Evaluation of environmental and comfort improvements on affective welfare in heifer calves on smallholder dairy farms.

A controlled trial on zero-grazed smallholder dairy farms was conducted to determine the effect of environmental and comfort improvements on sucking and lying behaviours in heifer calves on Kenyan smallholder dairy farms. The study involved 187 heifer calves from 150 farms in two Kenyan counties, 75 farms per county. Farms in one county received animal welfare training and improvements in the calf pen that included: 1) placement of rubber mats on the lying area; 2) fixing gaps/holes in the flooring and roofing; and 3) attaching a rubber nipple on the wall of the calf pen. During the 16-month data collection period, bimonthly farm visits were used to collect data on lying time (using accelerometers) and other animal- and farm-level factors. Multilevel mixed-effects linear regression was used to model daily lying times and frequency of lying bouts, with the animal as a random effect. Over the visits, daily lying times and lying bout durations averaged 12.6-86.7 min/bout, respectively, while the median for the frequency of lying bouts was between 30-46/day. Provision of rubber nipples for non-nutritive sucking lowered proportions of cross-sucking, self-sucking and object-sucking behaviours slightly but not significantly. In a final daily lying time model, superficial lymph node enlargement, body condition score and use of wood shaving/ sawdust/ crop waste as beddings had positive associations. In contrast, group housing and rubber mat use had negative associations with daily lying time. In an interaction term, lying time was significantly higher for calves on clean versus dirty floors if the age was <190 days but this difference diminished significantly in older animals. In a second interaction term, lying time was lower for calves with leaking versus non-leaking roofs, regardless of the pen floor level, but lying time was higher on elevated than non-elevated floors if the roof was intact. In the final model of the frequency of lying bouts, the use of a rubber mat, the years of experience in dairy farming, and calf body weight had negative associations. In contrast, body condition score had a positive association. In an interaction, the frequency of daily lying bouts was lower on clean floors than dirty floors, irrespective of tethering status, but when the floor was dirty, the lying bouts were higher for animals not tethered than the ones sometimes tethered. We conclude that the comfort improvements enhanced the welfare and lying experience of heifer calves on smallholder dairy farms.

Management factors associated with time-to-onset and fecal egg/oocyst counts of gastrointestinal parasites in heifer calves on Kenyan smallholder farms.

A prospective cohort study on zero-grazed smallholder dairy farms was conducted to determine factors associated with onset and counts of gastrointestinal parasitism in heifer calves. The researchers recruited 187 newborn heifer calves from 150 farms in Kenya. Over 16 months, farm visits every two months were used to collect rectal fecal samples and animal- and farm-level measures. Fecal samples underwent centrifugal fecal flotation with Sheather's sugar to determine counts of strongyle-type eggs and coccidia oocysts. Cox proportional hazard (Cox PH) analysis and mixed-effects negative binomial (MeNB) regression determined factors associated with time-to-onset and counts of strongyle-type eggs and coccidia oocysts, respectively (P < 0.05). The incidence rate of strongyles was 0.0011 cases/animal-day while coccidia was 0.0073 cases /animal-day. Incidence risks of strongyles and coccidia over the study period were 28.3 % (53/187) and 87.7 % (164/187), respectively. For infected calves, median time-to-onset for strongyles and coccidia was 78 (interquartile-range: IQR 38-117) and 43 (IQR 29-92) days, respectively. In the final Cox PH model for strongyles, breed (Ayrshires and Jerseys) and weaned calves had a greater hazard of infection than Friesians and preweaned calves, respectively. Calf tethering outside the pen was associated with a higher hazard of strongyle infection. In the final Cox PH model for coccidia, calves with watery and/or hemorrhagic diarrhea had a higher hazard compared with those with hard or soft feces. Weaning status and birth weight (kg) were time-varying covariates, leading to increased hazard over time. In the final MeNB model for strongyles, weaned animals had higher counts than those still on milk. In an interaction variable, the predicted strongyle-type egg counts increased with longer duration of farm operation when herd size was less than five cattle, but decreased when herd size was more than five. In the final MeNB model for coccidia, calves sometimes tethered outside their pens had higher counts than those continuously enclosed in the pen. Calf pen floors with either scant manure or moderate slurry had higher predicted counts than those on a clean pen floor. Calves with watery or hemorrhagic diarrhea and fed fresh Napier grass (Pennisetum purpureum) had higher counts compared with soft or hard feces and those not given fresh Napier, respectively. In an interaction variable, calves experiencing diarrhea and raised on elevated slatted floors had lower oocyst counts compared with those having diarrhea but not on elevated floors. The identified management practices associated with onset and counts of gastrointestinal parasitism should be considered in control efforts.

Histopathological, ultrastructural and molecular phylogenetic analysis of a novel microsporidium in a loggerhead sea turtle Caretta caretta.

Microsporidial spores were identified in the musculature of a loggerhead sea turtle Caretta caretta found dead on the shore in New Brunswick, Canada. Gastroenteritis was diagnosed on gross postmortem examination, with no gross abnormalities detected in the skeletal muscle. Histologically, the microsporidial spores were associated with inflammation and muscular necrosis and measured 1.1-1.7 × 2.2-3.4 µm. Spores were typically identified within sporophorous vesicles and, less often, in sporophorocysts and were weakly Gram positive, had punctate PAS staining, and were occasionally strongly acid-fast. Ultrastructural characteristics included 7-10 polar filament coils and other standard features of microsporidial spores. PCR for the microsporidial small subunit rRNA gene sequence was performed on DNA extracted from the muscle and small intestine, and the resulting amplicon was sequenced and queried against published microsporidial genomes. DNA sequences shared 98.2-99.8% sequence identity to Clade III of the Marinosporidia. This is the first report of a microsporidial infection contributing to the mortality of a sea turtle.

Complete resolution and remodeling of chronic recurrent multifocal osteomyelitis on MRI and radiographs.

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare condition thought to be under-diagnosed, with a true prevalence of more than the 1 in 10,000 estimated. It is a condition that is classically described as polyostotic with a relapsing and remitting course, preferentially affecting the metaphyses of tubular bones in the pediatric population. Lesions have characteristic appearances of cortical hyperostosis and mixed lytic/sclerotic medullary appearances radiographically, with active osteitis and periostitis best seen with fluid-sensitive sequences on magnetic resonance imaging (MRI). There are reports of lesions resolving on follow-up radiographs and MRI scans, but no supporting images. In particular, although the marrow appearances and degree of osteitis have been shown to improve on MRI, complete resolution and remodeling back to normal has never been demonstrated. We present a case of a lesion that has completely healed and remodeled back to normal appearances on both radiographs and MRI, and consider this the standard for the often loosely used terms "normalization" and "resolution". We discuss the implications of this for our understanding of the natural history of CRMO, and how this adds weight to the condition being significantly under-diagnosed. It provides a "gold standard" to be aimed for when assessing treatments for CRMO, and the optimal outcomes that are possible. It also provides further insight into the potential of pediatric bone to recover and remodel when affected by inflammatory conditions.

Ultrastructure of trichocysts in Hematodinium spp. infecting Atlantic snow crab, Chionoecetes opilio.

Trichocyst morphology and development were explored using transmission electron microscopy in Hematodinium spp. isolated directly from Atlantic snow crab (Chionoecetes opilio) hemolymph and from in vitro cultures. Appearance of trichocysts defines the initiation of a morphological transition in the parasites life cycle from vegetative stage to the transmission stage. Trichocysts within sporonts were found in distinct clusters near the nucleus in close apposition to the Golgi. As cells transitioned to more mature dinospores however, trichocysts were found randomly distributed throughout the cytoplasm. Clusters contained both primordial and maturing trichocysts at various stages indicating an asynchronous development. The random distribution of mature trichocysts suggests deployment to the cell membrane for future extrusion. Mature trichocysts of Hematodinium spp. appeared structurally similar to trichocysts from photosynthetic dinoflagellates. Hematodinium spp. trichocysts differed by the presence of peripheral tubules associated with novel cuboidal appendages in the apical region rather than a network of central electron dense fibres as found in photosynthetic dinoflagellates. Additionally, the trichocyst membrane of Hematodinium spp. was in close apposition to the square crystalline core. Trichocyst expulsion was not observed during our study which along with features of development and maturation within Hematodinium life stages should provide insight into proposed roles in host attachment or defense that could further our understanding of the mechanisms of pathogenesis and transmission of the parasite.

Limited prevalence of gaffkaemia (Aerococcus viridans var. homari) isolated from wild-caught European lobsters Homarus gammarus in England and Wales.

Gaffkaemia, caused by Aerococcus viridans var. homari, causes fatal infections in Homarus spp. (clawed lobsters). Despite its high economic significance to the lobster fisheries in the USA and northern Europe, data on its prevalence in captured and wild populations, particularly in Europe, is scarce. Following an outbreak of gaffkaemia in a European lobster holding facility in South Wales (UK), a base-line survey was conducted for gaffkaemia in wild populations of European lobster Homarus gammarus around the coast of England and Wales. In addition, isolates recovered from the original outbreak and the survey were typed using pulsed-field gel electrophoresis (PFGE) and compared with previously characterised isolates from the USA, UK and Canada. Locally caught H. gammarus were sampled at 30 sites from around the coast of England and Wales between March 2006 and October 2008. Results confirmed that the prevalence of gaffkaemia in populations of H. gammarus was low, with only 9 positive isolates recovered from 952 samples examined. PFGE analysis showed that the isolates from the outbreak investigation shared the same pulsotype as A. viridans var. homari isolates from the USA, Norway and Canada, as well as an isolate (NCIMB 1119) reportedly recovered from an outbreak of European lobsters in England in the 1960s. This confirms earlier studies that suggest virulent strains of A. viridans var. homari show very limited geographical or temporal genetic variation and were introduced into the UK with American lobsters H. americanus.

Giardia and Cryptosporidium on dairy farms and the role these farms may play in contaminating water sources in Prince Edward Island, Canada.

BACKGROUND: Cattle represent a reservoir for Giardia and Cryptosporidium and may contaminate water sources. OBJECTIVES: To determine the distribution of Cryptosporidium and Giardia on dairy farms and in water bodies near the farms. FARMS AND WATER SOURCES: Twenty dairy farms and 20 wells and 13 surface water samples associated with dairy farms. METHODS: Proportions of samples positive for Cryptosporidium or Giardia were determined by a direct immunofluorescence assay. Fecal and water samples were taken at different times. RESULTS: Thirty-two (95% CI: 29-35%) and 14% (95% CI: 12-17%) of fecal samples, and 100 (95% CI: 96-100) and 55% (95% CI: 32-77%) of herds, were positive for Giardia and Cryptosporidium, respectively. Giardia duodenalis assemblage E was detected in high proportions (90%) of fecal samples. Cryptosporidium bovis predominated (51%) in all cattle. C. andersoni predominated in adult cattle (53%), whereas the predominant species in animals < 2 months and 2-6 months was C. bovis, respectively. Only calves < 2 months of age were positive for C. parvum. In 46% (95% CI: 19-75%) and 85% (95% CI: 55-98%) of surface water, concentrations of Giardia cysts and Cryptosporidium oocysts were higher in downstream, than in upstream, locations of farms, whereas only 1 groundwater sample was positive for Cryptosporidium. CONCLUSIONS: This sample of dairy cattle was predominantly infected with nonzoonotic species and genotypes of Cryptosporidium, Giardia, or both. More studies are needed to determine if the presence of Giardia or Cryptosporidium in surface water was associated with shedding in animals from nearby farms.

Molecular epidemiology of Cryptosporidium and Giardia in humans on Prince Edward Island, Canada: evidence of zoonotic transmission from cattle.

To determine the zoonotic potential of Cryptosporidium and Giardia in Prince Edward Island (PEI), Canada, 658 human faecal specimens were screened that were submitted to the Queen Elizabeth Hospital diagnostic laboratory. Overall, 143 (22%) samples were Cryptosporidium positive, while three (0.5%) were positive for Giardia. Successful genotyping of 25 Cryptosporidium isolates by sequence analysis of the HSP70 gene revealed that 28 and 72% were C. hominis and C. parvum, respectively. Cryptosporidium isolates from humans and previously genotyped C. parvum from beef cattle were subtyped by sequence analysis of the GP60 gene. Subtyping identified three subtypes belonging to the family IIa. All three subtypes IIaA16G2RI (55%), IIaA16G3RI (22%) and IIaA15G2RI (22%) were found in the animal isolates, while two of the subtypes found in the animals, IIaA16G2RI (80%) and IIaA15G2RI (20%), were also identified in the human isolates. Cryptosporidium infection in humans peaked in April-June. Molecular epidemiological analysis of the human data showed a C. parvum peak in the spring and a relatively smaller peak for C. hominis in July-September. The majority (57%) of human Cryptosporidium isolates were found in children between 5 and 10 years of age. All three Giardia isolates were identified as G. duodenalis assemblage A. The overall Cryptosporidium prevalence in our human samples was high relative to other studies, but because the samples were submitted to a hospital diagnostic laboratory, the results may not be representative of the general population. Further, the presence of the same zoonotic C. parvum subtypes in cattle and human isolates implies that transmission is largely zoonotic and cattle may be a source of sporadic human infections on PEI. The presence of Giardia in people on PEI is rare, and the assemblage A found in humans might originate from humans, livestock or other domestic or wild animals.

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences.

Aerococcus viridans expression of Cpn60 is associated with virulence during infection of the American lobster, Homarus americanus Milne Edwards.

The Gram-positive bacterium Aerococcus viridans var. homari is a well-documented causative agent of the lethal systemic disease gaffkemia in both the American lobster, Homarus americanus, and the European lobster, Homarus gammarus. Previous phenotypic characterization has been unsuccessful at differentiating avirulent from virulent strains without performing lethal animal infection trials. Recent genetic characterization of A. viridans strains through 16S rRNA sequencing and random amplification of polymorphic DNA fingerprinting has revealed the presence of two subtypes. However, subtype 1 contains both virulent and avirulent strains which are genetically identical. The purpose of this study was to determine the proteomic mediators of virulence in A. viridans. Quantitative proteomic mapping of these two strains has revealed 29 differentially expressed protein spots, seven of which are only expressed in the virulent strain and could act as virulence factors. One protein, chaperonin 60 (Cpn60), is uniquely expressed in the virulent strain and has been shown to act as a virulence factor in many other bacteria. The proteomic mapping strategy employed in this study is the first to show phenotypic differences between virulent and avirulent strains. Cpn60 expression represents a potentially useful tool for identifying the virulent strains of A. viridans in epidemiological studies.

Reverse transcriptase activity in tissues of the soft shell clam Mya arenaria affected with haemic neoplasia.

Since all retroviruses possess reverse transcriptase (RT) enzyme, reverse transcriptase activity has been the main supportive evidence of retroviral etiology of haemic neoplasia (HN) in soft shell clams, Mya arenaria. The objective of the present study was to search for a putative retrovirus in various tissues of diseased clams following quantification of RT activity (biochemical indicator of retroviral infection). The clams were assessed by flow cytometry (FCM) for diagnosis of HN. RT activity was quantified by TaqMan-product enhanced reverse transcriptase (TM-PERT) assay in four different organs, gonad, gills, digestive gland, and mantle, at various stages of HN. The digestive gland, the organ with the highest RT activity, and haemocytes, the target cell of HN, were assessed by EM for presence of retroviruses. All organs were assessed by histology. The results of this study demonstrated that although all organs of healthy clams have some background RT activity, the activity observed in most of organs of diseased clams was significantly increased (p<0.05). An association was observed between the degree of neoplastic cell infiltration and the level of RT activity. Digestive gland showed the highest and most consistent RT activity in both healthy and diseased clams. No evidence for the existence of a retrovirus like particle was found by positive staining EM. The presence of RT activity without indications of retroviral particles in digestive gland and haemocytes suggests a probable endogenous source of RT.

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria.

Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R2 = 0.974, p < 0.001). Compared to HN-negative clams (<5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at approximately 10 to approximately 20% tetraploid cells, and the second at approximately 30 to approximately 80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at approximately 30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, > or = 30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

Anti-lipopolysaccharide factors in the American lobster Homarus americanus: molecular characterization and transcriptional response to Vibrio fluvialis challenge.

Two partial mRNA sequences predicted to encode anti-lipopolysaccharide factors (ALFs) were identified among expressed sequence tags generated from the American lobster Homarus americanus and complete cDNA sequences were obtained from library clones. Comparison of the translated amino acid sequences to those publicly available confirmed similarity to arthropod anti-lipopolysaccharide factors. Both protein sequences, designated ALFHa-1 and ALFHa-2, contained an N-terminal signal peptide and two half-cysteines participating in a disulfide bridge, features conserved in other ALFs. Predicted secondary structures were similar to that described for the ALF from the horseshoe crab Limulus polyphemus. As part of an exploratory study of immunity in H. americanus, lobsters were injected with the bacterium Vibrio fluvialis and gill, hematopoietic, and hepatopancreas tissues were sampled for analysis of gene expression of ALFHa-1 and ALFHa-2 by quantitative PCR. The relative abundance of ALFHa-2 mRNA was not significantly affected by Vibrio injection in any of the three tissues tested. In contrast, ALFHa-1 mRNA levels in gills were increased by the treatment some 17-fold. Our results support a molecularly specific regulation of antimicrobial proteins in response to bacterial infection in H. americanus.

The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs.

In this prospective study, feces of dogs with diarrhea were compared with feces of normal dogs for the presence of Clostridium difficile, C difficile toxins A and B, C perfringens, and C perfingens enterotoxin (CPE). C difficile toxins A, B, or both were present in feces of 18 of 87 (21%) dogs with diarrhea and 4 of 55 (7%) normal dogs (P = 0.03), whereas CPE was present in the feces of 24 of 87 (28%) dogs with diarrhea and 3 of 55 (5%) normal dogs (P = 0.01). C difficile was isolated from 2 of 87 (2%) dogs with diarrhea but was not isolated from the feces of 55 normal dogs, possibly because of poor survival of the organism in fecal samples. C perfringens was isolated from the feces of 23 of 24 (96%) CPE-positive dogs with diarrhea, 52 of 63 (83%) CPE-negative dogs with diarrhea, and 39 of 55 (71%) CPE-negative dogs with normal feces. No correlation was found between C perfringens spore number and the presence of CPE.

Analysis of intergenic spacer transcripts suggests 'read-around' transcription of the extrachromosomal circular rDNA in Euglena gracilis.

We report here the sequence of the 1743 bp intergenic spacer (IGS) that separates the 3'-end of the large subunit ribosomal RNA (rRNA) gene from the 5'-end of the small subunit (SSU) rRNA gene in the circular, extrachromosomal ribosomal DNA (rDNA) of Euglena gracilis. The IGS contains a 277 nt stretch of sequence that is related to a sequence found in ITS 1, an internal transcribed spacer between the SSU and 5.8S rRNA genes. Primer extension analysis of IGS transcripts identified three abundant reverse transcriptase stops that may be analogous to the transcription initiation site (TIS) and two processing sites (A' and A0) that are found in this region in other eukaryotes. Features that could influence processing at these sites include an imperfect palindrome near site A0 and a sequence near site A' that could potentially base pair with U3 small nucleolar RNA. Our identification of the TIS (verified by mung bean nuclease analysis) is considered tentative because we also detected low-abundance transcripts upstream of this site throughout the entire IGS. This result suggests the possibility of 'read-around' transcription, i.e. transcription that proceeds multiple times around the rDNA circle without termination.

Recurrent diarrhea associated with enterotoxigenic Clostridium perfringens in 2 dogs.

Two dogs were diagnosed with enterotoxigenic Clostridium perfringens-associated diarrhea. Diarrhea was responsive to antimicrobial therapy, but recurred after treatment was ceased. Clostridium perfringens enterotoxin was present in feces during diarrheic episodes but not when feces were normal. Both dogs responded to a prolonged course of oral cephalexin and dietary modification.

Complete sequence of the mitochondrial genome of Tetrahymena pyriformis and comparison with Paramecium aurelia mitochondrial DNA.

We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames ("ciliate-specific") are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events. Phylogenetic analyses using concatenated sequences of several mtDNA-encoded proteins provide high bootstrap support for the monophyly of alveolates (ciliates, dinoflagellates and apicomplexans) and slime molds.

U1 small nuclear RNA and spliceosomal introns in Euglena gracilis.

In the flagellated protozoon Euglena gracilis, characterized nuclear genes harbor atypical introns that usually are flanked by short repeats, adopt complex secondary structures in pre-mRNA, and do not obey the GT-AG rule of conventional cis-spliced introns. In the nuclear fibrillarin gene of E. gracilis, we have identified three spliceosomal-type introns that have GT-AG consensus borders. Furthermore, we have isolated a small RNA from E. gracilis and propose, on the basis of primary and secondary structure comparisons, that it is a homolog of U1 small nuclear RNA, an essential component of the cis-spliceosome in higher eukaryotes. Conserved sequences at the 5' splice sites of the fibrillarin introns can potentially base pair with Euglena U1 small nuclear RNA. Our observations demonstrate that spliceosomal GT-AG cis-splicing occurs in Euglena, in addition to the nonconventional cis-splicing and spliced leader trans-splicing previously recognized in this early diverging unicellular eukaryote.

Processing of precursor rRNA in Euglena gracilis: identification of intermediates in the pathway to a highly fragmented large subunit rRNA.

We have identified and characterized the stable steady-state intermediates that appear during formation of the cytoplasmic rRNA in Euglena gracilis. A 10.2 kb RNA is the precursor to both the small subunit (SSU) rRNA and 14 discrete fragments that comprise the large subunit (LSU) rRNA. The SSU rRNA is produced via two intermediates of 4.4 kb and 3.2 kb, whereas the LSU rRNA is generated by way of two RNA species of 5.8 kb and 5.3 kb. A number of unique intermediates are associated with a novel processing pathway by which the 14 mature fragments of the LSU rRNA are produced. Analysis of transcripts mapping within ITS1, the internal transcribed spacer separating the SSU and LSU rRNA coding regions, revealed that the LSU1 (=5.8S) rRNA is heterogeneous at its 5'-end, with a major cluster of primer extension products terminating approx. 4-5 nucleotides upstream from the predominant, mature 5'-end and a second, low-level extension product appearing further upstream within ITS1. The results reported here define the pre-rRNA processing pathway in E. gracilis and provide the basis for further studies of the mechanism of excision of the novel ITSs in this system.

Expression of hepatocyte low-density lipoprotein receptor-related protein is post-transcriptionally regulated by extracellular matrix.

The low-density lipoprotein receptor-related protein (LRP) is a multifunctional member of the low-density lipoprotein receptor family that has been implicated in a variety of physiologic and pathologic processes. However, little is known about LRP regulation at the molecular level, and the factors that might mediate LRP have not yet been characterized. This is particularly true of hepatocytes, an important site of LRP expression. Hepatocyte gene expression is known to be dependent on extracellular matrix composition, although the effect of extracellular matrix on lipoprotein receptor expression has not yet been investigated. Also, the mechanisms by which the extracellular matrix affects hepatocyte gene expression are not well understood. In this study, we show that hepatocyte LRP expression decreases rapidly at the mRNA, protein, and functional levels on collagen type I, but remains high on an Engelbreth-Holm-Swarm sarcoma matrix-preparation, Matrigel. LRP function was assessed with ligand binding studies and a novel cytotoxicity assay, using Pseudomonas exotoxin A. Investigation of the mechanism of LRP down-regulation revealed a two-fold longer LRP mRNA half-life in hepatocytes cultured on Matrigel relative to collagen. Taken together, these studies reveal that LRP expression in primary hepatocytes is dependent on the extracellular matrix, and that matrix-dependent differences in hepatocyte LRP mRNA expression are primarily due to changes in mRNA stability, indicating for the first time that the expression of LRP is subject to post-transcriptional regulation.