An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

Stromal populations and fibrosis in human long-term bone marrow cultures.

An immunofluorescence study of the adherent layer of human long-term bone marrow cultures (HLTBMC) revealed the following surface markers on the different stromal cell populations: stromal fibroblastic cells CD10+, FIB86.3+, CD13+, CD71+; adipocytes CD10+, FIB86.3-, CD13+, CD71-/+; and macrophages CD10-/+, FIB86.3+, CD13+, CD71-/+, CD14+, CD33+, CD25+, HLA-DR+, CD4+, CD19+, CD45+. The markers of the stromal fibroblastic cells in HLTBMC were similar to those of twice-passaged fibroblasts not only from bone marrow and spleen, but also from a hemopoietic non-supportive organ such as the skin. Some of the cultured human umbilical vein endothelial cells used as controls were found to be CD25+, demonstrating for the first time the interleukin-2 receptor p55 chain on normal non-hemopoietic cells. The stromal fibroblastic cells are overrepresented compared to the small non-macrophage hemopoietic cell population in the adherent layer of HLTBMC. In addition, silver staining revealed an increased reticulin content in most of the HLTBMC. An excessive growth of stromal fibroblastic cells and an excessive deposition of their product, the reticulin fibers, are the hallmark of myelofibrosis. The finding of equivalent observations in HLTBMC suggests that the hitherto unexplained, premature quenching of hemopoiesis in HLTBMC might at least partly be due to mechanisms similar to those operating in myelofibrosis in vivo.

Costimulation of CD4+ and CD8+ T cells through CD26: the ADA-binding epitope is not essential for complete signaling.

It was previously shown that CD26 (DPP IV, EC 3.4.14.5) is a binding site for adenosine deaminase (ADA, EC 3.5.4.4) on T cells and that costimulation by some anti-CD26 monoclonal antibodies (mAbs) and anti-CD3 induces CD4+ T cell proliferation. The CD26 epitopes involved in costimulation, the precise sequence of the events preceding proliferation, and the response of CD8+ compared with CD4+ T cells to CD26 were not extensively studied. We therefore compared the effects of the novel TA5.9 anti-CD26 mAb, recognizing an ADA-binding epitope, and the clearly distinct anti-Ta1 reference anti-CD26 mAb for their costimulatory properties in various T cell subsets. Both purified CD4+ and CD8+ T cells proliferated upon costimulation with anti-CD3 and either anti-CD26 mAb, but anti-TA5.9 mAb induced a more potent response than anti-Ta1. Either anti-CD26 mAb, together with anti-CD3, caused a similar sequential up-regulation of CD69, CD25 (IL-2R alpha), and CD71 (transferrin receptor) expression on CD4+ and CD8+ T cells. The activation markers appeared faster on the CD45R0+ than on the CD45R0- subsets. After costimulation, CD4+ T cell cultures contained significant amounts of the Th1 cytokines IL-2, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). In CD8+ T cell cultures relatively more IFN-gamma and TNF-alpha but almost no IL-2 was measured after triggering of CD3 and CD26. Our data demonstrate that the recognition of the ADA-binding epitope is not a prerequisite for the costimulatory capacity of anti-CD26 mAbs. Both CD4+ and CD8+ T cells and their CD45R0- and CD45R0+ subsets are sensitive to various aspects of activation via CD26, but the magnitude and/or kinetics differ according to the anti-CD26 used and the T cell subset studied.

Decreased serum dipeptidyl peptidase IV activity in major depression.

It has been recently shown that severe depression is characterized by immune dysfunctions such as blunted mitogen-induced blast transformation, which is linked to interleukin-2 (IL-2) mechanisms, and to autoimmune responses. In order to explore one of the putative pathophysiological mechanisms underlying both factors, we have measured the predexamethasone and postdexamethasone serum dipeptidyl-peptidase IV (DPP IV) activity in depressed inpatients and normal controls. This enzyme is an important mediator of IL-2-related blast proliferation, and it may play a role in autoimmunity. We found significantly lower DPP IV levels in major depressives as compared with healthy controls, and melancholics exhibited significantly lower enzyme activity than minor depressives. There was a significant negative correlation between serum DPP IV activity and the severity of illness. However, we were unable to detect any significant relationships between DPP IV on the one hand, and mitogen-induced blast transformation, soluble IL-2 receptor accumulation in PHA culture supernatant, total number of leukocytes and lymphocytes, T lymphocytes, CD4+ and CD25+ cells, on the other. Men exhibited significantly higher serum DPP IV levels than women.

Cloning and sequence analysis of the gene encoding human lymphocyte prolyl endopeptidase.

The human cDNA encoding prolyl endopeptidase, a cytoplasmic endoprotease which hydrolyses the peptide bond at the C-terminal side of proline, was sequenced. After the isolation of the 3' terminal fragment of the pep cDNA sequence from a human lymphocyte cDNA library, an approach based on the polymerase chain reaction (PCR) was undertaken to obtain the complete pep cDNA. Overlapping DNA fragments were generated by PCR from cDNA synthesized from human lymphocyte mRNA. The DNA fragments were subcloned and sequenced. The complete cDNA is 2562 nucleotides (nt) in length and contains an open reading frame coding for a protein of 710 amino acids (aa). Comparison of the primary PEP sequences from human lymphocyte and pig brain shows 97% identify. The aa sequence analysis shows homology with bacterial PEPs and with protease II from Escherichia coli. Asp641 probably participates in the active site of PEP.

DNA fingerprints revealing common and divergent human DNA methylation patterns.

We compared DNA fingerprints of different cell populations from the same individuals, after separate digestion with the isoschizomers MboI and Sau3A. Methylation differences were observed within every individual when comparing fingerprints of Sau3A- with MboI-digested DNA, and of Sau3A-digested sperm with somatic DNA. In some cases, differences were also detected between fingerprints of Sau3A-digested somatic DNA originating from various cell sources. Methylation patterns common to all cell populations examined, including the germline, were observed with a higher frequency than divergent ones. These 'common methylations' are most likely to find their origin during early embryogenesis.

Decreased expression of the memory marker CD26 on both CD4+ and CD8+ T lymphocytes of HIV-infected subjects.

Using a novel anti-CD26 (or anti-dipeptidyl peptidase IV) monoclonal antibody, we showed that the absolute numbers and the proportions of T4 and T8 cells expressing CD26 were significantly lower in HIV-infected persons than in controls. The absolute number of CD26+ T4 cells decreased according to disease progression, whereas the number of CD26+ T8 cells was low throughout all clinical stages. These trends were similar in CD26 dim and bright positive T-cell subsets. In both controls and HIV-positive subjects, the CD26 bright positive T cells were restricted to the CD45RO+ subset and preferentially co-expressed CD25 but largely lacked HLA-DR and CD38. Recall antigen-responsive cells from seronegative individuals were shown to co-express CD26 and CD45RO. The deficient CD26 expression on T8 cells from HIV-infected subjects could be normally upregulated after in vitro stimulation. In contrast to decreased T-cell-bound CD26, the enzymatic activity of plasma CD26/dipeptidyl peptidase IV was unchanged in HIV-infected patients compared with controls. We conclude that HIV infection leads to a deficient in vivo co-expression of CD26 bright and CD45RO on T cells. We speculate that this deficiency might play a part in the decrease of immunological memory during HIV infection.

Proteases and their inhibitors: today and tomorrow.

A major incentive in inhibitor research is that control of limited proteolysis constitutes a valuable pharmacological tool. Protease inhibitors have proved to be successful in influencing pathogenesis in many experimental models but a breakthrough to use in human therapy has mainly been restricted to aprotinin and angiotensin converting enzyme (ACE) inhibitors. However, the success of ACE inhibitors as pharmacological tools in hypertension has proved to be a strong stimulant for new protease inhibitor approaches to drug therapy. While emphasis in the search for next generations of ACE inhibitors may move from the circulation renin-angiotensin system to the local tissue systems, including heart, brain and genital tract, persistent and insightful design of renin inhibitors has already yielded highly specific molecules with potent activities in several in vivo models. The development of orally effective long-acting inhibitors will finally allow an evaluation to be made of their therapeutic profile with regard to the family of ACE inhibitors. The close relationship between renin and HIV-1 protease presents an exceptional opportunity for transfer of the knowledge acquired in renin inhibitor development during the past decade, to an accelerated generation of specific HIV-1 protease inhibitors as effective agents in treatment of AIDS. The self-assembly of 2 identical monomers into a symmetrical structure in HIV-1 protease is not only an elegant way to create an active enzyme while encoding a minimal amount of genetic information, but is also in concordance with the bilobular active-site found in mammalian aspartic proteases.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of immobilized adenosine deaminase (EC 3.5.4.4) for the rapid purification of native human CD26/dipeptidyl peptidase IV (EC 3.4.14.5).

The leukocyte differentiation antigen CD26 identified as dipeptidyl peptidase IV.(EC 3.4.14.5), cleaves off N-terminal dipeptides from peptides when a proline or alanine is located at the penultimate position. Seminal plasma and especially prostasomes, prostate-derived organelles which occur freely in seminal plasma, contain high amounts of CD26/dipeptidyl peptidase IV and therefore are suitable sources for the purification of the protein. The use of adenosine deaminase (EC 3.5.4.4) affinity chromatography for its purification is described. CD26/dipeptidyl peptidase IV was purified from human seminal plasma and prostasomes by a two step procedure. Ion exchange chromatography on DEAE-Sepharose, followed by affinity chromatography on adenosine deaminase-Sepharose resulted in the pure, native protein with an overall yield ranging from 35 to 55%. The N-terminal sequence of the amphiphilic enzyme purified from human prostasomes was determined to be Met-Lys-Thr-Pro-Trp-Lys-Val-Leu. The preparation obtained was free of contaminating aminopeptidase activity and proved to be very stable (up to 1 month at 37 degrees C). The calf intestinal adenosine deaminase we used is commercially available and can be employed for the purification of human, bovine and rabbit CD26/dipeptidyl peptidase IV. High affinity binding of porcine dipeptidyl peptidase IV was not observed. The availability of a source with high specific activity and the introduction of adenosine deaminase affinity chromatography permits the rapid purification of milligram quantities of natural mammalian CD26/dipeptidyl peptidase IV.

Kininase activity in human platelets: cleavage of the Arg1-Pro2 bond of bradykinin by aminopeptidase P.

A proline-specific peptidase aminopeptidase P (APP, EC 3.4.11.9) that cleaves the Arg1-Pro2 bond of bradykinin was isolated from human platelets by liquid chromatography. The enzyme was purified 557 times. The native molecule has a M(r) of 223,000. Human platelet APP exists as a trimer with a subunit M(r) of 71,000. The apparent Km of platelet APP is 66 mumol/L for bradykinin and 47 mumol/L for the internally quenched fluorogenic substrate Lys (2,4-dinitrophenyl)-Pro-Pro-NH-CH2-CH2-NH-2-aminobenzoyl. 2HCl which is used for the routine determination of the enzyme activity. The optimum pH for hydrolysis of the fluorogenic substrate is 8.0, and the optimum temperature is 43 degrees. Platelet APP is inhibited by 1,10-phenanthroline and activated by Mn2+, thus confirming its metalloprotease nature. Cu2+, Zn2+ and Hg2+ are strongly inhibitory. Inhibition by cysteine protease inhibitors suggests the presence of a thiol group essential for enzymatic activity. Serine protease inhibitors do not affect the enzyme activity.

Antibody binding profile of purified and cell-bound CD26. Designation of BT5/9 and TA5.9 to the CD26 cluster.

The CD26 activation antigen (Ag) which is expressed on a subpopulation of human T cells has been characterized as dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). In this paper, we describe the antibody binding profile of CD26/DPP IV, purified from human peripheral blood lymphocytes. The purified molecule binds to the anti-Ta1, anti-1F7 and anti-134-2C2 monoclonal antibodies (mAb), reported to react with cell-bound CD26 Ag. Among unclustered mAb recognizing T cell antigens, two, anti-BT5/9 and anti-TA5.9 were found to react with purified and cell-bound CD26 Ag. The classification of the BT5/9 Ag, the functional properties of the BT5/9+ T cell subset, as well as the in vivo effect of anti-BT5/9 mAb administration, are re-interpreted in the light of its specificity. Applying the anti-TA5.9 mAb in three color FACS analyses, we demonstrated that CD26+bright cells co-express CD45RO but not HLA-DR and CD38.

Lower serum dipeptidyl peptidase IV activity in treatment resistant major depression: relationships with immune-inflammatory markers.

Previous research in this laboratory has shown that major depression is accompanied by decreased serum activity of dipeptidyl peptidase IV (DPP IV), a serine protease that cleaves N terminal dipeptides from peptides with penultimate proline or alanine. DPP IV is involved in the metabolism of peptides, T cell activation and proliferation, including the production of cytokines, such as interleukin-1 (IL-1) and IL-2. The aim of this study was to examine (i) serum DPP IV activity in major and treatment resistant depression (TRD) in relation to other established immune and inflammatory markers of that illness, and (ii) the effects of antidepressive treatment on DPP IV activity. Serum DPP IV activity was significantly lower in major depression and TRD than in normal controls. In normal and major depressed subjects, there were significant and positive relationships between serum DPP IV activity and total serum protein, serum albumin, zinc, iron and transferrin. In the group of depressed subjects, there were significant and positive relationships between serum DPP IV activity and number of CD4+T cells and CD4+/CD8+ T cell ratio. There were no significant effects of subchronic treatment with antidepressants on serum DPP IV activity. The findings suggest that: (i) lower serum DPP activity may occur in chronic depression, TRD as well as in the acute phase of major depression; (ii) lower serum DPP IV accompanies the 'chronic' acute phase response in depression; and (iii) serum DPP IV activity is tightly coupled to increased number of CD4+ T cells in depressed subjects, but not in normal controls. Our results do not exclude the possible effects of longer-term treatment with antidepressants on serum DPP-IV activity.

Localization and characterization of aminopeptidase P in bovine adrenal medulla.

Aminopeptidase P (EC 3.4.11.9) is demonstrated for the first time in the cytosolic fraction of chromaffin cells of the bovine adrenal medulla. The enzyme is inhibited by metal chelators and by sulfhydryl-reactive agents, which suggests that both a tightly bound metal ion and a cysteine residue are necessary for enzymatic activity. Aminopeptidase P might be important for the modulation of the biological activity of neuropeptides. Its occurrence in the adrenal chromaffin cells provides a useful tool for studying the function of this unique proline-specific peptidase in neuropeptide processing and secretion.

Aminopeptidase P and dipeptidyl peptidase IV activity in human leukocytes and in stimulated lymphocytes.

Human white blood cells were shown to contain high aminopeptidase P activity. The specific activities found in the high-speed supernatant of the extracts of granulocytes, lymphocytes and monocytes ranged from 30 to 70 units per mg protein. Culturing lymphocytes during 7 days in the presence of phytohaemagglutinin resulted in a 70-200% increase in the specific aminopeptidase P activity and a 200% increase in the specific activity of dipeptidyl peptidase IV. The time-course of the activity of both aminopeptidase P and dipeptidyl peptidase IV during the stimulation of human T-lymphocytes by phytohaemagglutinin indicates an involvement of these two enzymes in the proliferative process of these immunocompetent cells. Due to their substrate specificity their potential substrates must have the N-terminal Xaa-Pro sequence known to be present in several immunologically important polypeptides.

Characterization of dipeptidyl peptidase IV (CD26) from human lymphocytes.

The membrane-bound dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) has been purified 5,400-fold from human peripheral blood mononuclear cells. The purification procedure included detergent solubilization and successive chromatography on DEAE Sepharose Fast Flow, Con A Sepharose, Cu2+ loaded metal-chelating Sepharose, Sephacryl S-300 High Resolution and Q Sepharose Hiload. The molecular mass of the native, detergent solubilized enzyme estimated by gel filtration was 264.kDa. Chromatofocusing indicated a pI of approximately 5.0. The pI optimum was 8.7. The enzymatic activity of the purified preparation was irreversibly inhibited by N-(H-Phe-Pro)-O-(4-nitrobenzoyl)hydroxylamine hydrochloride in the micromolar range. The binding of purified DPP IV to CD26 monoclonal antibodies confirmed the identity between CD26 and dipeptidyl peptidase IV. The purification and characterization of lymphocytic dipeptidyl peptidase IV is of great value for the identification of its natural substrates and for the study of its physiological significance in the T-lymphocyte function.

Exopeptidases in human platelets: an indication for proteolytic modulation of biologically active peptides.

The determination in human platelets of four exopeptidases--aminopeptidase P, dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme--by means of fluorometric or liquid chromatography techniques was carried out. The results obtained show that the specific activities of dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme in intact and disrupted platelets are small compared to their specific activities in serum. However, for aminopeptidase P the specific activity of this enzyme is much higher in platelets than in serum. This suggests that circulating platelets may have a significant role as scavengers for circulating peptides containing bonds susceptible for aminopeptidase P.

Living with locked-in syndrome: an explorative study on health care situation, communication and quality of life.

PURPOSE: to explore and describe the health care situation, the use of aids, the way of communicating and the quality of life of Locked-in syndrome patients in Flanders (Belgium) and to collect information on their fulfilled and unfulfilled needs. METHOD: in depth interviews with eight LIS-patients by means of an extensive questionnaire consisting of five parts: (i) general information and medical history, (ii) health care, rehabilitation and follow-up, (iii) speech and communication, (iv) quality of life, (v) needs and problems experienced. RESULTS: the patients' condition, mostly caused by ischemic stroke, persisted for a mean period of 6 years 8 months. Their mean age was 41;10 years. At the moment of our study all the patients were living at home. Care was provided by an extensive care team. Some recovery of head and neck movements was mentioned, recovery of upper and lower limb mobility however was very limited. Most patients use an alphabet system to communicate, all of them had access to and made use of a PC with internet connection. Except for the domain of physical functioning, the quality of life scores of our patient group are rather high. As for unfulfilled needs, half of the patients experience a lack of information on their condition and a lack of appropriate information on (communication) aids. CONCLUSION: most results seem to be in line with those of other studies, though larger scale and follow-up studies are needed to confirm these findings.

A sensitive method for the assay of serum prolyl endopeptidase.

A new fluorimetric assay for the determination of prolyl endopeptidase (EC 3.4.21.26) was developed. The synthetic substrate Z-glycyl-prolyl-4-methylcoumarinyl-7-amide (0.2 mmol/l; 5 microliters), K-phosphate buffer (100 mmol/l, pH 7.5; 100 microliters), and serum (10 microliters) are incubated for 120 min at 37 degrees C. The reaction is stopped with acetic acid (1.5 mol/l; 500 microliters) and the released 7-amino-4-methylcoumarin is measured fluorimetrically. The mean value of prolyl endopeptidase catalytic activity concentration in serum for 120 healthy volunteers was 0.455 (SD = 0.092) mumol of 7-amino-4-methylcoumarin released per litre of serum per minute. The proposed procedure is sensitive, robust, and economical.

Distribution of prolyl oligopeptidase in human peripheral tissues and body fluids.

Prolyl oligopeptidase (EC 3.4.21.26) activity was measured in human tissue homogenates and body fluids. The enzyme was ubiquitously present, revealing high activity in renal cortex, epithelial cells, fibroblasts, testis, lymphocytes and thrombocytes. The activity in the body fluids was low. Prolyl oligopeptidase activity was significant higher in tumours of prostate, lung and sigmoid, than in the healthy tissues. Sera of individuals suffering from HIV infection, malaria, prostate cancer or benign prostate hypertrophy contained lowered activity. Interestingly, the low serum activity during prostate carcinoma increased upon medical treatment with anti-androgens. This suggests hormonal control of the gene transcript. A positive correlation with angiotensin converting enzyme activity in hypertensive patients was demonstrated and this further supports the possible involvement of prolyl oligopeptidase in the renin-angiotensin system and in the pathogenesis of hypertension.