Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures.

In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3). Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.

Reproducibility of repeated human regional splenic blood flow measurements using [15O] water and positron emission tomography.

The reproducibility of measurement of regional splenic blood flow by dynamic positron emission tomography (PET) using [15O]water was evaluated. In 19 patients, the correlation between the first and second of two serial dynamic measurements was significant (P=1.78 x 10(-6); r=0.858). The regression equation was y = 1.06x, and the slope of the line described had a 95% confidence interval of 0.09. The error apparent between the two measurements was 0.129 (95% confidence interval 0.059). The results demonstrated sufficiently good reproducibility for measurements of regional splenic blood flow with PET and [15O]water to suggest use of this method for serial measurements intended to detect change, including drug effects.

Using the spleen for time-delay correction of the input function in measuring hepatic blood flow with oxygen-15 water by dynamic PET.

By the spleen, we calculated a time-delay correction of the input function for quantitation of hepatic blood flow with oxygen-15 water and dynamic positron emission tomography. The time delay (deltat) between the sample site and the spleen was calculated based on nonlinear multiple regression analysis when splenic blood flow was determined. Then hepatic blood flow was quantified by a method using the input function and incorporating deltat, which was assumed to be equal to the time delay between the sample site and the liver. Then hepatic arterial and portal blood flows were estimated separately as well as the delay time for passage within the organs of the portal circulation. The mean coefficient of variation and the mean sum of squares of errors decreased to about 70% when total hepatic blood flow was calculated from the results for regions of interest in three slices of the same liver segment. We concluded that using the spleen for time-delay correction of the input function for measuring hepatic blood flow by this method gave satisfactory results.

Possible regulation of epidermal growth factor-receptor tyrosine autophosphorylation by calcium and G proteins in chemically permeabilized rat UMR106 cells.

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (< or = 10(-6) M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5'-O-(2-thiodiphosphate) (GDPbetas). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca2+- and G protein-mediated pathways in UMR106 cells, revealing a previously unrecognized modulation of EGF signalling in osteoblast-like cells that contrasts with the simpler regulatory mechanisms found in A431 cells.

Errors in the spleen-blood partition coefficient for water in C15O2 steady-state PET.

The aim of this study was to clarify if the value of 0.93, determined for patients with normal livers, is useful as a pathological spleen-blood partition coefficient for water when the splenic blood flow is quantified by the C15O2 steady-state method. A steady-state PET scan with continuous inhalation of C15O2 and a dynamic PET scan with a H(2)15O bolus injection were performed. From 157 patients, 392 slices were chosen as having planes that encompassed the spleen and provided regions of interest with full signal imaging. A comparison of the results of the steady-state and dynamic methods was performed. When 0.93 was adopted as the spleen-blood partition coefficient for water, an error of about 25% was seen in the splenic blood flow of patients with cirrhosis. When measuring splenic blood flow, the H(2)15O dynamic method is necessary. However, a rough estimate of splenic blood flow is possible by the C15O2 PET steady-state method, if this error is known.

[Complete remission of liver metastasis from rectal cancer following continuous oral administration of tegafur-uracil after intrahepatic arterial single injection of CDDP and 5-FU].

An intrahepatic arterial injection of CDDP, 5-FU, followed by ten months of oral tegafur-uracil administration (2g/day), induced remission for 3 months or more in a 72-year-old male with rectal cancer and synchronous liver metastasis subsequent to anterior resection of the rectum. Tegafur-uracil showed an excellent anticancer effect against colorectal metastatic liver cancers without loss of QOL because a single-low dose of intraarterial anticancer injection was followed by continuous oral administration of tegafur-uracil, and the chemotherapy could be managed to obtain complete remission of the hepatic lesion.

Bacillus subtilis RNase III cleaves both 5'- and 3'-sites of the small cytoplasmic RNA precursor.

Bacillus subtilis small cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family. It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA. In the precursor, the 5'- and 3'-flanking regions form a stable double-stranded structure based on their complementary sequence. This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNase III. The B. subtilis enzyme that has similar activity to Escherichia coli RNase III has been purified and is designated Bs-RNase III. Recently, B. subtilis rncS has been shown to encode Bs-RNase III (Wang, W., and Bechhofer, D. H. (1997) J. Bacteriol. 179, 7379-7385). We show here that Bs-RNase III and the purified His-tagged product of rncS cleave pre-scRNA at both 5'- and 3'-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3'-site is less efficient. The 5'-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3'-end. Bs-RNase III cleavage yields a two-base 3'-overhang, which is consistent with the manner in which E. coli RNase III cleaves. We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant. It is concluded that Bs-RNase III is an enzyme that processes pre-scRNA.

Up-regulation of inducible nitric oxide (NO) synthase and NO production in HL-60 cells stimulated to differentiate by phorbol 12-myristate 13-acetate plus 1,25-dihydroxyvitamin D3 is not obtained with dimethylsulfoxide plus 1,25-dihydroxyvitamin D3.

In previous studies we found that the calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] augments the action of either prostaglandin E1 (PGE1) or NaF to induce differentiation of human promyelocytic HL-60 cells, a process that features increased generation of nitric oxide (NO) via up-regulation of inducible nitric oxide synthase (iNOS). We have now examined the short-term interaction of 1,25(OH)2D3 with phorbol 12-myristate 13-acetate (PMA) and dimethylsulfoxide (DMSO) in these cells. PMA (100 nM) alone generally up-regulated several classical indices of macrophagic differentiation and stimulated cellular production of interleukin (IL)-1alpha, IL-6, tumor-necrosis factor (TNF)-alpha, PGE2, and NO. Increased generation of NO primarily resulted from increased expression of cellular iNOS. When 1,25(OH)2D3 (10 nM) was added to PMA treatments, most PMA-induced changes, particularly its effects to up-regulate iNOS-dependent NO production and change cell morphology, were multiplicatively augmented. In contrast, DMSO (1.3%) alone, an inducer of granulocytic differentiation, increased cytokine production, but failed to stimulate NO production or induce iNOS. In contrast to its striking interaction with PMA, 1,25(OH)2D3 could not augment DMSO's differentiative effects. Changes in cellular cytokine production were eliminated as the driving force in HL-60 differentiation when specific neutralizing antibodies failed to produce any attenuation of iNOS up-regulation or of the shifts in cell morphology. However, indomethacin (30 microM) blocked the synergistic interaction between 1,25(OH)2D3 + PMA to shift cell morphology and stimulate NO production. Subsequently adding PGE2 (1 ng/ml) to indomethacin-treated cells restored the ability of 1, 25(OH)2D3 + PMA to interactively increase cellular NO production, but failed to fully replicate the strong shift in cell morphology typical of PMA + 1,25(OH)2D3 treatments. Our findings suggest that interaction between 1,25(OH)2D3 and PMA to induce macrophagic differentiation increases iNOS-dependent NO production by a mechanism involving a cyclooxygenase product(s), possibly PGE2.

Identification of a region required for binding to presecretory protein in Bacillus subtilis Ffh, a homologue of the 54-kDa subunit of mammalian signal recognition particle.

Bacillus subtilis Ffh protein is a homologue of the 54-kDa subunit of mammalian signal recognition particle (SRP54). It contains three highly hydrophobic regions (h1, h2, and h3) in the C-terminal methionine-rich domain (M-domain). Two of the hydrophobic regions, h2 and h3, are essential for small cytoplasmic RNA (scRNA) binding [Kurita, K., Honda, K., Suzuma, S., Takamatsu, H., Nakamura, K., & Yamane, K. (1996) J. Biol. Chem. 271, 13,140-13,146]. Using purified presecretory proteins and mutant Ffh proteins, we identified a region required for presecretory protein binding in B. subtilis Ffh. Deletion of this region, which consisted of residues Ser311-Gly362 of B. subtilis Ffh, including a hydrophobic sequence (h1), reduced precursor binding activity. In contrast, deletions of residues Leu121-Lys279, Lys364-Met446, or Leu338-Ser397 of B. subtilis Ffh did not. We also analyzed the mutant B. subtilis Ffh proteins, FfhQQQR and FfhQQQQ having wild-type residues 398-401 (Arg-Arg-Lys-Arg) replaced with Gln3Arg and Gln4, respectively. FfhQQQR bound to both scRNA and presecretory protein. Although the FfhQQQQ mutation prevented binding to scRNA, binding to the precursor was not affected. FfhQQQR restored the growth of B. subtilis DF46 strain in which ffh gene expression is regulated by an inducible promoter in the absence of an inducer, whereas FfhQQQQ did not. These results indicate that the region including h1 is required for B. subtilis Ffh to bind to presecretory protein. The results also suggest that scRNA is required for the complete function of the B. subtilis SRP-like particle in vivo, although this protein is intrinsically capable of binding a signal peptide free from scRNA.

Isolated maxillary bending in CL/FR strain mice: observation of craniofacial deformity and inheritance pattern.

OBJECTIVE: The CL/Fr mouse, known as a strain with spontaneous cleft lip and/or palate (CL/P), has been used as an animal model to investigate etiology in CL/P. METHOD: We examined a facial asymmetry mutant discovered in a CL/Fr mouse colony that was not associated with CL/P and was shown to be inheritable in subsequent generations. Facial asymmetry became apparent with postnatal growth, whereas it was not detectable at birth, and was termed "maxillary bending" (MB) based on the characteristic bending of the maxilla. RESULTS: As a result of selective breeding, an 'MB line', In which MB was observed in 21.68% (67/309) in addition to CL/P in 17.80% (55/309) of the offspring, was developed in the CL/Fr colony. In mating experiments between the MB line and C57BL/6J, all F1 progeny showed the normal phenotype. MB was observed in 0.72% (1/139) of the F2 generation, and the backcross generation showed segregation of MB in 6.25% (22/352) and CL/P in 1.42% (5/352). These instances suggested the occurrence of an additional mutation in the CL/Fr mouse genome controlled by an autosomal recessive gene with low penetrance. However, since the CL/Fr mouse primarily has a developmental deficiency in the maxilla, the possibility that CL/P and MB share common etiologic factors cannot be completely ruled out. CONCLUSION: The maxillary bending retains significance, as this mutant can serve as an animal model of abnormal facial growth. Elucidation of the etiologic relationship between MB and CL/P may provide clues to clarifying the deficiency in first branchial arch in the mouse.

Characteristics of NaF-induced differentiation of HL-60 cells.

Sodium fluoride (NaF) is known to stimulate osteoblastic bone formation, but little attention has been given to the possibility that NaF also affects bone resorption and the differentiation of osteoclastic progenitor cells. When human promyelocytic HL-60 cells were treated with NaF (0.5 mM, 0-4 days), cell proliferation was inhibited, and the addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10nM, 0-4 days) augmented this antiproliferative effect. NaF increased cellular reduction of nitroblue tetrazolium (NBT), and this effect was strongly augmented by 1,25(OH)2D3. In addition, NaF produced marked changes in cellular morphology, increased cellular adhesion to plastic, reduced the nuclear/cytoplasmic ratio, and increased cellular expression of chloroacetate esterase, but failed to alter cellular nonspecific esterase activity. Furthermore, NaF increased expression of CD11b and CD66b, and this stimulation was enhanced by adding 1,25(OH)2D3. The sum of these changes in classical promyelocytic cellular indices suggest: (1) that NaF stimulates the early stages of HL-60 differentiation toward a granulocyte-like cell and (2) that 1,25(OH)2D3 promotes these actions of NaF. Additional experiments aimed at further understanding the NaF-induced conversion of HL-60 cells identified further changes. NaF also increased cellular production of prostaglandin E2 (PGE2) and nitric oxide (NO) and induced expression of inducible nitric oxide synthase (iNOS); 1,25(OH)2D3 once again augmented these NaF-induced effects. Similarly, NaF stimulated the production of interleukin 1 alpha (IL-1 alpha), IL-6, and tumor necrosis factor-alpha, and 1,25(OH)2D3 again strongly enhanced these effects. Indomethacin completely blocked stimulation of NBT reduction, NO production, and iNOS expression induced by NaF plus 1,25(OH)2D3; adding exogenous PGE2 (0.1-10 ng/ml) to these indomethacin-blocked cultures dose-dependently restored NO production. These additional findings together with the observed slow onset (24-48 h) of NaF and 1,25(OH)2D3 interaction strongly suggest that 1,25(OH)2D3 acts as a cofactor with NaF primarily through interaction with an endogenous NaF-induced cyclo-oxygenase product(s), quite possibly PGE2 itself. Such a mechanism for NaF and 1,25(OH)2D3 interaction would be strongly analogous to the interaction we have recently demonstrated between 1,25(OH)2D3 and PGE1 on the differentiation of HL-60 cells.

Bacillus subtilis Ffh, a homologue of mammalian SRP54, can intrinsically bind to the precursors of secretory proteins.

We analyzed the binding activity of B. subtilis Ffh to the precursors of secretory proteins by purifying mature and precursor proteins of beta-lactamase derived from pUC18 and its derivatives, of which the signal peptide region was replaced with that of E. coli OmpA, B. subtilis AprE, PBP5* or an alkalophilic Bacillus sp. #1011 CGTase. Each of them was mixed with purified B. subtilis Ffh in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The tested precursor proteins, including those of E. coli, of which the signal sequences differ from those of B. subtilis in the number of charged amino acids and hydrophobicity, cross-linked with Ffh, whereas mature proteins did not. The addition of scRNA, the B. subtilis counterpart of mammalian SRP 7S RNA, into the mixture did not affect the complex formation. These findings suggest that B. subtilis Ffh intrinsically binds to several precursor proteins.

Quantitative measurement of human tissue hepatic blood volume by C15O inhalation with positron-emission tomography.

In order to estimate the tissue liver function, tissue hepatic blood volume was measured quantitatively and non-invasively using C15O inhalation in conjunction with positron-emission tomography. Fifty-eight patients with normal liver function, 14 patients with chronic hepatitis, 28 patients with hepatic cirrhosis, and 4 patients with obstructive jaundice were studied by positron-emission tomography scan after the single breath inhalation of 20 mCi of high specific activity 15O-labeled carbon monoxide. The mean tissue hepatic blood volume was significantly greater in patients with normal livers than in patients with chronic hepatitis or hepatic cirrhosis (mean: 20.5, 18.2, and 16.1 ml per 100 cm3, respectively, p = 8.6 x 10(-8)). Tissue hepatic blood volume (tHBV) correlated with the reaction of the mesenchymal system and protein synthesis, because there was a potent correlation between tHBV and hepatic fibrosis. In normal livers, we were able to demonstrate significant differences in tissue hepatic blood volume among liver segments.

The effect of Srb, a homologue of the mammalian SRP receptor alpha-subunit, on Bacillus subtilis growth and protein translocation.

To determine the signal recognition particle (SRP)-SRP receptor (Srb) system in Bacillus subtilis (Bs), we cloned the Bs srb gene, which encodes a homologue of the mammalian SRP receptor alpha-subunit [Oguro et al., DNA Res. 2 (1995) 95-100]. We sequenced a 6098-bp DNA containing srb and analyzed the gene organization. Primer extension experiment and Northern blot analysis revealed that srb constitutes an operon with two additional ORFs. A database search of known proteins revealed that one encodes a homologue of Escherichia coli RNase III [36.0% identical amino acids (aa)] and the other encodes a homologue of yeast Smc1 (26.6% identical aa). We then constructed a Bs mutant in which srb expression was induced by IPTG. The depletion of Srb caused a defect in the cell growth and the cells became filamentous and twisted. Furthermore, pulse-chase experiments using this mutant revealed that the 17% of the beta-lactamase precursor accumulated in the cell after a 4-min chase in the absence of IPTG, although almost all of the precursors were converted into the mature from after a 1-min chase in the presence of IPTG.

Analysis of models for quantification of arterial and portal blood flow in the human liver using PET.

OBJECTIVE: The purpose of our study was to quantify arterial and portal hepatic arterial blood flows. MATERIALS AND METHODS: Four models were developed using PET. The first model consisted of the components of the liver and the portal system. The second applied "curve analysis" to this model. The third model introduced a portosystemic shunt factor, whereas the last model introduced a coefficient for circulation time within the portal organs. In 51 patients (34 men and 17 women), PET scans of the liver were performed using the H2 15O dynamic method. RESULTS: Under all four models, the arterial and portal hepatic arterial blood flows of 504 regions of interest were calculated using the nonlinear least-squares method, and results were compared by the sum of the squares of errors. Additionally, results from the H2 15O dynamic method were compared by results from the C15O2 steady-state method. CONCLUSION: Of the four models, the last model produced curves with the best fit. When hepatic blood flow was quantified using PET and the H2 15O dynamic method, a model applying "curve analysis" and components related to portosystemic shunting and circulation time was found to be most accurate.

Determination of the spleen-blood partition coefficient for water with oxygen-15-water and oxygen-15-carbon dioxide dynamic PET steady-state methods.

METHODS: Regional splenic blood flow (SBF) was quantified by PET using a steady-state method with 15O-carbon dioxide. SBFs were determined using 104 tomographic planes obtained from 49 patients. RESULTS: When the spleen-blood partition coefficient for water (p) was > or = 0.85, significant correlations (p < 0.005) were found between SBF values determined by the steady-state and dynamic methods. The best correlation between SBFs determined by the two methods (r = 0.571) was found when p = 0.93. The best regression line, however, was thought to be the line when p = 0.93. The regression line between SBF calculated by the steady-state method (y) and SBF determined by the dynamic method (x) was y = 0.57 x + 0.03 with an F ratio of 48.75 (d.f. = 103, p = 5.0 x 10(-8)%, by ANOVA) when p = 0.92. CONCLUSION: A quick evaluation of SBF can be made by using the newly defined regression line.

Acquisition of azide-resistance by elevated SecA ATPase activity confers azide-resistance upon cell growth and protein translocation in Bacillus subtilis.

We isolated four azide-resistant secA mutants of Bacillus subtilis and found that all of them were the result of a single amino acid replacement of threonine 128 of SecA by alanine or isoleucine. In the presence of 1.5 mM sodium azide, cell growth and protein translocation of the wild-type strain were completely inhibited, but those of the azide-resistant mutant strains were not. Wild-type and two mutant SecA proteins were purified. Both the basal level and the elevated ATPase activity of the mutant SecA proteins were threefold higher than those of the wild-type SecA. The elevated ATPase activity of the SecA mutants was reduced upon the addition of 1.5 mM sodium azide by only 5-10% as compared with 40% for that of the wild-type. These results indicate that the elevated ATPase activity of the SecA mutants is resistant to sodium azide and that is also required for the protein translocation process of B. subtilis.

srb: a Bacillus subtilis gene encoding a homologue of the alpha-subunit of the mammalian signal recognition particle receptor.

We cloned a Bacillus subtilis gene (srb) encoding a homologue of the mammalian signal recognition particle receptor alpha-subunit (SR alpha). The gene is 987 bp in length and encodes a 329-amino acid protein. The deduced amino acid sequence of the protein shared 26.6, 36.2 and 49.7% identity with those of mammalian SR alpha, archaebacterial DP alpha and Escherichia coli FtsY, respectively. The protein contains three conserved GTP-binding elements like the other three SRP receptor proteins, though the N-terminal portion of the putative B. subtilis protein was shorter than the others. Secondary structure prediction showed than an amphipathic alpha-helix is positioned in the N-terminal region. A defect in srb inhibited cell growth and protein translocation.