Sequential passage of alpha2mu-globulin through the hepatic endoplasmic reticulum and Golgi apparatus during secretion.

Studies on the synthesis and secretion of the sex-dependent urinary protein, alpha2mu-globulin, have been extended by establishing its sequential passage from the rough to the smooth endoplasmic reticulum and Golgi-rich fractions of the liver of adult male rats. After injection of 14C-labeled amino acids, the maximum radioactivity of alpha2mu occurred at 20 min in the rough, 25 min in the smooth microsomes and 30 or 35 min in the Golgi-rich fractions. Radioactive alpha2mu-globulin appeared in the bloodstream and kidneys after a lag of 20--25 min. Results indicate that alpha2mu-globulin follows a secretory pathway similar to that of serum albumin.     

Using SIB and API kits to identify and biotype enterobacteria of different origin

A collection of 26 Enterobacteriaceae reference strains provided by Reference Centres in Moscow (USSR) and Copenhagen (Denmark) as well as a collection of 660 freshly isolated cultures of Gram-negative bacteria of different origin were investigated using SIB indicator systems manufactured at the Gorky Institute of Epidemiology and Microbiology (USSR) and API-20E, Rapid-20E and API-10S kits (API, France) with the aim of species determination. In analyzing freshly isolated cultures, API-20E, API-10S and SIB-B kits proved to be of approximately equal efficiency, whereas the Rapid-20E system enabled species identification in no more than 78% of the tested cultures. In a model biotyping of 284 E. coli cultures of different origin, SIB-B and API-20E kits in combination with the Analytical Profile Index enabled sufficiently rapid and standard identification of Enterobacteriaceae biovars.     

On choice of substrate and habitat in brachionid rotifers

Information on the distribution of 28 rotifers of the family Brachionidae from diverse waters in south and central Sweden was analyzed to reveal their relationships to substrate and habitat. Some brachionids are preferably planktic, others periphytic and/or benthic. Some non-planktic habitats are utilized more than others, but there is no evidence of a chemical attraction from any substrate. Instead, some substrates seem to be avoided, possibly depending on a poorer flora of periphytic algae. Besides substrate type, the following factors are found to be important for creating separate ecological niches in the brachionid family: temperature, oxygen content, trophic degree, chemical environment, food choice and sensitivity to predation. It is possible to delineate separate ecological niches for all brachionid rotifers, implying that Hutchinson's ideas about the plankton paradox are contradicted. Some species are specialists, other are generalists, the latter being characterized by a great morphological variation. The species are adapted in different ways to their preferential habitats, as regards foot, egg-carrying, protrusions and other lorical structures etc. Longer spines, for instance, are generally found in more transparent water, being a supposed protection against visual predators.     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

The crystallization of outer membrane proteins from Escherichia coli. Studies on lamB and ompA gene products

The outer membrane protein LambB from Escherichia coli has been crystallized from detergent-containing solutions. Several different crystal habits can be obtained under the same ionic and precipitant conditions by altering the detergent head group composition of the protein-detergent mixed micelle or by adding polar organic compounds. Two crystal forms have been partially characterized as P1 and C2221, the former diffracting to beyond 4 A resolution and the latter to 6 A. The detergents used were beta-octyl glucoside, octyl tetraoxyethylene, and octyl polyoxyethylene (polydisperse) either alone or as mixtures. In some experiments, the addition of small nonionic amphiphiles having n-butyl alkyl tails significantly influenced crystallization. The experiments suggest that the detergent region of the mixed micelle plays a critical role in crystal formation. Using the methods developed here for LamB and also for matrix porin (Garavito, R. M., Jenkins, J. A., Jansonius, J. N., Karlsson, R., and Rosenbusch, J. P. (1983) J. Mol. Biol. 164, 313-327), an additional protein from the outer membrane, OmpA, has been obtained as a microcrystalline preparation.     

High yield and stable transformation of the unicellular green alga Acetabularia by microinjection of SV40 DNA and pSV2neo

SV40 DNA and pSV2neo were microinjected into isolated nuclei of Acetabularia mediterranea. The injected nuclei were implanted into anucleate cell fragments of the same species. Such combinations not only survived but also formed progeny. The F₁, F₂ and F₃ generations of these combinations were analyzed. In the case of SV40-treated cells T-antigen was expressed and accumulated in the nuclei of all three generations studied as shown by indirect immunofluorescence. Nuclear exchange experiments revealed expression of the T-antigen only if a transformed nucleus but not if only a transformed cytoplasm was involved. Transformation by pSV2neo, a chimeric gene with a selectable marker was demonstrated by the induction of G-418 resistance as well as immunofluorescence. Genomic DNA was isolated from gametes, originating in cysts from the F₁, F₂ and F₃ generations of injected cells, and subjected to Southern analysis. These experiments demonstrated that both types of DNA are integrated into the host genome.     

Biosynthesis of D-alanyl-lipoteichoic acid: role of diglyceride kinase in the synthesis of phosphatidylglycerol for chain elongation.

Lipophilic and hydrophilic D-alanyl-lipoteichoic acids are elongated in Lactobacillus casei by the transfer of sn-glycerol 1-phosphate units from phosphatidylglycerol to the poly(glycerophosphate) moiety of the polymer. These sn-glycerol 1-phosphate units are added to the end of the poly(glycerophosphate) which is distal to the glycolipid anchor; 1,2-diglyceride results from this addition. The presence of a diglyceride kinase was suggested by the ATP-dependent phosphorylation of 1,2-diglyceride to phosphatidic acid. Inorganic phosphate was used to initiate the synthesis of lipophilic lipoteichoic acid (LTA) and the elongation of both lipophilic and hydrophilic LTA. Three observations suggest that phosphate and other anions play a role in the in vitro synthesis of LTA and its precursors. First, the conversion of 1,2-diglyceride to phosphatidic acid by diglyceride kinase was stimulated. Second, the synthesis of phosphatidylglycerol was increased. Third, the elongation of lipophilic and hydrophilic LTA was enhanced. These observations indicated that one effect of phosphate might be to enhance the utilization of 1,2-diglyceride for the synthesis of phosphatidic acid. This phospholipid is a precursor of phosphatidylglycerol, the donor of sn-glycerol 1-phosphate for elongation of LTA.     

Potentiation of antibody responses by specific IgM: specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.     

Biosynthesis of peptidoglycan in Gaffkya homari: role of the peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide

The incorporation of N-acetylmuramyl (MurNAc)-peptides from nucleotide-activated precursors (reference: uridine diphosphate [UDP]MurNAc-Ala(1)-dGlu(2)-Lys(3)- dAla(4)-dAla(5)) with incomplete or modified peptide subunits into peptidoglycan was studied with membrane preparations from Gaffkya homari. The effectiveness of their utilization at low and high concentrations was compared on the basis of the values of V(max)/K(m) and V(max), respectively. At low concentration, replacement of alanine by glycine in position 5 has a small effect on the activity of the peptidoglycan synthesizing system, whereas it has a significantly larger effect in positions 1 and 4. The importance of d-alanine in position 4 at low substrate concentrations is also observed with the incomplete UDP-MurNAc-peptides. For UDP-MurNAc-tripeptide and -tetrapeptide, V(max)/K(m) is 0.06 and 0.55, respectively, of the value for the -pentapeptide. At high substrate concentration, replacement of d-alanine by glycine in either position 1 or 5 decreases the activity to 0.37 of the value for the reference nucleotide, whereas replacement in position 4 has a smaller effect (0.74). The profiles established from V(max) and V(max)/K(m) with UDP-MurNAc-tripeptide, -tetrapeptide, and -pentapeptide show good correlation. At low concentration the specificity profiles of phospho-MurNAc-pentapeptide translocase, catalyzing the initial membrane reaction, are similar to those for the peptidoglycan synthesizing system; at high concentration, however, the profiles differ. The translocase appears to provide a primary specificity barrier at high substrate concentration for UDP-MurNAc-Ala-dGlu-Lys-dAla-dAla and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla, and at low concentration for UDP-MurNAc-Ala-dGlu-Lys and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla. Moreover, it is suggested that an additional specificity barrier exists in the peptidoglycan synthesizing system for certain nucleotides. Thus, the cytoplasmic enzymes and the membrane-associated enzyme(s) cooperate to insure the formation of functioning peptidoglycan in this organism.