A dimer of a single polypeptide chain catalyzes the terminal four reactions of the L-tryptophan pathway in Euglena gracilis.

In Euglena gracilis the terminal four enzyme activities of the tryptophan biosynthetic pathway were found to be associated with a protein with an estimated molecular weight of 325,000 +/- 20,000. The protein was purified approximately 2,000-fold with relatively proportional recoveries of all four enzyme activities. The purified material was homogeneous by the criteria of analytical disc gel electrophoresis and gel isoelectric focusing. Disc gel electrophoresis after denaturation with sodium dodecyl sulfate gave a single protein band with a molecular weight of 155,000 +/- 5,000. Disc gel electrophoresis in 8 M urea also gave rise to a single protein band. We interpret these results as evidence for a single species of subunit. The pathway in Euglena is the only one known to the present in which the terminal enzyme, tryptophan synthase, is not a separate molecular species.     

Substrate Binding and Active Site Residues in RNases E and G: ROLE OF THE 5��-SENSOR*

The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5′-sensor, the S1 domain, and the Mg⁺²/Mn⁺² binding sites. The effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the k_cat decrease ∼40- to 100-fold) and RNA binding consistent with a key role for the 5′-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition.     

A Human Immunodeficiency Virus Screening Algorithm to Address the High Rate of False-Positive Results in Pregnant Women in Japan

Background

Prenatal human immunodeficiency virus (HIV) testing is essential for the prevention of mother-to-child transmission. However, false-positive results of screening testing are a concern as they may cause unnecessary emotional stress to pregnant women waiting for confirmatory test results. In regions with an extremely low prevalence, the positive predictive values of screening are unacceptably low rate. Here, we propose a HIV screening algorithm consisting of serial two fourth-generation enzyme immunoassays to reduce the number of false-positive screening results.

Methodology/Principal Findings

When 6461 pregnant women presenting to two maternity hospitals located in the Tokyo metropolitan area of Japan from September, 2004 to January, 2006 were tested using Enzygnost HIV Integral as a first screening test, 27 showed positive reactions. When these positive reaction samples were tested using VIDAS HIV DUO Quick as a second screening test, only one of them had a positive reaction, and the remaining 26 were nonreactive. Confirmatory Western blots and nucleic acid amplification test also showed that one was positive and the remaining 26 were negative; the subject who was positive with the confirmatory tests was identical to the subject who was positive with the second screening test. Thus, by adding the second screening test, the false-positive rate was improved from 0.4% to 0%, and the positive predictive value from 3.7% to 100%, compared with the single screening test.

Conclusion

By applying our serial screening algorithm to HIV testing in maternity hospitals, many uninfected pregnant women would not need to receive confirmatory tests and be subjected to emotional turmoil while waiting for their confirmatory test results. This algorithm would be suitable for HIV testing of pregnant women living in low prevalence regions such as Japan.     

Neuropsin (Opn5): a novel opsin identified in mammalian neural tissue

We have cloned and characterised the expression of a new opsin gene, neuropsin (Opn5), in mice and humans. Neuropsin comprises seven exons on mouse chromosome 17. Its deduced protein sequence suggests a polypeptide of 377 amino acids in mice (354 in humans), with many structural features common to all opsins, including a lysine in the seventh transmembrane domain required to form a Schiff base link with retinaldehyde. Neuropsin shares 25-30% amino acid identity with all known opsins, making it the founding member of a new opsin family. It is expressed in the eye, brain, testis and spinal cord.     

Involvement of calcitonin gene-related peptide in control of human fetoplacental vascular tone

Calcitonin gene-related peptide (CGRP), one of the most potent endogenous vasodilators known, has been implicated in vascular adaptations and placental functions during pregnancy. The present study was designed to examine the existence of CGRP-A receptor components, the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1), in the human placenta and the vasoactivity of CGRP in the fetoplacental circulation. Immunofluorescent staining of the human placenta in term labor using polyclonal anti-CRLR and RAMP1 antibodies revealed that labeling specifically concentrated in the vascular endothelium and the underlying smooth muscle cells in the umbilical artery/vein, chorionic artery/vein, and stem villous vessels as well as in the trophoblast layer of the placental villi. In vitro isometric force measurement showed that CGRP dose dependently relaxes the umbilical artery/vein, chorionic artery/vein, and stem villous vessels. Furthermore, CGRP-induced relaxation of placental vessels are inhibited by a CGRP receptor antagonist (CGRP8-37), ATP-sensitive potassium (KATP) channel blocker (glybenclamide), and cAMP-dependent protein kinase A inhibitor (Rp-cAMPS) and partially inhibited by a nitric oxide inhibitor (Nomega-nitro-l-arginine methyl ester). We propose that CGRP may play a role in the control of human fetoplacental vascular tone, and the vascular dilations in response to CGRP may involve activation of KATP channels, cAMP, and a nitric oxide pathway.     

The primary visual pathway in humans is regulated according to long-term light exposure through the action of a nonclassical photopigment

BACKGROUND: The mammalian eye shows marked adaptations to time of day. Some of these modifications are not acute responses to short-term light exposure but rely upon assessments of the photic environment made over several hours. In the past, all attempts at a mechanistic understanding have assumed that these adaptations originate with light detection by one or other of the classical photoreceptor cells (rods or cones). However, previous work has demonstrated that the mammalian eye contains non-rod, non-cone photoreceptors. This study aimed to determine whether such photoreceptors contribute to retinal adaptation. RESULTS: In the human retina, second-order processing of signals originating in cones takes significantly longer at night than during the day. Long-term light exposure at night is capable of reversing this effect. Here, we employed the cone ERG as a tool to examine the properties of the irradiance measurement pathway driving this reversal. Our findings indicate that this pathway (1) integrates irradiance measures over time periods ranging from at least 15 to 120 min; (2) responds to relatively bright light, having a dynamic range almost entirely outside the sensitivity of rods; (3) acts on the cone pathway primarily through a local retinal mechanism; and (4) detects light via an opsin:vitamin A photopigment (lambda(max) approximately 483 nm). CONCLUSIONS: A photopigment with a spectral sensitivity profile quite different from those of the classical rod and cone opsins but matching the standard profile of an opsin:vitamin A-based pigment drives adaptations of the human primary cone visual pathway according to time of day.     

Some Physical Characteristics of the Enzymes of l-Tryptophan Biosynthesis in Higher Plants

Anthranilate synthetase, phosphoribosyltransferase, phosphoribosyl anthranilate isomerase, and indoleglycerol phosphate synthetase were examined in partially purified extracts of the monocotyledon, Zea mays and the dicotyledon, Pisum sativum. The plant extracts were chromatographed on DEAE-cellulose and Sephadex G150. The molecular weights of the enzymes were determined and found to be similar to those observed for many bacteria. None of the plant tryptophan enzyme activities was aggregated in vitro as is also the case with most bacteria. This is in contrast with the complex aggregation patterns observed in other eucaryotic organisms that have been examined (fungi and Euglena gracilis). The tryptophan enzymes from peas and corn were generally similar but some differences in stability were observed.     

Molecular cloning of biologically active proviral DNA of the anemia-inducing strain of spleen focus-forming virus.

Previously, we have molecularly cloned proviral DNA of a polycythemia-inducing strain of the spleen focus-forming virus (SFFVp). In this paper, we report that unintegrated proviral DNA of the anemia-inducing strain of SFFV (SFFVA) has been molecularly cloned into pBR322. This molecularly cloned DNA retains the biological activity of SFFVA, as infectious SFFV can be recovered from the DNA clone by marker rescue using a previously described two-stage cotransfection assay (Linemeyer et al., J. Virol. 35:710-721, 1980). The recovered SFFV retains an important property of the initial SFFVA which distinguishes SFFVA from SFFVP, namely, the ability of SFFVA to cause proliferation of erythroid cells in which hemoglobin synthesis is erythropoietin dependent. By utilizing a marker rescue technique, the splenomegaly and anemia characteristic of SFFVA-induced disease have been traced to a DNA fragment of SFFVA containing sequences coding for the env gene product. gp52. The results suggest that the differences in pathogenicity between SFFVP disease and SFFVA disease are an intrinsic property of the env gene products of these two variants of Friend virus, and future studies with the molecular clones of each strain should allow us to map regions of each env gene responsible for common and distinctive features of the erythroproliferative diseases induced by each virus.