Major depressive disorder in older adolescents: prevalence, risk factors, and clinical implications

In this article we summarize our current understanding of depression in older (14-18 years old) adolescents based on our program of research (the Oregon Adolescent Depression Project). Specifically, we address the following factors regarding adolescent depression: (a) phenomenology (e.g., occurrence of specific symptoms, gender and age effects, community versus clinic samples); (b) epidemiology (e.g., prevalence, incidence, duration, onset age); (c) comorbidity with other mental and physical disorders; (d) psychosocial characteristics associated with being, becoming, and having been depressed; (e) recommended methods of assessment and screening; and (f) the efficacy of a treatment intervention developed for adolescent depression, the Adolescent Coping With Depression course. We conclude by providing a set of summary statements and recommendations for clinicians.     

Physical interaction between specific E2 and Hect E3 enzymes determines functional cooperativity

The cellular protein E6AP functions as an E3 ubiquitin protein ligase in the E6-dependent ubiquitination of p53. E6AP is a member of a family of functionally related E3 proteins that share a conserved carboxyl-terminal region called the Hect domain. Although several different E2 ubiquitin-conjugating enzymes have been shown to function with E6AP in the E6-dependent ubiquitination of p53 in vitro, the E2s that cooperate with E6AP in the ubiquitination of its normal substrates are presently unknown. Moreover, the basis of functional cooperativity between specific E2 and Hect E3 proteins has not yet been determined. Here we report the cloning of a new human E2, designated UbcH8, that was identified in a two-hybrid screen through specific interaction with E6AP. We demonstrate that UbcH7, an E2 closely related to UbcH8, can also bind to E6AP. The region of E6AP involved in complex formation with UbcH8 and UbcH7 was mapped to its Hect domain. Furthermore, we show that UbcH5 and UbcH6, two highly homologous E2s that were deficient for interaction with E6AP, could associate efficiently with another Hect-E3 protein, RSP5. Finally, only the E6AP-interacting E2s could function in conjunction with E6AP in the ubiquitination of an E6 independent substrate of E6AP, whereas the noninteracting E2s could not. Taken together, these studies demonstrate for the first time complex formation between specific human E2s and the Hect domain family of E3 proteins and suggest that selective physical interaction between E2 and E3 enzymes forms the basis of specificity for functionally distinct E2:E3 combinations.     

Early versus delayed angiotensin-converting enzyme inhibition therapy in acute myocardial infarction. The healing and early afterload reducing therapy trial

BACKGROUND: Although ACE inhibitor therapy has been shown to reduce mortality in patients with acute myocardial infarction (MI), the optimal dose and the timing of its initiation have not been determined. METHODS AND RESULTS: In a double-blind trial of 352 patients with anterior MI, we compared the safety and effectiveness of early (day 1) versus delayed (day 14) initiation of the ACE inhibitor ramipril (10 mg) on echocardiographic measures of left ventricular (LV) area and ejection fraction (EF). An early, low-dose ramipril (0.625 mg) arm was also evaluated. Clinical events did not differ. During the first 14 days, the risk of manifesting a systolic arterial pressure of < or = 90 mm Hg was increased in both ramipril groups. LVEF increased in all groups during this period, but the early, full-dose ramipril group had the greatest improvement in EF (increase: full, 4.9 +/- 10.0; low, 3.9 +/- 8.2%; delayed, 2.4 +/- 8.8%; P for trend < .05) and was the only group that did not demonstrate a significant increase in LV diastolic area. CONCLUSIONS: The results of the present study demonstrated that in patients with anterior MI, the early use of ramipril (titrated to 10 mg) attenuated LV remodeling and was associated with a prompter recovery of LVEF. The use of low-dose regimen did not prevent hypotension and had only intermediate benefits on LV size and function. The more favorable effects on LV topography of the early use of full-dose ramipril support the results of the major clinical trials, which have demonstrated an early survival benefit of ACE inhibition.     

Familial hyperamylasaemia

A six year old boy underwent extensive investigation for recurrent abdominal pain and was found to have a persistently raised serum amylase. Endoscopic retrograde cholangiopancreatography was normal and macroamylasaemia was excluded. Serum amylase concentrations were found to be raised in other family members spanning three generations, all of whom were asymptomatic. Clearance studies suggested no evidence of a renal tubular defect and serum lipase concentrations were normal. This is the first report of apparently familial hyperamylasaemia and the mode of inheritance is consistent with an autosomal dominant pattern.     

Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.     

Site-specific, photochemical proteolysis applied to ion channels in vivo

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the "signature" Cys128-Cys142 disulfide loop of the nAChR alpha subunit.     

CASP, a novel, highly conserved alternative-splicing product of the CDP/cut/cux gene, lacks cut-repeat and homeo DNA-binding domains, and interacts with full-length CDP in vitro

Human CDP/cut and its murine counterpart, cux1/CDP are homeodomain repressor proteins in the family of Drosophila Cut. Northern blot analysis reveals complex alternative splicing, including forms too small to encode the full 1505 amino acid protein. We have characterized a CDP/cut alternatively spliced cDNA (CASP) of 3.4 kb. Human CASP, a predicted 678 amino acid polypeptide, shares 400 amino acids with CDP, but has an alternate N terminal exon of 20 aa, and the C-terminal 258 amino acids diverge from CDP/cut entirely. As the unique C-terminus of CASP lacks the three 'cut-repeats' and homeodomain of CDP/cut, we predict it does not bind DNA. Murine CASP, 96% similar to human, shares these features. Database searches identify homologs in chicken (86% identical to human CASP) and yeast (29% identical to human). Murine CASP mRNA is ubiquitous in mouse tissues and in tissue-culture cell lines. We generated a specific antiserum against the unique C-terminus of CASP, and used this reagent to demonstrate that CASP protein is expressed as an approx. 80 kDa protein in human and murine cells. Co-translation of in vitro-translated CDP and CASP mRNA, followed by immunoprecipitation with specific anti-CASP IgG, shows that CASP polypeptide can from a complex with CDP. Studies of the intron/exon structure of the murine cux/CDP/mCASP locus (> 100 kb) reveal that the unique 3' exons of CASP are interposed between cut-repeats 2 and 3 of the cux gene. We speculate that a primordial CASP-like gene captured a cut-repeat-homeobox gene to give rise to the eukaryotic Cut/CDP family of proteins.     

Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH 3T3 cells

Protein kinase C (PKC) isozymes exhibit important differences in terms of their regulation and biological functions. Not only may some PKC isoforms be active and others not for a given response, but the actions of different isoforms may even be antagonistic. In NIH 3T3 cells, for example, PKCdelta arrests cell growth whereas PKCepsilon stimulates it. To probe the contribution of the regulatory and the catalytic domains of PKC isozymes to isozyme-specific responses, we prepared chimeras between the regulatory and the catalytic domains of PKCalpha, -delta, and -epsilon. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in mouse fibroblasts. A major objective was to characterize the growth properties of the cells that overexpress the various PKC constructs. Our data demonstrate that both the regulatory and the catalytic domains play roles in cell proliferation. The regulatory domain of PKCepsilon enhanced cell growth in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and, in the presence of PMA, all chimeras with the PKCepsilon regulatory domain also gave rise to colonies in soft agar; the role of the catalytic domain of PKCepsilon was evident in the PMA-treated cells that overexpressed the PKC chimera containing the delta regulatory and the epsilon catalytic domains (PKCdelta/epsilon). The important contribution of the PKCepsilon catalytic domain to the growth of PKCdelta/epsilon-expressing cells was also evident in terms of a significantly increased saturation density in the presence of PMA, their formation of foci upon PMA treatment, and the induction of anchorage-independent growth. Aside from the growth-promoting effect of PKCepsilon, we have shown that most chimeras with PKCalpha and -delta regulatory domains inhibit cell growth. These results underscore the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.     

X-ray structure of 5-aminolaevulinate dehydratase, a hybrid aldolase

5-Aminolaevulinate dehydratase (ALAD) is a homo-octameric metallo-enzyme that catalyses the formation of porphobilinogen from 5-aminolaevulinic acid. The structure of the yeast enzyme has been solved to 2.3 A resolution, revealing that each subunit adopts a TIM barrel fold with a 39 residue N-terminal arm. Pairs of monomers wrap their arms around each other to form compact dimers and these associate to form a 422 symmetric octamer. All eight active sites are on the surface of the octamer and possess two lysine residues (210 and 263), one of which, Lys 263, forms a Schiff base link to the substrate. The two lysine side chains are close to two zinc binding sites one of which is formed by three cysteine residues (133, 135 and 143) while the other involves Cys 234 and His 142. ALAD has features at its active site that are common to both metallo- and Schiff base-aldolases and therefore represents an intriguing combination of both classes of enzyme. Lead ions, which inhibit ALAD potently, replace the zinc bound to the enzyme's unique triple-cysteine site.