individuals may clarify issues with significant legal and social ramifications for individuals as well as for the community. In such cases methods for estimating age should fulfil the following specific demands: (1) they must have been presented to the scientific community, as a rule by publication in peer-reviewed journals, (2) clear information concerning accuracy of age estimation by the method should be available, (3) the methods need to be sufficiently accurate and (4) in cases of age estimation in living individuals principles of medical ethics and legal regulations have to be considered. We have identified and summarized the methods that essentially fulfil these specific demands. In childhood and adolescence morphological methods based on the radiological examination of dental and skeletal development are to be recommended. In adulthood, the accuracy of most morphological methods is much reduced. Here a biochemical method based on aspartic acid racemization in dentine provides the most accurate estimates of age, followed by special morphological dental and skeletal methods. The choice of method has to take account of the individual circumstances of each case. Most methods require either the consultation of specialised and trained scientists or an adequate calibration by the "user". Very few attempts have been made to find common standardisation, calibration and evaluation procedures or to develop means of quality assurance for methods of age estimation. Efforts in these directions are necessary to guarantee quality standards and adequate answers to the important legal and social issue of age estimation in forensic medicine.

The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the Lisomer has been widely used in archaeology and geochemistry as a tool for dating. The method has proved controversial, particularly when used for bones. The non-linear kinetics of Asp racemization have prompted a number of suggestions as to the underlying mechanism(s) and have led to the use of mathematical transformations which linearize the increase in D-Asp with respect to time. Using one example, a suggestion that the initial rapid phase of Asp racemization is due to a contribution from asparagine (Asn), we demonstrate how a simple model of the degradation and racemization of Asn can be used to predict the observed kinetics. A more complex model of peptide bound Asx (Asn + Asp) racemization, which occurs via the formation of a cyclic succinimide (Asu), can be used to correctly predict Asx racemization kinetics in proteins at high temperatures (95^140 8C). The model fails to predict racemization kinetics in dentine collagen at 37 8C. The reason for this is that Asu formation is highly conformation dependent and is predicted to occur extremely slowly in triple helical collagen. As conformation strongly in£uences the rate of Asu formation and hence Asx racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question.In the case of archaeological bone, we argue that the D:L ratio of Asx re£ects the proportion of nonhelical to helical collagen, overlain by the e¡ects of leaching of more soluble (and conformationally unconstrained) peptides. Thus, racemization kinetics in bone are potentially unpredictable, and the proposed use of Asx racemization to estimate the extent of DNA depurination in archaeological bones is challenged.

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Estimates of the age of living and dead individuals, obtained in order to answer legal or social questions, require minimum quality standards in order to guarantee data quality. We present an outline strategy (with recommendations) for the attainment of quality assurance in age estimation based on aspartic acid racemisation. The strategy is based on a definition of minimum standards for laboratories, including documentation of procedures, methodology and levels of expertise, and the formulation of guidelines for intralaboratory and interlaboratory quality control.

To study the late events of cell wall assembly in Mycobacterium smegmatis, specific in vivo radiolabelling of exponentially growing liquid cultures over periods of less than one cell generation were carried out. N-Acetyl-[ 14 C]glucosamine was used to label peptidoglycan and [ 14 C]glucose to label arabinogalactan and arabinomannan. Over periods of several generations, radioactive cell wall material was turned over as soluble autolysis products into the culture fluid. However, turnover of newly synthesized and labelled cell wall was delayed for about one cell generation, implying inside-to-outside growth of the wall as observed in Bacillus. Little radioactive wall material was released into the culture fluid during the first generation of labelling in growing cultures, but the addition of amoxicillin plus the β-lactamase inhibitor clavulanic acid, at the minimum inhibitory concentration of amoxicillin, led to the release of radioactive peptidoglycan that could be isolated by gel filtration chromatography and contained nearly 3 mol alanine per glutamic acid residue, indicating that it was linear, un-crosslinked peptidoglycan that had never been substantially cross-linked to the cell wall due to inhibition of transpeptidation by amoxicillin. This peptidoglycan had no covalently attached arabinogalactan. Radioactive arabinogalactan was synthesized and released from the amoxicillin-treated bacteria without attachment to peptidoglycan. The results indicate that during growth, incorporation of arabinogalactan into the cell wall requires its ligation to newly synthesized peptidoglycan and that the peptidoglycan must be undergoing concomitant cross-linking to the inner surface of the cell wall. Inhibition of peptidoglycan transpeptidation prevents ligation of arabinogalactan to peptidoglycan and its consequent incorporation into the wall.