Role of cytosolic carbonic anhydrase Ca17a in cardiorespiratory responses to CO2 in developing zebrafish (Danio rerio).

The sensing of environmental fluctuations and initiation of appropriate physiological responses is crucial to homeostasis. Neuroepithelial cells (NECs) in fishes are putative chemoreceptors, resembling mammalian Type I (glomus) cells, that respond in vitro to changes in O2, CO2, NH3, and pH. Cytosolic carbonic anhydrase (Ca17a) is thought to be involved in CO2 sensing owing to its presence in NECs. Zebrafish (Danio rerio) lacking functional Ca17a were generated via CRISPR/Cas9 technology and used to assess the role of Ca17a in initiating the cardiorespiratory responses to elevated CO2 (hypercapnia). Unfortunately, the homozygous knockout mutants (ca17a-/-) did not survive more than ∼12-14 days postfertilization (dpf), restricting experiments to early developmental stages (4-8 dpf). Changes in ventilation (fV) and cardiac (fH) frequency in response to hypercapnia (1% CO2) in wild-type (ca17a+/+), heterozygous (ca17a+/-) and ca17a-/- fish were used to investigate Ca17a-dependent CO2 sensing and downstream signaling. Wild-type fish exhibited hyperventilation during hypercapnia as indicated by an increase in fV. In the ca17a-/- fish, the hyperventilatory response was attenuated markedly but only at 8 dpf. Hypercapnic tachycardia was observed for all genotypes and did not appear to be influenced by the absence of Ca17a. Interestingly, ca17a-/- fish exhibited a significantly lower resting fH that became more pronounced as the fish aged. The decrease in resting fH was prevented ("rescued") when ca17a-/- embryos were injected with ca17a mRNA. Collectively, the results of this study support a role for Ca17a in promoting hyperventilation during hypercapnia in larval zebrafish and suggest a previously unrecognized role for Ca17a in determining resting heart rate.

The effect of d-amphetamine and amitriptyline administered to pregnant rats on the locomotor activity and neurotransmitters of the offspring.

d-Amphetamine and amitriptyline (AT) were administered daily to female rats from day 7 of pregnancy until birth of the litters. Changes in the concentration of the biogenic amines, some of their metabolites, GABA, and the activities of glutamate decarboxylase, acetylcholinesterase (AChE), and choline acetyltransferase were determined in the whole brain of the offspring. The offspring of the amphetamine-treated rats showed a marked increase in serotonin concentration and that of its metabolite on postnatal day 1. Changes in the concentration of GABA were apparent on days 15 and 21 and were inversely correlated with changes in the activity of the synthesizing enzyme: Choline acetyltransferase and AChE activities were also increased at this time. Changes in neurotransmitter metabolism were not so evident in the offspring of rats treated with AT. The locomotor activity of the 8-, 15-, and 21-day offspring was also assessed. The offspring of the amphetamine-treated rats showed enhanced locomotor activity initially, but the activity decreased relative to the age-matched controls in the 21-day group. Offspring from the AT-treated group showed reduced locomotor activity.

[Cytokine therapy and the endocrine system: is it necessary to monitor more than the thyroid gland?]. / Zytokintherapie und Endokrinium: Ist es sinnvoll, mehr als die Schilddrüse zu untersuchen?

UNLABELLED: Cytokines and Endocrine System: Is it Meaningful to Monitor More than the Thyroid Gland? AIM: It was the aim of this study to evaluate the occurrence, the degree, and clinical relevance of immunological phenomena in endocrine and non-endocrine organs as seen under therapy with interferon-alpha (IFN-alpha) and/or interleukin-2 (Il-2). PATIENTS AND METHODS: In 61 patients (age: 50.2 +/- 12.8 years) receiving cytokines as treatment for hepatological or hemato-oncological diseases, parameters of the thyroid gland, the gonadal system, the adrenal gland the pituitary gland, parameters of water and electrolyte-balance, and of bone metabolism were measured. All patients were treated with interferon in a mean dosage of 15.3 +/- 10.5 mio U subcutaneously per week. Additionally, 15 patients were treated with interleukin-2 (36.5 +/- 22.3 mio U subcutaneously per week). Among other assays, patients "sera were screened for the existance of autoantibodies against 30 different antigenic substrates using an indirect immunofluorescence technique (IIF). RESULTS: Pts, treated with IFN-alpha and IL-2 (n = 13) showed a significant induction of thyroid antibodies against thyroglobulin (anti-TG): 61 +/- 82 U/ml vs. 1000 +/- 2352 U/ml (p < 0.05) and against thyroid-peroxidase (anti-TPO): 162 +/- 538 vs. 468 +/- 1071 U/ml (p < 0.05). In pts, only treated with IFN-alpha (n = 26), the increase of anti-TG from 69 +/- 97 to 134 +/- 178 U/ml and of anti-TPO from 20 +/- 21 to 21 +/- 19 U/ml was insignificant. Patients with a combined treatment of IFN-alpha and Il-2 were mainly affected: 2 patients developed hypo- and 5 hyperthyroidism. Thus, regular controls of thyroid function during the entire duration of therapy is justified. We were unable to show any influence of the duration of treatment or dosage of cytokines on the endocrine and non-endocrine parameters investigated. In addition, 8 different AAB (against smooth/striated muscles, parietal cells, nuclear parts, myelin, keratin, endothel, and neuroendothel) were detected by IIF which did not change under therapy. Two patients newly developed AAB during therapy, however none of them suffered from clinical symptoms. AAB against endocrine organs other than the thyroid gland were not detected. No other endocrine system showed alterations of its function. CONCLUSION: Based on the data of this study we conclude that the thyroid gland should be regularly monitored for immunological and functional changes during cytokine therapy. The results do not support a general recommendation for a routine screening of other, non-thyroidal endocrine systems for autoimmune phenomena and alterations of function in each patient.

[Effect of light deprivation on GABA metabolism in subcellular fractions of the rabbit visual system]. / Vliianie svetovoi deprivatsii na obmen GAMK v subkletochnykh fraktsiiakh zritel'noi sistemy krolikov.

Specific activity of glutamate decarboxylase was decreased in subfractions of light and heavy synaptosomes, and gamma-aminobutyric acid (GABA) transaminase activity--also in subfractions of free mitochondria, isolated from visual zone of brain cortex and of anterior mesencephalon but not from locomotive region of brain cortex of the light-deprived rabbits. Decrease in the ratio GABA transaminase/glutamate decarboxylase indicated distinct inhibition of GABA degradation as compared with its synthesis due to absence of sensory impulsation within the early periods of postnatal ontogenesis of the animals.

Effect of monocular deprivation on uptake and binding of [3H]glutamate in the visual system of rat brain.

[3H]Glutamate uptake and binding studies were performed in the visual cortices, lateral geniculate nuclei (LGN), and superior colliculi of 3-month-old rats with one eyelid surgically closed from postnatal day 10 (monocular deprivation). Uptake and binding were highest in the lateral geniculate nucleus followed by the visual cortex (69% and 15%, respectively compared to LGN values) and the superior colliculus (32% and 59% of LGN values). Monocular deprivation did not affect [3H]glutamate uptake in any of the visual regions examined. However, a 46% decrease in [3H]glutamate binding in the lateral geniculate nucleus ipsilateral to the sutured eye was detected. Binding levels in other regions were not affected.

Geographical distribution gradients of the major PKU mutations and the linked haplotypes.

Analysis of 81 phenylketonuria families from Bulgaria, Lithuania and eastern Germany demonstrated a high frequency of haplotype 2 and the associated Arg408----Trp408 substitution. Haplotype 3 and the splicing mutation in intron 12 are rare or absent in the groups studies. Pooling the data on European populations suggests a Balto-Slavic origin of the defect in codon 408 of the phenylalanine hydroxylase gene and a geographical gradient in the distribution of both major PKU mutations which may contribute to the higher incidence of classic PKU in northern Europeans.

Identification of crossovers in Wilson disease families as reference points for a genetic localization of the gene.

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. A minimum recombinant analysis using D13S22, ESD, RB1, D13S31, D13S55, D13S26, D13S39, and D13S12, all localized at 13q14-q22, has been carried out in 20 WD families of Northwest-European origin. No inconsistencies have been observed with respect to locus order or location of the WD locus (WND) compared with previous linkage studies. D13S31 was mapped as the closest marker proximal to WND, whereas D13S55 and D13S26 were mapped as the closest markers distal to WND. We have identified a crossover between WND and D13S31 in one family and a crossover between WND and D13S55 in another. These crossover sites can be used as reference points for new chromosome 13q14-q21 markers, and are therefore important for a more accurate mapping of the WD locus.

The phenylketonuria G272X haplotype 7 mutation in European populations.

We have compiled data on the frequencies of the phenylketonuria G272X mutation in European populations. This mutation occurs north of the Alps. It has a particularly high frequency in the Oslo Fjord region of Norway with the adjacent Bohuslän region of Sweden. An intermediate frequency was noted in a separate area, the eastern part of Germany with the adjacent western part of Czechoslovakia. The G272X mutation was associated with phenylalanine hydroxylase haplotype 7, except for one case with haplotype 3. Genealogical studies going back eight to nine generations revealed no common source for this mutation, but there was some geographical convergence to the Bohuslän region. These findings suggest a single origin for this mutation, with at least one founding population in south-eastern Norway/adjacent Sweden.

Deletion analysis of DMD/BMD families from the German Democratic Republic and selected regions of Czechoslovakia and Hungary.

Over the last two years we have screened 183 DMD/BMD families requesting prenatal diagnosis. Using cDNA probes cf56a,b we have detected exon deletions in 72 of them. In 62 cases the deletion was also detectable with currently available PCR primers. Deletion analysis for exons 8, 17, and 19, using either PCR or Southern blotting techniques, was performed for 65 of the 111 families which showed no deletions with cf56a,b. Eight of them were deleted for one or more of these exons. PCR offers new possibilities for deletion analysis in families without a living patient using either Guthrie papers or histologically conserved material from the dead patient. In 20 of 25 patients, we observed concordance between the clinical picture and the molecular deletion analysis in accordance with the open reading frame hypothesis. Five patients, however, presented with DMD in spite of our analysis showing an in frame deletion. Carrier determination in families in which DMD is caused by a deletion using linkage, dosage, or breakpoint analysis is discussed.

Unstable DNA may be responsible for the incomplete penetrance of the myotonic dystrophy phenotype.

Myotonic dystrophy (DM) is associated with the expansion and instability of a trinucleotide (CTG) repeat in a sequence encoding a cAMP-dependent protein kinase. The normal copy number of 5-35 repeats is exceeded in DM patients, with the size of the expansion broadly correlating with the severity of symptoms experienced. In most families reported, the unstable DNA sequence has increased in size on transmission to affected offspring, thereby providing a molecular explanation for the phenomenon of anticipation in DM, i.e. an increase in the severity of symptoms associated with an earlier age at onset of the disease in successive generations of a family. Here we present the first reported case of a family where the transmission of the affected chromosome from father to son is accompanied by a reduction in the size of the triplet expansion, such that it falls within the normal range. As the son remains asymptomatic, this type of molecular event may provide an explanation for the incomplete penetrance of the disease phenotype reported for this disorder. The implications for genetic counselling of DM families and the mechanistic considerations of the trinucleotide instability are discussed.