Full monosomy 21: echocardiographic findings in the third molecularly confirmed case.

Monosomy 21 is a rare chromosomal abnormality, with only nine cases reported in the literature. Affected infants display multiple dysmorphic features as well as skeletal, ocular, pulmonary, cardiac, renal, and genitourinary abnormalities. All monosomies are lethal except monosomy 21, but not all monosomy 21 fetuses survive to term. This report describes the echocardiographic findings and the congenital heart defects associated with the third case of molecularly confirmed full monosomy 21 in the literature. The cardiac defects included a mildly hypoplastic and hypertrophied left ventricle, a large ostium secundum atrial septal defect, a small anterior muscular ventricular septal defect, an interrupted inferior vena cava with azygos continuation, a parachute mitral valve, a bicuspid aortic valve, and a tortuous descending aorta. It also is the first description of a left pulmonary artery aneurysm and decreased left ventricular function as a component in the spectrum of defects found in full monosomy 21.

Polycyclic aromatic hydrocarbons are enriched but bioaccessibility reduced in brownfield soils adhered to human hands.

The health risk associated with exposure to urban brownfields is often driven by the incidental ingestion of soil by humans. Recent evidence found that humans likely ingest the fraction of soil that passes a 45-microm sieve, which is the particle size adhered to the hands. We evaluated if PAH concentrations were enriched in this soil fraction compared to the bulk soil and if this enrichment lead to an increase in bioaccessibility and thus an increase in incremental lifetime cancer risk for exposed persons. Soils (n=18) with PAH concentrations below the current Canadian soil quality guidelines for human health were collected from an Arctic urban site and were sieved to pass a 45-microm sieve. Soil PAH profiles were measured and bioaccessibility was assessed using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). PAHs were significantly enriched in the <45 microm size fraction (3.7-fold) and this enrichment could be predicted according to the fugacity capacity of soil (Enrichment=2.18-0.055Zsoil, r2=0.65, p<0.001). PAH release in the stomach and small intestine compartments of the SHIME was low (8%) and could not be predicted by PAH concentrations in 45-microm sieved soil. In fact, PAH release in the SHIME was lower from the <45 microm size fraction despite the fact that this fraction had higher levels of PAHs than the bulk soil. We postulate that this occurs because PAHs adsorbed to soil did not reach equilibrium with the small intestinal fluid. In contrast, PAH release in the colonic compartment of the SHIME reached equilibrium and was linked to soil concentration. Bioaccessibility in the SHIME colon could be predicted by the ratio of fugacity capacity of soil to water for a PAH (Bioaccessibility=0.15e(-6.4x10E-7Zsoil/Zwater), r2=0.53, p<0.01). The estimated incremental lifetime cancer risk was significantly greater for the <45 microm soil fraction compared to the bulk fraction; however, when bioaccessible PAH concentrations in a simulated small intestine were used in the risk assessment calculations, cancer risk was slightly lower in the <45 microm soil fraction for these soils. Our results highlight the importance of using a small soil size fraction, e.g. 45 microm, for contaminated site human health risk assessment. However, further work is needed to estimate the bioavailability of this size fraction in an in vivo model and to assess the correlation between in vitro and in vivo gastrointestinal models.

Estrogen receptor alpha-mediated adiposity-lowering and hypocholesterolemic actions of the selective estrogen receptor modulator acolbifene.

OBJECTIVE: The selective estrogen receptor (ER) modulator (SERM) acolbifene (ACOL), a potent and pure antiestrogen in the mammary gland and uterus, exerts beneficial pro-estrogenic actions on energy balance, insulin sensitivity and lipid metabolism. ACOL binds ERs alpha and beta, both of which have been involved in the metabolic actions of estrogen. This study aimed at determining the identity of the ER involved in the beneficial metabolic actions of ACOL. DESIGN AND MEASUREMENTS: ACOL was administered for 4 weeks to male and female wild-type and ERalpha knockout (KO) mice, and indices of energy balance as well as plasma and liver lipid concentrations were determined. RESULTS: ERalpha KO mice were heavier, gained more fat mass and had larger adipose depots than their wild-type counterparts. In both genders, ACOL decreased fat gain (50%) and white adipose tissue mass in male and female wild-type, but not in ERalpha KO mice. ACOL reduced plasma cholesterol in female wild-type mice (-27%), whereas the compound remained ineffective in their ERalpha KO counterparts. Plasma triglycerides were unaffected by ACOL. Finally, ACOL decreased liver cholesterol and triglyceride concentrations only in wild-type female animals. CONCLUSION: The beneficial metabolic actions of the SERM ACOL on adiposity and on plasma and liver lipids are entirely due to its interaction with the ERalpha.

Type and location of fluorescent probes incorporated into the potent mu-opioid peptide [Dmt]DALDA affect potency, receptor selectivity and intrinsic efficacy.

The dermorphin-derived tetrapeptide H-Dmt-d-Arg-Phe-Lys-NH(2) (Dmt = 2',6'-dimethyltyrosine) ([Dmt(1)]DALDA) is a highly potent and selective mu-opioid agonist capable of crossing the blood-brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt(1)]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6-dimethylamino-2'-naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea-pig ileum and mouse vas deferens assays, and in mu-, delta- and kappa-opioid receptor-binding assays. The analogues showed various degrees of mu receptor-binding selectivity, but all of them were less mu-selective than the [Dmt(1)]DALDA parent peptide. Most analogues retained potent, full mu-agonist activity, except for one with fluorescein attached at the C-terminus (3a) (partial mu-agonist) and one containing beta-(6'-dimethylamino-2'-naphthoyl)alanine (aladan) in place of Phe(3) (4) (mu- and kappa-antagonist). The obtained data indicate that the receptor-binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence-labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group (2a) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.

Cyclic enkephalin analogs containing various para-substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists.

The cyclic enkephalin analog H-Tyr-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) is a highly potent opioid agonist with IC(50)s of 35 pm and 19 pm in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe(1)-analog of this peptide showed 370-fold and 6790-fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr(1) hydroxyl-group in the interaction with mu and delta opioid receptors. In the present study, the effect of various substituents (-NH(2), -NO(2), -CN, -CH(3), -COOH, -COCH(3), -CONH(2)) introduced in the para-position of the Phe(1)-residue of H-Phe-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) on the in vitro opioid activity profile was examined. Most analogs showed enhanced mu and delta agonist potencies in the two bioassays, except for the Phe(pCOOH)(1)-analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH(3))(1)- and the Phe(pCONH(2))(1)-analogs. The latter compound showed subnanomolar mu and delta agonist potencies and represents the most potent enkephalin analog lacking the Tyr(1) hydroxyl-group reported to date. Taken together, these results indicate that various substituents introduced in the para-position of Phe(1) enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure-activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.

A chimeric opioid peptide with mixed mu agonist/delta antagonist properties.

There is evidence to indicate that opioid compounds with mixed mu agonist/delta antagonist properties are analgesics with low propensity to produce tolerance and physical dependence. A chimeric peptide containing the potent and selective mu agonist H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA) (Dmt=2',6'-dimethyltyrosine) and the potent and selective delta antagonist H-Tyr-TicPsi[CH2-NH]Cha-Phe-OH (TICP[Psi]) (Cha=cyclohexylalanine), connected 'tail-to-tail' via a short linker, was synthesized using a combination of solid-phase and solution techniques. The resulting peptide, H-Dmt-->D-Arg-->Phe-->Lys-NH-CH2-CH2-NH-Phe<--Cha[NH-CH2]PsiTic<--Tyr-H, showed the expected mu agonist/delta antagonist profile in the guinea-pig ileum and mouse vas deferens assays. Its mu and delta receptor binding affinities were in the low nanomolar range, as determined in rat brain membrane binding assays.

The effects of topiramate and sex hormones on energy balance of male and female rats.

OBJECTIVE: The effects of topiramate (TPM) on components of energy balance were tested in male and female rats that were (i) left intact, (ii) castrated or (iii) castrated with replacement therapies consisting of testosterone administration in orchidectomized (OCX) rats and of estradiol or progesterone treatments in ovariectomized (OVX) rats. METHODS: TPM was mixed into the diet and administered at a dose of 60 mg per kg of body weight. Male and female rats were treated for 28 and 35 days, respectively. At the end of the treatment period, variables of energy balance and determinants of lipid and glucose metabolism were assessed. RESULTS: TPM reduced energy and fat gains in both male and female rats either in the absence or in the presence of hormone replacement therapies. In male rats, it also decreased food intake, protein gain and energetic efficiency. In female animals, TPM reduced energetic efficiency while it stimulated lipoprotein lipase activity in brown adipose tissue. TPM also reduced plasma glucose and plasma leptin levels in female rats as well as plasma insulin and liver triglycerides in male animals. As expected, castration and sex hormones also strongly influenced energy balance. In male rats, OCX led to a decrease in energy and protein gains that was blocked by treatment with testosterone. In female rats, OVX caused increases in energy, fat and protein gains that were prevented by treatment with estradiol. CONCLUSION: In female rats, the effects of TPM on fat and energy gains were clearly not influenced by the sex hormone status of the rats. In male animals, there was also no interaction of TPM and the status of sex hormones on energy balance, suggesting that OCX and testosterone minimally interfere with the action of TPM on energy balance. The effects of TPM on energy balance were accounted for by a decrease in energetic efficiency, resulting from an effect exerted by the drug on both energy intake and thermogenesis. The present results also suggest that TPM can enhance insulin sensitivity.

Pseudoproline-containing analogues of morphiceptin and endomorphin-2: evidence for a cis Tyr-Pro amide bond in the bioactive conformation.

Analogues of the opioid peptides [D-Phe(3)]morphiceptin (H-Tyr-Pro-D-Phe-Pro-NH(2)) and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH(2)) containing the pseudoproline (Psi Pro) (4R)-thiazolidine-4-carboxylic acid (Cys[Psi(R1,R2)pro]) or (4S)-oxazolidine-4-carboxylic acid (Ser[Psi(R1,R2)pro]) in place of Pro(2) were synthesized. The pseudoproline ring in these compounds was either unsubstituted (R(1), R(2) = H) or dimethylated (R(1), R(2) = CH(3)) at the 2-C position. 2-C-dimethylated pseudoprolines are known to be quantitative or nearly quantitative inducers of the cis conformation around the Xaa(i-1)-Xaa(i)[Psi(CH(3),CH)(3)pro)] imide bond. All dihydropseudoproline-containing analogues (R(1), R(2) = H) showed good mu opioid agonist potency in the guinea pig ileum (GPI) assay, high mu receptor binding affinity in the rat brain membrane binding assay, and, like their parent peptides, excellent mu receptor binding selectivity. (1)H NMR spectroscopic analysis of the Cys[Psi(H,H)pro](2)- and Ser[Psi(H,H)pro](2)-containing analogues in DMSO-d(6) revealed that they existed in a conformational equilibrium around the Tyr-Xaa[Psi(H,H)pro] peptide bond with cis/trans ratios of 40:60 and 45:55, respectively. The dimethylated thiazolidine- and oxazolidine-containing [D-Phe(3)]morphiceptin- and endomorphin-2 analogues (R(1), R(2) = CH(3)) all retained full mu agonist potency in the GPI assay and displayed mu receptor binding affinities in the nanomolar range and high mu receptor selectivity. As expected, no conformers of the latter analogues with a trans conformation around the Tyr-Xaa[Psi(CH(3),CH(3)pro)] imide bond were detected by (1)H NMR spectral analysis, indicating that in these compounds the cis conformation is highly predominant (>98%). These results represent the most direct evidence obtained so far to indicate that morphiceptin and endomorphin-2 have the cis conformation around the Tyr-Pro peptide bond in their bioactive conformations.

[2',6'-Dimethyltyrosine]dynorphin A(1-11)-NH2 analogues lacking an N-terminal amino group: potent and selective kappa opioid antagonists.

Recent studies showed that dermorphin and enkephalin analogues containing two methyl groups at the 2',6'-positions of the Tyr(1) aromatic ring and lacking an N-terminal amino group were moderately potent delta and mu opioid antagonists. These results indicate that a positively charged N-terminal amino group may be essential for signal transduction but not for receptor binding and suggested that its deletion in agonist opioid peptides containing an N-terminal 2',6'-dimethyltyrosine (Dmt) residue may represent a general way to convert them into antagonists. In an attempt to develop dynorphin A (Dyn A)-derived kappa opioid antagonists, we prepared analogues of [Dmt(1)]Dyn A(1-11)-NH2 (1), in which the N-terminal amino group was either omitted or replaced with a methyl group. This was achieved by replacement of Tyr(1) with 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp]. Compounds were tested in the guinea pig ileum and mouse vas deferens bioassays and in rat and guinea pig brain membrane receptor binding assays. All analogues turned out to be potent kappa antagonists against Dyn A(1-13) and the non-peptide agonist U50,488 and showed only weak mu and delta antagonist activity. The most potent and most selective kappa antagonist of the series was [(2S)-Mdp(1)]Dyn A(1-11)-NH2 (5, dynantin), which showed subnanomolar kappa antagonist potency against Dyn A(1-13) and very high kappa selectivity both in terms of its K(e) values determined against kappa, mu, and delta agonists and in terms of its ratios of kappa, mu, and delta receptor binding affinity constants. Dynantin is the first potent and selective Dyn A-derived kappa antagonist known and may complement the non-peptide kappa antagonists norbinaltorphimine and GNTI as a pharmacological tool in opioid research.

Chromosomal and comparative mapping of rat oxytocin, oxytocin receptor and vasopressin genes.

Oxytocin and its receptor are potentially important for cardiovascular functions. In the present paper, we report their chromosome locations in the rat and their comparative mapping with the mouse and human. They are located in chromosome regions previously known to contain quantitative trait loci for blood pressure in various genetic crosses. Thus, they have become valid candidate genes for genetic hypertension.

Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada.

During a 2-year surveillance program (1996 to 1998) in Quebec, Canada, 442 strains of Candida species were isolated from 415 patients in 51 hospitals. The distribution of species was as follows: Candida albicans, 54%; C. glabrata, 15%; C. parapsilosis, 12%; C. tropicalis, 9%; C. lusitaniae, 3%; C. krusei, 3%; and Candida spp., 3%. These data, compared to those of a 1985 survey, indicate variations in species distribution, with the proportions of C. glabrata and C. parapsilosis increasing by 9 and 4%, respectively, and those of C. albicans and C. tropicalis decreasing by 10 and 7%, respectively. However, these differences are statistically significant for C. glabrata and C. tropicalis only. MICs of amphotericin B were > or =4 microg/ml for 3% of isolates, all of which were non-C. albicans species. Three percent of C. albicans isolates were resistant to flucytosine (> or =32 microg/ml). Resistance to itraconazole (> or =1 microg/ml) and fluconazole (> or =64 microg/ml) was observed, respectively, in 1 and 1% of C. albicans, 14 and 9% of C. glabrata, 5 and 0% of C. tropicalis, and 0% of C. parapsilosis and C. lusitaniae isolates. Clinical data were obtained for 343 patients. The overall crude mortality rate was 38%, reflecting the multiple serious underlying illnesses found in these patients. Bloodstream infections were documented for 249 patients (73%). Overall, systemic triazoles had been administered to 10% of patients before the onset of candidiasis. The frequency of isolation of non-C. albicans species was significantly higher in this group of patients. Overall, only two C. albicans isolates were found to be resistant to fluconazole. These were obtained from an AIDS patient and a leukemia patient, both of whom had a history of previous exposure to fluconazole. At present, it appears that resistance to fluconazole in Quebec is rare and is restricted to patients with prior prolonged azole treatment.

Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases.

Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein-DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.

Stereospecific synthesis of (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) and its incorporation into an opioid peptide.

To examine the effect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological activity, a stereospecific synthesis of the tyrosine analogue (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) was performed. The enkephalin analogue (2S)-Mdp-D-Ala-Gly-Phe-Leu-NH2 turned out to be a quite potent delta opioid antagonist and a somewhat less potent mu antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non for the binding of opioid peptides to delta and mu receptors but may be required for signal transduction.

Synthesis and in vitro opioid activity profiles of DALDA analogues.

The tetrapeptide DALDA (H-Tyr-D-Arg-Phe-Lys-NH2) is a polar and selective mu agonist showing poor penetration of the placental and blood-brain barriers. In an effort to enhance the potency of DALDA, analogues containing 2',6'-dimethyltyrosine (Dmt), N,2',6'-trimethyltyrosine (Tmt), 2'-methyltyrosine (Mmt) or 2'-hydroxy,6'-methyltyrosine (Hmt) in place of Tyr1, or Orn or alpha,gamma-diaminobutyric acid (A2bu) in place of Lys4, were synthesized. All compounds displayed high mu receptor selectivity in the rat and guinea pig brain membrane binding assays and most of them were more potent mu agonists than DALDA in the mu receptor-representative guinea pig ileum assay, with [Dmt1]DALDA showing the highest potency. Because of its extraordinary mu agonist potency, high mu selectivity, polar character (charge of 3 + ) and metabolic stability, [Dmt1]DALDA has potential for use in obstetrical or peripheral analgesia.

Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins.

Endonuclease assays of the H-N-H proteins encoded by two group I introns in the Chlamydomonas moewusii chloroplast psbA gene revealed that the CmpsbA.1 intron specifies a site-specific DNA endonuclease, designated I-CMOE:I. Like most previously reported intron-encoded endonucleases, I-CMOE:I generates a double-strand break near the insertion site of its encoding intron, leaving 3' extensions of 4 nt. This enzyme was purified from Escherichia coli as a fusion protein with a His tag at its N-terminus. The recombinant protein (rI-CMOE:I) requires a divalent alkaline earth cation for DNA cleavage (Mg(2+) > Ca(2+) > Sr(2+) > Ba(2+)). It also requires a metal cofactor for DNA binding, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date. rI-CMOE:I binds its recognition sequence as a monomer, as revealed by gel retardation assays. K:(m) and k(cat) values of 100 +/- 40 pM and 0.26 +/- 0.04 min(-1), respectively, were determined. Replacement of the first histidine of the H-N-H motif by an alanine residue abolishes both rI-CMOE:I activity and binding to its substrate. We propose that this conserved histidine residue plays a role in binding the metal cofactor and that such binding induces a structural modification of the enzyme which is required for DNA recognition.

The complete mitochondrial DNA sequence of Scenedesmus obliquus reflects an intermediate stage in the evolution of the green algal mitochondrial genome.

Two distinct mitochondrial genome types have been described among the green algal lineages investigated to date: a reduced-derived, Chlamydomonas-like type and an ancestral, Prototheca-like type. To determine if this unexpected dichotomy is real or is due to insufficient or biased sampling and to define trends in the evolution of the green algal mitochondrial genome, we sequenced and analyzed the mitochondrial DNA (mtDNA) of Scenedesmus obliquus. This genome is 42,919 bp in size and encodes 42 conserved genes (i.e., large and small subunit rRNA genes, 27 tRNA and 13 respiratory protein-coding genes), four additional free-standing open reading frames with no known homologs, and an intronic reading frame with endonuclease/maturase similarity. No 5S rRNA or ribosomal protein-coding genes have been identified in Scenedesmus mtDNA. The standard protein-coding genes feature a deviant genetic code characterized by the use of UAG (normally a stop codon) to specify leucine, and the unprecedented use of UCA (normally a serine codon) as a signal for termination of translation. The mitochondrial genome of Scenedesmus combines features of both green algal mitochondrial genome types: the presence of a more complex set of protein-coding and tRNA genes is shared with the ancestral type, whereas the lack of 5S rRNA and ribosomal protein-coding genes as well as the presence of fragmented and scrambled rRNA genes are shared with the reduced-derived type of mitochondrial genome organization. Furthermore, the gene content and the fragmentation pattern of the rRNA genes suggest that this genome represents an intermediate stage in the evolutionary process of mitochondrial genome streamlining in green algae.

Symptomatic and health status outcomes in the Canadian randomized MAC treatment trial (CTN010). Canadian HIV Trials Network Protocol 010 Study Group.

Our objective was to compare the effect of 2 regimens for treatment of Mycobacterium avium complex (MAC) bacteraemia in an HIV-positive population on symptoms and health status outcomes using a substudy of an open-label randomized controlled trial. The study was conducted in 24 hospital-based human immunodeficiency virus (HIV) clinics in 16 Canadian cities. Patients had HIV infection and MAC bacteraemia and were given either rifampin 600 mg, ethambutol 15 mg/kg daily, clofazimine 100 mg daily and ciprofloxacin 750 mg twice daily (4-drug arm) or rifabutin 600 mg daily (amended to 300 mg daily in mid-trial), ethambutol 15 mg/kg daily and clarithromycin 1000 mg twice daily (3-drug arm). The primary health status outcome was the change on the 8-item symptom subscale of the Medical Outcome Study (MOS)-HIV Health Survey adapted for MAC. Changes on other MOS-HIV subscales and on the Karnofsky score were also evaluated. Patients on the 3-drug arm had better outcomes on the MOS-HIV symptom subscale at 16 weeks (P=0.06), with statistically significant differences restricted to night sweats and fever and chills (P < 0.001). The proportion of patients improving on the symptom subscale relative to baseline was 55% on the 3-drug arm and 40% on the 4-drug arm. Patients on the 3-drug arm also had better Karnofsky score at 16 weeks (P < 0.001) and better outcomes on the social function, mental health, energy/fatigue, health distress and cognitive function subscales of the MOS-HIV. The 3-drug arm is superior to the 4-drug arm in terms of impact on MAC-associated symptoms, functional status and other aspects of health status.

Ancestral chloroplast genome in Mesostigma viride reveals an early branch of green plant evolution.

Sequence comparisons suggest that all living green plants belong to one of two major phyla: Streptophyta (land plants and their closest green algal relatives, the charophytes); and Chlorophyta (the rest of green algae). Because no green algae are known that pre-date the Streptophyta/Chlorophyta split, and also because the earliest diverging green algae show considerable morphological variation, the nature of the unicellular flagellate ancestor of the two green plant phyla is unknown. Here we report that the flagellate Mesostigma viride belongs to the earliest diverging green plant lineage discovered to date. We have sequenced the entire chloroplast DNA (118,360 base pairs) of this green alga and have conducted phylogenetic analyses of sequences derived from this genome. Mesostigma represents a lineage that emerged before the divergence of the Streptophyta and Chlorophyta, a position that is supported by several features of its chloroplast DNA. The structure and gene organization of this genome indicate that chloroplast DNA architecture has been extremely well conserved in the line leading to land plants.

Novel ligands lacking a positive charge for the delta- and mu-opioid receptors.

Recently we reported using minilibraries to replace Lys(9) [somatostatin (SRIF) numbering] of the potent somatostatin agonist L-363,301 (c[-Pro-Phe-D-Trp-Lys-Thr-Phe-]) to generate the potent neurokinin receptor (NK-1) antagonist c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]. This novel cyclic hexapeptide did not bind the SRIF receptor. Thus, a single mutation converted L-363,301, a SRIF agonist with potency ca. 2-8 times the potency of SRIF in laboratory animals,(24) into a selective NK-1 receptor antagonist with an IC(50) of 2 nM in vitro. During the screening of the same libraries for ligands of the delta-opioid receptor, we identified four compounds (1-4) which represent a new class of delta-opioid antagonists, some of which were also NK-1 receptor antagonists. The most potent delta-opioid antagonist, c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-] (2), showed a K(e) value of 128 nM in the mouse vas deferens assay and a delta-receptor binding affinity constant of 152 nM in the rat brain membrane binding assay. These results are of interest because they represent a novel class of delta-opioid antagonists and, like two previously reported delta-opioid antagonists, they lack a positive charge. To examine further the requirement for a positive charge in the delta-opioid ligands, we prepared two analogues of the beta-casomorphin-derived mixed mu-agonist/delta-antagonist, H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] (7), in which we eliminated the positive charge either through formylation of the primary amino group (5) or by the deletion of this N-terminal amino group (6). These latter compounds proved to be delta-opioid antagonists with K(e) values in the 16-120 nM range, as well as fairly potent mu-opioid antagonists (K(e) approximately 200 nM). These six compounds provide the most convincing evidence to date that there is no requirement for a positive charge in mu- and delta-opioid receptor antagonists. In addition, cyclic hexapeptide 4 lacks a phenolic hydroxyl group. Taken together, these data suggest that the prevailing assumptions about delta- and mu-opioid receptor binding need revision and that the receptors for these opioid ligands have much in common with the NK-1 and somatostatin receptors.

The complete mitochondrial DNA sequences of Nephroselmis olivacea and Pedinomonas minor. Two radically different evolutionary patterns within green algae.

Green plants appear to comprise two sister lineages, Chlorophyta (classes Chlorophyceae, Ulvophyceae, Trebouxiophyceae, and Prasinophyceae) and Streptophyta (Charophyceae and Embryophyta, or land plants). To gain insight into the nature of the ancestral green plant mitochondrial genome, we have sequenced the mitochondrial DNAs (mtDNAs) of Nephroselmis olivacea and Pedinomonas minor. These two green algae are presumptive members of the Prasinophyceae. This class is thought to include descendants of the earliest diverging green algae. We find that Nephroselmis and Pedinomonas mtDNAs differ markedly in size, gene content, and gene organization. Of the green algal mtDNAs sequenced so far, that of Nephroselmis (45,223 bp) is the most ancestral (minimally diverged) and occupies the phylogenetically most basal position within the Chlorophyta. Its repertoire of 69 genes closely resembles that in the mtDNA of Prototheca wickerhamii, a later diverging trebouxiophycean green alga. Three of the Nephroselmis genes (nad10, rpl14, and rnpB) have not been identified in previously sequenced mtDNAs of green algae and land plants. In contrast, the 25,137-bp Pedinomonas mtDNA contains only 22 genes and retains few recognizably ancestral features. In several respects, including gene content and rate of sequence divergence, Pedinomonas mtDNA resembles the reduced mtDNAs of chlamydomonad algae, with which it is robustly affiliated in phylogenetic analyses. Our results confirm the existence of two radically different patterns of mitochondrial genome evolution within the green algae.