Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Synthesis and possible role of carbohydrate moieties of yeast glycoproteins

The pathways for protein N- and O-glycosylation in yeast cells are summarized. Evidence is presented that the terminal glucosyl residues of the dolichyl-PP-oligosaccharide intermediate are responsible for decreasing the Km for the peptide to be N-glycosylated. A liposomal model system is introduced that allows the study of a dolichyl phosphate (Dol-P) dependent transmembrane transport of mannosyl residues. The results obtained so far suggest that the mannosylation of Dol-P and the transmembrane translocation of Dol-P-Man are catalysed by the enzyme more or less simultaneously. However, only about 8-10% of the enzyme molecules incorporated into the liposomes seem to carry out the 'coupled' reaction. The glycosylation of carboxypeptidase Y is not required for this protein to reach the vacuole, its target organelle. In the presence of low concentrations of tunicamycin, however, yeast cells do stop growth. This does not seem to be due to the inhibition of secretion of glycoproteins like external invertase. It is postulated that protein glycosylation is crucial for a cell cycle event during the G1 phase.     

An error estimation of Michaelis-Menten (Monod)-type kinetics

Due to research on biochemistry and genetic engineering, mathematical models of microbial growth have become more complicated but Michaelis-Menten or Monod type expressions have still been used for conversion rates, uptake rates, etc. It is worth examining the error that can be caused by these quasi-steady-state-hypotheses. This paper presents a simple but very effective rationale function that describes the error of the quasi-steady-state hypothesis in enzyme kinetics. A simplified fermentation kinetic model was used for comparison of microbial growth but no analytical error function has been found for batch cultivation. In the case of continuous fermentation the error can be given in an analytical form. Many simulations, based on real SCP experiments, show a significant effect of the quasi-steady-state hypothesis. Since the rate constants of intracellular events are not really known, we have to be very careful when taking into account Michaelis-Menten type expressions in the building of complicated models. Correspondence to: L. Szigeti     

Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes

The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.     

The complete amino acid sequence of the human erythrocyte membrane anion-transport protein deduced from the cDNA sequence.

1. We have isolated cDNA clones corresponding to the red cell membrane anion-transport protein (Band 3). 2. The cDNA clones cover 3475 bases of the mRNA and contain the entire protein-coding region, 150 bases of the 5' untranslated region and part of the 3' non-coding region, but do not extend to the 3' end of the mRNA. 3. The translated protein sequence predicts that the human red cell anion transporter contains 911 amino acids. 4. The availability of the amino acid sequence allows the interpretation of some of the many studies on the chemical and proteolytic modification of the human protein aimed at examining the structure and mechanism of this membrane transport protein.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.