The development of neutrophilic polymorphonuclear leukocytes in human bone marrow.

Neutrophilic leukocytes (PMN) and their precursors from normal human marrow and blood were examined by histochemical staining and by electron microscopy and cytochemistry in order to determine the origin and nature of their cytoplasmic granules. Human neutrophils contain two basic types of granules, azurophils and specifics, which differ in morphology, contents, and time of origin. Azurophils are large and may be spherical or ellipsoid, the latter with a crystalline inclusion. They are produced in the first secretory stage (promyelocyte), contain peroxidase and various lysosomal enzymes, and thus correspond to modified primary lysosomes. Specifics are smaller, may be spherical or elongated, and are formed during a later secretory stage (myelocyte). They lack lysosomal enzymes and contain alkaline phosphatase and basic protein; their contents remain largely undetermined. Specifics outnumber azurophils in the mature PMN because of reduction in numbers of azurophils per cell by cell division in the myelocyte stage. The findings indicate that the situation is basically the same as described previously in the rabbit, insofar as the origin, enzymic activity, and persistence in the mature cell of the two types (azurophil and specific) of granules are concerned. The main difference between PMN of the two species is in the morphology (size, shape, and density) of the granules, especially the azurophils.

Early and discrete changes in permeability of Escherichia coli and certain other gram-negative bacteria during killing by granulocytes.

Rapid killing of Escherichia coli by intact or disrupted rabbit granulocytes or by granulocyte fractions was found to be accompanied by an equally rapid increase in permeability of the E.coli envelope. This increase in permeability was detected by determining entry of substances that normally do not cross E.coli's permeability barrier, namely actinomycin D and o-nitrophenyl-beta-D-galactopyranoside (ONPG), a substrate for cytoplasmic beta-galactosidase. Because E.coli continue to incorporate radioactively labeled precursors into bacterial RNA and protein for at least 1 h, despite rapid killing by granulocytes, entry of actinomycin D could be measured by its inhibitory effect on macromolecular synthesis. Entry was evident within minutes after exposure to granulocytes or granulocyte fractions and is independent of pH over a range of 6.5-9.0. The effect of disrupted granulocytes or partially purified fractions on susceptibility of E.coli to actinomycin D and entry of ONPG is dose dependent. That the entry of actinomycin D and ONPG was not caused by gross destruction of the envelope is indicated by two sets of observations: (a) net influx of (42)K was maintained for at least 15 min, even though efflux of potassium was immediately accelerated upon addition of bactericidal concentrations of granulocyte fractions; (b) beta-galactosidase did not leak out of E.coli under conditions that produce maximal inhibition by actinomycin D. Different species of gram-negative bacteria exhibited different susceptibilities to the bactericidal and permeability effects of granulocyte fractions. Thus, three strains of E.coli and one strain of Salmonella typhimurium were highly susceptible to both the bactericidal and the permeability enhancing effects of granulocyte fractions, whereas two strains of Serratia marcescens and one strain of Pseudomonas aeruginosa were resistant to both effects. Another strain of P. aeruginosa was rendered susceptible to actinomycin D without being killed and two strains of S. typhimurium remained insensitive to actinomycin D while being killed by granulocytes.

Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.

Biochemical cytology of trichomonad flagellates. I. Subcellular localization of hydrolases, dehydrogenases, and catalase in Tritrichomonas foetus.

To determine the localization of several enzymes in Tritrichomonas foetus, the axenic KV-1 strain was grown in Diamond's medium with bovine serum, homogenized in 0.25 M sucrose, and subjected to analytical differential and isopycnic centrifugation. The fractions were assayed for their enzymatic composition and examined electron microscopically. NADH and NADPH dehydrogenases, about 90% of the catalase, and two hydrolases, alpha-galactosidase and manganese-activated beta-galactosidase I are in the nonsedimentable part of the cytoplasm. alpha-Glycerophosphate and malate dehydrogenases are associated with a large particle, whose equilibrium density in sucrose gradients is 1.24. This particle corresponds to that population of the paracostal and paraxostylar granules which, having a uniform granular matrix surrounded by a single membrane, resemble microbodies from other organisms. The small sedimentable portion of catalase (about 10% of the total activity) is not associated with these granules and equilibrates at density 1.22. The nature of the subcellular entity carrying catalase could not be ascertained. Hydrolases with a pH optimum around 6-6.5 (protease, beta-N-acetylglucosaminidase, beta-N-acetylgalactosaminidase, and cation-independent beta-galactosidase II), as well as a large part of acid phosphatase, are associated with a population of large particles which equilibrate at densities from 1.15 to 1.20. The hydrolases in these granules lose their structure-bound latency easily after freezing and thawing. These particles correspond to another population of the paracostal and paraxostylar granules which have varied shape and inhomogeneous content with frequent myelin figures, indicating a digestive function. The rest of the phosphatase and most of the acid beta-glucuronidase activity are in a smaller granule fraction with an equilibrium density around 1.18. The latency of these enzymes is quite resistant to freezing and thawing. This particle population consists of smaller, very often flattened vesicles and granules, many of which are clearly fragments of the prominent Golgi apparatus of the cell.

Resolution of granules from rabbit heterophil leukocytes into distinct populations by zonal sedimentation.

Postnuclear supernates from homogenates of essentially pure rabbit heterophil leukocytes were fractionated by means of zonal differential centrifugation through a discontinuous sucrose gradient at various speeds. Three distinct groups of granules were characterized biochemically and morphologically. They were, in order of decreasing sedimentation coefficient: (a) Large, relatively dense granules, identified morphologically as the azurophil or primary granules, and containing essentially all of the myeloperoxidase activity of the preparations, about one-third of their lysozyme activity, and between 50 and 80% of their content in five acid hydrolases typically associated with lysosomes in other cells; (b) smaller, less dense granules, with the morphological appearance of the specific or secondary granules, and carrying most of the alkaline phosphatase and the remainder of the lysozyme activity of the preparations; (c) a second group of lysosome-like particles, associated with a morphologically heterogeneous fraction, and containing the remainder of the acid hydrolases, but little or no myeloperoxidase. When p-nitrophenyl phosphate was used instead of beta-glycerophosphate for the assay of acid phosphatase, only small proportions of the total activity accompanied the two main lysosomal bands, and considerable activity was found in a zone slightly retarded with respect to the slowly moving band of acid hydrolases.

Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. I. Histochemical staining of bone marrow smears.

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, beta-galactosidase, beta-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.

Coordinated development of -glucuronidase and -galactosidase in mouse organs.

Changing concentrations of beta-glucuronidase and beta-galactosidase are coordinated during the development of mouse liver, heart, and brain. Although coordinate, the developmental patterns for the two enzymes are under independent control by genetic elements apparently linked to the respective structural genes.

Intestinal enzymes: indicators of proliferation and differentiation in the jejunum.

Some intestinal enZymes were assayed which were related to: (i) Cellular proliferation, for example, aspartate carbamoyltransferase, thymidine kinase, uridine kinase, and dihydroorotase; (ii) cellular differentiation, for example, lactase, invertase, maltase, alkaline phosphatase, and dipeptidase; and (iii) lysosomes, for example, beta-glucuronidase, acid beta-galactosidase, and acid phosphatase. These enzymatic determinations can be used to distinguish the crypt from the villus during healthy or diseased states.

Hurler's syndrome: deficiency of a specific beta galactosidase isoenzyme.

A marked deficiency of a specific thermolabile beta-galactosidase isoenzyme (pH optimum 3 to 5) was found in liver and kidney tissues of five patients with the Hurler's syndrome (types 1 to 3).

Tay-Sachs disease: generalized absence of a beta-D-N-acetylhexosaminidase component.

Two hexosaminidase components, separable by starch-gel electrophoresis and possessing both beta-D-N-acetylglucosaminidase and beta-D-N-acetylgalactosaminidase activity, are present in human tissues. One of these, hexosaminidase component A, is absent in brain, liver, kidney, skin, cultured skin fibroblasts, blood plasma, and leukocytes from nine patients with Tay-Sachs disease. Hexosaminidase assay may facilitate the early diagnosis of individuals homozygous for Tay-Sachs disease.

Neuronal GM1 gangliosidosis in a Siamese cat with beta-galactosidase deficiency.

A juvenile Siamese cat with severe, progressive motor disability was shown to have extensive neuronal degeneration caused by accumulation of GM(1) ganglioside. Tissues from brain and kidney were markedly deficient in beta-galactosidase activity. The disease in this cat is thought to be inherited as an autosomal recessive trait, and is strikingly similar to juvenile GM(1) gangliosidosis of children.

Mucopolysaccharidosis: secondarily induced abnormal distribution of lysosomal isoenzymes.

Total activities of acid hydrolases in liver of two patients with mucopolysaccharidosis are decreased for beta-galactosidase, alpha-galactosidase, and arylsulfatase A; total activities of four other hydrolases are normal or increased. The isoenzyme distribution of five hydrolases (beta-glucuronidase, alpha-glucosidase, beta- galactosidase, N-acetyl-beta-glucosaminidase, and alpha-galactosidase) is ábnormal in that the isoelectric points (by isoelectric focusing) of these enzymes are more acid than in control liver. Along with the isoenzyme abnormalities different kinds of glycolipids were stored in kidney, liver, and brain. The isoenzyme abnormalities can be reproduced in vitro by addition of chondroitin sulfate to a homogenate of normal liver, suggesting that stable binding occurs between mucopolysaccharides and the hydrolase molecules. After the addition of chondroitin sulfate, the total activity of beta-galactosidase is inhibited, whereas other hydrolases are affected only slightly or not at all.

Jejunal perfusion of simple and conjugated folates in celiac sprue.

The intestinal absorption of [3H]-pteroylmonoglutamate (simple folic acid) and pteroyl-micron[14C]glutamyl-gamma-hexaglutamate ([14C]PG-7, conjugated folic acid) was assessed by the method of jejunal perfusion in five patients with proven celiac sprue who were studied after a gluten-containing or a gluten-free diet, and in nine normal subjects. The luminal disappearance of each folate was markedly impaired after exposure of the patients to dietary gluten and improved by gluten restriction, but not to within the range found in the normal subjects. The luminal disappearance of each folate was markedly impaired after exposure of the patients to dietary gluten and improved by gluten restriction, but not to within the range found in the normal subjects. In each experiment, column chromatography of the luminal aspirates revealed similar spectra of hydrolytic products of [14C]PG-7, whereas the fraction of the distal aspirate chromatogram appearing as pteroyl-micron[14C]glutamyl-gamma-monoglutamate ([14C]-PG-1) was similar in all three groups. By accounting for the variable effects of absorption on the luminal appearance of [14C]PG-1 and by correcting for mucosal hydrolysis which was not followed by release of [14C]PG-1 to the luminal contents, the calculated rate of in vivo hydrolysis of [14C]PG-7 to [14C]PG-1 was found impaired in both celiac sprue groups, with significant improvement on treatment. In mucosal biopsies from the sprue patients, the in vitro activity of folate conjugase in whole homogenates was higher and the activity of disaccharidase lower than in a group of 12 normal mucosal biopsies. These in vitro data suggest that the predominant cellular location of mucosal folate conjugase is different from that of disaccharidase, whereas comparison with the results of in vivo hydrolysis suggests that measurement of the enzyme in whole mucosal homogenates overestimates its significant digestive activity. The present studies indicate that (a) the mucosal lesion of celiac sprue significantly limits the intestinal absorption of both simple and conjugated folate, and (b) malabsorption of conjugated folate results from a combination of impaired hydrolysis and decreased mucosal uptake of hydrolytic product.

Targeting of products of genes to tumor sites using adoptively transferred A-NK and T-LAK cells.

Despite successes in animals, cytokine gene expression selectively in human tumors is difficult to achieve owing to lack of efficient delivery methods. Since interleukin (IL)-2-activated natural killer (A-NK) and phytohemagglutinin and IL-2 activated killer T (T-LAK) cells, as previously demonstrated, localize and accumulate in murine lung tumor metastases following adoptive transfer, we transduced them to test their ability to deliver products of genes selectively to tumors. Assessments of transduction efficiency in vitro demonstrated that adenoviral transduction consistently resulted in high (>60%) transduction rates and substantial expression of transgenes such as GFP, Red2, luciferase, beta-galactosidase and mIL-12 for at least 4 days. In vivo experiments illustrated that Ad-GFP transduced A-NK and Ad-Red2 (RFP) transduced T-LAK or mIL-12 transduced A-NK cells localized 10-50-fold more or survived significantly better than mock transduced cells, respectively, within lung metastases than in the surrounding normal lung tissue. Most importantly, mIL-12 transduced A-NK cells provided a significantly greater antitumor response than non-transduced A-NK cells. Thus, adoptive transfer of A-NK and T-LAK cells represents an efficient method for targeting products of genes to tumor sites.

Midgut protease activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri.

We assayed the relative activities of midgut proteolytic enzymes in individuals of the fourth (L(4)) and fifth (L(5)) instar of Apollo larvae, inhabiting Pieniny Mts (southern Poland). The comparisons between midgut tissue with glicocalyx (MT) and liquid midgut contents with peritrophic membrane (MC) were made. Optimal media pHs of the assayed proteolytic enzymes in P. apollo midgut samples were similar to those of other lepidopteran species. Endopeptidases, as well as carboxypeptidases, digested effectively in alkaline environment, while aminopeptidases were active in a broad pH range. Trypsin is probably the main endoprotease (correlation with caseinolytic activity in MC of L(5) larvae: r=0.606; p=0.004); however, its activity was low as compared with that in other leaf-eating Lepidoptera. This suggests a minor role of trypsin and chymotrypsin in protein digestion in Apollo larvae, probably due to limited availability of the leaf proteins. Instead, due to very high carboxypeptidase A activity in midgut tissue, the larvae obtain exogenous amino acids either directly or from oligopeptides and glycoproteins. High and significant positive correlations between the enzyme activity and glucosidase as well as galactosidase activities strongly support this opinion.

Elevated thymic and serum galactosyltransferase with low-molecular-weight acceptor activity in murine leukemia.

Galactosyltransferase activity (UDP-galactose:N-acetyl-D-glucosamine-D-galactosyltransferase) could be measured in thymus and sera from different strains of mice. Total thymic homogenates or thymocyte preparations obtained from thymoma carrying AKR/J mice exhibited higher enzyme activity compared to nonleukemic control mice. A similar difference was also noted in Swiss mouse thymus which develop thymic leukemia upon a single injection of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboximide. Galactosidase, which was 25 times less active than galactosyltransferase, was not responsible for this difference. These observations were extended to an evaluation of the serum level of the enzyme as a potential tumor biomarker. A 3- to 4-fold increase in the activity of galactosyltransferase was detected in serum samples obtained from both leukemic mice models (AKR/J and Swiss) compared to the controls, whereas the sera from P388 tumor-bearing DBA/2 mice showed a statistically nonsignificant increase of only 20%. The data indicate that serum galactosyltransferase (that accepts the low-molecular-weight acceptor, N-acetyl-D-glucosamine) levels are elevated in the presence of thymic leukemia, and suggest the possibility of shedding of this enzyme from the tumor cells to the systemic circulation of the host. The implications, including the potential diagnostic significance of the results, are discussed.

Uridine 5'-diphosphate-galactose:glycoprotein galactosyltransferase activity in the ovarian cancer patient.

Uridine 5'-diphosphate-galactose:glycoprotein galactosyltransferase activity was demonstrated in homogenates of normal ovary and ovarian epithelial adenocarcinomas. The specific activity of the enzyme in ovarian tumors was 3 to 5 times higher than in normal ovaries when the enzyme was assayed under identical conditions. The glycoprotein fetuin, from which terminal sialic acid and penultimate galactose were removed (fetuin minus N-acetylneuraminis acid and galactose), acted as an excellent exogenous acceptor. Galactosyltransferase from normal ovary and ovarian tumor cells had similar properties. Both required Mn2+ and Triton X-100 and had broad pH optima between 5.5 and 7. Galactosyltransferase activity was also measured in serum samples from ovarian cancer patients and normal healthy individuals in the presence of fetuin minus N-acetylneuraminic acid and galactose as exogenous acceptor. The enzyme levels were significantly elevated in the sera of ovarian cancer patients as compared to normal controls. The differences in the levels of this enzyme in the tissues and sera of normal individuals and ovarian cancer patients were not due to differential levels of the degrading enzymes such as uridine 5'-diphosphate-galactose pyrophosphatase or beta-D-galactosidase. Serial determinations were carried out on the sera of 5 ovarian cancer patients over a long period of time. The serum level of galactosyltransferase activity appeared to correlate with tumor volume as well as with the clinical status of the patient, which suggests possible leakage of the tumor enzyme into the host sera. Serial determination of this enzyme level in ovarian cancer patients seems promising in measuring tumor progression or success of therapeutic approaches.

A versatile two-photon fluorescent probe for ratiometric imaging E. coliß-galactosidase in live cells and in vivo.

We have described the design, synthesis, spectroscopy and biological applications of NI-ßGal, a versatile fluorescent probe to detect E. coliß-galactosidase in live cells and mice sensitively and directly, which holds great promise for its application in biomedical research such as gene therapy for cancer in the future.

IMMUNO-MODULATORY PROPERTIES OF PREBIOTICS EXTRACTED FROM vernonia amygdalina.

BACKGROUND: Vernonia amygdalina, commonly called bitter-leaf, is widely consumed in many parts of Africa, and Nigeria, in particular. The leaf extract has been reported to have antimicrobial, anti-plasmodial, anti-helminthic, as well as prebiotic properties, but its immuno-modulatory effects have not been well-studied, neither have the prebiotics been identified. This study evaluated the immuno-modulatory properties of the aqueous leaf extract and identified the prebiotic components. METHODS: The immuno-modulatory potential was evaluated by monitoring the effects of oral administration of the extract on immunological, haematological and lipid profiles of Rattus norvegicus, while the prebiotic components were identified by thin layer chromatography (TLC), following liquid-liquid fractionation of the extract. RESULTS: Consumption of the extract caused significant increases in CD4+-, white blood cell-, total lymphocyte- and high density lipid (HDL) counts; decreases in low density lipid (LDL) and triglycerides and no significant effect on haemoglobin (Hb) and packed cell volume (PCV) in the blood of test animals. The water-soluble fraction of the extract contained most of the phyto-constituents of the extract and Thin Layer Chromatographic analysis of the fraction revealed the presence of fructo-oligosaccharide and galacto-oligosaccharide prebiotics. CONCLUSION: The results from this study have shown that the aqueous leaf extract of V. amygdalina has positive immune-modulatory and haematologic effects and contains some important prebiotic compounds.

Cellular origin of lactase decline in postweaned rats.

Intestinal lactase activity falls at weaning reaching low levels in the adult rat. Present work measures cellular migration rates and lactase activity along villi of intestines taken from rats of different ages to determine the cellular basis for this developmentally regulated fall in enzyme expression. An early fall in lactase activity taking place 15 to 23 days after birth was found to be associated with a shortening of the time available for lactase expression in brush-border membranes. A later fall in lactase activity occurring 23 to 46 days after birth was caused by an additional reduction in the rate at which lactase activity appeared in the brush-border membrane. These results are discussed in relation to previous work describing lactase biosynthesis in post-weaned rats.