Cleavage of proteoglycan aggregate by leucocyte elastase.

The partial degradation of proteoglycan aggregate by human leucocyte elastase yielded products that banded with Mr 190,000, 140,000, 88,000, and 71,000 when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Analysis of these bands revealed that the 190,000- and 140,000-Da bands contained chondroitin and keratan sulfate stubs and had N-terminal amino acid sequences corresponding to a sequence starting at residue 398 of the core protein of rat or human aggrecan. With increased time of digestion, the staining intensities of the 190,000-, 140,000-, and 88,000-Da bands decreased relative to the 71,000-Da band. Analysis of the 88,000- and 71,000-Da bands showed that they contained peptides substituted only with keratan sulfate stubs and that each band contained two peptides with different N-terminal sequences. One of these corresponded to a sequence that started at residue 398 of rat or human aggrecan and the other to the N-terminal sequence of bovine aggrecan. Under conditions of complete digestion, bands of 71,000 and 56,000 Da which contained only keratan sulfate stubs were observed on SDS-polyacrylamide electrophoresis. The 71,000-Da band was shown to have a single sequence similar to that starting at residue 398 of human and rat aggrecan and thus represents the globular domain 2 (G2) of the core protein of aggrecan. The 56,000-Da band was shown to have a sequence similar to that of the N-terminal sequence of bovine aggrecan indicating that this peptide corresponds to the globular domain 1 (G1) of the molecule. These results suggest that leucocyte elastase cleaves the core protein of aggrecan between valine 397 and isoleucine 398, which are located in the interglobular domain linking the G1 and G2 domains of the core protein of aggrecan. Further digestion of the proteoglycan aggregate with elastase resulted in the cleavage of the core protein within the chondroitin sulfate attachment domains.     

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.

     

FMRFamide-like Peptides in the Nervous and Endocrine Systems of the Digenean Helminth Echinostoma liei

FMRFamide-like immunoreactivity has been demonstrated in the digenean trematode Echinostoma liei. The functions of FMRFamide-like substances appear to be many and varied within the invertebrates, where they are involved in neurotransmission, cardiovascular regulation, muscular contraction and/or relaxation, and in co-ordination of growth and maturation. It is clearly indicated that FMRFamide-like substances function as neurotransmitter/neuromodulator in E. liei by the abundance of positively stained nerve fibres and perikarya seen throughout the CNS and PNS. A single endocrine-like cell also showing FMRFamide-like immunoreactivity is situated within the muscular cirrus pouch.     

Effect of dietary copper sulfate, Aureo SP250, or clinoptilolite on ureolytic bacteria found in the pig large intestine

The predominant ureolytic bacteria in the pig large intestine were determined while growing pigs were fed a basal diet or basal diet plus copper sulfate, Aureo SP250, or clinoptilolite. Fecal samples were collected from four pigs fed each diet at 3, 9, and 14 weeks and analyzed for total colony counts and percent ureolytic bacteria. Fecal urease activity, ammonia nitrogen, and identity of the ureolytic bacteria were determined at 14 weeks. Copper sulfate and Aureo SP250 reduced the number of ureolytic organisms, with a marked decrease occurring in the Streptococcus spp., which made up 74% of the ureolytic isolates from the pigs on the basal diet. Other ureolytic species detected at lower concentrations were Staphylococcus spp., Selenomonas ruminantium, Bacteroides multiacidus, and Eubacterium limosum. Copper sulfate also reduced fecal urease activity (P less than 0.10). Fecal ammonia concentrations were not different between pigs fed the various diets. These data suggest that the streptococci are the most numerous ureolytic species in the pig intestinal tract and are significantly reduced by copper sulfate and Aureo SP250; however, only copper sulfate reduced intestinal urease activity.     

Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins

Specific antisera were prepared to the inclusion body protein (gene VI product) and the gene I product of cauliflower mosaic virus (CaMV). Translational fusions between the lacZ gene and gene VI or gene I were constructed by cloning the relevant DNA fragments into the expression vectors pUR290, pUR291 or pUR292. Large amounts of fusion protein were synthesized when the inserted DNA fragment was in frame with the lacZ gene of the expression vector. These fusion proteins were used to raise specific antisera to gene VI and gene I proteins of CaMV. Antiserum to the gene VI product detected a range of proteins in crude extracts and in a subcellular fraction enriched for virus inclusion bodies. This range of proteins was further shown to be related to gene VI by Staphylococcus aureus V8 partial proteolysis. Antiserum to the gene I product detected viral specific proteins of 46, 42 and 38 K in preparations of CaMV replication complexes from infected plants but not in any other subcellular fraction.