Water transport and estimated transmembrane potential during freezing of mouse oocytes

Summary Steady-state currents at hyperpolarized membrane potentials were studied in the homologous giant neurons, LP1 and R2, ofAplysia using two-electrode voltage clamp. Nearly half of the steady-state current at voltages more hyperpolarized than –70 mV had characteristics similar to the inwardly rectifying potassium current (I _R) described previously inAplysia neurons. The pharmacological agents 4-bromophenacylbromide, indomethacin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate were found to modulateI _R.I _R was stimulated with BPB and indomethacin and inhibited with TPA. These agents alteredI _R by a mechanism independent ofcAMP, which can also modulateI _R. The effects of these modulators are consistent with their actions on arachidonic acid (AA) metabolism inAplysia nervous system, suggesting AA may constitutively inhibitI _R. When ganglia were perfused for 12 hr with medium containing BSA to absorb extracellular fatty acids,I _R was increased nearly twofold. This increase was partially inhibited by addition of AA to the perfusion medium, and completely inhibited by pretreatment of ganglia with BPB. Although no direct effect of shortterm exposure to exogenous AA was observed, long term exposure to exogenous AA and several other unsaturated fatty acids was accompanied by a decrease inI _R.     

Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354

The rates of breakdown and renewal of individual lipids in cultures of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354 were investigated by means of a pulse labelling technique using palmitate-1-¹⁴C. The results indicated that in growing cultures of both strains phospholipids were broken down, and cardiolipin had a very rapid turnover. In chase experiments, almost 45% and 40% of the radioactivity of this component were lost respectively from M. smegmatis and M. phlei during one generation time of the cell. The other two major components, phosphatidyl ethanolamine and phosphatidylinositol mannosides showed relatively low turnover. The loss of radioactivity from phosphatidylinositol mannosides was greater in M. phlei than in M. smegmatis but the loss of radioactivity from phosphatidyl ethanolamine was higher in M. smegmatis. The pattern of loss of radioactivity from lipids was almost the same in both strains, the difference being only in the extent of loss. The differences in the cellular localization of the phospholipids indicate their different roles within the cell. Results obtained with the glyceride fraction indicated a very rapid turnover of triglycerides in both strains.Abbreviations CL Cardiolipin - PE Phosphatidyl ethanolamine - PIMx phosphatidylinositol mannosides - PIM₂A phosphatidylinositol dimannoside tetra acylated - PIM₂B phosphatidylinositol dimannoside tri acylated - PIM₅ phosphatidylinositol pentamannoside tetra acylated     

Cell cycle-dependent changes in arachidonic acid and glycerol metabolism in Swiss 3T3 cells stimulated by platelet-derived growth factor

Quiescent Swiss 3T3 cells stimulated to divide by human platelet-derived growth factor (PDGF) were used to investigate cell cycle-dependent changes in arachidonic acid, stearic acid, and glycerol metabolism. PDGF at 12 ng/ml stimulated incorporation of labeled arachidonic and stearic acid into phosphatidic acid and phosphatidylinositol within 60 min. With similar kinetics PDGF stimulated glycerol incorporation into phosphatidic acid and phosphatidylinositol indicating early growth factor-dependent stimulation of de novo phosphatidylinositol synthesis. This early effect of PDGF was specific for the phosphatidylinositol synthesis pathway since no comparable changes were noted in other glycerolipids. After a lag of 4-6 h, PDGF strongly stimulated arachidonic acid incorporation into triacylglycerol: at 6 h, arachidonate radioactivity in triacylglycerol exceeded that in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. This effect of PDGF was not associated with de novo triacylglycerol synthesis since no increase in the rate of glycerol incorporation into this lipid was noted. Finally, PDGF stimulated incorporation of glycerol into all major phospholipids and triacylglycerol during S-phase. These results disclose three novel effects of PDGF on glycerolipid metabolism in Swiss 3T3 cells: 1) early selective activation of the phosphatidylinositol synthesis pathway; 2) delayed strong stimulation of arachidonic acid incorporation into triacylglycerol; and 3) late induction of de novo phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol synthesis. These PDGF effects are likely to play important roles in phosphatidylinositol metabolism, membrane biosynthesis, and fatty acid turnover in rapidly growing cells.     

Ca2(+)-dependence of arachidonic acid redistribution among phospholipids of cultured mouse keratinocytes

Mouse keratinocytes cultured in a medium containing less than 0.1 mM Ca2+ (low Ca2+) incorporated [1-14C]arachidonic acid (AA) into phospholipids by kinetics including; (i) a rapid labelling of phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and both acid-stable and alkenylacyl forms of phosphatidylcholine (PtdCho); and (ii) a slow but long-lasting radiolabel incorporation into both acid-stable and alkenylacyl forms of phosphatidylethanolamine (PtdEtn), partly associated with a net radioactivity loss from acid stable-PtdCho. Under low Ca2+ conditions no radioactivity transfer apparently occurred between PtdIns and other phospholipid classes. When cells were prelabelled for 24 h with [1-14C]AA and reincubated in label-free medium containing 1.2 mM Ca2+ (normal Ca2+), an early and extensive loss of radioactivity from PtdIns was observed, reasonably in connection with Ca2+ stimulation of phosphoinositide turnover. Cell shift to normal Ca2+ did not result in an increased synthesis of labelled eicosanoids, but was consistent with an increase of radioactivity incorporation into diacylglycerol (DAG) and with a complex pattern of [1-14C]AA redistribution, eventually leading to a marked radioactivity incorporation into acid stable-PtdEtn (but not into alkenylacyl-PtdEtn) and to a labelling decrease of acid stable-PtdCho. The possible mechanisms driving AA recycling after cell shift to normal Ca2+ are discussed.     

Secretagogue-responsive and -unresponsive pools of phosphatidylinositol in pancreatic islets

The effect of glucose on phosphatidylinositol turnover was studied. Phosphatidylinositol of rat pancreatic islets was labeled with myo[2-3H]inositol in the presence of various secretagogues (16.7 mM D-glucose, 22 mM D-mannose, 20 mM D-glyceraldehyde) and nonsecretagogues (3.3 mM D-glucose, 20 mM pyruvate, 16.7 mM D-galactose, 16.7 mM L-glucose). Upon subsequent stimulation with 16.7 mM D-glucose, only the islets that were labeled in the presence of secretagogues showed a loss of radioactivity from phosphatidylinositol. No loss of radioactivity from phosphatidylinositol occurred in the presence of 3.3 mM D-glucose even after labeling in the presence of secretagogues. A comparison of the subcellular distribution of labeled phosphatidylinositol in islets before and after stimulation with insulinotropic glucose revealed a loss of radioactivity from the plasma membrane fraction as judged by subcellular fractionation with a sucrose gradient. These results support a hypothesis advanced previously that pancreatic islets contain a unique pool of phosphatidylinositol that undergoes rapid turnover only in the presence of insulinotropic concentrations of D-glucose or other secretagogues [R. S. Rana, R. J. Mertz, A. Kowlura, J. F. Dixon, L. E. Hokin, and M. J. MacDonald (1985) J. Biol. Chem. 260, 7861-7867]. On the basis of the subcellular fractionation studies reported here, the secretagogue-responsive phosphatidylinositol pool appears to be located primarily in the plasma membrane of pancreatic islets.     

1,25-Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast-like cells

The effects of treatment with the osteotropic steroids 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 17β-estradiol, or dexamethasone on [1-¹⁴C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast-like (hOB) cells were investigated. Total uptake of [1-¹⁴C]AA was decreased in cells treated with dexamethasone when assayed after a 24-, 48-, or 96-h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48-h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1-¹⁴C]AA was diminished by 96-h treatment with 1,25(OH)₂D₃ (79 ± 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 ± 2% of control, P < 0.05). The 1,25(OH)₂D₃-dependent decrease in total uptake and in phosphatidylinositol incorporation of [1-¹⁴C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)₂D₃ was without effect on either total [1-¹⁴C]AA uptake or the specific [1-¹⁴C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)₂D₃ treatment decreased hOB cell uptake of [1-¹⁴C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)₂D₃-dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17β-Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.     

Chemotaxonomy of theSymphytum officinale agg. (Boraginaceae)

In a chemotaxonomic study of the genusSymphytum pyrrolizidine alkaloids and triterpenes were used as chemotaxonomical markers. A micro-extraction methods was developed for screening compounds of very small pieces of herbarium material. The occurrence of the pyrrolizidine alkaloids symphytine and (acetyl-)lycopsamine is very general forSymphytum taxa. Echimidine is present in someS. officinale L. plants and inS. tanaicense Steven. The triterpene isobauerenol is present inS. officinale, S. bohemicum Schmidt,S. tanaicense and inS. officinale var.lanceolatum Weinm. The chemotaxonomic hypothesis, proposed byGadella and collaborators, based on the presence of the triterpene isobauerenol inS. officinale and its absence inS. asperum Lepech. and the presence of the pyrrolizidine alkaloid echimidine inS. asperum and its absence inS. officinale, can no longer be applied absolutely to theS. officinale species complex. The pyrrolizidine alkaloid and triterpene pattern ofS. officinale (2n = 24) andS. bohemicum (2n = 24) is identical.S. bohemicum is morphologically, cytologically and phytochemically very similar toS. officinale. Furthermore, it readily crosses with the white flowered W. European diploids ofS. officinale. Therefore it seems likely that these two taxa are conspecific.S. tanaicense shows a pyrrolizidine alkaloid and triterpene pattern similar toS. officinale (2n = 40). Also on morphological and cytological grounds they are very similar. It seems highly probable thatS. tanaicense is conspecific withS. officinale (2n = 40) and represents an intraspecific variant only.S. officinale var.lanceolatum contained no pyrrolizidine alkaloids but did contain isobauerenol. This feature points to an origin fromS. officinale.     

The biosynthesis of indole-3-acetic acid byFrankia

Summary High perfomance liquid chromatography (HPLC) of the products of [5-³H] tryptophan metabolism byFrankia sp. Avc I1 indicates that small amounts of [³H] indole-3-acetic acid (IAA) are excreted into the growth medium.Frankia has a limited capacity for the catabolism of [2-¹⁴C]IAA and the product that accumulates is different from that detected inRhizobium japonicum cultures following inoculation with [2-¹⁴C]IAA. The data imply that the rate of turnover of IAA is much more rapid inRhizobium thanFrankia and that the two organisms employ different routes for the catabolism of IAA.     

Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identifiedHelix neurons in cell culture

Hemolymph of adultAplysia californica significantly affects neurite outgrowth of identified neurons of the land snailHelix pomatia. The metacerebral giant cell (MGC) and the motoneuron C3 from the cerebral ganglion and the neuron B2 from the buccal ganglion ofH. pomatia were isolated by enzymatic and mechanical dissociation and plated onto poly-l-lysine-coated dishes either containing culture medium conditioned byHelix ganglia, or pre-treated withAplysia hemolymph. To determine the extent of neuronal growth we measured the neurite elongation and the neuritic field of cultured neurons at different time points.Aplysia hemolymph enhances the extent and rate of linear outgrowth and the branching domain ofHelix neurons. With the hemolymph treatment the MGC neuron more consistently forms specific chemical synapses with its follower cell B2, and these connections are more effective than those established in the presence of the conditioned medium.     

Effects of insulin secretagogues and inhibitors on phospholipid metabolism in Langerhans' islets of rat pancreas

Glucose stimulation of [32P]-prelabelled pancreatic islets induced an increased incorporation of radioactivity into phosphatidylinositol. Glucagon and agents that increases cAMP in the islet did not affect phosphatidylinositol turnover, in spite of increased release of insulin. Furthermore, the potent inhibitor of insulin release, somatostatin did not alter phospholipid metabolism. Colchicine inhibited glucose-stimulated turnover of phosphatidylinositol as well as insulin release. These results may suggest that: (1) cAMP and phosphatidylinositol turnover are involved in different transmembrane control system for regulating insulin release; and (2) the function of microtubules modulate phospholipid metabolism.     

Formation of sperm cells inGalium mollugo (Rubiaceae),Trichodiadema setuliferum (Aizoaceae), andAvena sativa (Poaceae)

The formation of sperm cells has been examined ultrastructurally in the tricellular pollen grains ofGalium mollugo L. (Rubiaceae).Trichodiadema setuliferum Schwantes (Aizoaceae), andAvena sativa L. (Poaceae). After detachement from the intine the generative cell of all three species lies free within the vegetative cytoplasm. The two sperm cells are built inTrichodiadema andAvena by a single separating wall, while inGalium mollugo two independent walls are formed. However, both mechanisms separate the two male gametes completely.     

The high turnover rate of phosphatidylinositol in chicken heart ventricular cells during the earlier stages of development

The number of the chicken ventricular cells develops exponentially up to Day 12 in the developmental stages of embryos, after which the number gradualy decreases. The phosphatidylinositol metabolism at various stages in development (Days 5 to 21) has been studied. Ventricular cells were incubated in a physiological solution containing ³²P_i, or [1,3-³H]glycerol. Radioactivities incorporated into the phosphatidylinositol were estimated. The specific activity of [1,3-³H]glycerol, taken into phosphatidylinositol, at Day 12, was shown to be approximately equal to that of the other classes of phospholipids. However, the rate of labeling of ³²P_i into the phosphatidylinositol was extremely high in comparison with the other classes of phospholipids in the same ventricles. These results show that there is a rapid turnover of the phosphorylinositol moiety in the phosphatidylinositol in the earlier stages of development. This high turnover rate of the phosphatidylinositol was observed up to Day 12, after which it began to decrease. This turning point of the phosphatidylinositol metabolism coincided well with the decrease of the rate of cell proliferation. Therefore, this rapid turnover of phosphatidylinositol could have a specific functional role related to cell division. This rapid turnover of the phosphorylinositol moiety of the phosphatidylinositol associated with ventricular cell proliferation at different embryonic stages is reported for the first time.     

Ribosomal RNA genes structure in somePopulus spp. (Salicaceae) and their hybrids

The tandemly repeated multigene families encoding 18S and 25S rRNAs were studied at the restriction enzyme level inPopulus alba L.,Populus deltoides Bartr. exMarsh.,Populus trichocarpa Torr. & Gray and in the hybrids between the last two mentioned species. The analysis of single and double digestion with EcoRI, BamHI, XbaI, and SstI endonucleases showed the presence of single repetitive unit types of 12.25 and 11.75kb inP. alba andP. trichocarpa, respectively.P. deltoides showed two rDNA gene types having the same length (12.25Kb) but different nucleotide sequence in the IGS. The rDNAs genes ofP. deltoides andP. triochocarpa are inherited codominantly in their hybrids.     

Evidence for glucose-responsive and -unresponsive pools of phospholipid in pancreatic islets

The effect of glucose on the metabolism of phospholipids in pancreatic islets was studied with three radioactive phospholipid precursors, [32P]orthophosphate, [3H]myoinositol, and [3H]arachidonic acid, to determine the conditions necessary for studying the breakdown of prelabeled phospholipids. Islets were incubated in the presence of a radioactive precursor for 60 or 90 min and in the presence of either 3.3 or 16.7 mM glucose to prelabel phospholipids. To study the breakdown of prelabeled phospholipid, the unincorporated precursor was removed and the islets were reincubated for 15 or 20 min under conditions that either did or did not stimulate insulin release. Prelabeling in the presence of a noninsulinotropic concentration of glucose (3.3 mM) supported the incorporation of precursors into almost all islet phospholipids studied. Prelabeling in an insulinotropic concentration of glucose (16.7 mM) increased the incorporation of precursors into a number of phospholipids even more; and reincubation in 16.7 mM glucose caused a rapid loss of radioactivity from specific phospholipids (phosphatidylinositol and/or phosphatidylcholine, depending on the precursor). This breakdown was observed only when islets had been prelabeled in 16.7 mM glucose. The amount of radioactivity lost from phospholipid corresponded roughly to the additional amount incorporated during the prelabeling in the high concentration of glucose. Radioactivity in phospholipids in islets prelabeled in 3.3 mM glucose or in nonsecretagogue metabolic fuels, such as malate plus pyruvate, did not decrease when the islets were subsequently exposed to 16.7 mM glucose, nor did it decrease in 3.3 mM glucose when these islets had been prelabeled in 16.7 mM glucose. Glyceraldehyde, an insulin secretagogue, but not galactose or L-glucose which are not insulin secretagogues, stimulated phospholipid breakdown in islets that had been prelabeled in 16.7 mM glucose. Depriving islets of extracellular calcium, a condition that inhibits insulin release, inhibited phospholipid breakdown. The results suggest that pancreatic islets contain a glucose-responsive and a glucose-unresponsive phospholipid pool. The glucose-responsive pool becomes labeled and undergoes rapid turnover only under stimulatory conditions and may play a role in the stimulus-secretion coupling of insulin release.     

Measuring Brain Uptake and Incorporation into Brain Phosphatidylinositol of Plasma myo-[2H6]Inositol in Unanesthetized Rats: An Approach to Estimate In vivo Brain Phosphatidylinositol Turnover

The in vivo rate of turnover of phosphatidylinositol (PtdIns) in brain is not known. In brain, certain receptor-mediated signal transduction involves metabolism of PtdIns and a method to measure its turnover in awake animals is useful in studying the effect of lithium and other therapeutic agents. In a method described here, rats were infused subcutaneously with myo-[²H₆]inositol (Ins*) using an osmotic pump and, at 1 and 8 weeks, concentrations of free myo-inositol (Ins) and Ins* in plasma and brain were measured by GC-MS (chemical ionization). Also, PtdIns and PtdIns* together in brain were isolated, and Ins and Ins* from their headgroups were released enzymatically and specific activity of incorporated inositol was measured. The specific activity of inositol reached a steady state in plasma within 1 week of infusion, but not in brain even at 8 weeks. However, in brain, the specific activity of phosphatidylinositol was same as that of inositol at both time-points, suggestive of fast turnover of PtdIns. The animal experiment and the analytical methodology described here should be useful for measuring the rate of turnover of brain PtdIns in pathological and drug treatment conditions.     

13C and31P NMR spectroscopic studies on glucose metabolism inTribolium confusum

Nuclear magnetic resonance (NMR) technology was applied to study the glucose metabolism inTribolium confusum (Coleoptera).¹³C signals of D-(1-¹³C)glucose eaten by beetles were clearly detected in such metabolites of the glucose metabolism as glycogen, trehalose, triacylglycerol, alanine and proline by¹³C-NMR. After glucose feeding the³¹P-NMR spectra ofT. confusum showed the signal intensity increases in arginine-phosphate, sugar-phosphate and uridine diphosphoglucose. The results demonstrated the potential of NMR analysis for the study of glucose metabolism inT. confusum.     

Effect of Hypoxia and Dopamine on Arachidonic Acid Metabolism in Superior Cervical Ganglion

Superior cervical ganglion (SCG) may play a modulatory role on ventilatory control through its efferent sympathetic fibres, which innervate cells in the carotid bodies. In this study the in vivo effect of acute hypoxia versus normoxia on arachidonic acid (AA) metabolism was investigated in cat SCG. Using SCG homogenate AA was incorporated into glycerolipids of normoxic SCG in the following order: neutral glycerolipids > phosphatidylcholine (PtdCh) > phosphatidylinositol (PtdIns) > phosphatidylethanolamine (PtdE) > phosphatidylserine (PtdS) > and phosphatidic acid (PA). In vivo hypoxic treatment caused a significant decrease in incorporation of [1-¹⁴C]AA into PtdIns. Hypoxia had no significant effect on the level of AA radioactivity in diacylglycerol (DAG) as compared to control but significantly enhanced the level of arachidonoyl-CoA (AA-CoA) radioactivity. It was observed that dopamine (DA) one of the most important neurotransmitter in SCG decreases AA uptake into phospholipids of normoxic SCG. In normoxic SCG, DA significantly decreased, AA incorporation into PtdCh, PtdIns and DAG. Moreover, DA decreased the level of AA-CoA radioactivity. Hypoxia and dopamine has no effect on AA metabolism in medulla oblongata isolated from the same animals. These results indicate that arachidonic acid metabolism in SCG is sensitive to hypoxia and dopamine action. Moreover, these results indicate that hypoxia inhibits selectively AA incorporation on the level of acylCoA-lysophosphatidylinositol-acyltransferase.     

A reconsideration of the genusRabdosiella (Lamiaceae,Nepetoideae, Ocimeae)

The two generaPlectranthus andIsodon are compared and found to be very dissimilar.Isodon ist considered to be misplaced inOcimeae subtribePlectranthinae and apparently is more closely related to subtribeHyptidinae. The disjunct genusRabdosiella is compared to these two genera and regarded to be polyphyletic. The AfricanR. calycina (Benth.)Codd is returned toPlectranthus and calledP. calycinus Benth., while the AsianR. ternifolia (D. Don)Codd is placed inIsodon sect.Pyramidium and calledI. ternifolius (D. Don)Kudo.     

Production of α-amylase fromHalobacterium halobium

Maximum production of extracellular -amylase activity inHalobacterium halobium was at 40°C in a medium containing 25% (w/v) NaCl, 1% (w/v) soluble starch and 1% (w/v) peptone, in presence of 0.1mm ZnSO₄ after 5 days in shaking cultures. The amylase had optimal activity at pH 6.5 in the presence of 1 to 3% (w/v) NaCl at 53°C.S. Patel, N. Jain and D. Madamwar are with the Post Graduate Department of Biosciences, Sadar Patel University, Vallabh Vidyanagar-388120, India.     

Utilization of amino acids by bacteria from the pig small intestine

This study determined the utilization of amino acids (AA) by bacteria from the lumen of the pig small intestine. Digesta samples from different segments of the small intestine were inoculated into media containing 10 mmol/L each of select AA (l-lysine, l-threonine, l-arginine, l-glutamate, l-histidine, l-leucine, l-isoleucine, l-valine, l-proline, l-methionine, l-phenylalanine or l-tryptophan) and incubated for 24 h. The previous 24-h culture served as an inoculum for a subsequent 24-h subculture during each of 30 subcultures. Results of the in vitro cultivation experiment indicated that the 24-h disappearance rates for lysine, arginine, threonine, glutamate, leucine, isoleucine, valine or histidine were 50–90% in the duodenum, jejunum or ileum groups. After 30 subcultures, the 24-h disappearance rates for lysine, threonine, arginine or glutamate remained greater than 50%. The denaturing gradient gel electrophoresis analysis showed that Streptococcus sp., Mitsuokella sp., and Megasphaera elsdenii-like bacteria were predominant in subcultures for utilizing lysine, threonine, arginine and glutamate. In contrast, Klebsiella sp. was not a major user of arginine or glutamate. Furthermore, analysis of AA composition and the incorporation of AA into polypeptides indicated that protein synthesis was a major pathway for AA metabolism in all the bacteria studied. The current work identified the possible predominant bacterial species responsible for AA metabolism in the pig small intestine. The findings provide a new framework for future studies to characterize the metabolic fate of AA in intestinal microbes and define their nutritional significance for both animals and humans.