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1.
Article in English | MEDLINE | ID: mdl-37888912

ABSTRACT

Candida is one of the most common opportunistic fungal pathogens in humans. Its adhesion to the host cell is required in parasitic states and is important for pathogenesis. Many studies have shown that there is an increased risk of developing candidiasis when normal tissue barriers are weakened or when immune defenses are compromised, for example, during cancer treatment that induces immunosuppression. The mechanical properties of malignant cells, such as adhesiveness and viscoelasticity, which contribute to cellular invasion and migration are different from those of noncancerous cells. To understand host invasion and its relationship with host cell health, we probed the interaction of Candida spp. with cancerous and noncancerous human cell lines using atomic force microscopy in the single-cell force spectroscopy mode. There was significant adhesion between Candida and human cells, with more adhesion to cancerous versus noncancerous cell lines. This increase in adhesion is related to the mechanobiological properties of cancer cells, which have a disorganized cytoskeleton and lower rigidity. Altered geometry and cytoskeletal disruption of the human cells impacted adhesion parameters, underscoring the role of cytoskeletal organization in Candida-human cell adhesion and implicating the manipulation of cell properties as a potential future therapeutic strategy.

2.
PLoS One ; 17(11): e0277097, 2022.
Article in English | MEDLINE | ID: mdl-36383525

ABSTRACT

The essential oil from Rosmarinus officinalis L., a composite mixture of plant-derived secondary metabolites, exhibits antifungal activity against virulent candidal species. Here we report the impact of rosemary oil and two of its components, the monoterpene α-pinene and the monoterpenoid 1,8-cineole, against Candida albicans, which induce ROS-dependent cell death at high concentrations and inhibit hyphal morphogenesis and biofilm formation at lower concentrations. The minimum inhibitory concentrations (100% inhibition) for both rosemary oil and 1,8-cineole were 4500 µg/ml and 3125 µg/ml for α-pinene, with the two components exhibiting partial synergy (FICI = 0.55 ± 0.07). At MIC and 1/2 MIC, rosemary oil and its components induced a generalized cell wall stress response, causing damage to cellular and organelle membranes, along with elevated chitin production and increased cell surface adhesion and elasticity, leading to complete vacuolar segregation, mitochondrial depolarization, elevated reactive oxygen species, microtubule dysfunction, and cell cycle arrest mainly at the G1/S phase, consequently triggering cell death. Interestingly, the same oils at lower fractional MIC (1/8-1/4) inhibited virulence traits, including reduction of mycelium (up to 2-fold) and biofilm (up to 4-fold) formation, through a ROS-independent mechanism.


Subject(s)
Oils, Volatile , Rosmarinus , Eucalyptol/pharmacology , Candida albicans , Reactive Oxygen Species , Virulence , Oils, Volatile/pharmacology , Microbial Sensitivity Tests , Antifungal Agents/pharmacology , Monoterpenes/pharmacology
3.
Microbiol Spectr ; 10(6): e0318322, 2022 12 21.
Article in English | MEDLINE | ID: mdl-36394350

ABSTRACT

Candida albicans is part of the normal human flora but is most frequently isolated as the causative opportunistic pathogen of candidiasis. Plant-based essential oils and their components have been extensively studied as antimicrobials, but their antimicrobial impacts are poorly understood. Phenylpropenoids and monoterpenes, for example, eugenol from clove and citral from lemon grass, are potent antifungals against a wide range of pathogens. We report the cellular response of C. albicans to eugenol and citral, alone and combined, using biochemical and microscopic assays. The MICs of eugenol and citral were 1,000 and 256 µg/mL, respectively, with the two exhibiting additive effects based on a fractional inhibitory concentration index of 0.83 ± 0.14. High concentrations of eugenol caused membrane damage, oxidative stress, vacuole segregation, microtubule dysfunction and cell cycle arrest at the G1/S phase, and while citral had similar impacts, they were reactive oxygen species (ROS) independent. At sublethal concentrations (1/2 to 1/4 MIC), both oils disrupted microtubules and hyphal and biofilm formation in an ROS-independent manner. While both compounds disrupt the cell membrane, eugenol had a greater impact on membrane dysfunction. This study shows that eugenol and citral can induce vacuole and microtubule dysfunction, along with the inhibition of hyphal and biofilm formation. IMPORTANCE Candida albicans is a normal resident on and in the human body that can cause relatively benign infections. However, when our immune system is severely compromised (e.g., cancer chemotherapy patients) or underdeveloped (e.g., newborns), this fungus can become a deadly pathogen, infecting the bloodstream and organs. Since there are only a few effective antifungal agents that can be used to combat fungal infections, these fungi have been exposed to them over and over again, allowing the fungi to develop resistance. Instead of developing antifungal agents that kill the fungi, some of which have undesirable side effects on the human host, researchers have proposed to target the fungal traits that make the fungus more virulent. Here, we show how two components of plant-based essential oils, eugenol and citral, are effective inhibitors of C. albicans virulence traits.


Subject(s)
Candida albicans , Oils, Volatile , Infant, Newborn , Humans , Eugenol/pharmacology , Antifungal Agents/pharmacology , Reactive Oxygen Species , Oils, Volatile/pharmacology , Oils, Volatile/chemistry , Biofilms , Plant Oils/pharmacology , Microbial Sensitivity Tests
4.
Microorganisms ; 10(10)2022 Oct 08.
Article in English | MEDLINE | ID: mdl-36296264

ABSTRACT

Plant-based essential oils are promising anti-virulence agents against the multidrug-resistant opportunistic pathogen Candida albicans. Gas chromatography-mass spectrometry of Cinnamomum zeylanicum (cinnamon) leaf and Eugenia caryophyllus (clove) flower bud essential oils revealed eugenol (73 and 75%, respectively) as their major component, with ß-caryophyllene, eugenyl acetate, and α-humulene as common minor components. Cinnamon leaf and clove essential oils had minimum inhibitory concentrations of 600 and 500 µg/mL, respectively against the C. albicans RSY150 reference strain and 1000 and 750 µg/mL, respectively for the clinical reference strain ATCC 10231. The combined oils are additive (FICI = 0.72 ± 0.16) and synergistic (0.5 ± 0.0) against RSY150 and the clinical reference strain, respectively. Mycelial growth was inhibited by sublethal concentrations of either essential oil, which abolished colony growth. At half of the lowest combined lethal concentration for the two oils, the yeast-to-hyphal transition and mycelial growth was potently inhibited. Mutant strains als1Δ/Δ, als3Δ/Δ, hwp1Δ/HWP1+, and efg1Δ/Δ were sensitive to either or both oils, especially efg1Δ/Δ. In conclusion, oils of cinnamon leaf and clove and their combination significantly impact C. albicans virulence by inhibiting hyphal and mycelial growth.

5.
Antibiotics (Basel) ; 10(1)2021 Jan 15.
Article in English | MEDLINE | ID: mdl-33467766

ABSTRACT

Candida albicans is the causative agent of fatal systemic candidiasis. Due to limitations of antifungals, new drugs are needed. The anti-virulence effect of plant essential oils (EOs) was evaluated against clinical C. albicans isolates including cinnamon, clove, jasmine and rosemary oils. Biofilm, phospholipase and hemolysin were assessed phenotypically. EOs were evaluated for their anti-virulence activity using phenotypic methods as well as scanning electron microscopy (SEM) and atomic force microscopy (AFM). Among the C. albicans isolates, biofilm, phospholipase and hemolysins were detected in 40.4, 86.5 and 78.8% of isolates, respectively. Jasmine oil showed the highest anti-biofilm activity followed by cinnamon, clove and rosemary oils. SEM and AFM analysis showed reduced adherence and roughness in the presence of EOs. For phospholipase, rosemary oil was the most inhibitory, followed by jasmine, cinnamon and clove oils, and for hemolysins, cinnamon had the highest inhibition followed by jasmine, rosemary and clove oils. A molecular docking study revealed major EO constituents as promising inhibitors of the Als3 adhesive protein, with the highest binding for eugenol, followed by 1,8-cineole, 2-phenylthiolane and cinnamaldehyde. In conclusion, EOs have a promising inhibitory impact on Candida biofilm, phospholipase and hemolysin production, hence EOs could be used as potential antifungals that impact virulence factors.

6.
Can J Microbiol ; 66(12): 723-732, 2020 Dec.
Article in English | MEDLINE | ID: mdl-32762636

ABSTRACT

CcpN is a transcriptional repressor in Bacillus subtilis that binds to the promoter region of gapB and pckA, downregulating their expression in the presence of glucose. CcpN also represses sr1, which encodes a small noncoding regulatory RNA that suppresses the arginine biosynthesis gene cluster. CcpN has homologues in other Gram-positive bacteria, including Enterococcus faecalis. We report the interaction of CcpN with DivIVA of B. subtilis as determined using bacterial two-hybrid and glutathione S-transferase pull-down assays. Insertional inactivation of CcpN leads to cell elongation and formation of straight chains of cells. These findings suggest that CcpN is a moonlighting protein involved in both gluconeogenesis and cell elongation.


Subject(s)
Bacillus subtilis/cytology , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Cell Cycle Proteins/metabolism , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Catabolite Repression/genetics , Gene Expression Regulation, Bacterial/genetics , Gluconeogenesis/genetics
7.
Front Microbiol ; 11: 83, 2020.
Article in English | MEDLINE | ID: mdl-32117116

ABSTRACT

DivIVA plays multifaceted roles in Gram-positive organisms through its association with various cell division and non-cell division proteins. We report a novel DivIVA interacting protein in Enterococcus faecalis, named EF1025 (encoded by EF1025), which is conserved in Gram-positive bacteria. The interaction of EF1025 with DivIVAEf was confirmed by Bacterial Two-Hybrid, Glutathione S-Transferase pull-down, and co-immunoprecipitation assays. EF1025, which contains a DNA binding domain and two Cystathionine ß-Synthase (CBS) domains, forms a decamer mediated by the two CBS domains. Viable cells were recovered after insertional inactivation or deletion of EF1025 only through complementation of EF1025 in trans. These cells were longer than the average length of E. faecalis cells and had distorted shapes. Overexpression of EF1025 also resulted in cell elongation. Immuno-staining revealed comparable localization patterns of EF1025 and DivIVAEf in the later stages of division in E. faecalis cells. In summary, EF1025 is a novel DivIVA interacting protein influencing cell length and morphology in E. faecalis.

8.
Sci Rep ; 8(1): 8305, 2018 05 29.
Article in English | MEDLINE | ID: mdl-29844489

ABSTRACT

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.


Subject(s)
Microscopy, Atomic Force/methods , Microscopy, Confocal/methods , 2,4-Dichlorophenoxyacetic Acid/toxicity , Candida albicans/metabolism , Escherichia coli/metabolism , HEK293 Cells , Humans , Oxidative Stress/drug effects
9.
Article in English | MEDLINE | ID: mdl-29456868

ABSTRACT

BACKGROUND: Cinnamon (Cinnamomum zeylanicum) bark extract exhibits potent inhibitory activity against Candida albicans but the antifungal mechanisms of this essential oil remain largely unexplored. RESULTS: We analyzed the impact of cinnamon bark oil on C. albicans RSY150, and clinical strains isolated from patients with candidemia and candidiasis. The viability of RSY150 was significantly compromised in a dose dependent manner when exposed to cinnamon bark oil, with extensive cell surface remodelling at sub inhibitory levels (62.5 µg/mL). Atomic force microscopy revealed cell surface exfoliation, altered ultrastructure and reduced cell wall integrity for both RSY150 and clinical isolates exposed to cinnamon bark oil. Cell wall damage induced by cinnamon bark oil was confirmed by exposure to stressors and the sensitivity of cell wall mutants involved in cell wall organization, biogenesis, and morphogenesis. The essential oil triggered cell cycle arrest by disrupting beta tubulin distribution, which led to mitotic spindle defects, ultimately compromising the cell membrane and allowing leakage of cellular components. The multiple targets of cinnamon bark oil can be attributed to its components, including cinnamaldehyde (74%), and minor components (< 6%) such as linalool (3.9%), cinamyl acetate (3.8%), α-caryophyllene (5.3%) and limonene (2%). Complete inhibition of the mitotic spindle assembly was observed in C. albicans treated with cinnamaldehyde at MIC (112 µg/mL). CONCLUSIONS: Since cinnamaldehyde disrupts both the cell wall and tubulin polymerization, it may serve as an effective antifungal, either by chemical modification to improve its specificity and efficacy or in combination with other antifungal drugs.

10.
Reproduction ; 147(6): 875-83, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24625359

ABSTRACT

The coxsackievirus and adenovirus receptor (CXADR (CAR)) is a cell adhesion molecule expressed mainly in epithelial cells. Numerous evidence indicate that CXADR has an important role in testis development and function of the blood-testis barrier (BTB) in vitro. The role of CXADR in testis physiology in vivo has, however, not been addressed. We therefore constructed a conditional CXADR knockout (cKO) mouse model in which CXADR can be depleted at any chosen timepoint by the administration of tamoxifen. We report for the first time that testicular depletion of CXADR in adult and pubertal mice does not alter BTB permeability or germ cell migration across the BTB during spermatogenesis. Adult cKO mice display normal junctional ultra-structure and localization of the junctional proteins claudin-3, occludin, junction-associated molecule-A (JAM-A), and ZO1. The BTB was intact with no leakage of biotin and lanthanum tracers into the tubular lumen. Adult CXADR cKO mice were fertile with normal sperm parameters and litter size. Breeding experiments and genotyping of the pups demonstrated that CXADR-negative sperm could fertilize WT eggs. In addition, knocking down CXADR from postnatal day 9 (P9) does not affect testicular development and BTB formation. These cKO mice were analyzed at P49 and P90 and display an intact barrier and uncompromised fertility. We conclude that CXADR possesses no direct role in testicular physiology in vivo.


Subject(s)
Blood-Testis Barrier/metabolism , Coxsackie and Adenovirus Receptor-Like Membrane Protein/deficiency , Spermatogenesis , Spermatozoa/metabolism , Age Factors , Animals , Blood-Testis Barrier/ultrastructure , Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics , Female , Fertility , Intercellular Junctions/metabolism , Litter Size , Male , Mice, Inbred C57BL , Mice, Knockout , Permeability , Pregnancy , Sexual Maturation , Tight Junction Proteins/metabolism
11.
PLoS One ; 6(6): e20203, 2011.
Article in English | MEDLINE | ID: mdl-21674029

ABSTRACT

To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.


Subject(s)
Gene Silencing , Phenotype , Receptors, Virus/deficiency , Receptors, Virus/genetics , Animals , Atrioventricular Block/genetics , Atrophy/genetics , Behavior, Animal/drug effects , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Female , Fertility/drug effects , Fertility/genetics , Gene Knockout Techniques , Gene Silencing/drug effects , Intestinal Mucosa/metabolism , Intestines/drug effects , Intestines/pathology , Male , Mice , Motor Activity/drug effects , Motor Activity/genetics , Pancreas, Exocrine/drug effects , Pancreas, Exocrine/metabolism , Pancreas, Exocrine/pathology , Receptors, Virus/metabolism , Tamoxifen/pharmacology , Thymus Gland/cytology , Thymus Gland/drug effects , Thymus Gland/metabolism
12.
J Virol Methods ; 160(1-2): 38-47, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19406160

ABSTRACT

The diversity and extent of sequence variations between hepatitis C virus (HCV) isolates from Pakistan were studied and the probable effects of these variations were assessed on secondary viral structures. Sequencing and phylogenetic analysis was performed on 33 samples, of which 25 were typed as genotype 3 by RFLP (restriction fragment length polymorphism) and 8 remained unresolved. Rooted neighbour-joining (NJ) tree revealed that 28 isolates were HCV type 3a and 5 isolates were typed as 3b. The majority of unresolved samples clustered in a different branch of genotype 3, supported by a bootstrap value of 71%. Another, cluster, cluster I, was found to have a bootstrap value of 81%. Genetic distance values showed significant diversity of isolates in these two clusters compared to the reference sequences. Pair-wise comparison showed the presence of additional restriction sites of HaeIII and RsaI in unresolved isolates. In conclusion, unique sequence variability was observed in the 5'-UTR of HCV type 3 isolates from Pakistan. One of the reasons for this sequence variability is the presence of mutations, which are additional restriction sites in the 5'-UTR. These mutations were also responsible for failure of conventional RFLP to type some of the HCV isolates.


Subject(s)
5' Untranslated Regions , Hepacivirus/genetics , Hepatitis C/epidemiology , Hepatitis C/virology , Polymorphism, Genetic , Cluster Analysis , Female , Genotype , Hepacivirus/classification , Humans , Male , Middle Aged , Molecular Epidemiology , Molecular Sequence Data , Nucleic Acid Conformation , Pakistan/epidemiology , Phylogeny , Polymorphism, Restriction Fragment Length , RNA, Viral/genetics , Sequence Analysis, DNA , Sequence Homology
13.
J Coll Physicians Surg Pak ; 16(1): 55-60, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16441992

ABSTRACT

This review focuses on the role of the cytokine interleukin-1alpha (IL-1alpha) in the testis; elaborating upon its importance during the complex process of spermatogenesis while relating this cytokine to some of the pathophysiological states affecting the testis. IL-1alpha, a proinflammatory cytokine, is expressed constitutively by the intact adult rat testis where it acts on germ, Sertoli and Leydig cells to regulate germ cell proliferation and steroidogenesis. The sequence identity of testicular IL-1alpha matches with the one secreted by activated macrophages in systemic immunity. The classical macrophage IL-1alpha is produced as 32 kDa precursor protein which is processed to mature 17 kDa IL-1alpha and a 16 kDa propiece. The rat testicular IL-1alpha, mainly secreted by Sertoli cells, was found to have molecular heterogeneity that can be observed both at the transcriptional and the translational levels. In the rat testis, two transcripts were found to be expressed with 941 bp and 767 bp (that lacks 174 bp) which were translated into 32 kDa and 24 kDa precursor proteins, respectively. The 32 kDa precursor protein is processed to the 17 kDa mature IL-1alpha. Identical transcripts are also shown to be present in cat, dog and pig. Most of the functional role is assigned to the mature 17 kDa IL-1alpha isoform. However, functional analysis of recombinant rat IL-1alpha isoforms showed that there was a clear biopotency difference between these forms in order of 17 kDa IL-1alpha>32proIL-1alpha>24proIL-1alpha. Furthermore, the mature 17 kDa tIL-1alpha has also been implicated in pathologies such as orchitis, relapse of acute lymphoblastic leukemia (ALL) in the testis and infertility disorders in men. Thus, tIL-1alpha may play an important functional role both in coordination of normal testicular physiology as well as in contributing to the disease states in the testis.


Subject(s)
Interleukin-1/physiology , Testicular Diseases/etiology , Testis/metabolism , Testis/physiopathology , Animals , Cats , Dogs , Humans , Male , Rats , Spermatogenesis/physiology
14.
Pediatr Res ; 57(6): 896-901, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15845639

ABSTRACT

hCG has been reported to cause an inflammation-like effect in the testis, although the background and consequences of this phenomenon remain to be understood. This investigation reveals that a single injection of hCG (100 U) induces a transient surge in pro-inflammatory cytokine expression in the adult rat testis. Reverse transcriptase PCR analysis demonstrated onset of testicular expression of IL-1beta and IL-6 mRNA and increases in the levels of mRNA encoding the constitutively expressed cytokines IL-1alpha, IL-1 receptor antagonist, and tumor necrosis factor-alpha 4 h after hCG injection and a maximal response after 8-12 h. These increases were accompanied by a transient increase in testicular IL-1 bioactive protein. Twenty-four hours after administration of hCG, the levels of all cytokine mRNA had decreased, although most were still elevated above control. Immunohistochemical staining revealed that the IL-1beta protein was undetectable in normal testes but was seen to be localized to interstitial macrophages but not Leydig cells after hCG treatment. Testes devoid of Leydig cells after pretreatment with ethane dimethane sulphonate exhibited normal staining for interstitial macrophages but failed to respond to hCG with increases in IL-1beta mRNA and protein expression. We conclude that hCG induces testicular inflammation via local activation by Leydig cells of the production of pro-inflammatory cytokines by resident macrophages. It remains to be investigated whether the high-dose hCG regimens used for treatment of boys with cryptorchidism could induce similar increases of pro-inflammatory cytokines in the human testis and if such treatments could adversely affect future testicular function.


Subject(s)
Chorionic Gonadotropin/administration & dosage , Cytokines/genetics , Inflammation Mediators/metabolism , Testis/drug effects , Testis/immunology , Animals , Base Sequence , Cell Movement , DNA, Complementary/genetics , Gene Expression/drug effects , Humans , Injections, Subcutaneous , Interleukin-1/genetics , Interleukin-1/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Leukocytes/physiology , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Testosterone/blood
15.
Int J Androl ; 26(1): 37-45, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12534936

ABSTRACT

Interleukin-1alpha (IL-1alpha) is constitutively expressed in an age- and stage-dependent manner by rat Sertoli cells. However, the mechanism of regulation of IL-1alpha is unclear in testis. We studied this regulation at the level of the enzyme calpain, a potential regulator that cleaves 32 kDa proIL-1alpha to produce mature 17 kDa IL-1alpha. Both calpain I and II were found to cleave recombinant rat testis 32proIL-1alpha in vitro. A temporary age-related increase in messenger RNA (mRNA) levels of calpain I was found in testis of 20- and 25-day-old rats, coinciding with important events of spermatogenesis and a gradual increase in IL-1alpha, while calpain II expression was constant. In response to lipopolysaccharide (LPS), calpain I protein levels were down-regulated in the seminiferous tubules, while calpain II was less affected. By contrast, the liver after LPS treatment showed up-regulated calpain I and II immunoreactive protein and reverse transcriptase chain reaction (RT-PCR) signal. Depleting Leydig cells by ethane 1,2-dimethane sulphonate treatment resulted in down-regulated calpain I mRNA and protein expression, whereas calpain II remained unchanged. In summary, there is a differential expression of calpain I and II under pathological conditions induced either by endotoxin stimuli or Leydig cell depletion, which may produce a differential effect on IL-1alpha processing.


Subject(s)
Calpain/genetics , Calpain/metabolism , Interleukin-1/metabolism , Protein Precursors/metabolism , Testis/enzymology , Age Factors , Animals , Gene Expression Regulation, Enzymologic , Inflammation/enzymology , Leydig Cells/enzymology , Lipopolysaccharides/pharmacology , Male , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Seminiferous Tubules/enzymology , Sertoli Cells/enzymology , Spermatogenesis/physiology , Testis/cytology
16.
Biochem Biophys Res Commun ; 297(3): 492-7, 2002 Sep 27.
Article in English | MEDLINE | ID: mdl-12270120

ABSTRACT

The present study characterizes constitutively expressed rat testicular interleukin-1alpha (IL-1alpha) proteins. IL-1 bioactivity of crude testis protein was completely neutralized by IL-1alpha antiserum, IL-1 receptor antagonist, and soluble type I IL-1 receptor. Upon non-denaturating gel permeation chromatography, bioactive IL-1 eluted at molecular sizes of 45, 31, and 17kDa and at charges of pH 5.7 and 6.0 after chromatofocusing. SDS-PAGE/Western blot analysis of proteins extracted from whole testis, seminiferous tubules, interstitial, and seminiferous tubule fluids all demonstrated IL-1alpha immunoreactivity at 45, 24, and 19kDa. Activated macrophages and tissue proteins from endotoxin treated rats showed immunoreactive 31 and 19kDa IL-1alpha. The results indicate that the testis produces three isoforms of IL-1alpha proteins that are secreted into the interstitial compartment and tubular lumen where they may exert paracrine functions. The testicular IL-1alpha isoforms may represent posttranslationally modified precursor, mature IL-1alpha, and a 24-kDa alternate splice form.


Subject(s)
Interleukin-1/biosynthesis , Testis/immunology , Animals , Cell Division , Chromatography, High Pressure Liquid , DNA/biosynthesis , Interleukin-1/isolation & purification , Interleukin-1/metabolism , Male , Mice , Molecular Weight , Rats , Rats, Sprague-Dawley
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