The genetic composition of populations of cynomolgus macaques (Macaca fascicularis) used in biomedical research.

BACKGROUND: The genetic composition of cynomolgus macaques used in biomedical research is not as well-characterized as that of rhesus macaques. METHODS: Populations of cynomolgus macaques from Sumatra, Corregidor, Mauritius, Singapore, Cambodia, and Zamboanga were analyzed using 24 STRs. RESULTS: The Sumatran and Cambodian populations exhibited the highest allelic diversity, while the Mauritian population exhibited the lowest. Sumatran cynomolgus macaques were the most genetically similar to all others, consistent with an Indonesian origin of the species. The high diversity among Cambodian animals may result from interbreeding with rhesus macaques. The Philippine and Mauritian samples were the most divergent from other populations, the former due to separation from the Sunda Shelf by deepwater and the latter due to anthropogenic translocation and extreme founder effects. CONCLUSIONS: Investigators should verify their research subjects' origin, ancestry, and pedigree to minimize risks to biomedical experimentation from genetic variance stemming from close kinship and mixed ancestry as these can obscure treatment effects.

Molecular ABO phenotyping in cynomolgus macaques using real-time quantitative PCR.

Macaques are commonly used in biomedical research as animal models of human disease. The ABO phenotype of donors and recipients plays an important role in the success of transplantation and stem cell research of both human and macaque tissue. Traditional serological methods for ABO phenotyping can be time consuming, provide ambiguous results and/or require tissue that is unavailable or unsuitable. We developed a novel method to detect the A, B, and AB phenotypes of macaques using real-time quantitative polymerase chain reaction. This method enables the simple and rapid screening of these phenotypes in macaques without the need for fresh blood or saliva. This study reports the distribution of the A, B, and AB phenotypes of captive cynomolgus macaques that, while regionally variable, closely resembles that of rhesus macaques. Blood group B, as in rhesus macaques, predominates in cynomolgus macaques and its frequency distribution leads to a probability of major incompatibility of 41%. No silencing mutations have been identified in exon 6 or 7 in macaques that could be responsible for the O phenotype, that, although rare, have been reported. The excess homozygosity of rhesus and cynomolgus macaque genotypes in this study, that assumes the absence of the O allele, suggests the possibility of some mechanism preventing the expression of the A and B transferases.

Use of abbreviations by healthcare professionals: what is the way forward?

OBJECTIVE: To assess the understanding of commonly used abbreviations in the medical records among healthcare professionals. METHODS: A selection of abbreviations from surgical inpatient admissions (gathered over a 10 day period in October 2008), in the form of a standard questionnaire, was shown to different members of a multidisciplinary team to examine interpretation and knowledge. RESULTS: 209 questionnaires were analysed. The average correct response was 43%. Foundation Year 1 (F1) doctors scored the highest with 57% correct responses, whereas dieticians fared worst (20% correct). Among different abbreviations, NAD (91%) and SCBU (93%) were most often correctly answered, whereas CIC (3%) and STS (0.5%) were the most incorrectly answered. Certain abbreviations which are mostly used by nurses (eg, OTT) achieved a 75% correct response by them compared to only 11% by F1 and 10% by F2 doctors (p<0.001). Similarly, abbreviations such as COBH (p=0.025) and LUTS (p<0.001), although mostly correctly answered by junior doctors, were very poorly answered by nurses. CONCLUSION: The majority of healthcare professionals have a very poor knowledge of commonly used abbreviations. Use of unambiguous and approved list of abbreviations is suggested in order to ensure good communication in patient care.

Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.

The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.

Antioxidant and antiinflammatory effect of Epilobium parviflorum Schreb.

Epilobium parviflorum Schreb. (Onagraceae) is used for the treatment of benign prostatic hyperplasia (BPH), but its biological action is not entirely identified. This paper aims to report data on E. parviflorum with respect to its antioxidant and antiinflammatory effects. The aqueous acetone extract of E. parviflorum showed higher antioxidant effect in the DPPH assay than well known antioxidants and inhibited the lipid peroxidation determined by the TBA assay (IC(50) = 2.37 +/- 0.12 mg/mL). In concentrations of 0.2-15.0 microg/mL the extract possessed a protective effect, comparable to catalase (250 IU/mL), against oxidative damage, generated in fibroblast cells. In the COX inhibition assay E. parviflorum decreased the PGE(2) release, so showing inhibition of the COX-enzyme (IC(50) = 1.4 +/- 0.1 microg/mL).

Design and implementation of electron diverters for lobster eye space-based X-ray optics.

Micropore optics have recently been implemented in a lobster eye geometry as a compact X-ray telescope. Fields generated by rare-earth magnets are used to reduce the flux of energetic electrons incident upon the focal plane detector in such a setup. We present the design and implementation of the electron diverters for X-ray telescopes of two upcoming missions: the microchannel X-ray telescope onboard the space-based multiband astronomical variable objects monitor and the soft X-ray instrument onboard the solar wind magnetosphere ionosphere link explorer. Electron diverters must be configured to conform to stringent limits on their total magnetic dipole moment and be compensated for any net moment arising from manufacturing errors. The two missions have differing designs, which are presented and evaluated in terms of the fractions of electrons reaching the detector, as determined by relativistic calculations of electron trajectories. The differential flux of electrons to the detector is calculated, and the integrated electron background is determined for both designs.

Tyrosinase inhibition by extracts and constituents of Sideroxylon inerme L. stem bark, used in South Africa for skin lightening.

OBJECTIVE: To investigate the stem bark of Sideroxylon inerme L. and its compounds for tyrosinase-inhibition activity and to evaluate the mechanism involved of the most potent compounds in tyrosinase inhibition. MATERIALS AND METHODS: Three different extracts (acetone, methanol and dichloromethane) of Sideroxylon inerme L. were evaluated for their inhibitory effect in vitro on the monophenolase and diphenolase activated forms of tyrosinase, using a colorimetric procedure. This test was used for bioactivity-guided isolation of two active compounds using column chromatography and TLC. Active extracts were also investigated for their inhibitory effect on melanogenesis in cultured B16 melanoma cells. Antioxidant activities of the methanolic extract of Sideroxylon inerme and purified compounds were investigated using the 1,2-diphenyl-2-picrylhydrazyl (DPPH) antioxidant assay. The inhibition of tyrosinase activity relative to the inhibition of its activity at the transcriptional level was also studied by determination of the degree of expression of mRNAs for this gene by using extract of Sideroxylon inerme-treated cells (B16F10) and semi-quantitative RT-PCR. RESULTS: Methanolic and acetonic extracts of the stem bark of Sideroxylon inerme showed significant inhibition of monophenolase activity (IC50 values of 63 microg/ml and 82 microg/ml, respectively). The methanolic extract also exhibited 37% reduction of melanin content at 6.2 microg/ml in melanocytes without being significantly toxic to the cells. Examination for inhibition of monophenoloxidase in situ on TLC, followed by column chromatographic purification of the stem bark extract of Sideroxylon inerme, resulted in the isolation of two active compounds, epigallocatechin gallate and procyanidin B1, with IC50 values against monophenolase of 30 microg/ml and > 200 microg/ml, respectively. Epigallocatechin gallate exhibited a greater anti-tyrosinase activity than arbutin. Sideroxylon inerme bark extracts, epigallocatechin gallate and procyanidin B1 exhibited antioxidant DPPH radical scavenging activities with EC50 values of 1.54 microg/ml, 1.33 microg/ml and 1.68 microg/ml, respectively and were not particularly cytotoxic. During mechanism studies it was evident that at the transcription level, Sideroxylon inerme (25 microg/ml) was acting as a potent tyrosinase inhibitor compared to controls (untreated cells and kojic acid). CONCLUSION: The bark extract of Sideroxylon inerme and the two isolated compounds warrant further investigation in clinical studies to be considered as skin-depigmenting agents.

mTOR and cancer therapy.

Proteins regulating the mammalian target of rapamycin (mTOR), as well as some of the targets of the mTOR kinase, are overexpressed or mutated in cancer. Rapamycin, the naturally occurring inhibitor of mTOR, along with a number of recently developed rapamycin analogs (rapalogs) consisting of synthetically derived compounds containing minor chemical modifications to the parent structure, inhibit the growth of cell lines derived from multiple tumor types in vitro, and tumor models in vivo. Results from clinical trials indicate that the rapalogs may be useful for the treatment of subsets of certain types of cancer. The sporadic responses from the initial clinical trials, based on the hypothesis of general translation inhibition of cancer cells are now beginning to be understood owing to a more complete understanding of the dynamics of mTOR regulation and the function of mTOR in the tumor microenvironment. This review will summarize the preclinical and clinical data and recent discoveries of the function of mTOR in cancer and growth regulation.

Antisense-mediated reduction in insulin-like growth factor-I receptor expression suppresses the malignant phenotype of a human alveolar rhabdomyosarcoma.

The expression of the insulin-like growth factors (IGFs) and their receptors has been linked to cellular proliferation and tumorigenicity in a number of model systems. Since rhabdomyosarcoma cells express IGF-I receptors, an autocrine or paracrine loop involving this receptor and its ligands could be responsible in part for the growth characteristics of this tumor. To assess directly the role of the IGF-I receptor in rhabdomyosarcoma cell growth and tumorigenicity, a human alveolar rhabdomyosarcoma cell line with high IGF-I receptor expression was transfected with an amplifiable IGF-I receptor antisense expression vector. Four unique, transfected clones were analyzed and found to have reduced IGF-I receptor expression relative to the parental line. Integration of the antisense sequence was demonstrated by Southern blot analysis, and expression of antisense message in these clones was shown by S1 nuclease protection assay. Reduced IGF-I receptor surface expression in the transfectants was shown by decreased immunofluorescence with an IGF-I receptor monoclonal antibody and by decreased IGF-I binding as measured by Scatchard analysis. These clones had markedly reduced growth rates in vitro, impaired colony formation in soft agar, and failed to form tumors in immunodeficient mice when compared with vector-transfected clones. These results demonstrate that reduction of IGF-I receptor expression can inhibit both the in vitro and in vivo growth of a human rhabdomyosarcoma cell line and suggest a role for the IGF-I receptor in mediating neoplastic growth in this mesenchymally derived tumor.

Downregulation of Bim, a proapoptotic relative of Bcl-2, is a pivotal step in cytokine-initiated survival signaling in murine hematopoietic progenitors.

Two distinct signaling pathways regulate the survival of interleukin-3 (IL-3)-dependent hematopoietic progenitors. One originates from the membrane-proximal portion of the cytoplasmic domain of the IL-3 receptor (betac chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor and is involved in the regulation of Bcl-x(L) through activation of STAT5. The other pathway emanates from the distal region of the betac chain and overlaps with downstream signals from constitutively active Ras proteins. Although the latter pathway is indispensable for cell survival, its downstream targets remain largely undefined. Here we show that the expression of Bim, a member of the BH3-only subfamily of cell death activators, is downregulated by IL-3 signaling through either of two major Ras pathways: Raf/mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/mammalian target of rapamycin. Akt/phosphokinase B does not appear to play a significant role in this regulatory cascade. Bim downregulation has important implications for cell survival, since enforced expression of this death activator at levels equivalent to those induced by cytokine withdrawal led to apoptosis even in the presence of IL-3. We conclude that Bim is a pivotal molecule in cytokine regulation of hematopoietic cell survival.

Uses and abuses of in vitro tests in ethnopharmacology: visualizing an elephant.

Although in vivo models give a more accurate reflection of the activity of substances used in traditional medicine, their use in many countries is severely restricted due to economic and ethical concerns, and this has resulted in the widespread use of in vitro tests in ethnopharmacological studies. Such tests are very useful where the identity of compounds responsible for the biological activity of an extract is being investigated and where limited supplies of material are available, but it is important to consider a variety of factors before making over-predictive claims of that activity in one particular system explains the traditional use. The use of only one bioassay gives a very incomplete picture of the effect of the extract on the whole system involved. A symptom may be due to a number of disease states and, consequently, a variety of mechanisms may serve as targets for bioassays. In a similar way, it is very unusual for there to be only one target for a particular disease so a variety of test systems must be employed. Examples are given of batteries of test systems used to test plants and other materials with a reputation of being useful in wound-healing, diabetes, cancer and to treat cognitive decline associated with old age. In addition, consideration must be given to factors such as absorption into the body and metabolism of any substances present, either to decrease or increase the effect of the 'actives'.

In vitro cytotoxicity activity on several cancer cell lines of acridone alkaloids and N-phenylethyl-benzamide derivatives from Swinglea glutinosa (Bl.) Merr.

The methanol extract from the stems and fruits of Swinglea glutinosa (Rutaceae) afforded 11 known acridone alkaloids and three N-phenylethyl-benzamide derivatives, glycocitrine-IV, 1,3,5-trihydroxy-4-methoxy-10-methyl-2,8-bis(3-methylbut-2-enyl)acridin-9(10H)-one, 1,3,5- trihydroxy-2,8-bis(3-methylbut-2-enyl)-10-methyl-9-acridone, citbrasine, citrusinine-II, citrusinine-I, 5-dihydroxyacronycine, pyranofoline, 3,4-dihydro-3,5,8-trihydroxy-6-methoxy-2,2,7-trimethyl-2H-pyrano[2,3-a]acridin-12(7H)-one, 2,3-dihydro-4,9-dihydroxy-2-(2-hydroxy-propan-2-yl)-11-methoxy-10-methylfuro[3,2-b]acridin-5(10H)-one, bis-5-hydroxyacronycine, N-(2-{4-[(3,7-dimethylocta-2,6-dien-1-yl)oxy]phenyl}ethyl)benzamide, N-(2-{4-[(3,7-dimethyl-4-acethyl-octa-2,6-dien-1-yl)oxy]phenyl}ethyl)benzamide, and severine acetate. All compounds isolated were examined for their activity against three cancer cell lines: human lung carcinoma (COR-L23), human breast adenocarcinoma (MCF7), human melanoma (C32), and normal human fetal lung cell line, MRC-5. The acridones tested exhibited weak cytotoxicity but the amides showed moderate nonselective cytotoxic activity.

ABO blood group phenotype frequency estimation using molecular phenotyping in rhesus and cynomolgus macaques.

A much larger sample (N = 2369) was used to evaluate a previously reported distribution of the A, AB and B blood group phenotypes in rhesus and cynomolgus macaques from six different regional populations. These samples, acquired from 15 different breeding and research facilities in the United States, were analyzed using a real-time quantitative polymerase chain reaction (qPCR) assay that targets single nucleotide polymorphisms (SNPs) responsible for the macaque A, B and AB phenotypes. The frequency distributions of blood group phenotypes of the two species differ significantly from each other and significant regional differentiation within the geographic ranges of each species was also observed. The B blood group phenotype was prevalent in rhesus macaques, especially those from India, while the frequencies of the A, B and AB phenotypes varied significantly among cynomolgus macaques from different geographic regions. The Mauritian cynomolgus macaques, despite having originated in Indonesia, showed significant (P ≪ .01) divergence from the Indonesian animals at the ABO blood group locus. Most Mauritian animals belonged to the B blood group while the Indonesian animals were mostly A. The close similarity in blood group frequency distributions between the Chinese rhesus and Indochinese cynomolgus macaques demonstrates that the introgression between these two species extends beyond the zone of intergradation in Indochina. This study underscores the importance of ABO blood group phenotyping of the domestic supply of macaques and their biospecimens.

alpha-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus.

Extracts of six selected Malaysian plants with a reputation of usefulness in treating diabetes were examined for alpha-amylase inhibition using an in vitro model. Inhibitory activity studied by two different protocols (with and without pre-incubation) showed that Phyllanthus amarus hexane extract had alpha-amylase inhibitory properties. Hexane and dichloromethane extracts of Anacardium occidentale, Lagerstroemia speciosa, Averrhoa bilimbiPithecellobium jiringa and Parkia speciosa were not active when tested without pre-incubation. Extraction and fractionation of Phyllanthus amarus hexane extract led to the isolation of dotriacontanyl docosanoate, triacontanol and a mixture of oleanolic acid and ursolic acid. Dotriacontanyl docosanoate and the mixture of oleanolic acid and ursolic acid are reported from this plant species for the first time. All compounds were tested in the alpha-amylase inhibition assay and the results revealed that the oleanolic acid and ursolic acid (2:1) mixture was a potent alpha-amylase inhibitor with IC(50)=2.01 microg/ml (4.41 microM) and that it contributes significantly to the alpha-amylase inhibition activity of the extract. Three pure pentacyclic triterpenoids, oleanolic acid, ursolic acid and lupeol were shown to inhibit alpha-amylase.

Anti-inflammatory activities of the methanol extracts and an isolated furanoditerpene constituent of Sphenocentrum jollyanum Pierre (Menispermaceae).

Sphenocentrum jollyanum crude extracts and an isolated constituent were evaluated for anti-inflammatory activity using the carrageenan-induced hind paw oedema of healthy adult albino rats and utilizing the oral route of administration. The fruit methanol extract (79.58% inhibition at 200 mg kg(-1)) gave a higher anti-inflammatory activity than the root extract (53.75% inhibition at 200 mg ml(-1)). Further purification of the most active fruit methanol extract (MFE) led to the isolation of three furanoditerpenes identified as columbin, isocolumbin, fibleucin (uv, ir, nmr and ms) as well as a flavonoid-rich fraction (FDE). Both columbin (67.08% inhibition at 20 mg kg(-1), p<0.05) and FDE (76.25% inhibition at 200 mg kg(-1); p<0.05) gave significant anti-inflammatory activities in comparable range with reference acetylsalicylic acid (72.5% inhibition at 100 mg kg(-1)). The results provide some justification for the folkloric uses of Sphenocentrum jollyanum in the treatment of inflammatory-based diseases across the West African sub-region.

Fibroblast growth stimulation by extracts and compounds of Onosma argentatum roots.

The roots of Onosma argentatum are used traditionally in Turkey for wound healing and burns. The n-hexane-dichloromethane extract of the roots, and four shikonin derivatives (deoxyshikonin, acetyl shikonin, 3-hydroxy-isovaleryl shikonin and 5,8-O-dimethyl acetyl shikonin) isolated from the n-hexane-dichloromethane extract were investigated for their ability to stimulate the growth of human amnion fibroblasts. A range of concentrations was studied and the extract found to stimulate the growth of human amnion fibroblasts in vitro at 0.1 microg/mL whilst 5,8-O-dimethyl acetyl shikonin had the same effect at 0.05-5 microg/mL, although cytotoxicity was observed at 50 microg/mL for all samples. The extract and all the other isolated compounds showed cytotoxicity at 10 microg/mL with the extract and 3-hydroxy-isovaleryl shikonin showing cytotoxicity at 5 microg/mL. It is suggested that any wound healing effect of the roots of Onosma argentatum might be partly due to an additive effect of the shikonin derivatives present.

Growth and characterization of childhood rhabdomyosarcomas as xenografts.

Eleven surgical specimens of childhood rhabdomyosarcoma (RMS), two bone marrow samples, and cells from ascitic fluid from 1 patient were implanted sc into immune-deprived inbred CBA/CaJ mice. From these, seven lines of RMS were obtained as xenografts, each retaining the histologic characteristics of the tumor of origin and human lactate dehydrogenase isozymes; these represented 6 of the 11 surgical specimens, whereas one line originated from the transplanted cells from ascitic fluid. It was concluded that children RMS has a fairly high frequency of heterotransplantability in this system and that such a laboratory model may prove useful in the development of new therapeutic regimens in this disease.

Cloning efficiency of cultured human tumor cell lines measured with the use of a Coulter particle counter.

A technique is described by which colony formation in agarose may be rapidly and reproducibly determined with the use of a modified Coulter particle counter (CPC). The cloning efficiency of RD human rhabdomyosarcoma cells after exposure to vincristine sulfate or cisplatin has been compared with the CPC method or by conventional visual counting. These techniques give very similar results. In combination with a Coulter Channelyzer, the CPC technique can be used to evaluate drug effects in colony sizes from a median of 20 to greater than 290 cells. The integrity of colonies formed by 3 colon carcinoma cell lines was examined to determine if this method could be applied to other systems.

Relationship between topotecan systemic exposure and tumor response in human neuroblastoma xenografts.

BACKGROUND: Topotecan is a topoisomerase I inhibitor with activity against xenografts of childhood solid tumors and established clinical activity against neuroblastoma and rhabdomyosarcoma. We have studied the relationship between systemic exposure to and the antitumor activity of topotecan lactone (the active form of the drug) in the xenograft models. Furthermore, we determined whether the responses seen in these models occur at systemic exposure levels that are tolerable in children. METHODS: Neuroblastoma xenografts derived from the tumors of six different patients were established subcutaneously in immune-deprived mice. Topotecan was administered by intravenous bolus injection 5 days a week for 2 consecutive weeks, repeated every 21 days for three cycles. The minimum daily doses that induced complete responses (CRs) and partial responses (PRs) were determined. Topotecan lactone pharmacokinetic studies were performed in both tumor-bearing and nontumor-bearing mice. RESULTS: The minimum doses associated with CRs and PRs in four of the six neuroblastoma xenografts were 0.61 and 0.36 mg/kg body weight, respectively. The topotecan lactone single-day systemic exposures associated with these doses were 88 and 52 ng x hr/mL, respectively. There was an approximately sixfold difference in topotecan lactone systemic exposure (290 ng x hr/mL versus 52 ng x hr/mL) associated with achieving CRs in the least-sensitive and most-sensitive tumors, respectively. CONCLUSIONS: Neuroblastoma xenografts are highly sensitive to topotecan therapy, and responses in mice are achieved at systemic exposures similar to those that are clinically effective and tolerable in children. These results support the concept of deriving preclinical data relating systemic exposure to antitumor activity in xenograft models. Such data may be valuable in making informed decisions regarding the clinical development of new agents.

Bioactive extracts from Senecio samnitum Huet.

The antimicrobial and cytotoxic activities of extracts from Senecio samnitum Huet are reported. Extracts from S. samnitum were able to inhibit the in vitro proliferation of four human tumor cell lines. The dichloromethane extract demonstrated effective cytotoxic activity with IC50 of 22.89 microg mL(-1) on the Caco-2 cell line and the EtOAc extract had IC50 value of 11.91 microg mL(-1) against the COR-L23 cell line. The n-hexane extract displayed the best antibacterial activity against Gram positive bacteria, particularly Staphylococcus aureus. The antifungal activity of all extracts was also seen, particularly against the dermatophytes Trichophyton tonsurans and Microsporum gypseum for the methanol and n-hexane extracts.