Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation released from platelets

Platelets stimulated with thrombin release an inhibitor of plasminogen activator (PAI), which has been shown previously to be neutralized by activated protein C (APC). The requirements for optimal neutralization of PAI activity were investigated. The releasate of gel-filtered human platelets stimulated with thrombin served as a source of PAI. When 6 X 10(8) platelets/mL were incubated with thrombin (1 IU/mL), the releasate contained 18 to 26 ng/mL PAI as determined by incubation of the releasate with urokinase and measurement of residual urokinase activity on plasminogen (S2251). Preincubation of PAI with up to 4 micrograms/mL APC for two hours yielded less than 20% neutralization of PAI activity. In the presence of protein S, phospholipid, and Ca2+, neutralization of PAI activity was time-dependent with 50% neutralization occurring in two hours with 1 microgram/mL APC. The cofactor effects of protein S and phospholipid were concentration- dependent with half-maximal acceleration at approximately 3 micrograms/mL protein S and 10 micrograms/mL phospholipid when the experiments were performed at 1 microgram/mL APC. Diisopropylfluorophosphate-inactivated APC, gla-domainless APC, and thrombin-cleaved protein S had no effect on PAI activity, indicating requirement for preservation of the APC active site and of the Ca2+ binding ability of both APC and protein S. These results suggest coordinate binding of APC and protein S onto phospholipid membrane as a prerequisite for optimal expression of PAI neutralized by APC.     

Bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells do not contribute to vasculogenesis in Lewis lung carcinoma

The development of tumor vasculature is thought to occur through two complementary processes: sprouting angiogenesis from preexisting blood vessels of the host, and vasculogenesis, which involves the spontaneous development of vessels through specific recruitment, differentiation, and vascular incorporation of circulating endothelial cells (EC), endothelial progenitor cells (EPC), or potentially bone marrow-derived cells. Recent reports, however, have challenged the belief that bone marrow-derived cells contribute to tumor neovascularization, claiming an exclusive role for sprouting angiogenesis in tumor blood vessel development.In the present study, we explored the recruitment behavior of bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells to subcutaneously implanted Lewis lung carcinoma in a syngeneic bone marrow transplantation model. We observed that although lin(-)c-kit(+)Sca-1+ and their derived cells demonstrate significant recruitment to carcinomas in vivo, they do not appear to functionally contribute to tumor neovascularization. Furthermore, our results support the hypothesis that new vessel formation in carcinomas occurs primarily through endothelialization from adjacent and preexisting vasculature.     

Nucleolar characteristics of reducing bodies in reducing body myopathy

Reducing body myopathy is a rare muscle disease defined by abnormal inclusions in affected muscle fibers that can be stained with menadione-nitroblue tetrazolium. The origin of these inclusions has not been determined. Here we show that reducing bodies bear characteristics of nucleoli. Ultrastructurally, muscle biopsy specimens of a patient with adult-onset reducing body myopathy showed granular structures of reducing bodies with features similar to the granules of nucleoli, which consisted of pre-ribosomes. In addition, reducing bodies were positive for histochemistry of argyrophilic nucleolar organizer regions (a method for detecting the areas where ribosomal RNA is actively transcribed in the nucleolus), and for antibodies against nucleoli and nuclear ribonucleoprotein. The current findings suggest that reducing bodies contain pre-ribosomes and their associated proteins of the nucleolus and that formation of reducing bodies may result from defects of processing and assembly of ribosomes.     

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water- based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well- established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.      

Noninvasive measurement of cerebral blood flow in adults using near-infrared spectroscopy and indocyanine green: a pilot study

This pilot study was designed to determine the feasibility of measuring cerebral blood flow noninvasively after an intravenous bolus of indocyanine green using near-infrared spectroscopy and pulse dye-densitometry. Feasibility aside, this study did not attempt to validate the measured values of cerebral blood flow against an established method of measurement. Twelve healthy volunteers were investigated after peripheral intravenous injection of indocyanine green. Arterial and cerebral changes in indocyanine green concentration were measured using pulse dye-densitometry and near-infrared spectroscopy, respectively. Two methods of calculating cerebral blood flow were used, and a blood flow index was also estimated. Absolute cerebral blood flow was calculated using a modification of the Fick principle and a deconvolution algorithm to derive the impulse residue function. Mean (range) estimated cerebral blood flow for the Fick method was 8.2 mL/100 g/min (4.2-16.2 mL/100 g/min) and 8.3 mL/100 g/min (4.7-15.3 mL/100 g/min) for the impulse residue function method. The impulse residue function method provided a more precise intrasubject estimation of cerebral blood flow compared with the modified Fick principle, with a coefficient of variation of 10.1% versus 25.5%. The blood flow index was 8.6 mg/sec (range: 5.6-17.3 mg/sec) with an intrasubject coefficient of variation of 12.0%. Estimation of cerebral blood flow using near-infrared spectroscopy and pulse dye-densitometry can be made at the bedside after intravenous injection of indocyanine green, and the precision can be improved using a deconvolution algorithm. Notwithstanding the low values obtained for absolute cerebral blood flow, further investigation and validation of this bedside technique is warranted.     

Unequal crossing-over in unique PABP2 mutations in Japanese patients: a possible cause of oculopharyngeal muscular dystrophy

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset autosomal dominant muscle disease with a worldwide distribution. Recent findings reveal the genetic basis of this disease to be mutations in the polyA binding-protein 2 (PABP2) gene that involve short expansions of the GCG trinucleotide repeat encoding a polyalanine tract. The underlying mechanism causing the triplet-expansion mutation in PABP2 remains to be elucidated, although the DNA slippage model is thought to be a plausible explanation of that. METHODS AND RESULTS: We analyzed PABP2 using polymerase chain reaction analysis and DNA sequencing in Japanese patients with pathologically confirmed OPMD, and found mutated (GCG)(6)GCA(GCG)(3)(GCA)(3)GCG and (GCG)(6)(GCA)(3)(GCG)(2)(GCA)(3)GCG alleles instead of the normal (GCG)(6)(GCA)(3)GCG allele. These mutated alleles could be explained by the insertions or duplications of (GCG)(3)GCA and (GCG)(2)(GCA)(3), respectively, but not by the simple expansion of GCG repeats. The clinical features of our patients were compatible with those of other Japanese patients carrying PABP2 that encodes a polyalanine tract of the same length, but were not compatible with those of Italian patients. CONCLUSIONS: The mutated alleles identified in our Japanese patients with OPMD were most likely due to duplications of (GCG)(3)GCA and (GCG)(2)(GCA)(3) but not simple expansions of the GCG repeats. Therefore, unequal crossing-over of 2 PABP2 alleles, rather than DNA slippage, is probably the causative mechanism of OPMD mutations. All mutations that have been reported in patients with OPMD so far can be explained with the mechanism of unequal crossing-over. On the other hand, comparison of the clinical features of our patients with those of other patients in previous reports suggests that specific clinical features cannot be attributed to the length of the polyalanine tract per se.