Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.     

High gene flow and complex treeline dynamics of <Emphasis Type="Italic">Larix</Emphasis> Mill. stands on the Taymyr Peninsula (north-central Siberia) revealed by nuclear microsatellites

Arctic treelines are facing a strong temperature increase as a result of recent global warming, causing possible changes in forest extent, which will alter vegetation-climate feedbacks. However, the mode and strength of the response is rather unclear, as potential changes are happening in areas that are very remote and difficult to access, and empirical data are still largely lacking. Here, we assessed the current population structure and genetic differentiation of Larix Mill. tree stands within the northernmost latitudinal treeline reaching ~?72° N in the southern lowlands of the Taymyr Peninsula (~?100° E). We sampled 743 individuals belonging to different height classes (seedlings, saplings, trees) at 11 locations along a gradient from ‘single tree’ tundra over ‘forest line’ to ‘dense forest’ stands and conducted investigations applying eight highly polymorphic nuclear microsatellites. Results suggest a high diversity within sub-populations (H_E?=?0.826–0.893), coupled, however, with heterozygote deficits in all sub-populations, but pronounced in ‘forest line’ stands. Overall, genetic differentiation of sub-populations is low (F_ST?=?0.005), indicating a region-wide high gene flow, although ‘forest line’ stands harbour few rare and private alleles, likely indicating greater local reproduction. ‘Single tree’ stands, located beyond the northern forest line, are currently not involved in treeline expansion, but show signs of a long-term refuge, namely asexual reproduction and change of growth-form from erect to creeping growth, possibly having persisted for thousands of years. The lack of differentiation between the sub-populations points to a sufficiently high dispersal potential, and thus a rapid northward migration of the Siberian arctic treeline under recent global warming seems potentially unconstrained, but observations show it to be unexpectedly slow.     

Autosomal dominant intellectual disability

Intellectual disability (ID) is a heterogeneous entity defined as a substantial impairment of cognitive and adaptive function with an onset in early childhood and an IQ measure of less than 70. During the last few years, the next generation technologies, namely whole exome (WES) and whole genome sequencing (WGS), have given rise to the identification of many new genes for autosomal dominant (ADID), autosomal recessive (ARID) and X?linked forms of ID (XLID). The prevalence of ID is 1.5–2% for milder forms (IQ?<?70) and 0.3–0.5% for more severe forms of ID (IQ?<?50). Up to now, about 650 genes for ADID have been reported and it is expected that there are at least 350 genes still unidentified. Although the ADID genes can easily be classified according to the associated clinical findings, e.?g. different kind of seizures, abnormal body measurements, an advanced selection of reasonable genes for analyses is challenging. Many different panels for ID genes have been developed for a first diagnostic step, but more meaningful is the use of trio exome sequencing in individuals with sporadic ID. Using trio WES the mutation detection rate for de novo mutations in ID varies from 20 to 60%.Further research is needed for the identification of the remaining ID genes, a deeply understanding of the pathways and the development of therapies for the mostly rare causes of ID.     

Microangiopathic hemolytic anemia in patients with diseases of the hematopoietic and lymphatic systems

Microangiopathic haemolytic anaemia was diagnosed in the course of haematopoietic and lymphatic disorders such as chronic granulocytic leukemia, chronic myelofibrosis, chronic lymphatic leukemia, Osler's disease, chronic monocytic leukemia, and lymphoplasmocytic lymphoma, in 11 patients (6 women and 5 men) aged between 33 and 81 years (mean age 58.8 years) treated at the Haematological Out-Patient Clinic of the Postgraduate Medical Education Centre within 1977-1987. The following laboratory tests were carried out: 1) morphology of the peripheral blood and bone marrow, especially some haematological parameters concerning erythrocytes and blood platelets; 2) biochemical tests reflecting erythrocytes disintegration; 3) haemostasis. All examined patients suffered from haemolytic anaemia of various degree with characteristic changes in erythrocyte shape (helmets, tear-drops etc.). Haemolytic origin of anaemia was confirmed by the increased LDH activity. In the majority of patients no compensative stimulation of haematopoiesis (reticulocytosis, red blood cells hyperproliferation in bone marrow) was seen. Clinical symptoms of haemostatic disorders such as haemorrhagic diathesis and vein thrombosis were diagnosed in 50% of the patients. Blood platelet counts ranged from markedly decreased to significantly increased. Bone marrow smears did not show increased number of megacariocytes. Bleeding time was prolonged in the majority of examined patients while prothrombin index--decreased). Abnormal fibrinogen levels (decreased or increased) were found in the majority of patients with fibrin degradation products. Microangiopathic haemolytic anaemia in these patients differ from the typical Moschowitz's disease clinically probably due to the lack of compensative stimulation of erythropoiesis and lower thrombocytopenia.     

Amino acid sequences of ovomucoid third domain from 25 additional species of birds

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowskiet al. (1987,Biochemistry 26, 202–221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro¹⁴ Ser¹⁴ replacement in weka, an Ala¹⁴Thr¹⁵ replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile¹⁸ and Gly¹⁸ at the P₁ reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu³⁴) rather than positive (Lys³⁴ or Arg³⁴) amino acid residue at the NH₂ terminus of the helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genusGallus, in the five species genusSyrmaticus, and in the two species generaAix andPavo.     

Metabolic effects of long-term arterialization of portal blood

In nine splenectomized male dogs a splenic artery, -splenic vein shunt was made. Before splenectomy and 3, 6 and 18 months after arterialization of portal blood, different metabolic and endocrine parameters were estimated. Long-term arterialization of portal blood was followed by only insignificant increase of portal vein pressure but a significant drop of pCO2 and increase of pO2 in portal blood was recorded. Simultaneously, a significant decrease of the erythrocyte count, hematocrit value, serum cholesterol and uric acid levels, and a shortening of the T1/2 of insulin and glucagon were found. In contrast, long-term arterialization of portal blood was followed by a significant increase of serum triglycerides, alpha2-globulins, plasma renin activity, cortisol, gastrin and 25-hydroxyvitamin D, and by slight carbohydrate intolerance. No morphological abnormalities in the liver and kidney tissue were found. Data presented in this paper suggest usefulness of a splenic artery-splenic vein shunt in the treatment of some metabolic disorders and of the failing hepatocytes.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

Primary structure of V-ATPase subunit B from Manduca sexta midgut.

The amino acid sequence of a vacuolar-type ATPase (V-ATPase) subunit B has been deduced from a cDNA clone isolated from a Manduca sexta larval midgut library. The library was screened by hybridization with a labeled cDNA encoding subunit B of Arabidopsis thaliana tonoplast V-ATPase. The M. sexta V-ATPase subunit B consists of 494 amino acids with a calculated M(r) of 54,902. The amino acid sequence deduced for V-ATPase subunit B of M. sexta is between 98% and 76% identical with that of seven other V-ATPase subunits B and greater than 52% identical with three archaebacterial ATPase subunits B.