Ocular albinism type 1: more than meets the eye

Ocular albinism type 1 (OA1) is an X-linked recessive disorder characterized by a severe reduction of visual acuity, and hypopigmentation of the retina that leads to nystagmus, strabismus, and photophobia/photodysphoria. Microscopic examination of both retinal pigment epithelium and skin melanocytes in OA1 reveals the presence of macrome-lanosomes, suggesting that the OA1 gene product plays a role in melanosome biogenesis. Studies of mutations identified from OA1 patients and an Oa1 knock-out mouse model further implicate OA1 protein function in the late stage of melanosome development. Because its effects are primarily limited to the eye, OA1 represents an ideal model system to study the relationship between pigmentation and visual development. Based upon sequence homology and biochemical studies, OA1 may represent a novel intracellular G-protein coupled receptor. Understanding the function of OA1 will contribute greatly to our understanding of melanosome biogenesis and the role of pigmentation in visual development.     

Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT(1) receptors

Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions. It was hypothesized that LPS directly induces cardiac apoptosis. In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h. Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT(1)) inhibitor (losartan), but not by inhibitors of AT(2) receptors (PD-123319), tumor necrosis factor-alpha (TNFRII:Fc), or nitric oxide (N(G)-monomethyl-L-arginine). Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects. LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1-3 days, which dissipated after 1-2 wk. Losartan (23 mg. kg(-1). day(-1) in drinking water for 3 days) blocked LPS-induced in vivo apoptosis. In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT(1) receptors in myocytes.     

Human cell DNA replication is mediated by a discrete multiprotein complex

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.     

A back migration from Asia to sub-Saharan Africa is supported by high-resolution analysis of human Y-chromosome haplotypes

The variation of 77 biallelic sites located in the nonrecombining portion of the Y chromosome was examined in 608 male subjects from 22 African populations. This survey revealed a total of 37 binary haplotypes, which were combined with microsatellite polymorphism data to evaluate internal diversities and to estimate coalescence ages of the binary haplotypes. The majority of binary haplotypes showed a nonuniform distribution across the continent. Analysis of molecular variance detected a high level of interpopulation diversity (PhiST=0.342), which appears to be partially related to the geography (PhiCT=0.230). In sub-Saharan Africa, the recent spread of a set of haplotypes partially erased pre-existing diversity, but a high level of population (PhiST=0.332) and geographic (PhiCT=0.179) structuring persists. Correspondence analysis shows that three main clusters of populations can be identified: northern, eastern, and sub-Saharan Africans. Among the latter, the Khoisan, the Pygmies, and the northern Cameroonians are clearly distinct from a tight cluster formed by the Niger-Congo-speaking populations from western, central western, and southern Africa. Phylogeographic analyses suggest that a large component of the present Khoisan gene pool is eastern African in origin and that Asia was the source of a back migration to sub-Saharan Africa. Haplogroup IX Y chromosomes appear to have been involved in such a migration, the traces of which can now be observed mostly in northern Cameroon.     

Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer

Metallic surfaces can have unusual effects on fluorophores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy transfer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the silver islands so that there are a range of fluorophore-to-metal distances. We show that proximity of fluorophores to the silver islands results in increased fluorescence intensity, with the largest enhancement for the lowest-quantum-yield fluorophores. Importantly, the metal-induced increases in intensity are accompanied by decreased lifetimes and increased photostability. These effects demonstrate that the silver islands have increased the radiative decay rates of the fluorophore. For solvent-sensitive fluorophores the emission spectra shifted to shorted wavelengths in the presence of the silver islands, which is consistent with a decrease of the apparent lifetime for fluorophores near the metal islands. We also observed an increased intensity and blue spectral shift for the protein human glyoxalase, which displays a low quantum yield for its intrinsic tryptophan emission. In this case the blue shift is thought to be due to increased emission from a buried low-quantum-yield tryptophan residue. Increased intensities were also observed for the intrinsic emission of the nucleic acid bases adenine and thymine and for single-stranded 15-mers poly(T) and poly(C). And finally, we observed increased RET for donors and acceptors in solution and when bound to double-helical DNA. These results demonstrate that metallic particles can be used to modify the emission from intrinsic and extrinsic fluorophores in biochemical systems.     

Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding

Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS. Glu143 is positioned near the P-loop and interacts with the Mg(2+) of Mg NTP-binding proteins. We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+). The structure showed a water molecule at the place where Mg(2+) bound to the wild type enzyme. Mutant proteins E143A, and even E143D and E143Q with conservative mutations, lacked enzyme activity and failed to complement the methionine auxotrophy of the E. coli folC mutant SF4, showing that Glu143 is an essential residue. Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally. The E. coli E146Q mutant FPGS bound ADP with the same affinity as the wild type enzyme but bound ATP with much lower affinity and had higher ATPase activity than the wild type enzyme. The mutant enzyme was defective in forming the acyl-phosphate reaction intermediate from ATP and dihydropteroate. The E. coli FPGS requires activation by dihydropteroate or tetrahydrofolate binding to allow full activity. In the absence of a pteroate substrate, only 30% of the total enzyme binds ATP. We suggest that dihydropteroate causes a conformational change to allow increased ATP binding. The mutant enzyme was similarly activated by dihydropteroate resulting in increased ADP binding.