Foetal age determination and development in elephants

Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown-rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85-90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation.     

Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.     

Decreased serum levels of 1,25-(OH)2 vitamin D during 1 year of sunlight deprivation in the Antarctic

French Antarctic territories harbor bases that are devoted to scientific and technical work. Living and working conditions during 1-year sojourns in such an environment are quite acceptable, but the confinement and the drop in ultraviolet B radiation exposure during winter months raise the problem of preservation of normal vitamin D status. Seasonal variations in 25-hydroxyvitamin D [25(OH)D] levels have been well documented, but the effect of sunshine deprivation on 1,25 dihydroxyvitamin D [1,25(OH)2D] levels is quite controversial. The aim of this study was to address this question under the exceptional conditions of lack of sunshine exposure. Fifteen male Caucasian subjects participating in a 1-year mission in Antarctica were investigated. They were subjected to seven blood samplings, one before and six during their sojourn. Serum levels of 25(OH)D, 1,25(OH)2D, osteocalcin, and ICTP were measured. We found that levels of 25(OH)D and 1,25(OH)2D significantly decreased in these subjects during the mission, minimum levels being observed 10 months after their departure from France. ICTP concentrations did not change throughout this study, but osteocalcin levels were found to be higher at the end of the sojourn than before departure, which could argue for the existence of bone remodeling changes. Further studies are now needed to fully investigate bone metabolism changes and to address the question of vitamin D supplementation during this kind of sojourn.     

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6′)-Ii that encodes a 6′-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m⁵C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m⁵C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m⁵C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes'' respective specificities for nucleotides C1404 and C1407.     

Use1p is a yeast SNARE protein required for retrograde traffic to the ER

SNAREs on transport vesicles and target membranes are required for vesicle targeting and fusion. Here we describe a novel yeast protein with a typical SNARE motif but with low overall amino acid homologies to other SNAREs. The protein localized to the endoplasmic reticulum (ER) and was therefore named Use1p (unconventional SNARE in the ER). A temperature-sensitive use1 mutant was generated. use1 mutant cells accumulated the ER forms of carboxypeptidase Y and invertase. More specific assays revealed that use1 mutant cells were defective in retrograde traffic to the ER. This was supported by strong genetic interactions between USE1 and the genes encoding SNAREs in retrograde traffic to the ER. Antibodies directed against Use1p co-immunoprecipitated the SNAREs Ufe1p, myc-Sec20p and Sec22p, which form a SNARE complex required for retrograde traffic from the Golgi to the ER, but neither Bos1p nor Bet1p (members of the SNARE complex in anterograde traffic to the Golgi). Therefore, we conclude that Use1p is a novel SNARE protein that functions in retrograde traffic from the Golgi to the ER.     

Crystallization and preliminary X-ray analysis of Escherichia coli methionyl–tRNA formyltransferase

Methionyl–tRNA formyltransferase from Escherichia coli, a monomer of 34kDa, was overexpressed from its cloned gene fmt (Guillon, J.M., Mechulam, Y., Schmitter, J.M., Blanquet, S., and Fayat, G., J. Bacteriol. 174:4294–4301, 1992) and crystallized using ammonium sulphate as precipitant. The crystals are trigonal and have unit cell parameters a = b = 151.0Å, c = 81.8Å. They belong to space group P3₂21 and diffract to 2.0Å resolution. The structure is being solved by multiple isomorphous replacement. © 1996 Wiley-Liss, Inc.     

The chromatin remodelling complex NoRC safeguards genome stability by heterochromatin formation at telomeres and centromeres

Constitutive heterochromatin is crucial for the integrity of chromosomes and genomic stability. Here, we show that the chromatin remodelling complex NoRC, known to silence a fraction of rRNA genes, also establishes a repressive heterochromatic structure at centromeres and telomeres, preserving the structural integrity of these repetitive loci. Knockdown of NoRC leads to relaxation of centromeric and telomeric heterochromatin, abnormalities in mitotic spindle assembly, impaired chromosome segregation and enhanced chromosomal instability. The results demonstrate that NoRC safeguards genomic stability by coordinating enzymatic activities that establish features of repressive chromatin at centromeric and telomeric regions, and this heterochromatic structure is required for sustaining genomic integrity.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli.

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.     

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3–43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K_D = 220 pM) and HER3-expressing tumor cells with EC₅₀ values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3–43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3–43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.     

A TEST OF THE SIB-COMPETITION HYPOTHESIS FOR OUTCROSSING ADVANTAGE IN IMPATIENS CAPENSIS

One of the potential selective mechanisms invoked in discussions of breeding-system evolution is that competition within sibships increases the fitness of outcrossed progeny relative to selfed progeny. We tested this sib-competition hypothesis using cleistogamous (CL) and chasmogamous (CH) seeds of Impatiens capensis in a large greenhouse experiment. The experimental design was a double replacement series which also allowed us to test for inbreeding depression and overall resource partitioning among sibships. We found no evidence for strong inbreeding depression in the study population; although plants from CH seeds had a slight advantage over plants from CL seeds in total flower and pod production, CL plants had slightly higher growth. We also could not detect significant resource partitioning among sibships nor any evidence to support the sib-competition hypothesis for outcrossing advantage. CH sibships were not significantly more variable than CL sibships in any of the phenotypic traits measured. These results suggest that sibling competition may have little importance in the evolution of Impatiens breeding systems.