Coniferyl Alcohol, a Lignin Precursor, Stimulates Rhizobium rhizogenes A4 Virulence

Rhizobium rhizogenes, a soil bacterium, is the causative agent of the neoplastic disease hairy root. Upon incubation of Rhizobium rhizogenes A4 with coniferyl alcohol, a lignin precursor, bacterial virulence on cotton cotyledon slices was stimulated. This was observed both in numbers of root hairs produced and in their length. Stimulation was maximized after exposure of bacteria to 150 g/mL of coniferyl alcohol for 4 h. This was shown to be at the early log phase of bacterial growth.     

Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors

Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10-30 microM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.     

X-linked reticulate pigmentary layer. Report of a new patient and demonstration of a skewed X-inactivation

We report a boy, born to healthy first cousin parents, with diffuse hyperpigmentation of the skin and guttate hypomelanotic lesions, photophobia, abnormal hair, developmental delay, and recurrent bronchitis. Skin histology showed pigmentation incontinence with numerous melanophages. Electron microscopy showed a very high number of melanosomes and some degenerating keratinocytes. These features correspond to a rare genodermatosis, the X-linked reticulate pigmentary disorder with systemic manifestations. Skewed X-inactivation patterns were detected in the mother's lymphocytes.     

Lipoxins and lipoxin analogs in asthma

The pathobiology of asthma is characterized by production of eicosanoids, a diverse family of bioactive fatty acids that play important roles in regulating airway inflammation and reactivity. Lipoxins (LXs) are products of arachidonic acid metabolism that are distinct from leukotrienes (LTs) and prostaglandins (PGs) in structure and function. Unlike the pro-inflammatory PGs and LTs, LXs display counter-regulatory actions. Cell-type specific biological actions have been uncovered for LXs and LX stable analogs that promote resolution of acute inflammatory responses. At least two classes of receptors, CysLT1 receptors and LXA4 receptors (named ALX), can interact with LXA4 and LXA4 analogs to mediate their biological actions. LXs are generated during asthma and LXA4 signaling blocks asthmatic responses in humans and experimental model systems. Of interest, respiratory diseases of increased severity, such as aspirin-intolerant asthma, cystic fibrosis and steroid-dependent, severe asthma, display defective generation of these protective lipid signals. Together, these findings indicate a pivotal role for LXs in mediating airway homeostasis.     

N-glycosylation at one rabies virus glycoprotein sequon influences N-glycan processing at a distant sequon on the same molecule

Rabies glycoprotein (RGP(WT)) contains N-glycosylation sequons at Asn(37), Asn(247), and Asn(319), although Asn(37) is not efficiently glycosylated. To examine N-glycan processing at Asn(247) and Asn(319), full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn(247) sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn(319) in RGP(--3) are high-mannose. When both sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at sequons present individually or together suggests that glycosylation of one sequon affects glycosylation at another, distant sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn(247) and Asn(319). However, Asn(247) had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn(319). Moreover, when comparing the N-glycans at Asn(319) on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn(247) allows additional N-glycan processing to occur at Asn(319), yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one sequon influences N-glycan processing at a distant sequon on the same glycoprotein remains to be determined.     

A new seed-based assay for meiotic recombination in Arabidopsis thaliana

Meiotic recombination is a fundamental biological process that plays a central role in the evolution and breeding of plants. We have developed a new seed-based assay for meiotic recombination in Arabidopsis. The assay is based on the transformation of green and red fluorescent markers expressed under a seed-specific promoter. A total of 74 T-DNA markers were isolated, sequenced and mapped both physically and genetically. Lines containing red and green markers that map 1-20 cM apart were crossed to produce tester lines with the two markers linked in cis yielding seeds that fluoresced both in red and green. We show that these lines can be used for efficient scoring of recombinant types (red only or green only fluorescing seeds) in a seed population derived from a test cross (backcross) or self-pollination. Two tester lines that were characterized during several generations of backcross and self-pollination, one in the background of ecotype Landsberg and one in the ecotype Columbia, are described. We discuss the number of plants and seeds to be scored in order to obtain reliable and reproducible crossing over rate values. This assay offers a relatively high-throughput method, with the benefit of seed markers (similar to the maize classical genetic markers) combined with the advantages of Arabidopsis. It advances the prospect to better understand the factors that affect the rate of meiotic crossover in plants and to stimulate this process for more efficient breeding and mapping.     

Iron-ascorbic acid-induced oxidant stress and its quenching by paraoxonase 1 in HDL and the liver: comparison between humans and rats

Paraoxonase 1 (PON1) is a serum enzyme closely associated with high-density lipoprotein (HDL), which may protect against atherosclerosis by hydrolyzing lipid peroxides and several organophosphorus compounds. The purpose of the present study was to test the hypothesis that lipid peroxidation modifies the activity and protein mass of PON1 in humans and rats. Our findings revealed that the bulk of the activity monitored by the hydrolysis of paraoxon and phenyl acetate was confined to liver intracellular endoplasmic reticulum-derived microsomes and was mostly recovered in circulating HDL3. Confirmation was obtained by the determination of PON1 expression by Western blot. It is noteworthy that PON1 levels were consistently decreased in human sera, HDL, and liver microsomes compared with rat counterparts. Concomitant with iron-ascorbate-mediated lipid peroxidation, there was a decline in PON1 activity and protein in both HDL3 and microsomes, which was attenuated by butylated hydroxytoluene antioxidant treatment. The current data indicate that PON1 localization in microsomes and HDL3 could represent a selective cellular and lipoprotein response to oxidative stress. This was tested by the iron-ascorbate oxygen-radical generating system. It is also proposed that the increased PON1 level may have a function related to the well-known atherosclerosis resistance of rats.     

Substitution of feline leukemia virus long terminal repeat sequences into murine leukemia virus alters the pattern of insertional activation and identifies new common insertion sites

The recombinant retrovirus, MoFe2-MuLV (MoFe2), was constructed by replacing the U3 region of Moloney murine leukemia virus (M-MuLV) with homologous sequences from the FeLV-945 LTR. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. MoFe2 integrated infrequently (0 to 9%) near common insertion sites (CISs) previously identified for either parent virus. Using three different strategies, CISs in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parent virus in wild-type animals. Two of the newly identified CISs had not previously been implicated in lymphoma in any retrovirus model. One of these, designated 3-19, encodes the p101 regulatory subunit of phosphoinositide-3-kinase-gamma. The other, designated Rw1, is predicted to encode a protein that functions in the immune response to virus infection. Thus, substitution of FeLV-945 U3 sequences into the M-MuLV long terminal repeat (LTR) did not alter the target tissue for M-MuLV transformation but significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma. These observations support a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. The findings also demonstrate the oligoclonal nature of retrovirus-induced lymphomas by demonstrating proviral insertions at CISs in subdominant populations in the tumor mass. Finally, the findings demonstrate the utility of novel recombinant retroviruses such as MoFe2 to contribute new genes potentially relevant to the induction of lymphoid malignancy.