Blood–Brain Transfer of Glucose and Glucose Analogs in Newborn Rats

Little is known of the selectivity of the blood-brain barrier at birth. Hexoses are transported through the barrier by a facilitating mechanism. To study the capacity of this mechanism to distinguish between analogs of D-glucose, we compared the transport of fluorodeoxyglucose, deoxyglucose, glucose, methylglucose, mannose, galactose, mannitol, and iodoantipyrine across the cerebral capillary endothelium in newborn Wistar rats. Cerebral blood flow, glucose consumption, and the blood-brain permeabilities of the hexoses were 25-50% of the adult values but the ratios between the permeabilities of the individual hexoses were similar to the ratios observed in adult rats. The mannitol clearance into brain was considerably higher than in adult rats (about 10-fold), indicating a higher endothelial permeability to small polar nonelectrolytes. The brain water content was higher in newborn than in adult rats and was associated with a higher steady-state distribution of labeled methylglucose between brain and blood. Hexose concentrations were determined relative to whole blood because the apparent erythrocyte membrane permeability to glucose was as high as in humans and thus considerably higher than in adult rats. The half-saturation concentration of glucose transport across the blood-brain barrier was considerably higher than in adult rats, about three-fold, suggesting that net blood-brain glucose transfer is less sensitive to blood glucose fluctuation in newborn than in adult rats.     

Amino acid sequence of Manduca sexta adipokinetic hormone elucidated by combined fast atom bombardment (FAB)/tandem mass spectrometry

Combination of Fast Atom Bombardment Tandem Mass Spectrometry with Amino Acid Analysis assigns the amino acid sequence of the Manduca sexta adipokinetic hormone as pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-GlyNH2. Similarities and differences with other invertebrate hormones and with mammalian glucagon are discussed.     

Influence of the H-2u haplotype on immune function in F1 hybrid mice. I. Antigen presentation

Previously, we reported that myelin basic protein (MBP) peptide 1-37 contains an encephalitogenic epitope for PL/J mice, and MBP peptide 89-169 is encephalitogenic for SJL/J mice. (SJPL)F1 hybrid mice do not respond to immunization with these peptides in a co-dominant manner because the encephalitogenic response to peptide 1-37 dominates. To examine this phenomenon more closely, we tested the ability of MBP-primed parental or F1 T cells to respond to MBP or MBP peptides in the context of PL, SJL, or F1 antigen-presenting cells (APC). It was found that the F1 T cells responded to either the protein or the peptides when these were presented in the context of F1 or PL APC. However, F1 T cells would not respond to MBP in the context of SJL APC, although the latter cells were functionally intact. This effect was not antigen-specific because SJL APC would not present ovalbumin or PPD to primed F1 T cells. F1 T cells from mice immune to the strongly antigenic bacterium Listeria monocytogenes responded to bacterial antigens presented by SJL APC, although at a significantly lower level compared to the results obtained when these antigens were presented by F1 or PL APC. This finding implied that unbalanced antigen presentation was a quantitative rather than a qualitative phenomenon. When F1 hybrid mice from other strain combinations were tested, a similar effect was observed whenever one of the parental strains was PL/J. This effect was mapped to the MHC in MHC-congenic B10 mice.     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

Amplified expression of streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product

The endo-beta-N-acetylglucosaminidase H (Endo H) gene from Streptomyces plicatus has been cloned into the Escherichia coli plasmid pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature 272, 128-132), thus placing expression of this gene under control of the strong lambda promoter pL. The construction, pKCE3, which includes a properly positioned E. coli ribosome binding site from the lac operon (Robbins, P.W., Trimble, R. B., Wirth, D.F., Hering, C., Maley, F. Maley, G. F., Das, R., Gibson, B.W., and Biemann, K. (1984) J. Biol. Chem. 259, 7577-7583), was used to transform an E. coli strain lysogenic for a lambda prophage containing a temperature-sensitive repressor. By shifting cultures of pKCE3 lysogens to 42 degrees C, the production of Endo H commenced and was linear for about 1 h. Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S. plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure. Although most of the cloned Endo H was secreted into the periplasmic space by E. coli, its 4 kDa leader sequence peptide (Robbins et al. (1984] was only partially removed during processing. As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S. plicatus. Despite the residual approximately 2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E. coli or from S. plicatus. Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants.     

Refinement of HLA gene mapping with induced B-cell line mutants

The lymphoma cell line BJAB.B95.8.6 was gamma-irradiated to induce mutations of major histocompatibility complex (MHC) encoded genes. Cloned wild-type cells were phenotyped HLA-A1, A2, B 13, 1335, Bw4, Bw6, Cw4, DR5, DRw52, DQwl, DQw3, DPw2, DPw4, GLO1^*1, PGM3^*2-1, and ME1^*0 and possessed two apparently normal chromosome 6s prior to mutagenesis. Loss mutants were selected 5 days after 3 Gy gamma-irradiation employing three complement-fixing monoclonal antibodies specific for HLA-A2 (TÜ101) and Bw4 (TÜ48, TÜ109). Fifteen independently arising mutants were isolated and cloned. Typing with monospecific alloantisera and cell-mediated lympholysis revealed the presence of HLA-A1, 835, Bw6, Cw4, DR5. DRw52, DQw3, and DPw4 specificities on all mutant clones. HLA-A2, B13, and Bw4 were absent. Mutants differed in their expression of class 11 antigens. One group retained DQw1 and DPw2, another was DQw1^–, DPw2⁺, and a third was DQw1^–, DPw2^–. Karyotyping of the wild-type line and selected mutant clones showed that the loss of HLA specificities correlated with deletions which map the HLA-A and -B loci directly to the distal part of the 6p2l.33 region and the class II genes to the region 6p21.33 (proximal) to 6p21.31 (distal) on the short arm of chromosome 6.Abbreviations used in this paper: CML cell-mediated lympholysis - CTX cytotoxicity - DBBA direct bacterial binding assay - EBV Epstein-Barr virus - GLO glyoxalase - IBBA indirect bacterial binding assay - LU lytic units - ME1 cytoplasmic malic enzyme - MHC major histocompatibility complex - MOAB monoclonal antibody - NADP nicotinamide-adenine dinucleotide phosphate - PGM3 phosphoglucomutase isozyme 3 In partial fulfillment of Ph.D. thesis requirements.     

Protoplast transformation of group N streptococci with cryptic plasmids

Abstract By using an extension to group N streptococci of a contransformation procedure we have introduced 4 different-sized cryptic plasmids for Streptococcus lactis into the plasmid-free S. lactis IL1403. A mixture of 4 cryptic plasmids with an indicator plasmid (pHV1301) conferring erythromycin resistance was used for IL1403 protoplast transformation. Under such conditions, 41.5% of the erythromycin-resistant transformants were contransformed with one of the cryptic plasmids in addition to pHV1301. Indicator plasmid pHV1301 was later spontaneously segregated from doubly transformed cells. This protocol should be very useful for constructing lactic streptococcal strains bearing any phenotypically cryptic plasmid.     

Phytoplankton population dynamics of a small reservoir: physical/biological coupling and the time scales of community change

This paper describes the day-to-day changes in species compositionthat resulted from intermittent wind mixing of the surface watersof a small reservoir. Two major scales of community change weredetected: a short-term (>1 day) scale associated with theredistribution of cells within the basin, and a 5–14-dayscale associated with growth responses. The physical scalesof change were found to be almost identical to the biologicalscales: wind stress caused changes in the temperature gradientof surface waters at scales of a day, and major vertical mixingevents occurred at scales of 10–14 days. The presenceof buoyant species ensured that rapid advection of populationsfollowed wind events. Community change was a function of bothadvection and growth, so that both real and apparent changesin abundance occurred. The observed seasonal succession wasboth a true succession and a changing sequence of populationsdependent on horizontal advection of water within the basin.Consistent stratification was present throughout the summerperiod and phytoplankton diversity was low. Even so, the seasonalsuccession was best described as a series of allogenic perturbationsfollowed by biological restructuring of the community. Dailysampling was necessary to document fully the mechanisms drivingthe seasonal succession of species.     

Electro-fusion of mesophyll protoplasts ofAvena sativa

In order to assay the viability of electrically fused mesophyll protoplasts ofAvena sativa a technique was developed to determine adenylate levels in single protoplasts and fusion products. The results demonstrate that the intracellular ATP/ADP ratios are identical before and after fusion (values between 1.4 and 1.8) and that the time of the rounding up process is directly related to the ATP level of the hybrid. This was shown by the manipulation of the intracellular ATP/ADP ratio in the light using different effectors. Hybrids with an ATP/ADP ratio of 2.3 needed 54 s to round up completely; in the presence of antimycin (inhibition of both oxidative and light-dependent cyclic electron flow: ATP/ADP=1.1) or dibromothymoquinone (plastoquinone antagonist: ATP/ADP=1.0) the time for rounding up was slightly increased (64 s and 76 s respectively), whereas after preincubation with antimycin, dichlorophenyldimethylurea (inhibition of oxidative and light-dependent electron flow) or uncouplers (ATP/ADP=0.19–0.32) this process needed 128–153s for completion. These results are discussed in relation to the viability of electrically induced fusion products and to energy-dependent events involved in the process of fusion.