Synthetic analogs of the active site of cytochrome P-450cam characterization of thiolpeptide fragment-hemin complexes by optical and ESR spectrometries

Thiol-containing penta (Leu-Ala-Cys-Ser-Leu) and nona (Leu-Ala-Cys-Ser-Leu-Ile-Glu-Ser-Leu) peptides corresponding to residues 132-136 and 132-140, respectively, of apo-P450 from Psuedomonas putida were synthesized to examine heme-binding by the enzymes in the oxidized (ferric) form. The peptide-hemin complexes prepared in solution were characterized by their optical and ESR spectra. In these complexes without nitrogenous ligands, no ferric complexes in the low-spin state were observed, suggesting that simultaneous axial coordination of Cys-134 (thiolate) and Ser-139 (hydroxyl) of apo-P450cam to the heme is unlikely to occur. In the presence of nitrogenous ligands, such as py, Im and MeIm, the resulting complexes were good models of apo-P450cam-nitrogenous ligand adducts in the low-spin ferric state retaining a thiolate-Fe(III)-nitrogen axial coordination mode, as judged by their spectral pattern as well as by crystal field analysis of ESR g-values.     

Alloantigen presentation by B cells: two types of alloreactive T cell hybridomas, B cell-reactive and B cell-nonreactive

T cell-depleted, Sephadex G-10-passed unstimulated splenic B cells from C57BL/6 mice stimulated splenic T cells from CKB mice to produce IL 2 and to proliferate. The stimulatory ability of the unstimulated B cells was eliminated by 4000 rad irradiation of the unstimulated stimulator B cells. LPS-activated B cells could stimulate responder T cells more efficiently than unstimulated B cells. For further analysis of allostimulation by B cells, we established a series of alloreactive T cell hybridomas. Forty-five percent of these alloreactive T cell hybridomas could be stimulated to produce IL 2 by either macrophage-dendritic cells or unstimulated B cells. Fifty-five percent of these alloreactive T cell hybridomas could be stimulated by macrophage-dendritic cells but not by unstimulated B cells. T cell hybridomas that were not reactive with unstimulated B cells were also nonreactive to LPS-activated B cells. Analysis of two representative I-Ab-reactive T cell hybridoma clones, B cell-reactive clone CB-11.4 and B cell-nonreactive clone HTB-9.3, revealed again that the stimulatory ability of unstimulated B cells was sensitive to 4000 rad irradiation in the activation of CB-11.4 clone and that CB-11.4 could be stimulated more efficiently by LPS-activated B cells than by unstimulated B cells, but HTB-9.3 could not be stimulated by LPS-activated B cells. Thus, there may be two distinct types of T cells in the alloreaction: B-cell-reactive and B cell-nonreactive.     

Circadian rhythms of urinary excretions of water and electrolytes in patients receiving total parenteral nutrition (TPN)

In order to determine whether the usual feeding pattern actually modifies the circadian rhythms of urinary excretion of water and electrolytes, we compared the circadian rhythm characteristics in patients receiving total parenteral nutrition (TPN group) with those in patients on an ordinary hospital diet (control group). Statistically significant circadian rhythms were detected in all of the urinary variables investigated herein by using the population mean-cosinor method in both groups. In addition, there were no statistically significant differences of the mesor, the %-amplitude and the acrophase between the two groups. These results suggest that the usual feeding pattern is not a main determinant in forming the circadian rhythm characteristics of human urinary variables.     

1,2-α-D-Mannosidase from a wood-rotting basidiomycete, Pycnoporus sanguineus

An acidic α-D-mannosidase has been isolated from the culture filtrate of a wood-rotting Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme determined. The extracellular mannosidase was homogeneous on PAGE at pH 9.4. The M_r as determined by SDS-polyacrylamide disc gel electrophoresis was 64000, and the pI was pH 4.7 using electrofocusing. The purified enzyme had a pH optimum of 4.5 with Baker's yeast mannan and had no activity towards p-nitrophenyl-α-mannoside. The K_m and k_cat values for Manα1-2Man at pH 4.5 and 30° were 0.9 mM and 1.9 sec. the enzyme had no activity towards Manα1-3Manα1-2Man, and it cleaved specifically the 1,2-α-linked side chain of yeast α-mannan, producing free α-D-mannose.     

In vitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cells and dermal fibroblasts

Rat glomerular mesangial cells were cultured and their antigens were compared with those of aortic vascular smooth muscle cells and dermal fibroblasts. Glomeruli, aortic, and dermal explants were cultured for 3 weeks and subcultured in the same conditions. These cultured cells were evaluated by indirect immunofluorescence studies using antibodies against Thy-1 antigen, desmin, and chicken gizzard actin. Most of mesangial cells were positive for Thy-1, desmin, and actin. On the other hand, fibroblasts were negative for desmin, and smooth muscle cells stained Thy-1 scarcely, and were negative for desmin. In the latter two cells, actin-positive fibrils were thinner and fainter than mesangial cells. These results indicated that mesangial cells could be distinguished in vitro from vascular smooth cells and fibroblasts by immunofluorescence microscopy.     

Quantitative hybridization-arrest of mRNA in Xenopus oocytes using single-stranded complementary DNA or oligonucleotide probes.

The expression of heterologous mRNA in Xenopus oocytes was quantitatively inhibited by coinjection of single-stranded complementary DNA or synthetic complementary oligonucleotides. The lymphokines Interleukin-2 (IL-2) and Interleukin-3 (IL-3) were used as model systems to test the effectiveness of this procedure. Messenger RNA samples were hybridized to single stranded complementary DNA or oligonucleotides, injected into oocytes and the oocyte incubation medium assayed for the presence or absence of specific translation products 48 hours later. When IL-2 mRNA was hybridized to a large excess of long (490 bases) single stranded complementary DNA, the expression of IL-2 was effectively blocked (greater than 98%). Complementary oligonucleotides (18-23 bases) were almost as effective as the polynucleotide in inhibiting IL-2 activity (greater than 95%). Oligonucleotides derived from the 5' end, middle or 3' end of the coding sequence were all effective in arresting IL-2 mRNA translation. Oligonucleotide hybrid-arrest was effective even when no NaCl was present in the hybridization buffer, indicating that the annealing reaction could occur within the oocyte after injection. Definite proof that hybrid-arrest could occur in vivo was shown by the fact that oligonucleotides injected before or after mRNA injection, while not as effective as co-injection, still showed substantial inhibition of specific mRNA translation. The oligonucleotide hybrid-arrest method was equally effective in the case of IL-3, demonstrating its general applicability.     

Augmentation of lung antineutrophil elastase capacity with recombinant human alpha-1-antitrypsin

To evaluate the potential use of recombinant DNA-produced alpha-1-antitrypsin (alpha-1-AT) to augment the lung antineutrophil elastase defenses in alpha-1-AT deficiency, we compared the kinetics of intravenously administered recombinant produced alpha-1-AT (r alpha-1-AT) and purified normal human plasma alpha-1-AT (p alpha-1-AT) in the blood and lung of rhesus monkeys. The r alpha-1-AT was produced in yeast transformed with an expressing plasmid containing a full-length human alpha-1-AT complementary deoxyribonucleic acid and purified to greater than 99% homogeneity. The r alpha-1-AT has a molecular weight of 45,000, no carbohydrates, and is identical in sequence to normal plasma alpha-1-AT except for an additional N-terminal acetylmethionine. Despite its lack of carbohydrates, the r alpha-1-AT inhibited human neutrophil elastase with an association rate constant similar to that of p alpha-1-AT. Rhesus monkeys were infused intravenously with 120 mg/kg of r alpha-1-AT (n = 13) or p alpha-1-AT (n = 12) and the serum, urine, and lung epithelial lining fluid (ELF) concentrations of these molecules quantified at various intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of human pancreas

The developmental sequence of human pancreatic secretory proteins has not previously been studied in detail. We applied immunohistochemistry to study 20 fetal and neonatal pancreas' (8th to 39th gestational weeks) using antisera against the following pancreatic secretory proteins: pancreatic secretory trypsin inhibitor (PSTI), serine proteinases (trypsin, chymotrypsin, and elastase I), and amylase. PSTI was first detected in developing buds of the pancreas during the 8th gestational week, and proteinases were observed in acinar cells during the 14th week of gestation. Immunoreactivity for both PSTI and proteinases was found in most acinar cells soon after their appearance. Immunoreactivity for amylase could not be detected in fetal or neonatal pancreas tissue. PSTI was also found in developing islets during the 14th gestational week, but the number of immunoreactive cells had decreased by term. Cells positive for serine proteinases were occasionally in contact with islets in second-trimester fetuses. In discussing these results, we give particular attention to the nonparallel appearance of secretory products in the fetal pancreas, and the significance of cells immunoreactive for secretory proteins in endocrine islets.     

Electrophoretic heterogeneity and age-related change of serine proteinase fromAspergillus sojae

Five forms of serine proteinase (EC 3.4.21.14) have been purified fromAspergillus sojae. This paper reports heterogeneity of electrophoretic mobilities, isoelectric points, and kinetic parameters of multiple forms of serine proteinase fromAspergillus sojae, including agerelated changes in thek _cat /K _m of electrophoretic species.     

Molecular and enzymatic properties of 1,2-α-d-mannosidase fromAspergillus saitoi

The molecular properties, such as molecular weight, N-and C-terminal amino acids, amino acid composition, and circular dichroism, of 1,2--mannosidase isolated from the culture filtrate ofAspergillus saitoi were determined.The enzyme had aK _m of 0.67 mM andk _cat of 1.27/s with mannobiose at pH 50.0 and 30°C. The anomeric configuration of the reaction products of the enzyme was examined by studying the -anomer. A single Manl2Man linkage in intact Taka-amylase A fromAspergillus oryzae was hydrolyzed, producing free mannose.