Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation.

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.

Structural and functional interactions of transcription factor (TF) IIA with TFIIE and TFIIF in transcription initiation by RNA polymerase II.

A topological model for transcription initiation by RNA polymerase II (RNAPII) has recently been proposed. This model stipulates that wrapping of the promoter DNA around RNAPII and the general initiation factors TBP, TFIIB, TFIIE, TFIIF and TFIIH induces a torsional strain in the DNA double helix that facilitates strand separation and open complex formation. In this report, we show that TFIIA, a factor previously shown to both stimulate basal transcription and have co-activator functions, is located near the cross-point of the DNA loop where it can interact with TBP, TFIIE56, TFIIE34, and the RNAPII-associated protein (RAP) 74. In addition, we demonstrate that TFIIA can stimulate basal transcription by stimulating the functions of both TFIIE34 and RAP74 during the initiation step of the transcription reaction. These results provide novel insights into mechanisms of TFIIA function.

Long-term efficacy and safety of a new olive oil-based intravenous fat emulsion in pediatric patients: a double-blind randomized study.

BACKGROUND: A new intravenous lipid emulsion (ILE) prepared from a mixture of soybean and olive oils contains only long-chain triacylglycerols, with a low proportion (20%) of polyunsaturated fatty acids and 60% monounsaturated fatty acids. OBJECTIVE: The goal of this randomized, double-blind clinical trial was to assess in children the efficacy and safety of this new ILE compared with a control group receiving a soybean-oil emulsion. DESIGN: Eighteen children received for 2 mo 24% of nonprotein energy (1.80 g kg (-)(1) d(-)(1)) either as the new ILE or a soybean oil-based emulsion. Assessments were performed on days -30, 0, 30, and 60 and the changes (day 60 - day 0) assessed by analysis of variance. RESULTS: There were no significant differences in triacylglycerol, apolipoproteins A-I and B, or HDL cholesterol between the 2 groups, whereas total and LDL cholesterol were higher in the soybean oil group on day 60. The pattern of 20:4n-6 in erythrocyte membranes did not change significantly, nor did the ratio of 20:3n-9 to 20:4n-6. On day 60, 18:1n-9 was significantly higher in the olive oil group, the ratio of Sigma(n)-6 > C(18) + 18:3n-6 to 18:2n-6 was 2.20 +/- 0.09 in the olive oil group and 1.33 +/- 0.16 in the soybean-oil group, and Sigma(n)-3 > C(18) was 3.83 +/- 0.30 in the olive oil group and 4. 03 +/- 0.33 in the soybean-oil group. The peroxidation index was lower after the olive oil treatment. CONCLUSIONS: The olive oil-based emulsion was well tolerated, maintained a normal EFA status, and may be more suitable for prevention of lipid peroxidation than the soybean-oil-based emulsion.

Topological localization of the carboxyl-terminal domain of RNA polymerase II in the initiation complex.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) functions at multiple stages of transcription and is involved in the coupling of transcription to pre-mRNA processing. We have used site-specific protein-DNA photocross-linking to determine the position of the CTD along promoter DNA in the transcriptional pre-initiation complex. Comparison of the promoter contacts made by RNAP II with or without the CTD indicate that the CTD approaches promoter DNA downstream of the transcriptional initiation site between positions +16 and +26. Incubation of pre-assembled initiation complexes with antibodies to the CTD prior to UV irradiation led to specific photocross-linking of the IgG heavy chain to nucleotide +17, indicating that the CTD is accessible for protein-protein interactions in a complex containing RNAP II and the general initiation factors. In conjunction with previously published observations, our structural data are fully compatible with the notion that DNA wrapping around RNAP II places the CTD and the RNA exit channel into juxtaposition and provide a rationale for contacts between the SRB-mediator complex and core RNAP II observed in the RNAP II holoenzyme.

Wrapping of promoter DNA around the RNA polymerase II initiation complex induced by TFIIF.

The formation of the RNA polymerase II (Pol II) initiation complex was analyzed using site-specific protein-DNA photo-cross-linking. We show that the RAP74 subunit of transcription factor (TF) IIF, through its RAP30-binding domain and an adjacent region necessary for the formation of homomeric interactions in vitro, dramatically alters the distribution of RAP30, TFIIE, and Pol II along promoter DNA between positions -40 and +26. This isomerization of the complex, which requires both TFIIF and TFIIE, is accompanied by tight wrapping of the promoter DNA for almost a full turn around Pol II. Addition of TFIIH enhances photo-cross-linking of Pol II to a number of promoter positions, suggesting that TFIIH tightens the DNA wrap around the enzyme. We present a general model to describe transcription initiation.

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box.

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

Pregnancy in women with impaired renal function.

The outcome and consequences of pregnancy in women with impaired renal function are still debated. To assess the benefit of recent advances in coordinated obstetrical and nephrologic management, we analyzed fetal and maternal outcome of 43 pregnancies in 30 women with various types of primary renal disease and moderate to severe renal failure at conception defined by serum creatinine concentration (Scr) ranging from 0.11 to 0.49 mmol/l. All pregnancies took place during the 20-year period from 1975 through 1994 and were prospectively followed jointly by our Nephrology Unit and Obstetric and Neonatology Units of University Hospitals. Of the 43 pregnancies (45 fetuses), 13 ended in fetal death (including 5 first-trimester abortions and 8 fetal deaths beyond the 20th gestational week). There were 32 live births, a success rate of 82% not considering first-trimester abortions. Successful pregnancies were significantly more frequent in the last decade than in the preceding one (91 vs 65%, p = 0.05). Overall live birth rate was higher in pregnancies started with Scr < 0.20 mmol/l than in those with Scr > 0.20 mmol/l (80% vs 53%, p = 0.02). The upper preconception Scr value associated with a successful fetal outcome was 0.27 mmol/l. Hypertension was the major factor of fetal prognosis, as the relative risk of fetal loss was 10.6 times higher when hypertension was present at conception or early in pregnancy than when blood pressure was spontaneously normal or well-controlled by therapy. An accelerated course toward end-stage renal failure was observed in 7 patients (23%), all of whom had severe hypertension and heavy proteinuria at conception. We conclude that fetal outcome in patients with impaired renal function has been improved in recent years, due to advances in obstetrics and neonatology, improved blood pressure control and close co-operation between nephrologists and obstetricians, but that a risk of fetal loss and of accelerated deterioration of maternal renal disease still persists when Ccr at conception is lower than 25-30 ml/ min/1.73 m2.

Analysis of WT1 gene expression during mouse nephrogenesis in organ culture.

The temporal and spatial expression patterns of the Wilms tumor gene, WT1, were studied during the organogenesis of the mouse kidney in vitro. In situ hybridization and immunocytochemistry localized cellular expression of WT1 in whole kidney organ cultures to the induced metanephric mesenchyme and developing podocytes. Organ cultures were further characterized immunocytochemically with antibodies that specifically labeled the different tubular epithelial components and supporting mesenchyme of the developing nephrons. In organ cultures, the WT1 expression pattern could be visualized in induced metanephric mesenchyme and entire cell cohorts of differentiating podocytes. Expression of WT1 and cell specific markers were retained in short-term monolayer cultures of dissociated kidneys. The development of the metanephric kidney in vitro involves a highly restricted temporal and spatial cellular expression pattern of WT1 which closely follows that observed in tissue sections from gestational kidney isolated during organogenesis in the mouse.

Localization of subunits of transcription factors IIE and IIF immediately upstream of the transcriptional initiation site of the adenovirus major late promoter.

The assembly of a preinitiation complex containing RNA polymerase II on promoter DNA is a complex process that involves several general transcription factors. Using 5-[N-(p-azidobenzoyl)-3-aminoallyl] photocross-linking, we previously determined the locations of the two large subunits of transcription factor (TF) IIA (A35 and A21), TATA box-binding protein (TBP), RNA polymerase II-associated protein (RAP) 30, and TFIIB along the Ad2 ML promoter. We have now localized TFIIE34 and RAP74 just upstream of the transcription start site. The two subunits of TFIIF, RAP74 and RAP30, cross-linked to nucleotides that probed adjacent spaces on the same face of the DNA helix beginning just downstream of TBP at -19 and extending to -5. Specific photocross-linking of TFIIE34 required the presence TFIIE56. In addition, TFIIE and RAP74 strongly stimulated cross-linking of RAP30 and the large subunits of RNA polymerase II to position -19. Our topological data support the idea that RAP74 and TFIIE34 may be involved in melting of the promoter DNA upstream of the initiation site.

An evaluation of the safety of mixed micelles in healthy subjects.

BACKGROUND: Fat and water soluble vitamins are an essential part of i.v. nutrition (IVN). However, they are unstable in solution and may adhere to the bags and tubing containing the IVN. This study has examined the safety and side-effect profile of mixed micelles (mixed bile-salt lecithin micelles) used to solubilize water and fat soluble vitamins for i.v. administration. METHODS: Two groups of six healthy male subjects received either placebo or mixed micelles daily for 5 days by i.v. infusion in a randomized crossover design with a 9-day washout period separating the two treatment periods. RESULTS: Infusion of mixed micelles resulted in a significant increase in serum glycocholic acid from a median of 26.5 micrograms/dL (interquartile range 18 to 38) to 115 micrograms/dL (70 to 155) postinfusion. Glycocholic acid may have a lytic effect on cell membranes; however, in this study there was no evidence of hemolysis or increase in serum transaminases during mixed micelle infusion. There was no increase in reported side effects during mixed micelle infusion compared with placebo. CONCLUSION: Mixed micelles can be used safely for the solubilization of fat- and water-soluble vitamins and drugs that are to administered by i.v. injection.

Influence of pregnancy on the course of primary chronic glomerulonephritis.

According to some nephrologists, pregnancy has damaging effects on renal function in primary glomerulonephritis, but the evidence is conflicting. We evaluated the effect of pregnancy on the occurrence of end-stage renal failure (ESRF) in 360 patients with various histological forms of primary glomerulonephritis but with normal renal function (serum creatinine < or = 0.11 mmol/L) at presentation. In actuarial analyses, overall ESRF-free survival did not significantly differ between women who became pregnant after clinical onset of renal disease (n = 171) and those who did not conceive (n = 189). Furthermore, in a case-control study pregnancy did not emerge as a risk factor for progression to ESRF (odds ratio 1.15 [95% CI 0.61-2.18]), whereas the type of glomerulonephritis and hypertension were major determinants. We conclude that pregnancy does not affect the course of renal disease in patients who have normal renal function at conception.

Construction of a ribozyme directed against human interleukin-6 mRNA: evaluation of its catalytic activity in vitro and in vivo.

We have designed a ribozyme (Rz) that cleaves human interleukin-6 (IL-6) mRNA in vivo. This Rz was tested in vitro, and was found to give expected size fragments. It was then incorporated into a mammalian expression vector containing the constitutive cytomegalovirus (CMV) immediate early promoter and transfected into human U amniotic cells (UAC). Cell clones that stably express this catalytic RNA have been obtained. Some of them displayed a marked reduction of tumor necrosis factor (TNF)-induced IL-6 production. Their reduced ability to express IL-6 was related to the amount of Rz they produced and to the extent of IL-6 mRNA cleavage as observed by a ribonuclease protection assay. These data provide a method to study further the role of IL-6 production in various biologic situations, and suggest the feasibility of developing Rzs directed against various cytokines to study their biologic role and mechanism of action.

Home parenteral nutrition in children: bioavailability of vitamins in binary mixtures stored for 8 days.

Vitamin supply in children on long-term parenteral nutrition depends on the specific age-related needs and on the bioavailability of vitamins when introduced into nutritional bags. The present study aimed to investigate the vitamin status in children on home TPN receiving nutritional bags which had been stored during a prolonged period of 8 instead of 4 days and where the new vitamin preparation Cernevit has been introduced. 19 children aged from 5 months to 11 years receiving home parenteral nutrition, for 42 months on average, were studied. Daily vitamin supply for children above 2 years of age was: A 1050 ug, D 5.5 ug, E 10.2 mg plus 0.6 mg/g lipid (Intralipid), C 125 mg, B1 3.5 mg, B2 4.1 mg, B6 4.5 mg, biotine 69 mug; children who were younger than 2 years received half of these intakes. Water soluble vitamin status was only measured in children over 3 years old. Plasma levels remained stable and adequate for age, for most of the studied vitamins. Vitamin A concentration was inferior to 200 mug/l in 1 patient with hepatopathy. Plasma concentrations of vitamin E, which were initially below 6 mg/l in 4 patients, returned to normal during the study. Plasma levels of vitamin C were below 6.2 mg/l in several infants either temporarily (5 patients) or during the whole study period (2 patients). These results support a prolongation of the intervals between preparing batches of nutritional bags and also between deliveries. This results in a considerable reduction of costs, provided that plasma vitamin levels, specially vitamin C, are regularly monitored.

Action of spontaneously produced beta interferon in differentiation of embryonal carcinoma cells through an autoinduction mechanism.

In the current study, we have addressed the role of interferons (IFNs) in controlling the differentiation of pluripotent P19 embryonal carcinoma (EC) cells. Blocking IFN activity in the culture medium of differentiating cells with antibodies leads to a strong decrease in the degree of differentiation. The antibodies are active for a relatively short time. During this time, IFN-beta mRNA can be detected in the differentiating cells, as can increases of IFN stimulation response element-binding activity and NF-KB. The timing of IFN action also coincides with the accumulation of cytoplasmic double-stranded RNA (dsRNA) and with a drop in dsRNA unwindase-modificase activity. A model for the involvement of autoinduction of IFN by intracellular dsRNA in the control of differentiation in this system is presented.

Supplemented low-protein diets protect the rat kidney without causing undernutrition.

Low-protein diets supplemented with keto-analogues and essential amino acid (KA-EAA) mixtures or with EAA have been widely used to retard renal deterioration without affecting nutrition. These assumptions have recently been challenged in clinical studies and rest on little or no experimental data. The effects of EAA and KA-EAA supplementations have not been compared. We compared three groups of rats with subtotal nephrectomy that were fed (1) a 16% casein reference (R) diet, (2) a 6% casein plus EAA (A) diet, or (3) a 6% casein plus KA-EAA (K) diet with KA as amino acid salts. The three diets had the same energy and mineral contents, and they induced comparable growth. The two supplements had the same nitrogen content. The only difference found until month 3 was higher proteinuria and plasma urea levels in group R rats. Renal biopsies performed at month 3 showed more severe glomerular sclerosis and tubular changes in R rats than in A and K rats. From months 3 through 7, R rats developed higher plasma creatinine levels than did A and K rats (final median values: 167, 106, and 83 mumol/L; p < 0.04), had more proteinuria (232, 56, and 84 mg/day), and showed greater mortality rates. At the time the rats were killed, 2 R, 6 A, and 5 K rats had survived while receiving the diets. Examination of the remnant kidneys, regardless of time of death, showed that renal lesions were significantly worse in R than in A and K rats, with sclerosis affecting more than 50% of the glomeruli in 7 of 13 R, 4 of 14 A, and 4 of 15 K rats, and less than 25% glomeruli in 2 of 13 R, 10 of 14 A, and 10 of 15 K rats (A and K vs R: p < 0.03). In conclusion, restriction of nonessential amino acids compensated by EAA or by KA-EAA mixtures retards renal damage without affecting growth, but no real benefit of KA or EAA has been observed.

Specific controversies concerning the natural history of renal disease in pregnancy.

Whether or not pregnancy adversely affects the natural course of underlying primary renal disease, and whether fetal outcome is influenced by the type of renal disease per se are controversial issues. We retrospectively analyzed the fetal and maternal outcome in 148 women with various, biopsy-proven histological types of primary chronic glomerulonephritis (GN), including IgA GN (52 patients), membranous GN ([MGN] 20 patients), membranoproliferative type 1 GN ([MPGN] 58 patients), focal and segmental glomerulosclerosis ([FSGS] 13 patients), and minimal change nephrotic syndrome ([MCNS] 22 patients), who were pregnant (with a total of 290 pregnancies) after the clinical onset of GN, and in 104 women with reflux nephropathy (with a total of 254 pregnancies). Fetal outcome was poor in the presence of uncontrolled hypertension, nephrotic range proteinuria, and/or impaired renal function at conception or early in gestation, whatever the type of renal disease. An accelerated, more rapid than expected, worsening of maternal renal function was observed in five GN patients of whom four (two IgA, two MPGN) had serum creatinine (Scr) levels greater than 160 mumol/L (1.8 mg/dL) early in gestation, and in five patients with reflux nephropathy whose Scr at conception ranged from 180 to 490 mumol/L (2.0 to 5.5 mg/dL).(ABSTRACT TRUNCATED AT 250 WORDS)

[Fetal and maternal risk of pregnancy in women with primary chronic glomerulonephritis]. / Risques foetal et maternel de la grossesse chez les femmes atteintes de glomérulonéphrite chronique primitive. (I).

This review focuses on the reciprocal influence of underlying primary glomerular disease on fetal outcome and of pregnancy on the course of maternal nephropathy, based on most recent data in the literature and on a personal series of 240 pregnancies in 122 women with biopsy-proven chronic glomerulonephritis (CGN) followed at Necker Hospital. The first part analyzes the fetal outcome in the various histopathologic types of CGN and points out the major influence as risk factors for fetal outcome, of the presence of nephrotic syndrome, high blood pressure and/or impaired renal function at conception. The second part deals with the debated problem of the influence of pregnancy on maternal renal disease. It has become evident that pregnancy has no deleterious effect per se on the course of maternal disease when renal function is normal or near normal at conception, whereas an accelerated course is often observed when plasma creatinine is in excess of 0.18 mmol/l at conception, whatever the type of CGN. In the third part are described preconception counselling and the practical rules of maternal and fetal surveillance.