Structural studies on the murine Ia alloantigens. VI. Evidence that both subunits of the I-A alloantigen are encoded by the I-A subregion.

The alpha and beta subunits of the murine I-A alloantigens from several H-2 haplotypes were examined by comparative tryptic peptide mapping by using double label (3H and 14C) techniques. Significant structural variation between alleles was detected in both subunits. Tryptic digests of the alpha polypeptides from s, b, and d showed only 65% co-elution with k; beta-chains from s, b, d, and r were about 50% similar to the k beta subunit. Peptide analysis of the Ak subunits from intra-H-2 recombinant strains indicated that both the alpha and beta polypeptides are encoded within the I-A subregion.     

Phosphonate utilization by bacteria.

Bacteria able to use at least one of 13 ionic alkylphosphonates of O-alkyl or O,O-dialkyl alkylphosphonates as phosphorus sources were isolated from sewage and soil. Four of these isolates used 2-aminoethylphosphonic acid (AEP) as a sole carbon, nitrogen, and phosphorus source. None of the other phosphonates served as a carbon source for the organisms. One isolate, identified as Pseudomonas putida, grew with AEP as its sole carbon, nitrogen, and phosphorus source and released nearly all of the organic phosphorus as orthophosphate and 72% of the AEP nitrogen as ammonium. This is the first demonstration of utilization of a phosphonoalkyl moiety as a sole carbon source. Cell-free extracts of P. putida contained an inducible enzyme system that required pyruvate and pyridoxal phosphate to release orthophosphate from AEP; acetaldehyde was tentatively identified as a second product. Phosphite inhibited the enzyme system.     

Quercetin inhibits hexose transport in a human diploid fibroblast

Summary The flavonol quercetin, a phloretin analog, inhibits transport of 2-deoxyglucose and 3-O-methylglucose in a cultured human diploid fibroblast. This inhibition is related to transport itself and not to the reported effects of flavonoids on membrane-bound ATPases. From concentration-inhibition curves at several pH's we conclude that uncharged (acid) quercetin (pK=7.65) is the inhibitory form of the molecule (K _I =10m). Quercetin, unlike phloretin, is rapidly degraded in 0.1n NaOH; the degradation products are weakly inhibitory to hexose transport.     

Characterization of HeLa 5'-nucleotidase: a stable plasma membrane marker.

5'-Nucleotidase, assayed as 5'-AMPase, has been extensively characterized and established as a stable, quantitative plasma membrane marker in HeLa S3 cells. The membrane 5'-AMPase has a Km of 7.0 microM. Relative affinities of the other 5'-mononucleotides for the enzyme are 5'-GMP > 5'-TMP > 5'-UMP > 5'-CMP. There are activity optima at pH7 and 10; the latter is Mg(2+)-dependent. The membrane preparations have a small amount of acid phosphatase activity that is distinct from 5'-AMPase activity but no alkaline phosphatase. AOPCP, ADP, and ATP are strongly inhibitory. Mg2+, Ca2+, or Co2+ additions do not affect the pH 7.0 activity; Mn2+ activates slightly, whereas Zn2+, Cu2+, and Ni2+ are inhibitory. EDTA slowly inactivates, but removal of the EDTA without the addition of divalent cations restores activity. The inactivation is also substantially reversed by Co2+ or Mn2+, but reactivability by divalent cations decreases with time in EDTA. ConA strongly inhibits, and alpha-methyl-D-mannoside or glucose (the latter much less efficiently) relieves the inhibition, indicating that the 5'-AMPase is a glycoprotein. Histidine is also inhibitory. Ouabain, phloretin, cytochalasin B, cysteine, phenyl-alanine, MalNEt, and IAA are without effect. 5'-AMPase activity codistributes with pulse-bound [3H]ouabain when either of two cell fractionation procedures are used. The 5'-AMPase activity per cell is constant at different cell densities in exponentially growing cells, and activity per unit cell volume remains constant throughout the cell cycle. These properties, together with its absence in other organelles, its stability to storage, its insensitivity to certain experimental manipulations, and its general insensitivity to inhibitors of specific transport systems, make 5'-AMPase a useful quantitative marker in studies on the regulation of HeLa membrane transport systems. Key Words: HeLa, 5'-nucleotidase, plasma membrane marker, non-specific phosphatases, divalent ions, ConA, AOPCP, cell cycle, mitochondria, transport inhibitors.     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.     

Clearance and fate of leukemia-inhibitory factor (LIF) after injection into mice

Leukemia-inhibitory factor (LIF) elicits effects on a broad range of cell types, including cells of the monocytic and megakaryocytic series, embryonal stem cells, hepatocytes, adipocytes, and osteoblasts. Native and recombinant LIF, injected intravenously into adult mice, had an initial half-life of 6-8 min and a more prolonged second clearance phase. Clearance of 125I-LIF from the circulation was paralleled by a rapid accumulation in the kidneys, liver, lungs, and spleen and a more gradual accumulation in the thyroid gland. Labeling of the renal glomerular tufts, parenchymal hepatocytes, splenic red pulp, alveolar pneumocytes, and thyroid follicular cells as well as of megakaryocytes and osteoblasts in the bone cavities, placental trophoblasts, and cells of the choroid plexus was demonstrable autoradiographically. The appearance of a large amount of nonprecipitable 125I in the urine suggested that the kidneys were the major route of LIF clearance from the body.     

Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.