Solubilization of lignin by the ruminal anaerobic fungus Neocallimastix patriciarum.

The ability of the ruminal anaerobic phycomycete Neocallimastix patriciarum to digest model lignin compounds and lignified structures in plant material was studied in batch culture. The fungus did not degrade or transform model lignin compounds that were representative of the predominant intermonomer linkages in lignin, nor did it solubilize acid detergent lignin that had been isolated from spear grass. In a stem fraction of sorghum, 33.6% of lignin was apparently solubilized by the fungus. Solubilization of ester- and either-linked phenolics accounted for 9.2% of the lignin released. The amounts of free phenolic acids detected in culture fluid were equivalent to the apparent loss of ester-linked phenolics from the sorghum substrate. However, the fungus was unable to cleave the ether bond in hydroxycinnamic acid bridges that cross-link lignin and polysaccharide. It is suggested that the majority of the solubilized lignin fraction was a lignin carbohydrate complex containing ether-linked hydroxycinnamic acids. The lignin carbohydrate complex was probably solubilized through dissolution of xylan in the lignin-xylan matrix rather than by lignin depolymerization.     

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.     

Use of 16S-rRNA Based Techniques to Investigate the Ecological Succession of Microbial Populations in the Immature Lamb Rumen: Tracking of a Specific Strain of Inoculated Ruminococcus and Interactions with Other Microbial Populations in Vivo

Abstract The establishment of microorganisms in the rumen is a critical step if rumen manipulation is to be accomplished by use of microbial inoculants. Microbial populations in the maturing rumen undergo successional changes and, while in a state of flux, provide a possible opportunity for the introduction of specific strains of bacteria. While the rumen of the young lamb was maturing, we measured changes in several microbial populations with 16S-rRNA specific oligonucleotides: Rumincoccus, Fibrobacter, eukaryotes, Gram-positive bacteria, the Bacteroides–Porphromonas–Prevotella group, and anaerobic rumen fungi. In this study we repeatedly dosed 15 lambs with approximately 3.4 × 10⁸ to 0.8 × 10⁹ Ruminococcus cells dose^-1, twice a week, for 7 wk from 23 d to 63 d of age. Of the five Ruminococcus strains dosed (R. albus SY3 and AR67, and R. flavefaciens Y1, LP9155, and AR72) the most specific primers (based on 16S rDNA) were obtained for strain SY3. There was an increase in the eukaryotic population during dosing, and it was hypothesized that protozoal predation contributed to the disappearance of strain SY3. At the end of dosing PCR amplification showed that SY3 were approximately 10⁹ cells ml^-1, but decreased to below the detection limit of the PCR system (8.6 × 10⁴ ml^-1) within 28 d postdosing. These experiments showed that fibrolytic populations increased significantly (P < 0.1) above the controls during the dosing period and were elevated for several days postdosing. This suggests that dosing of highly fibrolytic bacteria makes more of the fiber available to other organisms able to degrade fiber, and in so doing increases the overall fibrolytic activity of the rumen. Examination of the succession of gram-positive bacteria and the Bacteroides–Porphromonas–Prevotella group showed a decline in relative abundance as the lambs matured. Received: 13 April 1999; Accepted: 14 July 1999; Online Publication: 15 February 2000     

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).     

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

AIMS: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function. METHODS AND RESULTS: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides-Porphyromonas-Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly. CONCLUSION: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.     

Butyrivibrio spp. and other xylanolytic microorganisms from the rumen have cinnamoyl esterase activity

High concentrations of hydroxycinnamic acids in the hemicellulosic fraction of dry season tropical grasses may influence the rate of microbial degradation of arabinoxylans by ruminant animals. The ability of 22 strains of Butyrivibrio fibrisolvens, other ruminal bacteria (Ruminococcus albus SY3, Ruminococcus flavefaciens RF1,Prevotella ruminicola AR20) and the ruminal phycomycete Neocallimastix patriciarum CX to digest the tropical grass Heteropogon contortus(spear grass) and hydrolyse esterified ferulic and p-coumaric acid was examined. Significant digestion (8-36%) of spear grass occurred with the B. fibrisolvens strains H17c, A38, LP92-1-1, 49,R. albus SY3 and N. patriciarum. Hydrolysis of ester-linked ferulic and p-coumaric acid occurred with all organisms except B. fibrisolvens strains GS113, OB156 and LP1028 and P. ruminicola AR20. The ratio of ferulic to p-coumaric acid hydrolysed by different strains of Butyrivibrio spp. varied markedly from 0.96 for AR 51 to 0.16 for A38. Butyrivibrios which were fibrolytic (H17c and A38) had higher extracellular cinnamoyl esterase activity than bacteria that did not digest spear grass fibre (LP 91-4-1 and AR 20) which had low activities or only produced cell associated enzyme. Cell associated and extracellular esterase activity were induced when Butyrivibrio spp. strains H17c, A38 and E14 and the Ruminococcus spp. were grown on birchwood xylan but induction did not occur to the same extent with N. patriciarum. This is the first reported observation of cinnamoyl esterase activity in the genus Ruminococcus. The fungus N. patriciarum had significantly higher digestibility of spear grass and solubilisation of phenolic acids than the bacteria. The study shows that high levels of extracellular cinnamoyl esterases are characteristic of a selection of fibre-degrading ruminal bacteria and fungi which probably indicates that these enzymes are common amongst xylanolytic ruminal microorganisms.     

New features of the cell wall of the radio-resistant bacterium Deinococcus radiodurans

We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.