Spinal cord involvement by atrophy and associations with disability are different between multiple sclerosis and neuromyelitis optica spectrum disorder.

BACKGROUND AND PURPOSE: The cervical and thoracic cross-sectional spinal cord area (CS-SCA) in multiple sclerosis (MS) correlates with disability, whilst such a correlation remains to be established in neuromyelitis optica spectrum disorder (NMOSD). Our aim was to clarify differences between MS and NMOSD in spinal cord segments where CS-SCA is associated with disability. METHODS: The CS-SCA at C2/C3, C3/C4, T8/T9 and T9/T10 vertebral disc levels was measured in 140 MS patients (111 with relapsing-remitting MS and 29 with progressive MS) and 42 NMOSD patients with anti-aquaporin-4 immunoglobulin G. Disability was evaluated by Expanded Disability Status Scale (EDSS) scores. Multivariate associations between CS-SCA and disability were assessed by stepwise forward multiple linear regression. RESULTS: Thoracic CS-SCA was significantly smaller in NMOSD patients than in MS patients even after adjusting for age, sex and disease duration (P = 0.002 at T8/T9), whilst there was no difference in cervical CS-SCA between the two diseases. Cervical and thoracic CS-SCA had a negative correlation with EDSS scores in MS patients (P < 0.0001 at C3/C4 and P = 0.0002 at T8/T9) whereas only thoracic CS-SCA correlated with EDSS scores in NMOSD patients (P = 0.0006 at T8/T9). By multiple regression analyses, predictive factors for disability in MS were smaller cervical CS-SCA, progressive course, older age and a higher number of relapses, whilst those in NMOSD were smaller thoracic CS-SCA and older age. CONCLUSIONS: Thoracic CS-SCA is a useful predictive marker for disability in patients with NMOSD whilst cervical CS-SCA is associated with disability in patients with MS.

Interkingdom signal indole inhibits Pseudomonas aeruginosa persister cell waking.

AIMS: Persister cells are stressed cells that have transient tolerance to antibiotics; these cells undergo no genetic change, but instead, their tolerance is due to reduced metabolism. Unfortunately, little is known about how persisters resuscitate, so we explored the waking of cells in the presence of the interkingdom signal indole. METHODS AND RESULTS: To generate a large population of persister cells, we induced the persister phenotype in the opportunistic pathogen Pseudomonas aeruginosa by pretreating cells with carbonyl cyanide m-chlorophenylhydrazone to reduce translation by depleting ATP levels, and found, via single cell observations, that proline is sufficient to wake the persister cells. P. aeruginosa is often present in the gastrointestinal tract, and indole from commensal bacteria such as Escherichia coli has been shown to inhibit P. aeruginosa quorum sensing and pathogenicity without influencing growth. Furthermore, indole is not toxic to P. aeruginosa persister cells. However, we find here that physiological concentrations of indole inhibit P. aeruginosa persister cell resuscitation with an efficiency of higher than 95%. Critically, when contacted with E. coli stationary-phase cultures, the indole produced by E. coli completely inhibits persister cell resuscitation of P. aeruginosa. CONCLUSIONS: Therefore, E. coli has devised a method to outcompete its competitors by preventing their resuscitation with indole. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides insight into why indole is produced by commensal bacteria.

Economic evaluation of a preemptive treatment strategy for invasive fungal infection in neutropenic patients with hematological diseases.

We compared the expected medical costs of empirical and preemptive treatment strategies for invasive fungal infection in neutropenic patients with hematological diseases. Based on the results of two clinical trials with different backgrounds reported by Oshima et al. [J Antimicrob Chemother 60(2):350-355; Oshima study] and Cordonnier et al. [Clin Infect Dis 48(8):1042-1051; PREVERT study], we developed a decision tree model that represented the outcomes of empirical and preemptive treatment strategies, and estimated the expected medical costs of medications and examinations in the two strategies. We assumed that micafungin was started in the empirical group at 5 days after fever had developed, while voriconazole was started in the preemptive group only when certain criteria, such as positive test results of imaging studies and/or serum markers, were fulfilled. When we used an incidence of positive test results of 6.7 % based on the Oshima study, the expected medical costs of the empirical and preemptive groups were 288,198 and 150,280 yen, respectively. Even in the case of the PREVERT study, in which the incidence of positive test results was 32.9 %, the expected medical costs in the empirical and preemptive groups were 291,871 and 284,944 yen, respectively. A sensitivity analysis indicated that the expected medical costs in the preemptive group would exceed those in the empirical group when the incidence of positive test results in the former was over 34.4 %. These results suggest that a preemptive treatment strategy can be expected to reduce medical costs compared with empirical therapy in most clinical settings.

Risk factors for pre- and post-engraftment bloodstream infections after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Bloodstream infections (BSI) are frequently observed after allogeneic hematopoietic stem cell transplant (HSCT), and could cause morbidity and mortality. METHODS: We retrospectively evaluated the incidence, characteristics of, and risk factors for BSI at both pre- and post-engraftment in 209 adult HSCT patients at our institute between June 2006 and December 2013. The median age at transplantation was 45 years (range, 15-65). A total of 122 patients received bone marrow, 68 received peripheral blood stem cells, and 19 received umbilical cord blood. RESULTS: The cumulative incidences of pre- and post-engraftment BSI were 38.9% and 17.2%, respectively. Nine patients had both pre- and post-engraftment BSI. In the pre- and post-engraftment periods, respectively, 67.4% and 84.1% of isolates were gram-positive bacteria (GPB), 28.3% and 11.4% were gram-negative bacteria (GNB), and 4.3% and 4.5% were fungi. Coagulase-negative staphylococci were the most commonly isolated GPB, while Stenotrophomonas maltophilia and Pseudomonas aeruginosa were the most commonly isolated GNB. Pre-engraftment BSI was associated with an increased risk of death. Overall survival at day 180 for patients with or without pre-engraftment BSI was 70.0% and 82.7%, respectively (P = 0.02). CONCLUSIONS: Risk factors for BSI in the pre-engraftment period were the interval between diagnosis and transplantation (261 days or more), engraftment failure, and high-risk disease status at HSCT in a multivariate analysis. No significant risk factor for BSI in the post-engraftment period was identified by a univariate analysis. These findings may be useful for deciding upon empiric antibacterial treatment for HSCT recipients.

Allotype analysis to determine the origin of cytomegalovirus immunoglobulin-G after allogeneic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) reactivation still remains a major problem following allogeneic hematopoietic stem cell transplantation (HSCT). PATIENTS AND METHODS: In this study, we analyzed an immunoglobulin allotype, IgG1m(f), in CMV-seropositive HSCT recipients and their donors to distinguish donor-derived antibody from recipient-derived antibody. Eight donor-recipient pairs were informative regarding the appearance of donor-derived immunoglobulin-G (IgG), as the recipients were homozygous null for the IgG1m(f) allotype and the donors were IgG1m(f) positive. In these patients, total IgG, IgM, and allotype-specific IgG against CMV were measured by enzyme-linked immunosorbent assay. All subjects were monitored for at least 9 months after HSCT with (n = 5) or without (n = 3) CMV reactivation. RESULTS: Donor-derived CMV IgG tended to be elevated earlier in patients with CMV-seropositive donors than in those with CMV-seronegative donors. In 1 patient with a CMV-negative donor, donor-derived CMV IgG was not detected until late CMV reactivation. In 3 patients without CMV reactivation, donor-derived CMV IgG was also elevated within 1-6 months after HSCT. CONCLUSION: In conclusion, the CMV serostatus of the donor may be related to the timing of the appearance of donor-derived CMV IgG and the reconstitution of humoral immunity against CMV, regardless of the CMV antigenemia level after HSCT.

Persistence of recipient-derived as well as donor-derived clones of cytomegalovirus pp65-specific cytotoxic T cells long after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV)-specific CD8(+) cytotoxic T lymphocytes (CMV-CTLs) play a crucial role in preventing CMV disease. However, the actual in vivo dynamics of CMV-CTL clones after allogeneic hematopoietic stem cell transplantation (alloHCT) are still unclear. METHODS: Using a single-cell T-cell receptor repertoire analysis, we monitored clones and chimerism of CMV-CTLs in 3 CMV-seropositive alloHCT recipients from CMV-seronegative donors, with or without CMV reactivation. RESULTS: Nearly all of the CMV-CTLs during follow-up were CD45RA(-) CCR7(-) effector memory/CD45RA(+) CCR7(-) effector T cells, and were highly matured. In each case, the use of BV gene families was restricted, especially in BV5, 7, 28, and 29. Although no common predominant CMV-CTL clones were found, several shared motifs of complementarity-determining region-3 were identified among the 3 cases; QGA in all, TGE and TDT in Case 1 and Case 2, and RDRG in Case 2 and Case 3. In all cases, CMV-CTL clones that were detected for the first time after alloHCT persisted as the dominant clones. In Case 1, without CMV reactivation, recipient-derived CMV-CTLs exclusively persisted as a dominant clone, while all CMV-CTLs in the other 2 cases, with CMV reactivation, were donor derived. CONCLUSION: Clone monitoring and chimerism analyses should help to further clarify novel aspects of immuno-reconstitution after alloHCT.

Low-dose acyclovir prophylaxis for the prevention of herpes simplex virus disease after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Currently, acyclovir (ACV) at 1000 mg/day is widely used as prophylaxis in the early phase of hematopoietic stem cell transplant (HSCT) in Japan. However, low-dose ACV (200 mg/day) has been shown to prevent varicella zoster virus reactivation in the middle and late phases of HSCT. METHODS: Therefore, in this study, we decreased the dose of ACV to 200 mg/day in the early phase after HSCT. We analyzed 93 consecutive herpes simplex virus (HSV)-seropositive patients who underwent allogeneic HSCT for the first time in our center between June 2007 and December 2011. RESULTS: Before August 2009, 38 patients received oral ACV at 1000 mg/day (ACV1000) until day 35 after HSCT, whereas 55 patients received oral ACV at 200 mg/day (ACV200) after September 2009. We compared the cumulative incidence of HSV infection in the 2 groups. Oral ACV was changed to intravenous administration because of intolerance in 66% and 45% of the patients in the ACV1000 and ACV200 groups, respectively (P = 0.060). The probability of severe stomatitis (Bearman grade II-III) was 76% and 60% in the ACV1000 and ACV200 groups, respectively (P = 0.12). The number of patients who developed HSV disease before day 100 after HSCT was 0 in the ACV1000 group and 2 in the ACV200 group, with a cumulative incidence of 3.6% (P = 0.43). HSV disease in the latter 2 patients was limited to the lips and tongue and was successfully treated with ACV or valacyclovir at a treatment dose. CONCLUSION: ACV at 200 mg/day appeared to be effective for preventing HSV disease in the early phase after HSCT.

Influence of HLA-DRB1 alleles on the susceptibility and resistance to multiple sclerosis in Japanese patients with respect to anti-aquaporin 4 antibody status.

BACKGROUND: Epistatic interactions between human leukocyte antigen (HLA)-DRB1 alleles alter multiple sclerosis (MS) risk in Caucasians. Such interactions have never been studied in Asian MS patients. OBJECTIVE: To investigate the influence of HLA-DRB1 alleles, including epistatic interactions at this locus, in Japanese MS patients with and without the anti-aquaporin 4 (AQP4) antibody. METHODS: The HLA-DRB1 locus was genotyped in 108 MS patients and 127 healthy controls. MS patients were further classified into two groups according to anti-AQP4 antibody status (27 positive and 81 negative). RESULTS: HLA-DRB1*09 (adjusted odds ratio (OR) = 0.243, 95% confidence interval (CI) 0.099-0.533) and HLA-DRB1*01 (adjusted OR = 0.327, 95% CI 0.103-0.873) decreased the incidence of anti-AQP4 antibody-negative MS. By contrast, HLA-DRB1*12 increased the risk of anti-AQP4 antibody-positive MS (adjusted OR = 3.691, 95% CI 1.233-10.565). Individuals with HLA-DRB1*09/15 decreased the risk of anti-AQP4 antibody-negative MS (adjusted OR = 0.164, 95% CI 0.026-0.593), while those with HLA-DRB1*12/15 increased the risk of anti-AQP4 antibody-positive MS (adjusted OR = 10.870, 95% CI 2.004-81.752). CONCLUSIONS: The ability of HLA-DRB1*09 to reduce the risk of anti-AQP4 antibody-negative MS may arise from an interaction with HLA-DRB1*15. By contrast, HLA-DRB1*12 increases susceptibility to anti-AQP4 antibody-positive MS, possibly via an interaction with HLA-DRB1*15.

Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.

We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

Small number of HTLV-1-positive cells frequently remains during complete remission after allogeneic hematopoietic stem cell transplantation that are heterogeneous in origin among cases with adult T-cell leukemia/lymphoma.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide long-term remission for patients with adult T-cell leukemia/lymphoma (ATLL) caused by human retrovirus, human T-lymphocyte virus (HTLV-1). To understand how HTLV-1-positive cells including ATLL cells were suppressed by allo-HSCT, we examined HTLV-1 provirus load and residual ATLL cells in peripheral blood of transplant recipients using PCR-based tests. We found that the copy number of HTLV-1 genome, called provirus, became very small in number after allo-HSCT; however, in most cases, provirus did not disappear even among long-term survivors. Tumor-specific PCR tests demonstrated that most of HTLV-1-positive cells that remained long after transplantation were not primary ATLL cells but donor-derived HTLV-1-positive cells. We also found a case having very low amount of residual disease in peripheral blood even long after transplantation. There was only one recipient in whom we failed to show the presence of HTLV-1 genome and antibody against HTLV-1 even with an extensive search, which strongly suggested the elimination of HTLV-1 after allo-HSCT. These results demonstrated that after allo-HSCT the small amount of residual HTLV-1-positive cells were heterogeneous in origin and that long-term disease control for ATLL could be obtained without the complete elimination of HTLV-1.

Protein kinase A phosphorylates titin's cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes.

beta-Adrenergic stimulation of cardiac muscle activates protein kinase A (PKA), which is known to phosphorylate proteins on the thin and thick filaments of the sarcomere. Cardiac muscle sarcomeres contain a third filament system composed of titin, and here we demonstrate that titin is also phosphorylated by the beta-adrenergic pathway. Titin phosphorylation was observed after beta-receptor stimulation of intact cardiac myocytes and incubation of skinned cardiac myocytes with PKA. Mechanical experiments with isolated myocytes revealed that PKA significantly reduces passive tension. In vitro phosphorylation of recombinant titin fragments and immunoelectron microscopy suggest that PKA targets a subdomain of the elastic segment of titin, referred to as the N2B spring element. The N2B spring element is expressed only in cardiac titins, in which it plays an important role in determining the level of passive tension. Because titin-based passive tension is a determinant of diastolic function, these results suggest that titin phosphorylation may modulate cardiac function in vivo.

Somatic cytogenetic and azoospermia factor gene microdeletion studies in infertile men.

The objective of the present study was to determine the frequency of somatic chromosomal anomalies and Y chromosomal microdeletions (azoospermia factor genes, AZF) in infertile males who seek assisted reproduction. These studies are very important because the assisted reproduction techniques (mainly intracytoplasmic sperm injection) bypass the natural selection process and some classical chromosomal abnormalities, microdeletions of AZF genes or some deleterious genic mutations could pass through generations. These genetic abnormalities can cause in the offspring of these patients male infertility, ambiguous external genitalia, mental retardation, and other birth defects. We studied 165 infertile men whose infertility was attributable to testicular problems (60 were azoospermic, 100 were oligospermic and 5 were asthenospermic). We studied 100 metaphases per patient with GTG banding obtained from temporary lymphocyte culture for chromosomal abnormality detection and performed a genomic DNA analysis using 28 Y chromosome-specific sequence-tagged sites for Y AZF microdeletion detection. Karyotyping revealed somatic anomalies in 16 subjects (16/165 = 9.6%). Of these 16, 12 were in the azoospermic group (12/60 = 20%) and 4 were in the oligospermic group (4/100 = 4%). The most common chromosomal anomaly was Klinefelter syndrome (10/165 = 6%). Microdeletions of AZF genes were detected in 12 subjects (12/160 = 7.5%). The frequencies detected are similar to those described previously. These results show the importance of genetic evaluation of infertile males prior to assisted reproduction. Such evaluation can lead to genetic counseling and, consequently, to primary and secondary prevention of mental retardation and birth defects.

Loss of the same alleles of HRAS1 and D11S151 in two independent pancreatic cancers from a patient with multiple endocrine neoplasia type 1.

The DNAs from two independent pancreatic cancers (tumors 1 and 2) in a patient with multiple endocrine neoplasia type 1 were analyzed. No amplification or gross rearrangement of 19 protooncogenes was observed. However, Southern blot analysis using polymorphic DNA probes revealed loss of heterozygosity at loci on chromosome 11p in both tumors. In tumor 1, an extensive region including the HRAS1, PTH, CALCA, and D11S151 loci was deleted, while in tumor 2 loss of heterozygosity was limited at the HRAS1 and D11S151 loci. Because loss of heterozygosity at other chromosomal loci in the two tumors was quite rare, loss of genes on 11p might be nonrandom. It is noteworthy that the same allele at the HRAS1 locus and also the same allele at the D11S151 locus were lost in the two independent tumors. These results suggest that loss of genes at the HRAS1 and/or D11S151 loci plays an important role unmasking the remaining sequences probably having a recessive mutation.

Single molecular observation of the interaction of GroEL with substrate proteins.

To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins.

Epitope expression and partial structural characterization of F62 lipooligosaccharide (LOS) of Neisseria gonorrhoeae: IgM monoclonal antibodies (3F11 and 1-1-M) recognize non-reducing termini of the LOS components.

F62 LOS of Neisseria gonorrhoeae consists of two components. The higher molecular weight (MW) component is recognized by monoclonal antibody (MAb) 1-1-M and the smaller MW component by MAb 3F11. Epitope expression of the two LOS components and their partial structures were investigated by treating the F62 LOS with several glycosidases and then monitoring their antigenicity with the two mouse IgM MAbs. The 1-1-M-defined LOS component was cleaved with both beta-N-acetylhexosaminidase and endo-beta-galactosidase, and each cleavage resulted in the loss of expression of the 1-1-M-defined epitope. The N-acetylhexosamine (HexNAc) released by the hexosaminidase was found to be GalNAc, and the smaller oligosaccharide released by the endo enzyme was identified to be a dimer GalNAc beta----Gal. In contrast, the MAb 3F11-defined LOS component was not digested by the endo galactosidase, but it was cleaved with alpha and beta-galactosidase, and expression of the MAb 3F11-defined LOS epitope expression of the MAb 3F11-defined LOS was abolished by the treatment with each of two exo enzymes. MAb 3F11 bound to the 1-1-M-defined LOS component resulting from the removal of the beta-GalNAc residue, and the resulting LOS was further cleaved with beta-galactosidase, but not with alpha-galactosidase. From these results, we conclude the following: (1) MAbs 1-1-M and 3F11 both recognize the non-reducing termini of the LOS components; (2) the 1-1-M-defined LOS component has the GalNAc beta----Gal beta 1----4-Glc (or GlcNAc) structure, and the GalNAc beta----Gal residue is involved in the MAb 1-1-M-defined epitope; (3) the MAb 3F11-defined LOS component may not have a Gal beta 1----4GlcNAc beta 1----4Gal beta 1----4Glc structure within the molecule. However, it has beta-Gal residue at its non-reducing terminus, and this residue is involved in the MAb 3F11-defined epitope; (4) the two LOS components share a similar antigenic structure, and the 3F11-defined epitope structure is present in the MAb 1-1-M-defined LOS component. Expression of this epitope within the 1-1-M-defined LOS molecule is blocked by the beta-GalNAc residue; however, the beta-GalNAc residue at the non-reducing end may be not the only structural difference between the two components.

Epitope expression of gonococcal lipooligosaccharide (LOS). Importance of the lipoidal moiety for expression of an epitope that exists in the oligosaccharide moiety of LOS.

Antigenic expression of lipooligosaccharide (LOS) of strain F62 of Neisseria gonorrhoeae, was investigated with mouse monoclonal IgM antibody 3F11. F62 LOS was modified in various ways in order to understand structural requirements for expression of the 3F11-defined epitope. When the LOS was partially deacylated by treating it with 50 mM NaOH at 80 degrees C for 20 min or with anhydrous hydrazine at 80 degrees C for 20 min, the binding of 3F11 to those deacylated LOS samples decreased significantly. Removal of phosphate groups by treatment of the LOS with HF (4 days at 4 degrees C) did not affect the antigenicity at all. Neither did reduction of carboxyl groups in the LOS molecule (by activation of carboxyl groups with a carbodiimide followed by treatment with NaBH4) alter epitope expression. On oxidation with NaIO4, the LOS lost its antigenicity completely. The presence of Mg2+ did not change the circular dichroism (CD) behavior of F62 LOS. However, the partially deacylated LOS samples showed significantly different CD patterns in the 190-200 nm region compared with F62 LOS, which suggests conformational changes of F62 LOS due to the loss of fatty acids in the lipoidal moiety. Oligosaccharide (OS) and lipoidal components obtained after hydrolysis of F62 LOS with 1% acetic acid, were not recognized by the antibody. The antigenicity of OS was not retained by non-stereospecific acylation of OS with decanoyl chloride. We conclude the following: (1) 3F11-defined epitope exists in the OS moiety of F62 LOS; however, for it to be expressed, the carbohydrate moiety must be in a certain conformation that is defined by an overall structure of the LOS molecule. This structure is significantly influenced by some of the fatty acids in the lipoidal moiety of the LOS molecule; (2) the presence of phosphate or 3-deoxy-manno-2-octulosonic acid (dOclA) is not essential for expression of the 3F11-defined epitope; (3) the presence of divalent cations does not affect epitope expression.

Isolation of tissue plasminogen activator from skin lesions with allergic vasculitis.

A tissue plasminogen activator was extracted from skin lesions with allergic vasculitis and purified by successive column chromatography on Sephadex G-200, DEAE-cellulose, Hydroxyaptite-cellulose and polyacrylamide gel electrophoresis. By these procedures, 160 micrograms of enzyme with a specific activity of 843.8 international units/mg protein was obtained from 5 g of original skin. The purified material was homogeneous as ascertained by sodium dodecyl sulfate polyacrylamide gel electrophoresis and had an apparent molecular weight of 110,000 as measured by gel filtration on Sephadex G-200. Its identity with human urokinase was investigated and was found to possess the same plasminogen activator activity as that of urokinase. It had high amindolytic activity, but only slight N-alpha-acetyl-glycyl-L-lysine methyl ester esterolytic activity. This tissue plasminogen activator was confirmed to be immunologically identical to human urokinase.

Ectopic production of parathyroid hormone by small cell lung cancer in a patient with hypercalcemia.

Severe hypercalcemia (serum calcium, 4.37-4.84 nmol/L) was found in a 70-yr-old man who had a small cell carcinoma of the lung with multiple metastases. The plasma immunoreactive PTH concentration was markedly elevated, as measured in three different PTH assays [N-terminal PTH, 4,650 ng/L (normal, 230-630); midregion PTH, 13,850 ng/L (normal, 180-560); C-terminal PTH, 9,900 ng/L (normal, less than 1,300)], but at autopsy the parathyroid glands were histologically normal. The PTH concentration of a liver metastasis was 503.5 ng/g wet wt (normal liver, less than 4.2-5.9), and the PTH in the tumor extract eluted at nearly the same position as synthetic human PTH-(1-84) on gel filtration chromatography. Northern blot analysis revealed PTH mRNA in the tumor as a single band of 0.9 kilobase. These results indicate that the ectopic PTH production by the lung cancer was the cause of hypercalcemia in this patient.