Mdm30 is an F-box protein required for maintenance of fusion-competent mitochondria in yeast

Mitochondrial fusion and fission play important roles for mitochondrial morphology and function. We identified Mdm30 as a novel component required for maintenance of fusion-competent mitochondria in yeast. The Mdm30 sequence contains an F-box motif that is commonly found in subunits of Skp1-Cdc53-F-box protein ubiquitin ligases. A fraction of Mdm30 is associated with mitochondria. Cells lacking Mdm30 contain highly aggregated or fragmented mitochondria instead of the branched tubular network seen in wild-type cells. Deltamdm30 cells lose mitochondrial DNA at elevated temperature and fail to fuse mitochondria in zygotes at all temperatures. These defects are rescued by deletion of DNM1, a gene encoding a component of the mitochondrial division machinery. The protein level of Fzo1, a key component of the mitochondrial fusion machinery, is regulated by Mdm30. Elevated Fzo1 levels in cells lacking Mdm30 or in cells overexpressing Fzo1 from a heterologous promoter induce mitochondrial aggregation in a similar manner. Our results suggest that Mdm30 controls mitochondrial shape by regulating the steady-state level of Fzo1 and point to a connection of the ubiquitin/26S proteasome system and mitochondria.     

Somatotropic axis in hypocretin-deficient narcoleptic humans: altered circadian distribution of GH-secretory events

Narcolepsy is a sleep disorder caused by impaired hypocretin (orexin) neurotransmission. Growth hormone (GH) secretion may be altered in narcolepsy for various reasons. Slow-wave sleep episodes, which are closely associated with GH-secretory events, are more randomly dispersed over 24 h in narcoleptics. Furthermore, hypocretins may inhibit pituitary GH release. We assessed the function of the somatotropic axis in narcolepsy by deconvolving 24-h (10-min sampling interval) plasma GH concentration profiles in seven hypocretin-deficient narcoleptic patients and in seven healthy controls matched for age, sex, and body weight. Both basal and pulsatile GH secretion rate and secretagogue-induced GH release were similar in patients and controls. However, narcoleptics secreted approximately 50% of their total production during the daytime, whereas controls secreted only 25% during the day. Also, the GH output pattern of narcoleptics was significantly less regular. We propose that hypocretin deficiency disrupts the circadian distribution of hypothalamic GH-releasing hormone release in narcoleptic patients to simultaneously cause daytime GH release and promote their propensity to fall asleep during the day.     

Influence of prolonged endurance cycling and recovery diet on intramuscular triglyceride content in trained males

Intramuscular triglycerides (IMTG) are assumed to form an important substrate source during prolonged endurance exercise in trained males. This study investigated the effects of endurance exercise and recovery diet on IMTG content in vastus lateralis muscle. Nine male cyclists were provided with a standardized diet for 3 days, after which they performed a 3-h exercise trial at a 55% maximum workload. Before and immediately after exercise and after 24 and 48 h of recovery, magnetic resonance spectroscopy (MRS) was performed to quantitate IMTG content. Muscle biopsies were taken after 48 h of recovery to determine IMTG content by using quantitative fluorescence microscopy. The entire procedure was performed two times; in one trial, a normal diet containing 39% energy (En%) as fat was provided (NF) and in the other a typical carbohydrate-rich athlete's diet (LF: 24 En% fat) was provided. During exercise, IMTG content decreased by 21.4 +/- 3.1%. During recovery, IMTG content increased significantly in the NF trial only, reaching preexercise levels within 48 h. In accord with MRS, fluorescence microscopy showed significantly higher IMTG content in the NF compared with the LF trial, with differences restricted to the type I muscle fibers (2.1 +/- 0.2 vs. 1.4 +/- 0.2% area lipid staining, respectively). In conclusion, IMTG content in the vastus lateralis muscle declines significantly during prolonged endurance exercise in male cyclists. When a normal diet is used, IMTG contents are subsequently repleted within 48 h of postexercise recovery. In contrast, IMTG repletion is impaired substantially when a typical, carbohydrate-rich athlete's diet is used. Data obtained by quantitative fluorescence microscopy correspond well with MRS results, implying that both are valid methods to quantify IMTG content.     

Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.     

The N and C termini of the splice variants of the human mitogen-activated protein kinase-interacting kinase Mnk2 determine activity and localization

The cap-binding eukaryotic initiation factor eIF4E is phosphorylated by the mitogen-activated protein (MAP) kinase-interacting kinases (Mnk's). Three forms of the Mnk's exist in human cells: Mnk1, Mnk2a, and Mnk2b. These last two are derived from the same gene by alternative splicing and differ only at their C termini. While Mnk2a contains a MAP kinase-binding site in this region, Mnk2b lacks such a sequence and is much less readily activated by MAP kinases in vitro. Expression of Mnk2b in mammalian cells leads to increased phosphorylation of eIF4E, showing that it acts as an eIF4E kinase in vivo. While Mnk2a is cytoplasmic, a substantial amount of Mnk2b is found in the nucleus. Both enzymes contain a stretch of basic residues in their N termini that plays a role in binding to eIF4G and functions as a nuclear localization signal. Binding of eIF4G or nuclear import appears to be regulated by the C terminus of Mnk2a. Furthermore, the MAP kinase-binding site of Mnk2a regulates nuclear entry. Within the nucleus, Mnk2b and certain variants of Mnk2a that are present in the nucleus colocalize with the promyelocytic leukemia protein PML, which also binds to eIF4E.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.     

Profilin I attached to the Golgi is required for the formation of constitutive transport vesicles at the trans-Golgi network

Profilin I was identified, by mass spectrometric sequencing and immunoblotting, as a component of purified Golgi cisternae from HepG2 cells. Binding to the Golgi was verified by indirect immunofluorescence in MT-1 cells showing that a fraction of profilin I colocalizes with TGN38, a marker of the trans-Golgi network (TGN). Studying the formation of constitutive exocytic vesicles at the TGN in a cell-free system demonstrated that cytosolic profilin I has no effect, while incubation of Golgi cisternae with a profilin I-specific antibody reduced vesicle formation by about 50%. Notably, the antibody displaces a fraction of the Golgi-bound dynamin II indicating that profilin I may indirectly promote vesicle formation by supporting the binding of dynamin II to the Golgi membrane. The impact of dynamin II on vesicle formation is demonstrated by incubating the Golgi with the proline-rich domain of dynamin II which concomitantly displaces dynamin II and inhibits vesicle formation. The data provide evidence that profilin I attaches to the Golgi apparatus and is required for the formation of constitutive transport vesicles.