Probing single molecule dynamics.

The room temperature dynamics of single sulforhodamine 101 molecules dispersed on a glass surface are investigated on two different time scales with near-field optics. On the 10(-2)- to 10(2)-second time scale, intensity fluctuations in the emission from single molecules are examined with polarization measurements, providing insight into their spectroscopic properties. On the nanosecond time scale, the fluorescence lifetimes of single molecules are measured, and their excited-state energy transfer to the aluminum coating of the near-field probe is characterized. A movie of the time-resolved emission demonstrates the feasibility of fluorescence lifetime imaging with single molecule sensitivity, picosecond temporal resolution, and a spatial resolving power beyond the diffraction limit.

A retrospective study of preimplantation embryos diagnosed with monosomy by fluorescence in situ hybridization (FISH).

This report is a retrospective study of preimplantation embryos diagnosed with monosomy for chromosomes 13, 15, 16, 18, 21, 22, X and Y on day 3 to determine the rate of true positives, false positives and/or mosaicism and to assess if these embryos are suitable for in vitro fertilization (IVF) transfer. In a one year period, 80 patients went through preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Monosomy was diagnosed in 51 embryos. Fluorescence in situ hybridization (FISH) was then performed on the blastomeres at day 5-7 with commercially available probes using the same probe set that initially identified monosomy for chromosomes 13, 16, 21 and 22 or chromosomes 15, 18, X and Y. Based on FISH analysis, the monosomy diagnosed during routine PGD-AS analysis was confirmed in 17 of the 51 embryos. A euploid result for the specific chromosomes tested was observed in 16 of the 51 embryos while mosaicism was found in the remaining 18 embryos. This results in an estimated false positive rate of 3.8% for a diagnosis of monosomy. Reanalysis of these embryos demonstrates that the majority of monosomy diagnoses represents true monosomy or mosaicism and should be excluded for transfer in IVF. Furthermore, improved understanding from recent emerging data regarding the fate of oocytes in women with advanced maternal age undergoing IVF to the development of early embryos may provide a valuable insight into the mechanism of chromosome mosaicism.

Changes in the secondary structure of apolipoprotein B-100 after Cu2+-catalysed oxidation of human low-density lipoproteins monitored by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectra have been obtained of human low-density lipoproteins (LDL) in H2O and 2H2O buffers. The absorption bands are assigned to vibrations of the lipid and apolipoprotein B-100 components. The analysis of second-derivative spectra allowed an assignment of individual protein bands to alpha-helical, random, coil or beta-structure and beta-turn conformations. Changes in the FTIR spectra after Cu2+-catalysed oxidation of the LDL particles indicate that the structure of apolipoprotein B-100 becomes less ordered, with some alterations of alpha-helical and beta-turn conformation. The main beta-structure absorption at 1620 cm-1 is unaffected by oxidation. Taking into account the resistance to oxidation and the slow H-2H exchange it is suggested that the beta-structure is hidden from external factors whereas other structures are mostly present on the surface of the LDL particle. Oxidation affects mainly the surface region of apolipoprotein B-100 and leads to a structural rearrangement which consequently changes the receptor specificity of the LDL.

Effects of a transposable element insertion on alcohol dehydrogenase expression in Drosophila melanogaster.

Variation in the DNA sequence and level of alcohol dehydrogenase (Adh) gene expression in Drosophila melanogaster have been studied to determine what types of DNA polymorphisms contribute to phenotypic variation in natural populations. The Adh gene, like many others, shows a high level of variability in both DNA sequence and quantitative level of expression. A number of transposable element insertions occur in the Adh region and one of these, a copia insertion in the 5' flanking region, is associated with unusually low Adh expression. To determine whether this insertion (called R142) causes the low expression level, the insertion was excised from the cloned R142 Adh gene and the effect was assessed by P-element transformation. Removal of this insertion causes a threefold increase in the level of ADH, clearly showing that it contributes to the naturally occurring variation in expression at this locus. Removal of all but one LTR also causes a threefold increase, indicating that the mechanism is not a simple sequence disruption. Furthermore, this copia insertion, which is located between the two Adh promoters and their upstream enhancer sequences, has differential effects on the levels of proximal and distal transcripts. Finally, a test for the possible modifying effects of two suppressor loci, su(wa) and su(f), on this insertional mutation was negative, in contrast to a previous report in the literature.

An analysis of transvection at the yellow locus of Drosophila melanogaster.

Studies of a wide variety of organisms have shown that homologous sequences can exert a significant impact on each other, resulting in changes in gene sequence, gene expression, chromatin structure, and global chromosome architecture. Our work has focused on transvection, a process that can cause genes to be sensitive to the proximity of a homologue. Transvection is seen at the yellow gene of Drosophila, where it mediates numerous cases of intragenic complementation. In this article, we describe two approaches that have characterized the process of transvection at yellow. The first entailed a screen for mutations that support intragenic complementation at yellow. The second involved the analysis of 53 yellow alleles, obtained from a variety of sources, with respect to complementation, molecular structure, and transcriptional competence. Our data suggest two ways in which transvection may be regulated at yellow: (1) a transcriptional mechanism, whereby the ability of an allele to support transvection is influenced by its transcriptional competency, and (2) a structural mechanism, whereby the pairing of structurally dissimilar homologues results in conformational changes that affect gene expression.

Calcium regulation of nuclear pore permeability.

The nuclear envelope is an integral part of the structural framework of the nucleus, and is involved in organizing intranuclear events. It serves as a selective barrier, actively transporting proteins required for normal nuclear function and exporting RNA. The movement of molecules across the nuclear envelope is critical for cellular homeostasis, and it allows cells to respond to external events. The only known pathway for direct communication between the cytoplasm and the nucleoplasm of a cell is through the nuclear pore complex. In the past decade, rapid advances have been made in elucidating the structure and function of the nuclear pore complex. Yet, researchers are just beginning to identify some of the regulatory mechanisms controlling transport through the pore complex. The nucleus is surrounded by a Ca2+ storage compartment, which sequesters and releases Ca2+ in response to intracellular second messengers, Recent evidence suggests that the nuclear Ca2+ store may indirectly regulate passive diffusion through the nuclear pore complex. The evidence for Ca2+ regulation of the nuclear pore complex will be discussed, along with the introduction of the simplest, testable model to describe the observations.

Low-density lipoproteins increase intracellular calcium in aequorin-loaded platelets.

Low-density lipoproteins activate isolated human platelets. The mechanism of this activation is unknown, but may involve increased phosphoinositide turnover. We have examined the effect of low-density lipoproteins on intracellular calcium concentrations in platelets loaded with the photoprotein aequorin. The lipoproteins induced concentration-dependent increases in intracellular calcium, associated with shape change and aggregation. These responses could be partially inhibited by the removal of extracellular calcium and by pre-incubation with acetylsalicylic acid. They were also antagonised by agents which increase cellular concentrations of cyclic adenosine and guanosine monophosphates. It is not clear whether the platelet-lipoprotein interaction involves a 'classical' lipoprotein receptor.

The postnatal development of corticotrigeminal projections in the cat.

The postnatal development of corticotrigeminal projections was studied in kittens following 3H-amino acid injections into the face area of the primary somatosensory cortex. Corticofugal axons grow into the brainstem and form the pyramidal tract prenatally. Corticotrigeminal projections begin to develop at the end of the first postnatal week. The earliest corticotrigeminal axons grow out of the pyramidal tract caudally and project into laminae III-V of the spinal trigeminal (Vs) nucleus caudalis. During the second postnatal week, corticotrigeminal axons grow out of the pyramidal tract in a caudal to rostral sequence and project up to the ventromedial borders of Vs-interpolaris, Vs-oralis, and to the principal trigeminal nucleus. Corticotrigeminal axons pause at the periphery of these nuclei for 1-2 days before penetrating the trigeminal neuropil and forming terminal arborizations in a centripetal direction. Coincident with the development of cortical projections to the principal trigeminal nucleus, some of the labeled axons which were in lamina III of Vs-caudalis project into lamina I and terminate. This sequence of development of corticotrigeminal projections closely parallels, albeit at a later time, the sequence of formation of the trigeminal nuclei, suggesting that the temporal sequence of cytogenesis of trigeminal neurons may be a factor which regulates their order of innervation by afferents. Corticotrigeminal projections develop bilaterally and, during the second postnatal week, are relatively equal in density in the ipsilateral and contralateral nuclei. Many of the ipsilateral corticotrigeminal projections are lost, however, after the second postnatal week, so that by the fourth postnatal week, corticotrigeminal projections are mainly contralateral and adultlike in their distribution. It remains to be determined whether the transience of ipsilateral corticotrigeminal projections is due to selective elimination of axon collaterals or to neuronal death.

Tubal pregnancy with normal hysterosalpingogram and negative serum pregnancy test.

A patient undergoing evaluation for primary infertility with regular menses had a normal hysterosalpingogram in the follicular phase and underwent diagnostic laparoscopy the next day, with the finding of a tubal pregnancy. The patient had a negative serum pregnancy test. Although a normal hysterosalpingogram and negative serum pregnancy test are each rare with a tubal pregnancy, the combination of the two occurring in a tubal pregnancy is previously unreported and did not exclude the diagnosis in this case.

Prevention of postoperative adhesions in the rat by in situ photopolymerization of bioresorbable hydrogel barriers.

OBJECTIVE: To assess the efficacy of a novel resorbable hydrogel barrier for preventing postoperative adhesions in animals. METHODS: A hydrogel barrier was formed in situ by photopolymerizing a solution of a macromolecular prepolymer in buffered saline using long-wavelength ultraviolet light. Two models in the rat were evaluated. In a primary adhesion model, devascularization and serosal injury were performed on the uterine horns using bipolar electrocautery. The prepolymer solution was applied to the horns and illuminated to form the barrier. On the seventh postoperative day, the fraction of the length of the horns involved in adhesions was scored, as was the quality of the adhesions. In a readhesion model, adhesions were formed as described and were surgically lysed on the seventh day, then were treated subsequently with the barrier and scored after 7 additional days. Each group in both models consisted of seven animals per treatment condition. Four prepolymer concentrations were examined in the primary adhesion model, and the optimal one was examined in the readhesion model. RESULTS: A conformal hydrogel barrier coating was formed upon in situ photopolymerization and adhered to the treated tissues. No residual hydrogel barrier was observed 7 days after application of the optimal gel concentrations. In the primary adhesion model, the mean fraction of the horns involved in adhesions was reduced significantly, from 76% in controls to 10% (P < .0001), and treatment with a 10% solution of prepolymer was determined to be optimal (P = .025). In the readhesion model, surgical lysis of adhesions alone did not reduce adhesions significantly (from 86% to 79%; P = .3), whereas lysis with barrier treatment did (79% to 28%; P = .002). CONCLUSIONS: In situ photopolymerization allowed the formation of adherent, conformal barriers, which demonstrated high efficacy in the prevention of adhesion formation and reformation in animals. This efficacy and ease of use warrant clinical evaluation.

The corticotrigeminal projection in the cat. A study of the organization of cortical projections to the spinal trigeminal nucleus.

The projection from the cerebral cortex to the spinal trigeminal nucleus has been studied light microscopically in adult cats. Both orthograde degeneration and orthograde intra-axonal labeling techniques have been applied. Our results indicate that the projection from the coronal gyrus (face area of primary somatosensory cortex) to the spinal trigeminal complex is somatotopically organized. In subnucleus caudalis this somatotopy is organized dorsoventrally and appears to match the somatotopic distribution of the divisional trigeminal afferents. Hence cortical fibers originating from the posterior coronal gyrus (upper representation) project ventrolaterally into caudalis where division I trigeminal afferents terminate. Likewise cortical fibers from the anterior coronal gyrus (jaw and tongue representation) terminate dorsomedially in caudalis to overlap with division III trigeminal afferents. In contrast, the distribution of corticofugal afferents to the rostral spinal trigeminal subnuclei (pars interpolaris and oralis) is organized mediolaterally. Therefore in these subnuclei the cortical projection does not appear to overlap the dorsoventral lamination of the divisional trigeminal afferents. In addition, our results suggest that the cortical projection to subnucleus caudalis includes fibers which terminate in the marginal zone (lamina I) and its extensions into the spinal trigeminal tract (the interstitial cells of Cajal). We have been unable to document a projection from the proreate gyrus to the spinal trigeminal complex.

Sensorimotor cortical projections to the marginal zone of the trigeminal subnucleus caudalis.

An ultrastructural investigation of the marginal zone (lamina I) of the spinal trigeminal subnucleus caudalis was carried out in 7 adult cats at 30 h through 7 days after ablations of face area of the contralateral sensorimotor cortex. Corticofugal boutons were observed to undergo electron-dense degeneration in the marginal zone beginning 4 days after the cortical lesion. These boutons were small (1--2 micrometers), widely dispersed and made synaptic contacts onto small dendrites or dendritic spines. These new observations indicate that cortical inhibition and facilitation of ascending orofacial sensation may be mediated in part by a direct pathway to the marginal zone.

Synapse development within the spinal trigeminal nucleus.

Pars interpolaris of the spinal trigeminal nucleus of kittens has been studied with the electron microscope at birth and at several subsequent ages during the first month of life. Attention has been given to ultrastructural maturational changes that occur in this neuropil, especially events in synaptogenesis. The results of this investigation include the following observations: (1) the neuropil, even at the earliest ages studied (three-hour-old kittens), is strikingly mature, necessitating a quantitative assessment in order to determine subtle developmental changes in synaptic patterns; (2) the number of axoaxonic contacts at birth are few, and their emergence is essentially a postnatal phenomenon; (3) it appears that the immature Gray type II or symmetrical synapse possesses distinct cleft material and dense, parallel membrane specializations. Synaptic vesicle accumulation at this contact appears to occur after the membrane specializations have formed. A previous study by Kerr26 has shown a reduced potential for primary afferent reorganization with the spinal trigeminal nucleus when kittens are subjected to trigeminal rhizotomy after three days of age. Our observations on the development of axoaxonic synaptic arrangements in the neonatal period may provide an explanation for these earlier results.

A morphometric study of the effects of maturation and aging on synaptic patterns in the spinal trigeminal nucleus of the cat.

Morphometric methods have been used to study the synaptic and terminal patterns in cat trigeminal nucleus, pars interpolaris, during development and aging. Ages 1, 3, 6, 11, 16, 21, 27, 110, 600 days and 8 and 11 years were studied. Both proportions and densities (number per unit area) of certain terminals and synapses showed significant changes with age. Axoaxonic synapses especially showed two major periods of increase (3-6 days and 21-27 days). The values of most parameters increased in the 21-27 day period to peak levels and then decreased gradually with age. The results indicate two separate critical synaptogenic periods of development and a loss of synaptic elements in aging. Factors contributing to these changes are discussed as is the potential for plasticity in the different afferents at each period.

Recombinant tissue plasminogen activator reduces adhesion formation in a rabbit uterine horn model.

To evaluate the potential benefit of recombinant tissue plasminogen activator (rt-PA) as an agent for reducing postoperative adhesions, a rabbit uterine horn model was studied. Fifty-five rabbits underwent laparotomy, at which time the uterus was abraded with scalpel and a thermal injury was induced with electrocautery. Before abdominal closure, rt-PA was applied topically in various dosages. Adhesions were evaluated at a second laparotomy performed 2 weeks later. Treatment significantly reduced both adhesion quantity (P less than 0.001) and adhesion density (P less than 0.001). In the second phase of the study, the efficacy of rt-PA as an adjunct to surgical adhesiolysis was investigated. Again, a dose-related treatment effect was observed (P less than 0.001). No wound healing or bleeding complications were seen.

Short-term Lupron or danazol therapy for pelvic endometriosis.

OBJECTIVE: To compare the efficacy of a 3-month trial of leuprolide acetate (LA; Lupron; TAP Pharmaceuticals, Deerfield, IL) versus danazol (Danocrine; Scenofi Winthrup Pharmaceuticals, New York, NY) therapy on laparoscopically proven endometriosis. DESIGN: Endometriosis severity was assessed at the time of laparoscopy and patients were randomized to receive 0.1 mg SC LA (n = 22) or 800 mg danazol orally (n = 18) daily for 3 months. A second laparoscopy and/or laparotomy was performed to assess the change in the extent of endometriosis and for surgical therapy. SETTING: Private practice at a university-affiliated hospital. PATIENTS: Forty patients with mild, moderate, or severe endometriosis. Ten patients were excluded. INTERVENTION: Three-month treatment randomly assigned to either LA or danazol. MAIN OUTCOME MEASURES: Prospective measurement of American Fertility Society endometriosis scores and size of ovarian endometriomata before and after therapy via laparoscopy. RESULTS: The mean endometriosis score including adhesions decreased significantly from 36 +/- 4.9 to 29 +/- 5.0 (mean +/- SEM) with LA and from 34 +/- 6.4 to 29 +/- 6.5 with danazol. The mean laparoscopic endometriosis score not including adhesions decreased from 27 +/- 3.5 to 22 +/- 3.4 with LA and 22 +/- 3.5 to 19 +/- 3.1 with danazol. Seven of 18 (39%) endometriomata responded to LA and 6 of 15 (40%) endometriomata responded to danazol. CONCLUSION: We conclude that both danazol and LA will reduce endometriosis scores after a 3-month course of therapy. Larger clinical trials are needed to compare short courses of therapy with the more established 6-month trials. A 3-month versus a 6-month course of therapy offers obvious benefits including decreased cost and decreased side effects.

Growth factor effects on endometrial epithelial cell differentiation and protein synthesis in vitro.

OBJECTIVE: To develop a baseline for projected studies of a rat endometriosis model. DESIGN: We investigated the effects of two macrophage-related growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), on proliferation, in vitro differentiation, and protein secretion of uterine epithelial cells from immature rats. Uterine epithelial cells grown on matrix covered filters were treated with growth factors (GFs) or estrogen and/or P. Incorporation of [35S]methionine by polarized uterine epithelial cell proteins and secretion of labeled proteins into apical and basal culture medium were examined. SETTING: Department of Cell Biology, Baylor College of Medicine, Houston, Texas. MAIN OUTCOME MEASURES: Cell associated and secreted proteins were resolved by gel electrophoresis, fluorography, and immunoblotting. Proliferation was quantified by cell counts in parallel cultures by hemocytometer. RESULTS: Estrogen and P increase protein synthesis by uterine epithelium. Transforming growth factor-beta depressed protein synthesis and secretion in uterine epithelial cells. Platelet-derived growth factor appears to have no effect on epithelial protein synthesis or secretion and does not modulate the effect of TGF-beta. Estrogen and P increase complement component 3 (C3) production by epithelial cells. CONCLUSION: Macrophage-secreted GFs may play a role in the development and maintenance of ectopic endometrial tissue. Both TGF-beta and ovarian steroids may participate in the dynamic regulation of protein synthesis by ectopic uterine epithelium. These molecules may indirectly affect the macrophage-stromal axis through nonspecific modulation of C3 secretion. Platelet-derived growth factor appears to have no direct effect on uterine epithelial cells. The recognized effect of PDGF on ectopic endometrial tissue is most likely mediated via the stromal component.

Efficacy of a resorbable hydrogel barrier, oxidized regenerated cellulose, and hyaluronic acid in the prevention of ovarian adhesions in a rabbit model.

OBJECTIVE: To compare the efficacy of a resorbable hydrogel material with oxidized regenerated cellulose and hyaluronic acid in an ovarian adhesion model. DESIGN: Controlled, blinded, and randomized study involving female rabbits. SETTING: Academic research environment. INTERVENTIONS: A water-soluble hydrogen precursor was applied to the ovary as a liquid and converted to a hydrogel by exposure to long wavelength ultraviolet light, a 0.4% solution of hyaluronic acid was applied to the ovary, or an oxidized regenerated cellulose patch was applied to the ovary after wedge resectioning. MAIN OUTCOME MEASURES: Extent and severity of adhesion formation. RESULTS: Application of the hydrogel reduced adhesion formation by 88%. Neither oxidized regenerated cellulose nor hyaluronic acid reduced adhesion formation. CONCLUSION: The photopolymerized, resorbable hydrogel material is highly effective for the reduction of periovarian adhesions in this model.

Probing the spatial dependence of the emission spectrum of single human retinal lipofuscin granules using near-field scanning optical microscopy.

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale approximately 150 nm.

Anterior encephalocele.

Eighteen cases of anterior encephalocele treated at the University of Malaya Hospital between 1970 and 1980 are discussed, and the literature concerning this defect is reviewed. A detailed analysis of the microscopic abnormalities present in the surgical specimens is included, along with the relevant radiographic and demographic data. Anterior encephalocele is more common in Southeast Asia than elsewhere. The possible ethnographic and geographic implications are presented, as well as a discussion of the relevant embryology, in attempting to define possible etiologies for this malformation. The author's surgical approach to the repair of this defect and reasons for preferring a transcranial, intradural approach are described. Potential complications are enumerated.