Control of the antibody response of C57BL/6 Lyt-congenic strains to the Lyt-3.1 alloantigen by a gene linked to theLyt-2 locus

Congenic anti-Lyt-3.1 sera have recently been produced by immunizing B6-Lyt-2^a mice with thymocytes from either B6-Lyt-2^a, Lyt-3^a or B6-Lyt-2^a, Lyt-3^a, H-2^k mice (Boos et al. 1978). Surprisingly, mice of the congenic strain B6 failed to produce either anti-Lyt-2.1 or anti-Lyt-3.1 cytotoxic antibodies after identical immunizations. To determine the genetic basis for the difference in response to Lyt-3.1, (B6 × B6-Lyt-2^a)F_a mice and progeny of the backcross, (B6 × B6-Lyt-2^a)F₁ × B6-Lyt-2^a, were immunized with B6-Lyt-2^a, Lyt-3^a, H-2^k thymocytes. In addition, thymic biopsies of backcross progeny were performed and thymocytes tested for the Lyt-2.2 antigenic specificity. Results indicate that gene(s) governing the immune response to Lyt-3.1 is (are) linked to theLyt-2 locus, and that the responder allele (linked toLyt-2 ^a ) shows very poor penetrance in Lyt-2^a/Lyt-2^b mice.     

Amino-Terminal Charge Affects the Periplasmic Accumulation of Recombinant Heregulin/EGF Hybrids Exported Using theEscherichia coliAlkaline Phosphatase Signal Sequence

AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export.     

A histofluorescence study of events accompanying accumulation and migration of norepinephrine within locally cooled nerves

Glyoxylic acid was used to induce fluorescence in sections of rabbit sciatic nerve. In fresh nerves treated with this agent there were scattered finely beaded axons with a weak blue-green fluorescence. During local cooling, blue—green fluorescence accumulated steadily at the proximal boundary of the cooled region but never at its distal boundary. This accumulation gave rise to dilated axons that often swelled into brilliantly fluorescent balloon-like structures up to 10 μm in diameter. Axonal fluorescence was probably specific for norepinephrine, being enhanced by inhibition of the metabolism and diminished by inhibition of the synthesis or storage of this neurotransmitter. After local cooling of nerves for 1.5 hr, specific fluorescence was confined within 0.8 mm of the cooled region. Rewarming led to rapid removal of fluorescence from the cooled region and to disappearance of most of the balloon-like swellings. Simultaneously, rewarming caused brightly fluorescent fibers that were neither dilated nor swollen to appear in distal regions of nerve. As this wave of fluorescence migrated distally with increasing duration of rewarming, it was spread over increasingly broad regions of nerve, which suggests that axonal transport of norepinephrine may involve some kind of dispersive process.     

Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5′-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K_m for xylose of 66.7?mM and a specific activity of 1.41?μmol/min/mg. In comparison, the native xylose isomerase was found to have a K_m for xylose of 117.1?mM and a specific activity of 0.29?μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

EMBRYONIC GENETIC LOAD IN THE HIGHBUSH BLUEBERRY,VACCINIUM CORYMBOSUM (ERICACEAE)

Hand-pollinations of 28 autotetraploid V. corymbosum accessions from a single population resulted in lower self- than outcross seed set. Fertility varied widely, ranging from clones that were effectively female sterile to individuals with high seed yields in both matings. Self- and outcross fertility were highly correlated. A genetic load model was invoked to explain these phenomena. Reduced self-fertility was attributed to homozygosity for sublethal mutations at loci controlling embryo development, or to loss of heterotic interactions at these loci. Near zero cross-fertility in some clones may be evidence of partially dominant mutational load. Estimates of the number of lethal equivalents per zygote carried by individuals in this population ranged from 2.2 to 20.4, with a mean of 9.6. Embryonic genetic load at the individual level was significantly correlated with heterozygosity at nine enzyme loci. Low pollen viability and reduced receptivity to pollen from any source were also noted in the low fertility genotypes. It is suggested that gametic, gametophytic, and embryonic development are symptomatic of the amount of genetic load carried by individuals.     

Parasympathetic innervation of canine tracheal smooth muscle

To investigate whether efferent parasympathetic fibers to the trachealsmooth muscle course through the pararecurrent nerve rather than therecurrent or the superior laryngeal nerve, we stimulated all threenerves in anesthetized dogs. We also recorded the pararecurrentnerve activity response to bronchoconstrictor stimuli and compared itwith pressure changes inside a saline-filled cuff of an endotrachealtube. Electrical stimulation (30 s, 100 Hz, 0.1 ms, 10 mA) increasedtracheal cuff pressure by 21.0 ± 3.2 and 1.3 ± 0.7 cmH₂O for the pararecurrent and the recurrent laryngealnerve, respectively. Stimulation of the superior laryngeal nerveincreased tracheal cuff pressure before, but not after, sectioning ofthe ramus anastomoticus, which connects it to the pararecurrent nerve.Intravenous administration of sodium cyanide increased pararecurrentnerve activity by 208 ± 51% and tracheal cuff pressure by14.4 ± 3.5 cmH₂O. Elevation of end-tidalPCO₂ to 50 Torr increased pararecurrent nerveactivity by 49 ± 19% and tracheal cuff pressure by 8.4 ± 3.6 cmH₂O. Further elevation to 60 Torr increasedpararecurrent nerve activity by 101 ± 33% and tracheal cuffpressure by 11.3 ± 2.9 cmH₂O. These results lead usto the conclusion that parasympathetic efferent fibers reach the smoothmuscle of the canine trachea via the pararecurrent nerve.

     

Serologic evidence of a Rickettsia akari-like infection among wild-caught rodents in Orange County and humans in Los Angeles County, California.

We detected antibodies reactive with Rickettsia akari, the etiologic agent of rickettsialpox in humans and in 83 of 359 (23%) rodents belonging to several species, collected in Orange County, CA. Reciprocal antibody titers >1:16 to R. akari were detected in native mice and rats (Peromyscus maniculatus, P. eremicus, and Neotoma fuscipes) and in Old World mice and rats (Mus musculus, Rattus rattus, and R. norvegicus), representing the first time that antibodies reactive with this agent have been detected in four of these species and the first report of these antibodies in rodents and humans west of the Mississippi River. We then tested serum samples from individuals who used a free clinic in downtown Los Angeles and found that 25 of 299 (8%) of these individuals had antibody titers >1:64 to R. akari. Serologic evidence suggested that R. akari or a closely related rickettsia is prevalent among several rodent species at these localities and that infection spills over into certain segments of the human population. Isolation or molecular confirmation of the agent is needed to conclusively state that R. akari is the etiologic agent infecting these rodents.     

Ectopic Expression of the Tetratricopeptide Repeat Domain of SPINDLY Causes Defects in Gibberellin Response

The SPINDLY (SPY) protein of Arabidopsis is a negative regulator of gibberellin (GA) response. The SPY protein has 10 copies of the tetratricopeptide repeat (TPR) at the N terminus. TPR motifs function as protein-protein interaction domains. Several spy alleles are affected only in the TPR region suggesting that protein-protein interactions mediated by this domain are important for proper GA signaling. We have used a reverse genetics approach to further investigate the role of the TPR domain. The TPR domain of SPY was overexpressed in wild-type, gai, and spy plants. Expression of the TPR domain alone is not sufficient to rescue spy mutants. Expression of the TPR domain in a wild-type background produces phenotypes similar to those caused by loss-of-function spy mutants including resistance to GA biosynthesis inhibitors, short hypocotyl length, and early flowering. The dwarfing of the floral shoot internodes caused by the gai mutation was suppressed by expression of the TRP domain. Expression of the TPR domain had no effect on the abundance of endogenous SPY mRNA. The TPR domain was found to interact with SPY both in vitro and in yeast two-hybrid assays. These data indicate that the TPR domain of SPY can participate in protein-protein interactions and that these interactions are important for the proper functioning of SPY.