Enhanced expression of cellular prion protein gene by insulin or nerve growth factor in immortalized mouse neuronal precursor cell lines

In order to understand the fundamental and putative roles of PrP(c) in the central nervous system, neuronal cell lines were established. Cells were immortalized by recombinant retrovirus vector-mediated transduction of SV40 T-antigen gene. Among these, two cell lines were selected based on their RT-PCR expressions of neuron-specific neurofilament (NF-H, NF-M) and cell morphology. These cell lines showed the properties of neuronal progenitor cells in antigenicity, morphology and responses to differentiating agents. Expression of PrP(c) was detected by immunocytochemical analysis. These cell lines responded to differentiating agents such as dibutyl cyclic AMP (dcAMP) and phorbol 12-myristate 13-acetate (PMA) before developing into neuronal-like cells. Neurite extensions were observed 20 min after incubation with the differentiating agents. Treatment with nerve growth factor (NGF) and insulin induced cell differentiation and enhanced expression of PrP gene (Prnp) mRNA and protein. The latter phenomenon was not inhibited by wortmannin, which is a specific inhibitor of phosphatidylinositol 3-kinase. These results suggest that PrP(c) plays an important role in the differentiation-mediated classic signaling pathway of neuronal cell.     

Effects of salinomycin and vitamin B(6) on in vitro metabolism of phenylalanine and its related compounds by ruminal bacteria,protozoa and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (B(6)) on the production of phenylalanine (Phe) from phenylpyruvic acid (PPY) and phenylacetic acid (PAA) and of PAA from Phe and PPY by mixed rumen bacteria (B), mixed rumen protozoa (P) and their mixture (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrate mixture (3 : 1) twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analyzed by HPLC. When PPY was used as a substrate, it completely disappeared without additives and converted mainly to Phe and PAA on the average by 396 and 178, 440 and 189, and 439 and 147 &mgr;M in B, P and BP, respectively, during the 12 h incubation period. The rate of disappearance showed no significant differences between the microbial suspensions with and without SL and B(6) during the incubation period. The production of Phe from PPY with SL was enhanced (p<0.05) by 40, 20 and 19% in B, P and BP, respectively, while PAA production from PPY with SL was inhibited (p<0.05) by 35, 37 and 38% in B, P and BP, respectively, during the 12 h incubation period. On the other hand, with B(6), the production of Phe and PAA from PPY tended to be enhanced by 14 and 17, 9 and 11, and 7 and 22% in B, P and BP, respectively, during the 12 h incubation period. When PAA added as a substrate was incubated in the incubation medium without any additives, it disappeared by 483, 462 and 507 &mgr;M and converted mainly to Phe on the average by 231, 244 and 248 &mgr;M in B, P and BP, respectively. The disappearance of PAA with SL was inhibited (p<0.05) by 16, 15 and 20%, in B, P and BP, respectively, whereas the disappearance of PAA with B6 was almost the same as that without B(6) in B and BP suspensions but tended to be enhanced by more than 9% in P suspensions during the 12 h incubation period. The production of Phe from PAA with SL tended to be inhibited by 12, 11 and 8% in B, P and BP, respectively, during the 6 h incubation period, but the inhibition was weakened during the 12 h incubation period, whereas Phe production from PAA with B(6) tended to be enhanced by 13, 16 and 8% in B, P and BP, respectively. When Phe was added as a substrate, the net Phe disappearance without additives was 549, 365 and 842 &mgr;M and converted mainly to PAA on the average by 254, 205 and 461 &mgr;M in B, P and BP, respectively. The net disappearance of Phe with SL was inhibited (p<0.05) by 38, 28 and 46%, whereas the net disappearance of Phe with B(6) was enhanced (p<0.05) by 9, 8 and 7% in B, P and BP, respectively. The production of PAA from Phe with SL was inhibited (p<0.05) by 73, 54 and 76% in B, P and BP, respectively. On the other hand, with B(6), PAA production from Phe was enhanced (p<0.05) by 19, 18 and 20% in B, P and BP, respectively. Based on these results, it seems that SL inhibited Phe disappearance and enhanced the synthesis of Phe from PPY, though not from PAA, and accumulated free Phe in the medium, whereas B(6) also enhanced Phe synthesis both from PPY and PAA, which could provide additional amino N for animals.     

Recombination events across the atpA-associated repeated sequences in the mitochondrial genomes of beets

 The mitochondrial atpA gene sequence of the normal fertile sugarbeet (cv ‘TK81-0’) exists in one full-length version and one truncated version, both of which are present in normal stoichiometry and have a 406-bp segment in common. The PCR approach as well as prolonged exposure of Southern blots indicates that the products of the recombination across the 406-bp repeat are present in substoichiometric amounts in the ‘TK81-0’ genome. Intriguingly, one of these substoichiometric sequence arrangements was revealed to be preferentially amplified in an evolutionary lineage that led to a cytoplasmic male-sterile variant [I-12CMS(2)] in wild beets. We also found the 406-bp repeat to be part of a 6.5-kb repeat in the mitochondrial genome of I-12CMS(2). This 6.5-kb duplication is likely to involve recombination between two sets of repeats (the above-mentioned 406-bp repeat and a 7-bp repeat) in an ancestral beet mitochondria. Received: 4 October 1997 / Accepted: 31 October 1997     

Clinical Performance of a New Soluble CD14-Subtype Immunochromatographic Test for Whole Blood Compared with Chemiluminescent Enzyme Immunoassay: Use of Quantitative Soluble CD14-Subtype Immunochromatographic Tests for the Diagnosis of Sepsis

We previously reported that a soluble CD14-subtype (sCD14-ST) immunochromatographic test (ICT) for plasma is more convenient than chemiluminescent enzyme immunoassay (CLEIA), but plasma separation makes bedside measurements difficult. We developed a new sCD14-ST ICT for whole blood and investigated whether quantitative determinations of sCD14-ST by ICT were useful for diagnosing sepsis and severe sepsis/septic shock. We studied 20 patients who fulfilled two or more systemic inflammatory response syndrome (SIRS) criteria and 32 patients who had been diagnosed with sepsis or severe sepsis/septic shock. Whole blood was collected on day 0 (on admission) and day 7, and the sCD14-ST concentration was quantitatively measured by CLEIA and ICT for whole blood. The patients’ Acute Physiology and Chronic Health Evaluation (APACHE) II, Sequential Organ Failure Assessment (SOFA), and Mortality in Emergency Department Sepsis (MEDS) scores were also calculated. The cut-off values obtained by the quantitative measurements made by ICT were 464.5 pg/mL for sepsis and 762.7 pg/mL for severe sepsis/septic shock (P < 0.0001). A Bland–Altman plot showed that no fixed bias or proportional bias was detected between CLEIA and quantitative ICT for whole blood. sCD14-ST concentrations were significantly correlated with APACHE II, SOFA, and MEDS scores (P < 0.0001). These results suggest that the new sCD14-ST ICT for whole blood may be a useful tool for the convenient, rapid bedside diagnosis and treatment of sepsis.     

Generation of intracellular domain of insulin receptor tyrosine kinase by gamma-secretase

The proteolytic cleavage of a precursor protein into alpha- and beta-subunits by furin is required to form functional insulin receptor (IR). In this study, we examined if IR undergoes the additional presenilin (PS)/gamma-secretase-dependent processing. In cells treated with gamma-secretase inhibitors or expressing the dominant-negative PS1 variant led to the accumulation of an endogenous IR C-terminal fragment. In the presence of proteasome inhibitors, we detected a PS/gamma-secretase cleavage product of the IR, termed the IR intracellular domain (ICD). Cellular fractionation and confocal microscopy analyses showed that the IR-ICD is predominantly detected in the nucleus. These data indicate that IR is a tyrosine kinase receptor, which undergoes PS/gamma-secretase-dependent processing. We also show that the autophosphorylation levels of the IR beta-subunit upon insulin stimulation were decreased by the inactivation of PS/gamma-secretase, raising the possibility that the PS/gamma-secretase proteolysis of IR may play a modulatory role in insulin signaling.     

VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.     

Sterol biosynthesis by a prokaryote: first in vitro identification of the genes encoding squalene epoxidase and lanosterol synthase from Methylococcus capsulatus

Sterol biosynthesis by prokaryotic organisms is very rare. Squalene epoxidase and lanosterol synthase are prerequisite to cyclic sterol biosynthesis. These two enzymes, from the methanotrophic bacterium Methylococcus capsulatus, were functionally expressed in Escherichia coli. Structural analyses of the enzymatic products indicated that the reactions proceeded in a complete regio- and stereospecific fashion to afford (3S)-2,3-oxidosqualene from squalene and lanosterol from (3S)-2,3-oxidosqualene, in full accordance with those of eukaryotes. However, our result obtained with the putative lanosterol synthase was inconsistent with a previous report that the prokaryote accepts both (3R)- and (3S)-2,3-oxidosqualenes to afford 3-epi-lanosterol and lanosterol, respectively. This is the first report demonstrating the existence of the genes encoding squalene epoxidase and lanosterol synthase in prokaryotes by establishing the enzyme activities. The evolutionary aspect of prokaryotic squalene epoxidase and lanosterol synthase is discussed.     

Neurotoxic prion protein (PrP) fragment 106-126 requires the N-terminal half of the hydrophobic region of PrP in the PrP-deficient neuronal cell line

The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.