Analysis of length variation in the V1-V2 region of env in nonsubtype B HIV type 1 from Uganda

We optimized an assay for analysis of length variation in the V1-V2 region of HIV-1 env in plasma samples from Uganda. V1-V2 env length variation was analyzed in 31 plasma samples containing subtype A, C, D, or A/D recombinant HIV-1. DNA corresponding to the V1-V2 region was amplified by nested PCR. One of the primers in the second step of the PCR was fluorescently labeled. Successful amplification was confirmed by agarose gel electrophoresis. V1-V2 length variation of PCR products was analyzed with an ABI PRISM 3100 genetic analyzer and GeneScan software. A diversity score was generated for each sample on the basis of the degree of fragment length variation. The V1-V2 region was successfully amplified from 30 of 31 samples. Fragment length analysis was successful for all of those 30 samples. The diversity score and lengths of V1-V2 fragments were unique for each sample. This assay can be used for analysis of V1-V2 length variation in subtypes commonly found in Uganda. This assay may be helpful for studies examining the impact of env length diversity on HIV-1 transmission and pathogenesis in regions where these subtypes are prevalent.     

A possible role of cholesterol-sphingomyelin/phosphatidylcholine in nuclear matrix during rat liver regeneration

BACKGROUND/AIMS: Phospholipids and cholesterol in chromatin have been previously demonstrated. The lipid fraction changes during cell proliferation in relation to activation of enzymes of phospholipid metabolism. The aim of the present work is to clarify if chromatin lipids may derive or not from nuclear matrix and if they have different roles. METHODS: The subnuclear fractions were isolated from rat hepatocyte nuclei and the lipid fraction was extracted and analysed by chromatography in normal and regenerating liver. The phosphatidylcholine-sphingomyelin metabolism enzymes activity was assayed, by using radioactive substrates. RESULTS: In nuclear matrix, cholesterol and sphingomyelin are respectively five and three times higher than those present in chromatin; the amount of phosphatidylcholine, which it is enriched in saturated fatty acids, is lower, thus indicating a less fluid structure. The lower content in phosphatidylcholine may be justified by the phosphatidylcholine-dependent phospholipase C activity, which increases during liver regeneration, reaching a peak at the beginning of S-phase, when also cholesterol and sphingomyelin increase. CONCLUSIONS: The nuclear matrix lipids are independent from chromatin lipids; the ratio cholesterol-sphingomyelin/phosphatidylcholine is higher and, as a consequence, nuclear matrix is less fluid in relation to DNA synthesis, suggesting a specific role of nuclear matrix as a structure involved in DNA duplication.     

The m.3243A>G mitochondrial DNA mutation and related phenotypes. A matter of gender?

The m.3243A>G “MELAS” (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) mutation is one of the most common point mutations of the mitochondrial DNA, but its phenotypic variability is incompletely understood. The aim of this study was to revise the phenotypic spectrum associated with the mitochondrial m.3243A>G mutation in 126 Italian carriers of the mutation, by a retrospective, database-based study (“Nation-wide Italian Collaborative Network of Mitochondrial Diseases”). Our results confirmed the high clinical heterogeneity of the m.3243A>G mutation. Hearing loss and diabetes were the most frequent clinical features, followed by stroke-like episodes. “MIDD” (maternally-inherited diabetes and deafness) and “PEO” (progressive external ophthalmoplegia) are nosographic terms without any real prognostic value, because these patients may be even more prone to the development of multisystem complications such as stroke-like episodes and heart involvement. The “MELAS” acronym is convincing and useful to denote patients with histological, biochemical and/or molecular evidence of mitochondrial disease who experience stroke-like episodes. Of note, we observed for the first time that male gender could represent a risk factor for the development of stroke-like episodes in Italian m.3243A>G carriers. Gender effect is not a new concept in mitochondrial medicine, but it has never been observed in MELAS. A better elucidation of the complex network linking mitochondrial dysfunction, apoptosis, estrogen effects and stroke-like episodes may hold therapeutic promises.     

Expanding the spectrum of megalencephalic leukoencephalopathy with subcortical cysts in two patients with GLIALCAM mutations

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a heterogeneous neurodegenerative leukodystrophy caused by recessive mutations in MLC1 or GLIALCAM (types MLC1 and MLC2A) of by dominant mutations in GLIALCAM (MLC2B). GlialCAM functions as an auxiliary subunit of both MLC1 and ClC-2 chloride channel, increasing and modifying the function of the latter. Dominant mutations in GLIALCAM cause transient features of MLC but lacks clinical deterioration. Most recessive and dominant mutations in GLIALCAM studied so far affect the targeting of GlialCAM and its associated subunits. Here, we have investigated two patients with MLC2. The first patient has MLC2B disease, as shown by the improvement in MRI and clinical parameters. In this case, we identified a novel GLIALCAM mutation (p.Q56P) which affected the localization of GlialCAM and its associated subunits, however activating ClC-2 function as the wild-type protein. The second patient has MLC2A disease, as indicated by the lack of clinical improvement, even though, interestingly, the MRI of this patient shows a partial improvement. In this case, we found a recessive mode of inheritance, as the patient harbors two compound heterozygous mutations in GLIALCAM. One of them introduces a stop codon (p.Q56X), whereas the second mutation is a missense mutation (p.R73W), for which we could not identify any trafficking defect or an altered functional effect on ClC-2 in vitro.     

Tryptophan oxidation by singlet molecular oxygen [O2(1Deltag)]: mechanistic studies using 18O-labeled hydroperoxides, mass spectrometry, and light emission measurements

Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O2(1Deltag)]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from W18O18OH decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.