Cloned MPC 11 myeloma cells express two kappa genes: a gene for a complete light chain and a gene for a constant region polypeptide.

Cloned MPC 11 mouse plasmacytoma cells synthesize a complete kappa light chain and also a kappa light chain constant region fragment. Partial amino terminal sequences of the in vitro forms of these two proteins have been determined. Both in vitro products contain typical light chain leaders; leaders are defined as the amino terminal sequences present on in vitro products but absent from the in vivo products found in living cells. The in vitro form of the MPC 11 complete light chain contains a leader sequence plus variable and constant region sequences. The in vitro form of the MPC 11 light chain constant region fragment contains a different leader sequence attached directly to a complete constant ragion sequence and has no variable region sequences. Thus the MPC 11 light chain fragment is not a degradation product of the MPC 11 complete light chain (or of any other complete light chain) and must be coded by a separate gene. The results reveal two unusual features of MPC 11 cells: first, expression of a unique variant light chain gene coding the light chain constant region fragment, and second, expression of two different kappa light chain genes (coding the complete light chain and the variant constant region fragment) in a single cell. In addition, evidence is provided that the in vitro forms of kappa light chains, three of which are presented here for the first time, include a minimum of three partially homologous but quite different leader sequences.     

Development of a quantitative method for detecting enteroviruses in estuarine sediments.

Several investigators have reported on the detection of enteric viruses in marine sediments, but none determined the efficiency of their methods and only limited volumes of sediment were sampled. The purpose of this investigation was to develop a quantitative method for detecting enteroviruses in marine sediments so that their relative proportion to viruses freely suspended in estuarine water could be more accurately determined. Poliovirus was found to adsorb readily to natural marine sediments collected along the Texas Gulf coast. A number of substances were evaluated for their ability to elute adsorbed viruses. A solution of 10% fetal calf serum adjusted to pH 10.5 and 0.05M ethylenediaminetetraacetate (pH 11.0) were found to be the best eluents. Using ethylenediaminetetraacetate as an eluent, it was possible to elute virus from large volumes of sediment and reconcentrate the sediment eluate into an economically assayable volume (30 to 50 ml). Poliovirus could be recovered from the sediment with an overall efficiency of 50%. This method was found to be satisfactory for the recovery of naturally occurring animal viruses in estuarine sediments from the upper Texas Gulf coast.     

Effects of paraquat on selected microbial activities in soil

Paraquat, applied as Gramoxone, to a nonamended sandy loam soil at five times the suggested field application rate (10 lb/A 115g/cm²) increased the numbers of bacteria, actinomycetes, and fungi during a 14-day incubation at 25°C. This increase was attributed to the use of compounds in the Gramoxone formulation rather than the use of paraquat. Treatment at one and five times the normal rate reduced CO₂ evolution by 44% and 67%, respectively, in soil amended with 2% glucose during a 12-day incubation. Similar treatments reduced CO₂ evolution in 1% straw-amended soil by 39% and 58%, respectively, during a 28-day incubation. Cellulose decomposition of cotton duck containing 13 and 176g of paraquat per milligram of material was inhibited for 15 and 28 days, respectively, in soil containing a large population of cellulolytic microorganisms. A concentration of 5000g/gm of paraquat was necessary to inhibit nitrification in soil by 44% druing a 28-day incubation at 20°C. Paraquat inhibited C₂H₂ reduction in artificial aggregates of soil amended with 2% glucose and incubated anaerobically at 25°C. Nitrogenase activity in aggregates was inhibited by 43% and 52% at concentrations of 580 and 720g/gm of paraquat respectively. The inhibitory effects of the herbicide were reduced when soil was amended with organic matter in the form of peat or straw. The availability of paraquat controlled the toxicity of the herbicide to soil microorganisms.     

Defluorination of fluoroacetate in the rat

Rats given 5 ppm F as FAc (equivalent to 26 ppm of NaFac) in the drinking water for approximately four months deposited as much fluoride in the skeletal system as did rats receiving 5 ppm F as NaF in the water. Little evidence could be found for the presence of organically bound fluoride in bone after ingesting FAc, though an appreciable proportion of skeletal fluoride deposited when NaF was ingested was shown not to respond to the fluoride ion electrode. The daily urinary excretion of total fluoride after FAc was somewhat greater than after NaF; about two thirds of this fluoride responded to the electrode, whereas more than 90 percent of the total fluoride after NaF was ionic in nature. The data are interpreted as showing that the rat is capable of splitting the C-F bond in FAc and/or in its fluoride-containing metabolites, with subsequent skeletal storage and renal excretion of the released fluoride ion. The chronic administration of this low level of FAc caused an early but temporary retardation of growth. The Krebs cycle was interfered with, as evidenced by increased concentrations of citrate in the kidney and urine. At termination of the experiment, histological examination of the testes showed that the FAc had induced severe damage characterized by massive disorganization of the tubules, nearly total loss of functional cells, absence of sperm, and damage to the Sertoli cells.     

Effect of phenolic compounds on glucose-6-phosphate dehydrogenase isoenzymes

Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.     

In-vitro translation of messenger-RNA from developing bean leaves. Evidence for the existence of stored messenger-RNA and its light-induced mobilisation into polyribosomes

Poly(A)-containing messenger RNA was purified from polyribosomes isolated from the primary leaves of 7-day-old dark-grown seedlings of Phaseolus vulgaris var. Masterpiece. Analysis of the messenger RNA on 2.4% polyacrylamide gels showed that it consists of a heterogeneous population of molecules with an average molecular weight of 500,000. The nucleotide composition of the RNA was 16.0% cytidylic acid, 39.4% adenylic acid, 21.3% guanylic acid and 23.2% uridylic acid. Based on the degree of resistance of the RNA to digestion with ribonucleases A and T₁ the average length of the poly(A) sequence was calculated to be 120 nucleotides. No significant differences in mobility in polyacrylamide gels, nucleotide composition or polyadenylic acid content were found between the poly(A)-containing mRNA from polyribosomes of primary leaves of dark-grown plants and those given a 16 h white light treatment. Purified poly(A)-containing mRNA was shown to direct the incorporation of [³⁵S]methionine into proteins in an in vitro protein-synthesising system from wheat germ. The protein products were fractionated according to molecular size by electrophoresis in 15% polyacrylamide/urea/SDS gels and the protein bands were detected by fluorography. Messenger RNAs directing the synthesis of three polypeptides with molecular weights of 34,000, 32,000 and 25,000 were detected in polyribosomes of plants following white light treatment. These messenger RNAs were absent, or present in much lower amounts, in polyribosomal messenger RNA from leaves of dark-grown plants, although they were present in total cell poly(A)-containing RNA. This indicates that certain messenger RNAs may be stored in the dark and that light stimulates these RNAs to engage in polyribosome formation. Continuous far-red (730 nm) irradiation for 4 h also caused the appearance of these messenger RNAs in the polyribosomes although 5 min red light followed by 4 h darkness had little effect. This suggests that phytochrome acting in the high energy mode, may be the photoreceptor responsible for initiating the response.Abbreviations mRNA messenger-RNA - rRNA ribosomal RNA - oligo (dT) oligo (deoxythymidylic acid) - poly(A) polyadenylic acid - EDTA ethylenediamine-tetra-acetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - SDS sodium dodecyl sulphate     

The natural occurrence of insulin receptors in groups on adipocyte plasma membranes as demonstrated with monomeric ferritin-insulin

This study was designed to document whether the reported distribution of insulin receptors in small groups of receptor sites randomly distributed in the glycocalyx of adipocytes and isolated adipocyte plasma membranes was a naturally occurring phenomena or due to artifacts. Possible artifacts include: (1) oligomeric forms of ferritin in the ferritin-insulin preparation, (2) an uneven distribution of the glycocalyx on the plasma membrane, or (3) ligand-induced aggregation of occupied receptor complexes. Biogel A 1.5m chromatography of the ferritin-insulin conjugate revealed the ferritin in the ferritin-insulin complex to consist of 55% monomers, 15% dimers, and 30% oligomers. The monomer peak was purified (> 95%) for use in these studies. Cationic ferritin, a glycocalyx marker, when incubated with paraformaldehyde-fixed plasma membranes, was found to be uniformly distributed on the surface of the plasma membrane indicative of uniformly distributed glycocalyx. The ability to demonstrate and inhibit ligand-induced aggregation on the isolated plasma membrane was established with a multivalent ligand, ferritin-concanavalin A. More than 66% of the ferritin-concanavalin A receptors were found in large clusters of 5 or more and 34% as singletons or clusters of up to 4 when incubated at 24°C with fresh membranes. Only 38% of the ferritin-concanavalin A receptors were in large clusters; 62% were singletons or clusters up to 4 on membranes prefixed with paraformaldehyde before incubation. The distribution of the monomeric ferritin-insulin was similar on both adipocytes and purified adipocyte plasma membranes and was consistent with earlier reports with ferritin-insulin. The quantitative distribution of the monomeric ferritin-insulin as singletons or in groups of 2–6 was comparable between the intact cells and isolated membranes incubated at 24°C. The binding of 500 μUnits monomeric ferritin-insulin per ml to the isolated plasma membranes was studied under incubation conditions similar to those used with ferritin-concanavalin A. Under all three conditions, fresh membranes at 24°C and 0–4°C and prefixed membranes at 24°C, the pattern of distribution of the monomeric ferritin-insulin as singletons or groups of 2–6 was identical, indicating that the ligand was not causing aggregation into clusters as did the concanavalin A. Thus, the occurrence of insulin receptors in small groups appears to be a natural phenomenon in the plasma membrane structure of adipocytes.