The behavior of cathepsin D during milk processing and its contribution to bitterness in a model fresh cheese.

The bovine endopeptidase cathepsin D was investigated regarding its temperature-dependent inactivation and ability to form bitter peptides within a spiked model fresh cheese. Cathepsin D was found to be more susceptible than other milk endogenous peptidases to temperature treatments in skim milk. Inactivation kinetics revealed decimal reduction times of 5.6 min to 10 s in a temperature range from 60 to 80°C. High temperature and ultra-high temperature (UHT) treatments from 90 to 140°C completely inactivated cathepsin D within 5 s. A residual cathepsin D activity of around 20% was detected under pasteurization conditions (72°C for 20 s). Therefore, investigations were done to estimate the effect of residual cathepsin D activity on taste in a model fresh cheese. The UHT-treated skim milk was spiked with cathepsin D and acidified with glucono-δ-lactone to produce a model fresh cheese. A trained bitter-sensitive panel was not able to distinguish cathepsin D-spiked model fresh cheeses from the control model fresh cheeses in a triangle test. Model fresh cheese samples were also analyzed for known bitter peptides derived from casein fractions using a HPLC-tandem mass spectrometry (MS) approach. In accordance with the sensory evaluation, the MS analyses revealed that the bitter peptides investigated within the cathepsin D-spiked model fresh cheese were not found or were below the limit of detection. Even though cathepsin D may be present during the fermentation of pasteurized milk, it does not seem to be responsible for bitter peptide formation from milk proteins on its own.

Cancer biomarkers in atherosclerotic plaque: Evidenced from structural and proteomic analyses.

Atherosclerosis and cancer are the leading causes of mortality around the world that share common pathogenic pathways. The aim of this study is the investigation of the protein profile of atherosclerotic plaque in order to find similar biomarker between cancer and atherosclerosis. The small pieces of human coronary artery containing advanced atherosclerotic plaque is obtained from patients during bypass surgery. Structural characterization of type V plaque, including fibrous connective tissue, necrotic lipid core, cholesterol clefts and calcium deposits are performed using high resolution transmission electron microscopy (HR-TEM). The protein profile of atherosclerosis plaque is also analyzed using 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF). TEM analysis shows that vascular smooth muscle cells (VSMCs) exhibit different and uncommon morphologies in atherosclerotic plaque which is correlated to the proliferative state of the cells. The proteomics analysis reveals proteins related to atherosclerosis formation including Mimecan, Ras Suppressor Protein-1 (RSUP-1) and Cathepsin D which identified as biomarker of cancerous tumors. The expression of Mimecan and RSUP-1 is down-regulated in atherosclerotic plaque while the expression of Cathepsin D is up-regulated. These data support that atherosclerotic plaque presents some degree of tumorgenesis with the significant activity of VSMCs as the key player in atherogenesis.

The Relationship between the Concentration of Cathepsin A, D, and E and the Concentration of Copper and Zinc, and the Size of the Aneurysmal Enlargement in the Wall of the Abdominal Aortic Aneurysm.

BACKGROUND: Despite advances in diagnostics and treatment, aortic aneurysms are an important clinical problem, mainly due to the accompanying complications that may lead to direct loss of life, also the number of diagnosed and operated aneurysms is constantly increasing. The aim of this study is to determine the relationship between the concentration of lysosomal peptidases cathepsin A, D, and E in the wall of the abdominal aortic aneurysm and the concentration of copper and zinc, and the size of the aneurysm widening in the wall of the abdominal aortic aneurysm. METHODS: The study included 27 patients with abdominal aortic aneurysm from the Department of Vascular Surgery and Transplantation of the University Clinical Hospital in Bialystok. The research material was the wall of the abdominal aortic aneurysm collected intraoperatively. The control material consisted of fragments of the abdominal aorta obtained from organ donors for transplantation. The concentration of cathepsin A, D, and E was determined using enzyme-linked immunosorbent assays. Concentrations of copper and zinc were determined by flame atomic absorption spectrometry after prior mineralization of the samples. All patients were interviewed and asked about basic demographic data, comorbidities, and risk factors for cardiovascular disease to which they were exposed in the past. The statistical analysis was performed using Statistica 10 statistical package. Mann-Whitney U-tests were used and also Spearman's r correlation assuming a significance level of P < 0.05. RESULTS: The concentration of cathepsin A, D, and E was higher in the aortic wall altered by the aneurysm than in the wall of the control aorta (P < 0.05). The analysis of the data showed that there was a positive correlation between the concentration of cathepsin A and D and the width of the aneurysmal widening (r = 0.699 and 0.750, respectively). There was no correlation between cathepsin E concentration and aneurysm width. CONCLUSIONS: The higher contents of cathepsin A, D, and E in the wall of the aortic aneurysm than in the normal aortic wall, as well as a positive correlation between the concentration of cathepsin A and D and the width of the aneurysmal widening, allow to assume the participation of these enzymes in the pathogenesis of the aneurysm.

Immunohistochemical Expression of Cathepsin D in Primary and Recurrent Squamous Cell Carcinoma.

AIM: The aim of this study is to analyze and compare the immunohistochemical expression of cathepsin B in primary oral squamous cell carcinoma (OSCC) and recurrent OSCC. MATERIALS AND METHODS: A total of 50 cases were studied immunohistochemically for rabbit polyclonal antihuman cathepsin D expression. A total of 10 cases of breast carcinoma were taken as positive controls. Immunohistochemical staining was performed using labeled streptavidin-biotin technique. RESULTS: All the 45 cases of OSCC, both primary and recurrent cases included, showed varying grades of cathepsin D immu-noreactivity. Statistical significance at 5% level was observed in cathepsin D expression between the different grades of well, moderate, and poorly differentiated primary squamous cell carcinomas. In the comparison of cathepsin D staining intensity among primary squamous cell carcinomas with and without recurrence, a statistical significance between the groups was observed when the p-value was at 10%, but the same comparison was not significant when the p-value was at 5%. CONCLUSION: Cathepsin D expression in primary squamous cell carcinomas with recurrences was very variable as compared with primary squamous cell carcinomas without recurrences. Comparison of cathepsin D expression in primary with their recurrent counterparts showed mostly similar intensity of expression in recurrent carcinomas, thus suggesting its limited usefulness in predicting recurrence. CLINICAL SIGNIFICANCE: Although cathepsin D might have shown limited usefulness in predicting cancer recurrence, it, however, is a proven valuable tool to detect the aggressiveness of various other tumors, and if corroborated with a larger sample may hold the key to early, more effective, and more specific treatment modalities for cases of oral cancer also.

Identification of lysosomal Npc1-binding proteins: Cathepsin D activity is regulated by NPC1.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1-binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032-1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC-MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.

The senescence-associated secretory phenotype promotes benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is characterized by increased tissue mass in the transition zone of the prostate, which leads to obstruction of urine outflow and considerable morbidity in a majority of older men. Senescent cells accumulate in human tissues, including the prostate, with increasing age. Expression of proinflammatory cytokines is increased in these senescent cells, a manifestation of the senescence-associated secretory phenotype. Multiplex analysis revealed that multiple cytokines are increased in BPH, including GM-CSF, IL-1α, and IL-4, and that these are also increased in senescent prostatic epithelial cells in vitro. Tissue levels of these cytokines were correlated with a marker of senescence (cathepsin D), which was also strongly correlated with prostate weight. IHC analysis revealed the multifocal epithelial expression of cathepsin D and coexpression with IL-1α in BPH tissues. In tissue recombination studies in nude mice with immortalized prostatic epithelial cells expressing IL-1α and prostatic stromal cells, both epithelial and stromal cells exhibited increased growth. Expression of IL-1α in prostatic epithelial cells in a transgenic mouse model resulted in increased prostate size and bladder obstruction. In summary, both correlative and functional evidence support the hypothesis that the senescence-associated secretory phenotype can promote the development of BPH, which is the single most common age-related pathology in older men.

Cellular Composition of the Spleen and Changes in Splenic Lysosomes in the Dynamics of Dyslipidemia in Mice Caused by Repeated Administration of Poloxamer 407.

We studied the effect of dyslipidemia induced by poloxamer 407 (300 mg/kg twice a week for 30 days) on cellular composition of the spleen and splenocyte lysosomes in mice. Changes in blood lipid profile included elevated concentrations of total cholesterol, aterogenic LDL, and triglycerides most pronounced in 24 h after the last poloxamer 407 injection; gradual normalization of lipid profile was observed in 4 days (except triglycerides) and 10 days. The most pronounced changes in the spleen (increase in organ weight and number of cells, inhibition in apoptosis, and reduced accumulation of vital dye acridine orange in lysosomes) were detected on day 4; on day 10, the indices returned to normal. Cathepsin D activity in the spleen also increased at these terms. The relationship between changes in the cellular composition of the spleen and dynamics of serum lipid profile in mice in dyslipidemia caused by repeated administrations of relatively low doses of poloxamer 407 is discussed.

Proteomic profiling of N-linked glycoproteins identifies ConA-binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma.

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N-linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty-eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high-levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA-binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, "ConA-binding proCD" (ConA-pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5-5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA-pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA-pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.

Identification of chemoresistance-related cell-surface glycoproteins in leukemia cells and functional validation of candidate glycoproteins.

Chemoresistance remains the most significant obstacle to successful chemotherapy for leukemia, and its exact mechanism is still unknown. In this work, we used the cell-surface capturing method together with quantitative proteomics to investigate differences in the glycoproteomes of adriamycin-sensitive and adriamycin-resistant leukemia cells. Two quantitative methods, isotopic dimethyl labeling and SWATH, were used to quantify glycoproteins, and 35 glycoproteins were quantified by both methods. High correlation was observed between the glycoproteins quantified by the above two methods, and 15 glycoproteins displayed a consistent significant change trend in both sets of quantitative results. These 15 proteins included classical multidrug resistance-related glycoproteins such as ABCB1 as well as a set of novel glycoproteins that have not previously been reported to be associated with chemoresistance in leukemia cells. Further validation with quantitative real-time PCR and Western blotting confirmed the proteomic screening results. Subsequent functional experiments based on RNA interference technology showed that CTSD, FKBP10, and SLC2A1 are novel genes that participate in the acquisition and maintenance of the adriamycin-resistant phenotype in leukemia cells.

Can immunohistochemistry quantification of Cathepsin-D be useful in the differential diagnosis between vital and post-mortem wounds in humans?

Markers of skin wound vitality and the research methodology used for their determination are still matters of debate in forensic pathology. Cathepsin-D, a lysosomal enzyme, is the most expressed cathepsin in human skin, and although it seems to have the necessary requirements to be utilized as a vitality marker, past research has provided no definitive and clear response on its potential usefulness. Immunohistochemistry with monoclonal antibodies and image analysis has been employed to detect and quantify the expression of Cathepsin-D in human skin wounds. We analyzed skin fragments obtained from 20 living individuals (group A) and 20 persons deceased from natural causes (group B). For each case, five skin fragments were withdrawn at 0', 5', 10', 30', and 90' after abdominal incision. Once the samples were formalin-fixed and paraffin-embedded, we analyzed the expression of Cathepsin-D through the quantification of the immunohistochemistry signal by image analysis. Immunoreactivity was displayed in Pixels of positive area measured by image analysis and converted in micrometer squares. The average levels of Cathepsin-D were higher in group B than in group A, except in three cases which showed a lower expression, with a statistically significant difference of Cathepsin-D expression between the two groups (p < 0.0001). Group B showed unvaried levels among the progressive samples and group A revealed an increasing predominant trend at 30'. Due to the high levels of expression of Cathepsin-D found in the post-mortem injuries, our study definitively excludes any usefulness of immunohistochemistry quantification of this enzyme in the differentiation between vital and post-mortem injuries.

Azaspiracid-1 inhibits the maturation of cathepsin D in mammalian cells.

Azaspiracid-1 (AZA-1) inhibits endocytosis, but the consequences of this alteration on cellular processes are unknown. We hypothesized that the inhibition of endocytosis is a key step of the mode of action of AZA-1, leading to perturbation of cellular processes dependent on proper functioning of endocytic machinery. We tested this working hypothesis by probing whether AZA-1 can alter the maturation of cathepsin D in MCF-7 epithelial cells, as a model system. We found that cell treatment with AZA-1 inhibited the conversion of 52 kDa procathepsin D into the mature 30 kDa protein. The effects induced by AZA-1 were similar to those elicited by chlorpromazine and other agents preventing proper maturation of lysosomal enzymes, indicating that the inhibition of endocytic transfer of proforms to late endosomes/lysosomess is responsible for the effect induced by the toxin. By immunofluorescence microscopy, we found no colocalization of cathepsin D and the early endosomal marker EEA-1 in control cells, where most of cathepsin D resides in late endosomes/lysosomes. Co-localization of cathepsin D and EEA-1 immunoreactivity, in turn, was found in cells exposed to AZA-1, indicating that the toxin blocks protein maturation at the early steps of endocytosis, causing accumulation of procathepsin D in early endosomes. The molecular alteration induced by AZA-1 involved both secreted and intracellular pools of procathepsin D, showing that the toxin effect does not result from a general impairment of vesicular trafficking but is the outcome of a perturbed centripetal process. Furthermore, AZA-1 was found to inhibit procathepsin D maturation also in normal fibroblasts, showing that this molecular response is induced by this toxin in different cell types. The data we obtained corroborated our hypothesis and provide a unified molecular frame for the mode of action of AZAs in animal models, involving a primary alteration of endocytic processes.

Interactions of the proteins of neuronal ceroid lipofuscinosis: clues to function.

Neuronal ceroid lipofuscinoses (NCL) are caused by mutations in eight different genes, are characterized by lysosomal accumulation of autofluorescent storage material, and result in a disease that causes degeneration of the central nervous system (CNS). Although functions are defined for some of the soluble proteins that are defective in NCL (cathepsin D, PPT1, and TPP1), the primary function of the other proteins defective in NCLs (CLN3, CLN5, CLN6, CLN7, and CLN8) remain poorly defined. Understanding the localization and network of interactions for these proteins can offer clues as to the function of the NCL proteins and also the pathways that will be disrupted in their absence. Here, we present a review of the current understanding of the localization, interactions, and function of the proteins associated with NCL.

The potential prognostic value of cathepsin D protein in serous ovarian cancer.

OBJECTIVES: Overexpression of a ubiquitous lysosomal aspartyl protease cathepsin D (cath-D) is involved in the progression of certain cancer types. This study investigated the prognostic value of the cath-D expression and its association with other known clinicopathological parameters in serous ovarian carcinoma. MATERIALS AND METHODS: Cath-D was detected by immunohistochemistry in 49 serous ovarian carcinomas and compared to 50 benign serous ovarian tumors. The results were correlated with clinicopathological characteristics and survival outcomes. RESULTS: In both cath-D positive benign and malignant serous ovarian tumors, a specific granular cytoplasmic staining was observed in both epithelial and stromal cells. Cath-D expression levels were higher in serous ovarian carcinomas than in benign tumors (P < 0.01). Cath-D expression in tumor epithelial cells correlated with mesenchymal cell expression (P < 0.001). The cath-D expression levels of tumor epithelial cells correlated with residual tumor size (P = 0.027) and was not related to other factors (P > 0.05). Patients with higher cath-D expression in epithelial cells had longer disease-free survival time (DFST; P = 0.025) and overall survival time (OST; P = 0.030). Through the Cox regression test, we found that the response to treatment (P < 0.001), pathological stage (P < 0.001), and peritoneal cytology results (P = 0.018) were independent effect factors for DFST. The expression level of cath-D in epithelial cells (P = 0.025), response to treatment (P < 0.001), pathological stage (P = 0.001), and peritoneal cytology results (P = 0.002) were independent effect factors for OST. CONCLUSION: This showed that cath-D was an indicator of malignancy in serous ovarian carcinoma. It was expressed more highly in serous ovarian carcinoma than benign serous ovarian tumor. Additionally, our results suggested that high expression of cath-D in tumor epithelial cells was a favorable survival prognostic factor for serous ovarian carcinoma.

Rapid verification of candidate serological biomarkers using gel-based, label-free multiple reaction monitoring.

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 µL of serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.

PKCδ-dependent signaling mediates ethambutol-induced toxic effects on human retinal pigment cells.

PURPOSE: Our previous report demonstrated that ethambutol (EMB) might induce cytoplasmic vacuolization and reduce the uptake of photoreceptor rod outer segments (ROS) in retinal pigment epithelium (RPE) cells, which are mediated via a protein kinase C (PKC)-dependent pathway. In the present study, we sought to identify the PKC isozyme(s) involved. METHODS: EMB-induced cytoplasmic vacuolization and uptake of ROS were observed under a phase contrast microscope. Western blots were performed to observe the membrane translocation of PKC isozymes and cytoplasmic release of cathepsin D. Quantitative PCR were performed to analyze gene expression of PKCδ. Human RPE cell line RPE50 and ARPE19 cells were pretreated with specific inhibitors or transfected with shRNAs of various PKC isozymes, including PKCα, ß, ε, γ, and δ, to examine whether EMB-induced toxic effects were prevented. RESULTS: In RPE50 cells, gene expression of PKCδ on both mRNA and protein levels was induced by EMB within 30 min to 3 h. EMB-induced cytoplasmic vacuolization in both RPE50 and ARPE19 cells was prevented by pretreating the cells with a specific inhibitor of PKCδ, Rottlerin, or depletion of PKCδ by shRNA. EMB-triggered reduction of ROS uptake was also significantly suppressed by pretreatment with Rottlerin, or depletion of PKCδ by shRNA technology. In contrast, pretreatment of the cells with specific inhibitors of PKCα, ß, ε, or γ, or depletion of PKCα or ß didn't influence the aforementioned EMB-triggered toxic effects. In addition, in RPE50, EMB induced the release of lysosomal enzyme cathepsin D into cytosol within 30 min to 6 h, which was also prevented by Rottlerin. CONCLUSIONS: EMB-induced vacuole formation, cytoplasmic release of cathepsin D, and reduction of phagocytosis in RPE are intimately correlated and regulated by the PKCδ signal pathway.

Trafficking of Candida albicans through oral epithelial endocytic compartments.

Oral epithelial cells are the first cells that interact with C. albicans during the establishment of oropharyngeal candidiasis. Following initial adhesion, C. albicans invades oral epithelial cells by inducing its own endocytosis and gains access to epithelial vacuolar compartments. Epithelial endocytic pathways are key innate immune mechanisms in host defense. We examined the trafficking of C. albicans through oral epithelial endocytic compartments. We present evidence that C. albicans is internalized by oral epithelial cells through actin-dependent clathrin-mediated endocytosis and is taken into vacuolar compartments immediately following its internalization. C. albicans-containing endosomes transiently acquired early endosomal marker EEA1, but showed marked defects in acquisition of late endosomal marker LAMP1 and lysosomal marker cathepsin D. Defective endolysosomal maturation may partially explain the inability of oral epithelial cells to kill C. albicans.

Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells.

Fas ligand (FasL) triggers apoptosis during cytotoxicity mediated by cytotoxic T lymphocytes and during immune downregulation. The ability of T cells and natural killer cells to trigger apoptosis through this mechanism is controlled by the cell surface expression of FasL (ref. 2). Because FasL expression is up-regulated on activation, FasL was thought to be delivered directly to the cell surface. Here we show that newly synthesized FasL is stored in specialized secretory lysosomes in both CD4+ and CD8+ T cells and natural killer cells, and that polarized degranulation controls the delivery of FasL to the cell surface. In this way, FasL-mediated apoptosis is finely controlled by receptor-mediated target-cell recognition. The cytoplasmic tail of FasL contains signals that sort FasL to secretory lysosomes in hemopoietic cells. This pathway may provide a general mechanism for controlling the cell surface appearance of proteins involved in immune regulation.

Involvement of angiotensin II type 1 receptors in interleukin-1ß-induced interleukin-6 production in human gingival fibroblasts.

The renin-angiotensin system is thought to be involved in inflammatory processes such as periodontitis. However, its precise role is still unclear. Therefore, in the present study the expression of the angiotensin II type 1 receptor (AT1R) was investigated in inflamed human gingival tissue, and the possible involvement of the AT1R in interleukin-1ß (IL-1ß)-induced interleukin-6 (IL-6) production by cultured human gingival fibroblasts (HGFs) was also studied. Immunohistochemical staining revealed that inflammatory cells and fibroblast-like cells were positive for the AT1R. However, in healthy gingival tissue, AT1R staining was very weak. The levels of AT1R mRNA and AT1R protein increased in HGFs after stimulation with IL-1ß. The levels of IL-1ß-induced IL6 mRNA and IL-6 protein were significantly reduced in AT1R gene-silenced HGFs compared with control HGFs. The data suggest that the AT1R may be involved in the regulation of gingival inflammation by modulating IL-1ß-induced IL-6 production in HGFs.

A Comparison of Various Chips Used for the Manufacture of Biosensors Applied in Non-Fluidic Array SPRi, Based on the Example of Determination of Cathepsin D.

Non-fluidic array SPR imaging (SPRi) with appropriate biosensors is a new tool for the determination of various biomarkers in body fluids. Numerous biomarkers can be determined without signal enhancement or preliminarily preconcentration. The introduction of a new material solution of the chip may increase the scope of the application of this technique. Solutions with adhesive separating foil and an Ag/Au chip were compared with the previously used two-paint separating polymer and pure gold chip. These solutions were tested using the example of a biosensor for cathepsin D (Cath D), which consisted of pepstatin A (a Cath D inhibitor) immobilized via a cysteamine linker using the NHS/EDC protocol. Four material versions of the Cath D biosensor proved adequate in terms of range of linearity, LOQ, precision and recovery. All four versions of the biosensor were used for the determination of Cath D in the blood serum patients with glioblastoma and control samples, producing very similar results and showing an elevated biomarker concentration in the case of cancer. Therefore, the problem of determining the correct level of Cath D in the serum of healthy individuals has been resolved, correcting literature data which ranged over three orders of magnitude.

Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats.

Cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (BMPR-IB), follicle-stimulating hormone (FSHR), and inhibin α (INHA), were determined in granulosa cells. Results showed that CTSD was expressed in corpus luteum, follicle, and granulosa cells. Notably, CTSD expression in the monotocous group was significantly higher than that in the polytocous group. In addition, CTSD knockdown could improve granulosa cell proliferation, inhibit cell apoptosis, and significantly elevate the expression of proliferating cell nuclear antigen (PCNA) and B cell lymphoma 2 (Bcl-2), but it lowered the expression of Bcl-2-associated X (Bax) and caspase-3. Furthermore, CTSD knockdown significantly reduced the ratios of cells in the G0/G1 and G2/M phases but substantially increased the ratio of cells in the S phase. The expression levels of cyclin D2 and cyclin E were elevated followed by the obvious decline of cyclin A1 expression. However, the expression levels of BMPR-IB, FSHR, and INHA clearly increased as a result of CTSD knockdown. Hence, our findings demonstrate that CTSD is an important factor affecting the litter size trait in goats by regulating the granulosa cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific trait.