Use of flavour profile and consumer panels to determine differences between local water supplies and desalinated seawater.

The San Diego County Water Authority of California has initiated planning for coastal desalination facilities to augment their water supplies. Integration of the different water qualities from these facilities into existing pipelines must be achieved. This investigation determined whether, and to what degree, consumers can discriminate between desalinated seawater and imported water supplies and how these investigations can contribute to decision making regarding the need for construction of facilities to blend such supplies prior to delivery. Based upon the results of the flavour profile analysis panel and the consumer evaluation sessions, it was concluded that free chlorine versus chloramine disinfection or different concentrations of disinfectants did not significantly affect consumer perception of the taste and odour of desalinated seawater or blends with Colorado River water and State project water. Consumers were able to discern between desalinated seawater and imported water, preferring imported water when forced to make a choice. However, the investigators did not believe that the difference in consumer perception was significant enough to warrant special blending facilities to mitigate the relatively minor aesthetic quality differences between imported water supplies and desalinated seawater.

Characteristics of salt taste and free chlorine or chloramine in drinking water.

Salty taste with or without chlorine or chloramine flavour is one of the major consumer complaints to water utilities. The flavour profile analysis (FPA) taste panel method determined the average taste threshold concentration for salt (NaCl) in Milli-Q water to be 640 +/- 3 mg/L at pH 8. Chlorine and chloramine disinfectants have no antagonistic or synergistic effects on the taste of NaCl, salt, in Milli-Q water. The flavour threshold concentrations for chlorine or chloramine in Milli-Q water alone or in the presence of NaCl could not be estimated by the Weber-Fechner curves due to the chlorine or chloramine flavour outliers in the 0.2-0.8 mg/L concentration range. Apparently, NaCl is not equilibrated with the concentration of ions in the saliva in the mouth and the concentration of free chlorine or chloramines cannot be tasted correctly. Therefore, dechlorinated tap water may be the best background water to use for a particular drinking water evaluation of chlorine and chloramine thresholds. Laboratory FPA studies of free chlorine found that a 67% dilution of Central Arizona Project (CAP) (Tucson, AZ) water with Milli-O water was required to reduce the free chlorine flavour to a threshold value instead of a theoretical value of 80% (Krasner and Barrett, 1980). No synergistic effect was found for chlorine flavour on the dilution of CAP water with Milli-Q water. When Central Avra Valley (AVRA) groundwater was used for the dilution of CAP water, a synergistic effect of the TDS present was observed for the chlorine flavour. Apparently, the actual mineral content of drinking water, and not just NaCl in Milli-Q water, is needed for comparative flavour tests for chlorine and chloramines.

Life satisfaction among kidney transplant recipients: demographic and biological factors.

PURPOSE: The Transplant Learning Center is a program providing education and support for solid-organ transplant recipients taking cyclosporine (Neoral or Sandimmune). One goal of the program is to improve patients' quality of life, which may be influenced by demographic and biological factors, and in turn influences adherence to prescribed medication regimens. We analyzed the results from the initial survey of enrollees to better understand life satisfaction and to test the validity and reliability of the satisfaction scale used in the program. METHOD: Patients enter the program through self-selection: all enrollees who received a kidney transplant were included in this analysis. Satisfaction was measured using the Life Satisfaction Index, which includes 8 questions about aspects of satisfaction with the patient's life. Associations between the Life Satisfaction Index and demographic factors, comorbid medical conditions, adverse effects of immunosuppressants, and medication compliance were examined. RESULTS: All 3676 kidney transplant recipients who completed the initial survey were included. Mean satisfaction scores were highest in persons who were older than 64 years, men, and those who were married. Satisfaction scores were positively correlated with education and income. Mean satisfaction score was significantly lower among persons with medical comorbidities, persons who reported that adverse effects of medications were more frequent, and persons who reported noncompliance with their medication regimen. CONCLUSION: The Life Satisfaction Index is a transplant-specific measure of health-related quality of life that can be used to help detect areas of potential concern in the clinical management of kidney transplant recipients.

When knowing more means less: the effect of fan on metamemory judgments.

The authors used a "fan" paradigm (J. R. Anderson, 1974) to test the accessibility and competition models of metamemory using judgments of learning (JOLs). JOLs in this study reflect one's confidence level in subsequently recognizing newly learned material. The number of facts, or "fan," associated with JOL-queried concepts varied from 1 to 3 associates. Results of 3 experiments indicated that as the level of fan increased, the magnitude of JOLs decreased. This finding was observed even when the fan effect (i.e., slower recognition as number of facts increase) was attenuated on a verification task in 2 of the experiments by manipulating the organization of the multiple concepts. The results supported the competition hypothesis (T. A. Schreiber, 1998; T. A. Schreiber & D. L. Nelson, 1998) as an important determinant of JOLs.

Dipeptidyl peptidase I and granzyme A are coordinately expressed during CD8+ T cell development and differentiation.

Dipeptidyl peptidase I (DPPI) is a granule protease that plays a requisite role in processing the proenzyme form of the CTL granule serine proteases (granzymes). This study assesses DPPI mRNA and enzyme expression during T lymphocyte ontogeny and CTL differentiation. The most immature CD3- CD4- CD8- thymocytes were found to express >40-fold higher levels of DPPI mRNA, although levels of DPPI enzymatic activity in CD3- CD4- CD8- thymocytes were only modestly higher than those seen for CD4+ CD8+ or CD4+ CD8- thymocytes. More mature CD8+ CD4- thymocytes and CD8+ splenocytes expressed significantly higher levels of DPPI mRNA and enzymatic activity than CD4+ CD8+ or CD4+ CD8- thymocytes. Granzyme A mRNA expression was observed in DPPI expressing CD3- CD4- CD8- and CD8+ CD4- thymocytes and was also observed in CD8+ CD4- splenocytes; however, expression was not observed in CD4+ CD8+ or CD4+ CD8- thymocytes. Both DPPI mRNA and granzyme A mRNA expression in CD8+ T cells decreased to very low or undetectable levels during the first 48 h after allostimulation in MLCs. However, peak levels of both DPPI and granzyme A expression were observed later in the course of CD8+ T cell responses to alloantigen, with DPPI mRNA expression peaking on either day 3 or day 4 and granzyme A expression peaking at the end of a 5-day MLR. These data indicate that DPPI is expressed at all stages of T cell ontogeny and differentiation in which granzyme A mRNA is detected; consequently, DPPI appears to be available for the processing and activation of granzyme A during both CD8+ T cell development and differentiation.

Documenting progress in home care.

The home is an ideal environment for the practice of occupational therapy. However, the tumultuous health care environment of the late 1990s requires practitioners to take special care not only in delivering effective services, but also in documenting the delivery of skilled care. Documentation is the bridge between the delivery of occupational therapy services in the home and the approval for reimbursement of services by third-party payers. This article presents principles for writing reimbursable progress notes for home care that are based on Medicare documents related to occupational therapy. Application of these principles can improve efficiency and excellence in practitioners' documentation skills.

A selective inhibitor of dipeptidyl peptidase I impairs generation of CD8+ T cell cytotoxic effector function.

CTL express high levels of dipeptidyl peptidase I (DPPI), a granule thiol protease able to convert the zymogen precursors of granzymes A and B into active proteases. In the present studies, the effects of specific inhibition of DPPI on generation of CTL effector functions were examined. When T cell DPPI activity was inhibited by >95% throughout 5-day MLC, a significant reduction in the generation of CD8+ T cell BLT esterase activity (<30% of control) and cytolytic activity (<10% of control) was observed. DPPI inhibition during the second to fourth days of 5-day MLC also was associated with reduced proliferation of CD8+ T cells, but had no effect on CD4+ T cell proliferation or IL-2 production by either population. CTL generated in the continuous presence of DPPI inhibition also exhibited impaired lysis of anuclear erythrocyte targets and diminished killing of nucleated targets by perforin-independent pathways. In contrast, inhibition of DPPI during only the last 24 h of 5-day MLC was associated only with reduced generation of BLT esterase activity and reduced lysis of nucleated targets by perforin-dependent pathways. Repeated or delayed inhibition of DPPI in MLC containing granzyme B-deficient responder cells also impaired generation of cytotoxic activity. These results indicate that DPPI or other DPPI-like protease activities not only are required for the activation of granzymes, but also play a role in the expansion and differentiation of full CD8+ T cell cytolytic activity.

Cloning and characterization of the cDNA encoding mouse dipeptidyl peptidase I (cathepsin C).

The mouse dipeptidyl peptidase I cDNA has been cloned and sequenced and patterns of DPPI mRNA expression in various tissues characterized. Sequences encoding DPPI are highly conserved among mouse, rat and human and include an unusually long propeptide and a distinctive tyrosine adjacent to the active site cysteine.

IL-1 beta convertase (ICE) does not play a requisite role in apoptosis induced in T lymphoblasts by Fas-dependent or Fas-independent CTL effector mechanisms.

Both IL-1beta convertase (ICE) and other members of the ICE-like family of proteases have been reported to play a role in Fas-mediated apoptosis. Con A-stimulated T lymphoblasts generated from splenocytes isolated from ICE-deficient H-2b mice were found to be more susceptible than wild-type lymphoblasts to DNA fragmentation induced by H-2b-specific CTL derived from normal or Fas ligand-deficient gld/gld mice. Trinitrophenyl (TNP)-modified, H-2b target cell-specific CTL were generated from perforin-deficient mice and were found to induce DNA fragmentation only in target cells expressing functional Fas receptors. Similar rates of DNA fragmentation were induced in TNP-modified ICE -/- and ICE +/+ T lymphoblast targets by perforin -/- TNP-modified, H-2b target cell-specific CTL. In addition, anti-Fas Abs induced apoptosis in thymocytes, Con A-stimulated spleen T cells, LPS-stimulated spleen B cells, and thymocytes from ICE -/- mice. However, DNA fragmentation induced by either allospecific FasL-defective CTL, or by perforin-deficient, TNP-modified, H-2b target cell-specific CTL was prevented in ICE -/- target cells loaded by electroporation with Ac-DEVD-CHO, an inhibitor of CPP32 and related ICE family proteases. These findings indicate that ICE does not play a requisite role in Fas-dependent or Fas-independent mechanisms of apoptosis induced in peripheral T lymphoblasts by CTL. However, both major pathways of CTL-induced apoptosis appear to be dependent on the enzymatic activity of other ICE family proteases.

Functional roles for granzymes in murine epidermal gamma(delta) T-cell-mediated killing of tumor targets.

Granzymes, a family of serine proteases contained in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells, play a critical role in killing tumor targets by triggering rapid breakdown of DNA and subsequent apoptosis. We have reported previously that dendritic epidermal T cells, which are skin-specific members of the tissue-type gamma(delta) T-cell family in mice, are capable of killing selected tumor cell lines. Here we report that short-term cultured dendritic epidermal T-cell lines contain significant N-alpha-benzyloxycarbonyl-L-Lys-thiobenzyl esterase activity, produce granzyme A protein, and express constitutively mRNA for granzymes A and B. Messenger RNA expression for granzyme B was also confirmed in freshly procured Thy-1+ epidermal cells (i.e., dendritic epidermal T cells). Finally, preincubation of dendritic epidermal T cell lines with a granzyme inhibitor, dichloroisocoumarin, but not with a cysteine protease inhibitor, E-64, abrogated completely their capacity to trigger DNA breakdown in YAC-1 target cells. These results reinforce the concept that dendritic epidermal T cells represent skin-resident killer cells that share several functional properties with conventional killer leukocytes, thereby playing a local immunosurveillance role against tumor development.

Interleukin-15 expression by human endothelial cells: up-regulation by ultraviolet B and psoralen plus ultraviolet A treatment.

The purposes of the present study were to determine whether endothelial cells express IL-15 and to evaluate effects of ultraviolet B (UVB) and 8-methoxypsoralens plus UVA (PUVA) on such expression. Cultured human endothelial cells derived from dermis or umbilical veins were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analyses for the detection of IL-15 mRNA and protein, respectively. Both dermal and umbilical vein endothelial cells were shown to express IL-15 mRNA and protein, and these markers were upregulated following UVB or PUVA treatment (but not by UVA or 8-methoxypsoralens alone). Also using RT-PCR, dermal and umbilical vein endothelial cells were shown to express IL-2R gamma c mRNA. These results expand the sources of IL-15 in skin to include keratinocytes, dermal fibroblasts, and now endothelial cells. That IL-15 from all three skin cells can be upregulated by UV treatment suggests a role for this cytokine in photosensitive disorders. Finally, the possibility of an autocrine effect of IL-15 on endothelial cells is raised by the expression of IL-2R gamma c in these cells.

Expression of recombinant human granzyme B. A processing and activation role for dipeptidyl peptidase I.

Human granzyme B (hGrzB) is the key member of a family of granule serine proteases (granzymes) that participate in target cell death inflicted by cytotoxic lymphocytes. The proenzyme activation peptide predicted from the cDNA encoding hGrzB is composed of two residues. We have devised a PCR strategy to delete this activation dipeptide within hGrzB and express active recombinant hGrzB in mammalian COS cells. Lysates of COS cells transfected with modified hGrzB cDNA were able to hydrolyze tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (Boc-Ala-Ala-Asp-SBzl), whereas lysates transfected with unmodified hGrzB cDNA were inactive. Accordingly, active recombinant hGrzB displayed no significant activities toward substrates containing Met-, Lys-, or Phe- at P1. It has been suggested that the mechanism by which the amino-terminal dipeptide is normally cleaved to generate active GrzB involves dipeptidyl peptidase I (DPPI). Our studies demonstrated that lysates of COS cells cotransfected with unmodified hGrzB cDNA (inactive) and rat DPPI cDNA were able to hydrolyze Boc-Ala-Ala-Asp-SBzl. Similarly, lysates of COS cells transfected with unmodified hGrzB cDNA, and devoid of Asp-ase activity, were also activated upon the addition of bovine spleen DPPI in a pH and time-dependent fashion. These results suggest that the activation dipeptide, and more particularly DPPI, may play and important role in the normal post-translational processing and activation of hGrzB in cytotoxic lymphocytes.

Dipeptidyl peptidase I is enriched in granules of in vitro- and in vivo-activated cytotoxic T lymphocytes.

Recent studies have suggested that dipeptidyl peptidase I (DPPI) is the major post-translational processing enzyme responsible for generating activated myeloid and lymphoid granule serine proteases. The current studies assessed the relative levels of DPPI and granzyme A (BLT esterase) in B6 anti-H-2d-specific CTL generated in mixed lymphocyte cultures (in vitro-activated CTL), by infusion of B6 spleen cells into irradiated H-2d mice (graft-vs-host, GVH CTL) or by 1 degree and 2 degrees peritoneal immunization of B6 mice with P815 (H-2d) cells (PE CTL). In contrast to low levels of DPPI activity in unstimulated CD4+ spleen T cells, both unstimulated CD8+ spleen T cells and in vitro-activated CTL populations were several-fold enriched in DPPI activity, while PE CTL and GVH CTL expressed even higher levels of DPPI. Depletion of DPPI-enriched cells by treatment with Leu-Leu-OMe resulted in loss of cytolytic effector function from each CTL population. However, PE CTL and GVH CTL were more sensitive to the toxicity of Leu-Leu-OMe than were in vitro-activated CTL. While standard BLT esterase assays detected much higher levels of this serine protease activity in GVH CTL or in vitro-activated CTL than in PE CTL, levels of BLT esterase activity significantly above the basal levels present in unstimulated CD8+ or CD4+ T lymphocytes were found in association with immunoreactive granzyme A in lysates of PE CTL. In both PE CTL and in vitro-activated CTL, DPPI and BLT esterase activity co-localized in the granule fraction of cell lysates, and similar percentages of total cellular BLT esterase and DPPI were exocytosed upon cross-linking of surface CD3. Thus, both in vivo- and in vitro-activated CTL were found to possess functional granules containing readily detectable albeit somewhat different levels of DPPI and granzyme A activity.

Generation of active myeloid and lymphoid granule serine proteases requires processing by the granule thiol protease dipeptidyl peptidase I.

The proenzyme activation peptides predicted from cDNAs encoding each of the granule serine proteases synthesized by cytotoxic lymphocytes and myeloid and mast cells are composed of 2 residues. The mechanism by which these amino-terminal dipeptides are cleaved to generate the active enzymes has not been elucidated. The comparable distribution of dipeptidyl peptidase I (DPPI) and serine proteases and the ability of DPPI to hydrolyze relevant dipeptide sequences suggested a role for DPPI in the processing and activation of granule serine proteases. This study demonstrates that inhibition of DPPI activity is associated with impairment of the generation of granule serine protease activity in CD8(+) T cells, lymphokine-activated killer cells, P815 mastocytoma cells, and U-937 myeloid cells. Inhibition of DPPI resulted in impairment of the generation of cathepsin G enzymatic activity without reduction in the amount of immunoreactive cathepsin G produced. In U-937 cells pulsed with [3H]isoleucine, inhibition of DPPI activity was associated with the accumulation of the inactive proenzyme form of cathepsin G bearing an amino-terminal dipeptide extension to the isoleucine residue that normally occupies the amino terminus of the enzymatically active protein. These results indicate that DPPI plays a requisite role in the post-translational processing and activation of members of the family of granule serine proteases expressed in bone marrow-derived effector cells.

Use of adjunctive modalities in occupational therapy.

This article reminds us that as practice evolves, we must revisit and reaffirm the fundamental philosophy and precepts in which our profession is grounded. The face of practice is fluid. Its superficial appearance is molded by external forces and stresses. These include the changes in the needs of the persons we serve, the emergence of new and different treatment modalities, and the realities of the socioeconomic environment in which we work. Beneath the surface, however, are the basic structures that all of us hold in common. These are our philosophical beliefs that are articulated both in the professional literature and in the ethical principles that we espouse.

Purification and characterization of dipeptidyl peptidase I from human spleen.

The lysosomal hydrolase, dipeptidyl peptidase I (DPPI), was purified from human spleen and its enzymatic activity characterized. The enzyme was purified to apparent homogeneity by a combination of differential pH solubility, heat-treatment, affinity chromatography on concanavalin A-agarose and p-hydroxymercuribenzoate-agarose, and gel filtration chromatography on Sephacryl S-300. This procedure resulted in a 1100-fold purification of DPPI protein with a yield of approximately 2% of the total DPPI activity. The enzyme was characterized as a glycoprotein with a pI of 5.4, a molecular mass of 200,000 Da as determined by gel filtration under nondenaturing conditions, and a subunit size of 24,000 Da. Amino acid sequence analysis of peptides isolated from cyanogen bromide and trypsin digests of the 24,000-Da subunit revealed extensive sequence similarity between human and rat DPPI. Purified DPPI exhibited both hydrolytic and transpeptidase (polymerase) activity. DPPI exhibited activity against a variety of dipeptide substrates including peptides with either non-polar or polar residues in the P1 position. In contrast to the reported substrate specificity of bovine and murine DPPI, the human enzyme exhibited a modest preference for peptides with nonpolar residues in the P1 position. DPPI content was found to be highest among cytotoxic lymphocytes and myeloid cells. The high level of DPPI expression in these cell populations correlates with their sensitivity to the toxic effects of leucyl-leucine methyl ester, a substrate for DPPI.

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.

Relationships among the subunits of the high molecular weight proteinase, macropain (proteasome).

An analysis of the subunits of the high molecular weight proteinase, macropain (multicatalytic proteinase or proteasome) from human erythrocytes has been conducted using N-terminal amino acid sequencing, gel electrophoresis and reverse-phase peptide mapping. This analysis provided evidence for the existence of 13 subunits of different primary structure. Five subunits were susceptible to the Edman degradation and yielded unique N-terminal sequences. Similarities among these sequences, however, indicated that the subunits are homologous. Two-dimensional gel electrophoresis discriminated 10 major components, which included two of the subunits for which N-terminal sequences had been determined and eight N-terminally blocked subunits. Tryptic peptide mapping indicated that all 10 of these components have a different amino acid sequence. Tryptic peptides from some of the subunits were subjected to amino acid sequence analysis, and the data indicated that all the subunits tested in this way are related by common ancestry. The data suggest that at least nine of the total of 13 subunits are encoded by members of the same gene family; the remaining four subunits have not yet been investigated in sufficient detail to establish their relationships. No evidence for a close relationship with any previously investigated proteinase family has been found. Finally, through a comparison of the 'latent' and 'active' forms of macropain, the study established a close similarity in the subunit composition of these catalytically very different species, although proteolytic degradation of selected subunits appears in the active form of the enzyme.