Correlation Between Serum 25(OH)D and Abdominal Visceral Fat Area in Patients with Type 2 Diabetes Mellitus in the Context of different Bone Mass.

Objective: To investigate the correlation between serum levels of 25-hydroxy vitamin D [25(OH)D] and the visceral fat area of patients with type 2 diabetes mellitus (T2DM) in the context of different bone mass. Materials and Methods: A total of 180 patients with T2DM were randomly selected for bone mineral density (BMD) examination. According to the results, they were divided into three groups: T2DM normal bone group (group A); T2DM bone mass reduction group (group B); T2DM osteoporosis group (group C). Result: Serum 25(OH)D levels in NC group, A group, B group and C group decreased in turn, and Visceral fat area (VFA) in group B and group C were significantly higher than those in group A and NC [(29.41±4.87) vs. (22.76±4.23) vs. (17.78±3.61) vs. (9.70±3.01), P<0.05], [(117.76±38.79), (125.08±37.90) vs. (89.79±26.51), (97.53±28.61), P<0.05]. Pearson correlation analysis showed that L1-L4 lumbar vertebrae bone density was positively correlated with 25(OH)D and VFA; left femoral neck bone density was positively correlated with 25(OH)D, and negatively correlated with VFA. Conclusion: Serum 25(OH)D and VFA may be associated with the development of T2DM combined with OP.

[MiR-128, a key regulator of oncogenic properties].

MicroRNAs (miRNA) are small noncoding RNAs that are critical regulators of gene function. In the recent years, miRNAs have been increasingly noted for their capacity to regulate key malignant properties of tumor cells. MicroRNA-128 (miR-128) is a brain-enriched miRNA that is normally involved in the development of the nervous system and in the maintenance of neural physiological functions. In tumorcells, miR-128 expression is dysregulated through a variety of genetic and epigenetic events. Dysregulation: of miR-128 has profound effects on tumorigenesis and maintenance of tumor cells through alterations in cellular proliferation, differentiation, metabolism, and apoptosis. This article will review the latest advances in our understanding of miR-128, specifically in the context of clinical and fundamental cancer biology. Further characterization of miR-128 will likely identify its new roles in cancer biology. The use of miR-128 as a diagnostic and/or therapeutic tool may result in improvements in diagnosis, prognosis, and treatment of numerous cancers.

Nicotine increases the resistance of lung cancer cells to cisplatin through enhancing Bcl-2 stability.

BACKGROUND: Nicotine is able to activate mitogenic signalling pathways, which promote cell growth or survival as well as increase chemoresistance of cancer cells. However, the underlying mechanisms are not fully understood. METHODS: In this study, we used immunoblotting and immunoprecipitation methods to test the ubiquitination and degradation of Bcl-2 affected by nicotine in lung cancer cells. Apoptotic assay was also used to measure the antagonising effect of nicotine on cisplatin-mediated cytotoxicity. RESULTS: We demonstrated that the addition of nicotine greatly attenuated Bcl-2 ubiquitination and degradation, which further desensitised lung cancer cells to cisplatin-induced cytotoxicity. In this process, Bcl-2 was persistently phosphorylated in the cells cotreated with nicotine and cisplatin. Furthermore, Akt was proven to be responsible for sustained activation of Bcl-2 by nicotine, which further antagonised cisplatin-mediated apoptotic signalling. CONCLUSIONS: Our study suggested that nicotine activates its downstream signalling to interfere with the ubiquitination process and prevent Bcl-2 from being degraded in lung cancer cells, resulting in the increase of chemoresistance.

[Immunogenicity and safety of revaccination of 23-valent pneumococcal polysaccharide vaccine in people aged 60 years and above].

Objective: To evaluate the immunogenicity and safety of revaccination of 23-valent pneumococcal polysaccharide vaccine (PPV23) in elderly people aged ≥60 years. Methods: The elderly aged ≥60 years with 1 dose of PPV23 vaccination were selected as revaccination group and those without history of pneumococcal vaccine immunization were selected as the first vaccination group. One dose of PPV23 was administered to both groups, and the first blood samples were collected before vaccination while the second blood samples were collected on day 28-40 after vaccination. ELISA was used to detect the concentrations of anti-specific serotype Streptococcus pneumoniae podocyte polysaccharide immunoglobulin G, and the safety of the vaccination was evaluated after 30 days. Results: The geometric mean concentration (GMC) of antibody to 23 serotypes before the vaccination (0.73-13.73 µg/ml) was higher in revaccination group than in the first vaccination group (0.39-7.53 µg/ml), the GMC after the vaccination (1.42-31.65 µg/ml) was higher than that before the vaccination (0.73-13.73 µg/ml) in the revaccination group, and the GMC after the vaccination (1.62-43.76 µg/ml) was higher than that before the vaccination (0.39-7.53 µg/ml) in the first vaccination group; the geometric mean growth multiple in revaccination group (2.16-3.60) was lower than that in the first vaccination group (3.86-16.13); The mean 2-fold antibody growth rate was lower in revaccination group (53.68%, 95%CI: 52.30%-55.06%) than in the first vaccination group (93.16%, 95%CI: 92.18%- 94.15%), all differences were significant (P<0.001). After the vaccination, 13 serotypes of GMC were higher in the first vaccination group than in revaccination group (P<0.001), the differences were not significant for 10 serotypes of GMC (P>0.05). The incidence of local adverse reaction was 19.20% and 13.27% in revaccination group and the first vaccination group, respectively (P=0.174). Conclusions: The antibody level in ≥60 years people who received one dose of PPV23 after a 5-year interval was still higher than that in unvaccinated people. The antibody level decreased after 5 years of the first vaccination, and the antibody level could be rapidly increased by one more dose vaccination, but the overall immune response was lower than that of the first vaccination; revaccination with PPV23 has a good safety.

[Immunogenicity and safety of a boost dose of measles, mumps, and rubella combined vaccine for 4-6 years old children].

Objective: To investigate the immunogenicity and safety of a boost dose of measles, mumps, and rubella combined vaccine (MMR) for children 4 to 6 years old. Methods: Children, aged 4 to 6 years old, had vaccinated with 1 dose of measles and rubella combined vaccine(MR) at the age of 8 months and 1 dose of MMR vaccine at 18-months, were recruited in Shanxi, Inner Mongolia, and Beijing, respectively. All children were assigned into 4, 5 and 6-year-old group. The children who met inclusion and exclusion criteria were vaccinated with 1 dose MMR vaccine, and were collected blood samples before vaccination and 35 to 42 d after the vaccination. During the study period, adverse events were collected at 30 min, 1 d, 2 d, 3 d, 4-12 d, and 13 to 42 days after vaccination. Serum was tested for IgG antibodies against measles, mumps and rubella. Geometric mean concentrations (GMC) of measles, mumps, and rubella antibodies were compared among groups by analysis of variance or non-parametric test. Seropositive rates and adverse event rates were compared among groups by Chi-square test or Fisher exact test. Results: A total of 500 children were included in immunogenicity analysis and 535 children were included in safety analysis. The overall adverse event rate was 20.37%, the most of severity for adverse events was mild. The rates of local and systemic adverse events were 0.37% and 20.00%, respectively. Symptoms of local adverse events were redness. The main systemic adverse events were fever, followed by cough, rash and runny nose. Received a dose of MMR vaccine for booster immunization, the seropositive rates of measles antibody, mumps antibody and rubella antibody were above 99% for all 3 age groups, and there was no significant difference between groups. There were significant differences in mumps antibody GMC among groups (P=0.042), but no significant differences in measles and rubella antibodies GMC. Conclusion: The immunogenicity and safety of a boosted MMR vaccintion in children aged 4, 5 and 6 years were all similar good.

The apoptosis in arsenic-induced oxidative stress is associated with autophagy in the testis tissues of chicken.

The aim of this study is to investigate whether arsenic (As) could induce testicular poisoning and influence the oxidative stress, apoptosis and autophagy in chickens. Seventy-two 1-day-old male Hy-line chickens were divided into 4 groups which were exposed to 0, 0.625, 1.25, and 2.5 mg/kg body weight (BW) of arsenic trioxide (As2O3) for 30, 60 and 90 days, respectively. Histological and ultrastructural changes, antioxidant enzyme activity, mRNA and protein levels of apoptosis and autophagy-related genes were detected. Oxidative stress injuries were obvious in the testes exposure to As2O3, which resulted in the decreased activities of antioxidant enzymes, such as catalase (CAT) and superoxide dismutases (SOD). Meanwhile, the changes of mRNA and protein levels of apoptosis and autophagy-related genes showed that As2O3 exposure induced enhanced testicular apoptosis and increased the levels of autophagy markers such as Microtubule associated protein light chains 3-II (LC3-II), dynein, Beclin-1, Autophagy associated gene 5 (ATG5) and ATG4B but not LC3-I and mammalian target of rapamycin (mTOR), and demonstrated the cross-talk between apoptosis and autophagy. Histological and ultrastructural abnormalities confirm the changes of the above indicators. Taken together, our findings provide deeper insights into roles of excessive apoptosis and autophagy in the aggravation of testicular damage, which could contribute to a better understanding of As2O3-induced testicular poisoning in chickens.

Prostase/KLK-L1 is a new member of the human kallikrein gene family, is expressed in prostate and breast tissues, and is hormonally regulated.

By using the positional candidate gene approach, we were able to identify a novel serine protease gene that maps to chromosome 19q13.3-q13.4. Screening of expressed sequence tags allowed us to establish the expression of the gene and delineate its genomic organization (GenBank accession no. AF135023). We named this gene KLK-L1. Another group, by using a subtraction hybridization method, cloned the same gene and named it prostase (GenBank accession nos. AF113140 and AF113141). Here, we describe the precise mapping and localization of the prostase/KLK-L1 gene between the known genes KLK2 (human glandular kallikrein) and zyme (also known as protease M/neurosin). The direction of transcription of prostase/KLK-L1 is the same as that of zyme but opposite to that of KLK2 and prostate-specific antigen genes. Contrary to the initial impression, prostase/KLK-L1 is expressed at high levels not only in prostate tissue but also in testis, mammary gland, adrenals, uterus, thyroid, and salivary glands. We have further demonstrated with in vitro experiments with the breast carcinoma cell line BT-474 that this gene is expressed and that its expression is up-regulated by androgens and progestins. On the basis of information on other genes that are localized in the same region (prostate-specific antigen, KLK2, zyme, and normal epithelial cell specific-1 gene), we speculate that prostase/KLK-L1 may be involved in the pathogenesis and/or progression of prostate, breast, and possibly other malignancies.

Autologous hematopoietic stem cell transplantation in patients with end-stage liver disease: a 5-year follow-up study of 48 patients.

OBJECTIVE: To investigate the long-term therapeutic effect of autologous hematopoietic stem cell transplantation in patients with End-stage Liver Disease (ESLD). PATIENTS AND METHODS: Forty-eight ESLD patients underwent autologous CD34+ stem cell transplantation were retrospectively reviewed. Changes in clinical and biochemical data, complications, and quality of life were monitored at 3, 6, 12, 36, and 60 months following the stem cell transplantation. Liver biopsies were obtained for histopathological analysis using Ishak system. RESULTS: Marked improvement in clinical and biochemical data was observed during the long-term follow-up. Serum albumin was significantly increased (p<0.001), while total serum bilirubin, prothrombin time (PT), and international normalized ratio (INR) were all significantly decreased (p<0.001). Ishak inflammation and fibrosis scores were significantly decreased with the increased time (p<0.001). The number of patients with ascites, model of end-stage liver disease (MELD) score, Child-Pugh class, and indocyanine green (ICG) score were all markedly reduced with increased time. Meanwhile, the quality of life score of the patients was significantly increased (p<0.001). Six patients died during the 5-years follow-up, and complications occurred in 17 patients. The incidence of complications was significantly associated with mortality of the patients (p<0.05). CONCLUSIONS: The study provided the evidence that autologous CD34+ stem cell transplantation could offer a long-term therapeutic benefit to patients with ESLD. The complications occurred during the process was significantly associated with survival of the patients. Future studies on a large cohort of patients are needed to confirm the long-term effect of stem cell therapy on ESLD.

The new human kallikrein gene family: implications in carcinogenesis.

The traditional human kallikrein gene family consists of three genes, namely KLK1 [encoding human kallikrein 1 (hK1) or pancreatic/renal kallikrein], KLK2 (encoding hK2, previously known as human glandular kallikrein 1) and KLK3 [encoding hK3 or prostate-specific antigen (PSA)]. KLK2 and KLK3 have important applications in prostate cancer diagnostics and, more recently, in breast cancer diagnostics. During the past two to three years, new putative members of the human kallikrein gene family have been identified, including the PRSSL1 gene [encoding normal epithelial cell-specific 1 gene (NES1)], the gene encoding zyme/protease M/neurosin, the gene encoding prostase/KLK-L1, and the genes encoding neuropsin, stratum corneum chymotryptic enzyme and trypsin-like serine protease. Another five putative kallikrein genes, provisionally named KLK-L2, KLK-L3, KLK-L4, KLK-L5 and KLK-L6, have also been identified. Many of the newly identified kallikrein-like genes are regulated by steroid hormones, and a few kallikreins (NES1, protease M, PSA) are known to be downregulated in breast and possibly other cancers. NES1 appears to be a novel breast cancer tumor suppressor protein and PSA a potent inhibitor of angiogenesis. This brief review summarizes recent developments and possible applications of the newly defined and expanded human kallikrein gene locus.

Prognostic value of human kallikrein 10 expression in epithelial ovarian carcinoma.

PURPOSE: Human kallikrein 10 (hK10; also known as the normal epithelial cell-specific 1 gene and protein) is a secreted serine protease, which belongs to the human kallikrein family. It has been reported that hK10 is down-regulated in breast and prostate cancer cell lines and that it may function as a tumor suppressor. Recently, we developed a highly sensitive and specific immunoassay for hK10 and found that this protein is abundantly expressed in ovarian tissue. In this study, we measured quantitatively hK10 levels in ovarian cancer cytosolic extracts and evaluated the prognostic value of this biomarker in ovarian cancer. EXPERIMENTAL DESIGN: Specimens from eight normal ovarian tissues, eight ovarian tissues with benign disease, and 182 ovarian tumors were investigated. RESULTS: hK10 concentration in ovarian tumor cytosols ranged from 0 to 84 ng/mg of total protein, with a median of 2.6. This median was highly elevated in comparison with normal and benign ovarian tissues (P < 0.001). A cutoff of 1.35 ng/mg was selected to categorize tumors as hK10 high and hK10 low. With chi(2) test and Fisher's exact test, high concentration hK10 was found to be associated with advanced disease stage, serous histological type, suboptimal debulking, and large residual tumor (>1 cm; all P < 0.05). hK10 status was additionally correlated with clinical outcome, including progression-free (PFS) and overall survival (OS) using the Cox model. In univariate analysis, we found that patients with hK10 high tumors were more likely to die and relapse, in comparison with patients with hK10 low tumors (hazards ratios for PFS and OS were 1.93 and 2.42, respectively; P < 0.05). Although this correlation disappeared after the entire patient population was subjected to multivariate analysis, it remained significant in the subgroup of patients with stage III/IV ovarian cancer (hazards ratios for PFS and OS were 1.98 and 2.12, respectively; P < 0.05). CONCLUSIONS: Our results indicate that hK10 is a new, independent, unfavorable prognostic marker, especially for late-stage ovarian cancer.

The region in the mu opioid receptor conferring selectivity for sufentanil over the delta receptor is different from that over the kappa receptor.

We determined the binding domains of sufentanil and lofentanil in the mu opioid receptor by comparing their binding affinities to seven mu/delta and six mu/kappa chimeric receptors with those to mu, delta and kappa opioid receptors. TMHs 6 and 7 and the e3 loop of the mu opioid receptor were important for selective binding of sufentanil and lofentanil to the mu over the kappa receptor. TMHs 1-3 and the e1 loop of the mu opioid receptor conferred binding selectivity for sufentanil over the delta receptor. Thus, the region that conferred binding selectivity for sufentanil differs, depending on chimeras used. In addition, the interaction TMHs 1-3 and TMHs 6-7 was crucial for the high affinity binding of these two ligands. These two regions are likely to contain sites of interaction with the ligands or to confer conformations specific to the mu receptor.

The hNT human neuronal cell line survives and migrates into rat retina.

The present studies were undertaken to determine if hNT cells can survive in the vitreous of the eye and migrate into the retina. The hNT neuronal cell line represents a uniform source of human tissue that may be of use in retinal grafts. hNT cells stored in liquid nitrogen were thawed and labeled with the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). Thirty thousand cells in 1 microL were injected epiretinally in rat. At survival times of 3, 14, 28, or 56 days, retinal sections were examined quantitatively by epifluorescence to reveal DiI-labeled cells. hNT cells survived in the vitreous at all time points without evidence of vascularization. At 3 days, essentially no hNT cells were found in deep retina, and only very few were attached to retina. At days 14, 28, and 56, hNT cells were found to cluster on the vitreal/retinal interface, and in deeper layers. The clusters of hNT cells took on the shape of a funnel at 14 days, and inverted funnel at 28 days, and by 56 days, populated the photoreceptor layer as a stratum. It is possible that hNT cells took on the morphology and function of photoreceptors. These results suggest that hNT cells injected epiretinally survive in the vitreous at least 56 days, migrate to the retinal/vitreous interface, and may migrate through the retina. This system permits the independent and quantitative evaluation of survival and migratory trophic responses.

Human kallikrein 10: a novel tumor marker for ovarian carcinoma?

BACKGROUND: Human kallikrein 10 (hK10, encoded by KLK10 gene) is a recently discovered member of the human kallikrein family. hK10 is a secreted serine protease. With the development of a highly sensitive and specific immunoassay for hK10, quantification of hK10 in the circulation is now feasible. Our aim was to investigate whether hK10 concentration in serum changes in various malignancies. METHODS: We used a highly specific and sensitive immunofluorometric assay to quantify hK10 protein in 374 serum samples from healthy individuals and patients with various malignancies. RESULTS: Serum hK10 concentration was found to be significantly elevated in 56% of the ovarian cancer patients and such an increase was not observed in serum of healthy individuals or in serum of patients with other types of cancer, with the exception of approximately 15% of patients with gastrointestinal cancer. This hK10 elevation does not correlate well with CA 125. We have further demonstrated that hK10 concentration changes during ovarian cancer progression. CONCLUSION: This is the first report describing that hK10 serum concentration is significantly elevated in the majority of ovarian cancer patients. Our results indicate that hK10 may be a potential new serological marker for ovarian cancer diagnosis and monitoring.

Immunohistochemical localization of human kallikreins 6, 10 and 13 in benign and malignant prostatic tissues.

Human kallikreins 6, 10 and 13 (hK6, hK10 and hK13) are expressed by many normal, mainly glandular tissues, including prostatic epithelium. Some kallikreins may function as tumor suppressors or are downregulated during cancer progression. The aim of this study was to evaluate the immunoexpression of these kallikreins in benign and malignant prostatic tissues and correlate their expression with prostate cancer (PC) prognosis. Included in the study were 25 cases of nonmalignant prostate and 179 cases of PC. Among them, 122 PC cases were immunostained for hK6, 94 for hK10 and 113 for hK13, respectively. The follow-up period for a subset of 68 patients who had undergone radical prostatectomy (RP) was 1-58 months (mean=13.4 +/- 1.7 and median=8.0 months). A cutoff value of 0.2 microg/l of serum PSA was established as a biochemical recurrence threshold. Follow-up information was available for 26/55 RP cases stained for hK6, 14/32 cases stained for hK10 and 25/59 cases stained for hK13. Gleason score (GS) 7 carcinomas were stratified as 7a and 7b, according to the primary grade. PC with GS 2-7a were histologically categorized as low malignant (LM) and PC with GS 7b-10 as high malignant (HM). The immunohistochemical method of streptavidin-biotin-peroxidase using monoclonal and polyclonal antibodies was performed. In the benign prostate and in prostatic intraepithelial neoplasia, a cytoplasmic immunostaining of varying intensity was evident. In PC, the immunoexpression of all kallikreins was decreased: 102/122 cases (84%) were positive for hK6, 73/94 (78%) for hK10 and 97/113 (86%) for hK13, respectively. A statistically significant difference in expression was found, in comparison to nonmalignant prostates (P=0.029, 0.009 and 0.045, respectively). Also, a positive correlation was observed between the immunoexpression of these three kallikreins. Concerning the histological grade, HM-PC expressed all three kallikreins with a slightly higher percentage than LM-PC: 79 vs 88% for hK6, 76 vs 79% for hK10 and 76 vs 92% for hK13. These differences were statistically significant only in the case of hK13 (P=0.024). Serum PSA did not correlate with kallikrein immunoexpression in PC. Furthermore, there was no significant correlation between kallikrein expression and pathological stage or recurrence, in the cases of RP. All three kallikreins are expressed in the nonmalignant and malignant prostate, with cancer tissues demonstrating slightly lower expression. Expression levels did not correlate with aggressiveness and they do not seem to have value for prostate cancer prognosis.

The normal epithelial cell-specific 1 (NES1) gene is up-regulated by steroid hormones in the breast carcinoma cell line BT-474.

The normal epithelial cell-specific 1 (NES1) gene encodes a serine protease which was found to be down-regulated in breast cancer. There is evidence that NES1 acts as a tumor suppressor gene in breast cancer cells. To further understand its role in breast tumorigenesis, we investigated the effect of estrogens, androgens, and progestins on NES1 gene expression, in the breast cancer cell line BT-474, at the transcription level. The reverse transcriptase polymerase chain reaction method was used to monitor changes in the NES1 mRNA. Our experiments showed that NES1 gene expression is up-regulated promptly in response to 17 beta-estradiol, 5 alpha-dihydrotestosterone (DHT) and norgestrel stimulation. NES1 gene mRNA started to increase 2 hours after estradiol stimulation and 8 hours after DHT stimulation. The stimulation of NES1 by estradiol can be dramatically blocked by the estrogen antagonists ICI 182,780 and 4-hydroxytamoxifen. Mifepristone (a synthetic antiprogestin) can partially block the up-regulation of the NES1 gene by norgestrel. Dose-response experiments indicated that the lowest stimulatory concentration of 17 beta-estradiol, DHT, and norgestrel is 10(-11) M, 10(-10) M, and 10(-10) M, respectively. The production of NES1 mRNA increased coordinately with increasing concentration of the stimulants. These results suggest that the NES1 gene is primarily regulated by estrogen, but also by androgen and progestin in the breast cancer cell line BT-474. It appears that NES1 may be involved in a pathway that counter balances the action of estrogens and androgens in steroid hormone responsive tissues.

Identification of novel human kallikrein-like genes on chromosome 19q13.3-q13.4.

The human kallikrein gene family is localized on chromosome 19q13.3-q13.4 and currently includes three members: KLK1 or pancreatic/renal kallikrein, KLK2 or human glandular kallikrein and KLK3 or prostate-specific antigen (PSA). The latter two genes are almost prostate-specific and they are used for diagnosis and monitoring of prostate cancer and more recently, in breast cancer applications. In this paper, we analyzed a 300Kb genomic DNA region around chromosome 19q13.3-q13.4 in an effort to map known kallikrein or kallikrein-like genes and identify new kallikrein-like genes. Using the known kallikrein or kallikrein-like genes PSA, KLK2, enzyme and normal epithelial cell-specific 1 gene (NES1) as landmarks, we have identified another six novel genes of which, five have protein homologies and gene structure similarities with other kallikreins or kallikrein-like genes. We conclude, contrary to the current belief, that the human kallikrein gene locus contains a large number of kallikrein-like genes (at least thirteen). In this paper, we present a detailed description of the human kallikrein gene locus, encompassing the already known and newly identified genes. These new genes, like the already known kallikreins, may have utility for diagnosis, monitoring and therapeutics of various cancers including those of the breast, prostate and testis.

Origin and phylogeographical structure of Chinese cattle.

Complete mitochondrial D-loop sequences of 231 samples were used to explore the origin and genetic diversity of Chinese cattle. Phylogenetical analysis of these sequences revealed both Bos taurus and Bos indicus mitochondrial types in Chinese cattle. Four of the previously identified mitochondrial DNA lineages (T1-T4) were identified in the Bos taurus type, including lineage T1, which was found for the first time in Chinese cattle. Two lineages (I1 and I2) were identified in the Bos indicus type. Our results support the suggestion that the Yunnan-Guizhou Plateau is the domestication site of Chinese zebu. We also found evidence that Tibetan cattle originated from taurine and zebu cattle. The distribution pattern of Chinese cattle breeds was closely related to the geographical and climatic background. It was possible to divide Chinese cattle in this study into two major groups: northern and southern cattle.

Preliminary examination of time-resolved fluorometry for protein array applications.

The advantages of time-resolved fluorometry over conventional fluorometric analysis are well known. However, time-resolved fluorescence has not as yet found wide applications in protein microarray or other multiparametric methods of analysis. Here we describe a general method which is suitable for multiparametric and microarray analysis, based on time-resolved fluorometry. A polystyrene surface is coated with different monoclonal antibodies, specific for certain analytes. The analyte mixtures are then universally biotinylated, using an active biotin ester. After removing excess biotin, the biotinylated samples are applied on the polystyrene surface, incubated and the excess is washed away. The bound moieties are then quantified by adding a universal detection reagent containing streptavidin, labelled with a fluorescent europium chelate. After washing and drying of the solid surface, the immobilized moieties are detected by using solid-phase, laser-excited time-resolved fluorometric analysis. In a preliminary examination of this principle, we have demonstrated that we can correctly identify upregulation of three secreted proteins, following stimulation of a breast carcinoma cell line with various steroids. Our method should be suitable for high-density microarray analysis of proteins, captured by specific monoclonal antibodies or other binding reagents.