Two different genes encode ferrochelatase in Arabidopsis: mapping, expression and subcellular targeting of the precursor proteins

Six adolescents, five males with prolactin-secreting pituitary macroadenomas and one female with idiopathic hyperprolactinaemia, are described. Their ages at presentation ranged from 13 years 7 months to 16 years 6 months. Presenting symptoms included headache, visual field defect, arrested growth and puberty. Only two cases had galactorrhoea. Every case had an elevated serum prolactin level. Three had surgery before the results of serum prolactin were to hand. Each patient was treated with bromocriptine. Bromocriptine suppressed serum prolactin level to normal in four cases, but in the girl with idiopathic hyperprolactinaemia, bromocriptine was not useful. In two boys, serum prolactin was not suppressed with bromocriptine therapy alone, and they were subsequently treated with cabergoline, surgery and irradiation. Nevertheless, in children and adolescents with prolactin-secreting pituitary adenoma, bromocriptine should be the first line of treatment.     

The chronocorrection of disorders in the blood coagulation rhythm with kurantil in patients with decompensated rheumatic heart defects

The circadian pattern of hemocoagulation was studied in patients with decompensated rheumatic heart disease (DRHD) concurrent with stages I-II circulatory failure (CF) during complex treatment or medical treatment with disaggregants. Biorhythmological studies demonstrated that in patients with DRHD and CF chronotherapy with curantyl had some advantages over the traditional therapy during complex drug therapy. In these patients, the chronopatterns of circadian rhythms of hemocoagulative parameters tended to normalize under the influence of curantyl chronotherapy, by diminishing the signs of external desynchronization. Advantages of chronotherapy over the traditional treatment found in patients with DRHD and stages I-II CF, as manifested by its clinical effect in shorter periods (on days 4-5) when small daily and course doses of the drug were used. Based on the biorhythmological studies of hemostatic parameters, a method of curantyl chronotherapy was developed for patients with DRHD and stages I-II CF, which may optimize the therapeutical process in patients with this abnormality.     

Loss of protein kinase C inhibition in the beta-T594M variant of the amiloride-sensitive Na+ channel

We previously reported the presence of a novel variant (beta-T594M) of the amiloride-sensitive Na+ channel (ASSC) in which the threonine residue at position 594 in the beta-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein-Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the beta-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the beta-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.     

Ex vivo cytotoxic drug evaluation by DiSC assay to expedite identification of clinical targets: results with 8-chloro-cAMP

There is a pressing need to reduce the time and cost of developing new cytotoxic agents and to accurately identify clinically active agents at an early stage. In this study, the differential staining cytotoxicity (DiSC) assay was used to assess the efficacy of the novel antitumour cAMP analogue, 8-chloro-cAMP (8-Cl-cAMP) (and its metabolite 8-Cl-adenosine) against 107 fresh specimens of human neoplastic and normal cells. Diagnoses included chronic and acute leukaemias, myeloma, non-Hodgkin's lymphoma (NHL) and miscellaneous solid tumours. The aim was to identify targets for subsequent phase I, II and III trials. 8-Cl-cAMP was tested at 4-985 microM, along with standard chemotherapeutic drugs. 8-Cl-cAMP and its metabolite caused no morphologically observable cell differentiation but induced dose-dependent cytotoxicity. Compared with untreated patients, previously treated chronic lymphocytic leukaemia (CLL) patients showed no increase in ex vivo resistance to 8-Cl-cAMP (P = 0.878); minimal cross-resistance with other cytotoxic drugs was detected. Compared with normal cells (mean LC90 = 1803 microM), 8-Cl-cAMP showed significant ex vivo activity against CLL (117.0 microM; P < 0.0001) and NHL (140.0 microM; P < 0.0001), of which eight were mantle cell NHL (84.7 microM), and greatest activity against cells from patients with acute myeloid leukaemia (AML; mean LC90 = 24.3 microM; in vitro therapeutic index 74-fold, P < 0.0001). Solid tumour specimens were comparatively resistant to 8-Cl-cAMP. The results highlight the clinical potential of 8-Cl-cAMP, point to several new phase I, II and III trial possibilities and provide a rationale for the inclusion of ex vivo cytotoxic drug evaluation in the drug development process.     

Fluorescent labelled analogues of neuropeptide Y for the characterization of cells expressing NPY receptor subtypes

Porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, and subtype selective analogues have been synthesized by solid phase peptide synthesis. The peptides were labelled with Cy3, a commercially available fluorescent marker based on a cyanine dye, by solid phase strategy. During the cleavage a partial fragmentation of the fluorescent marker occurred. This has been investigated by means of HPLC and electrospray mass spectrometry. The labelled analogues of NPY showed high affinity to the NPY receptor subtypes Y1 and Y2. Thus, Cy3-NPY, Y1-selective Cy3-[Pro34] NPY and Y2 selective Cy3-[Ahx5-24] NPY were used to label SK-N-MC- and SMS-KAN-cells, which are stably expressing the Y1-(SK-N-MC) and the Y2-receptor subtype (SMS-KAN). The binding of the labelled analogues to the receptors was reversible and specific. The photoactivatable analogue, [(Tmd)Phe27] NPY, which showed high affinity to both receptor subtypes was labelled with Cy3 in solution. Whereas the fluorescent labelling of the cells with analogues without photoactivatable amino acid was reversible, successful photocrosslinking could be investigated by the irreversible staining of the cells using Cy3-[(Tmd)Phe27] NPY. These subtype selective analogues are exciting tools to trace receptors in tissues and to identify the pharmacologically characterized subtypes without radioactivity.