Intra- and extracellular forms of alpha-glucosidase from Aspergillus niger.

An intracellular α-glucosidase (α-glu₁) of Aspergillus niger was purified and its properties were compared to those of a secreted α-glucosidase (α-glu_E). The estimated molecular weight of α-glu_I was 95,000 by gel filtration (α-glu_E = 63,000); it is a glycoprotein possessing 29 mol of mannose, 6 mol of glucosamine, and 14 mol of glucose (α-glu_E has 5–6 and 2 mol of mannose and glucosamine, respectively). The K_m′s of α-glu₁ for p-nitrophenyl-α-d-glucopyranoside and maltose were 1.49 and 1.04, respectively, slightly lower than those of α-glu_E. In addition, at 65 °C α-glu_I enzymatic activity decayed fivefold faster than that of α-glu_E, and anti-α-glu_E antibody did not recognize α-glu_I. While some of these distinctions between the enzymes could be ascribed to conformational differences, the great dissimilarity in molecular weight (approximately 32,000) and lack of reactivity with anti-α-glu_E argue against α-glu_I being related to α-glu_E. The antibody covalently coupled to horseradish peroxidase (Ab-Px) was used as a probe to determine the cellular location of α-glu_E by electron microscopic immunocytology. It was found on both sides of the plasma membrane (pm) and in the outer of the two layers of the cell wall. This may mean that α-glu_E is synthesized at the inner surface of the pm, is extruded through the pm, becomes associated with the outer layer of the cell wall (perhaps as enzyme—substrate complex), and is eventually released into the growth medium.     

Specificity of P2 primary alkylsulphohydrolase induction in the detergent-degrading bacterium Pseudomonas C12B. Effects of alkanesulphonates, alkyl sulphates and other related compounds.

Primary alkanesulphonates were shown to serve as non-metabolizable (gratuitous) inducers of the P2 primary alkylsulphohydrolase enzyme in resting cell suspensions of Pseudomonas C12B. The effects of increasing concentrations of inducer on the production of enzyme were complex and suggestive of a multiphasic phenomenon. However, it was possible to determine Kinducer constants (analogous to Km or Ki) for alkanesulphonates of chain length from C7 to c12. these decreased with increasing chain length in a manner characteristic of an homologous series. Primary alkyl sulphates also served as good inducers of alkylsulphohydrolase, but valid kinetic values could not be obtained because these esters are good substrates for the enzyme and are therefore appreciably hydrolysed during the induction period. Small amounts of enzyme were also produced when cyprinol sulphate, dodecyltriethoxy sulphate C12H23-[O-CH2-CH2]3-O-SO3-Na+), Crag herbicide and some secondary alkyl sulphates were tested as inducers.     

Experimental autoallergic sialadenitis in the LEW rat. I. Parameters of disease induction

Experimental autoallergic sialadenitis (EAS) is an autoimmune mononuclear cell infiltration of the submandibular salivary gland that results in tissue destruction and glandular dysfunction. A previous report has described an animal model of induced EAS in LEW rats following sensitization with allogeneic WF submandibular gland (SMG). The present study extends this observation to an EAS disease model induced following sensitization of LEW rats with syngeneic LEW SMG. Furthermore, we describe the characterization of the mononuclear cells in the glandular infiltrates, evaluate the production of autoantibodies, and establish the parameters important for reproducible induction of EAS. Our results demonstrate that EAS can be induced in a completely syngeneic system and the histopathology of disease induction in the syngeneic and allogeneic model systems is similar. Helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) T-cell subsets are the dominant cell types in the salivary mononuclear cell infiltrate. An anti-duct autoantibody was found in the serum of virtually all LEW rats with EAS. Although closely associated with disease development, the presence of this antibody was not a prerequisite for development of histopathologic disease. Induction of disease in both the syngeneic and allogeneic models of EAS is dependent upon administration of Bordetella pertussis at the time of sensitization. Finally, the histopathology of the cellular infiltrates in both the allogeneic and syngeneic models of EAS resemble those observed in the salivary tissues of Sj?gren's patients. While there are several differences between EAS in the LEW rat and the full expression of Sj?gren's syndrome, EAS may serve as a model to study the salivary gland component of this complex human disease.     

Chromosomal integration of plasmid DNA by homologous recombination in Enterococcus faecalis and Lactococcus lactis subsp. lactis hosts harboring Tn919.

Integration of pCI192, a pBR322-derived vector plasmid containing homology to the chromosomally located conjugative transposon Tn919 was observed in two strains that harbor Tn919, namely, Enterococcus faecalis GF590 and Lactococcus lactis subsp. lactis CH919. Hybridization analysis indicated that single-copy integration of the plasmid had occurred at low frequency. The Tn919::plasmid structure was conjugated from an E. faecalis donor to a L. lactis recipient, although at lower frequencies than was Tn919. Segregation of the tetracycline and chloramphenicol resistance markers during conjugation was observed. The integration strategy described allows for DNA manipulations to be performed in an easily manipulated model host strain with the subsequent transfer of integrated structures by conjugation to any strain capable of receiving Tn919. The results indicate that homologous recombination events may be used to introduce plasmid-encoded genes to the lactococcal chromosome.     

Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions

The nucleocapsid (NC) protein (p15) of the human immunodeficiency virus (HIV) has been cloned and overproduced (under the control of a phage T7 promoter) in soluble form in an Escherichia coli host. The soluble NC protein is a fusion protein containing 15 amino acids from the T7 gene 10 and 7 amino acids from the HIV p24 protein at the N-terminus to make a protein of 171 amino acids. The plasmid containing the fusion gene is designated p15DF. A homogeneous product has been isolated from the induced cells and, when isolated under aerobic conditions, contains 0.3-0.5 mol of Zn/mol of protein and has only 2 titratable SH groups. Reduction and refolding in the presence of Zn(II) yields a protein containing 2.0 mol of Zn/mol of protein and 6 titratable SH groups. On the other hand, if the cells are sonicated in 2 mM CdCl2 and purified at pH 5.0, an unoxidized protein containing 2 mol of Cd/mol of protein is obtained. The Cd(II) ions can be exchanged with Zn(II), Co(II), or 113Cd(II). The Co(II)2 NC protein shows d-d electronic transitions at 695 nm [epsilon = 675 M-1 cm-1 per Co(II)] and 640 nm [epsilon = 825 M-1 cm-1 per Co(II)] compatible with regular tetrahedral geometry around both Co(II) ions. The Co(II)2 and Cd(II)2 NC proteins show intense charge-transfer bands in the near-UV, at 355 nm (epsilon = approximately 4000 M-1 cm-1) and 310 nm (epsilon = approximately 8000 M-1 cm-1) for the Co(II) protein and 255 nm (epsilon = approximately 10(4) M-1 cm-1) for the Cd(II)2 NC protein, compatible with -S- coordination. 113Cd NMR of the 113Cd(II)2 NC protein shows two 113Cd NMR signals at 659 and 640 ppm, respectively, each integrating to approximately 1 Cd(II) ion. The downfield chemical shifts suggest coordination of each 113Cd(II) ion to 3 sulfur donor atoms. The spectroscopic data fully support the prediction that the NC protein binds metal ions to each of the tandem repeats of the -Cys-X2-Cys-X4-His-X4-Cys- sequence contained in the N-terminal half of the molecule. 113Cd NMR shows, however, that the sites are not identical. Isolation of the NC protein under standard aerobic conditions results in oxidation of the sulfhydryl groups and loss of the coordinated Zn(II) ions, while preparation of the NC protein as the Cd(II) derivative at low pH protects the sulfhydryl groups from oxidation.     

Follicle-stimulating hormone pulse amplitude decreases with the onset of the breeding season in the mare

The relationship between daily mean FSH concentrations in serum and the pattern of FSH detected by frequent sampling for 12-h periods (samples every 15 min) was examined in five mares during the transition into the breeding season. The five mature anestrous mares were exposed to a natural increase in daylength. Blood samples were collected daily from February 1 until the first ovulation of the breeding season (April 14 +/- 3.7 days, Mean +/- SEM). Periods of frequent blood collection were performed every two weeks. Blood samples were obtained daily by jugular venipuncture or jugular cannula (frequent samples). Mean daily concentrations of FSH in serum determined by RIA decreased during seasonal transition. Patterns of FSH in serum detected by frequent sampling were pulsatile. FSH pulse amplitude decreased during seasonal transition, and the decrease in amplitude was associated with the decrease in mean serum FSH concentrations. This decrease in FSH pulse amplitude may reflect an involvement of a follicular product from developing follicles or a change in hypothalamic stimulation of pituitary FSH release.