Reciprocal "flipping" underlies substrate recognition and catalytic activation by the human 8-oxo-guanine DNA glycosylase

Both 8oxo-guanine and formamidopyrimidines are major products of oxidative DNA damage that can result in the fixation of transversion mutations following replication if left unrepaired. These lesions are targeted by the N-DNA glycosylase hOgg1, which catalyses excision of the aberrant base followed by cleavage of the phosphate backbone directly 5' to the resultant abasic site in a context, dependent manner. We present the crystal structure of native hOgg1 refined to 2.15 A resolution that reveals a number of highly significant conformational changes on association with DNA that are clearly required for substrate recognition and specificity. Changes of this magnitude appear to be unique to hOgg1 and have not been observed in any of the DNA-glycosylase structures analysed to date where both native and DNA-bound forms are available. It has been possible to identify a mechanism whereby the catalytic residue Lys 249 is "primed" for nucleophilic attack of the N-glycosidic bond.     

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.     

Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrafish

Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl.     

Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA

The mild phenotype associated with targeted disruption of the mouse OGG1 and NTH1 genes has been attributed to the existence of back-up activities and/or alternative pathways for the removal of oxidised DNA bases. We have characterised two new genes in human cells that encode DNA glycosylases, homologous to the bacterial Fpg (MutM)/Nei class of enzymes, capable of removing lesions that are substrates for both hOGG1 and hNTH1. One gene, designated HFPG1, showed ubiquitous expression in all tissues examined whereas the second gene, HFPG2, was only expressed at detectable levels in the thymus and testis. Transient transfections of HeLa cells with fusions of the cDNAs to EGFP revealed intracellular sorting to the nucleus with accumulation in the nucleoli for hFPG1, while hFPG2 co-localised with the 30 kDa subunit of RPA. hFPG1 was purified and shown to act on DNA substrates containing 8-oxoguanine, 5-hydroxycytosine and abasic sites. Removal of 8-oxoguanine, but not cleavage at abasic sites, was opposite base-dependent, with 8-oxoG:C being the preferred substrate and negligible activity towards 8-oxoG:A. It thus appears that hFPG1 has properties similar to mammalian OGG1 in preventing mutations arising from misincorporation of A across 8-oxoG and could function as a back-up repair activity for OGG1 in ogg1(-/-) mice.     

Intra-specific variation in social organization of gorillas: implications for their social evolution

We analysed intra-specific variation in the social organization of gorillas and ecological and social factors influencing them, based on recent data on diet, day journey length, home range size, group size and proportion of multi-male groups in three subspecies [western lowland gorillas (WLG); eastern lowland gorillas (ELG); mountain gorillas (MG)]. Median group size was similar across subspecies and across habitats, but the extraordinarily large group including >30 gorillas was only found in habitat with dense terrestrial herbaceous vegetation. Within-group competition may determine the upper limit of group size in frugivorous WLGs and ELGs in lowland habitats with scarce undergrowth. A frugivorous diet may be a causal factor of subgrouping in multi-male groups of WLGs and ELGs, while a folivorous diet may prevent subgrouping in multi-male groups of MGs. Social factors, rather than ecological factors, may play an important role in the formation of multi-male groups and their cohesiveness in MGs. High gregariousness of female gorillas and their prolonged association with a protector male are explained by their vulnerability to both infanticide (MGs) and predators (ELGs). Comparison of long-term changes in group composition and individual movements between ELGs in Kahuzi and MGs in the Virungas suggest that the occurrence of infanticide may promote kin-male association within a group. Threat of infanticide may stimulate MG females to transfer into multi-male groups to seek reliable protection and maturing MG males to stay in their natal groups after maturity. By contrast, the absence of infanticide may facilitate ELG females to associate with infants and other females at transfer and ELG males to establish large groups in a short period by taking females from their natal groups, by luring females from neighbouring groups, or by takeover of a widow group after the death of its leading male. These conditions may prevent ELG and WLG maturing males from remaining to reproduce in their natal groups and possibly result in a rare occurrence of multi-male groups in their habitats. Similar reproductive features of MG and ELG females suggest both female strategies have been adaptive in their evolutionary history.     

Role of TIMPs (tissue inhibitors of metalloproteinases) in pericellular proteolysis: the specificity is in the detail

Pericellular proteolysis represents one of the key modes by which the cell can modulate its environment, involving not only turnover of the extracellular matrix but also the regulation of cell membrane proteins, such as growth factors and their receptors. The metzincins are active players in such proteolytic events, and their mode of regulation is therefore of particular interest and importance. The TIMPs (tissue inhibitors of metalloproteinases) are established endogenous inhibitors of the matrix metalloproteinases (MMPs), and some have intriguing abilities to associate with the pericellular environment. It has been shown that TIMP-2 can bind to cell surface MT1-MMP (membrane-type 1 MMP) to act as a 'receptor' for proMMP-2 (progelatinase A), such that the latter can be activated efficiently in a localized fashion. We have examined the key structural features of TIMP-2 that determine this unique function, showing that Tyr36 and Glu192-Asp193 are vital for specific interactions with MT1-MMP and proMMP-2 respectively, and hence activation of proMMP-2. TIMP-3 is sequestered at the cell surface by association with the glycosaminoglycan chains of proteoglycans, especially heparan sulphate, and we have shown that it may play a role in the regulation of some ADAMs (a disintegrin and metalloproteinases), including tumour necrosis factor alpha-converting enzyme (TACE; ADAM17). We have established that key residues in TIMP-3 determine its interaction with TACE. Further studies of the features of TIMP-3 that determine specific binding to both ADAM and glycosaminoglycan are required in order to understand these unique properties.     

The effects of superior ovarian nerve sectioning on ovulation in the guinea pig

The effects on spontaneous ovulation associated with the unilateral or bilateral sectioning of the superior ovarian nerves (SON) were analyzed in guinea pigs at different time intervals of the estrous cycle. Day 1 of the estrous cycle was defined as the day when the animal presents complete loss of the vaginal membrane (open vagina). Subsequent phases of the cycle were determined by counting the days after Day 1. All animals were autopsied on the fifth day of the estrous cycle after surgery. Sectioning the right, left, or both SONs on day 5 (early luteal phase) resulted in a significant increase in the number of fresh corpora lutea. Ovulation increased significantly when the left SON (L-SON) was sectioned during late follicular phase (day 1) and medium luteal phase (day 8). When surgery was performed on days 1 or 8, neither sectioning the right SON (R-SON) nor sectioning the SON bilaterally had an apparent effect on ovulation rates. Similarly, ovulation rates were not affected when unilateral (right or left) or bilateral sectioning of the SON was performed during late luteal phase two (day 12). Unilateral or bilateral sectioning of the SON performed during the early luteal phase (day 5) was associated with a significant decrease in uterine weight. A comparable effect was observed when the L-SON was sectioned during late follicular phase (day 1), or medium luteal phase (day 8). No effects on uterine weight were observed when unilateral or bilateral sectioning of the SON was performed during late luteal phase. Our results suggest that in the guinea pig the SON modulates ovulation, and that the degree of modulation varies along the estrous cycle. The strongest influence of the SONs on ovulation occurs during early luteal phase, and decrease thereafter, being absent by late luteal phase. In addition, sectioning the left or the right SON caused different responses by the ovaries of adult guinea pigs. This paper discusses the mechanisms by which ovulation increased when the SON was surgically cut.     

Intersectin 2, a new multimodular protein involved in clathrin-mediated endocytosis

Intersectin 1 (ITSN1) is a binding partner of dynamin that has been shown to participate in clathrin-mediated endocytosis. Here we report the characterization of a new human gene, ITSN2, highly similar to ITSN1. Alternative splicing of ITSN2 generates a short isoform with two EH domains, a coiled-coil region and five SH3 domains, and a longer isoform containing extra carboxy domains (DH, PH and C2 domains), suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. ITSN2 expression analysis indicates that it is widely expressed in human tissues. Intersectin 2 isoforms show a subcellular distribution similar to other components of the endocytic machinery and co-localize with Eps15. Moreover, their overexpression, as well as the corresponding ITSN1 protein forms, inhibits transferrin internalization.     

Growth hormone size variants: changes in the pituitary during development of the chicken

There is considerable evidence for the existence of structural variants of growth hormone (GH). The chicken is a useful model for investigating GH heterogeneity as both size and charge immunoreactive-(ir) variants have been observed in the pituitary and plasma. The present study examined the size distribution of ir-GH in the pituitary gland of chicken, from late embryogenesis through adulthood. Pituitaries were homogenized in the presence of protease inhibitor, and the GH size variants were separated by SDS-PAGE, transferred by Western blotting, immunostained with a specific antiserum to chicken GH, and quantitated by chemiluminescence followed by laser densitometry (chemiluminescent assay). Under nonreducing conditions ir-GH bands of 15, 22, 25, 44, 50, 66, 80, 98, 105 and >110 kDa were observed. Both the relative proportion of the GH size variants and the total pituitary content varied with developmental stage and age. The proportion of the 15-kDa fragment was greatest in the embryonic stage, and then it decreased. The proportion of the monomeric 22-kDa form was lowest at 18 days of embryogenesis (dE) and highest at 20 dE. In contrast, the high MW forms (>/=66 kDa) were lowest in embryos, and they increased (P < 0.05) after hatching. The 22-, 44-, 66-, and 80-kDa forms were assayed for activity by radioreceptor assay following isolation by semipreparative SDS-PAGE. Only the 22-kDa GH variant showed radioreceptor activity. Under reducing conditions for SDS-PAGE, ir-GH bands of 13, 15, 18, 23, 26, 36, 39, 44, 48, 59 and 72 kDa were oberved, but most of the high MW form disappeared. There was a concomitant increase in the proportion of the monomeric band and of several submonomeric forms. The present data indicate that the expression, processing, and/or release of some if not all size variants are under some differential control during growth and development of the chicken.