Locatization of human herpesvirus type 8 in human sperms by in situ PCR

SummaryObjectives: Defining the mechanism of infection with human herpesvirus-8 (HHV-8) or Kaposi’s sarcoma associated herpesvirus (KSHV) is an important clinical issue. HHV-8 has been linked to Kaposi’s sarcoma (KS) development in HIV-1-infected individuals, and KS develops in 40% of those infected with both viruses. A series of epidemiological data suggest that sexual transmission is one of the routes of transmission for HHV-8. In our studies, we sought to assess the cellular reservoirs of HHV-8 DNA in the semen of HIV-1-infected men and the potential role of HHV-8 infected spermatozoa in horizontal transmission.Design and methods: A nested polymerase chain reaction (PCR), in situ PCR (ISPCR) and a sodium iodide (NaI) DNA isolation technique that extracts both nuclear and episomal DNA were utilized to amplify specific genes in vitro and within intact cells to evaluate the types of seminal cells infected with HHV-8 in HIV-1-infected and uninfected men.Results HHV-8 was present in the spermatozoa and mononuclear cells of the semen in 64 of 73 (88%) HIV-1 infected individuals. Both the sperms as well as the mononuclear cells of the semen specimens of HIV-1 infected men were found to be infected with HHV-8. Multiplex ISPCR revealed that a significantly higher percentage of semen cells were infected with HHV-8 than HIV-1 (p>0.001). Rare (less than one in a 100,000) sperm cells were co-infected with both viruses. A co-culture of HHV-8 infected sperm with uninfected 293 or Sup-T1 cell lines resulted in an abortive infection of these cells with HHV-8. DNA isolation by NaI yielded 73% of the positive sperm, whereas the standard phenol/chloroform method resulted in significantly lower positives (45%) from the same specimens.Conclusions: Design and methods: Our data strongly suggest a potential sexual/horizontal route of transmission of HHV-8, via the HHV-8 infected sperm and other semen cells, where a large percentage of HIV-1 infected men’s sperm and other semen cells are infected with HHV-8. Co-culture studies have further supported the observations that HHV-8 in the sperm cells is infectious and capable of transmission of the virus to uninfected cells.     

Naupliar and Metanaupliar Development of Thysanoessa raschii (Malacostraca,Euphausiacea) from Godth?bsfjord,Greenland, with a Reinstatement of the Ancestral Status of the Free-Living Nauplius in Malacostracan Evolution

The presence of a characteristic crustacean larval type, the nauplius, in many crustacean taxa has often been considered one of the few uniting characters of the Crustacea. Within Malacostraca, the largest crustacean group, nauplii are only present in two taxa, Euphauciacea (krill) and Decapoda Dendrobranchiata. The presence of nauplii in these two taxa has traditionally been considered a retained primitive characteristic, but free-living nauplii have also been suggested to have reappeared a couple of times from direct developing ancestors during malacostracan evolution. Based on a re-study of Thysanoessa raschii (Euphausiacea) using preserved material collected in Greenland, we readdress this important controversy in crustacean evolution, and, in the process, redescribe the naupliar and metanaupliar development of T. raschii. In contrast to most previous studies of euphausiid development, we recognize three (not two) naupliar (= ortho-naupliar) stages (N1-N3) followed by a metanauplius (MN). While there are many morphological changes between nauplius 1 and 2 (e.g., appearance of long caudal setae), the changes between nauplius 2 and 3 are few but distinct. They involve the size of some caudal spines (largest in N3) and the setation of the antennal endopod (an extra seta in N3). A wider comparison between free-living nauplii of both Malacostraca and non-Malacostraca revealed similarities between nauplii in many taxa both at the general level (e.g., the gradual development and number of appendages) and at the more detailed level (e.g., unclear segmentation of naupliar appendages, caudal setation, presence of frontal filaments). We recognize these similarities as homologies and therefore suggest that free-living nauplii were part of the ancestral malacostracan type of development. The derived morphology (e.g., lack of feeding structures, no fully formed gut, high content of yolk) of both euphausiid and dendrobranchiate nauplii is evidently related to their non-feeding (lecithotrophic) status.     

Binding of β-Amyloid (1-42) Peptide to Negatively Charged Phospholipid Membranes in the Liquid-Ordered State: Modeling and Experimental Studies

To explore the initial stages of amyloid β peptide (Aβ42) deposition on membranes, we have studied the interaction of Aβ42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L_o) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L_o state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5–5 mol %) than higher (10–20 mol %) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aβ42 binding than L_o ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of β-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aβ42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol % anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aβ42 and membrane.     

Aluminum affects glial system and behavior of rats

Aluminum (Al) has been associated with neuronal dysfunction. These neuronal changes may involve glial alterations. We intend to evaluate the consequence of Al on the glial system and the behavior of rats exposed chronically to 0.3% of aluminum chloride in drinking water during 4 months in adulthood (A) or since intra-uterine age (IU); animals from this latter group were sacrificed at four months of age. Our data show an intense glial fibrillary acidic protein (GFAP)-immunoreactivity with a high density of astrocytes in both treated groups compared with controls. However, in IU rats, astrocytes display prominent glial cell bodies and processes. A and IU rat groups perform a significantly reduced locomotor activity. However, using the dark/light box test, the IU rats prefer to spend more time in the enlightened compartment compared to other groups. Behavioral and glial changes caused by Al exposure bring support for the role of Al in brain dysfunction involving glial alterations.     

Dual RAS blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in Akita angiotensinogen-transgenic mice

We investigated the effects of dual renin-angiotensin system (RAS) blockade on angiotensin-converting enzyme-2 (Ace2) expression, hypertension, and renal proximal tubular cell (RPTC) apoptosis in type 1 diabetic Akita angiotensinogen (Agt)-transgenic (Tg) mice that specifically overexpress Agt in their RPTCs. Adult (11 wk old) male Akita and Akita Agt-Tg mice were treated with two RAS blockers (ANG II receptor type 1 blocker losartan, 30 mg·kg(-1)·day(-1)) and angiotensin-converting enzyme (ACE) inhibitor perindopril (4 mg·kg(-1)·day(-1)) in drinking water. Same-age non-Akita littermates and Agt-Tg mice served as controls. Blood pressure, blood glucose, and albuminuria were monitored weekly. The animals were euthanized at age 16 wk. The left kidneys were processed for immunohistochemistry and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess gene and protein expression. Urinary ANG II and ANG 1-7 were quantified by ELISA. RAS blockade normalized renal Ace2 expression and urinary ANG 1-7 levels (both of which were low in untreated Akita and Akita Agt-Tg), prevented hypertension, albuminuria, tubulointerstitial fibrosis and tubular apoptosis, and inhibited profibrotic and proapoptotic gene expression in RPTCs of Akita and Akita Agt-Tg mice compared with non-Akita controls. Our results demonstrate the effectiveness of RAS blockade in preventing intrarenal RAS activation, hypertension, and nephropathy progression in diabetes and support the important role of intrarenal Ace2 expression in modulating hypertension and renal injury in diabetes.     

Phytoplankton distribution in the upwelling areas of the Moroccan Atlantic coast localized between 32°30'N and 24°N

Marine phytoplankton was studied in January and July 2002 along of four transects: 32°30′N, 29°N, 24°30′N and 24°N. A total of 142 taxa were recorded in this area. The maximal specific richness, both in summer and in winter, was registered around Dakhla (24°N). The global spatiotemporal variability of species richness and specific diversity showed that the most structured and species-rich populations are situated in the coastal areas. The vertical variation of the two parameters showed homogeneity between depth levels. The maximal phytoplanktonic densities are recorded in the southern transects due to the permanent upwelling activity in the southern Atlantic coast. The bathymetric distribution of densities was more heterogeneous during summer, characterized by a high intensity of upwelling. The species Thalassionema nitzschioides, Asterionellopsis glacialis, Melosira, Chaetoceros and Leptocylindrus minimus are indicators of upwelling.     

Cholesterol reverts Triton X-100 preferential solubilization of sphingomyelin over phosphatidylcholine: A P-NMR study

The distribution of phosphatidylcholine (PC) and sphingomyelin (SM) between the solubilized (micellar) and non-solubilized (lamellar) fractions arising from bilayers composed of PC and SM, with or without cholesterol (Chol) has been measured under conditions of partial, incomplete solubilization by Triton X-100. Quantitation is achieved by ³¹P-NMR determination of the composition of mixed micelles in the range of bilayer-micelle coexistence. We find that the solubilized fraction of bilayers consisting of binary mixtures of PC and SM is rich in SM, as expected from previous data on solubilization of pure PC and pure SM liposomes. In contrast, after partial solubilization of ternary mixtures of PC, SM and Chol, the solubilized fraction becomes SM-poor, as observed in the partial solubilization of biomembranes.     

A comparative study of the early osmotic,ionic, redox and hormonal signaling response in leaves and roots of two halophytes and a glycophyte to salinity

Salt stress is one of the most important abiotic stress factors affecting plant growth and productivity in natural ecosystems. In this study, we aimed at determining possible differences between salt tolerant and salt sensitive species in early (within 72 h) salt stress response in leaves and roots. To this purpose, we subjected three Brassicaceae species, namely two halophytes—Cakile maritima and Thellungiella salsuginea—and a glycophyte—Arabidopsis thaliana— to short-term salt stress (400 mM NaCl). The results indicate that the halophytes showed a differential osmotic and ionic response together with an early and transient oxidative burst, which was characterized by enhanced hydrogen peroxide levels and subsequent activation of antioxidant defenses in both leaves and roots. In addition, the halophytes displayed enhanced accumulation of abscisic acid, jasmonic acid (JA) and ACC (aminocyclopropane-1-carboxylic acid, the precursor of ethylene) in leaves and roots, as compared to A. thaliana under salt stress. Moreover, the halophytes showed enhanced expression of ethylene response factor1 (ERF1), the convergence node of the JA and ethylene signaling pathways in both leaves and roots upon exposure to salt stress. In conclusion, we show that the halophytes C. maritima and T. salsuginea experience an early oxidative burst, improved antioxidant defenses and hormonal response not only in leaves but also in roots, in comparison to the glycophyte A. thaliana. This differential signaling response converging, at least in part, into increased ERF1 expression in both above- and underground tissues seems to underlay, at least in part, the enhanced tolerance of the two studied halophytes to salt stress.