An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).     

Mating behaviour and insemination in the hopping mouse (Notomys alexis)

Hopping mice have extremely small testes and a reduced complement of male accessory sex glands. Nevertheless, greater than 1000 spermatozoa populate the isthmus of the oviduct after each insemination and a range of morphological types is found similar to that in the male tract. When females are primed with gonadotrophins they sometimes lock with more than one of the males within the group and in the present study two inseminations sometimes took place at the one oestrus. There is thus the possibility for intermale sperm competition within the female tract of these animals.     

Correlation of concentrations of estriol-3-sulfate with those of potassium and sodium in human breast cyst fluid

Estriol-3-sulfate (E3-3S) was assayed in 92 specimens of human breast cyst fluid (BCF) obtained by needle aspiration from women with fibrocystic disease. The concentrations of K+ and Na+ were determined in the same samples. The median concentration of E3-3S in the fluids from premenopausal women under 51 years of age (69 cases) was 4.4 ng/mL. Based on the K+ levels the samples were divided into two groups, above 50 mM (Type I) and below 50 mM (Type II). Correlations were made between the concentrations of the estrogen conjugate and the univalent ions. In the premenopausal women, Type I cysts were associated with above median E3-3S and Type II cysts with below median E3-3S (P less than 0.01). A K+/Na+ ratio of more than one was also related to elevated E3-3S (P less than 0.025). The BCF obtained from postmenopausal women and women older than 50 years tended to be low in E3-3S (median 1.64 ng/mL) and high in K+ but there were too few cases to permit statistical comparisons to be made. Since fibrocystic disease constitutes a risk factor for the development of breast cancer, it will be of interest to determine retrospectively whether any of the above subsets of BCF may be useful in identifying a patient at such risk.     

Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-pi interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.     

Successful embryo transfer in a small dasyurid marsupial, Sminthopsis crassicaudata

A technique for the transfer of early embryos from the uterus of donors to recipient females of a small dasyurid marsupial, Sminthopsis crassicaudata , is described. When the recipients were killed 3 to 5 d later, a few expanded blastocysts were found to be present in the uteri of most of the females provided that corpora lutea were present in the ovaries. This study demonstrates successful embryo transfer in a small marsupial species and supports the view that secretion of the endometrium due to luteal activity may be necessary for development of the expanded unilaminar blastocyst stage.     

Cytological organization of the seminiferous epithelium in the Australian rodents Pseudomys australis and Notomys alexis

Absolute and relative testis mass of two species of conilurine rodents, the plains rat (Pseudomys australis) and the hopping mouse (Notomys alexis), were markedly different. In plains rats the testis comprised approximately 3% of body mass while in hopping mice they rarely accounted for more than 0.2%. In both species, 8 cellular associations, or cycle stages, were recognized in the seminiferous epithelium. The relative frequencies, and hence durations, of Stages I to VIII were 5.1, 7.0, 12.5, 10.5, 5.4, 25.9, 4.4 and 29.0% of one cycle for plains rats (N = 14), and 20.4, 10.9, 9.6, 8.1, 5.1, 20.4, 14.7 and 10.8% for laboratory-bred hopping mice (N = 35). Generally, the relative durations of the cycle stages were very similar between plains rat individuals, but were variable between hopping mice. Also, organization of the seminiferous epithelium was less rigid in hopping mouse testes, and several anomalies were observed. These included: the occurrence of 2-4 cellular associations in about 20% (range 11.3-33.9%) of tubular cross-sections, deviations in cellular composition in certain cycle stages from that usually observed, and an increased incidence of degenerating cells, together with the presence of multinucleated "giant' cells, within the seminiferous epithelium.     

Paracentric inversion inv(11) (q21q23) in the Netherlands

Summary We report the result of investigations from 20 families with 72 carriers of the paracentric inversion inv(11)(q21q23) in the Netherlands. There is no increase in the rate of spontaneous abortions among carriers of the inversion or their partners. Also, so far, there are no children with recombinant chromosomes arising from the inversion. It is doubtful whether prenatal diagnosis would be helpful to carriers of this inversion. The results of the genealogy study and geographical distribution are discussed; it is suggested that all the families have arisen from a single mutation.     

Morphological changes in the oocyte and its surrounding vestments during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata

This light and transmission electron microscopical study shows that the first polar body is given off before ovulation and that part of its cell membrane and that of the surrounding oocyte have long microvilli at the time of its ejection. Several layers of cumulus cells initially surround the secondary oocyte and first polar body, but the ovulated oocytes in the oviducts in the process of being fertilized do not have cumulus cells around them. Partly expelled second polar bodies occur in the oviduct; they are elongated structures that lack organelles and have electron-dense nuclei. A small fertilization cone appears to form around the sperm tail at the time of sperm entry into the egg and an incorporation cone develops around the sperm head in the egg cytoplasm. In three fertilized eggs a small hole was seen in the zona, which was presumably formed by the spermatozoon during penetration. Cortical granules, present in ovarian oocytes, are not seen in fertilized tubal or uterine eggs; release of their contents probably reduces the chances of polyspermy, although at least one polyspermic fertilized egg was seen and several other fertilized eggs had spermatozoa within the zona pellucida. In the zygote the pronuclei come to lie close together, but there was no evidence of fusion. A "yolk mass," which becomes eccentric before ovulation, is extruded by the time the two-cell embryos are formed, but many vacuoles remain in the non-yolky pole of the egg. A shell membrane of variable thickness is present around all uterine eggs but its origin remains undetermined.     

Morphological observations on sperm–egg interactions during in vivo fertilization in the dasyurid marsupial Sminthopsis crassicaudata

The aim of the present study was to determine the morphological changes that take place in the male and female gametes during in vivo fertilization in the Australian marsupial, the fat-tailed dunnart, Sminthopsis crassicaudata. Plastic sections were cut of sperm and eggs recovered from the oviducts of recently mated individuals, and light microscopy of thick, and transmission EM of thin, sections was carried out. It was found that, before penetration of the zona, the spermatozoon came to lie along the outer surface with its rostral tip forming a depression in the zona substance. During penetration, zona material was packed tightly around the spermatozoon, and no large hole was formed. A spermatozoon within the perivitelline space had made contact with the oolemma by way of its apical tip. In a spermatozoon partly incorporated into the ooplasm, fusion appeared to have taken place between its plasma membrane and that of the oolemma. Mucoid coat material became deposited outside the zona at this time; its existence and/or the release of cortical granule content probably prevented polyspermy. Once inside the egg cytoplasm, the sperm head sometimes travelled a considerable distance before chromatin decondensation occurred. In addition, it appeared to rotate somewhat on its axis at this time. Finally, some membranous structures were found around two condensed sperm heads in the ooplasm, which may have been part of the pronuclear envelope. Thus this study on in vivo fertilization in the dunnart documents, for the first time, some aspects of fertilization in an Australian marsupial as seen with the transmission electron microscope; it indicates a few differences from those previously found for the American opossum.