A GHF7 CELLULASE FROM THE PROTIST SYMBIONT COMMUNITY OF Reticulitermes flavipes ENABLES MORE EFFICIENT LIGNOCELLULOSE PROCESSING BY HOST ENZYMES

Termites and their gut microbial symbionts efficiently degrade lignocellulose into fermentable monosaccharides. This study examined three glycosyl hydrolase family 7 (GHF7) cellulases from protist symbionts of the termite Reticulitermes flavipes. We tested the hypotheses that three GHF7 cellulases (GHF7‐3, GHF7‐5, and GHF7‐6) can function synergistically with three host digestive enzymes and a fungal cellulase preparation. Full‐length cDNA sequences of the three GHF7s were assembled and their protist origins confirmed through a combination of quantitative PCR and cellobiohydrolase (CBH) activity assays. Recombinant versions of the three GHF7s were generated using a baculovirus‐insect expression system and their activity toward several model substrates compared with and without metallic cofactors. GHF7‐3 was the most active of the three cellulases; it exhibited a combination of CBH, endoglucanase (EGase), and β‐glucosidase activities that were optimal around pH 7 and 30°C, and enhanced by calcium chloride and zinc sulfate. Lignocellulose saccharification assays were then done using various combinations of the three GHF7s along with a host EGase (Cell‐1), beta‐glucosidase (β‐glu), and laccase (LacA). GHF7‐3 was the only GHF7 to enhance glucose release by Cell‐1 and β‐glu. Finally, GHF7‐3, Cell‐1, and β‐glu were individually tested with a commercial fungal cellulase preparation in lignocellulose saccharification assays, but only β‐glu appreciably enhanced glucose release. Our hypothesis that protist GHF7 cellulases are capable of synergistic interactions with host termite digestive enzymes is supported only in the case of GHF7‐3. These findings suggest that not all protist cellulases will enhance saccharification by cocktails of other termite or fungal lignocellulases.     

The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1

The Puf family of RNA-binding proteins directs cell fates by regulating gene expression at the level of translation and RNA stability. Here, we report that the Caenorhabditis elegans pumilio homolog, puf-9, controls the differentiation of epidermal stem cells at the larval-to-adult transition. Genetic analysis reveals that loss-of-function mutations in puf-9 enhance the lethality and heterochronic phenotypes caused by mutations in the let-7 microRNA (miRNA), while suppressing the heterochronic phenotypes of lin-41, a let-7 target and homolog of Drosophila Brat. puf-9 interacts with another known temporal regulator hbl-1, the Caenorhabditis elegans ortholog of hunchback. We present evidence demonstrating that puf-9 is required for the 3'UTR-mediated regulation of hbl-1, in both the hypodermis and the ventral nerve cord. Finally, we show that this regulation is dependent on a region of the hbl-1 3'UTR that contains putative Puf family binding sites as well as binding sites for the let-7 miRNA family, suggesting that puf-9 and let-7 may mediate hypodermal seam cell differentiation by regulating common targets.     

Adaptive amplification and point mutation are independent mechanisms: evidence for various stress-inducible mutation mechanisms

“Adaptive mutation” denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase lac⁻ Escherichia coli cells on lactose medium induces Lac⁺ revertants at higher frequencies than predicted by usual mutation models. These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies (500–10,000 cells), we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac⁺ cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v) lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.     

A novel regulatory element in the dnmt1 gene that responds to co-activation by Rb and c-Jun

Rb, c-Jun and dnmt1 play critical roles in the process of cellular differentiation. We demonstrate that a regulatory region of murine dnmt1 contains an element which is responsible for transactivation by Rb and c-Jun in P19 embryocarcinoma cells which is not observed in Y1 adrenocarcinoma cells. During differentiation of P19 cells, the induction of Rb and c-Jun coincides with an increase of dnmt1 mRNA. Using linker scanning mutagenesis we identify the element that is responsible for this activation to be a non-canonical AP-1 site. Our data is an example of how a proto-oncogene activates its downstream effectors by recruiting a tumor suppressor. This interaction of Rb and a proto-oncogene might play an important role in differentiation. The responsiveness of dnmt1 to this type of signal is consistent with an important role for regulated expression of dnmt1 during cellular differentiation.     

Abdominal B-type Hox gene expression in Xenopus laevis

Previously, we reported a zebrafish iroquois gene, ziro3, and its expression during early embryogenesis (Mech. Dev. 87 (1999) 165). In the present study, we have isolated two novel zebrafish iroquois genes, ziro1 and ziro5, homologs of mouse Irx1 and mouse Irx5, respectively. The expression of both genes is initiated in dorsal neuroectoderm and mesoderm during gastrulation. Later, their expression appears in the central nervous system (CNS), excluding the telencephalon and most of the diencephalon. ziro1 expression is complementary to that of ziro3 in the notochord and later in the gut. In contrast, ziro5 expression mostly overlaps with that of ziro3. Interestingly, all three iroquois zebrafish genes are expressed in the notochord while only Irx3 is active in the mouse notochord. Their expression in later stages of embryogenesis was also compared.     

Expression of gp130 and leukaemia inhibitory factor receptor subunits in adult rat sensory neurones: regulation by nerve injury

Members of the interleukin-6 (IL-6) family of cytokines have been implicated as major mediators of the response of the adult nervous system to injury. The basis for an interaction of IL-6 cytokines with adult sensory neurones has been established by analysing the levels and distribution of the two signal-transducing receptor subunits, glycoprotein 130 (gp130) and leukaemia inhibitory factor receptor (LIFR), in the dorsal root ganglion (DRG) of male adult rats before and following nerve injury. All sensory neurones express gp130-immunoreactivity (IR) in the cytoplasm and on the plasma membrane. Levels of gp130 and its intracellular distribution remained unchanged up to 14 days following sciatic nerve axotomy. LIFR-IR was largely absent from the cytoplasm and plasma membrane of sensory neurones, but confined almost exclusively to the nuclear compartment. However, following axotomy, punctate cytoplasmic LIFR-IR was detected which persisted up to 28 days following axotomy. The expression of cytoplasmic LIFR 2 days post-axotomy was proportionally greater in a subset of small diameter sensory neurones which expressed either the sensory neuropeptide CGRP or the cell surface marker isolectin B4. The coexpression of gp130 and LIFR in the same intracellular compartment following axotomy conveys potential responsiveness of injured sensory neurones to members of the IL-6 family of cytokines.     

How cells change their phenotype

Recent attention has focused on the remarkable ability of adult stem cells to produce differentiated cells from embryologically unrelated tissues. This phenomenon is an example of metaplasia and shows that embryological commitments can be reversed or erased under certain circumstances. In some cases, even fully differentiated cells can change their phenotype (transdifferentiation). This review examines recently discovered cases of metaplasia, and speculates on the potential molecular and cellular mechanisms that underlie the switches, and their significance to developmental biology and medicine.     

Similarities and differences in the propagation of slow waves and peristaltic waves

The relationship between slow waves and peristaltic reflexes has not been well analyzed. In this study, we have recorded the electrical activity of slow waves together with that generated by spontaneous peristaltic contractions at 240 extracellular sites simultaneously. Recordings were made from five isolated tubular and six sheet segments of feline duodenum superfused in vitro. In all preparations, slow waves propagated as broad wave fronts along the longitudinal axis of the preparation in either the aborad or the orad direction. Electrical potentials recorded during peristalsis (peristaltic waves) also propagated as broad wave fronts in either directions. Peristaltic waves often spontaneously stopped conducting (46%), in contrast to slow waves that never did. Peristaltic waves propagated at a lower velocity than the slow waves (0.98 +/- 0.25 and 1.29 +/- 0.28 cm/s, respectively; P < 0.001; n = 24) and in a direction independent of the preceding slow wave direction (64% in the same direction, 46% in the opposite direction). In conclusion, slow waves and peristaltic waves in the isolated feline duodenum seem to constitute two separate electrical events that may drive two different mechanisms of contraction in the small intestine.     

Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression

High levels of peripheral-type benzodiazepine receptor (PBR), the alternative-binding site for diazepam, are part of the aggressive human breast cancer cell phenotype in vitro. We examined PBR levels and distribution in normal tissue and tumors from multiple cancer types by immunohistochemistry. Among normal breast tissues, fibroadenomas, primary and metastatic adenocarcinomas, there is a progressive increase in PBR levels parallel to the invasive and metastatic ability of the tumor (p < 0.0001). In colorectal and prostate carcinomas, PBR levels were also higher in tumor than in the corresponding non-tumoral tissues and benign lesions (p < 0.0001). In contrast, PBR was highly concentrated in normal adrenal cortical cells and hepatocytes, whereas in adrenocortical tumors and hepatomas PBR levels were decreased. Moreover, malignant skin tumors showed decreased PBR expression compared with normal skin. These results indicate that elevated PBR expression is not a common feature of aggressive tumors, but rather may be limited to certain cancers, such as those of breast, colon-rectum and prostate tissues, where elevated PBR expression is associated with tumor progression. Thus, we propose that PBR overexpression could serve as a novel prognostic indicator of an aggressive phenotype in breast, colorectal and prostate cancers.