A large-scale assessment of lakes reveals a pervasive signal of land use on bacterial communities.

Lakes play a pivotal role in ecological and biogeochemical processes and have been described as "sentinels" of environmental change. Assessing "lake health" across large geographic scales is critical to predict the stability of their ecosystem services and their vulnerability to anthropogenic disturbances. The LakePulse research network is tasked with the assessment of lake health across gradients of land use on a continental scale. Bacterial communities are an integral and rapidly responding component of lake ecosystems, yet large-scale responses to anthropogenic activity remain elusive. Here, we assess the ecological impact of land use on bacterial communities from over 200 lakes covering more than 660,000 km2 across Eastern Canada. In addition to community variation between ecozones, land use across Eastern Canada also appeared to alter diversity, community composition, and network structure. Specifically, increasing anthropogenic impact within the watershed lowered diversity. Likewise, community composition was significantly correlated with agriculture and urban development within a watershed. Interaction networks showed decreasing complexity and fewer keystone taxa in impacted lakes. Moreover, we identified potential indicator taxa of high or low lake water quality. Together, these findings point to detectable bacterial community changes of largely unknown consequences induced by human activity within lake watersheds.

Fitness effects of new mutations in Chlamydomonas reinhardtii across two stress gradients.

Most spontaneous mutations affecting fitness are likely to be deleterious, but the strength of selection acting on them might be impacted by environmental stress. Such stress-dependent selection could expose hidden genetic variation, which in turn might increase the adaptive potential of stressed populations. On the other hand, this variation might represent a genetic load and thus lead to population extinction under stress. Previous studies to determine the link between stress and mutational effects on fitness, however, have produced inconsistent results. Here, we determined the net change in fitness in 29 genotypes of the green algae Chlamydomonas reinhardtii that accumulated mutations in the near absence of selection for approximately 1000 generations across two stress gradients, increasing NaCl and decreasing phosphate. We found mutational effects to be magnified under extremely stressful conditions, but such effects were specific both to the type of stress and to the genetic background. The detection of stress-dependent fitness effects of mutations depended on accurately scaling relative fitness measures by generation times, thus offering an explanation for the inconsistencies among previous studies.

In utero hematopoietic stem cell transplantation: a caprine model for prenatal therapy in inherited metabolic diseases.

OBJECTIVES: We explored the feasibility and efficacy of in utero hematopoietic stem cell transplantation in the caprine animal model system with the objectives of determining procedures for transplantation and establishing methods for detecting engraftment. METHODS: Male fetal liver hematopoietic stem cells were injected into female fetuses during the immunotolerant period, using either hysterotomy or ultrasound-guided injections. RESULTS: The rate of fetal death was much lower for the ultrasound-guided injections. Donor cells were observed in the peritoneal fluid of 4 fetuses 3 days after injection, but no donor cells were detected in tissues at longer time periods. CONCLUSIONS: Ultrasound-guided injection of hematopoietic stem cells into the abdomen of a developing fetus is safe and feasible. The parameters required for successful engraftment have not yet been identified.

Caprine mucopolysaccharidosis IIID: a preliminary trial of enzyme replacement therapy.

Mucopolysaccharidosis type IIID (MPS IIID) is a lysosomal storage disorder resulting from lack of activity of the lysosomal hydrolase N-acetylglucosamine 6-sulfatase (6S) (EC 3.1.6.14). The syndrome is associated with systemic and central nervous system (CNS) heparan sulfate glycosaminoglycan (HS-GAG) accumulation, secondary storage of lipids, and severe, progressive dementia. In this investigation, caprine MPS IIID, established as a large animal model for the human disease, was used to evaluate the efficacy of enzyme replacement therapy (ERT). Recombinant caprine 6S (rc6S) (1 mg/kg/dose) was administered intravenously to one MPS IIID goat kid at 2, 3, and 4 wks of age. Five days after the last dose, the uronic acid (UA) content and the composition of uncatabolized HS-GAG fractions in the brain of the ERT-treated MPS IIID kid were similar to those from a control, untreated MPS IIID animal. However, hepatic uronic acid levels in the treated MPS IIID kid were approximately 90% lower than those in the untreated MPS IIID control; whereas the composition of the residual hepatic HS-GAG was identical to that in the untreated animal. Marked reduction of lysosomal storage vacuoles in hepatic cells of the treated MPS IIID kid was observed, but ERT had no effect on CNS lesions. No residual 6S activity was detected in brain or liver. This preliminary investigation indicates that other treatment regimens will be necessary to ameliorate MPS III-related CNS lesions.

Human beta-mannosidase cDNA characterization and first identification of a mutation associated with human beta-mannosidosis.

Human beta-mannosidosis is an autosomal recessive, lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Unlike the severe clinical manifestation of the disease in ruminants, in which it leads to neonatal death, the human disease phenotype is generally milder. In addition, the phenotypic manifestation among the reported cases of human beta-mannosidosis is variable, even among members of the same family. To understand the molecular basis of the human disease and the mechanisms for such clinical variability, we sequenced the entire coding region of the human beta-mannosidase gene using a combination of cDNA library screening, RT-PCR and 5' rapid amplification of cDNA ends (RACE). The composite cDNA is 3293 nt, consisting of an 87 nt 5'-untranslated region, 2640 nt coding region and 566 nt 3'-untranslated region. The gene was localized to human chromosome 4q22-25. Analysis of a multiple tissue northern blot demonstrated a single 3.7 kb transcript. Mutation analysis of a Czech gypsy family with two siblings differently affected with beta-mannosidosis demonstrated a homozygous A-->G transition 2 bp upstream of a splice acceptor site. The associated cryptic splice site activation and exon skipping caused by this mutation resulted in two abnormally spliced mutant mRNA species in both siblings.

Caprine beta-mannosidase: sequencing and characterization of the cDNA and identification of the molecular defect of caprine beta-mannosidosis.

The complete sequence of the caprine beta-mannosidase cDNA coding region has been determined, and a mutation that is associated with caprine beta-mannosidosis has been identified. Reverse transcriptase-polymerase chain reactions were performed using primers based on bovine and, later, goat cDNA sequences to produce an overlapping series of amplicons covering the entire coding region. The composite cDNA codes for an 879-amino-acid peptide that has four potential N-glycosylation sites. Comparison of the caprine and bovine cDNAs reveals that 96.3% of the nucleotides and 95.2% of the deduced amino acids are identical. A single-base deletion at position 1398 of the coding sequence was identified in the cDNA isolated from a goat affected with beta-mannosidosis. This deletion results in a shift in the reading frame and a premature termination of translation, yielding a deduced peptide of 481 amino acids. An assay, developed to determine the presence or absence of this mutation, confirmed that animals affected with beta-mannosidosis were homozygous for the mutation and that obligate carriers in a caprine beta-mannosidosis colony were heterozygous. This assay accurately distinguished between mutation carrier and noncarrier goats and was used for prenatal diagnosis using DNA collected from fetal fluids. The assay also confirmed chimerism in a goat with an atypically mild beta-mannosidosis phenotype. Thus, this application enables assessment of the efficacy of engraftment of hematopoietic stem cells after prenatal transfer from donor sources.

Regulation of prostaglandin endoperoxide H synthase-2 expression by 2,3,7,8,-tetrachlorodibenzo-p-dioxin.

We have examined the molecular mechanisms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated prostaglandin synthesis in Mardin Darvey canine kidney cells (MDCK). TCDD stimulates prostaglandin synthesis in these cells, at least in part, by elevating prostaglandin endoperoxide H2 synthase-2 (PGHS-2) levels. TCDD-stimulated transcription of the PGHS-2 gene was maximal (6-fold) within 2 h and resulted in a 100-fold increase in PGHS-2 mRNA and a 25-fold increase in PGHS-2 protein levels by 4 h. Transient transfection experiments using luciferase-reporter plasmids demonstrated that control element(s) responsible for TCDD activation of the murine PGHS-2 promoter in MDCK cells are located in the first 965 nucleotides upstream from the PGHS-2 transcriptional initiation site. A canonical xenobiotic response element, similar to those that control transcription of other well-known TCDD-sensitive genes, is present at position -157, but does not appear to be sufficient for halogenated aromatic hydrocarbon (HAH) activation of the PGHS-2 promoter. TCDD failed to stimulate transcription from the PGHS-2 promoter when reporter plasmids were transfected into Hepa 1c1c7 cells, a line which contains the functional aryl hydrocarbon receptor. It seems likely that inappropriate expression of PGHS-2 may contribute to the toxic effects of TCDD and other HAHs. In particular, PGHS-2 expression may affect those toxic reactions that involve inappropriate cellular growth, such as dermal hyperplasia and tumor formation. It is also likely that elevated synthesis of prostaglandins, which are potent regulators of immune function, could play a role in the immunotoxicity associated with HAH exposure.

Prostaglandin endoperoxide synthase gene structure: identification of the transcriptional start site and 5'-flanking regulatory sequences.

The gene for the murine prostaglandin endoperoxide (PGH) synthase (8, 11, 14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) has been cloned. The gene was isolated from a mouse NIH 3T3 cell genomic library and is contained in four overlapping lambda FIXII bacteriophage clones. The gene spans approximately 22 kb and consists of 11 exons. Primer extension and RNAse protection assays indicate that transcription of the gene begins at an initiation site 63 nucleotides 5' to the ATG translation initiation codon. Neither TATA or CAAT boxes are present immediately upstream of the transcriptional start site, but SP1 binding sites are present at positions -47 to -42 and -30 to -25, relative to the transcription initiation site. Examination of the 5'-end and 2400 bp of the 5'-flanking sequence of the gene revealed sequences with homology to several transcriptional regulatory sequences. Three putative AP-1 binding sites were found, two within the first exon and intron and another at position -2097 to -2090. The AP-1 site at position -2097 is adjacent to a sequence with similarity to a negative glucocorticoid regulatory element (nGRE) (position -2123 to -2009). The presence of AP sites by themselves, or in conjunction with an nGRE sequence, suggests a possible interplay between jun/fos regulatory proteins and the glucocorticoid receptor for positive and negative regulation of the PGH synthase gene. An unexpected finding was the presence at position -403 to -385 of a putative dioxin responsive element, a sequence found to be responsible for the induction of transcription of the cytochrome P450IA1 gene (CYPIA1) and other genes involved in detoxification/activation of polycyclic aromatic hydrocarbons.

Molecular basis for the inhibition of prostanoid biosynthesis by nonsteroidal anti-inflammatory agents.

Many nonsteroidal anti-inflammatory drugs exert their effects by inhibiting the synthesis of prostanoids. More specifically, these agents block the synthesis of prostaglandin endoperoxide G2 from arachidonic acid by competing with arachidonate for binding to the cyclooxygenase active site of prostaglandin endoperoxide synthase. Studies of the molecular biology of prostaglandin endoperoxide synthase indicate that there is a single gene for the enzyme. Thus, tissue-specific effects of nonsteroidal anti-inflammatory drugs probably result from differences in drug distribution and/or metabolism and not from the existence of tissue-specific prostaglandin endoperoxide synthase isozymes. Aspirin causes inactivation of prostaglandin endoperoxide synthase by first binding to the cyclooxygenase active site and then acetylating the protein at Ser530. Although the cyclooxygenase activity is inactivated, the hydroperoxidase activity of prostaglandin endoperoxide synthase is unaltered by Aspirin or other nonsteroidal anti-inflammatory drugs. Replacement of Ser530 of the native enzyme with an alanine residue by site-directed mutagenesis yields a prostaglandin endoperoxide synthase with unaltered catalytic and substrate binding activities. Thus, the hydroxyl group of Ser530 is not essential for enzyme activity. Instead, it appears likely that acetylation of prostaglandin endoperoxide synthase by Aspirin simply places a bulky acetyl group at or near the cyclooxygenase active site, thereby interfering with arachidonic acid binding.

The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases.

Acetylation of Ser-530 of sheep prostaglandin endoperoxide (PGG/H) synthase by aspirin causes irreversible inactivation of the cyclooxygenase activity of the enzyme. To determine the catalytic function of the hydroxyl group of Ser-530, we used site-directed mutagenesis to replace Ser-530 with an alanine. Cos-1 cells transfected with expression vectors containing the native (Ser-530) or mutant (Ala-530) cDNAs for sheep PGG/H synthase expressed comparable cyclooxygenase and hydroperoxidase activities. Km values for arachidonate (8 microM) and ID50 values for reversible inhibition by the cyclooxygenase inhibitors, flurbiprofen (5 microM), flufenamate (20 microM), and aspirin (20 mM), were also the same for both native and mutant PGG/H synthases; however, only the native enzyme was irreversibly inactivated by aspirin. Thus, the "active site" Ser-530 of PGG/H synthase is not essential for catalysis or substrate binding. Apparently, acetylation of native PGG/H synthase by aspirin introduces a bulky sidechain at position 530 which interferes with arachidonate binding. In related studies, a cDNA for mouse PGG/H synthase was cloned and sequenced. A sequence of 35 residues with Ser-530 at the midpoint was identical in the two proteins. Thus, Ser-530 does lie in a highly conserved region, probably involved in cyclooxygenase catalysis. Sequence comparisons of mouse and sheep PGG/H synthase also provided information about the heme-binding site of the enzyme. The sheep HYPR sequence (residues 274-277), which had been proposed to form a portion of the distal heme-binding site, is not conserved in the mouse PGG/H synthase, suggesting that this region is not the distal heme-binding site. One sequence, TIWLREHNRV (residues 303-312 of the sheep enzyme), is very closely related to the sequence TLW(L)LREHNRL common to thyroid peroxidase and myeloperoxidase. The histidine in this latter sequence is the putative axial heme ligand of these peroxidases. We suggest that the histidine (His-309) of sheep PGG/H synthase sequence is the axial heme ligand of this enzyme.

Structure-function relationships in sheep, mouse, and human prostaglandin endoperoxide G/H synthases.

Our studies are designed to determine which amino acid residues are involved in catalyzing the cyclooxygenase and hydroperoxidase activities of prostaglandin endoperoxide (PGG/H) synthase. We have deduced from complementary (c)DNAs the amino acid sequences of the sheep and mouse PGG/H synthases, and a portion of the human PGG/H synthase. These enzymes have amino acid sequences which are about 90% identical. Sequence similarities with putative heme binding regions of myeloperoxidase and thyroid peroxidase suggest that the sequence TI(L)WLREHNRV of PGG/H synthase contains the histidine (His309) which is the proximal heme ligand; the distal heme ligand may be His226 which is found in the sequence 222-KALGH-226. Using site-directed mutagenesis, we have replaced Ser530, the serine residue which is acetylated by aspirin, with Ala530 and with Asn530; the Ala530 mutant has both cyclooxygenase and hydroperoxidase activity, while the Asn530 mutant lacks cyclooxygenase activity but retains hydroperoxidase activity. These results establish that the hydroxyl group of Ser530 is not essential for catalysis or substrate binding and suggest that a bulky group at position 530, such as that introduced by aspirin acetylation, prevents arachidonate binding to the cyclooxygenase active site. Finally, we have found that tetranitromethane causes irreversible inactivation of cyclooxygenase activity and that the enzyme is protected from inactivation when ibuprofen is included in the reaction mixture. These results suggest that there is an essential tyrosine at the active site of PGG/H synthase.