The influence of protein starvation on hydrolytic and transport characteristics of the rat small intestine in chronic experiments

Time dynamics of maltose, glycylglycine, glucose, and glycine hydrolysis and absorption in isolated loop of the small intestine was studied in chronic experiments on Wistar rats (group 1) after their transition from the standard diet to the protein-free one with enhanced content of carbohydrates. During protein starvation, there were different changes in the rates of glucose and glycine absorption, and glycylglycine hydrolysis and absorption in isolated intestinal loop, but to the end of the 2nd week they returned to the initial levels (for glucose and glycylglycine) or increased (for glycine). The rates of maltose hydrolysis and derived glucose absorption remained at the initial levels for the first days of protein starvation, decreased on the 5th day, and did not change afterwards. Maltase, alkaline phosphatase, and amino peptidase M activities, determined in homogenates of the small intestinal mucosa (per g of the tissue) after 2 weeks of protein starvation, were lower in the rats of group 1 in comparison with the rats of group 2, kept on the standard diet. Thus, under protein deficiency the hydrolytic and absorptive capacities of the small intestine correspond to both ingested food composition, and body requirements.     

Kinetic analysis of glycine and glycylglycine absorption in rat small intestine in chronic experiment

Kinetics of glycylglycine hydrolysis and absorption as well as that of free glycine absorption in isolated loop of the small intestine was studied in chronic experiments in two groups of rats. In the 1st group (n = 5), the isolated loop daily received for 1 or two hours a glucose load (25 mM), whereas in the 2nd group (n = 4)--a glutamic acid load (25 mM). The "true" values (i.e. corrected for the influence of the pre-epithelial layer) of the Michaelis constant for dipeptide transport were lower than those for the free glycine transport: 16 +/- 1.8 versus 36.3 +/- 3.7 mM (in the 1st group) and 15.9 +/- 2.2 versus 34.0 +/- 3.7 mM (in the 2nd group), whereas values of the maximal rate of active transport as calculated per 1 cm of the intestine length were, on the contrary, higher: 0.64 +/- 0.06 versus 0.42 +/- 0.10 mumol/(min.cm) 1st group and 0.86 +/- 0.13 versus 0.56 +/- 0.04 mumol/(min.cm) in the in the 2nd group. It has been shown that, under these conditions, regarded as the most physiological, over 90% of glycylglycine is absorbed via the peptide transport system. Only a small part of this dipeptide amount (less than 10%) splits during membrane hydrolysis with subsequent absorption of the derived glycine. It has also been found that glutamic acid solution as a regular substrate load is more effective (as compared with the glucose solution) in retarding the atrophic changes occurring in the isolated intestine loop and in preserving its structural and functional parameters on a higher level.     

A Sensitive and Simple Method for the Determination of Fatty Acid Hydroperoxides with Horseradish Peroxidase

Glycine, glycylglycine, glycine methyl ester and glycine ethyl ester were found to be effective for the production and release of γ-galactosidase by Escherichia coli. Addition of an appropriate concentration of glycine and glycylglycine to the culture increased total enzyme production 6 to 7-fold and extracellular enzyme production over 240-fold at 24 hr cultivation. The enzyme synthesis was stimulated even at the exponential period of growth, and 93% of enzyme was found in the culture fluid at 24 hr cultivation on addition of 1.2% glycine. A large amount of protein was also accumulated in the culture fluid. A micrograph showed glycine gave swollen and irregular cells, indicating that the cell surface was altered. Various amino acid analogues and some antibiotics had a small or no effect on the production and release of the enzyme as compared with glycine. Polypeptone or brain heart infusion was needed as a nitrogen source for efficient production of the enzyme.     

Effect of dipolar ions on the entropy-driven polymerization of tobacco mosaic virus protein

The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 degrees C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein.     

Evaluation of role of the peptide transport system in absorption of dipeptides in the rat small intestine in chronic experiments <Emphasis Type="Italic">in vivo</Emphasis>

Hydrolysis and absorption of glycylglycine and glycyl-L-leucine as well as absorption of glycine and leucine were studied in chronic experiments on rats with their isolated small intestine loop. Values of the “true” kinetic constants (with taking into account effect of the preepithelial layer) were determined to be as follows: (1) K _t = 46.7 ± 4.0 and 2.15 ± 0.59 mM, J _max = 0.74 ± 0.15 and 0.16 ± 0.03 μmol min^?1 cm^?1 (for transport of free glycine and leucine, respectively); (2) K _t = 4.4 ± 0.6 and 4.8 ± 0.9 mM, J _max = 0.24 ± 0.02 and 0.23 ± 0.02 μmol min^?1 cm^?1 (for transport of glycylglycine and glycyl-L-leucine, respectively); (3) K _M = 5.4 ± 1.0 and 38.2 ± 4.4 mM, V _max = 0.09 ± 0.02 and 0.24 ± 0.07 μmol min^?1 cm^?1 (for membrane hydrolysis of these dipeptides, respectively). According to our calculations, in the wide range of the initial glycylglycine concentrations (2.5–40 mM) a part of the peptide component in its total absorption accounts for 0.77–0.80. In the case of glycyl-L-leucine a part of the peptide component in the total glycine absorption decreases from 0.89 to 0.84, while in the total leucine absorption—from 0.86 to 0.71, the initial dipeptide concentration rising from 5 to 40 mM. The obtained results show that the peptide component prevails in absorption of the studied dipeptides in the rat small intestine, but its role is much lesser than what many authors believe. In the case of glycyl-L-leucine, the peptide component can achieve saturation in the range of high substrate concentrations, its part decreasing essentially to become compared with absorption of free amino acids formed as a result of the dipeptide membrane hydrolysis.     

Activity coefficients of the peptide and the electrolyte in ternary systems water+glycylglycine+NaCl, +NaBr, +KCl and +KBr at 298.2 K

The activity coefficients of glycylglycine in four aqueous electrolyte solutions (+NaCl, +NaBr, +KCl and +KBr) were obtained at 298.2 K. The mean ionic activity coefficient of the electrolyte in aqueous solutions containing the peptide was determined from measurements of the potential differences of a cation and an anion ion-selective-electrode, each vs. a double junction reference electrode. The results show that the nature of the anion has a major effect on the activity coefficients of glycylglycine. Comparison of activity coefficient data for glycylglycine with literature data for glycine, both in aqueous NaCl solutions, indicates that the effect of the electrolyte is larger for the peptide than for the amino acid. For the peptide, in all cases, the effect of the electrolyte is more important at low molalities of the electrolyte. The Wilson equation was used to correlate the activity coefficient data obtained. The correlation results were satisfactory for the region of concentrated electrolyte.     

Prebiotic synthesis of amino acids from formaldehyde and hydroxylamine in a modified sea medium

In the course of a study of a possible mechanism for chemical evolution in the primeval sea, we have found a novel reaction for peptide formation from glycine and urea in an aqueous solution. Glycine reacted with urea to give N-carbamylglycine, N-carbamylglycylglycine and glycylglycine. This reaction provides a new pathway for the prebiotic synthesis of peptides.     

Prebiotic synthesis of amino acids from formaldehyde and hydroxylamine in a modified sea medium

In the course of a study of a possible mechanism for chemical evolution in the primeval sea, we have found a novel reaction for peptide formation from glycine and urea in an aqueous solution. Glycine reacted with urea to give N-carbamylglycine, N-carbamylglycylglycine and glycylglycine. This reaction provides a new pathway for the prebiotic synthesis of peptides.     

Active transport of amino acids by gamma-glutamyl transpeptidase through Caco-2 cell monolayers.

The possible role of the gamma-glutamyl cycle in the transport of amino acids, using the Caco-2 cell monolayer as an in vitro model of the small intestine, has been investigated. The transport of [2-3H]glycine and [2-3H]glycylglycine through the Caco-2 monolayer has been shown to occur by two modes of action. Active transport is unidirectional from apical to basolateral region and is a carrier mediated system. The enzyme gamma-glutamyl transpeptidase seems to be involved in this process, since when the enzyme is inhibited, the active transport is also inhibited. However transport still takes place, and this occurs by a slower non-active process, which is bidirectional and is mediated by passive diffusion. The rate of transport of [2-3H]glycylglycine and [2-3H]glycine were 585 (+/- 24) and 287 (+/- 16) pmolcm-2min-1 respectively, while the non-active transport takes place at 87 (+/- 6) pmolcm-2min-1. Thus, amino acid translocation in Caco-2 cells is shown to occur by two methods, one of which involves the gamma-glutamyl cycle.     

High prevalence of diabetes in an urban population in south India.

An urban population in a township in south India was screened for diabetes with an oral glucose tolerance test, every fifth person aged 20 and over registered at the local iron ore company''s hospital being screened. Of 678 people (346 men and 332 women) who were tested, 34 (5%; 20 men and 14 women) had diabetes and 14 (2%; 8 men and 7 women) had impaired glucose tolerance. Thirteen subjects were already known to be diabetic. Diabetes was present in 21% (37/179) of people aged over 40. The peak prevalence (41%; 7/17) was in the group aged 55-64. A family history of diabetes was present in 16 of the 34 subjects with diabetes and nine of the 15 with impaired glucose tolerance. Diabetes was significantly related to obesity in women but not in men (57% (8/14) v 5% (1/20)). The plasma glucose concentration two hours after glucose loading was correlated to body mass index, age, and income in both sexes. The prevalence of diabetes was significantly higher in subjects whose income was above the mean. When the overall prevalence of diabetes was adjusted to the age distribution of the Indians living in Southall, London, and in Fiji it increased to 10% and 9%, respectively. The prevalence of diabetes is high among urban Indians and is comparable with the high prevalence seen in migrant Indian populations.     

Radiation protection of glycine and glycylglycine

The protective action of a series of organic sulfur compounds and amino acids has been investigated by using glycine and glycylglycine as substrates. A linear relationship is shown to exist between the reciprocal of the G-m value for the substrate in the presence of the protecting agent, and the concentration of this protecting agent. The "protecting activity" of the agents, expressed as the ratio of the rate constants for the reaction of radicals with the protecting agent and the substrate, is determined.     

epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus.

Staphylococcus capitis EPK1 produces a glycylglycine endopeptidase, ALE-1 (M. Sugai, T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S. Inoue, and H. Suginaka, J. Bacteriol. 179:1193-1202, 1997), which hydrolyzes interpeptide pentaglycine chains of cell wall peptidoglycan of S. aureus. Characterizations of the enzyme activity and cloning of ale-1 revealed that ALE-1 is very similar to prolysostaphin produced by S. simulans bv. staphylolyticus. Strain EPK1 is resistant to lysis by ALE-1 and by lysostaphin. A gene that renders the cells resistant to glycylglycine endopeptidase (epr) was found 322 bp upstream of and in the opposite orientation to ale-1. The deduced amino acid sequence of epr showed similarities to FemA and FemB, which have been characterized as factors essential for methicillin resistance of S. aureus. Inactivation of either femA or femB causes decreased resistance to methicillin, increased resistance to lysostaphin, and decreased glycine content in the interpeptide chains of peptidoglycan. Therefore, femAB is suggested to be involved in the addition of glycine to pentapeptide peptidoglycan precursor. S. aureus with epr on a multicopy plasmid had phenotypes similar to those of femAB mutants except that it did not alter resistance level to methicillin. These results suggest that epr and femAB belong to the protein family involved in adding amino acids to the pentapeptide peptidoglycan precursor and that epr is involved in the addition of serine to the pentapeptide.     

Interaction of the vanadyl (IV) cation with carnosine and related ligands

The interaction of the vanadyl (IV) (VO²⁺) cation with carnosine (the dipeptide β-alanyl-histidine) has been investigated by electron absorption spectroscopy at high ligand-to-metal ratios and at different pH values. The results show that in the range 6.0–8.5, the cation interacts with the imidazole group of four different carnosine molecules and points to the presence of an axially coordinated water molecule. These suppositions were confirmed by the behavior of the VO²⁺/imidazole system, which was investigated under similar experimental conditions, and supported by previous ENDOR (electron-nuclear double resonance) results. The study was complemented with additional measurements using the glycylglycine, glycylglycine/imidazole, and histidine systems as ligands.     

The sites of hydrolysis of dipeptides containing leucine and glycine by rat jejunum in vitro

1. The effect of peptides containing leucine and glycine on accumulation of leucine and glycine by everted jejunal rings was studied. 2. It was shown that, on a molar basis, leucyl-leucine is a more effective inhibitor of uptake of [(14)C]leucine than is either leucylglycine or glycyl-leucine. These latter dipeptides behave alike. 3. The concentration of the dipeptides and their constituent amino acids in both the incubation medium and the tissue has been followed in these experiments by amino acid analysis. No leucine-containing peptides were observed in the tissue. 4. The inhibitory effects of the mixed dipeptides are altered by pH changes in an analogous way to the alterations in peptidase activity. 5. The experimental results indicate that leucine-containing peptides are hydrolysed before the transport step. 6. Glycylglycine, on the other hand, has only a small effect on the accumulation of glycine, although large amounts of the peptide accumulate unchanged in the tissue. This suggests that glycylglycine is taken up by a different mechanism to that for the leucine dipeptides.     

Castoreum and Steel Traps in Eastern North America

Historians and anthropologists have credited the invention of trapping beaver with castoreum bait and steel traps to the Indians of southeastern Canada in the 1790s. However, English trappers in Virginia used this technique before 1728, and the Indians of southeastern Canada were using it by 1756. It is suggested that the widespread adoption of castoreum and steel traps by Northeastern Indians in the 1790's was due to the collapse of the world fur market after the French Revolution.     

Epithelial responses to glycinamide and glycylglycinamide in the frog (Rana catesbeiana) tongue.

1. Open-circuit potential difference and short-circuit current across the frog tongue epithelium in response to glycinamide and glycylglycinamide were investigated. 2. Response to both of these amides were larger than the responses produced by glycine, glycylglycine and NaCl, and were independent of Na+ in the mucosal medium but dependent on H+ in the stimulus. 3. The relationships of the magnitudes of both response to the stimulus were similar to that in the taste nerve. 4. The results indicate that H+-dependent transport of these amides is related to taste reception in frogs.     

Condensation of oligoglycines with trimeta- and tetrametaphosphate in aqueous solutions

The dehydration condensation of glycine with trimetaphosphate in aqueous solution has been reinvestigated. Although it has been reported that the condensation of glycine under the alkaline conditions was brought about through the formation of cyclic acylphosphoramidate and hence the condensation of polyglycines could not occur, we found that the condensation of oligoglycines with trimeta- and tetrametaphosphate in aqueous solution are possible through the formation of their acylphosphates under the neutral or weak acidic conditions.Aqueous solutions of 1.0 M glycylglycine and 1.0 M trimetaphosphate in the various pH from 4.0 to 9.0 were incubated at 38 °C. The solutions were analyzed by HPLC with ninhydrin reaction system. Tetraglycine and hexaglycine were detected and their maximum yields were given in the reaction carried out around pH 7. They are approximately 15% and 4% after 30 days, respectively. Analogous experiments were performed with tetrametaphosphate. The results showed a similar pH dependence for the condensation, but the yields were about one-tenth of those of corresponding experiments with trimetaphosphate.Relative rates of dimerization of glycine, diglycine and triglycine in the equimolar concentration were also investigated at pH 6.0 at 38 °C. The rates for digylcine and triglycine were approximately twice and four times as large as that for glycine.Relevance of the experiments to chemical evolution is discussed.     

Measurement of products from X-irradiated glycylglycine in oxygen-free aqueous solutions.

The product yields in X-irradiated aqueous solutions of glycylglycine (0.05 M and 1.0 M) were measured under deoxygenated conditions. Comparison was made between the results obtained from X- and 60Co gamma-irradiated glycylglycine solutions reported by Garrison, Sokol, and Bennett-Corniea (Radiat. Res. 53, 376-384, 1973). The mechanisms proposed by Garrison et al. were tested by evaluating the stoichiometric relationships. The two intermediate radicals, deamination and H-abstraction radicals, were produced in the initial interactions of glycylglycine with reactive species (e-aq, OH, H) formed in H2O. Although the difference was fairly large at 0.05 M, the production of deamination radicals agreed well with the consumption of the radicals at 1.0 M. The production and the consumption of H-abstraction radicals were within the estimated experimental error in dilute solutions. Among all the products only the G value of aspartic acid decreased with increasing concentration of glycylglycine. This could be attributed to the fact that more acetylglycine is formed at the expense of aspartic acid at 1.0 M than at 0.05 M glycylglycine solutions. Competitive reactions involved with deamination radicals under conditions of homogeneously distributed reactants are discussed to elucidate the radiation chemistry of glycylglycine.     

Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives

Summary. Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, γ-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [³H]taurine evoked by K⁺-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K⁺, 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K⁺. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by γ-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and γ-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life. Authors’ address: Prof. Simo S. Oja, Brain Research Center, Medical School, FI-33014 University of Tampere, Finland     

Catawba Indians: morphology, genetics, and history

The Catawba Indians of South Carolina appear to be descendants of the once flourishing Siouan tribe now admixed primarily with Whites of the area. Study of physical traits and blood types was made in 1962 at the time they terminated reservation status. The data permit comparisions with other populations and an estimation of the genetic contribution of presumed parental populations to the hybrid. Skeletal remains of Siouan Indians from archeological sites in the area provide a basis for morphological comparisons. Similar ties of the Catawba to the Indian Knoll people of Kentucky and to the Occaneechi people of early historic times are noted. Results of blood typings of these ancient bones also enable some tentative serological comparisons with the present day Catawba Indians. Certain unusual factors in the history of the present population have affected their isolation and admixture, and this interaction of culture and biology is explored.