Empowering patients with high myopia: The significance of education.

PURPOSE: To investigate the status of patient education among highly myopic individuals focusing on the presence, sources, content, timing of the education and impact on patients. METHODS: Self-reported data were collected through an online 13-item questionnaire consisting of open and multiple-choice questions. The questionnaire was sent to 250 highly myopic members of a patient organization in the Netherlands, of whom 128 (51%) responded. RESULTS: At least one acute event had occurred in 66% (84/128) of participants at the time of the questionnaire. Among all participants, 25% (32/128) had not received patient education regarding alarm symptoms for any of these events. Among those who had been informed, the ophthalmologist was the most frequent (57%, 73/128) source of information. Participants who visited the ophthalmologist annually were more frequently informed than participants without annual visits (53%, 26/49 versus 26%, 9/35, p = 0.002). Those not informed were more likely to have a more than 3 days patient delay (92%, 12/13). Doctors delay was also present; 26% (22/84) of the participants with alarm symptoms had to wait 2 or more days before the first appointment. Long-term consequences of myopia had been discussed with 102 participants (80%, 102/128), again with the ophthalmologist as the most frequent source (59%, 76/128). PERSPECTIVES: Many myopic individuals have not been educated about their increased risk of acute events, which can result in patient delay and serious consequences with respect to visual prognosis. These findings underscore the critical importance of integrating patient education across the entire ophthalmic care chain for myopia.

Serum mannose-binding lectin deficiency is associated with cryptosporidiosis in young Haitian children.

BACKGROUND: Mannose-binding lectin (MBL) is a component of the innate immune response and binds microbial surfaces through carbohydrate recognition domains. MBL deficiency may contribute to susceptibility to a variety of infectious diseases, particularly in young children. MBL binds to the Cryptosporidium sporozoite and may be important in resistance to cryptosporidiosis. METHODS: We studied the association of serum MBL levels and cryptosporidiosis in a case-control study of young Haitian children with cryptosporidiosis versus children who were control subjects. RESULTS: Ninety-nine children were enrolled, as follows: 49 children with cryptosporidiosis, 41 healthy controls, and 9 children with diarrhea from other causes. Case children were more malnourished than controls, and 49% had persistent or chronic diarrhea. At enrollment, mean serum MBL levels were markedly lower in children with cryptosporidiosis (P = .002), as was the number of children with an MBL deficiency of < or = 70 ng/mL (P = .005). In multivariate analysis, the association of cryptosporidiosis and MBL deficiency persisted (P = .002; adjusted odds ratio, 22.4), as did the association of cryptosporidiosis with general malnutrition. The subset of children with cryptosporidiosis and MBL deficiency were more likely to be male (P = .025). CONCLUSIONS: MBL may be an important component of innate immune protection against Cryptosporidium infection in young children. Additional studies are necessary to determine whether MBL intestinal losses, deficient epithelial expression, and/or genetic polymorphisms in the MBL gene contribute to MBL deficiency in cryptosporidiosis and other enteric infections in young children.

Growth, compatible solute and salt accumulation of five mycorrhizal fungal species grown over a range of NaCl concentrations.

The oil sand industry in northeastern Alberta produces vast areas of severely disturbed land. The sodicity of these anthropic soils is one of the principal constraints that impede their revegetation. Previous in vitro studies have shown that the ectomycorrhizal fungi Laccaria bicolor (Maire) Orton UAMH 8232 and Hebeloma crustuliniforme (Bull) Quel. UAMH 5247 have certain salt-resistant traits and thus are candidate species for the inoculation of tree seedlings to be outplanted on salt-affected soil. In this study, the in vitro development of these fungi was compared to that of three mycorrhizal fungi [Suillus tomentosus (Kauff.) Sing., Snell and Dick; Hymenoscyphus sp. and Phialocephala sp.] isolated from a sodic site created by Syncrude Canada Ltd. Their growth, osmotica and Na/Cl contents were assessed over a range (0, 50, 100, 200 mM) of NaCl concentrations. After 21 days, the two ascomycetes (Hymenoscyphus sp. and Phialocephala sp.) were shown to be more resistant to the NaCl treatments than the three basidiomycete species. Of the basidiomycetes, L. bicolor was the most sensitive to NaCl stress, while H. crustuliniforme showed greater water stress resistance, and the S. tomentosus isolate exhibited greater Na and Cl filtering capacities and had a better biomass yield over the NaCl gradient tested. Both ascomycetes used mechanisms other than carbohydrate accumulation to palliate NaCl stress. While the Hymenoscyphus isolate accumulated proline in response to NaCl treatments, the darker Phialocephala isolate may have used compounds such as melanin. The basidiomycete species accumulated mainly mannitol and/or proline in response to increasing concentrations of NaCl.

Mycorrhizal inoculum potentials of pure reclamation materials and revegetated tailing sands from the Canadian oil sand industry.

Recent improvements in the management of oil sand tailings used by the Canadian oil sand industry have resulted in the production of composite tailing sands (CT): a new challenging material for reclamation work. Jack pine (Pinus banksiana Lamb.), hybrid poplar (Populus deltoides Bartr. ex Marsh. xPopulus nigra L.) and red clover (Trifolium pratense L.) plants were used in an 8-week greenhouse bioassay to evaluate the mycorrhizal inoculum potential of CT. This inoculum potential was compared with that of three other reclamation materials [common tailing sands (TS), deep overburden (OB) and muskeg peat (MK)], and with three sites reclaimed in 1982 (R82), 1988 (R88) and 1999 (R99). CT was devoid of active mycorrhizal propagules while all other materials showed some level of inoculum potential. Arbuscular mycorrhizal fungi were observed on roots of clover or poplar grown in TS, OB, and all substrates containing peat (MK, R82, R88 and R99). Pine roots were also colonized by vesicle-forming hyphae of an unidentified fine endophyte and by dark septate fungi. Ectomycorrhizas (ECM) were observed on pine and poplar grown in OB, MK, and in soils from the two older reclaimed sites (R82 and R88). Using morpho- and molecular typing, six ECM fungi were identified to the genus or species level: Laccaria sp., Thelephora americana, Wilcoxina sp. (E-strain), Tuber sp. (I-type), a Sebacinoid, and a Pezizales species. Laccaria sp. and Wilcoxina sp. were the most frequently observed ECM species.

Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer.

The early detection of breast cancer is the best means to minimise disease-related mortality. Current screening techniques have limited sensitivity and specificity. Breast nipple aspirate fluid can be obtained noninvasively and contains proteins secreted from ductal and lobular epithelia. Nipple aspirate fluid proteins are breast specific and generally more concentrated than corresponding blood levels. Proteomic analysis of 1 microl of diluted nipple aspirate fluid over a 5-40 kDa range from 20 subjects with breast cancer and 13 with nondiseased breasts identified five differentially expressed proteins. The most sensitive and specific proteins were 6500 and 15 940 Da, found in 75-84% of samples from women with cancer but in only 0-9% of samples from normal women. These findings suggest that (1) differential expression of nipple aspirate fluid proteins exists between women with normal and diseased breasts, and (2) analysis of these proteins may predict the presence of breast cancer.

Structure of murine Tcl1 at 2.5 A resolution and implications for the TCL oncogene family.

Tcl1 and Mtcp1, members of the Tcl1 family, are implicated in T-cell prolymphocytic leukemia. The crystal structure of a dimer of murine Tcl1 has been determined at 2.5 A resolution with an R factor of 0.225. Murine Tcl1, human Tcl1 and Mtcp1 share very similar subunit structures, with RMS differences of 0.6 and 1.4 A for C(alpha) atoms, respectively, while the sequences share 50 and 36% identity, respectively. These structures fold into an eight-stranded beta-barrel of unique topology and high internal symmetry of 1.1-1.3 A for the two halves of human and murine Tcl1 and 1.7 A for Mtcp1, despite the low 12-13% sequence identity. The molecular surfaces of all three structures showed a common planar region which is likely to be involved in protein-protein interactions.

Purification and characterization of recombinant forms of murine Tcl1 proteins.

The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells. The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1). The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases. The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues. The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies. Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-phase column. The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers. The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy. Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X-ray crystallographic methods of structure determination.

Improving the diffraction quality of MTCP-1 crystals by post-crystallization soaking.

Significant improvement in the resolution and quality of the X-ray diffraction of crystals of MTCP-1 protein was observed on post-crystallization soaking. The MTCP-1 crystals grown from 1.5 M ammonium sulfate diffracted to only 3.0 A resolution with some disorder in the diffraction. After post-crystallization soaking in a solution containing 2.0 M ammonium sulfate, the disorder was eliminated and diffraction extended to better than 2.0 A resolution. Both native and selenomethionine-enriched crystals demonstrated better diffraction after soaking for several months. This simple technique may be useful to improve the diffraction quality of protein crystals generally.

S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme.

An enzyme isolated from rat liver cytosol (native molecular mass 78. 3 kDa; polypeptide molecular mass 42.5 kDa) is capable of catalysing the NADH/NADPH-dependent degradation of S-nitrosoglutathione (GSNO). The activity utilizes 1 mol of coenzyme per mol of GSNO processed. The isolated enzyme has, as well, several characteristics that are unique to alcohol dehydrogenase (ADH) class III isoenzyme: it is capable of catalysing the NAD+-dependent oxidations of octanol (insensitive to inhibition by 4-methylpyrazole), methylcrotyl alcohol (stimulated by added pentanoate) and 12-hydroxydodecanoic acid, and also the NADH/NADPH-dependent reduction of octanal. Methanol and ethanol oxidation activity is minimal. The enzyme has formaldehyde dehydrogenase activity in that it is capable of catalysing the NAD+/NADP+-dependent oxidation of S-hydroxymethylglutathione. Treatment with the arginine-specific reagent phenylglyoxal prevents the pentanoate stimulation of methylcrotyl alcohol oxidation and markedly diminishes the enzymic activity towards octanol, 12-hydroxydodecanoic acid and S-hydroxymethylglutathione; the capacity to catalyse GSNO degradation is also checked. Additionally, limited peptide sequencing indicates 100% correspondence with known ADH class III isoenzyme sequences. Kinetic studies demonstrate that GSNO is an exceptionally active substrate for this enzyme. S-Nitroso-N-acetylpenicillamine and S-nitrosated human serum albumin are not substrates; the activity towards S-nitrosated glutathione mono- and di-ethyl esters is minimal. Product analysis suggests that glutathione sulphinamide is the major stable product of enzymic GSNO processing, with minor yields of GSSG and NH3; GSH, hydroxylamine, nitrite, nitrate and nitric oxide accumulations are minimal. Inclusion of GSH in the reaction mix decreases the yield of the supposed glutathione sulphinamide in favor of GSSG and hydroxylamine.

Crystal structure of MTCP-1: implications for role of TCL-1 and MTCP-1 in T cell malignancies.

Two related oncogenes, TCL-1 and MTCP-1, are overexpressed in T cell prolymphocytic leukemias as a result of chromosomal rearrangements that involve the translocation of one T cell receptor gene to either chromosome 14q32 or Xq28. The crystal structure of human recombinant MTCP-1 protein has been determined at 2.0 A resolution by using multiwavelength anomalous dispersion data from selenomethionine-enriched protein and refined to an R factor of 0.21. MTCP-1 folds into a compact eight-stranded beta barrel structure with a short helix between the fourth and fifth strands. The topology is unique. The structure of TCL-1 has been predicted by molecular modeling based on 40% amino acid sequence identity with MTCP-1. The identical residues are clustered inside the barrel and on the surface at one side of the barrel. The overall structure of MTCP-1 superficially resembles the structures of proteins in the lipocalin family and calycin superfamily. These proteins have diverse functions, including transport of retinol, fatty acids, chromophores, pheromones, synthesis of prostaglandin, immune modulation, and cell regulation. However, MTCP-1 differs in the topology of the beta strands. The structural similarity suggests that MTCP-1 and TCL-1 form a unique family of beta barrel proteins that is predicted to bind small hydrophobic ligands and function in cell regulation.

Purification and characterization of recombinant forms of TCL-1 and MTCP-1 proteins.

The TCL-1 gene which is located on chromosome 14 plays a major role in human hematopoeitic malignancies and encodes a 14-kDa protein whose function has not been determined. The TCL-1 gene is expressed in pre-B cells, in immature thymocytes, and at low levels in activated T cells but not in peripheral mature B cells and in normal cells. The TCL-1 protein is similar in its primary structure to a protein encoded by the mature T cell proliferation gene (MTCP-1). The MTCP-1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T cell proliferative diseases. The TCL-1 and MTCP-1 genes appear to be members of a family of genes involved in lymphoid proliferation and T cell malignancies. Our laboratory has undertaken the study of the TCL-1 and MTCP-1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, both purified TCL-1 and MTCP-1 proteins in forms with and without a six His tag sequence. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymeric-based reverse-phase column. The MTCP-1 recombinant proteins display greater solubility, do not form disulfide linked dimers or oligomers, and elute at a lower isopropanol concentration than the corresponding TCL-1 proteins. The purified recombinant TCL-1 and MTCP-1 proteins have been characterized by N-terminal sequence analysis, time of flight mass spectrometry, and circular dichroism spectroscopy. Initial results have indicated that the MTCP-1 protein with the His tag removed is suitable for both NMR and X-ray crystallographic methods of structure determination.

Purification of an Escherichia coli-expressed Nef protein from the human immunodeficiency virus-type 2.

The entire nef gene sequence of HIV-2, NIH-Z strain, has been cloned into the pJL6 expression vector and used for the synthesis of a 23-kDa protein in E. coli. The expressed protein is a fusion between the N-terminal 13 amino acids of the cII gene, 8 amino acids resulting from the ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef protein has been purified to apparent homogeneity on analytical scale (10-20 micrograms) by a combination of sequential detergent extraction, gel filtration, and reversed-phase high-performance chromatography. The expressed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by gel filtration and RP-HPLC. Larger amounts (> 100 micrograms) of the purified Nef protein have been produced by a purification procedure that employs sequential detergent extraction, chromatography on Q-Sepharose in the presence of 7 M urea, and chromatography on hydroxylapatite, also in 7 M urea. The purified HIV-2 Nef protein has been used for the production of polyclonal and monoclonal antibodies. The milder method of purification should facilitate structure-function studies of the Nef protein and its role in the life cycle of HIV.

Zeta-crystallin versus other members of the alcohol dehydrogenase super-family. Variability as a functional characteristic.

Species variability of the lens protein zeta-crystallin was correlated with those of alcohol dehydrogenases of classes I and III and sorbitol dehydrogenase in the same protein family. The extent of overall variability, nature of residues conserved, and patterns of segment variability, all fall within the limits typical of the 'variable' group of medium-chain alcohol dehydrogenases. This shows that zeta-crystallin is subject to restrictions similar to those of classical liver alcohol dehydrogenase and therefore derived from a metabolically active enzyme like other enzyme crystallins. Special residues at the active site, however, differ substantially, including an apparent lack of a zinc-binding site. This is compatible with altered functional properties and makes the spread within this medium-chain dehydrogenase family resemble the wide spread within the short-chain dehydrogenases. Schematic plotting is useful for illustrating the differences between 'variable' and 'constant' enzymes.

Dietary vitamin A, beta carotene and risk of epidermoid lung cancer in south-western France.

This hospital-based case-control study was designed to investigate the association of low dietary vitamin A and beta carotene consumption with epidermoid lung cancer. Cases were patients with histologically confirmed epidermoid lung cancer diagnosed in six selected hospitals of southwestern France in 1983-84. Controls were selected from patients admitted to the same hospitals during the same period with diagnoses other than cancer. Cases and controls were matched for sex, age, place of residence, occupation, professional exposure to carcinogens, tobacco and alcohol consumption. A total of 106 cases of epidermoid lung cancer and 212 controls were interviewed on their typical weekly intake of 80 food items rich in preformed vitamin A and beta carotene. Index measures of the vitamin A and beta carotene daily intakes were computed for each individual patient and expressed in retinol equivalent (RE). A statistically significant odds ratio (OR) was found for preformed vitamin A (OR = 4.3; 95% CI: 2.5-7.3) with the threshold of 1,000 RE. A similar result was found for beta carotene with the same threshold (OR = 4.1; 95% CI: 2.3-7.4). Using the conditional logistic regression, consumption of preformed vitamin A and consumption of beta carotene were significantly and independently associated with epidermoid lung cancer. While confirming the protective role of beta carotene against epidermoid lung cancer, this study also shows that preformed vitamin A might have a distinct and important protective effect.

Detection of heterologous fusion proteins in Escherichia coli with a monoclonal antibody.

Several laboratories have constructed expression vectors for the production of heterologous fusion proteins containing the N-terminal 13 amino acids of the bacteriophage lambda cII-coded protein in Escherichia coli. We have prepared a monoclonal antibody to a synthetic peptide having this CII amino acid sequence and have found that this antibody reacts with authentic CII protein in Western blot tests and with most CII peptide-containing fusion proteins in both radioimmunoprecipitation and Western blot assays. However, there are some CII-hybrid protein species with which the antibody does not react. Our findings indicate that this antibody is a valuable tool for detecting and purifying expressed proteins and in studying their structure and function.