Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.     

The kinetic equation for the chloride transport cycle of band 3. A 35Cl and 37Cl NMR study

The nature of a transmembrane transport process depends largely on the identity of the reaction that is rate-limiting in the transport cycle. The one-for-one exchange of two chloride ions across the red cell membrane by band 3 can be decomposed into two component reactions: 1) the binding and dissociation of chloride at the transport site, and 2) the translocation of bound chloride across the membrane. The present work utilizes 35 Cl NMR and 37 Cl NMR to set lower limits on the rates of chloride binding and dissociation at the saturated inward- and outward-facing band 3 transport sites (greater than or equal to 10(5) events site-1 s-1 in all cases). At both 0-3 and 37 degrees C, the NMR data specify that chloride binding and dissociation at the saturated transport sites are not rate-limiting, indicating that translocation of bound chloride across the membrane is the slowest step in the overall transport cycle. Using these results, it is now possible to describe many features of the kinetic equation for the ping-pong transport cycle of band 3. This transport cycle can be decomposed into two half-reactions associated with the transport of two chloride ions in opposite directions across the membrane, where each half-reaction is composed of sequential binding, translocation, and dissociation events. One half-reaction contains the rate-limiting translocation event that controls the turnover of the transport cycle; in this half-reaction, translocation must be slower than binding and dissociation. The other half-reaction contains the non-rate-limiting translocation event that in principle could be faster than binding or dissociation. However, when the following sufficient (but not necessary) condition is satisfied, both translocation events are slower than binding and dissociation: if the non-rate-limiting translocation rate is within a factor of 10(2) (0-3 degrees C) or 2 (37 degrees C) of the overall turnover rate, then translocation is rate-limiting in each saturated half-reaction. Thus, even though chloride appears to migrate through a channel that leads from the transport site to solution, the results support a picture in which the binding, dissociation, and channel migration events are rapid compared to the translocation of bound chloride across the membrane. In this case, chloride binding to the transport site can be described by a simple dissociation constant (KD = kappa OFF/kappa ON) rather than by a Michaelis-Menten constant (KM = (kappa OFF + kappa TRANSLOCATION)/KAPPA ON).     

Nucleotide sequence and gene organization of ColE1 DNA

The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein).     

The effect of total parenteral nutrition (TPN) on the enteroinsular axis in the rat

The effect of 6 days of total parenteral nutrition (TPN) on the enteroinsular axis was studied in vivo and in vitro in the rat. During the TPN period, blood samples were taken from control and TPN animals to determine the comparative pattern of GIP release. Glucose, insulin and GIP responses to oral glucose (OGTT) were compared in TPN and control rats. The effect of glucose and GIP on insulin release from the isolated perfused pancreas of the same animals was investigated to determine if TPN altered the sensitivity of the beta cell. In conjunction with these studies the number and distribution of GIP-containing cells were compared in control and TPN animals. TPN resulted in no change in basal levels of glucose, insulin and IR-GIP. An exaggerated insulin response to OGTT occurred after TPN whereas the glucose response was reduced. The IR-GIP response to glucose was normal following TPN. The isolated perfused pancreas showed a 30% increase in insulin release in response to GIP after TPN. The insulin response to glucose appeared normal as did the number and distribution of GIP cells. Fluctuations in GIP and insulin levels in control animals were diurnal in nature, whereas IR-GIP levels in TPN animals remained near fasting levels. It was hypothesized that the increase in beta cell sensitivity to GIP may be causally connected to the exposure of the pancreas to chronically low levels of GIP during TPN.     

Insertion of a foreign nucleotide sequence into mitochondrial DNA causes senescence in Neurospora intermedia

The kalilo variants of Neurospora contain a cytoplasmic genetic factor that causes senescence. This factor is a 9.0 kb transposable element (kalDNA) that lacks nucleotide sequence homology with mtDNA and is inserted into the mitochondrial chromosome, often at sites located within the open reading frame in the intron-DNA of the mitochondrial 25S-rRNA gene. Genomes containing the "foreign" DNA insert accumulate during growth, and death occurs as the cells become deficient in functional large and small subunits of mitochondrial ribosomes. The kalDNA transposon may be an "activator" element that causes breaks in mtDNA. Nonsenescing [+] strains of Neurospora do not contain kalDNA.     

Pilot-scale production of l-phenylalanine from d-glucose

Effects of inoculum size and total sugar content on both l-phenylalanine productivity and titre have been investigated using a tyrosine auxotrophic regulatory mutant of Escherichia coli. Fermentations were carried out in a 500 litre pilot fermenter with intermittent feeding of d-glucose plus phosphate. It was found that the productivity was not greatly affected by inoculum size. However, the l-phenylalanine titre was significantly affected by total sugar content. Relatively high productivities of up to 0.35–0.40 g l-phenylalanine l^?1 h^?1 have been achieved at l-phenylalanine titres of 14–15 g l^?1.     

Role of supernatant protein factor and anionic phospholipid in squalene uptake and conversion by microsomes

Supernatant protein factor (SPF), a cytosolic protein (Mr = 47,000) stimulates microsomal squalene epoxidase activity 4- to 10-fold in the presence of anionic phospholipid such as phosphatidylglycerol (PG) (Saat, Y., and Bloch, K. (1976) J. Biol. Chem. 251, 5155-5160). This effect has been ascribed to substrate translocation from inactive to active pools within the membrane of the endoplasmic reticulum (Friedlander, E. J., Caras, I. W., Lin, L. F. H., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045). Here we show that SPF and PG also stimulate squalene uptake per se by microsomes as well as stimulate squalene epoxidase. Microsomes preloaded with substrate in the presence of SPF and PG show full epoxidase activity. They do not require further addition of these factors during enzyme assay. Addition of SPF and PG to assay mixtures containing microsomes preloaded with substrate in the presence of SPF and PG did not further increase epoxidase activity. We also show that PG tightly binds to microsomes. This binding of PG is essential for the response of microsomal epoxidase to SPF. Solubilized microsomal enzymes have been reconstituted and show high epoxidase activity. In this system, SPF and PG do not stimulate the conversion of squalene into products.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglycemic (db/db) mice. Effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-3H]glucose, uptake of 3-O-[methyl-3H]glucose (methylglucose) and [2-14C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 +/- 18 and 180 +/- 9 microliter/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V- of methylglucose transport was decreased with no change in Km in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and 3H2O production from [5-3H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.