Cell wall strength of Hansenula polymorpha in continuous cultures in relation to the recovery of methanol oxidase (MOX)

Summary The changes in cell wall strength of Hansenula polymorpha have been investigated in continuous cultures with respect to the recovery of methanol oxidase (MOX). Cultures grown on several substrate mixtures that enable induction of MOX have been compared with cultures grown on methanol as the sole inducer. The effects of dilution rate (D) on lysis properties have been studied. The cell wall strength was consistently influenced by growth media and D. Media containing glycerol/methanol showed the slowest lysis kinetics, with a large fraction of non-degradable cell wall material. In continuous cultures grown on a mixture of glucose and methanol both the resistance to zymolyase and the mean cell wall thickness increased at D<0.1 h^–1. The yield of MOX by zymolyase lysis is reproducible and up to 100% higher than that of the standard ultrasonic treatment. The lysis kinetics indicated that zymolyase punctures the cell wall; since the release rate of MOX is lower than that of protein, the cell contents will leak through. At D-values>0.2 h^–1, both protein and MOX release rates increase, reflecting a change in lysis mechanism due to the increased fraction of thin daughter cells. Kinetic analysis of zymolyase lysis using both physical and enzymatic methods provides information for achieving optimal recovery of MOX.Abbreviations and symbols C _MOX MOX activity [MOX units·g X^–1] - D dilution rate [h^–1] - MOX methanol oxidase - k_c decay rate constant of A 610 nm [min^–1] - k_d decay constant of MOX activity [min^–1] - k_dis dissociation rate constant [min^–1] - k_MOX release rate constant of MOX activity [min^–1] - k_p release rate constant of protein [min^–1] - R recovery efficiency of enzyme [-] - St stability of enzyme [-]     

Regulation of the amylase secretion by the yeast Filobasidium capsuligenum and a 2-deoxy-d-glucose resistant mutant

Summary The regulation of extracellular amylase production by the basidiomycetous yeast Filobasidium capsuligenum CCY 64-5-1 was characterized using growing and resting cells. A basal level of amylolytic activity was produced with various carbon sources including glucose. Amylase secretion was repressed by glucose and, more severely, by 2-deoxy-d-glucose, whereas compounds with -1,4-linked glucose, such as methyl glucoside, maltose, -cyclodextrin and soluble starch, served as inducers. Repression was not relieved by exogenously added cAMP. The effects of several metabolic inhibitors on amylase secretion were studied. Following UV-mutagenesis a mutant strain (FC-5) capable of growing in a 2-deoxy-d-glucose supplemented corn starch medium was selected for further characterization. This strain produced more amylase, had acquired an increased resistance against repression by glucose, and retained a growth rate comparable to the wild type. FC-5 was also characterized by a reduced glucokinase activity and an increased hexokinase activity.     

Differences in the half-lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity

The different turnover rates of rat liver mitochondrial enzymes make autophagy unlikely to be the main mechanism for degradation of mitochondria. Although alternatives have been presented, hepatocyte heterogeneity has not been considered. Lighter hepatocytes isolated in a discontinuous Percoll gradient contain more glutamate dehydrogenase (GDH) (half-life 1 day) and a more active autophagic system than heavier hepatocytes. The latter contain more carbamoyl phosphate synthase (CPS) and ornithine carbamoyl transferase (OTC) (half-lives 8 days) but less lysosomal activity. As expected, isolated autophagic vacuoles contain, relative to the mitochondrial content, 3-times less OTC and CPS than GDH, probably reflecting a faster lysosomal engulfment of mitochondria in the light hepatocytes (which contain more GDH). These data may explain some of the half-life differences of the enzymes studied.     

Apparent identity of cerebral tyrosylsulfotransferase activities using either a cholecystokinin derivative or an acidic amino acid polymer as substrate

The tyrosylsulfotransferase activities of rat cerebral fractions transferring [35S]sulfate groups from 3'-phosphoadenosine 5'-[35S]phosphosulfate to either Boc-cholecystokinin-8 (in non-sulfated form) or the acidic amino acid polymer (Glu, Ala, Tyr)n (6:3:1) were compared. They appear similar regarding subcellular distribution (both being enriched in the microsomal fraction) and inhibition by an excess of the acidic amino acid polymer, NaCl or 2,6-dichloro 4-nitrophenol. These results obtained with artificial substrates suggest that identical (or closely similar) tyrosylsulfotransferases are responsible for sulfation of tyrosine residues of several secretory proteins from various tissues.     

Analysis by flow cytometry of rat hepatocytes from different acinar zones

Many functional, morphological and biochemical differences among hepatocytes from different acinar zones have been described. Therefore, it will facilitate studies on liver metabolism rapid, non-destructive procedures to isolate hepatocytes from these zones. Flow cytometry is a new powerful tool which, however, has not been used thus far to accomplish the separation of hepatocytes from different acinar zones. We describe here various cytometric parameters which characterize hepatocyte populations, separated by isopycnic centrifugation in Percoll gradients. The intraacinar origin of the different hepatocytes was assessed by enzymatic and morphological measurements.     

Ultrastructural immunocytochemical study of the massa caudalis of the subcommissural organ-Reissner's fiber complex in lamprey larvae (Geotria australis): Evidence for a terminal vascular route of secretory material

Summary The massa caudalis of the subcommissural organ-Reissner's fiber complex of lamprey larvae (Geotria australis) was studied immunocytochemically at the ultrastructural level by use of the immunoperoxidase-silver methenamine procedure. An antiserum raised against bovine Reissner's fiber was utilized as primary antibody.The caudalmost portion of the central canal and its ampulla caudalis communicate, via wide intercellular spaces in their dorsal wall, with large cavities or lacunae. In addition, distinct openings in the dorsal wall of the ampulla establish an open communication between the latter and the lacunae. The lacunae are lined by slender processes of cells of unknown nature. No junctional complexes can be observed between these cells, which lack a basal lamina. The lacunae communicate with structures resembling blood capillaries, however, they are devoid of a basal lamina. These peculiar vessels, in turn, are in direct communication with characteristic blood capillaries.Reissner's fiber (RF) and its massa caudalis are strongly immunoreactive with the antiserum used. The wide intercellular spaces in the dorsal wall of the central canal and the ampulla, as well as the lumina of the (i) lacunae, (ii) modified vessels and (iii) blood capillaries are filled with a flocculent, strongly immunoreactive material. No immunoreactive material was found outside these structures. Thus, the blood capillaries appear to represent the only final target of RF-material arriving at the ampulla caudalis.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, and Grant 6027 from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile. The authors express their gratitude to Mrs. Elizabeth Santibáñez and Mr. Julio Lamilla for providing the lamprey larvae and to Mr. Humberto Molina for preparing the three-dimensional drawing     

Presence of nucleosomes inPenicillium chrysogenum

We have studied the chromatin structure ofPenicillium chrysogenum. This fungus presents the typical nucleosomal repeat and the core DNA size characteristic of all the eukaryotes. The repeat length (about 180 base pairs) is in the range of those obtained for most fungi (160–180 base pairs) and shorter than in higher eukaryotes. Knowledge aboutP. chrysogenum chromatin structure opens the way to the study of the mechanisms of genetic regulation in this filamentous fungus.     

A complex balanced chromosomal rearrangement in repeated abortions

Summary A double balanced reciprocal translocation involving four chromosomes, t(1;19;6;14) (1p11; 19p11; 6q25; 14q21), was found in the phenotypically normal husband in a couple referred because of repeated abortions. Reciprocal translocations, t(6;14), had been transmitted by his mother, his father being apparently homozygous for a translocation comprising pairs 1 and 19-t(1;19)(1;19). The genetic consequences of this complex chromosomal rearrangement are analyzed.