Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells.

The mutant c-fgr protein (p58c-fgr/F523) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58c-fgr (p58c-fgr/wt) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive alpha-naphthyl butyrate esterase (alpha-NBE), a marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive alpha-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1 alpha,25-dihydroxyvitamin D3-treated WEHI-3B cells. Immunoblotting studies with antiphosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive alpha-NBE and cell transformation by p58c-fgr.     

Two monoclonal antibodies against small-cell lung cancer show existence of synergism in binding

Summary Murine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of¹²⁵I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC-1 or NKH-1. Unexpectedly, binding of¹²⁵I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK-3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy.     

Molecular cloning and expression ofThiobacillus ferrooxidans chromosomal ribulose bisphosphate carboxylase genes inEscherichia coli

The genes coding ford-ribulose-1,5-bisphosphate carboxylase (RuBPCase) from an iron-oxidizing bacterium,Thiobacillus ferrooxidans, were cloned into anEscherichia coli plasmid, pUC18. The recombinant plasmid, termed pTR11, contained a 4.0-kb PstI fragment including the entire coding regions for both large and small subunits of RuBPCase.Escherichia coli carrying pTR11 did not show any CO₂-fixing activity. However, a derivative plasmid with an appropriate deletion, which was placed under the control of atac promoter, conferred ribulose bisphosphate-dependent CO₂-fixing activity on the host cell. Analysis of gel-filtration chromatography of the RuBPCase synthesized inE. coli revealed that it had a hexadecameric form like the native enzyme ofT. ferrooxidans.     

Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency.

Oligonucleotide probes specific for the deleterious mutations harbored in the P-450(C21)A pseudogene and oligonucleotide probes specific for the corresponding sequences in the B gene were prepared to examine the molecular lesions in the P-450(C21) gene of P-450(C21)-deficient patients. Using these gene-specific probes, we performed Southern blot analyses of genomic DNAs from 11 patients and eight normal individuals. At least one allele of the B gene (the 3.7-kb TaqI fragment) in a patient was inactivated by mutations caused by recombination with the A gene. The A genes in normal individuals and patients seemed to be replaced frequently (i.e., 10/19 individuals) in their 3' portions by B gene sequences. All of these alterations occurred without changing the characteristic length (3.2 kb) of the TaqI fragment of the A gene, a result strongly suggesting that frequent gene conversions and/or intragenic recombinations have happened in the P-450(C21) genes. Densitometric analysis of the autoradiograms from hybridization experiments revealed extensive variation (from one to five copies) in the copy number of the A gene (the 3.2-kb TaqI fragment) whereas that of the B gene (the 3.7-kb TaqI fragment) was relatively constant at two or three copies.     

Electrical stimulation of the suprachiasmatic nucleus of the hypothalamus causes hyperglycemia

We have presented evidence suggesting that the suprachiasmatic nucleus (SCN) is involved in central regulation of glucose homeostasis. To elucidate this role of the SCN, we examined the effects of its electrical stimulation on glucose metabolism in male Wistar rats. During and shortly after this stimulation, we observed hyperglycemia associated with enhanced hyperglucagonemia but no immediate hyperinsulinemia. In addition, we detected significant increase in liver glycogen phosphorylase alpha activity and significant decrease in the liver glycogen content. These findings suggest that the SCN is important in control of glucose homeostasis through effects on glucagon and insulin secretions and liver glycogen metabolism.     

Hepatic accumulation of pyrophosphate during acetate metabolism

Accumulation of pyrophosphate induced by acetate administration was investigated in rat liver in situ and in perfused rat liver. Intraperitoneal injection of acetate into rats increased the pyrophosphate concentration in the liver to about 2 mumol/g liver, which was 200 times that in control liver. Perfusion of liver with acetate alone did not result in accumulation of pyrophosphate. However, the further addition of a Ca2+-mobilizing hormone, such as noradrenaline or angiotensin II, together with glucagon to the perfusion medium containing 1 mM acetate caused accumulation of pyrophosphate to a similar level to that observed in vivo. Acetate, glucagon and a Ca2+-mobilizing hormone were all required for accumulation of pyrophosphate in perfused liver. Omission of Ca2+ from the perfusion medium or addition of a Ca2+-antagonist reduced the accumulation significantly. The two kinds of hormones, glucagon and an alpha-agonist, either singly or in combination, did not affect the rate of acetate utilization. These results show that liver cells accumulate a large amount of pyrophosphate during acetate metabolism at high intracellular levels of Ca2+ that can be realized by the synergistic actions of the two kinds of hormones.     

Abnormal S-state turnovers in NH3-binding Mn centers of photosynthetic O2 evolving system

The inhibitory effects of NH3 on S-state turnovers were studied by curve fitting and deconvolution of thermoluminescence glow curves and low-temperature EPR spectroscopy. The following results were found: (i) High concentrations of NH3 upshifted the recombination temperatures of both S2QB- and S2QA- charge pairs, indicating formation of an abnormal S2 state having a lowered oxidation potential. (ii) The abnormal S2 was correlated to alterations in EPR multiline signal: high concentrations of NH3 induced the modified multiline signal having reduced hyperfine line spacing, accompanied by disappearance of the g = 4.1 signal, while low concentrations of NH3 reduced the line width of the g = 4.1 signal with a slight shift in its g value to 4.2 concomitant with suppression in amplitude of the normal multiline signal, both suggesting coordination of NH3 to the Mn center. (iii) More than half of the NH3-binding abnormal S2 centers underwent S-state turnover to yield S3QB- and S3QA- pairs having normal thermoluminever, the NH3-binding S3 was unable to undergo further S-state turnovers. (iv) The interruption of S-state turnover at S3 was assumed to be due to the inability of electron abstraction from the S3 state. Based on these, the mechanism of NH3 inhibition was discussed.     

Comparative structures of the apopolysialoglycoproteins from unfertilized and fertilized eggs of salmonid fishes

The complete amino acid sequence of the major polysialoglycoproteins (PSGPs) from two genera of salmonid fish eggs, Salvelinus and Oncorhynchus, has been determined. The occurrence of tandem repeats of a genus-specific dodeca- and tridecapeptide was found for the apoPSGP of Salvelinus leucomaenis pluvius (Slp) and Oncorhynchus masou ishikawai (Omi), respectively, their amino acid sequences being highly homologous with that of rainbow trout [Salmo gairdneri (Sg)] apoPSGP (*denotes the glycosylation site; mean value of N = approximately 25): H-PSGP(Slp): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-)N H-PSGP(Omi): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser-)N H-PSGP(Sg): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly-)N Within 5-7 min following fertilization H-PSGP is converted to the low-molecular-mass PSGP (L-PSGP) by a specific protease (PSGPase). We have purified L-PSGP from the fertilized eggs of S. leucomaenis pluvius and Oncorhynchus keta (chum salmon) and compared it with rainbow trout egg L-PSGP(Sg) by analysis of their amino acid sequence: L-PSGP(Slp): Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp L-PSGP(Ok): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser L-PSGP(Sg): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly The data support the conclusion that H-PSGP is degraded in vivo 5-7 min after fertilization to L-PSGP by proteolytic cleavage at the position two residues C-terminally to the Pro residue, i.e., -Pro-Ser-Xaa-Asp-(Xaa = either Gly, Ser, or Asp) by the action of PSGPase.     

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.     

Identification of 2-azelaoylphosphatidylcholine as one of the cytotoxic products generated during oxyhemoglobin-induced peroxidation of phosphatidylcholine

Cytotoxic product(s), which are responsible for inducing the release of acetylcholinesterase-enriched vesicles from human erythrocytes and cell lysis, are generated when 1-saturated-2-polyunsaturated glycerophosphocholine was incubated with oxyhemoglobin (Itabe, H., Kobayashi, T. and Inoue, K. (1988) Biochim. Biophys. Acta 961, 13-21). To identify the products, a model compound, 1-O-octadecyl-2-linoleoylglycerophosphocholine was incubated with oxyhemoglobin. The oxidation products were isolated by both straight-phase and reverse-phase HPLC. The products, which were responsible for inducing erythrocyte membrane damage, were analyzed by secondary ion mass spectrometry and 1H-NMR. One of the cytotoxic products isolated was identified as 1-O-octadecyl-2-azelaoylglycerophosphocholine. Methyl esterification of the product confirmed the proposed structure.