Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP

Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats 1, 2, or 4 days after adrenodemedullation. Adrenodemedullation reduced plasma epinephrine by 95% and norepinephrine by 35% but did not affect muscle norepinephrine content. In soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis increased by 15-20% by 2 days after surgery but returned to normal levels after 4 days. The rise in rates of protein degradation was accompanied by an increased activity of Ca(2+)-dependent proteolysis in both muscles, with no significant change in the activity of lysosomal and ATP-dependent proteolytic systems. In vitro rates of Ca(2+)-dependent proteolysis in soleus and EDL from normal rats decreased by ~35% in the presence of either 10(-5) M clenbuterol, a beta(2)-adrenergic agonist, or epinephrine or norepinephrine. In the presence of dibutyryl cAMP, proteolysis was reduced by 62% in soleus and 34% in EDL. The data suggest that catecholamines secreted by the adrenal medulla exert an inhibitory control of Ca(2+)-dependent proteolysis in rat skeletal muscle, mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.     

Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development

REV3 is the catalytic subunit of DNA polymerase ζ (pol ζ), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350 kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevis REV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340 kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65 pM is much lower than those of other replication and repair proteins including the accessory pol ζ subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol ζ in X. laevis.     

Regulation of the Rev1–pol ζ complex during bypass of a DNA interstrand cross‐link

DNA interstrand cross‐links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL (“approach”), a nucleotide is inserted across from the unhooked lesion (“insertion”), and the leading strand is extended beyond the lesion (“extension”). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site‐specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error‐free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1–pol ζ recruitment requires the Fanconi anemia core complex but not FancI–FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair‐associated mutagenesis.     

Calcareous nannofossil biostratigraphy of the Paleocene sediments of the Odessa Gas Field (NW Black Sea)

Calcareous nannofossils from Paleocene sediments of two boreholes (Odeska-6 and 20) from the north-western shelf of the Black Sea are examined. Five nannofossil Zones are identified according to the standard zonations of Martini (1971) and Quillévéré et al. (2002): the Chiasmolithus danicus Zone (NP3), the upper part of Ellipsolithus Macellus Zone (NP4b), the Fasciculithus tympaniformis Zone (NP5), the Heliolithus kleinpelli Zone (NP6) and the Heliolithus riedelii Zone (NP8). This biostratigraphical work allows us to correlate the Bilokamian and Kachian regional stages of the Stratigraphic Scheme of Southern Ukraine (Zernetskiy et al., 1993) to the standard nannofossil zonations and, therefore, to the International Chronostratigraphic scheme. The presence of an unconformity between the Bilokamian and Kachian regional stages in the borehole section of Odeska-6 is suggested by Linear Sedimentary Rates estimated for the two boreholes. This unconformity corresponds to the upper part of the Chiasmolithus danicus nannofossil Zone (NP3) and the lower part of Ellipsolithus Macellus (NP4a), and is estimated to last nearly 1.94 Ma.     

Living with primary ciliary dyskinesia: a prospective qualitative study of knowledge sharing, symptom concealment, embarrassment, mistrust, and stigma

Background

Primary ciliary dyskinesia (PCD) is a chronic respiratory disease for which there is little psycho-social research and no qualitative studies of individuals living with the condition. A questionnaire-based survey in 2003 found evidence of stigmatisation in some individuals with PCD. Although the questionnaire had face and construct validity, stigmatisation was not cross-validated against interviews. The present study had the twin aims of carrying out a qualitative study of the adult patients living with PCD, and using a structured design to validate the questionnaire measure of stigma.

Methods

Interviews were carried out with six pairs of individuals with PCD, matched for sex, situs, and age, one with a high stigma score in 2003 and the other with a low stigma score. Depth-qualitative interviews were conducted by one author to explore themes surrounding the psycho-social impact of PCD using a grounded theory analysis. The interviewer was blind to the stigma scores of participants, and after the qualitative analysis was completed, the interviewer made an assessment of which member of each pair seemed the more stigmatised, after which the code was broken.

Results

Interviews revealed a number of themes, including other people's knowledge of PCD, the sharing of knowledge about PCD, the concealment of symptoms of PCD, embarrassment at symptoms, changes of behaviour in response to PCD, mistrust of medical care, in particular in relation to problems in diagnosis, a mistrust of general practitioners who were seen as poorly informed, and the importance of expert care at tertiary referral centres. Although stigmatisation as such was rarely mentioned directly by respondents, when the interviewer's judgement on level of stigmatisation was correlated with stigma scores from 2003, it was found that the more stigmatised member had been correctly identified in all six pairs (p = .016).

Conclusion

Our results suggest that some people with PCD feel isolated through mistrust in medicine, and lack of knowledge surrounding PCD. Many responses to PCD can be explained in terms of stigmatisation, and in particular felt stigma. The correlation between questionnaire used several years previously, and the interviewer's judgements of stigmatisation suggest that the stigma questionnaire had both predictive validity and long-term stability. As in other chronic conditions, stigmatisation occurs only in some individuals with PCD, and the present study explores the basis of stigmatisation, and validate the questionnaire as a measure of difference in stigma.