Examining temporal trends in psychological distress and the co-occurrence of common substance use in a population-based sample of grade 7-12 students from 2013 to 2019.

PURPOSE: Characterizing trends and correlates of adolescent psychological distress is important due to observed global increases over the last 20 years. Substance use is a commonly discussed correlate, though we lack an understanding about how co-occurrence of these concerns has been changing over time. METHODS: Data came from repeated, representative, cross-sectional surveys of grade 7-12 students across Ontario, Canada conducted biennially from 2013 to 2019. Poisson regression with robust standard errors was used to examine changes in the joint association between psychological distress (operationalized as Kessler-6 [K6] scores ≥ 13) and substance use over time. Weighted prevalence ratios (PR) and their 99% confidence intervals were estimated, where p < 0.01 denotes statistical significance. RESULTS: The prevalence of psychological distress doubled between 2013 and 2019, with adjusted increases of about 1.2 times each survey year. This biennial increase did not differ based on sex, perceived social standing, school level, or any substance use. Students using substances consistently reported a higher prevalence of psychological distress (between 1.2 times and 2.7 times higher). There were similarly no differential temporal trends based on substance use for very high distress (K6 ≥ 19) or K6 items explored individually. CONCLUSION: Psychological distress steeply increased among adolescents and substance use remains important to assess and address alongside distress. However, the magnitude of temporal increases appears to be similar for adolescents reporting and not reporting substance use.

Expression of lysyl oxidase from cDNA constructs in mammalian cells: the propeptide region is not essential to the folding and secretion of the functional enzyme.

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32-2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including beta-aminopropionitrile, phenylhydrazine, ethylenediamine, alpha, alpha'-dipyridyl, and diethyldithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase.

Downregulation of lysyl oxidase in cadmium-resistant fibroblasts.

Lysyl oxidase, a copper-dependent metalloenzyme, plays a central role in crosslinking of collagen and elastin in the extracellular matrix. Notably, lung lysyl oxidase activity is markedly stimulated in rats exposed to cadmium vapors. To further understand the mechanism of cadmium toxicity, the mRNA expression, synthesis, post-translational processing, and catalytic activity of lysyl oxidase were examined in cadmium-resistant (CdR) cells and the cadmium-sensitive Swiss mouse 3T3 cells from which they were derived. These CdR cells synthesized and accumulated markedly elevated levels of metallothionein, a known marker for cadmium resistance, whereas the expression of lysyl oxidase was reduced considerably. In comparison to the parental, cadmium-sensitive cells, the suppression of enzyme production in the CdR cells was seen at the mRNA level, at the levels of intracellular proprotein production and mature enzyme secreted into the medium, and in terms of total enzyme activity in the culture. The presence of cupric chloride in the culture medium during the incubation of the CdR cells for 16 h significantly enhanced lysyl oxidase activity accumulating in the medium, suggesting that lysyl oxidase deficiency in CdR cells may be related to abnormal copper metabolism.

Regulation of lysyl oxidase expression in lung fibroblasts by transforming growth factor-beta 1 and prostaglandin E2.

The regulation of lysyl oxidase produced by cultured, lipid-enriched, neonatal rat lung fibroblasts was explored. The presence of 40 pM of transforming growth factor-beta 1 (TGF-beta 1) in overnight cultures increased levels of enzyme secreted into the medium by 1.6-fold while steady-state levels of lysyl oxidase mRNA increased similarly. In contrast, incubation of these cultures with 100 nM of prostaglandin E2 (PGE2) reduced enzyme activity levels by 40 to 50% although steady-state mRNA was not changed. Consistent with the effect of PGE2, the presence of indomethacin stimulated levels of secreted enzyme activity. When present in cultures simultaneously with TGF-beta 1, PGE2 prevented the stimulation beyond control levels seen with TGF-beta 1 alone. Densitometry of protein bands immunoprecipitated by antibody to lysyl oxidase indicated that the degree of conversion of the 50 kD proenzyme to the 29 kD enzyme was not significantly altered by TGF-beta 1 or PGE2. However, the net accumulation of all forms of lysyl oxidase protein was increased by TGF-beta 1 and decreased by PGE2. These results indicate that TGF-beta 1 and specific prostaglandin(s) exert opposing effects on the expression of lysyl oxidase in these lung fibroblasts.

Retinoic acid prevents downregulation of ras recision gene/lysyl oxidase early in adipocyte differentiation.

Adipocyte differentiation of 3T3-L1 cells is a complex process which is inhibited by retinoic acid (RA). Since RA acts by nuclear receptors which directly regulate gene expression, we postulate that the primary targets of RA action in this system are genes which are regulated early in adipose conversion. In this study, we demonstrate the use of the differential display technique to search for early events in adipose commitment which are sensitive to RA. A mRNA was identified on the basis of its RA-dependent gene expression 24 h after initiation of a standard differentiation protocol. Molecular cloning of the cDNA revealed it to be identical to the ras recision gene (rrg), for lysyl oxidase. Indeed, two mRNAs identical to those recognized by lysyl oxidase probes were expressed in preadipocytes and tandemly repressed with 24 h of exposure to differentiation conditions. Lysyl oxidase activity was similarly reduced in the media of differentiated cells. RA completely blocked the differ-entiation-related reduction in rrg/lysyl oxidase gene expression, although RA had no independent stimulatory effect on rrg/lysyl oxidase expression in cells not exposed to differentiating conditions. Thus, differential display has been successfully used to identify rrg/lysyl oxidase as an early marker for adipose conversion that is responsive to RA.

Oxidation of peptidyl lysine by copper complexes of pyrroloquinoline quinone and other quinones. A model for oxidative pathochemistry.

Various o- and p-quinones were assessed as oxidants of peptidyl lysine in elastin and collagen substrates in the presence and absence of divalent copper as paradigms of protein-lysine 6-oxidase (lysyl oxidase) which contains both quinone and copper cofactors. Pyrroloquinoline quinone was among the most active in the absence and the most active of the o- and p-quinones tested in the presence of copper. The optimal rate of elastin oxidation occurred at a 2:1 PQQ/Cu(II) ratio while Cu(II) itself oxidized elastin relatively slightly. Elastin oxidation by 2:1 PQQ/Cu(II) required aerobic conditions consistent with oxygen-dependent turnover of this catalytic pair. Dimethylsulfoxide and catalase individually or in combination inhibited elastin oxidation by PQQ/Cu(II) by approx. 50%, suggesting that oxygen free radical species participate in the reaction. Amino-acid analysis of elastin and collagen substrates oxidized by 2:1 PQQ/Cu and then reduced with borohydride revealed that alpha-aminoadipic-delta-semialdehyde and lesser amounts of covalent cross-linkages were generated by this oxidant. In contrast, lysine oxidase produced aldehydes and significantly greater quantities of cross-linkage products, consistent with the known specificity of the enzyme. These data, thus, indicate the potential for free quinones, such as PQQ, particularly when stimulated by appropriate metal ions, to act as adventitious oxidants of lysine side-chains in proteins.

Spatial localization of distinct rheumatic disease-associated epitopes and the RNA "cap" of the U1 snRNP particle.

The spatial organization of two rheumatic disease-associated epitopes and the RNA "cap" structure of the U1 small nuclear ribonucleoprotein (snRNP2) was analyzed both in situ and in vitro by two independent interference immuno-assays. Sm and RNP autoantibodies, associated with systemic lupus erythematosus and mixed connective tissue disease, respectively, were used to probe the epitope locations. The Sm epitope on the U1 snRNP structure was localized proximal to the RNP. Experiments with an anti-m7G (mRNA "cap") monoclonal antibody revealed that an in situ association of the Sm and RNP epitopes with the mRNA "cap" structure may exist. Our findings, together with previous observations by others, suggest a model for the spatial arrangement of these rheumatic disease-associated protein epitopes, and the U1 RNA within the U1 snRNP particle.

U1 SnRNP association with HnRNP involves an initial non-specific splice-site independent interaction of U1 SnRNP protein with HnRNA.

Precursor mRNA is complexed with proteins in the cell nucleus to form heterogeneous nuclear ribonucleoprotein (hnRNP), and these hnRNPs are found associated in vivo with small nuclear RNPs (snRNPs) for the processing of pre-mRNA. In order to better characterize the ATP-independent initial association of U1 snRNP with hnRNP, an important early event in assembly of the spliceosome complex, we have determined some of the components essential to an in vitro reassociation of U1 snRNP with hnRNP. U1 snRNP reassociated in vitro with 40S hnRNP particles from HeLa cells and, similar to the in vivo hnRNP/U1 snRNP association, the in vitro interaction was sensitive to high salt concentrations. U1 snRNP also associated with in vitro reconstituted hnRNP in which bacteriophage MS2 RNA, which lacks introns, was used as the RNA component. Purified snRNA alone would not associate with the MS2 RNA-reconstituted hnRNP, however, intact U1 snRNP did interact with protein-free MS2 RNA. This indicates that the U1 snRNP proteins are required for the hnRNP/U1 snRNP association, but hnRNP proteins are not. Thus, the initial, ATP-independent association of U1 snRNP with hnRNP seems to be mediated by U1 snRNP protein(s) associating with hnRNA without requiring a splice-site sequence. This complex may then be further stabilized by intron-specific interactions and hnRNP proteins, as well as by other snRNPs.

Induction of lung lysyl oxidase activity and lysyl oxidase protein by exposure of rats to cadmium chloride: properties of the induced enzyme.

Inspiration of CdCl2 results in a focally fibrotic response in rat lungs and markedly increases the activity of lung lysyl oxidase. Western blot analyses of urea-extractable rat lung proteins revealed that the levels of an immunoreactive, 32,000-Da protein were markedly increased in the cadmium-exposed rat lung tissue, consistent with the induction of lysyl oxidase protein. Anion exchange chromatography revealed low levels of multiple peaks of catalytically functional lysyl oxidase in control rat lung extracts, while the profile of cadmium-exposed rat lung extracts displayed markedly elevated levels of multiple peaks of enzyme activity indicating that the charge heterogeneity is expressed in the activated enzyme. The cadmium-induced enzyme was purified as a species of 32 kDa, without resolving individual ionic variants. The catalytic and physical properties of the isolated enzyme were very similar to those of previously well characterized basal enzyme of bovine aorta, including the presence of a pyrroloquinoline quinone-like carbonyl cofactor. The copper and cadmium content of the cadmium-induced enzyme indicated little if any replacement of tightly-bound copper by cadmium in the exposed lung.

Specific detection of quinoproteins by redox-cycling staining.

Quinones and related quinonoid substances catalyze redox cycling at an alkaline pH in the presence of excess glycine as reductant. With nitroblue tetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. This property of quinonoid compounds is used for the specific staining of quinoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The dopa-containing vitelline proteins and the 6-hydroxydopa-containing bovine serum amine oxidase are stained with the nitroblue tetrazolium/glycinate reagent. Also, the mammalian quinoproteins, diamine oxidase and lysyl oxidase, purported to contain pyrroloquinoline quinone, tested positive in this procedure. No quinonoid components were detected in three putative pyrroloquinoline quinone-containing quinoproteins, dopamine beta-hydroxylase, lipoxygenase, and peptidylglycine-amidating monoxygenase. Redox-cycling staining therefore confirms the presence of covalently bound quinones in the copper-dependent amine oxidases, but not in two putative quinoprotein oxygenases. Clarification of the biological significance of quinolation should be facilitated by identification of quinoproteins using this approach.

Antigenic variation in alkaline deoxyribonuclease induced by three different strains of Epstein-Barr virus.

To determine whether biological and/or biochemical variants exist between strains of Epstein-Barr virus (EBV), we superinfected Raji cells with the nontransforming lytic strain of EBV (HR-1), and two isolates that both transform B-lymphocytes and superinfect Raji cells, B95-8, and NPC-EBV. The superinfected cells were assayed for EBV specific DNase. A new electrophoretic form of DNase was observed in cells superinfected with B95-8 EBV as compared to the enzymes induced by the HR-1 and NPC-EBV isolates. There were antigenic differences in the DNase induced by the EBV strains. Since antibody to EBV DNase is a marker for nasopharyngeal carcinoma (NPC), these data may have implications for EBV-associated disease.

The autoimmune antigen Me is distinct and related to undifferentiated connective tissue disease.

Using prototype Me serum, a new autoantibody-antigen system has been identified by Ouchterlony immunodiffusion and indirect immunofluorescence. Although immunologically distinct, the Me antigen has physiochemical and biochemical properties similar to those of the Sm antigen. Immunoblot assays indicate that Me sera commonly recognize 4 peptides of molecular weights approximating 100K, 65K, 21K, and 16K. The last of these may be identical to the D peptide recognized by Sm antibodies. The Me antigen may be associated with the RNP-Sm macromolecular complex. Me-positive patients have few clinical symptoms, and the most common diagnosis is undifferentiated connective tissue disease.

Small nuclear ribonucleoprotein antigens are absent from 10S translation inhibitory ribonucleoprotein but present in cytoplasmic messenger ribonucleoprotein and polysomes.

A cytoplasmic 10S ribonucleoprotein particle (iRNP), which is isolated from chick embryonic muscle, is a potent inhibitor of mRNA translation in vitro and contains a 4S translation inhibitory RNA species (iRNA). The iRNP particle shows similarity in size to the small nuclear ribonucleoprotein (snRNP) particles. Certain autoimmune disease patients contain antibodies directed against snRNP antigenic determinants. The possibility that iRNP may be related to the small nuclear particles was tested by immunoreactivity with monospecific autoimmune antibodies to six antigenic determinants (Sm, RNP, PM-1, SS-A (Ro), SS-B (La), and Scl-70). By Ouchterlony immunodiffusion assays, the cytoplasmic 10S iRNP did not show any immunoreactivity. Also, a more sensitive hemagglutination inhibition assay for detecting Sm and RNP antigens failed to show reactivity with the 10S iRNP. Thus, the 10S iRNP particles are distinct from the similarly sized snRNP. However, free and polysomal messenger ribonucleoprotein (mRNP) particles and polysomes also isolated from chick embryonic muscle and analyzed by Ouchterlony immunodiffusion and hemagglutination inhibition for the presence of the antigenic determinants showed reactivity to Sm and RNP autoantibodies, but were not antigenic for the other four antibodies. Some of the Sm antigenic peptides of mRNP particles and polysomes were identical to those purified from calf thymus nuclear extract, as judged by Western blot analysis. The association of Sm with free and polysomal mRNP and polysomes suggests that Sm may be involved in some cytoplasmic aspects of mRNA metabolism, in addition to a nuclear function in mRNA processing.

Comparison of various preparations of nuclear antigens by hemagglutination inhibition (HAI).

A clinical laboratory carrying out tests for antinuclear antibodies requires an efficient, reliable preparation method to produce a high yield of nuclear antigens at low cost and a very sensitive, specific assay method for antigen activity. Various tissues were employed for preparation of small nuclear ribonucleoprotein (snRNP) and Sm antigens for these purposes. Fresh calf thymus cells and nuclei, commercially available calf and rabbit thymus acetone powders, fresh rat kidney and liver cells were used as sources of antigens prepared similarly by methods published previously. Preparations of antigens from whole calf thymus cell extracts were prepared with and without inhibitors to protease and RNase. snRNP and Sm antigens were assayed at each preparation step by hemagglutination inhibition (HAI). Using HAI it was possible to routinely assay snRNP and Sm at nanogram/ml quantities which was 10(6) fold more sensitive than Ouchterlony immunodiffusion. Results were expressed as relative specific activity as compared with calf thymus nuclear extract prepared by conventional methods. Protease and RNase inhibitors did not significantly increase yields. Thymus was the best source of snRNP and Sm. Fresh calf thymus extract produced a good, stable, reliable quantity of antigens, whereas calf and rabbit thymus acetone powders provided antigen at higher specific activity with less labor but slightly lower yields. Thus, considering the total cost of preparations, commercial sources may be superior to fresh sources in the clinical laboratory setting. These studies also revealed the utility of the sensitive HAI test not only in the clinical laboratory but also for further research endeavors.

Age-related ultrastructural changes in human embryonic lung fibroblasts.

Scanning and transmission electron microscopy were used to examine human embryonic lung fibroblasts at different population doubling levels. Scanning electron microscopy of cells at population doubling levels 26, 45 and 59 did not reveal a significant change in cell size with increasing age. However, transmission electron microscopy of cells at population doubling levels 19 and 45 showed an increase in nuclear lobes, a decrease in the number of ribosomes associated with rough endoplasmic reticulum, and changes to the internal structure of mitochondria on increasing population doubling level. No other previously reported age-related changes were found.