Cloning and characterization of cDNA encoding canine alpha-L-iduronidase. mRNA deficiency in mucopolysaccharidosis I dog.

alpha-L-Iduronidase is a lysosomal enzyme, the deficiency of which causes mucopolysaccharidosis I (MPS I); a canine MPS I colony has been bred to test therapeutic intervention. The enzyme was purified to apparent homogeneity from canine testis and found to consist of two electrophoretically separable proteins that had common internal peptides but differed at their amino termini. A 57-base oligonucleotide, corresponding to the most probable codons of the longest peptide, was used to screen a canine testis cDNA library. Three cDNAs were isolated, two of which lacked the 5'-end whereas the third was full-length except for a small internal deletion. The composite sequence encodes an open reading frame of 655 amino acids that includes all sequenced peptides. The amino terminus of the larger protein, glutamic acid 26, is at the predicted signal peptide cleavage site, whereas the amino terminus of the smaller protein is leucine 106. There are six potential N-glycosylation sites and a non-canonical polyadenylation signal, CTTAAA. A search of GenBank showed that the amino acid sequence of alpha-L-iduronidase has similarity to that of a bacterial beta-xylosidase. A full-length cDNA corresponding to the composite sequence was constructed (pcIdu) and inserted into the pSVL expression vector (pSVcIdu). Two days after Cos-1 cells were transfected with pSVcIdu, their intracellular and secreted level of alpha-L-iduronidase activity has increased 8- and 22-fold, respectively, over the endogenous activity. Fibroblasts of MPS I dogs, which have no alpha-L-iduronidase activity, lacked the normal alpha-L-iduronidase mRNA of 2.2 kilobases and contained instead a trace amount of a 2.8-kilobase species. Isolation and characterization of an expressible alpha-L-iduronidase cDNA represents the first step toward mutation analysis and replacement therapy.     

A life cycle assessment of Agaricus bisporus mushroom production in the USA

The International Journal of Life Cycle Assessment - Stakeholders across the food product supply chain are increasingly interested in understanding the environmental effects of food production....     

Printing of protein microarrays via a capillary-free fluid jetting mechanism

Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microm and approximately 500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer.     

Gamma371 Thr-->Ile substitution in the fibrinogen gammaD domain causes hypofibrinogenaemia.

Six members of a family with hypofibrinogenaemia had fibrinogen concentrations ranging from 0.5 to 1.1 mg/ml and, after sequencing the entire coding region and the intron exon boundaries of all three fibrinogen genes, a single heterozygous ACT-->ATT mutation was identified in the gamma gene. This novel mutation was not detected in normal family members or unrelated controls. The gamma371 Thr-->Ile substitution occurs at a conserved threonine in the gammaD domain, but molecules containing the new isoleucine were not present in circulating fibrinogen. The evidence for this was that purified gamma chains had a normal mass of 48375 Da compared to a control of 48374 Da, and tryptic peptide maps were entirely normal. The mutation predicts a mass increase of 12 Da in peptide T-36, but on mass mapping only the normal [M+2H] ion was detected, at 948 m/z. There was no new signal at 954 m/z that would indicate expression of variant chains. Also the normal 948 m/z signal was at the same intensity in digests from the proposita and controls. Crystal structures show a hydrogen bond from the threonine hydroxyl to the main chain and this case suggests this bond is critical in maintaining the structure of the gammaD domain.     

Two mutations produce intron insertion in mRNA and elongated beta-subunit of human beta-hexosaminidase

An elongated beta-subunit of the lysosomal enzyme beta-hexosaminidase was found in fibroblast strains derived from two patients with juvenile Sandhoff disease and two asymptomatic individuals sharing an unusual isoenzyme pattern: a low level of residual A (alpha beta) isoenzyme activity (3-6% of normal for the juvenile Sandhoff and 9-10% for the asymptomatic strains) without B (beta beta) isoenzyme activity. The elongated beta-subunit was abnormal in other ways: It reacted with antiserum against the unfolded polypeptide, it was not phosphorylated on mannose residues, it was not processed to the mature form, and it was degraded rapidly. The increased length of the beta-subunit was caused by two different mutations. Cells from two juvenile Sandhoff and one asymptomatic individuals had the previously described G----A transition in intron 12 that creates a splice site, causing an in-frame insertion of 24 intronic nucleotides into mRNA (Nakano, T., and Suzuki, K. (1989) J. Biol. Chem. 264, 5155-5158). The second mutation was found in cells from the asymptomatic girl whose A+B- isoenzyme pattern had been designated "Hexosaminidase Paris" (Dreyfus, J. C., Poenaru, L., Vibert, M., Ravise, N., and Boue, J. (1977) Am. J. Hum. Genet. 29, 287-293); duplication of a region straddling the junction of intron 13 and exon 14 generates an alternate splice site, causing an in-frame insertion of 18 nucleotides into mRNA. Although the two new splice sites are used preferentially, the normal sites may be used to some extent, accounting for the residual A isoenzyme activity.     

Regulation of boreal forest understory vegetation: the roles of resources and herbivores

To understand inter-trophic linkages between components of the boreal forest understory vegetation, three hypotheses were tested: survival, growth and abundance of grasses and legumes were controlled by (i) resource availability alone, (ii) by herbivores alone, and (iii) by both resource availability and herbivores. The hypotheses were tested using three experimental treatments – fertilization, herbivore exclusion, and fertilization plus herbivore exclusion – in three areas having different densities of resident herbivores, mostly snowshoe hares and ground squirrels. The highest density of snowshoe hares is comparable to natural levels during peaks in the snowshoe hare cycle. As the density of herbivores increased so too did the level of response by the measured variables – survival, growth of transplants and leaf area index of established vegetation. In general, fertilization resulted in a decrease in survival and growth of transplants, and fences increased survival and growth; both responses were more noticeable at higher herbivore densities. Fertilizer and herbivore exclosure fences had only negligible effects on established grass and legume abundance at all hare densities. We have shown that some hypotheses of vegetation regulation are over-simplified because different species groups (i.e., grasses and legumes) are regulated by different factors, at different life history stages, and sometimes these factors act in opposing directions. We argue that during the increase phase and peak of the snowshoe hare cycle (high herbivore density), growth and survival of establishing plants is regulated by herbivores. During the decline and low phases of the snowshoe hare cycle herbivores will have little impact on early life stages, whereas the established, mature, vegetation will be resource-regulated. Because of the variability in responses to the same manipulations we may begin to understand which plant life history stages are most vulnerable to consumer and resource regulation, the magnitudes of these sources of regulation at each of these stages, and how these vary among species groups and types of environments.     

Ligand binding to heme proteins: relevance of low-temperature data

Binding of carbon monoxide to the beta chain of adult human hemoglobin has been studied by flash photolysis over the time range from about 100 ps to seconds and the temperature range from 40 to 300 K. Below about 180 K, binding occurs directly from the pocket (process I) and is nonexponential in time. Above about 180 K, some carbon monoxide molecules escape from the pocket into the protein matrix. Above about 240 K, escape into the solvent becomes measurable. Process I can be observed up to 300 K. The low-temperature data extrapolate smoothly to 300 K, proving that the results obtained below 180 K provide functionally relevant information. The experiments show again that the binding process even at physiological temperatures is regulated by the final binding step at the heme iron and that measurements at high temperatures are not sufficient to fully understand the association process.