Zinc-induced decrease of the thermal stability and regeneration of rhodopsin

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn(2+) on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn(2+) concentrations (0-50 microm Zn(2+)). The thermal bleaching process is accelerated in the presence of Zn(2+) with k rate constants, at 55 degrees C, of 0.028 +/- 0.002 min(-1) (0 microm Zn(2+)) and 0.056 +/- 0.003 min(-1) (50 microm Zn(2+)), corresponding to t(12) values of 24.4 +/- 1.6 min and 11.8 +/- 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E(a) increase at 50 microm Zn(2+) for the process, with deltaG++ decrease and increase in deltaH++ and deltaS++ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn(2+) in normal and pathological retinal function.     

Temperature influence on embryonic development of Anopheles albitarsis and Anopheles aquasalis

Temperature influence on the embryonic development of Anopheles aquasalis and An. albitarsis was investigated. At 26 degrees C, 75% and 60% of respectively An. aquasalis and An. albitarsis eggs hatched, with one peak of eclosion, between the 2nd and 3rd day after oviposition. At 20 +/- 2 degrees C, around 66-70% of An. aquasalis eggs hatched, with one eclosion peak, on the 5th day. On the other hand, An. albitarsis eclosion at 21+/- 2 degrees C decreased to 10-22%, with two eclosion peaks, on the 4th-5th day and on the 9th-12th day. These data indicate a stronger temperature influence over An.albitarsis than over An. aquasalis embryos.     

Mouse novel Ly9: a new member of the expanding CD150 (SLAM) family of leukocyte cell-surface receptors

Human CS1, also known as novel Ly9, 19A24, or CRACC, is a member of the immunoglobulin gene superfamily (IgSF) expressed on natural killer cells and other leukocytes. Here we describe the cloning of the mouse homologue of this gene. The mouse novel Ly9 gene is shown to encode a transmembrane protein composed of two extracellular immunoglobulin-like domains, a transmembrane region and an 88-amino acid cytoplasmic domain. Mouse novel Ly9 is structurally similar to the extracellular domains of CD84 and CD229 (Ly9). Both mouse and human novel Ly9 genes mapped close to the CD229gene in a region where other members of the CD150 family have also been mapped, and analysis of their genomic sequences showed that they have an identical intron/exon organization. Northern blot analysis revealed that the expression of mouse and human novel Ly9 was predominantly restricted to hematopoietic tissues, with the exception of testis. Here we show that SAP (SH2D1A), an adapter protein responsible for the X-linked lymphoproliferative disease, binds to the phosphorylated cytoplasmic tail of human but not mouse novel Ly9. Taken together, these data indicate that mouse novel Ly9 is a new member of the expanding CD150 family of cell surface receptors.     

A retrospective hormonal and immunohistochemical evaluation of 47 acromegalic patients: prognostic value of preoperative plasma prolactin.

This study was performed to investigate the correlations between preoperative prolactin (PRL) plasma values, immunohistochemical picture and the clinical course in growth hormone (GH) secreting pituitary adenomas. In 47 patients (19 males and 28 females; mean age 40 years; range 13 - 70 years), we measured GH, IGF-1 and prolactin plasma values both before and after transsphenoidal surgery, and basal IGF-1 and GH after an oral glucose tolerance test (OGTT) during four years of follow-up. We considered those patients as "controlled" who presented an undetectable growth hormone after OGTT (GH < 1 microg/l), IGF-I plasma values in the normal range, matched for age and sex, and no clinical activity or neuroradiological recurrence after a four-year follow-up. We considered patients as "poorly controlled" who still showed elevated GH and IGF-I plasma levels, uninhibited GH after OGTT (GH > 1 microg/l), presence of clinical activity and/or radiological signs of adenoma recurrence, even if a reduction of tumor size had been demonstrated. RESULTS: Controlled patients (n = 22) exhibited mean preoperative PRL levels (+/- SEM) lower than the group of poorly controlled (n = 25) ones (21.40 +/- 5.51 vs. 38.44 +/- 5.16 microg/l; p < 0.03). From 3 to 12 months after surgery, postoperative PRL levels were also lower in the controlled patients compared to the poorly controlled ones (8.31 +/- 1.20 vs. 25.32 +/- 3.20 microg/l; p < 0.0001). Eighty percent (20/25) of poorly controlled patients showed both PRL and GH positivity after immunostaining. Only 3/22 (13.6 %) of controlled patients showed the same double positivity. In conclusion, preoperative hyperprolactinemia identifies a group of acromegalic patients at elevated risk of disease persistence after surgery. We hypothesize that most of these high-risk patients may have more aggressive mixed GH-PRL secreting adenomas.     

Simplifying amino acid alphabets by means of a branch and bound algorithm and substitution matrices

MOTIVATION: Protein and DNA are generally represented by sequences of letters. In a number of circumstances simplified alphabets (where one or more letters would be represented by the same symbol) have proved their potential utility in several fields of bioinformatics including searching for patterns occurring at an unexpected rate, studying protein folding and finding consensus sequences in multiple alignments. The main issue addressed in this paper is the possibility of finding a general approach that would allow an exhaustive analysis of all the possible simplified alphabets, using substitution matrices like PAM and BLOSUM as a measure for scoring. RESULTS: The computational approach presented in this paper has led to a computer program called AlphaSimp (Alphabet Simplifier) that can perform an exhaustive analysis of the possible simplified amino acid alphabets, using a branch and bound algorithm together with standard or user-defined substitution matrices. The program returns a ranked list of the highest-scoring simplified alphabets. When the extent of the simplification is limited and the simplified alphabets are maintained above ten symbols the program is able to complete the analysis in minutes or even seconds on a personal computer. However, the performance becomes worse, taking up to several hours, for highly simplified alphabets. AVAILABILITY: AlphaSimp and other accessory programs are available at http://bioinformatics.cribi.unipd.it/alphasimp     

Thermostable Pyrococcus furiosus DNA ligase catalyzes the synthesis of (di)nucleoside polyphosphates

DNA ligase from the hyperthermophilic marine archaeon Pyrococcus furiosus (Pfu DNA ligase) synthesizes adenosine 5'-tetraphosphate (p4A) and dinucleoside polyphosphates by displacement of the adenosine 5'-monophosphate (AMP) from the Pfu DNA ligase-AMP (E-AMP) complex with tripolyphosphate (P3), nucleoside triphosphates (NTP), or nucleoside diphosphates (NDP). The experiments were performed in the presence of 1-2 microM [alpha-32P]ATP and millimolar concentrations of NTP or NDP. Relative rates of synthesis (%) of the following adenosine(5')tetraphospho(5')nucleosides (Ap4N) were observed: Ap4guanosine (Ap4G) (from GTP, 100); Ap4deoxythymidine (Ap4dT) (from dTTP, 95); Ap4xanthosine (Ap4X) (from XTP, 94); Ap4deoxycytidine (Ap4dC) (from dCTP, 64); Ap4cytidine (Ap4C) (from CTP, 60); Ap4deoxyguanosine (Ap4dG) (from dGTP, 58); Ap4uridine (Ap4U) (from UTP, <3). The relative rate of synthesis (%) of adenosine(5')triphospho(5')nucleosides (Ap3N) were: Ap3guanosine (Ap3G) (from GDP, 100); Ap3xanthosine (Ap3X) (from XDP, 110); Ap3cytidine (Ap3C) (from CDP, 42); Ap3adenosine (Ap3A) (from ADP, <1). In general, the rate of synthesis of Ap4N was double that of the corresponding Ap3N. The enzyme presented optimum activity at a pH value of 7.2-7.5, in the presence of 4 mM Mg2+, and at 70 degrees C. The apparent Km values for ATP and GTP in the synthesis of Ap4G were about 0.001 and 0.4mM, respectively, lower values than those described for other DNA or RNA ligases. Pfu DNA ligase is used in the ligase chain reaction (LCR) and some of the reactions here reported [in particular the synthesis of Ap4adenosine (Ap4A)] could take place during the course of that reaction.     

PEX11alpha is required for peroxisome proliferation in response to 4-phenylbutyrate but is dispensable for peroxisome proliferator-activated receptor alpha-mediated peroxisome proliferation

The PEX11 peroxisomal membrane proteins promote peroxisome division in multiple eukaryotes. As part of our effort to understand the molecular and physiological functions of PEX11 proteins, we disrupted the mouse PEX11alpha gene. Overexpression of PEX11alpha is sufficient to promote peroxisome division, and a class of chemicals known as peroxisome proliferating agents (PPAs) induce the expression of PEX11alpha and promote peroxisome division. These observations led to the hypothesis that PPAs induce peroxisome abundance by enhancing PEX11alpha expression. The phenotypes of PEX11alpha(-/-) mice indicate that this hypothesis remains valid for a novel class of PPAs that act independently of peroxisome proliferator-activated receptor alpha (PPARalpha) but is not valid for the classical PPAs that act as activators of PPARalpha. Furthermore, we find that PEX11alpha(-/-) mice have normal peroxisome abundance and that cells lacking both PEX11alpha and PEX11beta, a second mammalian PEX11 gene, have no greater defect in peroxisome abundance than do cells lacking only PEX11beta. Finally, we report the identification of a third mammalian PEX11 gene, PEX11gamma, and show that it too encodes a peroxisomal protein.     

Analysis of 22 deletion breakpoints in dystrophin intron 49

Over 60% of Duchenne and Becker muscular dystrophies are caused by deletions spanning tens or hundreds of kilobases in the dystrophin gene. The molecular mechanisms underlying the loss of DNA at this genomic locus are not yet understood. By studying the distribution of deletion breakpoints at the genomic level, we have previously shown that intron 49 exhibits a higher relative density of breakpoints than most dystrophin introns. To determine whether the mechanisms leading to deletions in this intron preferentially involve specific sequence elements, we sublocalized 22 deletion endpoints along its length by a polymerase-chain-reaction-based approach and, in particular, analyzed the nucleotide sequences of five deletion junctions. Deletion breakpoints were homogeneously distributed throughout the intron length, and no extensive homology was observed between the sequences adjacent to each breakpoint. However, a short sequence able to curve the DNA molecule was found at or near three breakpoint junctions.