On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.

Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation.

Static ocular counterroll reflex in skew deviation.

OBJECTIVE: The static ocular counterroll (OCR) reflex generates partially compensatory torsional eye movements during head roll. It is mediated by the utricle in the inner ear. Skew deviation is a vertical strabismus thought to be caused by imbalance in the utriculo-ocular pathway. We hypothesized that if skew deviation is indeed caused by damage to this reflex pathway, patients with skew deviation would show abnormal OCR. METHODS: Eighteen patients with skew deviation caused by brainstem or cerebellar lesions and 18 normal participants viewed a target at 1 m. Ocular responses to static passive head roll-tilts of approximately 20° were recorded using search coils. Static OCR gain was calculated as the change in torsional eye position divided by the change in head position during sustained head roll. Perception of the subjective visual vertical (SVV) was also measured. RESULTS: Group mean OCR gain was reduced by 45% in patients. At an individual level, OCR gains were asymmetric between eyes and between torsional directions in 90% of patients. In addition, the hypotropic eye incyclotorting gain was lower than the hypertropic eye excyclotorting gain during head roll toward the hypotropic eye in 94% of patients. No consistent pattern of gain asymmetry was found during head roll toward the hypertropic eye. The SVV was tilted toward the hypotropic eye. CONCLUSION: Static OCR gain is significantly reduced in skew deviation. Interocular and directional gain asymmetries are also prevalent. The asymmetries provide further evidence that disruption of the utriculo-ocular pathway is a mechanism for skew deviation.

Patterns of ocular oscillation in oculopalatal tremor: imaging correlations.

OBJECTIVE: To determine patterns of nystagmus in oculopalatal tremor (OPT, also designated oculopalatal myoclonus) and correlate them with MRI changes in the inferior olivary nuclei (ION). Mixed torsional-vertical pendular nystagmus (PN) in OPT has been considered to signify unilateral brainstem damage and symmetric vertical nystagmus considered to indicate bilateral disease. METHODS: Ocular oscillations were analyzed in 22 patients with OPT, 20 from focal brainstem lesions, with or without cerebellar lesions, and two from the progressive ataxia and palatal tremor syndrome. MRI was performed in all patients. RESULTS: Patients had mainly vertical oscillations with varied combinations of torsional and horizontal components. Fourteen patients had binocular symmetry of PN and eight showed dissociated PN. MRI demonstrated ION signal change, unilateral in 14 and bilateral in eight. Unilateral olivary changes were associated with symmetric PN in six and with dissociated nystagmus in eight patients. Bilateral olivary changes were visible in eight patients with symmetric nystagmus. Dissociated PN was associated with MR pseudohypertrophy of ION on the side of the eye with greater vertical amplitude of oscillation. Notably, four patients never developed palatal tremor despite ION signal change. OPT resolved in one patient after 20 years and was markedly reduced in another patients after 6 years. CONCLUSIONS: Dissociated pendular nystagmus predicted asymmetric (unilateral) inferior olivary pseudohypertrophy on MRI with accuracy, but symmetric pendular nystagmus was associated with either unilateral or bilateral signal changes in the inferior olivary nucleus. Instability of eye velocity to position integration from damage to the paramedian tract projections and denervation of the dorsal cap of the inferior olive are proposed mechanisms of the pendular nystagmus.

Saccades in children with spina bifida and Chiari type II malformation.

BACKGROUND: Saccades are essential for optimal visual function. Chiari type II malformation (CII) is a congenital anomaly of the cerebellum and brainstem, associated with spina bifida. OBJECTIVE: To investigate the effects of CII on saccades and correlate saccadic parameters with brain MRI measurements. METHODS: Saccades were recorded in 21 participants with CII, aged 8 to 19, using an infrared eye tracker. Thirty-nine typically developing children served as controls. Participants made saccades to horizontal and vertical target steps. Nineteen participants with CII had MRI. Regression analyses were used to investigate the effects of spinal lesion level, number of shunt revisions, presence of nystagmus, and midsagittal MRI measurements on saccades. RESULTS: Saccadic amplitude gains, asymptotic peak velocities, and latencies did not differ between the control and CII groups (p > 0.01). No significant differences were found between saccadic gains, asymptotic peak velocities or latencies, and spinal lesion level, number of shunt revisions, presence of nystagmus, or MRI measurements. CONCLUSIONS: Saccades were normal in most participants with Chiari II malformation (CII). Neural coding of saccades is robust and is typically not affected by the anatomic deformity of CII.

Expression of lux genes in a clinical isolate of Streptococcus pneumoniae: using bioluminescence to monitor gemifloxacin activity.

A clinical isolate of Streptococcus pneumoniae was transformed with a plasmid containing the lux operon of Photorhabdus luminescens that had been modified to function in gram-positive bacteria. Cells containing this plasmid produced light stably and constitutively, without compromising the growth rate. Light output was correlated with measurements of optical density and viable counts during exponential growth and provided a sensitive, real-time measure of the pharmacodynamics of the fluoroquinolone gemifloxacin.

The vertical vestibulo-ocular reflex, and its interaction with vision during active head motion: effects of aging.

The effects of aging on the vertical vestibulo-ocular reflex (VOR), and its interactions with vision during active head motion had not been investigated. We measured smooth pursuit, combined eye-head tracking, the VOR, and its visual enhancement and cancellation during active head motion in pitch using a magnetic search coil technique in 21 younger (age < 65) and 10 elderly (age > or = 65) subjects. With the head immobile, subjects pursued a target moving sinusoidally with a frequency range of 0.125 to 2.0 Hz, and with peak target accelerations (PTAs) ranging from 12 to 789 degrees /s(2). Combined eye-head tracking, the VOR in darkness, and its visual enhancement during fixation of an earth-fixed target (VVOR) were measured during active sinusoidal head motion with a peak-to-peak amplitude of 20 degrees at frequencies of 0.25, 0.5, 1.0 and 2.0 Hz. The efficacy of VOR cancellation was determined from VOR gains during combined eye-head tracking. VOR and VVOR gains were symmetrical in both directions and did not change with aging, except for reduced gains of the downward VOR and VVOR at low frequency (0.25 Hz). However, in the elderly, smooth pursuit, and combined eye-head tracking gains and the efficacy of cancellation of the VOR were significantly lower than in younger subjects. In both the young and elderly groups, VOR gain in darkness did not vary with the frequency of active head motion while the gains of smooth pursuit, combined eye-head tracking, and VVOR declined with increasing target frequency. VOR and VVOR performance in the elderly implicates relative preservation of neural structures subserving vertical vestibular smooth eye motion in senescence.

Opsoclonus in three dimensions: oculographic, neuropathologic and modelling correlates.

Opsoclonus is a dyskinesia consisting of involuntary, arrhythmic, chaotic, multidirectional saccades, without intersaccadic intervals. We used a magnetic scleral search coil technique to study opsoclonus in two patients with paraneoplastic complications of lung carcinoma. Eye movement recordings provided evidence that opsoclonus is a three-dimensional oscillation, consisting of torsional, horizontal, and vertical components. Torsional nystagmus was also present in one patient. Antineuronal antibody study revealed the presence of anti-Ta (Ma2 onco-neuronal antigen) antibodies in one patient, which had previously been associated only with paraneoplastic limbic encephalitis and brainstem dysfunction, but not opsoclonus, and only in patients with testicular or breast cancer. Neuropathologic examination revealed mild paraneoplastic encephalitis. Normal neurons identified in the nucleus raphe interpositus (rip) do not support postulated dysfunction of omnipause cells in the pathogenesis of opsoclonus. Computer simulation of a model of the saccadic system indicated that disinhibition of the oculomotor region of the fastigial nucleus (FOR) in the cerebellum can generate opsoclonus. Histopathological examination revealed inflammation and gliosis in the fastigial nucleus. This morphological finding is consistent with, but not necessary to confirm, damage to afferent projections to the FOR, as determined by the model. Malfunction of Purkinje cells in the dorsal vermis, which inhibit the FOR, may cause opsoclonus by disinhibiting it.

Use of green fluorescent protein in visualisation of pneumococcal invasion of broncho-epithelial cells in vivo.

The pneumococcus is the principle cause of bacterial pneumonia and also a major cause of bacterial meningitis. The mechanisms and sites of pneumococcal adherence and invasion of the respiratory tract in vivo are not clear however. We have made pneumococci expressing green fluorescent protein (GFP) and used it to trace pneumococcal adherence and invasion in vivo. By using GFP pneumococci we have shown bacterial adherence and invasion of broncho-epithelial cells in vivo by 4 h post-infection, with increases in pneumococcal invasiveness by 24 h. Using confocal image analysis we have shown varying levels of pneumococcal penetration and internalisation into host cells, as well as translocation through epithelial layers. To our knowledge this is the first report of pneumococcal invasion and cellular translocation in vivo.

Central nystagmus induced by deep-brain stimulation for epilepsy.

PURPOSE: The goal of the present study was to describe the localization of central nystagmus induced as a side effect of electrical deep-brain stimulation for epilepsy. METHODS: Bilateral deep-brain stimulating electrodes were inserted in the centromedian nucleus of the thalamus to control seizures in a patient with intractable epilepsy. RESULTS: Cathodal high-frequency stimulation through the deepest contact of each electrode elicited cycles of slow ipsiversive conjugate eye deviations, each followed by rapid contralateral jerks. The involved electrode contacts were situated at the mesodiencephalic junction just inferior to the centromedian nucleus of the thalamus and rostral to the superior colliculus. Right-sided stimulation evoked left beating nystagmus and left-sided stimulation evoked right beating nystagmus. Stimulation through other electrode contacts did not induce nystagmus. Electronystagmography showed the nystagmus to have constant velocity slow phases. CONCLUSIONS: A central nystagmogenic area exists in humans that appears to be homologous to the nucleus of the optic tract, a region described in nonhuman primates to play a role in the generation of optokinetic nystagmus.

A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype.

Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with approximately 30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1(del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1(del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1(del34/+) animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease.

alpha-thalassemia resulting from a negative chromosomal position effect.

To date, all of the chromosomal deletions that cause alpha-thalassemia remove the structural alpha genes and/or their regulatory element (HS -40). A unique deletion occurs in a single family that juxtaposes a region that normally lies approximately 18-kilobase downstream of the human alpha cluster, next to a structurally normal alpha-globin gene, and silences its expression. During development, the CpG island associated with the alpha-globin promoter in the rearranged chromosome becomes densely methylated and insensitive to endonucleases, demonstrating that the normal chromatin structure around the alpha-globin gene is perturbed by this mutation and that the gene is inactivated by a negative chromosomal position effect. These findings highlight the importance of the chromosomal environment in regulating globin gene expression.

A comparison of tangent screen, goldmann, and humphrey perimetry in the detection and localization of occipital lesions.

OBJECTIVE: To compare manual kinetic perimetry with tangent screen and Goldmann techniques and automated static perimetry with the Humphrey Field Analyzer in the detection and localization of occipital lobe lesions. DESIGN: Prospective consecutive comparative case series. PARTICIPANTS: Twelve patients with well-defined occipital lobe infarcts on magnetic resonance (MR) imaging were studied. MAIN OUTCOME MEASURES: The patients were tested by tangent screen, Goldmann, and Humphrey perimetry (central 30-2 threshold program). The three visual fields were compared and correlated with MR images. RESULTS: All three perimetric techniques detected the presence of postchiasmal lesions. However, localization of lesions differed with perimetric technique. Visual fields obtained from tangent screen and Goldmann perimetry were similar and corresponded well with the location of lesions on MR images in all 12 patients. Humphrey perimetry inaccurately localized the lesion to the proximal part of the postchiasmal pathway by revealing incongruous fields in two patients, failed to detect sparing of the posterior occipital cortex or occipital pole in four patients, and estimated a larger extent of damage in one patient when compared with MR images and manual perimetry. CONCLUSIONS: All three perimetric techniques are satisfactory screening tests to detect occipital lesions. However, tangent screen and Goldmann perimetry provide information about the location and extent of lesions that is more consistent with prevailing knowledge of the effects of the lesion in the postgeniculate visual pathway.

Representation of the visual field in the human occipital cortex: a magnetic resonance imaging and perimetric correlation.

OBJECTIVES: To evaluate the retinotopic map of the human occipital cortex by correlating magnetic resonance imaging (MRI) findings with visual field defects in patients with occipital lobe infarcts and to assess the compatibility between our cliniconeuroimaging findings and the location of lesions predicted by the classic Holmes map and a revised map. METHODS: Magnetic resonance images were obtained in 14 patients with occipital lobe infarcts. Visual field analysis was performed with tangent screen, the Goldmann perimeter, and the Humphrey Field Analyzer. Based on the pattern of visual field deficit, the location of the lesion in the mesial occipital lobe in each patient was predicted using the Holmes map and other retinotopic maps of the occipital cortex. The predicted location of the lesion was then compared with its actual location shown on MRI to assess the compatibility between our data and the other maps. These maps determine retinotopic correlates of the medial occipital lobe, but they cannot establish correlates of the striate cortex (V1). The medial occipital representation of central vision was evaluated by regression analysis. RESULTS: The MRI correlations in this study confirmed gross estimates of the retinotopic organization of the occipital cortex. However, our findings did not correlate exactly with the Holmes map. We determined that the central 15 degrees of vision occupies 37% of the total surface area of the human medial occipital lobe. Based on our data, a refined retinotopic map is presented. CONCLUSIONS: The resolution of conventional MRI testifies to its considerable value in localizing occipital lobe lesions. Our findings support, and refine, the Holmes map of the human occipital cortex.

Ocular tracking of step-ramp targets by patients with unilateral cerebral lesions.

The relationship of sinusoidal smooth pursuit defects to pursuit defects with step-ramp targets in patients with cerebral lesions is unclear. We examined pursuit and saccades to both step-ramp and sinusoidal targets in 17 patients with unilateral cerebral lesions. Two types of pursuit defects were found. One group of three patients had ipsi-directional sinusoidal pursuit defects from lesions to the posterior internal capsule. Their chief abnormality with step-ramp targets was increased contra-directional pursuit. Their ipsi-directional step-ramp pursuit was often normal and disproportionately better than their ipsi-directional sinusoidal pursuit. Another patient with a parietal lesion had a second type of pursuit defect. He had low-normal sinusoidal pursuit bilaterally, but decreased ipsi- and contra-directional step-ramp pursuit. Also, he had an abnormal contra-directional drift after saccades to stationary targets. Despite these pursuit defects, saccadic accuracy did not show poor compensation for target motion in either patient type. The patient with the parietal lesion also had increased latencies for contralateral saccades. Recovery of pursuit was studied in one patient with an infarct of the posterior internal capsule. Initially he had a contra-directional bias that caused decreased ipsi-directional pursuit, increased contra-directional pursuit, and a contra-directional drift after saccades to stationary targets. Four months later, ipsi-directional pursuit and the post-saccadic drift to stationary targets had recovered, but contra-directional pursuit remained abnormally high. We conclude that lesions of descending pursuit tracts in the internal capsule are characterized by a contra-directional bias which recovers partly through a direction-specific adaptation. Lesions that affect the human homologue of posterior parietal cortex cause asymmetric bi-directional defects in pursuit initiation and increased contralateral saccadic latencies.

Understanding alpha globin gene expression: a step towards effective gene therapy.

During the past 20 years developments in molecular and cellular biology have kindled the hope that one might eventually ameliorate or even cure some serious genetic diseases by repairing or replacing the defective gene. Other articles deal with the formidable problems of isolating pluripotent hematopoietic stem cells; efficiently, safely, and stably transfecting them, and developing transplantation protocols to ensure that the corrected cells supplant the patient's abnormal stem cells after transplantation. Assuming that these hurdles can be overcome, it will also be important to establish the ideal segment of DNA to introduce into stem cells to ensure that, regardless of its position of integration in the genome, the gene in question will be appropriately regulated. In the case of the globin genes this is a particularly difficult task because in order to correct disorders of globin synthesis we need to obtain high levels of stable, tissue- and developmental-stage specific expression. Issues relevant to this problem arising from the analysis of the human beta globin cluster are discussed in the article in this issue by Grosveld. In this article we review our current understanding of how eukaryotic genes might be expressed from their normal chromosomal environment, using the human alpha globin cluster as a specific example. We also discuss how this information might be used in the development of strategies for gene therapy.

Cortical control of eye movements.

Event related cortical potentials and imaging provide information about roles of the cerebral hemispheres in generating voluntary and reflexive saccades. This review also discusses relationships between smooth pursuit and visual discrimination of motion direction and the phenomenon of blindsight. Cortical effects of strabismus on motion processing and smooth pursuit are reviewed.

Vestibulo-ocular reflex deficits to rapid head turns following intratympanic gentamicin instillation.

The response of the vestibulo-ocular reflex following unilateral vestibular deafferentation by gentamicin ablation was studied using transient stimuli. The response to these rapid passive head turns showed a strong asymmetry with permanent, reduced gains toward the side of lesion. These gain reductions have large variation (gains of 0.26 to 0.83), which may result from preferential sparing of regularly firing afferent fibers following gentamicin ablation. Based on the size and nature of the nonlinearity, an explanation based on Ewald's second law was discounted.

Smooth pursuit and saccades to moving targets in blind hemifields. A comparison of medial occipital, lateral occipital and optic radiation lesions.

We tested smooth pursuit and saccades to targets within the contralateral homonymous visual field defects of 10 patients with unilateral cerebral hemispheric lesions. Four patients had medial occipital lesions that spared the putative motion area in lateral occipitotemporal cortex and the optic radiations proximal to this site. The other six had lesions involving this area or the proximal optic radiations. From current hypotheses, that surviving extra-striate cortex is responsible for 'blindsight', we expected that the patients with lesions restricted to medial occipital cortex would be more likely to have residual ocular motor responses to moving targets. We found, however, that these patients with sparing of the lateral motion area did not show significantly better correlations of either post-saccadic eye velocity with target velocity or initial saccadic amplitude with the position of the moving target, compared with the other six patients. With increased target velocities against a background of darkness, two patients had weakly positive ocular motor correlations. However, in one patient this finding was replicated by a normal control stimulating the patient's scotoma and, in the other patient, the correlation disappeared when the eye with a residual monocular temporal crescents was occluded. These features indicate an artifact from light scatter. Only one other patient demonstrated a consistent, but weak, correlation of ocular tracking with target motion in the blind region; paradoxically, this patient had a lateral occipitotemporal lesion. We conclude that sparing of the lateral motion area is not sufficient condition for residual tracking of moving targets in homonymous visual field defects, and may not even be necessary.