Polymorphism of ADAM12, DPP6 and PRKN genes and their associations with milk production traits in Holstein.

Milk production traits as the most important economic traits of dairy cows, they directly reflect the benefits of breeding and the economic benefits of pasture. In this study, A disintegrin and metalloproteinase-12 (ADAM12), Parkinson's disease gene 2 (PRKN) and dipeptidyl peptidase-like protein subtype 6 (DPP6) polymorphism in 384 Chinese Holstein cows were detected by time-of-flight mass spectrometry and through statistical analysis using software such as Popgene 32, SAS 9.4 and Origin 2022, the relationship between single nucleotide polymorphisms (SNPs) of three genes with four milk production traits such as daily milk yield (DMY), milk fat percentage (MFP), milk protein percentage (MPP) and somatic cell score (SCS) was verified at molecular level. The results showed that four polymorphic loci (116,467,133, 116,604,487, 116,618,268 and 116,835,111) of DPP6 gene, two polymorphic loci (97,665,052 and 97,159,837) of PRKN gene and two polymorphic loci (45,542,714 and 45,553,888) of ADAM12 gene were detected. PRKN-97665052, DPP6-116467133, ADAM12-45553888, DPP6-116604487 and DPP6-116835111 were all in Hardy-Weinberg equilibrium state (p > .05). ADAM12-45542714, PRKN-97159837 and PRKN-97665052 were moderately polymorphic (0.25 ≤ PIC <0.50) in Holstein. It is evident that the selection potential and genetic variation of these five loci are relatively large, and the genetic richness is relatively high. The correlation analysis of different genotypes between these eight loci and milk production traits of Holstein showed that ADAM12-45542714 and DPP6-116835111 (p < .01) had an extremely significant effects on the DMY of Chinese Holstein in Ningxia, while PRKN-97665052 had an extremely significant effect on MFP (p < .01). The effect of PRKN-97665052 and DPP6-116467133 on MPP of Holstein were extremely significant (p < .01). DPP6-116618268 had an extremely significant effect on the SCS of Holstein in Ningxia (p < .01), and AA genotype individuals showed a higher SCS than GG genotype individuals; the other two loci (ADAM12-45553888 and DPP6-116604487) had no significant effects on milk production traits of Holstein (p > .05). In addition, through the joint analysis of DPP6, PRKN and ADAM12 gene loci, it was found that the interaction effect between the three gene loci could significantly affect the DMY, SCS (p < .01) and MPP (p < .05). In conclusion, several different loci of DPP6, PRKN and ADAM12 genes can affect the milk production traits of Holstein to different degrees. PRKN, DPP6 and ADAM12 genes can be used as potential candidate genes for milk production traits of Holstein for marker-assisted selection, providing theoretical basis for breeding of Holstein.

Monitoring circulating dipeptidyl peptidase 3 (DPP3) predicts improvement of organ failure and survival in sepsis: a prospective observational multinational study.

BACKGROUND: Dipeptidyl peptidase 3 (DPP3) is a cytosolic enzyme involved in the degradation of various cardiovascular and endorphin mediators. High levels of circulating DPP3 (cDPP3) indicate a high risk of organ dysfunction and mortality in cardiogenic shock patients. METHODS: The aim was to assess relationships between cDPP3 during the initial intensive care unit (ICU) stay and short-term outcome in the AdrenOSS-1, a prospective observational multinational study in twenty-four ICU centers in five countries. AdrenOSS-1 included 585 patients admitted to the ICU with severe sepsis or septic shock. The primary outcome was 28-day mortality. Secondary outcomes included organ failure as defined by the Sequential Organ Failure Assessment (SOFA) score, organ support with focus on vasopressor/inotropic use and need for renal replacement therapy. cDPP3 levels were measured upon admission and 24 h later. RESULTS: Median [IQR] cDPP3 concentration upon admission was 26.5 [16.2-40.4] ng/mL. Initial SOFA score was 7 [5-10], and 28-day mortality was 22%. We found marked associations between cDPP3 upon ICU admission and 28-day mortality (unadjusted standardized HR 1.8 [CI 1.6-2.1]; adjusted HR 1.5 [CI 1.3-1.8]) and between cDPP3 levels and change in renal and liver SOFA score (p = 0.0077 and 0.0009, respectively). The higher the initial cDPP3 was, the greater the need for organ support and vasopressors upon admission; the longer the need for vasopressor(s), mechanical ventilation or RRT and the higher the need for fluid load (all p < 0.005). In patients with cDPP3 > 40.4 ng/mL upon admission, a decrease in cDPP3 below 40.4 ng/mL after 24 h was associated with an improvement of organ function at 48 h and better 28-day outcome. By contrast, persistently elevated cDPP3 at 24 h was associated with worsening organ function and high 28-day mortality. CONCLUSIONS: Admission levels and rapid changes in cDPP3 predict outcome during sepsis. Trial Registration ClinicalTrials.gov, NCT02393781. Registered on March 19, 2015.

Circulating dipeptidyl peptidase-3 at admission is associated with circulatory failure, acute kidney injury and death in severely ill burn patients.

BACKGROUND: Dipeptidyl peptidase-3 (DPP3) is a metallopeptidase which cleaves bioactive peptides, notably angiotensin II, and is involved in inflammation regulation. DPP3 has been proposed to be a myocardial depressant factor and to be involved in circulatory failure in acute illnesses, possibly due to angiotensin II cleavage. In this study, we evaluated the association between plasmatic DPP3 level and outcome (mortality and hemodynamic failure) in severely ill burn patients. METHODS: In this biomarker analysis of a prospective cohort study, we included severely ill adult burn patients in two tertiary burn intensive care units. DPP3 was measured at admission (DPP3admin) and 3 days after. The primary endpoint was 90-day mortality. Secondary endpoints were hemodynamic failure and acute kidney injury (AKI). RESULTS: One hundred and eleven consecutive patients were enrolled. The median age was 48 (32.5-63) years, with a median total body surface area burned of 35% (25-53.5) and Abbreviated Burn Severity Index (ABSI) of 8 (7-11). Ninety-day mortality was 32%. The median DPP3admin was significantly higher in non-survivors versus survivors (53.3 ng/mL [IQR 28.8-103.5] versus 27.1 ng/mL [IQR 19.4-38.9]; p < 0.0001). Patients with a sustained elevated DPP3 had an increased risk of death compared to patients with high DPP3admin but decreased levels on day 3. Patients with circulatory failure had higher DPP3admin (39.2 ng/mL [IQR 25.9-76.1] versus 28.4 ng/mL [IQR 19.8-39.6]; p = 0.001) as well as patients with AKI (49.7 ng/mL [IQR 30.3-87.3] versus 27.6 ng/mL [IQR 19.4-41.4]; p = 0.001). DPP3admin added prognostic value on top of ABSI (added chi2 12.2, p = 0.0005), Sequential Organ Failure Assessment (SOFA) score at admission (added chi2 4.9, p = 0.0268), and plasma lactate at admission (added chi2 6.9, p = 0.0086) to predict circulatory failure within the first 48 h. CONCLUSIONS: Plasma DPP3 concentration at admission was associated with an increased risk of death, circulatory failure, and AKI in severely burned patients. Whether DPP3 plasma levels could identify patients who would respond to alternative hemodynamic support strategies, such as intravenous angiotensin II, should be explored.

An N-terminal Flag-tag impairs TPP1 regulation of telomerase function.

The shelterin protein complex protects natural chromosome ends from being recognized as DNA damage sites and also regulates the synthesis of telomeric repeats by telomerase. TPP1, a shelterin subunit that is essential for telomerase extension of telomeres, has been studied intensively in recent years. Many such studies utilize epitope tagged TPP1, but it is unclear how the tags may affect the multiple cellular functions of TPP1. Here we analyzed the effect of adding a 3x Flag epitope tag to the N- or C-terminus of TPP1. While the position of the tag did not affect TPP1's interaction within the shelterin complex or its localization to telomeres, the N-terminal Flag tag on TPP1 impaired telomerase function, resulting in reduced telomerase processivity in vitro and a failure to stimulate telomere elongation in vivo. The C-terminally Flag-tagged TPP1, in contrast, behaved similarly to untagged TPP1 in all functional aspects examined. These findings suggest that caution is required when utilizing epitope tagged TPP1 to study its regulation of telomerase function.

The amino terminus extension in the long dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus.

The intracellular prolyl peptidase DPP9 is implied to be involved in various cellular pathways including amino acid recycling, antigen maturation, cellular homeostasis, and viability. Interestingly, the major RNA transcript of DPP9 contains two possible translation initiation sites, which could potentially generate a longer (892 aa) and a shorter version (863 aa) of DPP9. Although the endogenous expression of the shorter DPP9 form has been previously verified, it is unknown whether the longer version is expressed, and what is its biological significance. By developing specific antibodies against the amino-terminal extension of the putative DPP9-long form, we demonstrate for the first time the endogenous expression of this longer isoform within cells. Furthermore, we show that DPP9-long represents a significant fraction of total DPP9 in cells, under steady-state conditions. Using biochemical cell fractionation assays in combination with immunofluorescence studies, we find the two isoforms localize to separate subcellular compartments. Whereas DPP9-short is present in the cytosol, DPP9-long localizes preferentially to the nucleus. This differential localization is attributed to a classical monopartite nuclear localization signal (K(K/R)X(K/R)) in the N-terminal extension of DPP9-long. Furthermore, we detect prolyl peptidase activity in nuclear fractions, which can be inhibited by specific DPP8/9 inhibitors. In conclusion, a considerable fraction of DPP9, which was previously considered as a purely cytosolic peptidase, localizes to the nucleus and is active there, raising the intriguing possibility that the longer DPP9 isoform may regulate the activity or stability of nuclear proteins, such as transcription factors.

Structurally Diverse Phenolic Amides from the Fruits of Lycium barbarum with Potent α-Glucosidase, Dipeptidyl Peptidase-4 Inhibitory, and PPAR-γ Agonistic Activities.

A total of nine new phenolic amides (1-9), including four pairs of enantiomeric mixtures (3-5 and 8), along with ten known analogues (10-19) were identified from the fruits of Lycium barbarum using bioassay-guided chromatographic fractionation. Their structures were elucidated by comprehensive spectroscopic and spectrometric analyses, chiral HPLC analyses, and quantum NMR, and electronic circular dichroism calculations. Compounds 5-7 are the first example of feruloyl tyramine dimers fused through a cyclobutane ring. The activity results indicated that compounds 1, 11, and 13-17 exhibited remarkable inhibition against α-glucosidase with IC50 of 1.11-33.53 µM, 5-150 times stronger than acarbose (IC50 = 169.78 µM). Meanwhile, compounds 4a, 4b, 5a, 5b, 13, and 14 exerted moderate agonistic activities for peroxisome proliferator-activated receptor (PPAR-γ), with EC50 values of 10.09-44.26 µM. Especially,compound 14 also presented inhibitory activity on dipeptidyl peptidase-4 (DPPIV), with an IC50 value of 47.13 µM. Furthermore, the banding manner of compounds 14 and 17 with the active site of α-glucosidase, DPPIV, and PPAR-γ was explored by employing molecular docking analysis.

Interactions of the proteins of neuronal ceroid lipofuscinosis: clues to function.

Neuronal ceroid lipofuscinoses (NCL) are caused by mutations in eight different genes, are characterized by lysosomal accumulation of autofluorescent storage material, and result in a disease that causes degeneration of the central nervous system (CNS). Although functions are defined for some of the soluble proteins that are defective in NCL (cathepsin D, PPT1, and TPP1), the primary function of the other proteins defective in NCLs (CLN3, CLN5, CLN6, CLN7, and CLN8) remain poorly defined. Understanding the localization and network of interactions for these proteins can offer clues as to the function of the NCL proteins and also the pathways that will be disrupted in their absence. Here, we present a review of the current understanding of the localization, interactions, and function of the proteins associated with NCL.

Identification of 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones: a new class of potent, selective, and orally active non-peptide dipeptidyl peptidase IV inhibitors that form a unique interaction with Lys554.

The design, synthesis, and structure-activity relationships of a new class of potent and orally active non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones, are described. We hypothesized that the 4-phenyl group of the isoquinolone occupies the S1 pocket of the enzyme, the 3-aminomethyl group forms an electrostatic interaction with the S2 pocket, and the introduction of a hydrogen bond donor onto the 6- or 7-substituent provides interaction with the hydrophilic region of the enzyme. Based on this hypothesis, intensive research focused on developing new non-peptide DPP-4 inhibitors has been carried out. Among the compounds designed in this study, we identified 2-[(3-aminomethyl-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-6-isoquinolinyl)oxy]acetamide (35a) as a potent, selective, and orally bioavailable DPP-4 inhibitor, which exhibited in vivo efficacy in diabetic model rats. Finally, X-ray crystallography of 35a in a complex with the enzyme validated our hypothesized binding mode and identified Lys554 as a new target-binding site available for DPP-4 inhibitors.

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation.

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation.

Direct association of adenosine deaminase with a T cell activation antigen, CD26.

CD26, the T cell activation molecule dipeptidyl peptidase IV (DPPIV), associates with a 43-kilodalton protein. Amino acid sequence analysis and immunoprecipitation studies demonstrated that this 43-kilodalton protein was adenosine deaminase (ADA). ADA was coexpressed with CD26 on the Jurkat T cell lines, and an in vitro binding assay showed that the binding was through the extracellular domain of CD26. ADA deficiency causes severe combined immunodeficiency disease (SCID) in humans. Thus, ADA and CD26 (DPPIV) interact on the T cell surface, and this interaction may provide a clue to the pathophysiology of SCID caused by ADA deficiency.

Novel Methods for the Quantification of Dipeptidyl Peptidase 3 (DPP3) Concentration and Activity in Human Blood Samples.

BACKGROUND: The ubiquitously expressed dipeptidyl peptidase 3 (DPP3) is involved in protein metabolism, blood pressure regulation, and pain modulation. These diverse functions of DPP3 are attributed to the degradation of bioactive peptides like angiotensin II. However, because of limitations in currently available assays for determination of active DPP3 in plasma, the exact physiological function of DPP3 and its role in the catabolism of bioactive peptides is understudied. Here, we developed 2 assays to specifically detect and quantify DPP3 protein and activity in plasma and validated DPP3 quantification in samples from critically ill patients. METHODS: Assay performance was evaluated in a sandwich-type luminometric immunoassay (LIA) and an enzyme capture activity assay (ECA). DPP3 plasma concentrations and activities were detected in a healthy, population-based cohort and in critically ill patients suffering from severe sepsis and septic shock. RESULTS: The DPP3-LIA and DPP3-ECA show an almost ideal correlation and very similar and robust performance characteristics. DPP3 activity is detectable in plasma of predominantly healthy subjects with a mean (±SD) of 58.6 (±20.5) U/L. Septic patients show significantly increased DPP3 plasma activity at hospital admission. DPP3 activities further increase in patients with more severe conditions and high mortality risk. CONCLUSION: We developed 2 highly specific assays for the detection of DPP3 in plasma. These assays allow the use of DPP3 as a biomarker for the severity of acute clinical conditions and will be of great value for future investigations of DPP3's role in bioactive peptide degradation in general and the angiotensin II pathway in specific.

Changes in spatio-temporal localization of tripeptidyl peptidase II (TPPII) in murine colon adenocarcinoma cells during aggresome formation: a microscopy study based on a novel fluorescent proteasome inhibitor.

Extralysosomal proteolysis is a multistep process involving the Ubiquitin- Proteasome System (UPS) and supplementary peptidases. Tripeptidyl peptidase II (TPPII) is the most extensively characterized enzyme, supplementing and sometimes substituting for proteasomal functions. In response to proteasome inhibition, polyubiquitinated proteins acting as proteasome substrates aggregate with proteasomes and form aggresomes. Several proteasome inhibitors are used as anti-cancer drugs. Thus, in our study, we used a novel fluorescent-tagged proteasome inhibitor BSc2118 to induce aggresome formation in C26 murine colon adenocarcinoma cells. It allowed us to obtain effective, inhibitor-based, proteasome staining in vivo. This method has been validated by standard post-fixed indirect immunostaining and also allowed co-immunodetection of TPPII and polyubiquitinated proteins under laser scanning confocal microscopy. We found that in the absence of the inhibitor, TPPII is diffusely dispersed within the cytoplasm of C26 cells. The proteasome and ubiquitin-rich perinuclear region failed to display enhanced TPPII staining. However, when proteasome function was impaired by the inhibitor, TPPII associated more closely with both the proteasome and polyubiquitinated proteins via TPPII recruitment to the perinuclear region and subsequently into emerging aggresomal structures. Furthermore, we have demonstrated the dynamic recruitment of TPPII into the developing aggresome: TPPII in the early aggresome was dispersed within the central part but subsequently aggregated on the surface of this structure. In the mature aggresome of C26 cells TPPII formed a spherical mantle, which surrounded the round core containing proteasomes and polyubiquitinated proteins. Our morphological data indicate that TPPII displays spatial localization with proteasomes especially upon proteasome inhibition in aggresomes of C26 cells.

Alteration in dipeptidyl peptidase activities in cultured human carcinoma cells.

For the investigation of the possibility of its being a marker enzyme for tumor cells, the activity of dipeptidyl peptidase (DPP) IV (EC 3.4.14.5), a membrane-bound enzyme, in cultured human carcinoma cells was examined. The homogenates of three carcinoma cell lines (HeLa, KB, and K-44) contained lower glycylprolyl methylcoumarinamide (Gly-Pro-MCA) hydrolase activities at pH 8.7 (assumed to be DPP IV) and higher activities of alkaline phosphatase and gamma-glutamyl transpeptidase, which are also membrane-bound enzymes, than those of normal human fibroblasts (HF). Examination of carcinoma cells for the subcellular localization and pH optimum of Gly-Pro-MCA hydrolase activity revealed that the activity of a lysosomal enzyme that hydrolyzes Gly-Pro-MCA at pH 6.4 was markedly increased in carcinoma cells, but not in normal cells. The separation and characterization of Gly-Pro-MCA hydrolases by gel filtration, affinity chromatography, and substrate specificity demonstrated that HF have three peaks indicating DPP IV, DPP II, and an unknown enzyme, whereas the three carcinoma cell lines gave a prominent peak indicating DPP II and a trace of DPP IV. The DPP II activity was 6- to 24-fold higher in carcinoma cell lines than in HF, and it also was 2.85- to 4.13-fold higher than the DPP IV activity in carcinoma cell lines but was 10-fold lower in HF. These clear enzymatic differences between carcinoma cells and normal HF may be useful as a marker of malignancy.

Effect of growth and sodium butyrate on brush border membrane-associated hydrolases in human colorectal cancer cell lines.

The activities of brush border membrane-associated hydrolases such as alkaline phosphatase (Alkpase), aminopeptidase, dipeptidyl aminopeptidase IV (DAP-IV), sucrase, lactase, and trehalase were studied in 14 different human colorectal cancer cell lines. The effect of sodium butyrate, a known differentiating agent, and cell growth on the activities of these enzymes was also examined. All 14 cell lines exhibited brush border membrane enzyme activities, and in general, the activity of Alkpase, aminopeptidase, and DAP-IV was much higher than the disaccharidases. However, the specific enzyme activities varied among different cell lines. The induction of Alkpase activity by sodium butyrate occurred in most of the 14 cell lines (2- to 123-fold), while induction of the other enzyme activities was observed in several (1.5- to 3.5-fold). In some instances, butyrate caused a decrease in enzyme activity. There was no statistically significant correlation between the induction of Alkpase activity and that of other enzyme activities by sodium butyrate. Levels of aminopeptidase and DAP-IV activity were found to be dependent on cell density and increased 3- to 4-fold by the tenth day in most of the cell lines. Sodium butyrate altered the subcellular distribution pattern of the disaccharidases, causing a significant increase in activity associated with the soluble (cytoplasmic) fraction. Other enzymes such as Alkpase and DAP-IV continued to be predominantly associated with the membrane fraction in butyrate-treated cells. These data suggest that brush border membrane hydrolase activity and the effect of sodium butyrate may provide useful information regarding the differentiation of human colorectal cancer cells.

[Autoimmune encephalitis due to mantle cell lymphoma]. / Auto-immune encefalitis door een mantelcellymfoom.

BACKGROUND: Autoimmune encephalitis is a rare condition characterized by subacute development of cognitive and psychiatric symptoms. A paraneoplastic syndrome involves autoimmune encephalitis caused by classic antibodies. Although this condition is often associated with cancer, no malignancy has yet been found in 70-90% of patients at the time of diagnosis. CASE DESCRIPTION: We saw a 58-year-old male patient with fatigue, diarrhoea and weight loss. He was also experiencing hyperekplexia, personality changes and an instable gait. PET-CT revealed generalised lymphadenopathy. Histopathological analysis of a lymph node showed mantle cell lymphoma. Further investigation of the fluid revealed anti-DPPX IgG antibodies. We treated the patient's mantle cell lymphoma with R-CHOP; he achieved complete remission and his neurological symptoms resolved almost completely. CONCLUSION: The presence of anti-DPPX IgG antibodies is rare. Although it has not been proven that these antibodies are related to malignancies, this is the third of 30 known cases in which anti-DPPX IgG antibodies and a lymphatic malignancy were found.

Comparison of dipeptidyl peptidase IV prepared from pig liver and kidney.

Dipeptidyl peptidase IV (dipeptidylpeptide hydrolase, EC 3.4.14.-) has been purified from the microsomal fraction of pig liver, using an immunoaffinity chromatography, and its properties compared with those of the enzyme purified from pig kidney. The amino acid compositions of both enzymes were similar. The same kinds of carbohydrates were found in both enzymes, but there were differences in the molar concentrations of individual sugars. The liver enzyme had greater concentrations of mannose, fucose and sialic acid than the kidney enzyme, while the concentrations of galactose and glucosamine were greater in the kidney enzyme. The carbohydrates accounted for approx. 18.3 and 22.7% of the weight of the kidney and liver enzymes, respectively. The pH optima, molecular weights, substrate specificities and Km values of the two enzymes and the effects of diisopropylfluorophosphate on their activities were nearly identical. The liver enzyme was heat- and pH-sensitive, but not attacked by proteinases.

Effect of triton X-100 on the electrophoretic mobility of solubilized intestinal brush border membrane dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (dipeptidylpeptide hydrolase, EC 3.4.14.-) was solubilized from a rat intestinal mucosal brush border membrane preparation in varying concentrations of Triton X-100. Samples of a solubilized supernatant fraction were subjected to polyacrylamide gel electrophoresis, and the activities of several brush border enzymes were measured in gel slices following elution of the enzymes from the gel. At low concentrations of Triton X-100 (0.5%), two peaks of dipeptidyl peptidase IV activity were observed suggesting the presence of two electrophoretically distinct enzymes. However with increasing Triton X-100 concentrations, the slower migrating species was converted to a faster migrating form and only one major band of activity was observed at 10% Triton. These results indicate that there is only one form of the enzyme and that care must be taken when interpreting the electrophoretic patterns of detergent solubilized membrane bound enzymes.

Coronary vascularization during development in the rat and its relationship to basic fibroblast growth factor.

OBJECTIVE: Our overall aims were to elucidate the temporal and spatial sequence of coronary vascularization during development in the rat, and to determine whether basic fibroblast growth factor expression corresponds to any phase of the vascularization process. METHODS: Immunohistochemical, histochemical, morphometric and in situ hybridization analyses were performed on prenatal and postnatal hearts of various ages. RESULTS: Coronary vascularization, which begins at embryonic day 13 (E13) with blood island-like structures in the epicardium, progresses from this layer toward the endocardium as indicated by a transmural gradient of vascular volume throughout the ventricles. Vascular smooth muscle first appears in E17 hearts at the time a capillary-like plexus coalesces and penetrates the aorta to form the main coronary arteries. These vessels maintain an anastomatic morphology and must undergo subsequent remodeling in order to assume adult branching characteristics. The early postnatal period is characterized by development of the arterial tree and the enzymatic differentiation of the arteriolar and venular ends of the capillary bed. Although bFGF is expressed both prenatally and postnatally, the highest mRNA expression was noted during the early period of vascularization (E14 and E15), and the early neonatal period (1-6 days) which corresponds to a period of substantial microvascular growth. CONCLUSIONS: Coronary vascularization follows a temporal sequence which includes transmural expansion of the capillary bed, arteriolar formation subsequent to vascular penetration of the aorta, and postnatal growth, differentiation, and remodeling. Since high levels of bFGF expression are correlated with key time points in coronary vascular growth, bFGF may play an important role in this process.

Is there a direct correlation between the activities of various lysosomal enzymes and prolactin secretion in the rat anterior pituitary?

The degree of endocrine activity by rat pituitary lactotrophs was manipulated and the lysosomal involvement in the release and intracellular degradation of PRL was monitored by concomitant assays of acid phosphatase, beta-glucuronidase, dipeptidyl peptidases I and II, and nonspecific esterases in the anterior lobe. The in vitro release of PRL by cell cultures was inhibited by 0.5 or 1.5 microM bromocriptine during 24 or 72 h of incubation and by 0.75 microM for 4 h. Long term treatment caused a 40% reduction in the total PRL content of cells and media; however, after 4 h of bromocriptine treatment, no reduction in PRL content was found. TRH (10 ng/ml) in medium for 4 h increased PRL release, whereas it produced intracellular hormone depletion. In vitro treatment of anterior lobes of diestrous rats with 1 microM dopamine decreased PRL release by 50%. No changes in lysosomal enzyme activities were observed after the inhibition of release or stimulation of the in vitro secretion of PRL. Haloperidol (2.5 mg/kg BW) caused a 90% increase in serum PRL concentrations in diestrous rats 1 h after sc injection. alpha-Methyl-p-tyrosine (200 mg/kg BW, ip) stimulated in vivo PRL secretion, monitored 1, 4, and 24 h after drug administration. The administration of 100 micrograms/rat bromocriptine for 3 consecutive days, a single dose of 100 micrograms/rat polyestradiol phosphate, and their combination resulted in the expected changes in PRL production in female rats. These in vivo treatments failed to alter lysosomal enzyme activities in the anterior pituitary. These findings suggest that the release, intracellular accumulation, or depletion of PRL occurred without concomitant changes in lysosomal enzyme activity in the anterior pituitary. We suggest that the system of lysosome-dependent hormone degradation may involve more specific enzymes than those monitored to date.