Binding of JAB1/CSN5 to MIF is mediated by the MPN domain but is independent of the JAMM motif

Macrophage migration inhibitory factor (MIF) binds to c-Jun activation domain binding protein-1 (JAB1)/subunit 5 of COP9 signalosome (CSN5) and modulates cell signaling and the cell cycle through JAB1. The binding domain of JAB1 responsible for binding to MIF is unknown. We hypothesized that the conserved Mpr1p Pad1p N-terminal (MPN) domain of JAB1 may mediate binding to MIF. In fact, yeast two hybrid (YTH) and in vitro translation/coimmunoprecipitation (CoIP) analysis showed that a core MPN domain, which did not cover the functional JAB1/MPN/Mov34 metalloenzyme (JAMM) deneddylase sequence, binds to MIF comparable to full-length JAB1. YTH and pull-down analysis in conjunction with nanobead affinity matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry demonstrated that MIF(50-65) and MPN are sufficient to mediate MIF-JAB1 interaction, respectively. Finally, endogenous CoIP of MIF-CSN6 complexes from mammalian cells demonstrated that MPN is responsible for MIF-JAB1 binding in vivo, and, as CSN6 does not contain a functional JAMM motif, confirmed that the interaction does not require JAMM.     

Changes in androgen receptor, estrogen receptor alpha, and sexual behavior with aging and testosterone in male rats

Reproductive aging in males is characterized by a diminution in sexual behavior beginning in middle age. We investigated the relationships among testosterone, androgen receptor (AR) and estrogen receptor alpha (ERα) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3 months) and middle-aged (12 months) rats were given sexual behavior tests, then castrated and implanted with vehicle or testosterone capsules. Rats were tested again for sexual behavior. Numbers of AR and ERα immunoreactive cells were counted in the anteroventral periventricular nucleus and the medial preoptic nucleus, and serum hormones were measured. Middle-aged intact rats had significant impairments of all sexual behavior measures compared to young males. After castration and testosterone implantation, sexual behaviors in middle-aged males were largely comparable to those in the young males. In the hypothalamus, AR cell density was significantly (5-fold) higher, and ERα cell density significantly (6-fold) lower, in testosterone- than vehicle-treated males, with no age differences. Thus, restoration of serum testosterone to comparable levels in young and middle-aged rats resulted in similar preoptic AR and ERα cell density concomitant with a reinstatement of most behaviors. These data suggest that age-related differences in sexual behavior cannot be due to absolute levels of testosterone, and further, the middle-aged brain retains the capacity to respond to exogenous testosterone with changes in hypothalamic AR and ERα expression. Our finding that testosterone replacement in aging males has profound effects on hypothalamic receptors and behavior has potential medical implications for the treatment of age-related hypogonadism in men.     

Crystal structure of the DUF16 domain of MPN010 from Mycoplasma pneumoniae

We have determined the crystal structure of the DUF16 domain of unknown function encoded by the gene MPN010 of Mycoplasma pneumoniae at 1.8 A resolution. The crystal structure revealed that this domain is composed of two separated homotrimeric coiled-coils. The shorter one consists of 11 highly conserved residues. The sequence comprises noncanonical heptad repeats that induce a right-handed coiled-coil structure. The longer one is composed of approximately nine heptad repeats. In this coiled-coil structure, there are three distinguishable regions that confer unique structural properties compared with other known homotrimeric coiled-coils. The first part, containing one stutter, is an unusual phenylalanine-rich region that is not found in any other coiled-coil structures. The second part is a highly conserved glutamine-rich region, frequently found in other trimeric coiled-coil structures. The last part is composed of prototype heptad repeats. The phylogenetic analysis of the DUF16 family together with a secondary structure prediction shows that the DUF16 family can be classified into five subclasses according to N-terminal sequences. Based on the structural comparison with other coiled-coil structures, a probable molecular function of the DUF16 family is discussed.     

Rapid detection of arbuscular mycorrhizae in roots and soil of an intensively managed turfgrass system by PCR amplification of small subunit rDNA

 Arbuscular mycorrhizal fungi (AMF) were detected in uninoculated soil and roots of turfgrass (Agrostis palustris) by direct extraction and PCR amplification of the small subunit rRNA gene. Sequence analysis of the cloned PCR product confirmed the identity of the amplified DNA as an AMF sequence having 95% identity to Glomus intraradices. The sensitivity of the method was gauged by comparison with the most probable number analysis of infective propagules in an intensively managed turfgrass system having 56 propagules per 100 g soil. In contrast to the heavily managed system, infective propagule numbers were high in systems under moderate and limited management. The method described may be useful for rapid investigations of genetic diversity and community structure of AMF. Accepted: 8 January 1999     

Reconsideration of the derivation of Most Probable Numbers,their standard deviations,confidence bounds and rarity values

Aims: The purpose of this work was to derive a simple Excel spreadsheet and a set of standard tables of most probable number (MPN) values that can be applied by users of International Standard Methods to obtain the same output values for MPN, SD of the MPN, 95% confidence limits and test validity. With respect to the latter, it is considered that the Blodgett concept of ‘rarity’ is more valuable than the frequently used approach of improbability (vide de Man). Methods and Results: The paper describes the statistical procedures used in the work and the reasons for introducing a new set of conceptual and practical approaches to the determination of MPNs and their parameters. Examples of MPNs derived using these procedures are provided. The Excel spreadsheet can be downloaded from http://www.wiwiss.fu‐berlin.de/institute/iso/mitarbeiter/wilrich/index.html . Conclusions: The application of the revised approach to the determination of MPN parameters permits those who wish to use tabulated values, and those who require access to a simple spreadsheet to determine values for nonstandard test protocols, to obtain the same output values for any specific set of multiple test results. The concept of ‘rarity’ is a more easily understood parameter to describe test result combinations that are not statistically valid. Provision of the SD of the log MPN value permits derivation of uncertainty parameters that have not previously been possible. Significance and Impact of the Study: A consistent approach for the derivation of MPNs and their parameters is essential for coherence between International Standard Methods. It is intended that future microbiology standard methods will be based on the procedures described in this paper.     

水华蓝藻噬藻体对不同条件培养的宿主细胞感染性分析

在从武汉东湖水样中培养分离水华蓝藻噬藻体(Planktothrix agardhii Virus from Lake Donghu,PaV-LD)的基础上,对在不同条件培养的宿主蓝藻细胞中,PaV-LD增殖效率及裂解作用进行了测定分析。分别将PaV-LD接种到生长期、半连续培养更新率或光照不同的宿主蓝藻液中,并采用稀释培养计数(Mostprobable number,MPN)方法与电镜观察,测定子代PaV-LD释放量及宿主细胞的裂解作用。结果显示:对数生长期宿主蓝藻单个细胞中子代PaV-LD的平均释放量为350感染单位(Infectious Units,IU/cell),显著高于稳定生长期的平均释放量110 IU/cell。在用新鲜培养基更新率为0%、35%、50%和65%的半连续培养宿主蓝藻中,接种PaV-LD 5d之后,噬藻体的释放量分别约为50 IU/cell、70 IU/cell、220 IU/cell或310 IU/cell,表明子代PaV-LD释放率随培养基更新率的增加而显著提高。在光照条件下感染3—4d后,宿主蓝藻细胞充分裂解,并释放大量子代PaV-LD,滴度可由初始7.00×103IU/mL快速增加到8.56×107IU/mL;但在遮光条件下,同样感染的蓝藻细胞未见裂解,也检测不到释放的子代噬藻体。电镜观察显示,在光照条件下感染的蓝藻细胞类囊体膜结构消失,而大量子代PaV-LD颗粒主要分布在原有类囊体的部位。显然,宿主蓝藻细胞的培养条件和状态可能对获得噬藻体纯培养有决定性影响。     

Structural and functional differences of SWIRM domain subtypes

SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.     

The crystal structure of the human Mov34 MPN domain reveals a metal-free dimer

The 26S proteasome is a large protein complex involved in protein degradation. We have shown previously that the PSMD7/Mov34 subunit of the human proteasome contains a proteolytically resistant MPN domain. MPN domain family members comprise subunits of the proteasome, COP9-signalosome and translation initiation factor 3 complexes. Here, the crystal structure of two C-terminally truncated proteins, MPN 1-186 and MPN 1-177, were solved to 1.96 and 3.0 A resolution, respectively. MPN 1-186 is formed by nine beta-strands surrounded by three alpha-helices plus a fourth alpha-helix at the C terminus. This final alpha-helix emerges from the domain core and folds along with a symmetrically related subunit, typical of a domain swap. The crystallographic dimer is consistent with size-exclusion chromatography and DLS analysis showing that MPN 1-186 is a dimer in solution. MPN 1-186 shows an overall architecture highly similar to the previously reported crystal structure of the Archaeal MPN domain AfJAMM of Archaeoglobus fulgidus. However, previous structural and biophysical analyses have shown that neither MPN 1-186 nor full-length human Mov34 bind metal, in opposition to the zinc-binding AfJAMM structures. The zinc ligand residues observed in AfJAMM are conserved in the yeast Rpn11 proteasome and Csn5 COP-signalosome subunits, which is consistent with the isopeptidase activity described for these proteins. The results presented here show that, although the MPN domain of Mov34 shows a typical metalloprotease fold, it is unable to coordinate a metal ion. This finding and amino acid sequence comparisons can explain why the MPN-containing proteins Mov34/PSMD7, RPN8, Csn6, Prp8p and the translation initiation factor 3 subunits f and h do not show catalytic isopeptidase activity, allowing us to propose the hypothesis that in these proteins the MPN domain has a primarily structural function.     

Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils in southeast Queensland

AIMS: To investigate the occurrence and levels of Arcobacter spp. in pig effluent ponds and effluent-treated soil. METHODS AND RESULTS: A Most Probable Number (MPN) method was developed to assess the levels of Arcobacter spp. in seven pig effluent ponds and six effluent-treated soils, immediately after effluent irrigation. Arcobacter spp. levels in the effluent ponds varied from 6.5 x 10(5) to 1.1 x 10(8) MPN 100 ml(-1) and in freshly irrigated soils from 9.5 x 10(2) to 2.8 x 10(4) MPN g(-1) in all piggery environments tested. Eighty-three Arcobacter isolates were subjected to an abbreviated phenotypic test scheme and examined using a multiplex polymerase chain reaction (PCR). The PCR identified 35% of these isolates as Arcobacter butzleri, 49% as Arcobacter cryaerophilus while 16% gave no band. All 13 nonreactive isolates were subjected to partial 16S rDNA sequencing and showed a high similarity (>99%) to Arcobacter cibarius. CONCLUSIONS: A. butzleri, A. cryaerophilus and A. cibarius were isolated from both piggery effluent and effluent-irrigated soil, at levels suggestive of good survival in the effluent pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide quantitative information on Arcobacter spp. levels in piggery effluent and to associate A. cibarius with pigs and piggery effluent environments.     

Improved aeroponic culture of inocula of arbuscular mycorrhizal fungi

We compared conventional atomizing disc aeroponic technology with the latest ultrasonic nebulizer technology for production of Glomus intraradices inocula. The piezo ceramic element technology used in the ultrasonic nebulizer employs high-frequency sound to nebulize nutrient solution into microdroplets 1 μm in diameter. Growth of pre-colonized arbuscular mycorrhizal (AM) roots of Sudan grass was achieved in both chambers used but both root growth and mycorrhization were significantly faster and more extensive in the ultrasonic nebulizer system than in the atomizing disc system. Shearing of the AM fungi (AMF) infected roots in both the systems did not reduce inoculum viability, as evident from the MPN data. However, sheared roots from the ultrasonic nebulizer system had significantly more infective propagules than those produced in the atomizing disc system. Thus, the latest ultra-sonic nebulizer aeroponic technology appears to be superior and an alternative to conventional atomizing disc or spray nozzle systems for the production of high-quality AMF inocula. These can be used in small doses to produce a large response, which is a prerequisite for commercialization of AMF technology. Accepted: 11 January 2000