Priority recommendations for the implementation of patient-reported outcomes in clinical cancer care: a Delphi study.

PURPOSE: The aim of this study was to develop priority recommendations for the service level implementation of patient-reported outcomes (PROs) into clinical cancer care. METHODS: Development of draft guidance statements was informed by a literature review, the Knowledge to Action (KTA) implementation framework, and discussion with PRO experts and cancer survivors. A two-round modified Delphi survey with key stakeholders including cancer survivors, clinical and research experts, and Information Technology specialists was undertaken. Round 1 rated the importance of the statements and round 2 ranked statements in order of priority. RESULTS: Round 1 was completed by 70 participants with round 2 completed by 45 participants. Forty-seven statements were rated in round 2. In round 1, the highest agreement items (>90% agreement) included those that focused on the formation of strong stakeholder partnerships, ensuring ongoing communication within these partnerships, and the use of PROs for improvement and guidance in clinical care. Items ranked as the highest priorities in round 2 included assessment of current staff capabilities and service requirements, mapping of workflows and processes to enable collection, and using collected PROs to guide improved health outcomes. CONCLUSIONS: This stakeholder consultation process has identified key priorities in PRO implementation into clinical cancer care that include clinical relevance, stakeholder engagement, communication, and integration within the existing processes and capabilities. IMPLICATION FOR CANCER SURVIVORS: Routine adoption of PRO collection by clinical cancer services requires multiple implementation steps; of highest priority is strong engagement and communication with key stakeholders including cancer survivors.

Correlates of epidermal nerve fiber densities in HIV-associated distal sensory polyneuropathy.

OBJECTIVE: To demonstrate the relationship between epidermal nerve fiber density (ENFD) in the leg and the phenotype of HIV-associated distal sensory polyneuropathy (HIV-DSP) in a multicenter prospective study (ACTG A5117). METHODS: A total of 101 HIV-infected adults, with CD4 cell count <300 cells/mm(3) and who had received antiretroviral therapy (ART) for at least 15 consecutive weeks, underwent standardized clinical and electrophysiologic assessment. All 101 subjects were biopsied at the distal leg (DL) and 99 at the proximal thigh (PT) at baseline. ENFD was assessed by skin biopsy using PGP9.5 immunostaining. Associations of ENFD with demographics, ART treatment, Total Neuropathy Score (TNS), sural sensory nerve action potential (SNAP) amplitude and conduction velocity, quantitative sensory testing (QST) measures, and neuropathic pain were explored. RESULTS: ENFD at the DL site correlated with neuropathy severity as gauged by TNS (p < 0.01), the level of neuropathic pain quantified by the Gracely Pain Scale (GPS) (p = 0.01) and Visual Analogue Scale (VAS) (p = 0.01), sural SNAP amplitude (p < 0.01), and toe cooling (p < 0.01) and vibration (p = 0.02) detection thresholds. ENFD did not correlate with neurotoxic ART exposure, CD4 cell count, or plasma HIV-1 viral load. CONCLUSIONS: In subjects with advanced HIV-1 infection, epidermal nerve fiber density (ENFD) assessment correlates with the clinical and electrophysiologic severity of distal sensory polyneuropathy (DSP). ENFD did not correlate with previously established risk factors for HIV-DSP, including CD4 cell count, plasma HIV-1 viral load, and neurotoxic antiretroviral therapy exposure.

Deletion of the cnxE gene encoding the gephyrin-like protein involved in the final stages of molybdenum cofactor biosynthesis in Aspergillus nidulans.

The Aspergillus nidulans cnxE gene, required for molybdenum cofactor biosynthesis, was isolated by functional complementation of an Escherichia coli mogA mutant strain. The deduced CnxE polypeptide consists of two domains which display similarity to the E. coli proteins MoeA and MogA, respectively, separated by a putative hinge region of around 58 amino acid residues which is notably histidine rich. A deletion mutant lacking the entire cnxE gene, including both MoeA-like and MogA-like domains, was identified. Compared to the wild type, a small increase in the intermediate precursor Z was observed in the deletion strain but was significant only under conditions in which the molybdoenzyme nitrate reductase was induced. Elevated levels of the pathway intermediate molybdopterin were found both under nitrate reductase-inducing and non-inducing conditions in the deletion mutant compared to the wild type. This increase is in contrast to previous results for cnxABC, cnxF, cnxG, and cnxH mutants, in which the levels of molybdopterin were substantially reduced, and therefore supports previously published classical genetic and biochemical studies that indicated that the CnxE protein is likely to be involved in the final stages of molybdenum cofactor biosynthesis. We have found no evidence during our chemical analysis for any involvement of this protein in the intermediate section of the molybdenum cofactor biosynthetic pathway (i.e. in the synthesis of molybdopterin from precursor Z), as has been suggested previously for E. coli MoeA. The 2.5-kb cnxE transcript is not abundant and appears to be expressed constitutively.

A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer.

Prostate-specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa) but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia, because both PCa and benign prostatic hyperplasia release PSA into the serum. We have identified previously a truncated form of precursor PSA (pPSA) in prostate tumor extracts consisting of PSA with a serine-arginine pro leader peptide ([-2]pPSA) instead of the normally expressed 7 amino acid pro leader peptide. In the current study we developed monoclonal antibodies to detect [-2]pPSA and other isoforms of pPSA for Western blot analysis. PSA was immunoaffinity purified from 100 to 200 ml of serum from each of five men with biopsy-proven cancer and three biopsy-negative men, all with total PSA levels in the diagnostically relevant range near 10 ng/ml. The truncated [-2]pPSA was estimated to range from 25 to 95% of the free PSA in the five PCa samples but only 6-19% of the free PSA in the biopsy-negative men. Immunohistochemical studies showed positive staining for [-2]pPSA in PCa epithelium and that [-2]pPSA was enriched in cancer cell secretions. In vitro activation studies showed that human kallikrein 2 and trypsin readily activated full-length pPSA but were unable to activate [-2]pPSA to mature PSA. Thus, [-2]pPSA, once formed, is a stable but inactive isoform of PSA. Truncated [-2]pPSA may represent an important new diagnostic marker for the early detection of PCa.

Development of a dual monoclonal antibody immunoassay for total human kallikrein 2.

BACKGROUND: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-alpha(1)-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. METHODS: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. RESULTS: The minimum detectable concentration in the thK2 assay was 0.008 microg/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and three different hK2-serum protease complexes. The median serum concentration of thK2 in control samples (0.013 microg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 microg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at -70 degrees C. CONCLUSIONS: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.

A case of sacro-coccygeal chordoma masquerading as pilonidal sinus.

Chordoma is a rare, slow-growing, but locally aggressive malignant tumour derived from the primitive notochord. Pilonidal sinus disease, characterised by communicating granulating natal cleft tracts and abscesses, is a common condition. We report a case of a sacro-coccygeal chordoma diagnosed incidentally on surgical exploration of a case of presumed pilonidal disease.

Seminal plasma contains "BPSA," a molecular form of prostate-specific antigen that is associated with benign prostatic hyperplasia.

BACKGROUND: We previously reported that levels of BPSA, a modified form of prostate-specific antigen (PSA), are significantly elevated in prostate transition-zone tissue exhibiting nodular hyperplastic changes associated with the presence of benign prostatic hyperplasia (BPH). BPSA was purified and found to contain a characteristic clip between Lys182 and Ser183. We now describe the identification of BPSA in seminal plasma. METHODS: PSA was purified from seminal plasma by immunoaffinity chromatography. The purified PSA was further resolved by hydrophobic interaction chromatography, and the individual PSA forms were analyzed by gel electrophoresis and N-terminal amino-acid sequencing. RESULTS: BPSA comprised about 8% of the PSA in pooled seminal plasma, and was identical to BPSA purified from prostate tissues. BPSA was cleanly resolved from all active and inactive forms of PSA. Other inactive forms of PSA in seminal plasma consisted largely of PSA clipped at Lys145, though about 30% of the inactive seminal plasma PSA was intact, mature PSA. CONCLUSIONS: BPSA represents a distinct form of inactive PSA in the seminal plasma that may represent a specific marker for the biochemical changes associated with nodular development in the prostate transition zone found in patients with BPH.

Different proportions of various prostate-specific antigen (PSA) and human kallikrein 2 (hK2) forms are present in noninduced and androgen-induced LNCaP cells.

BACKGROUND: Human prostate-specific antigen (PSA) and human kallikrein 2 (hK2) are expressed primarily by prostate epithelial cells. PSA and hK2 both exist as free protein and complexed with protease inhibitors (e.g., alpha1-antichymotrypsin, ACT) in serum. The expression of PSA and hK2 in LNCaP cells is upregulated by androgen. METHODS: LNCaP, a prostate cancer cell line that secretes both PSA and hK2, was used as a model to study the biosynthesis and processing of PSA and hK2 upon androgen induction. RESULTS: Precursor (zymogen or pro) forms of both PSA and hK2 were detected in spent media of induced and noninduced LNCaP cells, indicating that PSA and hK2 are secreted as proPSA (pPSA) and prohK2 (phK2), respectively, and are converted to the mature forms extracellularly. A 3-fold higher ratio of mature to pPSA was detected in the spent media of mibolerone-induced LNCaP cells compared to noninduced cells. In addition to the inactive proform of PSA, more than half of the mature unclipped PSA present in the spent media did not complex with exogenously added ACT. Spent media of mibolerone-induced LNCaP cells contained nearly 100% mature hK2, whereas the spent media of noninduced cells contained mostly phK2. CONCLUSIONS: These results indicate that androgens not only upregulate the expression of these kallikreins, but also have a significant effect on the processing of PSA and hK2. These results also show that LNCaP cells express a heterogeneous mixture of inactive PSA and hK2 forms that may serve as a model for the genesis of these forms in physiological fluids. These findings may also provide insights into the forms and ratios of PSA and hK2 in normal and malignant breast tissues.

Benign prostatic hyperplasia-associated prostate-specific antigen (BPSA) shows unique immunoreactivity with anti-PSA monoclonal antibodies.

We previously identified a modified molecular form of prostate-specific antigen that is significantly elevated in the nodular transition zone tissue of prostates with benign prostatic hyperplasia. This prostate-specific antigen form, designated BPSA, is inactive and contains clipped polypeptide bonds at amino-acid residues Lys145-146 and Lys182-183. BPSA is not elevated in prostate cancer tissues and may therefore be a prostate-specific antigen marker to better discriminate benign prostatic hyperplasia from early prostate cancer. In this work we characterize the immunoreactivity of BPSA in competition assays with prostate-specific antigen using anti-prostate-specific antigen mAb recognizing six different epitopes on the prostate-specific antigen molecule. One mAb showed > 50% loss of immunoreactivtiy with BPSA compared with prostate-specific antigen, while the binding of two mAbs was largely unaffected and three mAbs had intermediate reactivity. BPSA purified from prostate tissue and seminal plasma, as well as BPSA generated in vitro by mild trypsin-treatment were found to have a similar pattern of reactivity to the six mAbs. However, other forms of inactive seminal plasma prostate-specific antigen, either intact or clipped at Lys145 only, had immunoreactivity similar to total prostate-specific antigen. These results demonstrate that BPSA has unique immunological properties from other forms of prostate-specific antigen, which should allow the development of BPSA-specific mAbs for the study of benign prostatic hyperplasia. Measurement of BPSA levels in the serum may help discriminate benign prostatic hyperplasia from early prostate cancer.

"BPSA," a specific molecular form of free prostate-specific antigen, is found predominantly in the transition zone of patients with nodular benign prostatic hyperplasia.

OBJECTIVES: The biologic mechanism for the increased proportion of noncomplexed ("free") prostate-specific antigen (PSA) found in the serum of patients with benign prostate disease is unknown. We recently reported that most of the PSA found in benign, hyperplastic, and cancerous prostatic tissue is in the free form. To determine whether specific molecular forms of free PSA are associated differentially with normal, hyperplastic, or cancerous prostatic tissue, we have further characterized the free PSA in each type of prostatic tissue. METHODS: PSA was purified by immunoaffinity chromatography from matched prostatic tissue samples of peripheral zone cancer (PZ-C), PZ noncancer (PZ-N), and transition zone (TZ) tissue from 10 large-volume (greater than 50 g) and 8 small-volume (less than 25 g) radical prostatectomy specimens. Eight TZ specimens obtained during transurethral resection of the prostate for benign prostatic hyperplasia (BPH) were also analyzed. The different molecular forms of PSA were further resolved by high-performance hydrophobic interaction chromatography. Clipped forms of PSA were identified by N-terminal amino acid sequencing. RESULTS: More than 99% of the PSA in prostatic tissues was in the free, noncomplexed form. Specimens from the prostate TZ were found to contain elevated levels of an altered form of PSA, which we designated BPSA. Purified BPSA contained a distinctive cleavage at lysine 182. The median percent BPSA (%BPSA) was 11.4 in the TZ of specimens with nodular BPH compared with a %BPSA of 4.1 in the TZ of specimens without nodular BPH (P <0.0014). The median %BPSA levels of the PZ-N and PZ-C tissues ranged from 3.2 to 4.9 and were not significantly different from one another or from the %BPSA level of TZ tissues without nodular BPH. CONCLUSIONS: We have identified a specific molecular form of clipped free PSA, called BPSA, that is increased within the prostatic TZ of patients exhibiting nodular BPH. Higher levels of percent free PSA in serum have been found to correlate strongly with prostate volume, which in turn is closely associated with the progressive enlargement of nodular BPH tissue within the TZ of the prostate. Thus, it is possible that a proportion of the serum percent free PSA found in patients with BPH may be composed of BPSA released into the serum.

A precursor form of prostate-specific antigen is more highly elevated in prostate cancer compared with benign transition zone prostate tissue.

Prostate-specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but in the critical diagnostic range of 4-10 ng/ml it has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). PSA in serum is comprised of a variety of both "free" and "complexed" forms that have been used to improve the specificity of PSA for prostate cancer detection. We previously reported that pro PSA (pPSA), the zymogen or precursor form of PSA, is a component of free PSA in the serum of PCa patients. In the current study, we examined prostate tissues to understand the origin and specificity of pPSA. PSA was immuno-affinity purified from matched sets of prostate tissues including peripheral zone cancer (PZ-C); peripheral zone noncancer; and benign tissue from the transition zone (TZ), the primary site of BPH within the prostate. We found that pPSA is differentially elevated in PZ-C, but is largely undetectable in TZ. N-terminal sequencing revealed that the pPSA was comprised primarily of [-2]pPSA and minor levels of [-4]pPSA, containing pro leader peptides of 2 and 4 amino acids, respectively. The median value of pPSA was 3% in PZ-C and 0% (undetectable) in TZ (P < 0.0026). No pPSA was detected in 13 of 18 transition zone specimens (72%), but only 2 of the 18 matched cancer specimens (11%) contained no measurable pPSA. These results demonstrate that pPSA is more highly correlated with prostate cancer than with BPH. The pPSA in serum may represent a more cancer-specific form of PSA that could help distinguish prostate cancer from BPH.

Identification of a novel complex between human kallikrein 2 and protease inhibitor-6 in prostate cancer tissue.

Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation.

Prostatic human kallikrein 2 inactivates and complexes with plasminogen activator inhibitor-1.

Human kallikrein 2 (hK2) is a serine protease expressed predominantly in the prostate which has 80% homology to prostate-specific antigen (PSA). hK2 is an active trypsin-like protease which has been shown by immuno-histochemical staining to be more highly expressed in prostate carcinoma than in benign prostate tissue. Unlike PSA, hK2 activates pro-PSA , pro-hK2 and the zymogen form of urokinase-type plasminogen activator (uPA), an extracellular protease correlated with prostate cancer and metastasis. We show here that hK2 rapidly forms a complex with plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of uPA in tissues. In addition, hK2 inactivated 6 to 7 mol of PAI-1 by cleavage at Arg346-Met347 for every mole of hK2-PAI-1 complex formed. In contrast with hK2, PSA neither complexed with nor inactivated PAI-1. PAI-1 inhibited hK2 comparably with protein C inhibitor (PCI) and at least 20 times more rapidly than alpha1-anti-chymotrypsin (ACT). N-Terminal sequencing shows that hK2 forms a covalent complex with PAI-1, PCI and ACT after cleavage at Arg346-Met347, Arg354-Ser355 and Leu358-Ser359, respectively. During complex formation, hK2 inactivated PAI-1 but did not inactivate ACT or PCI. Our current results suggest that the increased hK2 expression in prostate cancer tissues could influence cancer biology not only by activation of uPA but also by inactivation of its primary inhibitor, PAI-1.

Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.

OBJECTIVES: Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility. METHODS: We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels. RESULTS: The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech). CONCLUSIONS: Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.

Failure of cytarabine in progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. AIDS Clinical Trials Group 243 Team.

BACKGROUND: Progressive multifocal leukoencephalopathy affects about 4 percent of patients with the acquired immunodeficiency syndrome (AIDS), and survival after the diagnosis of leukoencephalopathy averages only about three months. There have been anecdotal reports of improvement but no controlled trials of therapy with antiretroviral treatment plus intravenous or intrathecal cytarabine. METHODS: In this multicenter trial, 57 patients with human immunodeficiency virus (HIV) infection and biopsy-confirmed progressive multifocal leukoencephalopathy were randomly assigned to receive one of three treatments: antiretroviral therapy alone, antiretroviral therapy plus intravenous cytarabine, or antiretroviral therapy plus intrathecal cytarabine. After a lead-in period of 1 to 2 weeks, active treatment was given for 24 weeks. For most patients, antiretroviral therapy consisted of zidovudine plus either didanosine or stavudine. RESULTS: At the time of the last analysis, 14 patients in each treatment group had died, and there were no significant differences in survival among the three groups (P=0.85 by the log-rank test). The median survival times (11, 8, and 15 weeks, respectively) were similar to those in previous studies. Only seven patients completed the 24 weeks of treatment. Anemia and thrombocytopenia were more frequent in patients who received antiretroviral therapy in combination with intravenous cytarabine than in the other groups. CONCLUSIONS: Cytarabine administered either intravenously or intrathecally does not improve the prognosis of HIV-infected patients with progressive multifocal leukoencephalopathy who are treated with the antiretroviral agents we used, nor does high-dose antiretroviral therapy alone appear to improve survival over that reported in untreated patients.

Keratoconjunctivitis sicca associated with achalasia of the cardia, adrenocortical insufficiency, and lacrimal gland degeneration: Keratoconjunctivitis sicca secondary to lacrimal gland degeneration may parallel degenerative changes in esophageal and adrenocortical function.

OBJECTIVE/DESIGN: This study aimed to examine and describe three siblings with alacrima, the eldest of whom had associated achalasia and adrenocortical insufficiency. PARTICIPANTS: Three affected siblings and four age-matched control subjects participated. INTERVENTION/MAIN OUTCOME MEASURES: The three children underwent complete ophthalmologic examinations; computed tomographic scanning of brain, orbit, chest, and abdomen; and measurement of serum cortisol. All three were subjected to a short synacthen challenge. Lacrimal gland biopsies were performed on the two younger subjects, and specimens were studied by light and electron microscopy. RESULTS: All three children showed virtually absent tear secretion as tested by the Schirmer test. The resulting keratopathy was most severe in the oldest child, who developed bilateral corneal melting. The two younger children showed interpalpebral corneal staining with rose bengal. All three children improved after punctal occlusion. Addison's disease was present in the oldest child. Computed tomographic scanning showed absent lacrimal and shrunken adrenal glands in association with achalasia of the cardia in the oldest child. The lacrimal glands were found to be reduced in size in the next eldest child. When evaluated by electron microscopy, the lacrimal gland biopsy specimens from the two younger children showed neuronal degeneration associated with depletion of secretory granules in the acinar cells. CONCLUSION: In this disease, radiologic evidence of reducing lacrimal gland size with increasing age could represent a degenerative process. This may be paralleled by other signs and the possibility of adrenocortical insufficiency and achalasia of the cardia should be investigated in all children presenting with dry eyes. These children appear to have a progressive neuronal disease.

Extrusion of porous polyethylene orbital implant in recurrent retinoblastoma.

High-density porous polyethylene allografts are used as orbital implants in reconstruction of anophthalmic sockets. The use of this material for integrated buried implants is rather new, and studies on removed implants from humans are limited. We studied the case of a 6-year-old boy with an extruded porous polyethylene implant due to orbital recurrence of retinoblastoma. The clinical and morphological features are reported. Histopathology revealed fibrovascular downgrowth in the outer two thirds of the sphere. Scanning electron microscopy, however, documented further downgrowth of fibrous tissue into the central core. Transmission electron microscopy depicted a spectrum of collagen fiber orientations to polyethylene material, ranging from perpendicular to concentric. Immunohistochemistry studies were inconclusive.

Human glandular kallikrein, hK2, shows arginine-restricted specificity and forms complexes with plasma protease inhibitors.

BACKGROUND: Human glandular kallikrein (hK2) is a new potential marker for prostate cancer. It is a serine protease expressed in human prostate epithelial cells which has 78% sequence identity with prostate-specific antigen (PSA). PSA is a widely used biochemical marker for prostate cancer. METHODS: Recombinant hK2 expressed in mammalian cells was purified to homogeneity by immunoaffinity chromatography, using an anti-hK2 mAb. hK2 enzymatic specificity was determined on peptide substrates by N-terminal amino acid sequencing. hK2 complexes were analyzed by SDS-PAGE and Western blots. RESULTS: hK2 was found to cleave peptide substrates exclusively at selected arginine residues. An amidolytic activity of 4,100 pmol/min per microgram hK2 was obtained on the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide, while no activity was found on methoxysuccinyl-Arg-Pro-Tyr-p-nitroanilide, a chymotrypsin substrate used to measure PSA activity. hK2 complexed completely with alpha 1-antichymotrypsin and alpha 2-antiplasmin after 4 hr at 37 degrees C, but showed no detectable complex with antithrombin III and alpha 1-protease inhibitor under these conditions. hK2 also formed a rapid complex with alpha 2-macroglobulin. CONCLUSIONS: These results demonstrate that hK2 is an active protease with arginine-selective specificity, which forms covalent complexes with plasma protease inhibitors.

A precursor form of PSA (pPSA) is a component of the free PSA in prostate cancer serum.

OBJECTIVES: Prostate-specific antigen (PSA) is a widely used serum marker for human prostate cancer (PCa). The majority of PSA in serum is present as a complex with alpha-1-antichymotrypsin (ACT). In recent years, the ratio of free (uncomplexed) to total PSA has shown improved discrimination of PCa from benign prostatic hyperplasia. This study examines the nature of the free PSA from detected in PCa serum and shows that some of the uncomplexed PSA is an inactive precursor of PSA (pPSA). METHODS: Western blot analysis was used to detect clipped, fragment forms of PSA in sera and seminal fluid. Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) was used to identify forms of PSA present in the free PSA population. Pooled sera was passed over a PSA immunoaffinity column, and the eluted PSA components were further resolved by HIC-HPLC. RESULTS: Western blot analysis of whole sera showed complexed PSA and the intact, approximately 34 kilodalton free PSA. Only negligible levels of clipped or degraded forms of PSA, as found in seminal fluid, were detected. Column fractions measured for uncomplexed PSA using the Tandem-MP free PSA assay showed that about 25% of the free PSA eluted as pPSA beginning at the [-4]amino acid. Studies with purified recombinant [-4]pPSA showed that this proenzyme form is inactive and does not complex with ACT. CONCLUSIONS: These results suggest that the uncomplexed PSA in PCa serum is primarily unclipped PSA that contains a significant fraction of pPSA.

The Aspergillus nidulans cnxABC locus is a single gene encoding two catalytic domains required for synthesis of precursor Z, an intermediate in molybdenum cofactor biosynthesis.

The Aspergillus nidulans complex locus, cnxABC, has been shown to be required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway. The locus was isolated by chromosome walking a physical distance of 65-kilobase pairs from the brlA gene and defines a single transcript that encodes, most likely, a difunctional protein with two catalytic domains, CNXA and CNXC. Mutations (cnxA) affecting the CNXA domain, mutants (cnxC) in the CNXC domain, and frameshift (cnxB) mutants disrupting both domains have greatly reduced levels of precursor Z compared with the wild type. The CNXA domain is similar at the amino acid level to the Escherichia coli moaA gene product, while CNXC is similar to the E. coli moaC product, with both E. coli products encoded by different cistrons. In the wild type, precursor Z levels are 3-4 times higher in nitrate-grown cells than in those grown on ammonium, and there is an approximately parallel increase in the 2.4-kilobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway. Analysis of the deduced amino acid sequence of several mutants has identified residues critical for the function of the protein. In the CNXA section of the protein, insertion of three amino acid residues into a domain thought to bind an iron-sulfur cofactor leads to a null phenotype as judged by complete loss of activity of the molybdoenzyme, nitrate reductase. More specifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in binding the cofactor. This supports the proposition that these residues play an essential catalytic role. An insertion of seven amino acids between residues valine 139 and serine 140, leads to a temperature-sensitive phenotype, suggesting a conformational change affecting the catalytic activity of the CNXA region only. A single base pair deletion leading to an in frame stop codon in the CNXC region, which causes a null phenotype, effectively deletes the last 20 amino acid residues of the protein, indicating that these residues are necessary for catalytic function.