Human μ-opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous G_(i/o) proteins

Human μ-opioid receptor （HμOR） with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl （Ohm） were 9.1± 0.7 and 6.52±0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by μ-selective agonists [D-Ala2], N-methylPhe4, glyol5]enkephalin （DAGO）, Ohm, and morphine, but neither by δ nor by K selective agonist. Na+ （100 mM） and GTP （50 μM） could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed HμOR. μ-selective agonists DAGO and Ohm effectively stimulated [35S]GTPγS binding (EC50 = 2.7nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin （PTX）. These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.

Lateral olfactory tract usher substance(LOTUS) protein,an endogenous Nogo receptor antagonist,converts a non-permissive to permissive brain environment for axonal regrowth

It is well known that primates,including humans,hardly recover motor function after spinal cord injury（SCI）when compared with non-primate mammals such as rodents.This limited functional recovery is in part due to a non-permissive environment of the central nervous system（CNS）inhibiting axonal regrowth.

Endogenous mucin conveyed to the mucosa with microbes can assure lumen fermentation and large intestinal security-swine versus fowl

Endogenous protein leaving the ileum largely consists of accrued mucins from the upper gastrointestinal tract(GIT)that had resisted digestion.The amounts released rely on their mucosal generation during enteral feeding which vary with age as well as diet.These digestion resistant proteins of endogenous origin continue to be unavailable in the large intestine,whereas those of dietary origin provide amino acids that largely support the existing microbial population while denying limited amounts for absorption.Other mucins pre-exist within the large intestine as two layers at the lumen surface.A loose layer harboring a diverse microbial population is superimposed on the unstirred water layer(USWL)which simultaneously acts as an obstacle to microbes at the loose layer while performing as a molecular sieve for nutrients.The USWL is formed through interplay between enterocyte and goblet cells;however,the basis for presence of the loose layer is elusive.Large intestinal fermentation predominates within the colon of swine,whereas fowl employ their ceca.Motility within the colon of swine segregates fine materials into haustrae out-pocketings that parallel their placement within the ceca of fowl.Viscous mucins from small intestinal endogenous losses may envelop microbes within the large intestinal lumen to present successive adherents on the USWL that assemble its loose layer.The loose layer continually functions as a microbial reservoir in support of lumen fermentation.Microbial catabolism of mucin within the loose layer is known to be slow,but its proximity to the enterocyte is of advantage to enterocyte absorption with by-product amino acids fostering the USWL.

Basis for the diversity and extent in loss of digestible nutrients created by dietary phytin:Emphasis on fowl and swine

Phytin is the Ca^(2+)-Mg^(2+)-K^(+)salt of phytic acid that is created and deposited in the aleurone layer and/or germ of grains and legumes.Its high presence in feedstuffs for fowl and swine diets results in it being a universal and significant impediment to optimum performance.Phytin impairs gastrointestinal recovery of a wide array of nutrients,the effect varying with the nutrient concerned.On exposure to low pH during gastric digestion,phytin dissociates into phytic acid and solubilized Ca^(2+).Even at low gastric pH,phytic acid is negatively charged which forms the basis of its anti-nutritive behavior.Pepsinogen has extensive basic amino acids on its activation peptide that are presented as cations at low pH which are targeted by pepsin for activation.Partially crystalized Ca^(2+)near the enzyme?s active site further stabilizes its newly formed structure.Thus,phytic acid appears to interfere with gastric digestion by several mechanisms;interfering with pepsinogen activation by binding to the polypeptide?s basic amino acids;coordinating free Ca^(2+),destabilizing pepsin;binding some dietary proteins directly,further compromising gastric proteolysis.Upon digesta attaining neutrality in the duodenum,Ca^(2+)and other cations rebind with accessible anions,phytic acid being a significant contender.Phytate not only binds free cations but can also strip them from enzymes(e.g.Ca^(2+),Zn2+)which reduces their structural resistance to autolysis and ability as co-factors(e.g.Zn2+)to increase enzyme activity.Goblet cells initially employ Ca^(2+)as an electronic shield between mucin layers enabling granule formation and cell storage.After mucin granule release,Ca^(2+)is progressively displaced by Na^(+)to free the viscous mucins enabling its translocation.Mucin entangles with the glycocalyx of adjacent enterocytes thereby constructing the unstirred water layer(USWL).Excessive removal of Ca^(2+)from mucin by phytic acid increases its fluidity facilitating its loss from the USWL with its associated Na^(+).This partly explains increased mucin and Na^(+)losses noted with high phytate diets.This review suggests that phytic acid binding of Ca^(2+)and less so Zn2+is the basis for the diversity in nutrient losses encountered and that such losses are in proportion to dietary phytate content.

Large intestinal dynamics differ between fowl and swine:Anatomical modifications,microbial collaboration,and digestive advantages from fibrolytic enzymes

The large intestinal systems of fowl and swine recover nutrients from ileal indigesta by a strategically different manner.Indigesta with fowl enter a short colon where retro-peristalsis using urine from the urodeum carries small particulates and solutes into both ceca while coarse materials collect in the cloaca.Fowl repetitively add fine and soluble materials into both ceca to continue fermentation until complexity of the remainder exceeds microbial action,then contents apart from faeces are entirely evacuated.Indigesta with swine initially enter a short cecum followed by a lengthy progression through to the rectal ampulla.Wall out-pocketings of circular muscle or haustrae occur throughout the length of the pig's cecum and helicoidal colon.Each pocket carries contents acquired earlier in the cecum.Motility collects fines and solutes into haustrae during their progression through the colon whereas coarse particulates assemble in the core.Haustrae contents continually ferment during movement to the distal colon with resulting volatile fatty acids(VFA)and electrolytes being absorbed.Mucin loosely covers the lumen surface in caeca as well as helicoidal colon that may capture microbes from active intestinal contents as well as release others to sustain fermentation.The microbial community continually modifies to accommodate fibre complexity as encountered.Resistant starches(RS)and simple oligosaccharides rapidly ferment to yield VFA while encouraging butyric acid in the cecum and anterior colon,whereas non-starch polysaccharides(NSP)complexity requires extended durations through the remaining colon that enhance acetic acid.Residual fibre eventually results in undue complexity for fermentation and consolidates at termination of the colon.These compact pellets are placed on core contents to form faeces having a nodular surface.Acetic,propionic,and butyric acids represent the bulk of VFA and are derived from non-digestible carbohydrates.Fibrolytic enzymes,when supplemented to feed,may increase the proportion of oligosaccharides and simpler NSP to further the rate as well as extent of fermentation.Active absorption of VFA by mucosal enterocytes employs its ionized form together with Na^(+),whereas direct membrane passage occurs when non-dissociated.Most absorbed VFA favour use by the host with a portion of butyric acid together with by-products from protein digestion being retained to reform mucin and sustain mucosal integrity.