B cell subsets in spleens of BALB/c mice: identification and isolation of MMTV-expressing and MMTV-responding subpopulations

The DNA of BALB/c mice contains two genomic- and one subgenomic-size MMTV proviruses that appear to be preferentially expressed in their spleen cells, although intact MMTV virions cannot be detected in the tissues of these mice. This retrovirus antigen expression is restricted to a subpopulation of B lymphocytes, as determined by double label immunofluorescence studies. Nylon-adherent, SIg-positive spleen lymphocytes from BALB/c mice are capable of being stimulated by purified MMTV in lymphocyte transformation assays. Two possibilities were explored: the MMTV-positive cells are the responders to MMTV in the blastogenesis assay, or there exists two B lymphocyte subsets, one expressing the MMTV antigen(s) and the other responding to the virus. Depletion of MMTV-positive, nylon-adherent cells with anti-MMTV and complement resulted in no significant change in the blastogenesis to MMTV, indicating that the MMTV-negative lymphocytes are the responders in this reaction. These results were confirmed by positive selection experiments by using a fluorescence-activated cell sorter. Two populations of nylon-adherent cells, on the basis of the presence or absence of MMTV antigen in their surfaces, were obtained by a two-way sorting procedure and were used in lymphocyte transformation assays. MMTV-expressing lymphocytes were found to be nonresponsive to MMTV antigens, although high levels of [3H]thymidine incorporation were observed in the MMTV-negative, nylon-adherent cell cultures exposed to MMTV. These data indicate that in normal BALB/c mice, expression of endogeneous retrovirus genetic information is restricted to a nylon-adherent spleen cell subset that is different from the one responding in blastogenesis to the viral antigens.     

Notes on Algerian aquatic fungi: Pythium pythioides

Pythium pythioides (Roze & Cornu) Ramsbottom is recorded from Algeria. Since its original discovery in 1869 this species had not been found back. A brief history is given and morphological and reproductive characters are discussed.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

The oral assimilation of radiomanganese by the mouse

The metabolism of orally administered radiomanganese was studied in mice. Assimilation of absorbed manganese (Mn) was determined using whole body counting techniques. When⁵⁴MnCl₂ was administered, 2.7% of the dose was retained after 10 d compared with 1.2% from the⁵⁴Mn-nitrilotriacetate (NTA) complex. However, this difference was accounted for by the rapid and persistent adsorption of the Mn onto the teeth of the lower jaw when fed as the ionic salt at pH 2.0 compared with the NTA-chelate fed at pH 9.0. Once corrected for the amount adsorbed onto the teeth, the biodistribution and relative specific activity of the assimilated radiomanganese into a variety of tissues were similar for both forms of the metal.     

Chloride transport across the integumentary epithelium ofManduca sexta (Lepidoptera: Sphingidae)

Summary Moulting fluid ofManduca sexta contains high concentrations of potassium and bicarbonate (100 mM) and low concentrations of chloride (5 mM). This fluid begins to disappear from the exuvial space approximately 9–10 h before the actual shedding of the integument. During this time, the integument can be isolated in an Ussing cell and electrical properties measured in vitro. In a normal 32 mM KHCO₃ saline, potential difference (PD) is around 10 mV, exuvial side positive, and short-circuit current (SCC) is 15–20 A cm^–2. Substitution of chloride slightly reduces both PD and SCC, although resistance does not change significantly. Measurement of chloride transport in the absence of K⁺ indicates that 100% of the SCC can be accounted for by the net chloride flux (2 A cm^–2). TheK _m andJ _max for transepithelial chloride transport are 14 mM and 0.1 Eq cm^–2 h^–1. Bilateral potassium addition stimulates chloride transport, doubling net chloride flux as potassium concentration increases from 2 to 5 mM. Chloride net flux is not inhibited by the presence of furosemide (1 mM), nor in HCO ₃ ^– -free saline by thiocyanate (1 or 10 mM) or acetazolamide (0.1 mM), but is inhibited by 100% N₂. The pattern of chloride transport inM. sexta is similar to that previously reported for the rectum of locusts. As chloride is normally at low concentrations in the moulting fluid, it is suggested that this transport system acts to maintain low intracellular concentrations which may be necessary for enzymatic functions in the epidermal cells and has little importance in fluid transport.Abbreviations PD potential difference - PPI pharate pupal integument - SCC short circuit current In the time since this research was performed, A.M. Jungreis passed away. He will be missed by his friends and colleagues     

Folding patterns of porin and bacteriorhodopsin.

Porin spans the outer membrane of Escherichia coli with most of the protein embedded within the membrane. It lacks pronounced hydrophobic domains and consists predominantly of beta-pleated sheet. These observations require the accommodation of polar and ionizable residues in an environment that has a low dielectric constant. Owing to a currently limited understanding of the constraints governing membrane protein structure, a minimal approach to structure prediction is proposed that identifies segments causing polypeptides to reverse their direction (turn identification). The application of this procedure avoids hydrophobicity parameters and yields a model of porin which is in good agreement with all experimental data available. The presence of polar and ionizable residues within membrane boundaries implies a dense (saturating) network of hydrogen bond donor and acceptor groups. Application to a paradigm of hydrophobic membrane proteins, bacteriorhodopsin, reveals a pattern consistent with its alpha-helical folding. The postulated structure includes significantly more polar residues in the membrane domain than have been assumed previously, suggesting that there are also hydrogen bonding networks in bacteriorhodopsin. Extensive networks permeating protein interior and surfaces would explain the extraordinary stability and the tight interactions between functional units in the formation of crystalline arrays of both proteins.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase