A new variant of serum leucine aminopeptidase in the mouse: Its development and possible regulation

Serum leucine aminopeptidase (LAP) isozymes were compared in four strains of inbred mice during postnatal development, adult life, and pregnancy. In pregnancy, no changes in the maternal serum LAP pattern were observed, in contrast to human studies. One strain, DD/S, differs from the other three in serum LAP. Polymorphism in serum LAP has not been previously described in the mouse. Neonatal DD/S mice exhibit a single band of serum LAP upon starch gel electrophoresis; however, between 14 and 18 days of age, two distinct bands appear, which persist throughout adult life. In the strains C57BL/6J, BALB/cJ, and DBA/2J there is a single band of activity at all stages. Crosses and backcrosses between DD/S and C57BL/6J show that the double-band variant is inherited as an autosomal recessive. The variant is independent of both the supernatant malic enzyme (Mod-1) and the intestinal LAP (Lap-1) loci, which are known to be linked on chromosome 9. The serum LAP variant is linked to an intestinal alkaline phosphatase variant. The presence of a separate structural gene is suggested by the genetic independence of the serum LAP variant from Lap-1. Also, the two serum LAP bands of DD/S are not interconverted by treatment with neuraminidase, -mercaptoethanol, or heat or by mixing the sera of DD/S and C57BL/6J prior to electrophoresis. The level of serum LAP activity in DD/S is approximately twice that in C57BL/6J. While these observations imply two structurally distinct proteins, the absence of any trace of the second LAP band in the heterozygote strongly suggests that the LAP variant protein is not the result of a separate structural gene. Intestinal LAP in DD/S migrates with the same electrophoretic mobility as the serum LAP variant, implying that the variant might originate in the intestine and its appearance in the serum be modulated by some factor at an unlinked locus.This work was supported by National Institutes of Health Grant RR08117.     

Further studies on the genetic control of chicken plasma alkaline phosphatase isozyme.

The effects of neuraminidase treatment on the electrophoretic pattern of alkaline phosphatase (AP) isozymes and AP activity were investigated in chicken plasma. AP comprised three isozymes. The zymogram of an individual chicken plasma had two bands, either the faster (F) or the slower (S) moving band by isozyme types and the B band irrespective of isozyme types. Mobility of the S band and AP activity in chicken plasma were not affected by neuraminidase treatment. The treatment has a reduced migration rate of the F band equal to that of the S band and the B band of both types closer to the origin. The genetic control of these bands is discussed.     

Further studies on the genetic control of chicken plasma alkaline phosphatase isozyme

The effects of neuraminidase treatment on the electrophoretic pattern of alkaline phosphatase (AP) isozymes and AP activity were investigated in chicken plasma. AP comprised three isozymes. The zymogram of an individual chicken plasma had two bands, either the faster (F) or the slower (S) moving band by isozyme types and the B band irrespective of isozyme types. Mobility of the S band and AP activity in chicken plasma were not affected by neuraminidase treatment. The treatment has a reduced migration rate of the F band equal to that of the S band and the B band of both types closer to the origin. The genetic control of these bands is discussed.     

Genetic control of alkaline phosphatase isozymes in chicken duodenum

A genetic control of alkaline phosphatase (AP) in chicken duodenum was studied in a White Plymouth Rock strain. Unpurified chicken duodenum AP heated in an extraction procedure comprised either F or S band by isozyme types. On the other hand, chromatographically purified intestinal AP (NBCo, USA) had three bands, i.e., F', S' and B' bands. Characterization by urea, heat and neuraminidase treatments suggested that the genetic control of plasma AP isozymes may be applicable to the duodenum AP isozymes.     

A study of the candidate virulence factors of Bacteroides fragilis

Bacteroides fragilis strains were classified as virulent or avirulent on the basis of their clearance from the subcutaneous tissues of mice. To determine the factors which may contribute to the virulence of B. fragilis strains, we studied encapsulation, hydrophobicity, growth rate, serum sensitivity, agglutination with erythrocytes of different origin, and neuraminidase production. The strains of the virulent group displayed a higher growth rate in broth and a lower sensitivity to the bactericidal activity of serum than the strains of the avirulent group. They also agglutinated different types of erythrocytes more strongly than did the avirulent strains. No significant differences were found between the two groups of strains as regards encapsulation, hydrophobicity and neuraminidase activity.     

Dietary gangliosides positively modulate the percentages of Th1 and Th2 lymphocyte subsets in small intestine of mice at weaning.

We studied the influence of dietary gangliosides on the number of spontaneous cytokine-secreting cells from two intestinal lymphocyte populations: lamina propria lymphocytes and Peyer's patches lymphocytes in Balb/c mice for 28 days after weaning. Weanling mice were separated into two groups, designated as Control and BG. The Control group was fed with a semipurified diet without gangliosides and the BG group was fed with the semipurified diet supplemented with 47 mg/kg of a mixture of bovine brain gangliosides. Intestinal lymphocytes were isolated from mice killed at 3, 7, 14 and 28 days after weaning, and the percentages of spontaneous Th1 as well as Th2 cytokine-secreting lymphocytes were determined using the ELISPOT assay. The BG group animals showed an earlier development in the number of cytokine-secreting cells, which appeared one week later in Control animals. In addition, mice fed with the ganglioside-supplemented diet showed a significantly higher number of Th1 and Th2 cytokine-secreting lymphocytes than Control mice in lamina propria and Peyer's patches lymphocytes at the end of the experimental period (28 days). Our results suggest that dietary gangliosides influence the maturation process of the intestinal immune system that take place during weaning.     

Mice overexpressing CD97 in intestinal epithelial cells provide a unique model for mammalian postnatal intestinal cylindrical growth

Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein–coupled receptor CD97 shows an expression gradient along the crypt–villus axis in the normal human intestine. We here report that transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine. Intestinal enlargement involves an increase in length and diameter but does not affect microscopic morphology, as typical for cylindrical growth. The megaintestine is acquired after birth and before weaning, independent of the genotype of the mother, excluding altered availability of milk constituents as driving factor. CD97 overexpression does not regulate intestinal growth factors, stem cell markers, and Wnt signaling, which contribute to epithelial differentiation and renewal, nor does it affect suckling-to-weaning transition. Consistent with augmented cylindrical growth, suckling but not adult transgenic mice show enlarged crypts and thus more crypt fissions caused by a transient increase of the crypt transit-amplifying zone. Intestinal enlargement by CD97 requires its seven-span transmembrane/cytoplasmic C-terminal fragment but not the N-terminal fragment binding partner CD55. In summary, ectopic expression of CD97 in intestinal epithelial cells provides a unique model for intestinal cylindrical growth occurring in breast-fed infants.     

Purification and properties of neuraminidase isozymes in Arthrobacter ureafaciens mutant

An Arthrobacter ureafaciens mutant (M1057) capable of producing neuraminidase constitutively was isolated by NTG mutagenesis from A. ureafaciens KMS 3663. Four molecular species (L, M1, M2, and S) of neuraminidase isozymes were homogeneously purified from the mutant and parent strains by means of DEAE-cellulose, affinity chromatography, ammonium sulfate precipitation, chromatofocusing, and Ultrogel AcA44 gel filtration. The molecular weights of L, M1, M2, and S isozymes were shown to be approximately 88,000, 66,000, 66,000, and 52,000, respectively. The optimal pHs and Km values of these isozymes for N-acetylneuraminosyl-alpha,(2-6)-lactose were 4.5-5.5 and 0.6-0.8 mM. Neuraminidase L, M1, M2, and S were able to hydrolyze oligosaccharides, glycoproteins and gangliosides containing alpha,(2-3)-, alpha,(2-6)-, and alpha,(2-8)-linked N-acetylneuraminic acid. Among these isozymes isolated, isozyme S was most active on colominic acid.     

A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain

Indirect immunofluorescence was used to establish a domain-specific marker for hepatocyte plasma membranes. In frozen sections of fixed rat liver (0.5-4 microns), antibodies directed against rat intestinal leucine aminopeptidase (LAP) recognized an antigen that was restricted to the bile canalicular plasma membrane. Fluorescence was not observed on the sinusoidal or lateral membranes, and intracellular staining was not detected. The liver antigen was identified as LAP, based on its chemical similarity to intestinal LAP. First, immunoprecipitation experiments using trypsin-solubilized intestinal LAP (G-200 fraction, 91% pure) established a correlation between the loss of LAP enzyme activity from the soluble fraction and the appearance in the specific immunoprecipitates of polypeptides migrating on SDS PAGE between 110,000 and 130,000 daltons. The antigen precipitated from a detergent extract of liver plasma membranes had the same electrophoretic mobility. Second, the chymotryptic map of the major band in the liver immunoprecipitate was similar to that of purified intestinal LAP.     

A comparison of their virulence for mice and the enzymatic activity of Streptococcus pneumoniae strains

In most cases no correlation between the virulence of S. pneumoniae and their enzymatic activity was registered in 101 S. pneumoniae strains isolated in pneumococcal infections of different localization. Pneumococcal strains belonging to different serotypes and characterized by their low virulence for mice (LD50 = 10(6) colony-forming units) had the highest neuraminidase and protease-alzolase activity in comparison with highly virulent cultures of these bacteria. In pneumococcal cultures in the R-form avirulence for mice occurred mainly in combination with low enzymatic activity.     

Immune effect of heat-killed multistrain of Lactobacillus acidophilus against Salmonella typhimurium invasion to mice

AIMS: This study attempted to determine whether lactic acid bacteria (LAB) could have a better probiotic function when used as a multistrain mixture, i.e. Mix-LAB, than when used as a monostrain. To this end, three strains of Lactobacillus acidophilus, specifically strain LAP5, LAF1 and LAH7, were heat-killed and mixed. This heat-killed Mix-LAB was used to evaluate the effectiveness of multistrain in inhibiting Salmonella invasion into cultured cells and into organs (spleen and liver) of live mice. METHODS AND RESULTS: BALB/c mice were orally administered with heat-killed Mix-LAB or sterile normal saline (control) for seven consecutive days and then challenged with orally administered Salmonella typhimurium on day 8. Results showed that, at day 6 after the challenge, the mice which had received Mix-LAB exhibited lower rates (P < 0.05) of Salmonella invasion into liver and spleen than did the control mice. Also, before the Salmonella challenge, the serum tumour necrosis factor-alpha (TNF-alpha) levels were not significantly different (P > 0.05) between these two groups of mice. After the challenge, however, the serum TNF-alpha level was significantly elevated (P < 0.05) in the control group, but not significantly changed in the Mix-LAB fed mice. To investigate possible factors involved in heat-killed Mix-LABs antagonistic effect on Salmonella invasion of mouse organs, heat-killed single strain and Mix-LAB were evaluated for ability to inhibit Salmonella invasion into cultured human intestinal Int-407 and Caco-2 cells. Results showed that none of the heat-killed strains were able to protect these cultured cells from Salmonella invasion, even though strains of LAP5 and Mix-LAB were adherent to them. However, study of the activation of murine macrophage Raw 264.7 cells showed that heat-killed Mix-LAB stimulated TNF-alpha production, nitric oxide release, and increased phagocytic activity in macrophages. CONCLUSIONS: Our findings suggest that heat-killed Mix-LAB can inhibit Salmonella invasion of mouse organs through the immunomodulating role of activated macrophage. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of heat-killed Mix-LAB to prevent bacterial infection in mice was found to be more significant than that of viable monostrain. This effect may be due to the activation of the immune system rather than to the adherence of LAB to the intestine epithelium.     

The virulence of Streptococcus pneumoniae strains--the causative agents of pneumococcal infection at different sites]

A total of 256 S. pneumoniae strains, the causative agents of infectious processes with different localization, were studied for their virulence (in experiments on mice), neuraminidase and aldolase-protease activity (APA). In pneumococcal strains isolated 18-20 hours after intraperitoneal infection their virulence for mice increased, on the average, 1,000-fold and the average level of extracellular and cellular neuraminidase and APA increased 2- to 5-fold in comparison with the initial values. Pneumococcal strains causing acute pneumococcal infections with different localization, or the aggravation of such infections, exhibited higher virulence for mice and higher levels of neuraminidase and APA, while the inflammatory process at the period of clinical remissions was mainly maintained by S. pneumoniae cultures with low virulence.     

Increased Intestinal Epithelial Cell Turnover and Intestinal Motility in Gymnophalloides seoi-Infected C57BL/6 Mice

The changing patterns of goblet cell hyperplasia, intestinal epithelial cell turnover, and intestinal motility were studied in ICR and C57BL/6 mice infected with Gymnophalloides seoi (Digenea: Gymnophallidae). Whereas ICR mice retained G. seoi worms until day 7 post-infection (PI), C57BL/6 mice showed a rapid worm expulsion within day 3 PI. Immunosuppression with Depo-Medrol significantly delayed the worm expulsion in C57BL/6 mice. Goblet cell counts were increased in both strains of mice, peaking at day 1 PI in C57BL/6 mice and slowly increasing until day 7 PI in ICR mice. In C57BL/6 mice infected with G. seoi, newly proliferating intestinal epithelial cells were remarkably increased in the crypt, and the increase was the highest at day 1 PI. However, in ICR mice, newly proliferating intestinal epithelial cells increased slowly from day 1 to day 7 PI. Intestinal motility was increased in G. seoi-infected mice, and its chronological pattern was highly correlated with the worm load in both strains of mice. Meanwhile, immunosuppression of C57BL/6 mice abrogated the goblet cell proliferation, reduced the epithelial cell proliferation, and suppressed the intestinal motility. Goblet cell hyperplasia, increased intestinal epithelial cell turnover, and increased intestinal motility should be important mucosal defense mechanisms in G. seoi-infected C57BL/6 mice.     

Enzyme variation of Eimeria arizonensis from Peromyscus truei and P. boylii

Cricetid rodents, Peromyscus truei and P. boylii, were inoculated with sporulated oocysts of Eimeria arizonensis collected from wild P. truei maintained in the lab. In P. truei the prepatent period was 4-5 days, the patent period was 9-11 days, and sporulated oocysts were 21.5 x 25.0 (20-23 x 24-26) microns with sporocysts 7.7 x 12.0 (6-8 x 10-13) microns. In P. boylii the prepatent period was 6-7 days, the patent period was 8-9 days, and sporulated oocysts were 20.1 x 23.2 (18-22 x 21-24) microns with sporocysts 6.8 x 10.0 (5-8 x 9-12) microns. Sporulated oocysts from both host species were used in direct side-by-side comparison of isozyme banding patterns using protein electrophoresis. The parasite has polytypic loci for leucine aminopeptidase (LAP), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6-PGD). In oocysts from P. truei, LAP showed one band with fast migration and LDH and 6-PGD each showed two bands, one with fast and one with slow migration. In oocysts from P. boylii, LAP and LDH each had one band with slow migration and 6-PGD had one band with moderate migration. Oocysts of E. arizonensis collected from P. boylii were used to inoculate P. truei. The prepatent and patent periods, structural measurements, and isozyme banding patterns of the resultant oocysts were the same as those from P. truei when inoculated with oocysts from P. truei.     

A surface‐exposed neuraminidase affects complement resistance and virulence of the oral spirochaete Treponema denticola

Neuraminidases (sialidases) catalyse the removal of terminal sialic acid from glycoconjugates. Bacterial pathogens often utilize neuraminidases to scavenge host sialic acid, which can be utilized either as a nutrient or as a decorating molecule to disguise themselves from host immune attacks. Herein, a putative neuraminidase (TDE0471) was identified in Treponema denticola, an oral spirochaete associated with human periodontitis. TDE0471 is a cell surface‐exposed exo‐neuraminidase that removes sialic acid from human serum proteins; it is required for T. denticola to grow in a medium that mimics gingival crevice fluid, suggesting that the spirochaete may use sialic acid as a nutrient in vivo. TDE0471 protects T. denticola from serum killing by preventing the deposition of membrane attack complexes on the bacterial cell surface. Animal studies revealed that a TDE0471‐deficient mutant is less virulent than its parental wild‐type strain in BALB/C mice. However, it causes a level of tissue damage similar to the wild type in complement‐deficient B6.129S4‐C3^tm1Crr/J mice albeit the damage caused by both bacterial strains is more severe in these transgenic mice. Based on these results, we propose that T. denticola has evolved a strategy to scavenge host sialic acid using its neuraminidase, which allows the spirochaete to acquire nutrients and evade complement killing.     

Comparative characterization of pulmonary surfactant aggregates and alkaline phosphatase isozymes in human lung carcinoma tissue

Alkaline phosphatase (AP) isozymes are surfactant-associated proteins (SPs). Since several different AP isozymes have been detected in the pneumocytes of lung cancer patients, we attempted to identify the relationship between pulmonary surfactant aggregate subtypes and AP isozymes. Pulmonary surfactant aggregates were isolated from carcinoma and non-carcinoma tissues of patients with non-small cell carcinoma of the lung. Upon analysis, ultraheavy, heavy, and light surfactant aggregates were detected in the non-carcinoma tissues, but no ultraheavy surfactant aggregates were found in the carcinoma tissues. Surfactant-associated protein A (SP-A) was detected as two bands (a 27-kDa band and a 54-kDa band) in the ultraheavy, heavy, and light surfactant aggregates found in the non-carcinoma tissues. Although both SP-A bands were detected in the heavy and light surfactant aggregates from adenocarcinoma tissues, the 54-kDa band was not detected in squamous cell carcinoma tissues. Liver AP (LAP) was detected in the heavy and light surfactant aggregates from both non-carcinoma and squamous carcinoma tissues, but not in heavy surfactant aggregates from adenocarcinoma tissues. A larger amount of bone type AP (BAP) was found in light surfactant aggregate fractions from squamous cell carcinomas than those from adenocarcinoma tissues or non-carcinoma tissues from patients with either type of cancer. LAP, BAP, and SP-A were identified immunohistochemically in type II pneumocytes from non-carcinoma tissues and adenocarcinoma cells, but no distinct SP-A staining was observed in squamous cell carcinoma tissues. The present study has thus revealed several differences in pulmonary surfactant aggregates and AP isozymes between adenocarcinoma tissue and squamous cell carcinoma tissue.     

Genetic correlations between nicotine reinforcement‐related behaviors and propensity toward high or low alcohol preference in two replicate mouse lines

Common genetic factors may contribute to the high comorbidity between tobacco smoking and alcohol use disorder. Here, we assessed behavioral and biological effects of nicotine in replicate mouse lines selectively bred for high (HAP2/3) or low alcohol preference (LAP2/3). In Experiment 1, free‐choice (FC) oral nicotine and quinine intake were assessed in HAP2/3 and LAP2/3 mice. Effects of nicotinic acetylcholine receptor blockade by mecamylamine on nicotine intake in HAP2 mice were also examined. In Experiment 2, HAP2/3 and LAP2/3 mice were tested for differences in sensitivity to nicotine‐induced taste conditioning. In Experiment 3, the effects of a single nicotine injection on nucleus accumbens (NAc) and dorsal striatum monoamine levels in HAP2/3 and LAP2/3 mice were tested. In Experiment 1, HAP2/3 mice showed greater nicotine intake and intake ratio than LAP2/3 mice. There were no line differences in quinine intake. Mecamylamine reduced nicotine intake and intake ratio in HAP2 mice. In Experiment 2, HAP2/3 mice showed weaker nicotine‐induced conditioned taste aversion (CTA) compared with LAP2/3 mice. In Experiment 3, nicotine treatment increased NAc dopamine turnover across both HAP2/3 and LAP2/3 mouse lines. These results show that there is a positive genetic correlation between oral alcohol intake (high alcohol intake/preference selection phenotype) and oral nicotine intake and a negative genetic correlation between oral alcohol intake and sensitivity to nicotine‐induced CTA.     

Genetic variation of an intestinal leucine arylaminopeptidase (Lap-1) in the mouse and its location on chromosome 9

Electrophoretic variation for an intestinal enzyme that cleaves L-leucyl-beta-naphthylamide has been discovered among inbred mouse strains. Several strains including related strains C57BL/6J, C57BL/10J, C57BR/cdJ, C57L/J, and C58/J demonstrate an electrophoretic band of this enzyme that is absent in other strains and stocks thus far observed. The enzyme is tentatively being called leucine arylaminopeptidase (LAP) and the variant genetic locus Lap-1. The presence of the band is determined by an allele designated Lap-1a. Homozygotes for the alternate allele, Lap-1b, are without the band and heterozygotes are, under our electrophoretic conditions, indistinguishable from Lap-1a homozygotes. Data from recombinant inbred lines and a B6D2F1 X DBA/2J backcross established linkage of Lap-1 to dilute (d) and supernatant malic enzyme (Mod-1) on chromosome 9 in the following order: Lap-1-d-Mod-1. The Lap-1 to d map distance was estimated to be 21.3 +/- 4.6 cM from backcross data and 8.1 +/- 4.8 cM from recombinant inbred lines.     

Genetic variation in alkaline phosphatase of the house mouse (Mus musculus) with emphasis on a manganese-requiring isozyme

Genetic variation among inbred strains is described for electrophoretic migration of alkaline phosphatase from intestine, kidney, blood plasma, and three isozymes of liver. A manganese-requiring isozyme of liver and kidney unaffected by neuraminidase is described, and the locus controlling variation in this isozyme is designated Akp-1. Data from recombinant inbred strains place the locus on chromosome 1 at a distance of 3.6 +/- 2.9 cM from the M1s locus on the side distal to the centromere. Test-cross data show the following gene order and recombination percentages: Dip-1 19.0 +/- 3.8% Lp 7.4 +/- 2.2% Akp-1.