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Weak bases partially activate Xenopus eggs and permit changes in membrane conductance whilst inhibiting cortical granule exocytosis.

At extracellular pH values close to their pKa values the weak bases, ammonia and procaine, elicited a series of events in non-activated Xenopus eggs, some of which resembled those normally occurring at fertilization. These included: (1) a transient increase in membrane conductance; (2) modification of the microvilli; (3) thickening of the cortical cytoplasm and displacement of the cortical granules; (4) pigment accumulation; (5) contractions and shape changes. However, these eggs did not undergo the cortical reaction nor emit the second polar body. Cortical granule exocytosis of inseminated or artificially stimulated eggs was inhibited if the eggs had been previously treated for 15 min with the weak base and subsequently rinsed. Multiple sperm-entry sites were exhibited by the inseminated eggs, suggesting polyspermy. However, such eggs did not cleave and although sperm had fused with the egg membrane, they were not incorporated. Nevertheless, a transient increase in membrane conductance was evoked, which was longer in duration and had a slightly different form from that normally accompanying fertilization. In these eggs cortical granules were intact but displaced away from the plasma membrane. Prolonged contact with the weak base rendered eggs totally unresponsive to sperm or artificial stimuli but eggs recovered when rinsed sufficiently. These effects of weak bases on unfertilized Xenopus eggs or during fertilization were completely absent at pH 7.4. Although changes in intracellular pH or Ca2+ may be involved in these phenomena, direct action by the weak base itself cannot be ruled out.

Ammonium Chloride↗

Type I collagen permits invasive behaviour by retinal pigmented epithelial cells in vitro.

Epithelial cells cultured on type I collagen gels adopt a typical apical--basal polarity and undergo differentiation. We have compared the behaviour of chick embryo retinal pigmented epithelial (RPE) cells on collagen and on plastic with and without gelatin coats. RPE cell proliferation was similar on all three substrata, and post-confluent cultures exhibited multilayering. On plastic and gelatin-coated plastic, dome formation, typical of transporting epithelia, occurred. On type I collagen gels, however, dome formation did not occur, but rather invasion of the gel matrix by cords of epithelial cells took place. In contrast, invasive behaviour of the cells was markedly reduced on type IV coated collagen gels, particularly in the presence of laminin. These results illustrate the prominent role of the extracellular matrix on phenotypic expression by RPE cells and may represent a more general phenomenon.

Animals↗

Ultraviolet microbeam irradiation of chromosomal spindle fibres shears microtubules and permits study of the new free ends in vivo.

Irradiation of birefringent chromosomal spindle fibres in crane-fly spermatocytes in metaphase I or anaphase I produces an area of reduced birefringence (ARB) on the fibre. This ARB moves poleward and is lost at the pole. Ultrastructural and immunofluorescence analysis of ARBs obtained by irradiation with monochromatic ultraviolet light of wavelength 260 nm shows that the microtubules in the irradiated area are depolymerized, though the rest of the spindle appears unaffected. The area of microtubule depolymerization moves poleward with the ARB, and once the ARB reaches the pole the irradiated half-spindle appears normal. The motion of the ARB, therefore, appears to be due to the behaviour of the sheared microtubules in the chromosomal spindle fibre. The relative stability of the sheared microtubules shows that chromosomal fibre microtubules are not dynamically unstable, as are microtubules under certain conditions in vitro. However, ARB motion may be due to a moderated version of dynamic instability. Possible models for ARB motion are discussed.

Animals↗

The use of peripheral nerve grafts to enhance neuronal survival, promote growth and permit terminal reconnections in the central nervous system of adult rats.

During both development and regeneration, the survival of neurones and the growth of axons are controlled by inherent neuronal properties, conditions in the axonal environment, and the establishment of appropriately timed and specific functional contacts. To study the effects of extrinsic influences on the survival, growth and connectivity of axotomized neurones in the mature mammalian CNS, we replaced the optic nerve in adult rats with segments of autologous peripheral nerve (PN) and used morphometric techniques, neuroanatomical tracer substances and immunological cell markers to examine retinal ganglion cells (RGCs), their axons in the PN grafts and their terminals in the superior colliculi (SC) of these animals. We observed that: (1) the survival of axotomized RGCs was enhanced by the PN grafts; (2) in the PN-grafted eyes, approximately 20% of the surviving RGCs regrew their axons into the grafts and (3) some of the RGC axons that regenerated along the PN grafts bridging the eye and the tectum re-entered the SC, arborized and made synaptic contacts with tectal neurones. It is not known if the terminal connections established between RGCs and cells in the SC are appropriate, functional or capable of influencing the long-term survival of their cells of origin.

Animals↗

Isolation of an Escherichia coli mutant which permits the high-level expression of a streptomycin-resistance gene derived from Pseudomonas aeruginosa.

Pretreatment with streptomycin at a low concentration influenced the susceptibility to streptomycin of a strain of Escherichia coli carrying a streptomycin-resistance plasmid, pSA1700, derived from Pseudomonas aeruginosa. This phenomenon was due to a mutation that occurred at about 10(-8)-10(-10) of frequency in a regulatory gene involved in gene expression on the chromosome of E. coli. A product encoded by the regulatory gene on the chromosome of E. coli might normally repress gene expression by binding to part of the promoter region of the streptomycin-resistance gene derived from P. aeruginosa.

Cloning, Molecular↗

Identification of the nonA and nonB loci of Bacillus subtilis Marburg permitting the growth of SP10 phage.

Mutational inactivation of both nonA and nonB genes are required for the permissiveness of Bacillus subtilis Marburg cells to infection by phage SP10. By transformational analysis of the nonA strain with DNAs from gently lysed protoplasts carrying the integrative plasmid pMUTIN (em) insertions in every 20 kb along the whole chromosome, we have identified the nonA to be the cured state of endogenous prophage SPbeta. Direct DNA sequencing, on the other hand, revealed one nonsense mutation of nonB in ydiR, which is a component gene of the intrinsic restriction system BsuMR of B.subtilis Marburg. Introduction of the wild type ydiR into the nonB strain at aprE locus resulted in complementation of nonB. Furthermore, as the SP10 genome was found to possess multiple BsuM target sites, it is considered that SP10 can infect and multiply in B.subtilis cells, which are SPbeta free and possess a defective BsuMR restriction system.

Bacillus subtilis↗

Dot-blot hybridization using digoxigenin-labeled cDNA probe complementary to the S1 gene of avian infectious bronchitis virus permits discrimination between virus strains.

Digoxigenin-dUTP-labeled DNA probe was prepared from a cDNA clone complementary to the gene encoding S1 region of the spike protein of infectious bronchitis coronavirus (IBV) strain M41. The probe exclusively reacted with four strains at 56 degrees C which were grouped to the same serotype as the strain used for the probe. In contrast, at 68 degrees C, the probe reacted only with the homologous strain and did not react even with the strains belonging to the same serotype. The dot-blot hybridization thus appeared serotype-specific at 56 degrees C and strain-specific at 68 degrees C. In addition, it was revealed that the S1 gene has some nucleotide sequence variation even among strains in the same serotype. This technique should be applied to determining serotypes of the virus isolates and to differentiating field isolates from the vaccine strain.

Animals↗

Spontaneous, but not induced, puberty permits adequate bone mass acquisition in adolescent Turner syndrome patients.

Lumbar L2-L4 bone mineral density (BMD) values were measured in 37 adolescent and young adult Turner syndrome patients. Nine had developed spontaneous puberty and had had regular menses since menarche (12.55 years +/- 1.17 years) to the time of BMD evaluation (14.96 years +/- 1.26 years). In the other 28, puberty was induced with increasing doses of oral ethinyl estradiol (2.5-10.0 microg/day, for 2 years) and later administration of estrogen/gestagen therapy up to the time of BMD evaluation. In 18, the adolescent group, menarche appeared at 14.68 years +/- 0.63 years and BMD was evaluated at 17.77 years +/- 0.70 years, and in the other 10, the young adult group, menarche appeared at 14.47 years +/- 0.53 years and BMD was evaluated at 20.90 years +/- 0.68 year. BMD values were in the normal range in those who had developed spontaneous puberty (Z score values, -0.24 +/- 0.22) and in the osteopenia range in those in whom puberty was induced (Z score values, -2.09 +/- 0.79 and -2.18 +/- 0.32 for the adolescent and young adult groups, respectively) p < 0.0001. Height Z score values were similar in all three groups (-3.45 +/- 0.77, -3.15 +/- 0.83, and -3.08 +/- 0.33, respectively). No significant differences in calcium intake or physical activity were found among groups. Neither the karyotype distribution nor growth hormone (GH) therapy (five in the spontaneous puberty and six in the induced puberty groups had been treated for a 3.5- to 4.4-year period) explained the differences in BMD values. Because the main difference between groups was the availability of estrogens to bone tissue from infancy to menarche and of estrogens/gestagens from then on up to the time of BMD evaluation, our results suggest that normal gonadal function from infancy to adulthood may be required for adequate bone mass peaking. Early detection of osteopenia and improvement in general measures for adequate bone mass peaking (calcium intake and physical activity) should be considered mandatory in the health care of these patients.

Absorptiometry, Photon↗

Quantitative determination of oestradiol-17 beta hydroxysteroid dehydrogenase: increased sensitivity by HPLC separation of the hormones permits the measurement of enzyme activity in cryostat sections.

The activity of the oestradiol-17 beta hydroxysteroid dehydrogenase in human endometrial and breast cancer specimens was determined by the NAD-dependent conversion of oestradiol-17 beta to oestrone. The sensitivity of the determination was improved by the separation of the hormones by HPLC. We are now able to determine oestradiol-17 beta hydroxysteroid dehydrogenase quantitatively in cryostat sections. A clear correlation of serum progesterone levels and oestradiol-17 beta hydroxysteroid dehydrogenase activity in the endometrium was demonstrated, and we found a more than 30-fold increase in enzyme activity after the progesterone surge. In contrast, in breast cancer samples, we found no correlation between oestradiol-17 beta hydroxysteroid dehydrogenase and the measured serum parameters.

17-Hydroxysteroid Dehydrogenases↗

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion.

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.

Animals↗

The oral contraceptive pill effectively permits cycle batching for an intermittent in vitro fertilization programme in Trinidad and Tobago.

Assisted reproductive technology (ART) in small island states like Trinidad and Tobago is usually provided in batches so as to minimize the cost of providing the service. As a result, patients 'cycles have to be synchronized in order to coincide with the arrival of a visiting embryologist. This is a retrospective study which evaluates the experience of pre-treatment with an oral contraceptive pill (OCP) as a means of batching cycles for an intermittent ART programme. Seventy-four in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles in which OCP usage was employed (Group A), were compared with 121 cycles which did not require pharmaceutical manipulation (Group B). In both groups more than 50% of women were older than 36-years. Two cycles were cancelled in Group A and seven in Group B, because of poor ovarian response. Although the pregnancy rate per treatment cycle was higher in Group A than in Group B (26.3% vs 17.3%), this difference was not significant. More spontaneous miscarriages occurred in the non-OCP women and ovarian cyst formation was more common in these women. The authors experience indicates that the OCP is a simple, cheap and efficient means of batching patients for an intermittent ART programme and can be utilized in other small ART centres.

Adult↗

Absence of major histocompatibility complex class I on neural stem cells does not permit natural killer cell killing and prevents recognition by alloreactive cytotoxic T lymphocytes in vitro.

Potential applications of neural stem cells (NSCs) for transplantation requires understanding myosin heavy chain (MHC) expression and the ability of T cells and natural killer (NK) cells to recognize this progenitor population. Cells from the cortices of day-13 embryonic (E13) B6 (H-2(b)) mice were explanted and cultured to expand NSCs. Analysis of P2-P17-cultured cells using anti-MHC class I/II monoclonal antibodies (mAbs) showed marginal expression of both products. Although recombinant murine interferon-gamma (rmIFN gamma) exposure did not alter the multipotential capacity of these stem cells, titration of mrIFN gamma NSC cultures demonstrated that MHC molecules could be strongly upregulated after addition of 3 ng/ml rmIFN gamma for 60 hours. To assess the susceptibility of NSCs with low or absent versus high levels of MHC expression to lysis by cytotoxic T lymphocyte (CTL) and NK populations, untreated and rmIFN gamma-treated NSC target cells were examined. Untreated NSCs were not recognized by BALB/c (H-2(d)) allospecific anti-H-2(b) CTL, consistent with the mAb findings; however, upregulation of MHC products on both early and later passaged NSCs resulted in their efficient lysis by CTL. NK cells were prepared from syngeneic B6 or allogeneic BALB/c mice. Although NK cells effectively killed control YAC-1 target cells, these effectors did not kill MHC-deficient (or expressing) NSC targets. Thus, similar to hematopoietic, embryonic, and mesenchymal stem cell populations, unmanipulated NSCs are not readily killed by T and NK cells. These findings suggest that following transplant into syngeneic or allogeneic recipients, NSCs may exhibit diminished susceptibility to clearance by host T- and NK-cell populations.

Animals↗

Prolactin permits the expression of a circadian variation in lipogenic responsiveness to insulin in hepatocytes of the golden hamster (Mesocricetus auratus).

Lipogenesis was determined at two times (07.00 and 16.00 h) during a 14-h daily photoperiod (08.00-22.00 h) in freshly prepared hamster hepatocytes with or without addition of insulin. The hamsters were pretreated for 5 days with bromocriptine (to inhibit prolactin secretion), bromocriptine and prolactin replacement, or control saline injections. Lipogenesis was determined by incorporation of [14C]acetate into total cell lipids over a 30-min interval. Lipogenesis was three times greater at 07.00 than at 16.00 h and insulin was effective in stimulating further lipogenesis only at 07.00 h. Bromocriptine pretreatment severely reduced incorporation of radiolabel at 07.00 h to levels comparable with controls at 16.00 h and completely inhibited the stimulatory effect of insulin at 07.00 h. Prolactin replacement in bromocriptine-treated hamsters reversed the inhibitory effect of bromocriptine on hepatocyte lipogenesis and promoted dramatic lipogenic responses to insulin at 07.00 h. These results indicate that insulin stimulates hepatic lipogenesis only during some portion of a day and that prolactin facilitates the lipogenic response.

Animals↗

Prolactin permits the expression of a circadian variation in insulin receptor profile in hepatocytes of the golden hamster (Mesocricetus auratus).

Insulin receptor profile was determined in freshly isolated hamster hepatocytes at two times (07.00 and 16.00 h) during a 14-h daily photoperiod (08.00-22.00 h). The hamsters were pretreated for 5 days with bromocriptine (to inhibit prolactin release), bromocriptine and prolactin replacement, or control saline injections. The insulin receptor profile was determined by Scatchard plot analysis. The insulin receptor number (high and low affinity) was three times greater at 07.00 than at 16.00 h among saline-injected controls. However, their affinities did not differ. Bromocriptine pretreatment reduced (70%) both the high and low affinity receptor numbers and increased the affinity of the high affinity receptor at 07.00 h. Prolactin replacement in bromocriptine-treated hamsters restored the receptor profile at 07.00 h to control values. These data indicate that prolactin facilitates the expression of a circadian variation in insulin receptor profile.

Animals↗