Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase.

The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions.     

Respiration in Cells and Mitochondria of Male-Fertile and Male-Fertile and Male-Sterile Nicotiana spp

Three cytoplasmic male-sterile Nicotiana cultivars together with corresponding male-fertile progenitors and restored lines were investigated in order to find possible correlations between respiratory characteristics and male sterility. Oxygen consumption measurements were performed on cells from suspension cultures as well as on mitochondria isolated from green leaves. Inhibitors, which have been reported to specifically block either the cytochrome (KCN) or the alternative (propyl gallate and sali-cylhydroxamic acid [SHAM] respiratory pathways, were used in order to measure the capacity and activity of the two pathways. One of the inhibitors, SHAM, was found unsuitable to measure the activity of the alternative pathway due to the lack of specificity of SHAM for this pathway. A great difference in the capacity of the alternative pathway was detected between the two types of cell materials tested. Mitochondria isolated from green leaves showed a capacity of the alternative pathway of 5 to 20% of total mitochondrial repiration, while the capacity of cells from suspension cultures generally ranged from 50 to 80%. In addition to this, in organello synthesis of mitochondrial proteins revealed differences between mitochondria isolated from green leaves and from cell suspensions. No correlation, however, could be found between respiratory characteristics and male sterility.     

Biocorrosion and biodeterioration of antique and medieval glass

Over the past few years we have examined various antique and medieval glasses with regard to general biogenic damage, biopitting (crater erosion), bio‐crusts, and opalescent and white biogenic films. Experiments were carried out on pieces from Roman glass bottles excavated near Abu Tor, Sinai, some pieces of green and blue glass from Cologne Cathedral, some pieces from a little church in Evreux, glass samples from the fortress of the former Dukedom of Delmenhorst near Oldenburg, and some neolithic flint tools from the Negev Desert, Israel. Modern glass from a pigsty (19th century) additionally has been used for laboratory experiments on the attack of glass surfaces by fungi and bacteria. Some of the bacteria used in these experiments were isolated from the ancient pieces of glass. Biopitting with structures very similar to the biopitting of marble and limestone was found on almost all specimens. Lichens were not identified directly, but fungi and algae were observed in the pits as well as under the thin layers exfoliating from the Roman glass bottles. Initial steps of colonization and the potential for heavy‐metal accumulation by the isolated bacteria have been shown in laboratory experiments. A fractal dimension of diffusion‐limited disaggregation (DLD) is suggested as one possible explanation for the characteristic form and structure of the microbially induced and shaped biopitting patterns. A biopitting classification is suggested.     

Isolation and structure of guinea pig gastric and pancreatic somatostatin

Somatostatin-14 has been isolated from guinea pig pancreas and stomach and purified to homogeneity by gel filtration, ion-exchange chromatography and reverse phase HPLC. Amino acid composition and sequence analysis indicated that guinea pig somatostatin from both tissues has the same structure as somatostatin-14 from all other mammalian species yet studied. The study has demonstrated that somatostatin-14 from gastric tissue has the same structure as the peptide from hypothalamus and pancreas.     

Characterization and Stability of Ribosomes from Mesophilic and Thermophilic Bacteria

Ribosomes were isolated from three mesophilic and three thermophilic strains of Bacillus. The ribosomes consisted of about 55% protein and 45% ribonucleic acid. Average ratios for the absorbance at 260/235 and 260/280 mmu were 1.77 and 1.92 for the mesophiles and 1.63 and 1.84 for the thermophiles. Ultracentrifugation revealed mainly components with sedimentation coefficients of about 30, 50, 70, 100, and 120S. All the preparations were shown to contain a ribonuclease which, in the presence of ethylenediaminetetraacetic acid, led to ribosome breakdown as measured by the increase in acid-soluble nucleotides. The stability of the ribosomes from the thermophiles was consistently greater than that of the ribosomes from the mesophiles. After 5 hr at 37 C, the breakdown was about 80% for the ribosomes from the mesophiles and 55 to 70% for those from the thermophiles. At 60 C, the ribosomes from the mesophiles were broken down slightly more and at a faster rate than those from the thermophiles. At temperatures above 60 C, the breakdown was again more pronounced for the ribosomes from the mesophiles.     

Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes

A partial library of cloned human DNA was screened for sequences represented on and specific to the X chromosome. The library was constructed from Bam HI-digested human DNA from cells with X chromosome polyploidy, and was cloned in pBR322. The screening was performed by individually hybridizing ³²P-labeled cloned plasmids to Southern blots containing Bam HI-digested DNA from mouse-human hybrid cells having the human X chromosome and from derivative hybrids lacking the human X. Of 45 clones assayed, 33 contained sequences homologous to ones represented many times on the X. In situ hybridization to metaphase chromosomes demonstrated that at least four of these clones were homologous to autosomes as well. Only one of the 18 clones of this kind tested cross-hybridized with another. Two recombinant plasmids were shown to be derived from the X chromosome and to be X chromosome-specific by three criteria: they hybridized to a single band in the Southern blots of Bam HI-digested DNA from hybrid cells containing the X chromosome; they hybridized to a band of the same molecular weight as did the inserted DNA fragment; and they showed a dosage effect when hybridized to Southern blots of Bam HI-digested DNA from XY and XXX cells. One of these hybridized as a single-copy or low-order reiterated sequence in a Cot analysis using male DNA as driver. Our methods can be applied to the identification of any chromosome-specific clone. The two X-specific clones identified here should be useful in investigating the mechanism of X inactivation and in isolating a Barr body.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

The influence of ammonium and calcium lons on gluconeogenesis in isolated rat hepatocytes and their response to glucagon and epinephrine

The use of n-butylmalonate as an inhibitor of malate transport from mitochondria and of aminooxyacetate as an inhibitor of glutamate-aspartate transaminase indicated that rat liver hepatocytes employ the aspartate shuttle for gluconeogenesis from lactate which supplies reducing equivalents to the cytosolic NAD system. In contrast, malate is transported from mitochondria to cytosol for gluconeogenesis from pyruvate. This conclusion is corroborated by the finding that the addition of ammonium ions enhances gluconeogenesis from lactate but inhibits glucose formation from pyruvate. In hepatocytes, glucagon and epinephrine have relatively little effect on glucose synthesis from lactate. Ammonium ions permit both of these hormones to exert their usual stimulation of gluconeogenesis from lactate.Calcium ions (1.3 mm) enhance gluconeogenesis from lactate and from lactatepyruvate mixtures (10:1). The stimulatory effects of Ca²⁺ and NH₄⁺ are additive and, when lactate is the substrate, the rates of gluconeogenesis achieved are so high as to preclude further stimulation by glucagon.     

Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis

Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc2(6) and trimethoprim-resistant mutant mc2(26) was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Km values for H2folate and NADPH of 0.68 +/- 0.2 microM and 21 +/- 4 microM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 +/- 0.4 microM and 5.3 +/- 1.5 microM, respectively. A kcat of 4.5 sec-1 was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Ki value of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc2(26) strain of M. smegmatis.     

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.     

Genetics of whitefish and vendace in England and Wales

Proportion of loci polymorphic ( P ) and mean heterozygosity per locus ( H _exp) were P = 0.130 and H _exp= 0.096 for C. albula from the English Lake District and P = 0.217 and H _exp= 0.046 for C. lavaretus from the English Lake District and Llyn Tegid, Wales in 1991–2. Significant deviation from the Hardy-Weinberg model was observed at the superoxide dismutase ( sSOD* ) locus in C. albula from Bassenthwaite Lake. This could have been due to chance, to the Wahlund effect or to selection. Genetic distances between the two species were consistent with their taxonomy and genetic differentiation was much greater for the C. lavaretus populations than for the two geographically close C. albula stocks. Llyn Tegid C. lavaretus showed unique alleles at two loci and conservation of fish in this lake is therefore of great importance. Red Tarn C. lavaretus differed most from other Lake District populations probably due to founder effects from early stocking events and/or from selection due to the high altitude of this lake.     

Streptomycin and tetracycline resistance plasmids in Staphylococcus hyicus and other staphylococci

Plasmids associated with resistance to streptomycin, to streptomycin plus chloramphenicol or to tetracycline in Staphylococcus hyicus isolated from the skin of pigs have been compared, by restriction endonuclease digest patterns, with similar staphylococci from human sources and with published DNA base sequences. Several plasmids from Staph. hyicus have proved to have a very similar structure to those described from Staph. aureus but others appeared very dissimilar. This confirms the opinion that staphylococci from animal skin share a pool of plasmids with those from human skin but may also possess some of quite different structure.     

Molecules and Morphology,Phylogenetics and Genetics

Various explanations can be offered for the incongruence between phylogenetic hypotheses resulting from morphological and molecular data sets. Of these, the possibility that incongruence may result from the mutation of major morphogenetic genes leading to dramatic morphological divergence unaccompanied by equivalent change of the phylogenetic marker molecule(s) used is discussed in detail. As evidence for this hypothesis, several examples for such incongruence are surveyed. It seems possible that in many cases the genetic basis of the morphological characters responsible for the incongruence found may be simple, and that the genes involved may be homologous to genes known from mutant systems. It is suggested that: 1. the systematic documentation of incongruence between molecular and morphological phylogenies may help to assess the frequency of evolutionary change through the mutation of major morphogenetic genes, and that 2. the identification of major morphological characters distinguishing closely related taxa with mutant phenotypes known from mutant systems eventually may allow an experimental approach to the problem of evolutionary change resulting from major genes. Natural taxa suspected to be the result of such processes could be changed morphologically through transformation with the relevant genes.     

Differential uptake and metabolism of sitosterol and cholesterol by Achlya, Pythium, and Phytophthora species.

The relative ability of isolates of Achlya bisexualis and A. ambisexualis and isolates of Pythium and Phytophthora to take up and metabolize sitosterol and cholesterol was studied. Species of Pythium and Phytophthora took up cholesterol and sitosterol efficiently, whereas Achlya species took up booth sterols inefficiently. Species of Pythium and Phytophthora produced a polar metabolite and esters from sitosterol as they did from cholesterol. Achlya species did not produce the polar metabolite from either sterol. In these experiments Achlya species produced esters only from cholesterol; however, their failure to produce esters from sitosterol may have been due to the higher sitosterol than cholesterol concentration.     

Halo- and psychrotolerant Geomyces fungi from arctic cryopegs and marine deposits

Comparative characterization of Geomyces isolates was performed. The isolates were obtained from Arctic cryopegs and the surrounding ancient marine deposits, from nonsaline permafrost soils, and from temperate environments. Microbiological (cultural and morphological) and molecular criteria were used to confirm the identification of the isolates as Geomyces pannorum. The isolates from cryopegs and surrounding marine deposits were shown to differ from those obtained from nonsaline soils and temperate environments in their ability to grow at negative temperatures (-2 degrees C) under increased salt concentration (10%). The results are discussed in relation to the possible inheritance of the adaptive characteristics acquired in specific environments.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats.

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent. faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium-like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum, shared characteristics with Ent. columbae. The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol. Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Vitrification and rapid freezing of rabbit fetal tissues and skin samples from rabbits and pigs

Vitrification (3.58 M EG and 2.82 M DMSO in PBS with 20% FCS) and rapid-freezing (0.25 M sucrose, 2.25 M EG, and 2.25 M DMSO in PBS with 20% FCS) procedures were assayed to cryopreserve rabbit tissue samples from 12-day fetuses, and skin samples from live born pups and adult rabbits. These methods were also assayed to cryopreserve pig skin samples obtained from abattoir animals. The ability of rabbit tissue samples to attach and colonize the substratum by cell proliferation was not affected by the assayed cryopreservation procedures, regardless of specimen age. In porcines, sample attachment and cell proliferation capability of primary cultures were not affected by applied cryopreservation procedures. Almost all primary cultures from cryopreserved skin samples reached confluency (from 92 to 100%). Results reported here allow us to establish in both species, rabbit and pig, a cryobank of skin samples from adult specimens classified as outliers for longevity (in rabbits) and prolificacy (in pigs).     

Intercontinental and intracontinental biogeography patterns and methods

The study of biogeography has benefited from the exponential increase of DNA sequence data from recent molecular systematic studies, the development of analytical methods in the last decade concerning divergence time estimation and geographic area analyses, and the availability of large-scale distributiofi data of species in many groups of organisms. The underlying principle of divergence time estimation from DNA and protein data is that sequence divergence depends on the product of evolutionary rate and time. With their molecular clock hypothesis, Zuckerkandl and Pauling （1965） separated rates of molecular evolution from time by incorporating fossil evidence. Originally,     

Isolation and Characterization of Tubules and Plasma Membranes from Cytophaga columnaris

Tubular structures are released from cells of Cytophaga columnaris after lysis of the cells. To determine the nature of these tubules, they were purified and their composition was determined. Tubules were isolated after treating cell lysates with 1.0% sodium dodecyl sulfate at pH 8.1, which solubilizes all structural components except tubules. Plasma membranes from the same organism were isolated by discontinuous sucrose gradient centrifugation of lysed cells. Both tubules and membranes are composed of lipids and proteins. Lipids extracted from tubules and plasma membranes produced similar patterns when examined by thin-layer chromatography. Proteins solubilized from membranes were separated into 14 bands by polyacrylamide gel electrophoresis, whereas those solubilized from tubules separated into only 5 bands. The presence of lipids in tubules from C. columnaris supports the idea that they are derived from membranes of intact cells. In this respect they are similar to tubules produced by cells of Clostridium botulinum and different from other tubular structures ("rhapidosomes") found in cells of Saprospira grandis.     

Relation between Mycoplasmas and Leukaemia and Related Diseases

Forty-five bone marrow specimens from leukaemia patients and 40 marrows from nonleukaemia patients were cultured for mycoplasmas. No mycoplasmas were isolated. Sera from patients with leukaemia or reticuloses and from non-leukaemic subjects were examined for antibodies to the Negroni and 880 strains of Mycoplasma pulmonis and also to the K7, K10, and prototype (PG18) strains of M. fermentans. No significant differences were observed between the two groups of patients with respect to antibodies to these mycoplasmas. These findings support those of the majority of other workers in failing to show any relationship between mycoplasmas and neoplastic disease in man.