Using metrology in early prehistoric stone tool research: further work and a brief instrument comparison.

Early prehistoric research aims to discover the activities of our ancestors and piece together the process of evolution and sociocultural development. A key element in this process is the study of stone tools, particularly how these tools functioned in prehistory. Currently, there are no established quantitative methods that address stone tool function. This article provides a summary of previous studies using metrological methods in stone tool research and details the use of laser scanning confocal microscopy to conduct areal surface analysis using three-dimensional data sets. Research to-date is preliminary but promising and shows that microscopic metrological approaches can provide a quantitative method to identify how stone tools were used. A limited comparison of two metrological systems is presented, the results of which highlight a need for caution and further investigation on the comparability of related data sets.

Delta opioid receptors mediate glucose uptake in skeletal muscles of lean and obese-diabetic (ob/ob) mice.

Specific binding sites for [125I]beta-endorphin and the delta1-opioid [3H][D-pen(2), D-pen(5)]enkephalin (DPDPE) were quantified using autoradiography in soleus and extensor digitorum longus (EDL) muscles of lean and obese-diabetic (ob/ob) mice. The density of binding was significantly higher in obese-diabetic than lean mice. The uptake of 2-deoxy-D-[1-3H]deoxyglucose, a nonmetabolized glucose analogue, into isolated soleus and EDL muscles was stimulated by beta-endorphin, beta-endorphin 1-27, and DPDPE, but not by the delta2-opioid deltorphin II. Both beta-endorphin and DPDPE stimulated deoxyglucose uptake in obese-diabetic mice. Thus, glucose transport in skeletal muscle may be partly mediated via delta1-opioid receptors. The increased receptor density in obese-diabetic mice may be an adaptive response.

Short-term folic acid supplementation induces variable and paradoxical changes in plasma homocyst(e)ine concentrations.

Folic acid is presently the mainstay of treatment for most subjects with elevated plasma homocyst(e)ine concentrations [Plasma or serum homocyst(e)ine, or total homocysteine, refers to the sum of the sulfhydryl amino acid homocysteine and the homocysteinyl moieties of the disulfides homocystine and homocystein-cysteine, whether free or bound to plasma proteins.] Changes in homocyst(e)ine in response to folic acid supplementation are characterized by considerable interindividual variation. The purpose of this study was to identify factors that contribute to heterogeneity in short-term responses to folic acid supplementation. The effects of folic acid supplementation (1 or 2 mg per day) for 3 wk on plasma homocyst(e)ine concentrations were assessed in 304 men and women. Overall, folic acid supplementation increased mean plasma folate 31.5 +/- 98.0 nmol/L and decreased mean plasma homocyst(e)ine concentrations 1.2 +/- 2.4 micromol/L. There was evidence of substantial interindividual variation in the homocyst(e)ine response from -18.5 to +7.1 micromol/L, including an increase in homocyst(e)ine in 20% of subjects (mean increase 1.5 +/- 1.4 micromol/L). Basal homocyst(e)ine, age, male gender, cigarette smoking, use of multivitamins, methylene tetrahydrofolate reductase, and cystathionine beta-synthase polymorphisms accounted for 47.6% of the interindividual variability in the change in homocyst(e)ine after folic acid supplementation, but about 50% of variability in response to folic acid was not explained by the variables we studied.

A proteomic approach for the discovery of early detection markers of hepatocellular carcinoma.

Individuals chronically infected with hepatitis B or C virus (HBV, HCV) are at high risk for the development of hepatocellular carcinoma (HCC), with disease progression occurring relentlessly over many years. The diagnosis of HCC usually occurs at late stages in the disease when there are few effective treatment options and the prognosis for patients with HCC is very poor. The long latency period, together with clearly identified at risk populations, provide opportunities for earlier detection that will allow more timely and effective treatment of this devastating cancer. We are using a proteomic approach to test the hypothesis that changes in the amount of certain serum polypeptides, or changes in their post-translational modifications, can be used to predict the onset of HCC. Advances in the standardization of two dimensional gel electrophoresis (2DE) coupled with computerized image analysis now permit the reproducible resolution of thousands of polypeptides per run. Serum polypeptides from individuals at different stages in the disease continuum are being resolved by 2DE to identify those that change with disease progression. Polypeptides found by this method can be further characterized by mass spectrometry. In addition, the potential for changes in the glycan structure of certain polypeptides to serve as a marker for disease progression can be explored. The proteomic approach is expected to liberate us from the need to "cherry pick" or guess the best biomarkers and let the data tell us which are the best indicators of disease. Information may also be gleaned about the pathobiology of the disease process.

Polymorphisms in the CBS gene associated with decreased risk of coronary artery disease and increased responsiveness to total homocysteine lowering by folic acid.

Elevated total plasma homocysteine (tHcy) is an established risk factor for the development of vascular disease and neural tube defects. Total homocysteine levels can be lowered by folic acid supplements but individual response is highly variable. In this case-control study, involving 142 coronary artery disease (CAD) patients and 102 controls, we have typed six genetic polymorphisms in three homocysteine metabolizing genes and examined their relationship to the incidence of CAD, tHcy levels, and lowering of tHcy levels in response to folic acid supplementation. We found that two single nucleotide polymorphisms in the cystathionine beta synthase (CBS) gene, 699C --> T and 1080T --> C, are associated with decreased risk of CAD and increased responsiveness to the tHcy lowering effects of folic acid. Individuals homozygous for 699T were significantly underrepresented in CAD patients as compared to controls (4.9% vs 17.3%, P = 0.0015), as were individuals homozygous for the 1080C (29.6% vs 44.2%, P = 0.018). Additionally, 699T and 1080C homozygous individuals were the most responsive to folate supplementation. 699T homozygotes lowered tHcy levels 13.6% on average, compared to 4.8% lowering in 699C homozygotes (P = 0.009), while 1080C homozygotes lowered 12.9% compared to just 2.7% for 1080T homozygotes (P = 0.005). The two polymorphisms in CBS are third codon changes and would not be predicted to affect the underlying protein. However, there is strong linkage disequilibrium between these two positions, suggesting that they may also be linked to other as yet unidentified polymorphisms within the CBS gene. These observations suggest that specific CBS alleles are a risk factor for the development of vascular disease and that genetic information could be predictive of individual response to folic acid supplementation.

Gender differences in genetic susceptibility for lung cancer.

In contrast to men, the incidence of lung cancer among women has increased over the past decade. The basis for this increase among female smokers remains unknown. Surgical patients with a diagnosis of lung cancer and control subjects without a history of malignancy completed a smoking questionnaire and donated a blood sample. DNA was extracted from peripheral mononuclear cells and genotyped for polymorphisms in cytochrome P450 1A1 (CYP1A1) (exon 7) and glutathione S-transferase M1 (GSTM1) (null). No gender differences in either age at diagnosis or histological subtype were observed among lung cancer patients. In both patients (n = 180) and controls (n = 163), females smoked significantly less than males. The pack-year history associated with adenocarcinoma was smaller than that for squamous cell carcinoma. No significant association was observed between the GSTM1 null genotype and cancer risk. However, women had a larger cancer risk than men (odds ratio 4.98 vs. 1.37) if they possessed the mutant CYP1A1 genotype. Female cancer patients were significantly more likely than female controls to have both the CYP1A1 mutation and GSTM1 null genotype. The combined variant genotypes conferred an odds ratio of 6.54 for lung cancer in women versus 2.36 for men, independent of age or smoking history. These data suggest that polymorphisms in CYP1A1 and GSTM1 contribute to the increased risk of females for lung cancer.

Geographic variation in viral load among hepatitis B carriers with differing risks of hepatocellular carcinoma.

The risk of hepatocellular carcinoma (HCC) varies significantly among hepatitis B virus (HBV) carriers from different geographic regions. We compared serological markers of HBV infection in adult male carriers from Haimen City, China and Senegal, West Africa, where the prevalence of chronic infection is similar. HCC mortality among HBV carriers is much higher in Haimen City than it is in Senegal (age-standardized rate, 878 versus 68 per l0(5) person-years). A dramatic difference was observed when HBV DNA levels in serum were assessed among carriers by Southern blot. In the Senegalese group (n = 289), 14.5% were HBV DNA positive by Southern blot in their 20s, and this percentage declined in each subsequent decade of age to 3.3, 2.9, and 0% thereafter. In the Chinese group (n = 285), a higher prevalence of HBV DNA positivity and a less consistent reduction were seen; 29.4% were positive in their 20s, and 30.2, 23.6, and 20.6%, respectively, were positive in each subsequent decade of age. Among 102 male Asian-American HBV carriers, the prevalence of HBV DNA positivity was intermediate between the Chinese and Senegalese populations (36.8, 10.7, 3.0, and 4.6% in each subsequent decade of age). Viral titers were similar among those who were HBV DNA positive in all three populations [median value, 10(7) virions/ml (range, 10(6)-10(9) virions/ml)]. The presence of HBV DNA in serum was positively associated with serum glutathione S-transferase, a marker of liver damage. These findings suggest that the more prolonged maintenance of productive virus infection in the Chinese carriers compared with the Senegalese carriers may explain their higher risk of HCC. This profound difference in the natural history of chronic infection may be due to earlier age of infection in China or to as yet unknown environmental or genetic factors.

Spontaneous seroconversion in hepatitis B e antigen-positive chronic hepatitis B: implications for interferon therapy.

This study compared rates of spontaneous hepatitis B e antigen (HBeAg)-positive to -negative seroconversion in chronic carriers of hepatitis B virus (HBV) with rates reported during interferon-alpha therapy. Four hundred fifty-four Asian-American HBeAg-positive HBV carriers, followed for 1-10 years, were tested approximately every 6 months for HBeAg. Patients with alanine aminotransferase levels > or = 50 IU/mL at entry had 1067.3 seroconversions/10(5) person-months in the 5- to 19-year age group, 1753.3 in the 20- to 34-year group, and 1257.2 in the 35- to 50-year group. Published data indicate that 30% of children and 33% of adults seroconvert during interferon-alpha treatment and follow-up. In our study population, spontaneous seroconversion occurred in 15% of children (95% confidence interval [CI], 8%-27%), 23% of adults 20-34 years (95% CI, 15%-34%), and 17% of adults 35-50 years (95% CI, 10%-28%) during the same interval. The high rate of spontaneous seroconversion should be weighed in decisions to treat HBV carriers with interferon-alpha.

Glutathione S-transferase expression in hepatitis B virus-associated human hepatocellular carcinogenesis.

Hepatitis B virus (HBV) and aflatoxin B1 represent the main risk factors for the development of hepatocellular carcinoma (HCC) in areas endemic for liver cancer. The glutathione S-transferases (GSTs) are a family of Phase II detoxification enzymes that catalyze the conjugation of a wide variety of endogenous and exogenous toxins, including aflatoxin B1, with glutathione. This study characterizes the GST isoenzyme composition (alpha, mu, and pi) of both HBV-infected normal hepatic tissues and HCCs. Analysis of matched pairs of hepatic tissue (normal and tumor) from 32 HCC patients indicated that total GST activity was significantly higher in normal tissues than in tumor tissues, although the percentage of samples expressing GST alpha and pi was equivalent. GST mu was detected by Western blot in the normal tissue from 87.5% of the subjects possessing the GST M1 gene but only 28.6% of the corresponding tumor tissues. The GST activity of normal tissue from GST M1 null patients was significantly decreased as compared to that of subjects possessing the GST M1 gene (264.6 and 422.2 nmol/min/mg, respectively; P = 0.005). GST pi appeared to be overexpressed in the normal tissue of GST M1 null patients, a potential compensatory effect. Patients positive for HBV DNA had significantly lower GST activity than those who were HBV negative (302.1 versus 450.0 nmol/min/mg, respectively; P = 0.02). These results suggest that cellular protection within the human liver is compromised by HBV infection and further decreased during hepatocellular tumorigenesis.

Somatostatin receptor subtype mRNA expression in human colorectal cancer and normal colonic mucosae.

Somatostatin analogues may be useful novel agents in the systemic treatment of advanced colorectal cancer, as somatostatin inhibits proliferation in a wide variety of cell types. Here, we report the expression profiles of somatostatin receptor mRNAs in 32 pairs of malignant and normal colonic epithelia. Receptor subtype 2 (hSSTR2) mRNA was detected throughout nearly 90% of both malignant and normal tissue by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Subtype 5 (hSSTR5) mRNA was detected in 46% and 45% of tumour and mucosal samples respectively, but in 75% (9/12) of early-stage tumours (tubulovillous adenomas, Dukes' A and B) compared with 31% (5/16) of late-stage tumours (Dukes' C and 'D' tumours), 0.05>P>0.025 (chi2 with Yates' correction). There was also reduced expression of hSSTR5 in samples of metastatic tumour (11%, 1/9) compared with all tumour samples (56%, 18/32) 0.025>P>0.01 (chi2 with Yates' correction). Other hSSTRs (1, 3 and 4) were expressed infrequently. Thus, hSSTR2 expression is retained after malignant transformation in colonic epithelium and, although it may potentially be a target for antiproliferative therapy, its ubiquitous expression militates against this. hSSTR5 warrants investigation as a tumour suppressor.

Analysis of somatostatin receptor subtype mRNA expression in human breast cancer.

Somatostatin is a widely distributed inhibitory peptide with growth-inhibitory effects in several human tumours, including breast cancer, raising the possibility that it may have therapeutic potential. The effects of somatostatin are mediated via a family of cell-surface receptors that differ in their tissue distribution, pharmacological properties and intracellular response mediators, suggesting that they mediate different functions of the peptide. We have analysed the expression of somatostatin receptor subtype (SSTR1-5) mRNA in normal and malignant breast tissue. Receptor expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR) using receptor subtype-specific primers and by in situ hybridization (ISH) with riboprobes synthesized by in vitro transcription of cloned PCR products. A total of 51 breast carcinomas, 36 samples of matched normal tissue, two axillary node metastases and eight normal/benign breast tissue samples were analysed. SSTR2 expression was ubiquitous in both normal and malignant breast tissue. Expression of SSTR5 was detected in approximately one-third of tumour and normal tissue, but fewer than 13% of all tissues expressed SSTR1, 3 and 4. These data suggest that SSTR2 gene expression is ubiquitous in breast cancer. Although this is unlikely to have diagnostic or prognostic significance, SSTR2-specific somatostatin analogues may have therapeutic potential in breast cancer.

Evidence for a hormonal action of beta-endorphin to increase glucose uptake in resting and contracting skeletal muscle.

The uptake of 2-[3H]deoxyglucose, a non-metabolisable derivative of glucose, was studied in resting and contracting muscle. An isolated phrenic nerve/hemidiaphragm preparation of the mouse was used, and contractions of the muscle were elicited by electrical stimulation of the nerve. beta-Endorphin stimulated the uptake of 2-deoxyglucose in resting diaphragm muscle. The rate of uptake in the presence of the optimum concentration of beta-endorphin was similar to that in the presence of the optimum concentration of insulin over the short incubation period. beta-Endorphin also stimulated the uptake of 2-deoxyglucose in contracting muscle, but the optimum concentration of the peptide for this effect was three orders of magnitude lower than in resting muscle. The optimum concentration for insulin, however, was similar in resting and contracting muscle. An analogue of the C-terminal tetrapeptide of beta-endorphin also stimulated 2-deoxyglucose uptake, but this peptide was equally efficacious in resting and contracting muscle. It is suggested that beta-endorphin, which is released into the circulation during exercise, may have a hormonal action to increase the uptake of glucose during muscular activity. This peptide or its metabolites may be partly responsible for the insulin-independent uptake of glucose during exercise.

Gestational age and cell viability determine the effect of frozen storage on human fetal hematopoietic progenitor cell preparations.

We have assessed hematopoietic progenitor cell preparations (including CD34-positive stem cells) obtained from human fetal liver at 6-17 weeks of gestational age for total cell numbers, viability and the ability to tolerate (frozen) storage. Hematopoietic cell preparations obtained from the 16-to 17-week gestational age range had the highest total cell count and the greatest proportion of CD34-positive stem cells. However, these preparations were insufficiently robust to withstand the freeze/thaw protocol required. Cells obtained from the 13- to 15-week gestational age range showed optimal post-thaw viability and it is suggested that these cell preparations are the most applicable for in utero transplantation.

The epidemiology of hepatitis viruses B, C, and D.

Chronic viral hepatitis is caused mainly by chronic infection with hepatitis viruses B (HBV), C (HCV), or delta (HDV). Persons chronically infected with one or more of these viruses may develop chronic progressive hepatitis, cirrhosis, and liver failure. In addition, chronic HBV and HCV infections are major causal risk factors for hepatocellular carcinoma. Alcohol consumption accelerates the development of chronic liver disease among HCV-infected individuals and may have similar effects on persons chronically infected with HBV alone or HBV and HDV, but the reported studies are inconsistent.

Distribution of opioid peptide receptors in muscles of lean and obese-diabetic mice.

Autoradiography was used to demonstrate beta-endorphin and delta-opioid receptors in muscles of normal and obese-diabetic mice. The density of the receptors was significantly higher in the obese-diabetic mice. In both normal and diabetic mice, glycolytic and oxidative fibers exhibited the beta-endorphin receptors. However, a significantly greater density of beta-endorphin receptors was observed in the extensor digitorum longus muscles than in the soleus muscles in the diabetic mice. In normal muscles the beta-endorphin receptors were largely restricted to regions where endplates were present, but in the obese-diabetic mice they were densely distributed along the length of the muscle fibers.

Long-term storage of human fetal haematopoietic progenitor cells and their subsequent reconstitution. Implications for in utero transplantation.

Haematopoietic progenitor cells were isolated from human fetal liver, obtained between 6 and 15 weeks gestation. After preparation of a single cell suspension, the cells were stored using a stepwise freezing protocol; taking the cells from room temperature through -70 degrees C to liquid nitrogen. Viability (trypan blue exclusion), morphology (Leishman stain), identification of cell type (flow cytometry) and growth characteristics in semi-solid culture medium were assessed using the fresh cell suspension. We were able to confirm that the predominant cells in human fetal liver up to about 15 weeks gestation are those of the erythroid lineage. It was established that viability in excess of 75% was required to ensure adequate growth in culture after frozen storage and it was deemed important to ensure morphological integrity of the cell preparations. The colonies formed in culture were observed to be producing haemoglobin between 7 and 9 days after initial seeding. We have determined that cells can be stored in liquid nitrogen for up to 2 years without loss of (1) viability, (2) morphological features and (3) ability to form colonies and produce haemoglobin in culture. These findings offer encouragement for the implementation of a cell bank to support an in utero transplantation programme.