MKS1 mutations cause Joubert syndrome with agenesis of the corpus callosum.

Joubert syndrome (JS) is a clinically and genetically heterogeneous ciliopathy characterized by episodic hyperpnea and apnea, hypotonia, ataxia, cognitive impairment and ocular motor apraxia. The "molar tooth sign" is pathognomonic of this condition. Mutations in the MKS1 gene are a major cause of Meckel-Gruber syndrome (MKS), the most common form of syndromic neural tube defects, frequently resulting in perinatal lethality. We present the phenotype and genotype of a child with severe JS and agenesis of the corpus callosum (ACC). In our patient, a next generation sequencing (NGS) approach revealed the following two variants of the MKS1 gene: first, a novel missense variant [ c.240G > T (p.Trp80Cys)], which affects a residue that is evolutionarily highly conserved in mammals and ciliates; second, a 29 bp deletion in intron 15 [c.1408-35_1408-7del29], a founder mutation, which in a homozygous state constitutes the major cause of MKS in Finland. We review the MKS1-variants in all of the eleven JS patients reported to date and compare these patients to our case. To our knowledge, this is the first patient with Joubert syndrome and agenesis of the corpus callosum where a potentially causal genotype is provided.

Soluble substances released from postischemic reperfused rat hearts reduce calcium transient and contractility by blocking the L-type calcium channel.

OBJECTIVES: This study was designed to investigate the effects of cardiodepressant substances released from postischemic myocardial tissue on myocardial calcium-regulating pathways. BACKGROUND: We have recently reported that new cardiodepressant substances are released from isolated hearts during reperfusion after myocardial ischemia. METHODS: After 10 min of global ischemia, isolated rat hearts were reperfused, and the coronary effluent was collected for 30 s. We tested the effects of the postischemic coronary effluent on cell contraction, Ca2+ transients and Ca2+ currents of isolated rat cardiomyocytes by applying fluorescence microscopy and the whole-cell, voltage-clamp technique. Changes in intracellular phosphorylation mechanisms were studied by measuring tissue concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), as well as activities of cAMP-dependent protein kinase (cAMP-dPK) and protein kinase C (PKC). RESULTS: The postischemic coronary effluent, diluted with experimental buffer, caused a concentration-dependent reduction of cell shortening and Ca2+ transient in the field-stimulated isolated cardiomyocytes of rats, as well as a reduction in peak L-type Ca2+ current in voltage-clamped cardiomyocytes. The current reduction resulted from reduced maximal conductance--not from changes in voltage- and time-dependent gating of the L-type Ca2+ channel. The postischemic coronary effluent modified neither the tissue concentrations of cAMP or cGMP nor the activities of cAMP-dPK and PKC. However, the effluent completely eliminated the activation of glycogen phosphorylase after beta-adrenergic stimulation. CONCLUSIONS: Negative inotropic substances released from isolated postischemic hearts reduce Ca2+ transient and cell contraction through cAMP-independent and cGMP-independent blockage of L-type Ca2+ channels.

Cellular adhesion molecules on vascular smooth muscle cells.

Several studies during the last years have shown that, in addition to endothelial cells, vascular smooth muscle cells also express the cellular adhesion molecules ICAM-1 and VCAM-1 in atherosclerosis, restenosis and transplant vasculopathy. In vitro studies have characterized stimulatory and inhibitory factors that regulate the expression of ICAM-1 and VCAM-1 on cultured smooth muscle cells. There is evidence for a role of adhesion molecules on smooth muscle cells for leukocyte accumulation and activation of mononuclear cells. Some recent data suggest that the expression of adhesion molecules on smooth muscle cells are cell cycle-dependent and influence smooth muscle cell proliferation and differentiation. Therefore, ICAM-1 and VCAM-1 on smooth muscle cells may contribute to the inflammatory reaction in the vascular wall and may actively be involved in the progression and stability of atherosclerotic plaques.

Effects of platelet-activating factor on intracellular Ca2+ concentration and contractility in isolated cardiomyocytes.

We investigated the effects of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) on intracellular Ca2+ concentration ([Ca2+]i) and cell length in isolated and field-stimulated rat cardiomyocytes. [Ca2+]i and cell length of field-stimulated cells were determined simultaneously by confocal laser scan microscopy by using the fluorescent Ca2+ dye Fluo-3. PAF (10(-12)-10(-8) M) inhibited systolic [Ca2+]i increase in a time- and concentration-dependent manner. Maximal effects were observed after an incubation time of 6-8 min, resulting in a 17% (10(-12) M), 41% (10(-10) M), and 52% (10(-8) M PAF) inhibition of systolic [Ca2+]i increase. A time- and concentration-dependent decrease in simultaneously measured cell shortening also was demonstrated. Cell shortening was inhibited by 10% (10(-12) M), 32% (10(-10) M), and 50% (10(-8) M) after an incubation time of 8 min. The effects of PAF could be antagonized by the PAF-receptor antagonist WEB 2170. These data demonstrate that PAF receptor-dependently induces a negative inotropic effect, which is correlated with a decrease in systolic [Ca2+]i and is most likely not due to a decrease in myofilament sensitivity.

Regulation of tumor necrosis factor alpha- and interleukin-1-beta-induced induced adhesion molecule expression in human vascular smooth muscle cells by cAMP.

This study investigates the hypothesis that the elevation of intracellular cAMP may affect cytokine-induced expression of adhesion molecules on human vascular smooth muscle cells. In cultured human smooth muscle cells from coronary arteries and saphenous veins, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) induced the expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1), whereas interferon-gamma (INF-gamma) selectively stimulated the expression of ICAM-1. Adenylyl cyclase was stimulated either by the stable prostacyclin mimetic cicaprost or by forskolin. Adhesion molecules were detected by a cell surface enzyme immunoassay and the respective mRNA by reverse transcriptase polymerase chain reaction (rt-PCR). Cicaprost as well as forskolin significantly inhibited TNF-alpha- and IL-1 beta-induced cell surface expression of ICAM-1 and VCAM-1. Semiquantitative rt-PCR measurements showed a marked decrease of TNF-alpha- and IL-1 beta-induced mRNA levels of both adhesion molecules after preincubation with cicaprost. The stability of TNF-alpha-induced ICAM-1 and VCAM-1 expression at mRNA and protein level was not altered by cicaprost. The IFN-gamma-induced increase of cell surface expression of ICAM-1 and the respective mRNA levels, however, were not significantly altered by elevation of intracellular cAMP. Basal and stimulated cAMP levels, measured by radioimmunoassay, did not differ in TNF-alpha- and IFN gamma-treated cells. The present results demonstrate that the expression of adhesion molecules on human smooth muscle cells induced by cytokines is differentially modulated by activation of adenylyl cyclase.

A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle--dependent changes in mast cell--nerve fiber contacts in murine skin.

Close contacts between mast cells (MC) and nerve fibers have previously been demonstrated in normal and inflamed skin by light and electron microscopy. A key step for any study in MC-nerve interactions in situ is to simultaneously visualize both communication partners, preferably with the option of double labelling the nerve fibers. For this purpose, we developed the following triple-staining technique. After paraformaldehyde-picric acid perfusion fixation, cryostat sections of back skin from C57BL/6 mice were incubated with a primary rat monoclonal antibody to substance P (SP), followed by incubation with a secondary goat-anti-rat TRITC-conjugated IgG. A rabbit antiserum to CGRP was then applied, followed by a secondary goat-anti-rabbit FITC-conjugated IgG. MCs were visualized by incubation with AMCA-labelled avidin, or (for a more convenient quantification of close MC-nerve fiber contacts) with a mixture of TRITC- and FITC-labelled avidins. Using this simple, novel covisualization method, we were able to show that MC-nerve associations in mouse skin are, contrary to previous suggestions, highly selective for nerve fiber types, and that these interactions are regulated in a hair cycle-dependent manner: in telogen and early anagen skin, MCs preferentially contacted CGRP-immunoreactive (IR) or SP/CGRP-IR double-labelled nerve fibers. Compared with telogen values, there was a significant increase in the number of close contacts between MCs and tyrosine hydroxylase-IR fibers during late anagen, and between MCs and peptide histidine-methionine-IR and choline acetyl transferase-IR fibers during catagen.

Modulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 on human coronary smooth muscle cells by cytokines.

Adhesion molecules have been demonstrated immunohistochemically on smooth muscle cells in atherosclerotic plaques. In endothelial cells cytokines are potent modulators of adhesion molecule expression. We therefore investigated the effects of cytokines on adhesion molecule expression on cultured human coronary and pulmonary smooth muscle cells by cell ELISA and confocal microscopy. Human coronary and pulmonary smooth muscle cells expressed ICAM-1 and VCAM-1 but not E-selectin. ICAM-1 expression was upregulated by TNF alpha, Il-1 beta and IFN-gamma. VCAM-1 expression was increased by TNF alpha and weakly by Il-1 beta, IFN-gamma had no effect on VCAM-1 expression. Cytokine effects on ICAM-1 and VCAM-1 were based on de novo synthesis. These results demonstrate that cytokines regulate ICAM-1 and VCAM-1 expression on human coronary and pulmonary smooth muscle cells. These effects may play an important role in the immune mechanisms in atherosclerosis.

Phototaxic behavior and the retinotectal transport of horseradish peroxidase (HRP) in surgically created cyclopean salamander larvae (Ambystoma).

UNLABELLED: Negative phototaxis (NP) was used to evaluate the recovery of vision in albino axolotl larvae with one eye discarded and the other transplanted either to the orbit (orthoclops) or to the top of the head (cyclops). NP was assessed at approximately 1, 2 and 3 months postoperatively, using an automated, infrared monitor. Some 88% of the orthoclopes and 64% of the cyclopes recovered NP. However, among the cyclopes that did recover, the quantitative aspects of NP were virtually the same as those of the orthoclopes. That the cyclopean eye can regenerate retinotectal pathways was established by anterograde tracing of horseradish peroxidase (HRP). But where previously uninjured animals transported HRP to the contralateral tectum, both the cyclopes and the orthoclopes distributed the enzyme to the left and right tectal halves. Heavy deposits of HRP were found in the tecta of some animals that lacked NP. To find out if an optic tectum is actually required for NP, a series of ablation experiments were performed, using Ambystoma punctatum larvae. Tectectomy had the same effect on NP as bilaterally extirpating the eyes or intracranially severing both optic nerves, i.e. removing the tectum abolished NP. THE RESULTS: (1) confirm the efficacy of the ectopic eye in the cyclops preparation; (2) show that the ectopic eye can regenerate retinotectal pathways; (3) indicate that retinotectal contact is a necessary but insufficient condition for NP.

Retinoic acid treatment inhibits mitosis in the pre-existing spinal cord during tail regeneration of the axolotl larva, Ambystoma mexicanum.

During tail regeneration in the axolotl larva, Ambystoma mexicanum, retinoic acid reduced mitosis some 10-fold in the pre-existing spinal cord. This conclusion followed from comparisons of mitotic indices in the central grey matter of spinal cords, proximal to the amputation plane, of animals intracoelomically injected with (a) retinoic acid 2 days before amputation and the mitotic inhibitor, demecolcine 5 h before sacrifice; or (b) as for the latter but without retinoic acid; or (c) only the solvent for (a) and (b). Animals were sacrificed concurrently, 9 days after tail amputation. In addition, the demecolcine treatment revealed the true mitotic activity of the spinal cord of axolotl to be at least five times greater than might otherwise have been suspected.

Measures of phototaxis and movement detection in the larval salamander.

In this investigation, we describe two precise tests of visual function that integrate quasinatural situations with time-lapse video recording and infrared computerized monitoring of activity to assess movement detection and phototaxic tendencies, respectively. Four groups of larvae from A. punctatum, A. tigrinum, A. mexicanum, and a mutant albino axolotl were tested in an alley containing light and dark halves and lined with infrared sensors to monitor their phototaxic response. A. punctatum showed no phototaxic tendency, while the other three groups displayed a strong negative phototaxic response. Enucleation of the eyes in mutant albinos eliminated the negative phototaxis. Visual detection of motion was tested by videorecording the behavior of A. punctatum, A. mexicanum, and the mutant albino axolotl larvae while they explored a large bowl with 6 small vials on the perimeter, one of which contained white worms. A. punctatum rapidly approached the worm vial and engaged in intense predatory behavior. A. mexicanum responded to the presence of worms very slowly and rapidly lost interest. Albino axolotls displayed no visual recognition of the worms. The results indicate that visual function can be precisely determined in larval salamanders utilizing behavioral measures consistent with the animal's natural tendencies.

Tectectomy in the cyclopean salamander.

In an Ambystoma larva with both natural eyes removed and one eye grafted atop the head (Cyclops preparation), vision-dependent behavior usually recovers from the enucleation inherent in the operation, but the optically activated skin blanching reaction reappears in a very small number of instances. In the present studies, while the latter trend continued for the conventional Cyclops preparation, tectectomy concurrent with the ectopic eye transplantation resulted in a several-fold increase in the recovery of blanching competency. Some 60 percent of the tectectomized Cyclops animals exhibited the same Hogben-Slome pigmentation indices as larvae with one natural eye intact (controls). As measured planimetrically with an image analyzer, the pigment spots (melanosome containing portions of dermal melanocytes) contracted to the same extent in the blanch-competent Cyclops animals as in controls with a single natural eye.

Effects of retinoic acid on the muscle patterns produced during forelimb regeneration in larval salamanders (Ambystoma).

After systemic treatment with retinoic acid (RA), Ambystoma opacum and A. punctatum larvae regenerated forelimbs with a wide variety of skeletal and gross anatomical abnormalities. Yet the musculatures within the RA-treated limb regenerates were normal even in instances where the cartilages were deformed beyond recognition as components of the limb skeleton. RA is known to induce reduplication of limb structures, sometimes entire segments. When the latter condition occurred in the present study, the corresponding replicates exhibited limb musculatures which were perfect down to minute details, yet of opposite bilateral symmetry. The results attest, firstly, to the independence of myogenesis and chondrogenesis during limb regeneration. Secondly, RA treatment unmasked an otherwise hidden potential within postembryonic salamander limb tissue for the morphogenesis of the contralateral musculature.

Anterior decerebration blocks visual habituation in the larval salamander (Ambystoma punctatum).

Amputation of the rostral half of the cerebrum induces a compulsion-like reaction in larval Ambystoma punctatum towards Enchytraeus protected within glass vials. Normal and craniotomized larvae are visually attracted to worm-containing vials, as revealed by time-lapse video taping but, after several unsuccessful attempts to get the prey, habituate and depart. The video tapes revealed that anteriorly decerebrated animals spent as much as 100 of 120 minutes at the worm-containing vial, repeatedly but futilely attacking the glass. The data indicate that the telencephalon plays an active negative role in the salamander larva's visually guided behavior.

Two-eyed versus one-eyed salamanders: does binocularity enhance the optically evoked skin blanching reactions of Ambystoma larvae?

A wide variety of visual functions show increases attributable to binocularity, and the question pursued here was whether a second eye enhances the visually stimulated skin blanching reaction of the larval salamander. Dermal melanin spots (produced by the aggregations of melanosomes within dermal melanophores and which contract or expand to lighten or darken the skin) were measured in eyeless (controls), one-eyed and two-eyed Ambystoma punctatum larvae after chronic adaptation of the subjects to a white background (i.e., stimulus conditions for maximum blanching). The eyeless subjects showed no blanching (thus remained dark) in white cups, and they exhibited melanin spots 7 or 8 times the size of those of the other two groups. All one-eyed or two-eyed subjects exhibited blanching reactions; planometric comparison revealed a significantly larger melanin spot area for one-eyed than for two-eyed animals; i.e., the binocular condition permitted greater contraction of the pigment spots than did the monocular condition. Analytical data compared favorably with independently ascertained pigmentation indices. The results indicate that a second eye quantitatively elevates the blanching maximum of a larval salamander.

Immunological alterations inducible by mercury compounds. II. HgCl2 and gold sodium thiomalate enhance serum IgE and IgG concentrations in susceptible mouse strains.

We determined the levels of total IgE, IgG and IgM in the sera of HgCl2 and gold sodium thiomalate (GST) treated A.SW, C57Bl/6, and DBA/2 mice. In HgCl2 treated A.SW mice, IgE and IgG levels increased up to 30 times above normal. In C57Bl/6 mice HgCl2 induced a slight IgE increase, but no change in IgG. Strain DBA/2, by contrast, did not show significant increases in either IgE or IgG. A.SW mice also proved susceptible to the immunostimulatory effects of GST in that they responded by significant increases in IgE, IgG, and IgM. C57Bl/6 mice responded to GST by an increase in IgM alone, and DBA/2 mice again, were resistant. The same strain-specific responses have been reported with respect to the autoimmunizing effects inducible by HgCl2 and GST.

Transplanted eyes of foreign donors can reinstate the optically activated skin camouflage reactions in bilaterally enucleated salamanders (Ambystoma).

Bilateral enucleation immediately and irreversibly cancels the skin camouflage reactions of Ambystoma larvae. A heteroplastic eye transplant can reinstate the latter reaction. Recipients of a heteroplastic eye recovered the reaction as frequently and to the same extent as animals with one of their own eyes reattached.

Respiratory phase detection and delay determination for breath-by-breath analysis.

The delay between air flow and gas concentration signals is generally assumed to be constant within a breath as well as from breath to breath, but it was not possible to examine the constancy of the delay with the delay determination techniques so far available. Thus we developed new methods for respiratory phase detection and delay determination. The presented algorithm for the detection of the start of inspiration and expiration (phase detection) replaces the generally used valve assembly with two pneumotachographs. Now, the pneumotachograph is used in a bidirectional mode, but with a volume criterion for phase detection replacing the less reliable threshold criterion. To measure the delay between flow and gas concentration signals, a test gas is periodically injected as a marker. This test gas contains less N2 than ambient air. Therefore, the delay is determined as time between the moment of injection and the drop of N2. These two methods rendered it possible to examine delay variations and their consequences. The investigation of various breathing patterns demonstrated that the usually assumed errors caused by delay uncertainty are underestimated. We suggest reliance on a breath-by-breath delay determination to account for delay variations.

A pulsed diagonal-beam ultrasonic airflow meter.

The construction and specific function of a new ultrasonic flowmeter are described. The mean velocity of the respiratory airflow is calculated by measuring the transit times of short ultrasonic pulse trains, simultaneously transmitted upstream and downstream at a 500-Hz rate. The flowmeter system consists of a control unit and a separate flow head. The former includes the power supplies, a controlling microprocessor, most of the signal-processing circuitry, and three analog outputs for flow, volume, and temperature. The flow head contains the respiratory tube with a constant circular cross section (length 90 mm, diam 20 mm, dead space 35 ml), a fast temperature sensor, two electronic circuits for processing of flow and temperature data, and a sound transmission channel with two capacitive ultrasonic wide-band transducers. This respiratory airflow meter, suitable for spirometric maneuvers (vital capacity, forced vital capacity) as well as for long-term breath-by-breath respiratory analysis, is extremely fast (response time 1-2 ms) and accurate (volume accuracy with room air +/- 0.7%), with low noise (below 9 ml/s), a wide flow range (bidirectional from 0 to 9 l/s), and a flat frequency response up to 70 Hz.

[Hepatitis B screening in pregnant patients and their children--a Greifswald perinatal-pediatric study]. / Hepatitis-B-Screening bei Schwangeren und ihren Kindern--Eine Greifswalder perinatologisch-pädiatrische Studie. 1. Befunde bei Schwangeren und stillenden Fauen.

During a period of one year with systematic screening performed in 1,401 pregnant women 8 cases positive for HBs-antigen could be detected. For the investigation we used a counter electrophoresis method. In 500 serial specimens an additional examination was performed by aid of a RIA as well. With an incidence of 0.57% the number of HBs antigen positive pregnancies in the territory of Greifswald was found to be significantly higher than reported previously from other regions of the GDR. Our results claim for a mass screening for hepatitis B during pregnancy. Because of the possibility of virus excretion with human milk, HBs positive mothers should not be used as milk donors. Other results related to our investigations give evidence for the significance of obtaining specific hygienic regulations when medical care is given to asymptomatic HBsAg carriers. They also underline the necessity of interdisciplinary cooperation for teaching medical staff in health care centers and giving correct advice to those detected as hepatitis B carriers.