Tunicamycin-resistant mutants and chromosomal locations of mutational sites in Bacillus subtilis.

The types of tunicamycin-resistant mutants of Bacillus subtilis were analyzed, and their mutational sites on the chromosome were mapped. A type 1 mutation that simultaneously expressed hyperproductivity of extracellular alpha-amylase was located close to amy E. Type 2 mutations were near aroI.     

Inhibition of the synthesis of microbial cellulose by coumarin

Coumarin, a specific inhibitor of the biosynthesis of celluloseof higher plant cell walls, inhibited the cellulose formationof Acetobacter xylinum. The degree of inhibition reached 55%in the presence of 1 mM coumarin, which causes 70% inhibitionin the case of plant cellulose. (Received April 12, 1976; )     

Effects of CB-154 (2-Br-alpha-ergocryptine) on prolactin and growth hormone release in an acromegalic patient with galactorrhea.

An acromegalic patient with galactorrhea was treated with an ergot alkaloid, 2-Br-alpha-ergocryptine (CB-154). Serum prolactin decreased rapidly to normal level by CB-154 and the complete cessation of galactorrhea was noted. The inhibitory effect of CB-154 On growth hormone (GH) release was also noted, but slight. The mechanism of inhibitory action of CB-154 on both prolactin and GH secretion was discussed in connection with the experimental model of pituitary tumors, in which both hormones were produced by a single type of tumor cells. The discontinuation of CB-154 treatment was associated with the return of both prolactin and GH levels to the initial high values with resumption of galactorrhea.     

N-(2-Pyridyl)acetamide complexes of palladium(II), cobalt(II), nickel(II), and copper(II)

N-(2-Pyridyl)acetamide (aapH) complexes of palladium(II), cobalt(II), nickel(II), and copper(II) have been studied by means of magnetic susceptibilities, and infrared, electronic, and PMR spectra. In the octahedral complexes M(aapH)₂X₂(M = Co, Ni, Cu; X = Cl, Br, NCS, NO₃), bidentate aapH is chelated through the pyridine-N and amid-O atomes, whereas in the square-planar Pd(aapH)₂X₂ (X = Cl, Br) unidentate aapH is coordinated through the pyridine-N atom alone. Under alkaline conditions aapH is deprotonated in the presence of palladium(II) to form Pd(aap)₂·4H₂O, aap being an anionic bidentate ligand and chelating through the pyridine-N and amide-O atoms.     

Plasmic degradation of bovine fibrinogen and non-crosslinked fibrins in solution and in gel form.

1. Analysis of degradation processes of bovine fibrinogen by bovine plasmin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a study on the mode of changes of the properties related to clotting of digestion products as a function of time were performed. Gross features and patterns very similar to those which had been reported with human fibrinogen-plasmin systems were obtained. 2. Based on the molecular size of the degradation products and the mode of appearance and disappearance of the degradation products, the processes could tentatively be divided into three stages: stage 1, where fibrinogen (mol. wt 370 000) was degraded to produce fragments X1 (330 000) and X2 (290 000); stage 2, fragment X2 was degraded with appearance of Y (210 000) and D1 (140 000); stage 3, appearance of fragments D1, D2 (110 000), and D3 (100 000) sequentially and E (68 000) with concomitant disappearance of Y. 3. A microseparation method, which is a combination of dansylation and sodium dodecylsulfate-polyacrylamide gel electrophoresis, was devised to analyze the events of stage 1 in detail, and a molecular model for the process was proposed. 4. The plasmic degradation processes of bovine non-cross-linked fibrins in solution and in gel form were compared with that of fibrinogen and it was found that the state of the substrates, fibrins, could cause differences in the degradation patterns. With the former substrate, essentially the same sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns as those with fibrinogen were obtained. With the latter substrate, however, a distinct difference in the mode of degradation of beta chains was observed.     

The structure and function of acid proteases. VI. Effects of acid protease-specific inhibitors on the acid proteases from Aspergillus niger var. macrosporus.

1. The Type B acid protease from Aspergillus niger var. macrosporus was inactivated by reaction with diazoacetyl-DL-norleucine methyl ester (DAN), DL-1-diazo-3-tosylamido-2-heptanone (DTH), and L-1-diazo-3-tosylamido-4-phenyl-2-butanone (DTPB) in the presence of cupric ions. The reaction with DAN took place with 1:1 stoichiometry. The enzyme was also inactivated by reaction with 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) with concomitant incorporation of approximately two EPNP molecules per molecule of protein. Moreover, these reactions of DAN and of EPNP were markedly inhibited by pepstatin. These results seem to indicate that, as in the case of porcine pepsin [EC 3.4.23.1] and related acid proteases, the enzyme has two essential carboxyl groups at the active site, one reactive with DAN and related diazo reagents in the presence of cupric ions and the other reactive with EPNP, and that pepstatin binds in the vicinity of these residues. 2. The Type A acid protease from the same mold, on the other hand, was found to be markedly less sensitive to these specific inhibitors. Under conditions where the Type B enzyme was completely inactivated by DAN and related diazo reagents, only partial inactivation of this enzyme occurred. The effect of prior mixing of DAN and cupric ions on the pH profile of inactivation was also different from that for the Type B enzyme. Moreover, the Type A enzyme was not inactivated by EPNP. These results thus indicate that the nature of the active site of the Type A enzyme is rather different from that of the Type B enzyme and hence that the Type A enzyme belongs to a different class of acid proteases from the Type B enzyme.     

On the relationship between the frequency of two types of lethal-mutation and X-ray doses in drosophila

The dose-frequency relationship for each of 2 types of lethal mutations, fractional- and whole-lethal, was obtained using X-rays on Drosophila melanogaster. The results show that fractional-lethal mutations are induced by X-rays, and also that the proportion of fractional-lethal mutations in the total of mutations tends to decrease with increasing doses, namely, 61% at 0 R, 47% at 500 R, 37% at 1000 R and 20% at 2000 R. The same tendency is observed with visible mutations.In order to consider the problems related to the above results, the relationship between the true frequency and the observed frequency of the induced lethal mutations is discussed, taking into consideration the existence of the ontensible whole-lethal and the ontensible normal.     

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.     

Lectin Binding Assays for In-Process Monitoring of Sialylation in Protein Production

Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galβ1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(β1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galβ1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galβ1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.