Evidence for multiple xenogenous origins of plastids: comparison of psbA-genes with a xanthophyte sequence

Summary When only plastidic features are considered, it is difficult to distinguish between monophyletic and polyphyletic xenogenous origins of plastids. We suggest that a direct comparison of nuclear and plastidic sequence-similarity pattern will help to solve this problem. The D1 amino acid sequence of six major groups of photosynthetic eukaryotes and of the two groups of photosynthetic prokaryotes are now available, including the psbA-gene product from Bumilleriopsis filiformis, which is the first molecular sequence reported for a xanthophycean alga. Evidence is provided for an independent and polyphyletic origin of plastids from five out of the six major taxa of photosynthetic eukaryotes. This conclusion is reached by comparing a plastid-based pattern of D1 similarity with a nucleus-based similarity pattern published recently. Furthermore, the availability of D1 sequences from five eukaryotic algae led to a re-evaluation of the taxonomic position of Prochlorothrix.     

Growth promoting and metabolic activity of the human insulin analogue [Gly,Arg,Arg]insulin (HOE 901) in muscle cells

[Gly^A21,Arg^B31,Arg^B32]insulin (HOE 901) represents a biosynthetic human insulin analogue that, due to its isoelectric point, precipitates at neutral tissue pH leading to a retarded absorption rate and a corresponding longer duration of action. In the present investigation we have evaluated the growth promoting and metabolic activity of this analogue in muscle tissue using exponentially growing H9c2 cardiac myoblasts and adult rat ventricular cardiomyocytes. Equilibrium binding studies of ¹²⁵I-labelled IGF-I (insulin-like growth factor I) to differentiating myoblasts revealed the presence of 7×10³ IGF-I receptors per cell. In contrast, no specific binding of insulin could be detected. Competition binding experiments showed a slightly higher affinity of HOE 901 for the IGF-I receptor when compared to regular human insulin with IC₅₀ (half-inhibitory concentration) values of 70 and 101 nM, respectively. However, the supermitogenic insulin analogue [Asp^B10]insulin competed significantly more efficiently for IGF-I binding (IC₅₀: 44 nM). Maximum growth promoting activity of the peptides was then determined in serum-starved myoblasts by an incubation with the peptides (5×10⁻⁷ M) for 16 h in the presence of [³H]thymidine. [Asp^B10]Insulin produced a stimulation of DNA synthesis (about 3-fold) which was comparable to the effect of IGF-I and significantly (P<0.005) higher than the effect of HOE 901 with the latter being essentially equipotent to native insulin. Comparable results were obtained at lower concentrations of the peptides (10⁻⁹ to 10⁻⁸ M). Metabolic activity of HOE 901 was determined by measuring the dose-dependent stimulation of 3-O-methylglucose transport in adult cardiomyocytes. Maximum transport stimulation was identical for insulin and HOE 901 with EC₅₀ (half-effective concentration) values of 0.7×10⁻¹⁰ and 1.9×10⁻⁹ M, respectively. We concluded that the IGF-I receptor-mediated growth promoting activity of HOE 901 in muscle cells and the maximal metabolic activity of this analogue are not different from those of native human insulin. It is suggested that differential interaction with IGF-I receptors significantly contributes to the action profile of insulin analogues.     

Luminal transport step of para-aminohippurate (PAH): transport from PAH-loaded proximal tubular cells into the tubular lumen of the rat kidney in vivo

 Proximal tubular cells were loaded for 10 s with [³H]para-aminohippurate ([³H]PAH) by microperfusing the peritubular capillaries with Ringer solution containing 0.05 mmol/l PAH. Immediately thereafter [³H]PAH influx from cells into a column of equilibrium solution injected into the oil-filled tubular lumen was measured by re-aspirating the fluid after 1–10 s of contact time. The rise of luminal PAH concentration within 2 s of contact time was almost linear, reaching a luminal / capillary concentation ratio of 1.6 after 2 s and of 3.2 after 5 s. The 2-s PAH concentration ratio was not changed when different manoeuvres were applied to depolarize proximal tubular cells. Also, the 2-s PAH concentration ratio was not influenced by varying the luminal pH from 6.0 to 8.0 or the luminal Cl^–concentration from zero to 134 mmol/l or when either 5 mmol/l urate or 25 mmol/l lactate was in the luminal perfusate. A decrease in the 2-s PAH concentration ratio, i.e. trans-inhibition, was observed when 25 or 50 mmol/l HCO₃ ^–(–50%) was in the luminal perfusate. Trans-inhibition was also seen with 5 mmol/l of the following substituted benzoates: 2-hydroxy-benzoate (–58%), 2-methoxy-benzoate (–46%), 2-hydroxy-benzoate-acetyl ester (–36%), 2-hydroxy-3,5-dinitro-benzoate (–48%), 3,5-dichloro-benzoate (–49%), and 2,3,5-trichloro-benzoate (–45%). No effect was seen with benzoate, 3-hydroxy-benzoate, 2-chloro-benzoate, 2-nitro-benzoate, 2,5-dinitro-benzoate, 3-sulfamoyl-benzoate and 4-sulfamoyl-benzoate. However, analogues of the latter two compounds possessing two additional side groups, such as furosemide and piretanide, or a hydrophobic moiety, such as probenecid, were inhibitory (by –62, –41 and –49% respectively). Phenoxyacetate had no effect; however, it inhibited if in addition it had three chloro groups, as in 2,4,5-trichlorophenoxyacetate (–71%) or a hydrophobic carbamoyl side group, as in mersalylic acid (salyrgan, –75%). Benzene-sulfonate trans-inhibited (–33%), as did phenolsulfonphthalein (phenol red, –39%) and sulfofluorescein (–55%). However, the trans-inhibitory effect of the corresponding carboxy-compounds was absent (phenolphthalein) or weaker (fluorescein, –42%). The trans-inhibitory effect of the uricosurics ethacrynic acid (–53%), tienilic acid (–55%) indacrinone (–72%) and benzbromarone (–42%) could be attributed to two chloro or bromo side groups on the benzene ring. Other trans-inhibiting uricosuric substances were indomethacin (–42%), sulfinpyrazone (–38%), losartan (–80%) its metabolite EXP 3174 (–55%), and AA 193 (–65%). These organic acids, with pKa values between 2.8 and 4.9, possess chloro and sulfin groups, as well as heterocyclic 5-ring and hydrophobic ring or chain areas. No significant effect was seen with 5 mmol/l PAH, 2-oxo-glutarate, DIDS, cGMP, prostaglandin E₂, cortisol, benzylamiloride, pyrazinoic acid and 25 mmol/l lactate. Our data indicate that in situ the secretory luminal PAH transport proceeds in a non-rheogenic fashion, per exclusionem by anion exchange. The observed trans-inhibition of PAH secretion seems to correlate with the affinity for the luminal PAH transporter and, for uricosuric substances, with their uricosuric potency. Received: 15 October 1996 / Received after revision: 17 December 1996 / Accepted: 18 December 1996     

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15–25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80–150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20°C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.     

One-year results: palatal implants for the treatment of obstructive sleep apnea.

OBJECTIVE: To evaluate long-term effectiveness of palatal implants for treatment of mild to moderate obstructive sleep apnea (OSA). STUDY DESIGN: A prospective study of 26 referred patients with a pretreatment apnea-hypopnea index (AHI) of 10 to 30 and a body mass index of < or =30, representing an extended follow-up of a subset of 41 patients enrolled in previous short-term trials. RESULTS: Twenty-one of 26 patients (80.8%) experienced a decrease in AHI. Fifteen of 26 patients (57.7%) had a follow-up AHI <10 at 1 year, whereas 13 patients (50%) had a 50% or greater reduction to an AHI <10 at 1 year. Mean AHI was reduced from 16.5 +/- 4.5 at baseline to 12.5 +/- 10.5 at 3 months (P < 0.014) and to 12.3 +/- 12.7 at 1 year (P < 0.019). CONCLUSIONS: Patients initially responding to palatal implants with improved AHI maintained improvement through long-term follow-up at 1 year.     

Phenytoin–Lipid Conjugates: Chemical,Plasma Esterase-Mediated,and Pancreatic Lipase-Mediated Hydrolysis in Vitro

Phenytoin–lipid conjugates obtained by covalent binding of hydroxymethylphenytoin to diacylglycerides and to 3-acyloxy-2-acyl-oxymethylpropionic acids formed dispersions with a particle size of 10–200 µm when briefly sonicated in a sodium taurodeoxycholate-containing ethanol–water mixture. In contrast to the corresponding bis-deacyl derivatives, the lipids were not significantly hydrolyzed in aqueous buffers and in plasma. Incubation with pancreatic lipase yielded primarily the bis-deacyl compounds, which are comparable to monoglycerides, and subsequently liberated phenytoin. The glyceride-derived prodrugs were better substrates for the enzyme than the 3-acyloxy-2-acyloxymethyl-propionic acid derivatives. It is concluded that the phenytoin lipid conjugates are hydrolyzed by pancreatic lipase in a similar manner as natural triglycerides.     

Effect of etoposide (VP16-213) on lipid peroxidation and antioxidant status in a high-dose radiochemotherapy regimen

Summary A total of 13 patients receiving bone marrow transplants (BMT) for treatment of different haematological diseases were investigated. Conditioning therapy preceding BMT consisted of fractionated total-body irradiation (12 Gy) and high-dose chemotherapy with cyclophosphamide (2±60 mg/kg). Patients stratified to be at high risk for relapse (6/13) were additionally treated with etoposide (30 mg/kg). Plasma concentrations of absolute and lipid-standardized antioxidants (-tocopherol and -carotene) decreased following conditioning therapy, presumably as the result of an enhanced breakdown of these antioxidants. Etoposide treatment did not amplify the loss of essential anti-oxidants but significantly increased lipid hydroperoxide concentrations in serum. We suggest that the abnormal generation of lipid hydroperoxides is the result of free radical formation.