Clem Penny

Novel methods of molecular sex identification from skeletal tissue using the amelogenin gene

Sex identification from skeletal material is of vital importance in order to reconstruct the demographic variables of an individual in forensic genetics and ancient DNA (aDNA) analysis. When the use of conventional methods of sex identification are impossible, molecular analysis of the X and Y chromosomes provides an expedient solution. Two novel systems of molecular sex identification suitable for skeletal material using the amelogenin gene are described, beginning in intron 2-3, spanning exon 3 and ending in intron 3-4. This area was optimal for sexing, as it includes 14 sex-specific polymorphic regions in addition to an indel (insertion or deletion of nucleotides). Once optimised and working with 100% efficiency on the controls, these procedures were applied to a collection of miscellaneous archaeological skeletons (ex situ) sourced from the Raymond Dart Collection of Human Skeletons (Dart Collection). This collection was used to optimise these techniques for skeletal remains derived from an archaeological context. These methods produced 46.66% sex results for the ex situ sample, which is within the normal range for aDNA studies. These new techniques are optimal for sex identification, with both the inherent control of isolating many sex-specific features and combined with the use of sensitive micro-fluidic electrophoresis.

The effect of alkaline phosphatase inhibitors on intracellular lipid accumulation in preadipocytes isolated from human mammary tissue.

Background: A previous study has demonstrated that alkaline phosphatase (AP) may play a role in the control of intracellular lipid accumulation in the rodent preadipocyte cell line, 3T3-L1. The present study investigated whether AP may have a similar function in preadipocytes isolated from human mammary gland tissue. Methods: Preadipocyte maturation was induced in the presence or absence of the tissue non-specific AP inhibitors levamisole and histidine, and the tissue-specific AP inhibitor PheGlyGly. Cellular AP activity and adipogenesis were both assessed at 0 and 12 days post-induction of differentiation. Results: After differentiation, AP activity increased 5.1±1.3-fold in the absence and 8.9±2.8-fold (P<0.05) in the presence of levamisole. However, adipogenesis increased 1.95±0.11-fold in the absence but only 1.36±0.06-fold (P<0.001) in the presence of levamisole. There was a 4.2±2.2-fold increase in AP activity in the absence and a 0.51±0.46-fold (P<0.05) decrease in the presence of histidine. Adipogenesis increased 2.09±0.35-fold in the absence of histidine but only 1.22±0.30-fold (P<0.05) in the presence of histidine. PheGlyGly had no effects. Fluorescent microscopy showed AP activity was localized to the triglyceride-containing droplets of the cell. Conclusion: This is the first study to show that tissue non-specific AP inhibitors can block adipogenesis in human preadipocytes.

The effect of retinoic acid on the proportion of insulin cells in the developing chick pancreas.

SummaryWe assessed the potential role of all-trans-retinoic acid on the developing chick pancreas, specifically with regard to the proportions of insulin cells. The endodermal component of the dorsal pancreatic bud of 5-d-old chick embryos was cultured on Matrigel. Retinoic acid (10−6 or 10−5M) was added to a standard serum-free medium, Hams F12 containing insulin, transferrin and selenium (F12.ITS). Control grafts were cultured in F12.ITS alone or in F12.ITS with DMSO (the diluent for retinoic acid). After 7 d the explants were retrieved, freeze-dried, vapor-fixed, and embedded in resin. Endocrine cell types were identified by immunocytochemistry. The numbers of insulin cells were expressed as a proportion of the sum of insulin plus glucagon cells. Retinoic acid had a dose-related effect; the proportion of insulin cells in explants treated with the lower dose of retinoic acid (10−6M) was more than twice the proportion of insulin cells in explants treated with the higher dose (10−5M) of retinoic acid and more than three times that of the control grafts.

Mseleni joint disease: A potential model of epigenetic chondrodysplasia

OBJECTIVE In this paper past research on the natural history of Mseleni joint disease, a crippling endemic osteoarthritis, its socio-economic impacts, the demographics, diet, geology and the genetic background of affected people are reviewed. In addition, some new research ideas are suggested to continue the search for etiological avenues for this disease such as stable isotope analysis and epigenetic mechanisms. RESULTS Mseleni joint disease is a chondrodysplasia first described in 1970. It is geographically confined to a remote area in the Maputaland region in northern Kwazulu Natal, South Africa. This disease affects most joints but primarily those of the hip; it is a progressive condition beginning with pain and stiffness until the patients ability to walk becomes compromised. Mseleni joint disease is characterized by two distinct abnormalities, protrusio acetabuli that mainly affects females and increases in frequency with age, and hip dysplasia that is more frequent with age. Much research has been conducted on the people with the disease and their surrounding environment. CONCLUSION Despite intensive investigations into the etiology of Mseleni joint disease, it remains unknown. As a result the examination of epigenetic mechanisms and stable isotope analysis of teeth are suggested as a means of providing information on the etiology of the disease. These methods can also be applied to other chondroplasias of unknown etiology.

A high resolution SEM study of the effects of RU486, used as a postcoital contraceptive, on the rat uterus during early pregnancy

During the window of receptivity, a narrow range of time under the control of the ovarian hormones progesterone and oestrogen, when a blastocyst can attach to the uterine surface, the plasma membrane of the uterine epithelial cells undergoes a remarkable change in structure, known as ‘the plasma membrane transformation’ of early pregnancy. RU486, the controversial abortion drug (Mifegyne™), acts as a progesterone receptor antagonist, resulting in transcriptionally inactive progesterone receptors. In view of this, a change in the well‐documented sequences of the plasma membrane transformation is postulated. This study therefore aims to investigate the effects of RU486 on this sequence of events in the implantation and non‐implantation sites of the rat uterus. In both RU486 treated and control animals, on days 4.5, 5.5 and 6.5 of pregnancy, scanning electron microscopy revealed a distinct pattern of folding of the uterine surface in non‐implantation sites. In contrast, folding was not observed within the implantation sites. These results indicate that surface alterations are probably not under the control of progesterone signalling. The lack of folding at the implantation sites possibly ensures maximum close contact between the blastocyst and the maternal tissue thus promoting implantation. During early pregnancy, specifically on day 5.5, the microvilli of the uterine epithelial cells in the treated animals were more dense than those in the untreated animals. Such microvilli are characteristic of the uterine epithelial cells of a uterus under‐stimulated by hormones. Flattening of the apical cell borders usually seen at the time of blastocyst attachment and implantation was not observed following RU486 treatment. Large apical protrusions were observed in the RU486 treated animals only, possibly linked in someway to apoptosis. The antiprogestin properties of RU486 may further elucidate the progesterone effects associated with early pregnancy.

Retinoic acid increases the length and volume density of ducts in the rat embryonic pancreas

In this study, the role of all‐trans retinoic acid (RA) on the proliferation of rat embryonic pancreas ducts and on the proportion of insulin cells was investigated. All‐trans RA (10−6 m) was added to Hams F12.ITS serum‐free medium in which 12.5 day rat dorsal pancreatic buds were cultured on Matrigel. Control explants were cultured on Matrigel in Hams F12.ITS alone or in Hams F12.ITS containing ethanol (the diluent for RA). After a 7 day culture period, explants were incubated with bromodeoxyuridine (BrdU) for assessment of cell proliferation. Explants were processed for both morphometry and immunocytochemistry. The length density and volume density of the pancreatic ducts were assessed using an image analysis system. Cells positive for insulin, BrdU and glucagon were localized on adjacent serial sections. RA treatment caused a statistically significant increase in the volume density (P < 0.007) and length density (P < 0.008) of the ducts, as well as a 1.2‐fold increase (P < 0.0001) in the proportion of insulin to glucagon cells, compared to both control groups. Few insulin cells were BrdU positive, indicating that cells had a low proliferation rate. The increased proportion of insulin cells may relate to the increased volume density and length density of the ducts in RA‐treated explants. It is suggested that RA stimulated the production of additional progenitor cells and not proliferation of existing insulin cells.

Regulation of Embryonic Chick Insulin Cells: Effect of Retinoic Acid and Insulin-Like Growth Factor 1

We are interested in the regulation of early pancreatic differentiation, particularly with regard to factors that enhance insulin cell proliferation. Both retinoic acid and insulin-like growth factor 1 (IGF-1) are known to be important in the proliferation and differentiation of insulin cells. Individually, they have the ability to increase the proportion of insulin cells when added to cultures of chick dorsal pancreatic buds. The aim of this study was to define the action of retinoic acid (RA) in combination with IGF-1 on the proportion of insulin cells. The dorsal pancreatic bud of 5-day-old chick embryos was excised and the endodermal component, with minimal adherent mesenchyme, was explanted onto Matrigel. RA (10–6 M) and IGF-1 (50 ng/ml) were added to Ham’s F12 culture medium containing transferrin (5 µg/ml) and selenium (10–10 M) (F12.TS). Control explants were cultured in F12.TS alone or in F12.TS containing dimethyl sulphoxide (DMSO) [F12.TS (DMSO)]. After 7 days in culture, insulin and glucagon cells were localized immunocytochemically; numbers of insulin cells were expressed as a percentage of insulin plus glucagon cell counts. Addition of RA plus IGF-1 to the medium increased the proportion of insulin cells markedly (23.43%) compared with the proportions in control explants (11.3% with F12.TS (DMSO), 13.2% with F12.TS). This increase represents a more than twofold increase in the proportion of insulin cells over that of control explants.

REGULATION OF THE PROPORTION OF INSULIN CELLS IN EMBRYONIC CHICK PANCREAS: EFFECT OF A GROWTH FACTOR–REDUCED EXTRACELLULAR MATRIX IN COMBINATION WITH RETINOIC ACID

SummaryBoth retinoic acid (RA) and transforming growth factor (TGF)-β1 are known to be influential in the development of insulin cells. Respectively, they increase and decrease the proportion of insulin cells when added to cultures of embryonic chick dorsal pancreatic buds. The aim of this study was to define the action of RA in the presence of decreased levels of TGF-β1, as are found in growth factor-reduced Matrigel (GFRM), on the proportion of insulin cells. The endodermal component of 5-d chick dorsal pancreatic buds was explanted on the GFRM. Retinoic acid (10−6M) was added to Hams F12 culture medium containing insulin (5 μg/ml), transferrin (5 μg/ml), and selenium (10−10M) (F12.ITS). Control explants were cultured in F12.ITS alone or in F12.ITS containing dimethyl sulfoxide (DMSO). After 7 d in culture, insulin and glucagon cells were localized immunocytochemically; changes in numbers of insulin cells were expressed as a percentage of insulin plus glucagon cells. Medium containing RA or DMSO increased the proportion of insulin cells significantly compareo with the proportion in the explants cultured in F12.ITS medium alone

Exogenous activin increases the proportion of insulin cells in the developing chick pancreas in culture.

As activin is believed to be a key signalling factor during early pancreatic development, its influence on the proliferation and/or determination of insulin cells in the developing chick dorsal pancreatic bud was investigated. Dorsal pancreatic buds of 5‐day‐old chick embryos were explanted on to Matrigel and cultured in serum‐free medium (Hams F12.ITS), to which 1 or 10ng/ml activin was added. After 7 days in culture, the explants were processed for immunocytochemistry and the insulin‐positive cells were scored and expressed as a proportion of the sum of insulin and glucagon cells. When compared to the control cultures (Hams F12.ITS alone), activin treatment resulted in respective increases in the proportion of insulin cells of 1.6 and 1.9 fold. It is suggested that activin treatment favours differentiation of the insulin cell pathway relative to glucagon cells.

Brief communication: Minimally invasive bone sampling method for DNA analysis.

Obtaining a bone sample for DNA analysis has traditionally been a destructive practice, which has resulted in reluctance on behalf of curators for skeletal collections to allow invasive testing. A novel minimally invasive bone sampling method for DNA analysis is presented here. This method uses a conventional hand drill wherein the bone sample is extracted from the intercondylar fossa of the femur; it does not interfere with any known anthropometric landmarks and only leaves a small hole on the surface of the bone. The temperature of the drill is documented and it was established due to the minor increase in temperature, that this should not affect the molecular integrity of the sample. This method is easily replicated and is suitable for both human and other animal skeletal material and can be applied to rare specimens with little risk.