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.

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.

Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1.

OBJECTIVE: As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes. RESEARCH METHODS AND PROCEDURES: 3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis was measured using the triglyceride-specific dye, oil red O at 0, 3, 7 and 11 days after initiation of adipogenesis in the presence or absence of the alkaline phosphatase inhibitors, levamisole, histidine and Phe-Gly-Gly. Intracellular localisation of the enzyme was detected using ELF-phosphatase, a fluorescent substrate and alkaline phosphatase gene expression was assessed using RT-PCR. RESULTS: Alkaline phosphatase activity was detected in untransformed cells (1.91+/-0.62 mU/mg protein) and activity increased 11.5+/-1.4-fold after 11 days treatment with transformation medium and 5.3+/-0.3-fold in transformation medium containing levamisole (p<0.05). Triglyceride content of cells increased 3.1+/-0.2-fold after 11 days treatment with transformation medium and 2.1+/-0.3-fold in the presence of levamisole (p<0.005). Histidine inhibited adipogenesis and alkaline phosphatase to a greater extent than did levamisole, but Phe-Gly-Gly had no effect on these variables. Alkaline phosphatase was localised around the lipid droplets of the cells. Gene expression of alkaline phosphatase increased during adipogenesis. DISCUSSION: This study demonstrates that tissue-nonspecific alkaline phosphatase is present in 3T3-L1 cells and that it may play a role in the control of adipogenesis.

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 patient's 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 some way to apoptosis. The antiprogestin properties of RU486 may further elucidate the progesterone effects associated with early pregnancy.

The relationship between alkaline phosphatase activity and intracellular lipid accumulation in murine 3T3-L1 cells and human preadipocytes.

Alkaline phosphatase (ALP) is expressed in 3T3-L1 preadipocytes, and its activity increases during adipogenesis. The purpose of this study was to determine whether ALP activity could be used as a measure of intracellular lipid accumulation in human preadipocytes and 3T3-L1 cells and which of the factors that induce adipogenesis are responsible for stimulating ALP activity. Adipogenesis was initiated in 3T3-L1 cells by incubation with differentiation medium containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The effect of leaving out each of the differentiation medium components was studied. Adipogenesis was also assessed in human preadipocytes and 3T3-L1 cells in the presence of the ALP inhibitor histidine. ALP activity was measured using an automated colorimetric assay and intracellular lipid accumulation was measured using the lipid-specific dye oil red O. Removal of insulin or dexamethasone from the differentiation medium had little effect on either ALP activity or lipid accumulation in 3T3-L1 cells, while removal of IBMX blocked both. Histidine inhibited ALP activity and adipogenesis in human preadipocytes and 3T3-L1 cells. Pearson univariate correlation analysis demonstrated strong correlations between ALP activity and lipid accumulation in human preadipocytes (r=0.78, n=69) and in 3T3-L1 cells (r=0.92, n=27). These data suggest that ALP and fat storage are tightly linked during preadipocyte maturation and that the measurement of ALP activity may be a novel technique for the quantification of intracellular lipid accumulation that is more sensitive and rapid than currently used methods.