Normal Animal Tissues Research Articles

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats.

To investigate the stability of the seven housekeeping genes: beta-actin (ActB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18s ribosomal unit 5 (18s), cyclophilin A (CycA), hypoxanthine-guanine phosphoribosyl transferase (HPRT), ribosomal protein large P0 (36B4) and terminal uridylyl transferase 1 (U6) in the diabetic retinal tissue of rat model. The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two groups; normal control rats and streptozotocin-induced diabetic rats. The stability analysis of gene expression was investigated using geNorm, NormFinder, BestKeeper, and comparative delta-Ct (ΔCt) algorithms. The 36B4 gene was stably expressed in the retinal tissues of normal control animals; however, it was less stable in diabetic retinas. The 18s gene was expressed consistently in both normal control and diabetic rats' retinal tissue. That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats. Furthermore, there was no ideal gene stably expressed for use in all experimental settings. Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Somatic mutations in aging and disease.

Time always leaves its mark, and our genome is no exception. Mutations in the genome of somatic cells were first hypothesized to be the cause of aging in the 1950s, shortly after the molecular structure of DNA had been described. Somatic mutation theories of aging are based on the fact that mutations in DNA as the ultimate template for all cellular functions are irreversible. However, it took until the 1990s to develop the methods to test if DNA mutations accumulate with age in different organs and tissues and estimate the severity of the problem. By now, numerous studies have documented the accumulation of somatic mutations with age in normal cells and tissues of mice, humans, and other animals, showing clock-like mutational signatures that provide information on the underlying causes of the mutations. In this review, we will first briefly discuss the recent advances in next-generation sequencing that now allow quantitative analysis of somatic mutations. Second, we will provide evidence that the mutation rate differs between cell types, with a focus on differences between germline and somatic mutation rate. Third, we will discuss somatic mutational signatures as measures of aging, environmental exposure, and activities of DNA repair processes. Fourth, we will explain the concept of clonally amplified somatic mutations, with a focus on clonal hematopoiesis. Fifth, we will briefly discuss somatic mutations in the transcriptome and in our other genome, i.e., the genome of mitochondria. We will end with a brief discussion of a possible causal contribution of somatic mutations to the aging process.

Bovine Skeletal Muscle Satellite Cells: Isolation, Growth, and Differentiation.

Skeletal muscle in cattle occupies a large part of the animal's body mass and develops into an important source of nutrients for human nutrition. Recently, the attention on bovine myogenic cells is increased to develop strategies of cultured in vitro meat as an alternative food source, more sustainable, ethical, and healthy than traditional meat production. At present, investigating the proliferation and differentiation of bovine skeletal muscle myogenic cells in vitro maintains its importance in the study of the mechanisms underlying the physiological and pathological events affecting the skeletal muscle, but it is of particular interest in animal husbandry and the food industry fields.In cell-based biological research, cell lines are one of the favored experimental tools because a population of cells could proliferate indefinitely in vitro under different stimuli, but they are limited to addressing the relevant biological properties of a cell population. On the other hand, primary cells from normal animal tissues undergo a limited number of divisions in vitro before they enter senescence but preserve their original characteristics and functions, and researchers can acquire the opportunity to study the individual donors and not just cells.In this chapter, we provide a basic protocol to isolate satellite cells from the skeletal muscle of cattle to obtain a good number of myogenic cells that can grow in in vitro conditions and undergo multiple rounds of cell division (myoblasts) before entering differentiation (myotubes). Furthermore, the robust expansion of these cells leads to the possibility to investigate physiological events or disorders related to the skeletal muscle tissue.

Transposable elements and their role in aging.

Transposable elements (TEs) are an important part of eukaryotic genomes. The role of somatic transposition in aging, carcinogenesis, and other age-related diseases has been determined. This review discusses the fundamental properties of TEs and their complex interactions with cellular processes, which are crucial for understanding the diverse effects of their activity on the genetics and epigenetics of the organism. The interactions of TEs with recombination, replication, repair, and chromosomal regulation; the ability of TEs to maintain a balance between their own activity and repression, the involvement of TEs in the creation of new or alternative genes, the expression of coding/non-coding RNA, and the role in DNA damage and modification of regulatory networks are reviewed. The contribution of the derepressed TEs to age-dependent effects in individual cells/tissues in different organisms was assessed. Conflicting information about TE activity under stress as well as theories of aging mechanisms related to TEs is discussed. On the one hand, transposition activity in response to stressors can lead to organisms acquiring adaptive innovations of great importance for evolution at the population level. On the other hand, the TE expression can cause decreased longevity and stress tolerance at the individual level. The specific features of TE effects on aging processes in germline and soma and the ways of their regulation in cells are highlighted. Recent results considering somatic mutations in normal human and animal tissues are indicated, with the emphasis on their possible functional consequences. In the context of aging, the correlation between somatic TE activation and age-related changes in the number of proteins required for heterochromatin maintenance and longevity regulation was analyzed. One of the original features of this review is a discussion of not only effects based on the TEs insertions and the associated consequences for the germline cell dynamics and somatic genome, but also the differences between transposon- and retrotransposon-mediated structural genome changes and possible phenotypic characteristics associated with aging and various age-related pathologies. Based on the analysis of published data, a hypothesis about the influence of the species-specific features of number, composition, and distribution of TEs on aging dynamics of different animal genomes was formulated.

Establishment methods and research progress of livestock and poultry immortalized cell lines: A review.

An infinite cell line is one of the most favored experimental tools and plays an irreplaceable role in cell-based biological research. Primary cells from normal animal tissues undergo a limited number of divisions and subcultures in vitro before they enter senescence and die. On the contrary, an infinite cell line is a population of non-senescent cells that could proliferate indefinitely in vitro under the stimulation of external factors such as physicochemical stimulation, virus infection, or transfer of immortality genes. Cell immortalization is the basis for establishing an infinite cell line, and previous studies have found that methods to obtain immortalized cells mainly included physical and chemical stimulations, heterologous expression of viral oncogenes, increased telomerase activity, and spontaneous formation. However, some immortalized cells do not necessarily proliferate permanently even though they can extend their lifespan compared with primary cells. An infinite cell line not only avoids the complicated process of collecting primary cell, it also provides a convenient and reliable tool for studying scientific problems in biology. At present, how to establish a stable infinite cell line to maximize the proliferation of cells while maintaining the normal function of cells is a hot issue in the biological community. This review briefly introduces the methods of cell immortalization, discusses the related progress of establishing immortalized cell lines in livestock and poultry, and compares the characteristics of several methods, hoping to provide some ideas for generating new immortalized cell lines.

Clostridial Diseases of Horses: A Review.

The clostridial diseases of horses can be divided into three major groups: enteric/enterotoxic, histotoxic, and neurotoxic. The main enteric/enterotoxic diseases include those produced by Clostridium perfringens type C and Clostridioides difficile, both of which are characterized by enterocolitis. The main histotoxic diseases are gas gangrene, Tyzzer disease, and infectious necrotic hepatitis. Gas gangrene is produced by one or more of the following microorganisms: C. perfringens type A, Clostridium septicum, Paeniclostridium sordellii, and Clostridium novyi type A, and it is characterized by necrotizing cellulitis and/or myositis. Tyzzer disease is produced by Clostridium piliforme and is mainly characterized by multifocal necrotizing hepatitis. Infectious necrotic hepatitis is produced by Clostridium novyi type B and is characterized by focal necrotizing hepatitis. The main neurotoxic clostridial diseases are tetanus and botulism, which are produced by Clostridium tetani and Clostridium botulinum, respectively. Tetanus is characterized by spastic paralysis and botulism by flaccid paralysis. Neither disease present with specific gross or microscopic lesions. The pathogenesis of clostridial diseases involves the production of toxins. Confirming a diagnosis of some of the clostridial diseases of horses is sometimes difficult, mainly because some agents can be present in tissues of normal animals. This paper reviews the main clostridial diseases of horses.

Resveratrol-modified mesoporous silica nanoparticle for tumor-targeted therapy of gastric cancer

ABSTRACT Resveratrol (Res) has been shown to exhibit anti-cancer properties in gastric cancer. However, its clinical application is limited by its poor pharmacokinetics, stability, and low solubility. Hence, this study aimed to explore and verify a better delivery system for gastric cancer therapy. Using transmission electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and ultraviolet (UV) spectrometry, we observed the shape and encapsulation of resveratrol-modified mesoporous silica nanoparticles (MSN-Res) that were synthesized by chemical methods. To explore the anti-cancer effects of these MSN-Res in vivo and in vitro, we established AGS and HGC-27 tumor-bearing mouse models. Meanwhile, the proliferation of gastric cancer cells in vitro and in vivo was assessed by Cell Counting Kit-8, EdU, and Ki-67 immunohistochemical staining methods, while cellular apoptosis, and invasion and migration were detected by TdT-mediated dUTP nick end labeling (TUNEL) and Transwell assays, respectively. FTIR and UV results showed that we successfully synthesized and loaded drugs. Safety evaluation experiments showed that neither MSN-SH nor MSN-Res had toxic effects on the normal tissues of animals. Moreover, in vitro experiments revealed that MSN-Res significantly inhibited the proliferation, invasion, and migration of gastric cancer cells. Furthermore, TUNEL assay showed that MSN-Res promoted apoptosis in gastric cancer. These results were confirmed by the nude mouse tumorigenesis experiment. In conclusion, we demonstrated that MSN-Res showed better inhibitory effect on the development of gastric cancer than Res alone, indicating that MSN-Res could be a promising drug delivery system for gastric cancer treatment.

Development of proton beam irradiation system for small animals using FFAG accelerator

To evaluate the biological effect of boron compounds on normal tissues in boron neutron capture therapy, it is necessary to compare the radiation effects of neutron irradiation and gamma ray irradiation on small animals in nonclinical studies. Neutron beams and gamma rays are difficult to collimate; thus, it is impossible to administer a dose on each part of the normal tissues of small animals. Therefore, we have developed a method to compare biological effects by irradiating neutrons and gamma rays after irradiating a proton beam to the point just before the threshold dose at which the biological effects occur. We aim to develop an irradiation field that irradiates proton beams locally using a fixed-field alternating-gradient (FFAG) accelerator and a small ridge filter.An aluminum scatterer, transmission dose monitor, ridge filter, range shifter, collimator, and a proton beam from the FFAG accelerator were used to form an irradiation field with a spread out Bragg peak (SOBP) with a length of 10 mm and a size of 10 mm2.Dose measurement was performed using a Gafchromic film and an ionization chamber. The irradiation system was simulated using the PHITS Monte Carlo simulation code, and simulation results were compared with experimental results.We succeeded in forming an irradiation field with a dose rate of 14.3 ± 0.24 Gy/min, an SOBP length of 10 mm, and a size of 10 mm2.

Somatic Mutagenesis in Mammals and Its Implications for Human Disease and Aging.

DNA mutations as a consequence of errors during DNA damage repair, replication, or mitosis are the substrate for evolution. In multicellular organisms, mutations can occur in the germline and also in somatic tissues, where they are associated with cancer and other chronic diseases and possibly with aging. Recent advances in high-throughput sequencing have made it relatively easy to study germline de novo mutations, but in somatic cells, the vast majority of mutations are low-abundant and can be detected only in clonal lineages, such as tumors, or single cells. Here we review recent results on somatic mutations in normal human and animal tissues with a focus on their possible functional consequences.

ScanPAV: a pipeline for extracting presence-absence variations in genome pairs.

MotivationThe recent technological advances in genome sequencing techniques have resulted in an exponential increase in the number of sequenced human and non-human genomes. The ever increasing number of assemblies generated by novel de novo pipelines and strategies demands the development of new software to evaluate assembly quality and completeness. One way to determine the completeness of an assembly is by detecting its Presence–Absence variations (PAV) with respect to a reference, where PAVs between two assemblies are defined as the sequences present in one assembly but entirely missing in the other one. Beyond assembly error or technology bias, PAVs can also reveal real genome polymorphism, consequence of species or individual evolution, or horizontal transfer from viruses and bacteria.ResultsWe present scanPAV, a pipeline for pairwise assembly comparison to identify and extract sequences present in one assembly but not the other. In this note, we use the GRCh38 reference assembly to assess the completeness of six human genome assemblies from various assembly strategies and sequencing technologies including Illumina short reads, 10× genomics linked-reads, PacBio and Oxford Nanopore long reads, and Bionano optical maps. We also discuss the PAV polymorphism of seven Tasmanian devil whole genome assemblies of normal animal tissues and devil facial tumour 1 (DFT1) and 2 (DFT2) samples, and the identification of bacterial sequences as contamination in some of the tumorous assemblies.Availability and implementationThe pipeline is available under the MIT License at https://github.com/wtsi-hpag/scanPAV.Supplementary information Supplementary data are available at Bioinformatics online.

Molecular characterization, prevalence and clinical relevance of Phodopus sungorus papillomavirus type 1 (PsuPV1) naturally infecting Siberian hamsters (Phodopus sungorus).

Phodopus sungorus papillomavirus type 1 (PsuPV1), naturally infecting Siberian hamsters (Phodopus sungorus) and clustering in the genus Pipapillomavirus (Pi-PV), is only the second PV type isolated from the subfamily of hamsters. In silico analysis of three independent complete viral genomes obtained from cervical adenocarcinoma, oral squamous cell carcinoma and normal oral mucosa revealed that PsuPV1 encodes characteristic viral proteins (E1, E2, E4, E6, E7, L1 and L2) with conserved functional domains and a highly conserved non-coding region. The overall high prevalence (102/114; 89.5 %) of PsuPV1 infection in normal oral and anogenital mucosa suggests that asymptomatic infection with PsuPV1 is very frequent in healthy Siberian hamsters from an early age onward, and that the virus is often transmitted between both anatomical sites. Using type-specific real-time PCR and chromogenic in situ hybridization, the presence of PsuPV1 was additionally detected in several investigated tumours (cervical adenocarcinoma, cervical adenomyoma, vaginal carcinoma in situ, ovarian granulosa cell tumour, mammary ductal carcinoma, oral fibrosarcoma, hibernoma and squamous cell papilloma) and normal tissues of adult animals. In the tissue sample of the oral squamous cell carcinoma individual, punctuated PsuPV1-specific in situ hybridization spots were detected within the nuclei of infected animal cells, suggesting viral integration into the host genome and a potential etiological association of PsuPV1 with sporadic cases of this neoplasm.

Nuclear Glycogen Inclusions in Canine Parietal Cells.

Nuclear glycogen inclusions occur infrequently in pathologic conditions but also in normal human and animal tissues. Their function or significance is unclear. To the best of the authors' knowledge, no reports of nuclear glycogen inclusions in canine parietal cells exist. After initial observations of nuclear inclusions/pseudoinclusions during routine histopathology, the authors retrospectively examined samples of gastric mucosa from dogs presenting with gastrointestinal signs for the presence of intranuclear inclusions/pseudoinclusions and determined their composition using histologic and electron-microscopic methods. In 24 of 108 cases (22%), the authors observed various numbers of intranuclear inclusions/pseudoinclusions within scattered parietal cells. Nuclei were characterized by marked karyomegaly and chromatin margination around a central optically empty or slightly eosinophilic area. The intranuclear inclusions/pseudoinclusions stained positive with periodic acid-Schiff (PAS) and were diastase sensitive, consistent with glycogen. Several PAS-positive/diastase-sensitive sections were further examined by transmission electron microscopy, also using periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining to identify polysaccharides. Ultrastructurally, the nuclear inclusions were composed of electron-dense particles that were not membrane bound, without evidence of nuclear membrane invaginations or cytoplasmic organelles in the nuclei, and positive staining with PA-TCH-SP, confirming a glycogen composition. No cytoplasmic glycogen deposits were observed, suggesting that the intranuclear glycogen inclusions were probably synthesized in loco. Nuclear glycogen inclusions were not associated with gastritis or colonization by Helicobacter-like organisms ( P > .05). Our findings suggest that nuclear glycogen inclusions in canine parietal cells could be an incidental finding. Nevertheless, since nuclear glycogen is present in several pathologic conditions, further investigations could be warranted to determine their true significance.

“SMART” LASER SCALPELS FOR ROBOTIC SURGERY

Background: Elaboration of automatized and robotic systems for precision and minimally traumatic surgery is one of the main areas of modern surgery. The concept of the so-called “smart” laser scalpels seems a promising technical solution in this field. Aim: To develop organizational principles of a feedback smart surgical laser devices based on CO₂ and fiber lasers. Materials and methods: As laser sources, we used a one mode wave CO₂ laser with a power of up to 25 W, high frequency pumping of the active media and radiation wavelength of 10.6 mcm, as well as a one mode fiber Er laser with a power of up to 5 W and radiation wavelength of 1.54 mcm. The laser device feedback was organized with an autodynic control of laser evaporation of biological tissues. The “smart” laser scalpel effects were studied in the porcine tissues in vitro. The feedback laser devices were tested on normal and tumor animal tissues (white rats) in vitro and in vivo. Also, we tested the possibility of diagnostics of laser evaporation on human tumor tissues. Results: Taking the one mode CO₂ laser and one mode fiber Er laser as examples, it was shown that an autodynic signal arising during evaporation of various biological tissues has different spectral characteristics. This makes the bases for organization of a feedback in surgical devices functioning as a “smart” scalpel. A “smart” surgical feedback device based on CO₂ laser and a decoy of a “smart” surgical device based on a fiber Er laser were developed. We studied the possibilities of differential diagnostics of a type of a tissue being evaporated in vitro with the use of the data from laser scalpels. Also, pre-clinical trials of a CO₂ laser-based “smart” surgical device on biological tissues were performed. The trials showed that such a “smart” laser scalpel allows for intra-operative differentiation between normal and tumor tissues that would give the possibility to implement minimally traumatic surgery principles. Conclusion: The method of autodynic diagnostics of laser evaporation of biological tissues allows for development of surgical devices functioning as a “smart” laser. It gives the possibility of a real-time differentiation of various types of tissues, including normal and tumor ones.

Momordica charantia (bitter melon) decreases serum/tissue lipid parameters in hyperammonemic rats

Introduction: Ammonia is present in all living organisms as a byproduct of the degradation of proteins and other nitrogenous compounds. At high levels ammonia is toxic, causing functional disturbances in the central nervous system that could even lead to coma and death. The present study evaluates the efficacy of an ethanolic extract of Momordica charantia L. (MCE) fruits in decreasing serum and tissue lipids and body weight in rats with experimentally-induced hyperammonemia. Materials and Methods: Experimental hyperammonemia was induced in adult male Wistar rats (180-200 g) by intraperitoneal (i.p.) injections of ammonium chloride (100 mg/kg body weight) thrice a week. The effect of oral administration (thrice a week for eight consecutive weeks) of MCE (300 mg/kg body weight) on lipid profiles in serum and tissues (brain and liver) of normal and experimental animals were analyzed. Results and Discussion: The levels of serum and tissue cholesterol, triglycerides, free fatty acids, and phospholipids were significantly increased in ammonium chloride - induced hyperammonemic rats. The administration of MCE to ammonium chloride - treated rats significantly restored all these changes to almost normal levels. Ammonium chloride - treated rats showed increases in body weight compared to the control rats. The exact mechanism of this antihyperlipidemic effect of MCE needs to be investigated and the active constituents identified and isolated.

Allergen and Ozone Exacerbate Serotonin-Induced Increases in Airway Smooth Muscle Contraction in a Model of Childhood Asthma

Background: Serotonin (5-HT) modulates cholinergic neurotransmission and exacerbates airway smooth muscle (ASM) contraction in normal animal and nonasthmatic human tissue. Exposure to house dust mite allergen (HDMA) and ozone (O₃) leads to airway hyperreactivity and 5-HT-positive cells in the airway epithelium of infant rhesus monkeys. Research shows that concomitant exposure in allergic animals has an additive effect on airway hyperreactivity. Objectives: In this study, the hypothesis is that the exposure of allergic infant rhesus monkeys to HDMA, O₃ and in combination, acting through 5-HT receptors, enhances 5-HT modulation of postganglionic cholinergic ASM contraction. Methods: Twenty-four HDMA-sensitized infant monkeys were split into 4 groups at the age of 1 month, and were exposed to filtered air (FA), HDMA, O₃ or in combination (HDMA+O₃). At the age of 6 months, airway rings were harvested and postganglionic, and parasympathetic-mediated ASM contraction was evaluated using electrical-field stimulation (EFS). Results: 5-HT exacerbated the EFS response within all exposure groups, but had no effect in the FA group. 5-HT₂, 5-HT₃ and 5-HT₄ receptor agonists exacerbated the response. 5-HT concentration-response curves performed after incubation with specific receptor antagonists confirmed the involvement of 5-HT₂, 5-HT₃ and 5-HT₄ receptors. Conversely, a 5-HT₁ receptor agonist attenuated the tension across all groups during EFS, and in ASM contracted via exogenous acetylcholine. Conclusions: HDMA, O₃ and HDMA+O₃ exposure in a model of childhood allergic asthma enhances 5-HT exacerbation of EFS-induced ASM contraction through 5-HT₂, 5-HT₃ and 5-HT₄ receptors. A nonneurogenic inhibitory pathway exists, unaffected by exposure, mediated by 5-HT₁ receptors located on ASM.

Stroke as an emergency: Semmelweis redivivus?

Simply told, the story of tPA or ‘tissue-type plasminogen activator’ begins like this. In 1952, Danish biochemists isolated a fibrinolytic substance from normal animal tissues. It had been detected a few years before and was known to act through activation of plasminogen. In the mid-1970s, groups in New York and Leuven found that several malignant tumours also secrete a plasminogen activator, generally in proportion to their degree of invasive behaviour. The Leuven group obtained a highly productive melanoma cell line from an American patient and eventually purified it. The Belgian group also observed that the human plasminogen activator had a specific affinity for fibrin: in the absence of fibrin it had only a weak-activating effect on plasminogen, through competitive binding and ‘silencing’ of tPA by α2-antiplasmin. This selective action represented a great advantage—at least theoretically—over the non-specific plasminogen activators urokinase and streptokinase that were available at the time. Indeed in the early 1980s, animal experiments in Leuven and St Louis showed that tPA could dissolve thrombi of venous and arterial origin without systemic fibrinolysis. The first humans to be treated with tPA (from the melanoma cell line, in hindsight with low doses) were two patients in Rotterdam with thrombosis of renal and iliofemoral veins in the region of a transplanted kidney (Weimar et al ., 1981); one of them survives to this day. The clinical experiment resulted from a chance conversation at a scientific meeting between a nephrologist from Rotterdam and a biochemist from the Leuven group. After pilot experiments in patients with coronary thrombosis had proved successful, a proper clinical trial was on the agenda but would require far greater quantities of the substance than could be extracted from the tumour cell line. In collaboration with the Leuven group, scientists at the Genentech corporation cloned the complementary DNA for …

Magnetoacoustic imaging of human liver tumor with magnetic induction

Magnetoacoustic tomography with magnetic induction (MAT-MI) is an imaging technique under development to achieve imaging of electrical impedance contrast in biological tissues with spatial resolution close to ultrasound imaging. However, previously reported MAT-MI experimental results are obtained either from low salinity gel phantoms, or from normal animal tissue samples. In this study, we report the experimental study on the performance of the MAT-MI imaging method for imaging in vitro human liver tumor tissue. The present promising experimental results suggest the feasibility of MAT-MI to image electrical impedance contrast between the cancerous tissue and its surrounding normal tissues.

Characterization of a New Monoclonal Antibody Against PAX5/BASP in 1525 Paraffin-embedded Human and Animal Tissue Samples

We describe the newly generated DAK-PAX5 monoclonal antibody raised against a fixation-resistant epitope of the human PAX5/BSAP molecule. Following Western-blot, absorption, and chess-board titration tests, and optimization of antigen-retrieval and detection methods, DAK-Pax5 was used in parallel with a reference antibody (clone 24) on tissue micro-arrays (TMAs) constructed from normal human and animal tissues and from hematologic and nonhematologic human malignancies. Such TMAs were also tested with an anti-PAX2 antibody. DAK-Pax5 reacted with normal human and animal B-cells and with 460/473 B-cell non-Hodgkin lymphomas (B-NHLs). All plasmacytomas/plasmablastic tumors (n=13) and T/NK-cell neoplasms (n=264) turned out consistently negative as did acute myelogenous leukaemias (n=19) except 2 carrying t(8;21). Positivity was found in 6/6 and 155/169 lymphocyte predominant and classical HLs, respectively, although the staining intensity varied through cases. Among 521 nonhematologic malignancies, DAK-Pax5 reacted with 22/399 carcinomas (4/11 neuroendocrine, 2/4 Merkel-cell, 4/21 prostatic, 1/11 urothelial, 1/26 renal, 2/12 cervical squamous-cell, 3/13 ovarian, and 5/75 colonic). When compared with clone 24, DAK-Pax5 produced a stronger positivity in most if not all B-NHLs and HLs. No cross-reactivity with the anti-PAX2 antibody was recorded. DAK-Pax5 represents a new reliable tool for diagnostics and research.

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca(2+)-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mM Ca(2+) exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s(20,w)(0) of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca(2+), CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R(G) values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K(D) value of 21 microM in solution (140 mM NaCl, 2 mM Ca(2+)) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K(D) value of 23 microM (140 mM NaCl, 2 mM Ca(2+)), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mM Ca(2+) and 140 mM NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.

Protein Kinase D Is an Essential Regulator of C. elegans Innate Immunity

Protein kinase D (PKD) mediates signal transduction downstream from phospholipase C and diacylglycerol (DAG). PKDs are activated by hormones and stress in cell lines, but little is known about PKD functions and regulation in vivo. Here, we show that DKF-2, a C. elegans PKD, regulates innate immunity. Animals lacking DKF-2 were hypersensitive to killing by bacteria that are pathogens of C. elegans and humans. DKF-2 induced 85 mRNAs, which encode antimicrobial peptides and proteins that sustain intestinal epithelium. Induction of immune effector mRNAs by DKF-2 proceeded via PMK-1 (p38 Map-kinase)-dependent and -independent pathways. TPA-1, a PKCdelta homolog, regulated activation and functions of DKF-2 in vivo. Therefore, DKF-2 provides a molecular link that couples DAG signaling to regulation of immunity. This intersection between DAG-TPA-1-DKF-2 and PMK-1 pathways enables integrated immune responses to multiple stimuli. Thus, a PKD mobilizes activation of host immune defenses against pathogens by previously unappreciated signaling pathways and mechanisms.