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.     

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.     

Regulatory factors produced by lymphocytes. I. The occurrence of multiple alpha-lymphotoxins associated with ribonuclease activity.

Supernatant fluids of mitogen-activated human tonsil lymphocytes contain large amounts of a factor toxic to mouse L cells. This substance, with a m.w. of 80,000 +/- 5,000 daltons, is called alpha-lymphotoxin (alpha-LT), to differentiate it from another toxin elaborated by mitogen activated human blood lymphocytes, called beta-lymphotoxin (beta-LT), which differs from alpha-LT in size (45,000 +/- 5,000 daltons), antigenicity, and stability. Further purification of alpha-LT by sequential phosphocellulose and DEAE-cellulose chromatography and polyacrylamide gel electrophoresis (PAGE) identifies a series of cytotoxins differing in ion exchange characteristics and electrophoretic mobilities. The three PAGE fractions (PAGE Ia, Ib and II), recovered in 2, 4.6, and 21% yield from the starting serum-free culture supernatant, represent purifications of 24-, 24- and 1851-fold, respectively. Each cytotoxic fraction has a ribonuclease activity. Comparison of RNase and mouse L cell cytotoxic activities of the three alpha-LT fractions shows that both activities for all three fractions have a similar temperature stability pattern and that both are similarly inhibited by DNA, single strand forms better than double strands, by glycerol in 5 to 20% concentration, and by protein denaturing reagents. These observations suggest, but do not prove, that mouse L cell toxicity and RNase activity are mediated by the same substance, which appears to occur in multiple or isozymic forms.     

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.

     

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.     

Copper resistance determinants in bacteria.

Copper is an essential trace element that is utilized in a number of oxygenases and electron transport proteins, but it is also a highly toxic heavy metal, against which all organisms must protect themselves. Known bacterial determinants of copper resistance are plasmid-encoded. The mechanisms which confer resistance must be integrated with the normal metabolism of copper. Different bacteria have adopted diverse strategies for copper resistance, and this review outlines what is known about bacterial copper resistance mechanisms and their genetic regulation.     

Fertility after bilateral cryptorchidism. Evaluation by paternity, hormone, and semen data.

PURPOSE: Evaluation of the fertility of a cohort of formerly bilaterally cryptorchid men in comparison with a group of formerly unilaterally cryptorchid men, and a group of control men. MATERIALS AND METHODS: Using a detailed questionnaire concerning paternity and factors related to paternity, a cohort of formerly bilateral cryptorchid men were studied and compared with men who had undergone orchiopexy for unilateral cryptorchidism, and a group of control men. All study subjects had had surgery at the Children's Hospital of Pittsburgh, Pittsburgh, Pa., between 1955 and 1975. A subset of the full cohort underwent clinical evaluation that included a physical examination, serum hormonal determination and semen analyses. RESULTS: Paternity rates are significantly lower among the formerly bilaterally cryptorchid men who have attempted to father a child (65.3%) as compared to the formerly unilaterally cryptorchid (89.7%; p < 0.001) and control men (93.2%; p < 0.001). Differences in the ability to father children are also apparent when semen and hormone levels are compared between the three groups. The bilateral group has significantly lower sperm density and inhibin B levels, and higher FSH and LH levels, than the unilateral and control groups. CONCLUSIONS: Men born with bilateral cryptorchidism have severely compromised fertility in adulthood. This reduction in fertility is clearly shown in comparisons of both paternity rates, and in semen and hormone analyses, between the formerly bilateral, formerly unilateral, and control groups.