Fungal degradation of kraft lignin and lignin sulfonates prepared form synthetic 14C-lignins

Kraft lignins (KL), bleached kraft lignins (BKL), and lignin sulfonates (LS) were prepared from synthetic ¹⁴C-lignins labeled in the aromatic nuclei or in the propyl side chains. These and control lignins (CL) were incubated with the lignin-decomposing white-rot fungus, Phanerochaete chrysosporium Burds., in a defined culture medium containing cellulose as growth substrate. Decomposition was monitored by measuring the ¹⁴CO₂ evolved. Average percentages of the [ring-¹⁴C]- and [side chain-¹⁴C]-lignins, respectively, recovered as ¹⁴CO₂ at the cessation of ¹⁴CO₂ evolution were: KL, 41 and 31; BKL, 42 and 26; LS, 28 and 21; and CL, 26 and 24. Gel permeation chromatography of radiolabeled materials extracted from spent cultures showed that substantial degradation to nonvolatile products had occurred. The polymeric components in the extracts were further degraded in fresh cultures. These results indicate that industrial lignins are significantly bioalterable, and that under favorable conditions industrial lignins are substantially biodegradable.     

ULTRASTRUCTURE AND DIFFERENTIATION IN CLADOPHORA GLOMERATA. I. CELL DIVISION

Cladophora glomerata is a coenocytic, fresh-water green alga in which mitosis and cytokinesis occur independently. The mitotic spindle is centric, closed, and develops from two half-spindles which form from amorphous but well-defined MTOCs at each pole. The nucleolus is only partially dispersed during mitosis and structured kinetochores are evident on the chromosomes. Anaphase separation of chromosomes is asynchronous and results from spindle elongation plus shortening of the chromosome-to-pole distance. Neither a phycoplast nor a phragmoplast is present during cytokinesis. Microtubules are associated with the septum but whether they participate actively in its ingrowth is not clear. Two types of vesicles are associated with the growing septum. The membrane at its leading edge is thicker and more densely stained than elsewhere. The ultrastructure of nuclear and cell division in C. glomerata is sufficiently different from the data on other green algae that conclusions about phylogeny must await further study, especially of other coenocytic green algae.     

FROZEN THIN SECTIONS OF FRESH TISSUE FOR ELECTRON MICROSCOPY, WITH A DESCRIPTION OF PANCREAS AND LIVER

A simple method has been developed that allows frozen thin sections of fresh-frozen tissue to be cut on a virtually unmodified ultramicrotome kept at room temperature. A bowl-shaped Dewar flask with a knifeholder in its depths replaces the stage of the microtome; a bar extends down into the bowl from the microtome's cutting arm and bears the frozen tissue near its lower end. When the microtome is operated, the tissue passes a glass or diamond knife in the depths of the bowl as in normal cutting. The cutting temperature is maintained by flushing the bowl with cold nitrogen gas, and can be set anywhere from about -160°C up to about -30°C. The microtome is set for a cutting thickness of 540–1000 A. Sections are picked up from the dry knife edge, and are placed on membrane-coated grids, flattened with the polished end of a copper rod, and either dried in nitrogen gas or freeze-dried. Throughout the entire process the tissue is kept cold and does not come in contact with any solvent. The morphology seen in frozen thin sections of rat pancreas and liver generally resembles that in conventional preparations, although freezing damage and low contrast limit the detail that can be discerned. Among unusual findings is a frequent abundance of mitochondrial granules in material prepared by this method.     

PKX, the causative agent of proliferative kidney disease (PKD) in Pacific salmonid fishes and its affinities with the Myxozoa

Proliferative kidney disease (PKD), caused by an unclassified protozoan (PKX), is reported from Pacific salmon, Oncorhynchus tshawytscha (Walbaum) and O. kisutch (Walbaum), and steelhead trout, Salmo gairdneri Richardson, held at the Mad River Hatchery in California, USA. The cumulative mortality attributed to the disease was 95, 13, and 18% respectively. The mortalities were greatest at mean water temperatures of 12-14 degrees C during July 1983. The ultrastructure of the PKX organism and its associated pathology during clinical disease in all three species were consistent with those of the parasite in rainbow trout (Salmo gairdneri) as described in European outbreaks. Significant mortalities did not occur after August, at which time the parasite could no longer be detected in the salmon species. The steelhead continued to exhibit parasites in the kidney interstitium and epithelium and lumens of the tubules. Myxosporidan trophozoites and developing spores were also observed in the lumens of the kidney tubules of these fish. Although a mixed infection with another parasite may have occurred, evidence suggests that the myxosporidans are later stages of PKX. They were only observed in fish exposed to water with the infective stage and were particularly prominent in recovering fish. The PKX organism is similar to UBO, an unclassified protozoan of carp suspected to be an early stage of Sphaerospora renicola Dykovà & Lom. Both parasites infect the blood and kidney, divide by endogeny, and are released by disintegration of the primary mother cell. The intraluminal myxosporean forms show similarities to Sphaerospora spp. in that they are monosporous and sporoblasts are formed within pseudoplasmodia.(ABSTRACT TRUNCATED AT 250 WORDS)     

M?ssbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters

Active beef heart aconitase contains a [4Fe-4S] cluster. One iron of the cluster, Fea, is labile and can be removed easily by oxidation in air to yield the [3Fe-4S]1+ cluster of inactive aconitase. We have previously shown that substrate binds to Fea. We have continued our M?ssbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4S]2+, is mixed with substrate, two species (substrates or intermediates bound to Fea) labeled S1 and S2 are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species S2, but not S1, is observed, suggesting that S2 represents a carbanion transition state complex. We have prepared M?ssbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 degree C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 degrees C. Details of these experiments are given, although in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4S]1+; EPR at g = 1.94) in the presence of substrate with material selectively enriched with 57Fe in either Fea or the other three cluster sites. The spectra were analyzed with a spin Hamiltonian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the reduced [3Fe-4S]0 cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase containing a 3Fe cluster is not active in either oxidation state.     

Chromosome-mediated transfer of the murine Na,K-ATPase alpha subunit confers ouabain resistance.

We transferred murine NIH 3T3 metaphase chromosomes into monkey CV-1 cells to investigate the different ouabain sensitivities of rodent and primate cells. In 16 ouabain-resistant transferents, the mouse Na,K-ATPase alpha 1 subunit gene was detected, suggesting that structural differences between the rodent and primate alpha 1 subunits determine the different ouabain sensitivities.     

Effect of O-sulphate groups in lactose and N-acetylneuraminyl-lactose on their enzymic hydrolysis.

1. Lactose 6''-O-sulphate, N-acetylneuraminyl-(alpha 2 leads to 3)-D-lactose 6''-O-sulphate, N-acetylneuraminyl ?-O-sulphate-(alpha 2 leads to 3)-D-lactose 6''0-O-sulphate, N-acetylneuraminyl ?-O-sulphate-(alpha 2 leads to 6)-D-lactose and N-acetylneuraminyl-(alpha 2 leads to 3)- and -(alpha 2 leads to 6))-lactose 6''-O-sulphate were prepared by chemical sulphation of lactose, N-acetylneuraminyl-lactose and tis isomers by using pyridine-SO3 reagent. 2. Significant kinetic differences were observed in the enzymic hydrolysis of the sulphated derivatives compared with unsubstituted substrates. 3. In the case of reactions catalysed by rat liver lysosomal and Clostridium perfringens neuraminidases (EC 3.2.1.18), the presence of an O-sulphate group in the N-acetylneuraminyl moiety affected the reaction by decreasing the Km and the Vmax, its presence in the galactosyl moiety affected the reaction by decreasing the Km and increasing the Vmax. and its presence in both N-acetylneuraminyl and galactosyl moieties decreased the Km and the Vmax. of the reaction. 4. Mixed-substrate reaction kinetic data indicated competition between the sulphated and unsubstituted substrates for the same active sites on the neuraminidase molecule. 5. Lactose 6''-O-sulphate neither behaved as a substrate nor acted as an inhibitor with respect to unsubstituted lactose and p-nitrophenyl beta-D-galactopyranoside when tested with lactase of suckling rat intestine and Escherichia coli beta-D-galactosidase (EC 3.2.1.23). 6. Preliminary investigation also indicated that, whereas glucose 6-O-sulphate and glucose 3-O-sulphate were were neither substrate nor inhibitor of glucose oxidase (EC 1.1.3.4), galactose 6-O-sulphate was oxidized half as fast as unsubstituted galactose by galactose dehydrogenase (EC 1.1.1.48).     

Incorporation of N-fluoroacetyl-d-glucosamine into hyaluronate by rabbit tracheal explants in organ culture

1. Incubation of rabbit tracheal explants with N-[(3)H]acetyl-d-glucosamine and N-acetyl-d-[1-(14)C]glucosamine led to labelling of a number of soluble macromolecular products separable from the medium, after papain digestion, by ion-exchange chromatography. 2. With N-acetyl-d-[1-(14)C]glucosamine in the incubation medium, a neutral glycoprotein, two acidic glycoprotein fractions, hyaluronic acid and a glycosaminoglycan fraction were obtained and all were radioactively labelled. Similar labelling occurred with N-fluoroacetyl-d-[1-(14)C]glucosamine or N-fluoro[(3)H]acetylglucosamine as precursor. 3. Maximal labelling was obtained at 96h after incubation of cultures. N-Fluoroacetyl-glucosamine under these conditions was incorporated into hyaluronate less efficiently than N-acetylglucosamine. 4. With N-fluoroacetyl-d-[1-(14)C]glucosamine as precursor, a hyaluronate component was separated that on enzymic degradation by glycosidases (hyaluronidase, beta-glucuronidase and N-acetyl-beta-hexosaminidase) yielded a (14)C-labelled oligosaccharide fraction together with N-acetyl-d-[1-(14)C]glucosamine and N-fluoroacetyl-d-[1-(14)C]glucosamine, consistent with some exchange of N-acetyl groups having occurred. 5. The results on enzymic degradation of labelled macromolecules by glycosidases suggest that the presence of incorporated N-fluoroacetyl side chains may render the hyaluronate analogue more resistant to hyaluronidase.