Separation of galactolipid molecular species by high-performance liquid chromatography

A technique for the separation, detection, and quantification of molecular species of monogalactosyldiglyceride and digalactosyldiglyceride is described. Use of the technique to analyze the molecular species composition of the galactolipids isolated from Dunaliella salina chloroplasts is presented. The results indicate that the respective compositions of the two lipids are quite different. This suggests that the enzymes involved in galactolipid metabolism are very specific with respect to acyl chain composition and pairing, or that extensive retailoring of constituent acyl chains occurs following formation of digalactosyldiglyceride from monogalactosyldiglyceride.     

Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster

A plasmid library of Acinetobacter calcoaceticus HindIII fragments was constructed, and clones that complemented an Escherichia coli pabA mutant were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were obtained. We infer that complementation of E. coli pabA mutants was the result of the expression of the amphibolic anthranilate- synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that the plasmid insert carried the entire trpGDC gene cluster. In E. coli minicells, the plasmid insert directed the synthesis of polypeptides of 44,000, 33,000, and 20,000 daltons, molecular masses that are consistent with the reported molecular masses of phosphoribosylanthranilate transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase component II, respectively. A 3,105- bp nucleotide sequence was determined. Comparison of the A. calcoaceticus trpGDC sequences with other known trp gene sequences has allowed insight into (1) the evolution of the amphibolic trpG gene, (2) varied strategies for coordinate expression of trp genes, and (3) mechanisms of gene fusions in the trp operon.      

Heteronuclear 2D NMR studies of an engineered insulin monomer: assignment and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design

Insulin provides an important model for the application of genetic engineering to rational protein design and has been well characterized in the crystal state. However, self-association of insulin in solution has precluded complementary 2D NMR study under physiological conditions. We demonstrate here that such limitations may be circumvented by the use of a monomeric analogue that contains three amino acid substitutions on the protein surface (HisB10----Asp, ProB28----Lys, and LysB29----Pro); this analogue (designated DKP-insulin) retains native receptor-binding potency. Comparative 1H NMR studies of native human insulin and a series of three related analogues--(i) the singly substituted analogue [HisB10----Asp], (ii) the doubly substituted analogue [ProB28----Lys; LysB29----Pro], and (iii) DKP-insulin--demonstrate progressive reduction in concentration-dependent line-broadening in accord with the results of analytical ultracentrifugation. Extensive nonlocal interactions are observed in the NOESY spectrum of DKP-insulin, indicating that this analogue adopts a compact and stably folded structure as a monomer in overall accord with crystal models. Site-specific 2H and 13C isotopic labels are introduced by semisynthesis as probes for the structure and dynamics of the receptor-binding surface. These studies confirm and extend under physiological conditions the results of a previous 2D NMR analysis of native insulin in 20% acetic acid [Hua, Q. X., & Weiss, M. A. (1991) Biochemistry 30, 5505-5515]. Implications for the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogues.     

Computation of frequency-to-spatial transform by olfactory bulb glomeruli

A physiological simulation of 2.5% of the input and inhibitory neurons and 25% of the primary mitral/tufted cells in a single mammalian olfactory bulb glomerulus was constructed. This physiological simulation used the integrate-and-fire paradigm with realistic activation curves and synaptic delays. The dendritic integration incorporated non-linear interactive effects of individual cell excitatory and inhibitory post-synaptic potentials (PSPs) from both axodendritic and dendrodendritic synaptic contacts. Refractory periods for granule-cell inhibition of mitral/tufted cell activity lead to relatively fixed-frequency rhythmic activity in the glomerulus, independent of the input frequency from the olfactory nerve. Though the frequency of mitral/ tufted cell firing in bulb was approximately independent of input frequency, the number of cells active in the glomerulus was a roughly-linear function of input frequency to the glomerulus, indicating the mechanism's ability to function as a frequency-to-spatial encoder.     

Chromosome 1 Charcot-Marie-Tooth disease (CMT1B) locus in the Fcγ receptor gene region

Summary The Charcot-Marie-Tooth disease (hereditary motor and sensory neuropathy) loci have been reported to be on at least three chromosomes: 1 (CMT1B, HMSN1B), 17 (CMT1A), and X (CMTX). In this study multipoint linkage analysis of two Duffy-linked families given a combined LOD score of 8.65 to establish that the Duffy-linked CMT1B gene exists in the 18 centimorgan region between the antithrombin III gene and the Duffy/ sodium-potassium ATPase loci. The simultaneous segregation of polymorphisms near the CMT1A locus on chromosome 17 excludes linkage to this chromosome region in both families. Polymorphic sites that flank the CMT1B gene have been subchromosomally localized to the proximal chromosome-1 long arm (1q21.21q25) by spot blot analysis of sorted chromosomes, polymorphic deletion analysis, in situ hybridization, and multipoint linkage analysis.     

Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.

We have constructed hybrid retrovirus packaging cell lines that express the gibbon ape leukemia virus env and the Moloney murine leukemia virus gag-pol proteins. These cells were used to produce a retrovirus vector at over 10(6) CFU/ml, with a host range that included rat, hamster, bovine, cat, dog, monkey, and human cells. The gag-pol and env expression plasmids were separately transfected to reduce the potential for helper virus production, which was not observed. The NIH 3T3 mouse cells from which the packaging lines were made are not infectable by gibbon ape leukemia virus; thus, the generation and spread of possible recombinant viruses in the packaging cells is greatly reduced. These simian virus-based packaging cells extend the host range of currently available murine and avian packaging cells and should be useful for efficient gene transfer into higher mammals.     

Evidence for leucine zipper motif in lactose repressor protein

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.     

Phenotypic and functional analysis of the cellular response in regional lymphoid tissue during an acute virus infection

A phenotypic and functional analysis has been made of the cellular response in regional lymphoid tissue of C57BL/6J mice infected with lymphocytic choriomeningitis virus. Massive recruitment of nondividing cells occurred from 3 days after infection, with total numbers of CD8+ T lymphocytes, B220+ B cells, and Thy-1- B220- null cells being high from day 4 to day 6. In contrast, the peak counts for CD4+ T cells were recorded on day 4 and declined dramatically thereafter. Enhanced expression of IL-2R and Ly-24, both of which can be regarded as T cell activation markers, was found for both the CD4+ and the CD8+ subsets, being most prominent for the CD8+ T cells on day 6. Evidence of T cell proliferation was not recognized until days 5 and 6, coincident with enhanced responsiveness of the lymphocytes to rIL-2 and the development of virus-specific cytotoxic activity. Elimination of the CD4+ T cells by treatment of mice with mAb did not modify either the pathogenesis of lymphocytic choriomeningitis, or the expression of activation markers on the CD8+ T cells which are known to be the key effectors in this disease. Thus, the pattern of responsiveness for the CD8+ population is of recruitment to the lymph node, progressive increase in the expression of activation markers and enhanced sensitivity to rIL-2, with late proliferation and generation of cytotoxic activity. This model provides a system for the rigorous in vivo analysis of parameters influencing lymphocyte differentiation and activation in a virus infection.     

Geographic differences for delay of sexual maturation in Peromyscus leucopus: effects of photoperiod, pinealectomy, and melatonin

Effects of short-day photoperiod, pinealectomy, and melatonin on sexual maturation were tested in Peromyscus leucopus from either Connecticut (CT) or Georgia (GA). Laboratory reared-stocks from CT and GA were exposed to short daylength (photoperiod) from birth or 25 days of age. At 12 wk of age, delay in sexual maturation was indicated in most CT mice by decreased testis length, combined testes weight, and seminal vesicle weight. Conversely, GA animals did not delay sexual maturation when exposed to short-day photoperiod from either birth or 25 days of age. These results indicate that responses to short daylengths differ for juvenile CT and GA populations. In a second experiment, pinealectomized or sham-operated CT males were exposed to short-day (9L:15D) or long-day (16L:8D) photoperiod from birth. Pinealectomy blocked the effect of short daylength on reproduction. Therefore, the pineal must be involved in the delay of sexual maturation observed for short-day CT mice. The effects of melatonin, a pineal gland hormone, were tested with chronic s.c. implants or daily injections. In CT mice given either melatonin implants or afternoon injections, sexual maturation was delayed. GA mice were insensitive to all melatonin treatments. Further, no differences in circadian organization (phase angle, duration of activity, period under constant dark) between GA and CT animals were apparent. Collectively, these studies indicate that melatonin is involved in the mechanism responsible for delay of sexual maturation in CT mice. Short-day insensitivity of GA Peromyscus leucopus probably results from a deficiency in the melatonin effector pathway and is not due to a disruption of circadian organization.