Mass spectrometric measurements of methane and oxygen utilization by methanotrophic bacteria

Abstract A mass spectrometer with membrane inlet was used to measure methane and oxygen utilization rates at various methane concentrations in Methylosinus trichosporium and a locally isolated strain of a methane-oxidizing coccus (OU-4-1). The apparent K _m for methane was found to be 2 μM for M. trichosporium and 0.8 μM for strain OU-4-1. These K _m-values are 10–30 times lower than most previously reported values. The ratio of oxygen to methane utilization rates was 1.7 for M. trichosporium and 1.5 for strain OU-4-1 corresponding to a growth yield of 0.38 and 0.63 g dry weight/g methane, respectively.     

Multizooidal Budding in Parasmittina trispinosa (Johnston) (Bryozoa,Cheilostomata)

Two principally different wall types occur in the bryozoan colony: Exterior walls delimiting the super-individual, the colony, against its surroundings and interior walls dividing the body cavity of the colony thus defined into units which develop into sub-individuals, the zooids. In the gymnolaemate bryozoans generally, whether uniserial or multiserial, the longitudinal zooid walls are exterior, the transverse (proximal and distal) zooid walls interior ones. The radiating zooid rows grow apically to form “tubes” each surrounded by exterior walls but subdivided by interior (transverse) walls. The stenolaemate bryozoans show a contrasting mode of growth in which the colony swells in the distal direction to form one confluent cavity surrounded by an exterior wall but internally subdivided into zooids by interior walls. In the otherwise typical gymnolaemate Parasmittina trispinosa the growing edge is composed of a series of “giant buds” each surrounded by exterior walls on its lateral, frontal, basal and distal sides and forming an undifferentiated chamber usually 2–3 times as broad and 3 or more times as long as the final zooid. Its lumen is subdivided by interior walls into zooids 2–3, occasionally 4, in breadth. This type of zooid formation is therefore similar to the “common bud” or, better-named, “multizooidal budding” characteristic of the stenoleamates but has certainly evolved independently as a special modification of the usual gymnolaemate budding.     

Protein affinity chromatography reveals cell cycle dependent association of cellular factors with human DNA polymerase α

DNA polymerase α/primase (Polα) is the key replication enzyme in eukaryotic cells. This enzyme synthesizes and elongates short RNA primers at an unwound origin of replication. Polα was used as an affinity ligand to identify cellular replication factors interacting with it. Protein complexes between Polα and cellular factors were analyzed by co-immunoprecipitations with monoclonal antibodies directed against Polα and by protein affinity chromatography of cell extracts derived from pure G₁-and S-phase cell populations on Polα affinity columns. Co-immunoprecipitations resulted in the identification of a polypeptide with a molecular weight of 46 kDa. For Polα affinity chromatography, the ligand was purified from insect cells infected with a recombinant baculovirus encoding the catalytic subunit (p180) of Polα (Copeland and Wang, 1991). With 5×10⁸ infected Sf9 cells, a rapid one step purification protocol was used which yielded in five hours 0.6 mg pure enzyme with a specific activity of 140,000 units/mg. The G₁-and S-phase cell populations were generated by block, release and counterflow centrifugal elutriation of exponentially growing human MANCA cells. Starting with 2×10⁹ non synchronous cells, 5×10⁸ G₁-phase cells were isolated. Chromatography of cell extracts derived from G₁-or S-phase cells on Polα affinity columns resulted in identifying several polypeptides in the range of 40–70 kDa. Some of these polypeptides are more abundant in eluates derived from S-phase extracts than from G₁-phase extracts.     

Neuropeptide Y modulates the action of vasodilator agents in guinea-pig epicardial coronary arteries

Recently, we have demonstrated that guinea-pig epicardial coronary arteries are supplied by numerous nerve fibres containing neuropeptide Y (NPY) immunoreactivity. However, examination of vasomotor responses revealed that NPY did not elicit a contractile response in these arteries. In contrast, acetylcholine (ACh), calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) all relaxed precontracted arteries. In the present study, we have used histochemical, immunohistochemical and in vitro pharmacological techniques, in order to further investigate the possible role of NPY in guinea-pig epicardial coronary arteries. A double-immunofluorescence staining technique revealed that CGRP and substance P were co-localized in nerve fibres distinct from those displaying NPY immunoreactivity. Furthermore, using a method combining immunofluorescence and histochemical techniques, we observed that putative cholinergic nerve fibres (identified by their acetylcholinesterase content) and NPY-immunoreactive nerve fibres are two different nerve populations. An in vitro pharmacological method demonstrated that NPY markedly inhibited the relaxant responses mediated by ACh, VIP, substance P and isoprenaline but had no effect on CGRP. These results suggest that NPY-containing nerves associated with guinea-pig epicardial coronary arteries may be predominantly involved in modulating the action of vasodilator agents.     

Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast

Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.     

Sulfide-Dependent Methane Production and Growth of a Thermophilic Methanogenic Bacterium

Two strains of a thermophilic methanogenic bacterium, designated MT1 a and b, were isolated from a coastal surface sediment in the northern part of the Swedish West Coast. The two strains were identical in appearance and nutritional requirements and resembled Methanobacterium thermoautotrophicum. MT1 had an extraordinary requirement for sulfide. At sulfide levels below 0.1 mM, growth was poor and the methane production rate decreased. Other sources of sulfur, such as cysteine, sulfate, or thiosulfate, could not replace sulfide. The results indicate that a fast turnover rate of reduced sulfur compounds is involved in the energy metabolism of this organism.     

Accumulation of Glucosylceramide and Glucosylsphingosine (Psychosine) in Cerebrum and Cerebellum in Infantile and Juvenile Gaucher Disease

Abstract: Three major clinical variants of Gaucher disease have been defined: Type I, chronic nonneuronopathic; Type II, acute neuronopathic; and Type III, subacute neuronopathic. In a search for the underlying molecular basis of the neurological manifestations, the concentration and composition of cholesterol, phospholipids, neutral glycosphingolipids, and gangliosides were examined in cerebral and cerebellar cortices of five cases of Type II, eight cases of Type III, and one case of presumed Type I/III. In Type II the concentration of glucosylceramide was 140-530 μmol/kg in cerebral cortex and 51-450 μmol/kg in cerebellar cortex, the highest values found in the most fulminant cases. These concentrations were 20-80 times greater than normal in cerebral cortex and 5-40 times normal in cerebellar cortex. In Type III the concentration of glucosylceramide was 37-65 and 59-1750 μmol/kg in cerebral and cerebellar cortex, respectively. The highest concentrations were found in the cerebellum of patients who had survived splenectomy for several years. The ceramide composition of the accumulated glucosylceramide suggested that brain gangliosides were the major precursors of the glucosylceramide in brains of Type II but in cerebellar cortex in Type III was partly of extracerebral origin. The levels of lactosylceramide and oligohexaosylceramides were slightly raised in all brain specimens from the Gaucher cases. The ganglioside concentration was normal, whereas there was a certain increase in the proportion of GM2 and GM3 gangliosides. The brain glycosphingolipid changes in the Type I/III case were similar but slightly less than those in Type III cases of corresponding age. Glucosylsphingosine (psychosine), never detected in normal human brain, was demonstrated in brains from all the Gaucher cases. The psychosine concentration was highest in Type II cases, 3.8-8.8 and 3.9-12.3 μmol/kg in cerebral and cerebellar cortex, respectively, with the highest values found in the most fulminant cases. In Type III the psychosine concentration varied more widely, 0.8-4.6 and 1.4-6.3 μmol/kg in cerebral and cerebellar cortex, respectively. The lowest value, 0.7 μmol/kg, was found in the Type I/III case. Our method detected psychosine down to 0.01 μmol/kg, which means that the concentration of psychosine was increased at least 100- to 1000-fold in Gaucher grey matter. We suggest that the accumulation of the cell-toxic substance psychosine is the basis for the extensive neuronal cell loss in Gaucher disease, which is most striking in Type II disease.     

A Comparison of the Polypeptide Pattern Between a Brain Primary Culture and Fractions of Bulk-Isolated Glial Cells

Abstract: This paper reports on the electrophoretic protein/polypeptide pattern of a rat brain primary culture. For comparison, the polypeptide pattern of neuronal and glial enriched fractions from adult rat brain and cerebral hemispheres from newborn and adult rat have been analysed. Water-soluble and SDS-extractable polypeptide fractions appeared and/or increased in amount in the cultures until confluency. The polypeptide pattern of the cultures most resembled that of the glial cell fractions, showing some of this fraction's specificity. Removal of fetal calf serum and addition of 0.1 mM dibutyryl cyclic adenosine monophosphate (dB-cAMP) produced few changes in the electrophoretic pattern. The study thus provides evidence in favour of the astroglial nature of the brain primary culture. It also shows that the cells undergo some maturation in the culture.