A novel TNNI2 mutation causes Freeman–Sheldon syndrome in a Chinese family with an affected adult with only facial contractures

Distal arthrogryposes (DAs), a clinically and genetically heterogeneous group of disorders characterized by congenital contractures with predominant involvement of the hands and feet, can be classified into at least 12 different forms. These autosomal dominant disorders are of variable expressivity and reduced penetrance. Mutations in sarcomeric protein genes, including troponin I2 (TNNI2), troponin T3 (TNNT3), tropomyosin 2 (TPM2), embryonic myosin heavy chain 3 (MYH3), and myosin binding protein C1 (MYBPC1), have been identified in distal arthrogryposis type 1 (DA1, MIM 108120), type 2B (DA2B, MIM 601680) and type 2A (DA2A)/Freeman–Sheldon syndrome (FSS, MIM 193700). However, mutations causing FSS have only been reported in MYH3. Herein we describe a Chinese DA family whose members meet classical strict criteria for FSS, as well as one member of the family who has isolated facial features consistent with FSS. No disease-causing mutation was found in MYH3. Segregation of microsatellite markers flanking the TNNI2 and TNNT3 genes at 11p15.5 was compatible with linkage. Subsequent sequencing of TNNI2 revealed a novel mutation, c.A493T (p.I165F), located in the C-terminal region, which is critical for proper protein function. This mutation was found to cosegregate with the FSS phenotype in this family, and assessment using SIFT and PolyPhen-2 predicted a damaging effect. To the best of our knowledge, we report the first TNNI2 mutation in classical FSS and describe an atypical adult FSS case with only facial contractures resulting from somatic mosaicism. We infer that DA1, DA2B and FSS represent a phenotypic continuum of the same disorder and provide further genetic evidence for this hypothesis.     

Tyrosine protein kinase activity in purified rat Leydig cells

Tyrosine protein kinase activity has been estimated in purified testicular cells with the synthetic peptide substrate NH₂-GLU-ASP-ALA-GLU-TYR-ALA-ALA-ARG-ARG-ARG-GLY-COOH. High levels of enzyme specific activity (56–165 pmol/mg/min) were found in the two populations of Leydig cells isolated by Metrizamide gradient centrifugation. Some activity was also detected in germinal cells, red cells and seminiferous tubules from testis but at levels 6–20 times lower than those found in the Leydig cell fractions. Higher levels of tyrosine protein kinase specific activity were found in population I than in population II Leydig cells.     

Interaction between the urea cycle and the orotate pathway: studies with isolated hepatocytes

Two enzymes catalyze the synthesis of carbamylphosphate (CP) in the liver. One is intramitochondrial and utilizes ammonia to make CP for ureagenesis; the second is cytoplasmic and utilizes glutamine to produce CP for pyrimidine biosynthesis. The extent to which the metabolic independence of the two pathways is abridged by the use of a common precursor was examined with measurements of the incorporation of [14C]NaHCO3 into orotic acid, uridine nucleotides, and urea in isolated hepatocytes. Pyrimidine synthesis was markedly stimulated by physiological concentrations of ammonia, and the stimulation was antagonized by ornithine. At intracellular concentrations of ornithine and levels of ammonia found in the portal circulation, some 90% of pyrimidine synthesis was ammonia-dependent. When the glutamine-dependent activity was released from feedback inhibition with galactosamine, the ammonia-dependent incorporation still accounted for 2/3 of pyrimidine synthesis. These results do not support the widely held view that the cytoplasmic enzyme is the sole source of CP for pyrimidine biosynthesis in the liver. They suggest instead that the bulk of the CP incorporated into hepatic pyrimidines is of mitochondrial origin. However, an experiment with intact animals failed to provide decisive evidence on this interpretation. Pyrimidine biosynthesis was sharply inhibited by the addition of uridine, but ureagenesis was unaffected. When physiological levels of ammonia were provided, the sensitivity of pyrimidine biosynthesis to uridine was lost. Although inhibition of the ammonia-dependent enzyme by pyrimidines has been observed with cell-free preparations, it was not evident in the intact cell. Thus, to the extent that the CP consumed in pyrimidine biosynthesis is of mitochondrial origin, feedback control of the orotate pathway appears to be thwarted.     

Human brain monoamine oxidase: solubilization and kinetics of inhibition by octylglucoside

Complete solubilization of both the A and B forms of human brain monoamine oxidase (MAO) occurred when crude mitochondria were incubated in the presence of 50 mM octylglucoside (OG). Upon removal of this nonionic detergent by dialysis, approximately 100% of the starting activity was present in the dialysate. The effects of solubilization were examined by comparison of several properties of the membrane-bound and OG-treated oxidases. The percentage inhibition of phenylethylamine (PEA) and the 5-hydroxytryptamine (5-HT) deamination by deprenyl and clorgyline were identical. The Km values obtained for the deamination of PEA, a B-selective substrate, 5-HT, an A-selective substrate, and tyramine (TYR), a nonselective substrate, were also comparable. OG was found to inhibit type A (I50 = 8.1 mM) and B (I50 = 4.7 mM) MAO activities at concentrations at least 10-fold below those used to solubilize the oxidases. Kinetic studies revealed that OG was an apparent competitive inhibitor of PEA deamination whereas OG produced a mixed-type pattern of inhibition when 5-HT was the variable substrate. Inhibition of TYR deamination by either the A or B form of MAO produced a mixed pattern of inhibition. The findings herein suggest that solubilization of the A and B forms of MAO by OG does not significantly alter the substrate and inhibitor specificity of the oxidases following removal of detergent. However, in the presence of concentrations of OG 50 times less than the critical micellar concentration of this detergent, marked inhibition of deamination by both forms of human brain MAO is observed. Accordingly, the usefulness of OG is limited to situations where the detergent is completely removed before quantitation of MAO activity.     

Antigen-initiated B lymphocyte differentiation. V. Electrophoretic separation of different subpopulations of AFC progenitors for unprimed IgM and memory IgG responses to the NIP determinant.

Spleen cells from CBA mice were separated by continuous, free-buffer film cell electrophoresis, and the capacity of cells in different fractions to mount an adoptive immune response specific for the NIP hapten determined. Experimental conditions were such that AFC progenitor B cells were measured, rather than helper or suppressor T cells. The IgM response of unprimed animals (a virgin or antigen inexperienced population) and the IgG response of long-term hapten-primed animals (a B memory cell population) were compared. The results indicated physical and biological heterogeneity in splenic B cells, with AFC progenitors for unprimed IgM and memory IgG responses being extensively separated.AFC progenitors for a primary IgM response in normal, germ-free and athymic mouse spleen, and bone marrow, separated into three distinct populations. Two of these were of much higher mobility than the typical splenic B cells and separated in the T cell zone. These cells produced a relatively early peak response of AFC after stimulation.AFC progenitors for a secondary IgG response were predominantly typical low-mobility B cells. Three regions of activity were separated, one overlapping part of the IgM progenitors. The slowest migrating activity peaks corresponded to the mobility of some recirculating B cells. These cells produced a more delayed AFC response after stimulation.AFC from the spleens of immunised mice separated as a single, broad, mediummobility peak distinct from most B cells and AFC progenitors. IgM and IgG (memory) AFC had similar electrophoretic characteristics.     

[3H]8-Hydroxy-2-(Di-n-Propylamino)Tetralin Binding to Pre- and Postsynaptic 5-Hydroxytryptamine Sites in Various Regions of the Rat Brain

The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD approximately 2 nM) followed by the cerebral cortex (KD approximately 6 nM) and the striatum (KD approximately 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.     

The rise in chlorophyll a fluorescence yield and decay in delayed light emission in Tris-washed chloroplasts in the 6–100 μs time range after an excitation flash

Parallel measurements of the rise in chlorophyll a fluorescence yield and delayed light emission decay, after a 10 ns saturating excitation flash, have been made in tris(hydroxymethyl)aminomethane-washed chloroplasts. Various electron donor systems (Mn²⁺; ascorbate; reduced phenylenediamine and benzidine) were used in conjuction with different preillumination regimes to alter [P⁺-680], the oxidized form of the Photosystem II reaction center chlorophyll a. Conditions giving rise to high [P⁺-680] resulted in only a small rise in fluorescence yield, an inhibition of a 6 μs delayed light component, and an enhancement of a 60 μs component of delayed light emission. These results confirm the hypothesis that P⁺-680 acts as a quencher of fluorescence and that delayed light emission in the microsecond time range is due to the back reaction of P⁺-680 and Q^?. (Q is the first “stable” electron acceptor of Photosystem II.) Two preillumination flashes are required before the full effect of Tris washing is observed in the delayed light emission decay and fluorescence yield rise; this suggests that a capacity to hold two charges exists between the Tris block and P⁺-680. Tris washing has no direct effect on the movement of electrons from Z (the first electron donor to P⁺-680) to P⁺-680. Finally, Mn²⁺ donates electrons to P⁺-680 via Z.     

Localized alteration in lateral root development in roots colonized by an arbuscular mycorrhizal fungus

 The morphological responses of root systems to localized colonization by endophytes is not well understood. We examined the responses of lateral roots to the arbuscular mycorrhizal (AM) fungus Gigaspora margarita Becker & Hall inoculated locally into the soil. Peanut (Arachis hypogaea L.) and pigeon pea (Cajanus cajan (L.) Millsp.) were examined. Root boxes filled with nutrient-poor soil in were inoculated in one half with the fungus and in the other half with a sterilized inoculum. Responses were apparent after 30 days but not after 20 days. Overall, lateral root development was more advanced in inoculated soil. This was clearly observed for 2nd- and 3rd-order lateral roots, but less clear for 1st-order lateral roots in both species, although percentage of colonized root length was higher in 1st-order lateral roots. Whilst in peanut the responses were clearly evident at the level of lateral roots initiated on more proximal parts of the tap root axis, they occurred on more distal parts in pigeon pea. We conclude that plants under nutrient-poor conditions give priority to mycorrhizal roots when partitioning assimilation products within the root system. Thus, AM formation may induce local morphological alteration of root systems. Accepted: 29 August 1996     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.