Integration of hepatitis B virus: analysis of unoccupied sites.

Hepatitis B virus (HBV) sequences integrated in the PLC/PRF/5 cell line (Alexander cells), which was derived from a human primary liver carcinoma, were previously extensively studied. Here we describe the analysis of the unoccupied sites of two linearly integrated forms of HBV DNA, AL-14 and AL-26, that were characterized previously. No major cellular DNA rearrangements were seen at the integration sites except for small deletions of host sequences: 2 kilobases of DNA in AL-14 and 17 base pairs (bp) in AL-26. The unoccupied site of AL-26 was found to be missing 182 bp, which previously mapped next to the right end of the integration sites of several independent clones. These were believed to be of cellular origin, but we show here that these 182 bp are in fact from unusual HBV sequences. Surprisingly, a region of this newly detected HBV DNA sequence is more homologous to that of woodchuck HBV DNA. Our analysis shows that the normal counterparts of both AL-14 and AL-26 contain minisatellite-like repetitive sequences. Based on the data presented here and our previous finding of HBV DNA integration at satellite sequences, we propose that genomic simple repetitive sequences are hot spots for HBV DNA integration.     

Acute Uteroplacental Ischemic Embryo: Lactic Acid Accumulation and Prostaglandin Production in the Fetal Rat Brain

A new experimental model for studying the effects of acute ischemia on brain development in the near-term fetal rat has been devised. Ischemic conditions are achieved by complete clamping of blood vessels branching from the uterine vasculature into each individual fetus for designated times followed by removal of the clamps to permit reperfusion. Accumulation of lactic acid in the fetal brain depends on the length of the restriction period, reaching a plateau level of 29 mumol/g tissue at about 30 min. It also depends on the reperfusion time. Thus after a period of 15 min of restriction lactate levels show an increase over the next 30-min reperfusion to a value of 25.5 mumol/g followed by a rapid decrease to normal values by 3 h of reperfusion. Restriction of 15 min followed by reperfusion of 45 min causes an elevation of prostaglandin E2 (PGE2) level from 12.4 +/- 0.86 ng/g to 21.1 +/- 0.6 ng/g (p less than 0.001). This elevation in PGE2 level is less apparent after 20 min of restriction. No effects are seen on the level of PGF2 alpha. Both PGE2 and PGF2 alpha accumulate in vitro in a time-dependent manner by brain particulate fraction. In vitro synthesis of both PGE2 and PGF2 alpha is inhibited by indomethacin (100% and 68%, respectively) and AA861 (94% and 76%, respectively). BW755c and nordihydroguaiaretic acid do not affect PGE2 formation but enhance PGF2 alpha production by 112% and 152%, respectively. Particulate fractions from restricted brain produce less PGF2 alpha than control brains (6.38 +/- 1.62 versus 11.43 +/- 2.2, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts

A major nutritional drawback of many crop plants is their low content of several essential amino acids, particularly lysine. The biosynthesis of lysine in plants is regulated by several feedback loops. Dihydrodipicolinate synthase (DHPS) from Escherichia coli, a key enzyme in lysine biosynthesis, which is considerably less sensitive to lysine accumulation than the endogenous plant enzyme has been expressed in chloroplasts of tobacco leaves. Expression of the bacterial enzyme was accompanied by a significant increase in the level of free lysine. No increase in protein-bound lysine was evident. Free lysine accumulation was positively correlated with the level of DHPS activity in various transgenic plants. Compartmentalization of DHPS in the chloroplast was essential for its participation in lysine biosynthesis as no lysine overproduction was obtained in transgenic plants that expressed the bacterial enzyme in the cytoplasm. The elevated level of free lysine in the transgenic plants was sufficient to inhibit, in vivo, a second key enzyme in lysine biosynthesis, namely, aspartate kinase, with no apparent influence on lysine accumulation. The present report not only provides a better understanding of the regulation of lysine biosynthesis in higher plants but also offers a new strategy to improve the production of this essential amino acid.     

Thromboxane and Prostacyclin Levels in Fetal Rabbit Brain and Placenta After Intrauterine Partial Ischemic Episodes

The appearance of arachidonic acid (AA) oxidation products in fetal rabbit brain and placenta under normal or partial short-term ischemic episodes induced by placental blood vessel restriction was examined. Intracerebral administration of [3H]AA into close-to-term rabbit fetuses gave rise to radioactively labeled prostaglandin (PG) E2, thromboxane B2, and 6-keto-PGF1 alpha metabolites as detected by HPLC analysis. A significant increase of 20-30% of [3H]AA precursor into eicosanoids was detected in brain of fetuses after 2-h restriction. The thromboxane B2 and 6-keto-PGF1 alpha levels were determined by radioimmunoassay technique over a period of 48 h following ischemic episodes. Thromboxane B2 content in affected animals was higher by five- and twofold at 3 h over control fetal brain and placental tissue values, respectively, and remained significantly higher for 24 h. 6-Keto-PGF1 alpha levels reached a peak value that was greater by 2.5- and 1.5-fold at 6 h for the ischemic brain and placental tissue, respectively, compared with control fetuses. PGE2 levels were less affected, attaining a maximum of 1.9- and 1.1-fold in brain and placenta correspondingly. The thromboxane/prostacyclin ratio reached a maximum in the brain after approximately 3 h, while that in the placenta continued to rise even after 20 h. Persisting high levels of thromboxane are indicative of cerebral vasoconstriction and may suggest possible damaging effects.     

Zero-crossing detectors in primary visual cortex?

David Marr and others have hypothesized that the visual system processes complex scene information in stages, the first of which involves the detection of light intensity edges or zero-crossings (Marr, 1982). Ideal zero-crossing detector mechanisms have been described and modeled in terms of their possible physiological implementation (Marr and Hildreth, 1980; Poggio, 1983). We now present evidence of visual cortical receptive fields which resemble in spatial organizational terms the requirements of zero-crossing analysis. The linear and nonlinear summation within and between the receptive field subunits are described and compared with predicted processes. The relative subunit sizes and separations are analyzed in these terms. Our findings support the notion that receptive fields may correspond with zero-crossing filters rather than zero-crossing detector gates.     

Threonine Overproduction in Transgenic Tobacco Plants Expressing a Mutant Desensitized Aspartate Kinase of Escherichia coli

In higher plants, the synthesis of the essential amino acid threonine is regulated primarily by the sensitivity of the first enzyme in its biosynthetic pathway, aspartate kinase, to feedback inhibition by threonine and lysine. We aimed to study the potential of increasing threonine accumulation in plants by means of genetic engineering. This was addressed by the expression of a mutant, desensitized aspartate kinase derived from Escherichia coli either in the cytoplasm or in the chloroplasts of transgenic tobacco (Nicotiana Tabacum cv Samsun NN) plants. Both types of transgenic plants exhibited a significant overproduction of free threonine. However, threonine accumulation was higher in plants expressing the bacterial enzyme in the chloroplast, indicating that compartmentalization of aspartate kinase within this organelle was important, although not essential. Threonine overproduction in leaves was positively correlated with the level of the desensitized enzyme. Transgenic plants expressing the highest leaf aspartate kinase activity also exhibited a slight increase in the levels of free lysine and isoleucine, both of which share a common biosynthetic pathway with threonine, but showed no significant change in the level of other free amino acids. The present study proposes a new molecular biological approach to increase the limiting content of threonine in higher plants.