Search

Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.

Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has been discussed for its safety and efficacy in cancer treatments. For this reason, we have inquired into its use on triple-negative human breast cancer. Analyzing the biological effects of CBD on MDA-MB-231, we have demonstrated that both CBD dosage and serum concentrations in the culture medium influence its outcomes; furthermore, light scattering studies demonstrated that serum impacts the CBD aggregation state by acting as a surfactant agent. Pharmacological studies on CBD in combination with chemotherapeutic agents reveal that CBD possesses a protective action against the cytotoxic effect exerted by cisplatin on MDA-MB-231 grown in standard conditions. Furthermore, in a low serum condition (0.5%), starting from a threshold concentration (5 µM), CBD forms aggregates, exerts cytostatic antiproliferative outcomes, and promotes cell cycle arrest activating autophagy. At doses above the threshold, CBD exerts a highly cytotoxic effect inducing bubbling cell death. Finally, IGF-1 and EGF antagonize the antiproliferative effect of CBD protecting cells from harmful consequences of CBD aggregates. In conclusion, CBD effect is strongly associated with the physical state and concentration that reaches the treated cells, parameters not taken into account in most of the research papers.

Palmitoylethanolamide (PEA) is an endogenous lipid produced on demand by neurons and glial cells that displays neuroprotective properties. It is well known that inflammation and neuronal damage are strictly related processes and that microglia play a pivotal role in their regulation. The aim of the present work was to assess whether PEA could exert its neuroprotective and anti-inflammatory effects through the modulation of microglia reactive phenotypes. In N9 microglial cells, the pre-incubation with PEA blunted the increase of M1 pro-inflammatory markers induced by lipopolysaccharide (LPS), concomitantly increasing those M2 anti-inflammatory markers. Images of microglial cells were processed to obtain a set of morphological parameters that highlighted the ability of PEA to inhibit the LPS-induced M1 polarization and suggested that PEA might induce the anti-inflammatory M2a phenotype. Functionally, PEA prevented Ca2+ transients in both N9 cells and primary microglia and antagonized the neuronal hyperexcitability induced by LPS, as revealed by multi-electrode array (MEA) measurements on primary cortical cultures of neurons, microglia, and astrocyte. Finally, the investigation of the molecular pathway indicated that PEA effects are not mediated by toll-like receptor 4 (TLR4), on the contrary, a partial involvement of cannabinoid type 2 receptor (CB2R) was shown by using a selective receptor inverse agonist.

Simple Summary Cell-to-cell communication in the tumor microenvironment is a crucial process to orchestrate the different components of the tumoral infrastructure. Among the mechanisms of cellular interplay in cancer cells, tunneling nanotubes (TNTs) are dynamic connections that play an important role. The mechanism of the formation of TNTs among cells and the molecules involved in the process remain to be elucidated. In this study, we analyze several bladder cancer cell lines, representative of tumors at different stages and grades. We demonstrate that TNTs are formed only by mid or high-stage cell lines that show muscle-invasive properties and that they actively transport mitochondria and proteins. The formation of TNTs is triggered by stressful conditions and starts with the assembly of a specific multimolecular complex. In this study, we characterize some of the protein components of the TNTs complex, as they are potential novel molecular targets for future therapies aimed at counteracting tumor progression. Abstract RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor for RalA GTPase that is involved in several cellular processes, including cytoskeletal organization. Previously, we demonstrated that RalGPS2 also plays a role in the formation of tunneling nanotubes (TNTs) in bladder cancer 5637 cells. In particular, TNTs are a novel mechanism of cell–cell communication in the tumor microenvironment, playing a central role in cancer progression and metastasis formation. However, the molecular mechanisms involved in TNTs formation still need to be fully elucidated. Here we demonstrate that mid and high-stage bladder cancer cell lines have functional TNTs, which can transfer mitochondria. Moreover, using confocal fluorescence time-lapse microscopy, we show in 5637 cells that TNTs mediate the trafficking of RalA protein and transmembrane MHC class III protein leukocyte-specific transcript 1 (LST1). Furthermore, we show that RalGPS2 is essential for nanotubes generation, and stress conditions boost its expression both in 5637 and HEK293 cell lines. Finally, we prove that RalGPS2 interacts with Akt and PDK1, in addition to LST1 and RalA, leading to the formation of a complex that promotes nanotubes formation. In conclusion, our findings suggest that in the tumor microenvironment, RalGPS2 orchestrates the assembly of multimolecular complexes that drive the formation of TNTs.

Spermatids are haploid differentiating cells that, in the meantime they differentiate, translocate along the seminiferous epithelium towards the tubule lumen to be just released as spermatozoa. The success of such a migration depends on dynamic of spermatid–Sertoli cell contacts, the molecular nature of which has not been well defined yet. It was demonstrated that the vascular endothelial cadherin (VEC) is expressed transitorily in the mouse seminiferous epithelium. Here, we evaluated the pattern of VEC expression by immunohistochemistry first in seminiferous tubules at different stages of the epithelial cycle when only unique types of germ cell associations are present. Changes in the pattern of VEC localization according to the step of spermatid differentiation were analysed in detail using testis fragments and spontaneously released germ cells. Utilizing the first wave of spermatogenesis as an in vivo model to have at disposal spermatids at progressive steps of differentiation, we checked for level of looser VEC association with the membrane by performing protein solubilisation under mild detergent conditions and assays through VEC-immunoblotting. Being changes in VEC solubilisation paralleled in changes in phosphotyrosine (pY) content, we evaluated if spermatid VEC undergoes Y658 phosphorylation and if this correlates with VEC solubilisation and spermatid progression in differentiation. Altogether, our study shows a temporally restricted pattern of VEC expression that culminates with the presence of round spermatids to progressively decrease starting from spermatid elongation. Conversely, pY658-VEC signs elongating spermatids; its intracellular polarized compartmentalization suggests a possible involvement of pY658-VEC in the acquisition of spermatid cell polarity.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

Simple Summary Communication between cells helps tumors acquire resistance to chemotherapy and makes the struggle against cancer more challenging. Tunneling nanotubes (TNTs) are long channels able to connect both nearby and distant cells, contributing to a more malignant phenotype. This finding might be useful in designing novel strategies of drug delivery exploiting these systems of connection. This would be particularly important to reach tumor niches, where glioblastoma stem cells proliferate and provoke immune escape, thereby increasing metastatic potential and tumor recurrence a few months after surgical resection of the primary mass. Along with the direct inhibition of TNT formation, TNT analysis, and targeting strategies might be useful in providing innovative tools for the treatment of this tumor. Abstract Glioblastoma (GBM) is a particularly challenging brain tumor characterized by a heterogeneous, complex, and multicellular microenvironment, which represents a strategic network for treatment escape. Furthermore, the presence of GBM stem cells (GSCs) seems to contribute to GBM recurrence after surgery, and chemo- and/or radiotherapy. In this context, intercellular communication modalities play key roles in driving GBM therapy resistance. The presence of tunneling nanotubes (TNTs), long membranous open-ended channels connecting distant cells, has been observed in several types of cancer, where they emerge to steer a more malignant phenotype. Here, we discuss the current knowledge about the formation of TNTs between different cellular types in the GBM microenvironment and their potential role in tumor progression and recurrence. Particularly, we highlight two prospective strategies targeting TNTs as possible therapeutics: (i) the inhibition of TNT formation and (ii) a boost in drug delivery between cells through these channels. The latter may require future studies to design drug delivery systems that are exchangeable through TNTs, thus allowing for access to distant tumor niches that are involved in tumor immune escape, maintenance of GSC plasticity, and increases in metastatic potential.

Palmitoylethanolamide (PEA) belongs to the class of N-acylethanolamine and is an endogenous lipid potentially useful in a wide range of therapeutic areas, products containing PEA are licensed for use in humans as a nutraceutical, a food supplement, or food for medical purposes for its analgesic and anti-inflammatory properties demonstrating efficacy and tolerability. However, the exogenously administered PEA is rapidly inactivated, in this process, fatty acid amide hydrolase (FAAH) plays a key role both in hepatic metabolism and in intracellular degradation. So, the aim of the present study was the design and synthesis of PEA analogues that are more resistant to FAAH-mediated hydrolysis. A small library of PEA analogues was designed and tested by molecular docking and density functional theory calculations to find the more stable analogue. The computational investigation identified RePEA as the best candidate in terms of both synthetic accessibility and metabolic stability to FAAH-mediated hydrolysis. The selected compound was synthesized and assayed ex vivo to monitor FAAH-mediated hydrolysis and to confirm its anti-inflammatory properties. 1H-NMR spectroscopy performed on membrane samples containing FAAH in integral membrane protein demonstrated that RePEA is not processed by FAAH, in contrast with PEA. Moreover, RePEA retains PEA&rsquo, s ability to inhibit LPS-induced cytokine release in both murine N9 microglial cells and human PMA-THP-1 cells.

Increasing evidence supports a decisive role for neuroinflammation in the neurodegenerative process of several central nervous system (CNS) disorders. Microglia are essential mediators of neuroinflammation and can regulate a broad spectrum of cellular responses by releasing reactive oxygen intermediates, nitric oxide, proteases, excitatory amino acids, and cytokines. We have recently shown that also in ex-vivo cortical networks of neurons, astrocytes and microglia, an increased level of tumor necrosis factor-alpha (TNF-α) was detected a few hours after exposure to the bacterial endotoxin lipopolysaccharide (LPS). Simultaneously, an atypical "seizure-like" neuronal network activity was recorded by multi-electrode array (MEA) electrophysiology. These effects were prevented by minocycline, an established anti-inflammatory antibiotic. We show here that the same inhibitory effect against LPS-induced neuroinflammation is exerted also by natural plant compounds, polyphenols, such as curcumin (CU, curcuma longa), crocin (CR, saffron), and resveratrol (RE, grape), as well as by the glucagon like peptide-1 receptor (GLP-1R) agonist exendin-4 (EX-4). The drugs tested also caused per-se early transient (variable) changes of network activity. Since it has been reported that LPS-induced neuroinflammation causes rearrangements of glutamate transporters in astrocytes and microglia, we suggest that neural activity could be putatively increased by an imbalance of glial glutamate transporter activity, leading to prolonged synaptic glutamatergic dysregulation.

The β-amyloid peptide is generated by the proteolysis of the amyloid precursor protein (APP) by the action of β- and γ-secretase. The mechanisms underlying this process are poorly understood. Using a cell-based reporter gene assay we analysed the possible signals and pathways that could be involved in APP cleavage. We used the stable cell line HeLa AG that expresses the human APP(695) fused with the yeast transcription factor Gal4. This fusion protein is normally translocated into the plasma membrane and after APP-Gal4 cleavage, the AICD-Gal4 fragment released can activate the transcription of a luciferase reporter gene. Through this reporter system, we demonstrated that Ras GTPase, but not Ral and Rap, could promote APP-Gal4 cleavage. In addition HeLa AG cells stimulated with EGF or PDGF or overexpressing EGFR exhibit increased APP proteolysis in a Ras-dependent way. This process is also dependent on γ-secretase activity, being abolished by the γ-secretase inhibitor DAPT.

Ubiquitin-specific peptidase 8 (USP8) is a deubiquitinating enzyme that works as a regulator of endosomal sorting and vesicle morphology in cultured cells. Its function in vivo is, however, unknown as USP8 gene deletion leads to embryonic lethality. Previously, we have shown that USP8 is highly expressed in male germ cells. These cells develop a peculiar acidic vesicle that is indispensable for fertilization, the acrosome; USP8 might be involved in vivo in acrosomogenesis. The objective of this study was to test this hypothesis by determining if selective components of the early endosomal machinery interact functionally with USP8 during acrosomogenesis using protein-protein interaction assays and double/triple immunolabeling. Moreover, by exploiting the characteristic of USP8 that exhibits a microtubule interacting and trafficking/transport (MIT) domain, we verified whether USP8 effectively associates with spermatid microtubules by microtubule cosedimentation and binding assays. USP8 was able to interact with spermatid ESCRT-0 (endosomal-sorting complex required for transport-0) and microtubule structures; USP8/ESCRT-0-labeled vesicles, monitored by fluorescence microscopy, were found to contribute to acrosome formation while USP8 can directly link, via its MIT domain, the labeled vesicles/developing acrosome to microtubules, which could favor both acrosome assembly and shaping. VPS54, the vacuolar-sorting protein responsible for early endocytic retrograde transport, was here detected for the first time in male germ cells; VPS54 followed the intracellular route of USP8/ESCRT-0-labeled vesicles during acrosomogenesis. We concluded that in vivo USP8 has a role strongly associated with acrosome biogenesis and that the early endosome pathway is significantly involved in the process, which suggests that the acrosome could be a novel lysosomerelated organelle. acrosome, acrosome biogenesis, early endosome, endosomes, spermatid, spermatogenesis, STAM2, USP8, VPS54

The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPy(C748A) caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.

Msj-1 (mouse sperm cell-specific DnaJ first homologue) is a gene specifically expressed in germ cells at haploid stages. The protein first appears in round spermatids, accumulates in the periacrosomal region of elongating spermatids, and is maintained in spermatozoa. The msj-1 expression pattern is consistent with a role for this DnaJ protein in the spermiogenesis process. In this study, we used two experimental models, the anuran amphibian Rana esculenta and the wobbler mutant mouse, to explore the role of MSJ-1 during spermatogenesis, with a focus on spermiogenesis. Mice homozygous for the recessive mutation wobbler (wr/wr), a mutation of unknown identity, produce sperm cells characterized by a missing acrosome. In Rana esculenta testis, detection of high levels of MSJ-1 protein coincided with the appearance of postmeiotic germ cells during the annual sexual cycle. Conversely, elimination of the meiotic and postmeiotic stages, through gonadotropin administration at low temperature, abolished the MSJ-1 immunoreactive signal. In 20day-old mice, when postmeiotic germ cells appeared for the first time, MSJ-1 mRNA and protein were observed in 1/1 testis but were barely detectable in wr/wr testis. In adult testis, reduced MSJ-1 protein levels were observed in both 1/wr and wr/wr testis, as compared with 1/1 controls. Similarly, numbers of spermatids that stained by immunofluorescence for MSJ-1 appeared to be progressively reduced in adult 1/1, 1/wr, and wr/wr mouse testes, respectively. Characterization of the endocrine status of wobbler testis revealed reduced transcript levels of estrogen receptor a and reduced intratesticular androgen levels. However, androgen treatment did not affect MSJ-1 protein levels in either frogs or mice. In conclusion, our data in Rana esculenta and the wobbler mouse demonstrate a tight correlation between MSJ-1 protein expression and postmeiotic stages. In particular, the findings in the wobbler testis suggest a role for this protein in acrosomogenesis.

We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25(Mm). Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25(Mm) is exclusively expressed. Using a glutathione S-transferase fusion protein, we demonstrated an "in vitro" interaction between mUBPy and the N-terminal half (amino acids 1-625) of CDC25(Mm). In addition "in vivo" interaction was demonstrated after cotransfection in mammalian cells. We also showed that CDC25(Mm), expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25(Mm), thus regulating the level of this Ras-guanine nucleotide exchange factor.

The CDC25 gene product is an exchange factor for Ras proteins and it activates the Ras/cAMP pathway in the yeast Saccharomyces cerevisiae. The overexpression of the CDC25 gene in S. cerevisiae cells causes a partial glucose-derepressed phenotype which is particularly evident for expression of invertase. To define domains of Cdc25 protein relevant for this derepression and to test another glucose repressed system, different to invertase, we have overexpressed different regions of the CDC25 gene under the control of a GAL-promoter. We found that a derepression of both GAL-regulated promoters and invertase was related to the overexpression of CDC25 regions that contain a functional guanine nucleotide exchange (GEF) domain. The effect on GAL-promoters was particular evident when the CDC25 gene was under the control of a UAS(gal) element and operates at transcriptional level, although a moderate derepression was found also for UAS(gal)/lacZ reporter gene. Finally, the overexpression of the GEF domain of CDC25 also caused an increase in the expression of the GAL4 regulatory gene, while a constitutive activation of the Ras/cAMP pathway did not produce any increase in GAL4 expression. These findings indicate that the overexpression of the catalytic domain of CDC25 gene is necessary and sufficient to give a glucose-derepression of GAL promoters and of invertase. They also suggest that the derepression of GAL promoters occurs through an increase of GAL4 expression in a Ras/cAMP independent way

RalGPS2 is a guanine nucleotide exchange factor for RalA GTPase characterized by a C-terminal Pleckstrin Homology (PH) domain; this GEF is endogenously expressed in PC12 cells and in rat brain but its role in PC12 cells and in cell differentiation is actually unknown. Here we have shown that transient expression of RalGPS2-PH-PxxP domain in PC12 and PC12-TrkA cells induces high level of neurite outgrowth; this differentiation is comparable with that of PC12 cells treated with RalGPS2 siRNA. Stable PC12 cell lines expressing the PH-PxxP domain of RalGPS2 have been generated; in these cell lines the PH-PxxP domain acts as a dominant negative for RalA activation, promotes cells differentiation and re-directs NGF signals towards MAPKs. Furthermore it has been also demonstrated that the PH-PxxP domain of RalGPS2 induces microspikes formation a typical feature of cells in which the Cdc42 GTPase is constitutively activated.

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell–cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli–germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin–positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ–Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.

RalGPS2 is a murine guanine nucleotide exchange factor of the RalGPS family; it contains a Cdc25-like GEF domain and does not exhibit a Ras-binding domain. The main characteristic of RalGPS2 is its pleckstrin homology (PH) domain, present at the C terminus, that preferentially binds phosphatidylinositol-4,5-biphosphate and in HEK 293 cells localized in membranes, causing ruffling and vesiculation. Moreover, RalGPS2 contains a PxxP motif in the central part of the molecule. This motif binds in vitro and in vivo SH3 domains of Grb2 and PLCgamma. RalGPS2 and its GEF domain activate RalA in vivo while the PH-PxxP domains inhibited it behaving as a dominant negative for the RalA pathway; this activation was not inhibited by co-expression of a dominant negative Ras. RalGPS2 is physiologically expressed in testis and brain; when overexpressed, the whole RalGPS2 causes considerable morphological changes in HEK 293 cells, suggesting its possible role on cytoskeleton reorganization. This is further strengthened by data obtained in NIH3T3 cells where expression of PH-PxxP domain promotes actin depolymerization. Finally, RalGPS2 and its GEF domain induce Ras-independent transcriptional activation of the c-fos promoter in NIH3T3 cells.

Recombinant human nerve growth factor (rhNGF) is regarded as the most promising therapy for neurodegeneration of the central and peripheral nervous systems as well as for several other pathological conditions involving the immune system. However, rhNGF is not commercially available as a drug. In this work, we provide data about the production on a laboratory scale of large amounts of a rhNGF that was shown to possessin vivobiochemical, morphological, and pharmacological effects that are comparable with the murine NGF (mNGF), with no apparent side effects, such as allodynia. Our rhNGF was produced by using conventional recombinant DNA technologies combined with a biotechnological approach for high-density culture of mammalian cells, which yielded a production of ≈21.5 ± 2.9 mg/liter recombinant protein. The rhNGF-producing cells were thoroughly characterized, and the purified rhNGF was shown to possess a specific activity comparable with that of the 2.5S mNGF by means of biochemical, immunological, and morphologicalin vitrostudies. This work describes the production on a laboratory scale of high levels of a rhNGF within vitroand, more important,in vivobiological activity equivalent to the native murine protein.

Msj-1 (mouse sperm cell-specific DnaJ first homologue) is a gene specifically expressed in germ cells at haploid stages. The protein first appears in round spermatids, accumulates in the periacrosomal region of elongating spermatids, and is maintained in spermatozoa. The msj-1 expression pattern is consistent with a role for this DnaJ protein in the spermiogenesis process. In this study, we used two experimental models, the anuran amphibian Rana esculenta and the wobbler mutant mouse, to explore the role of MSJ-1 during spermatogenesis, with a focus on spermiogenesis. Mice homozygous for the recessive mutation wobbler (wr/wr), a mutation of unknown identity, produce sperm cells characterized by a missing acrosome. In Rana esculenta testis, detection of high levels of MSJ-1 protein coincided with the appearance of postmeiotic germ cells during the annual sexual cycle. Conversely, elimination of the meiotic and postmeiotic stages, through gonadotropin administration at low temperature, abolished the MSJ-1 immunoreactive signal. In 20-day-old mice, when postmeiotic germ cells appeared for the first time, MSJ-1 mRNA and protein were observed in +/+ testis but were barely detectable in wr/wr testis. In adult testis, reduced MSJ-1 protein levels were observed in both +/wr and wr/wr testis, as compared with +/+ controls. Similarly, numbers of spermatids that stained by immunofluorescence for MSJ-1 appeared to be progressively reduced in adult +/+, +/wr, and wr/wr mouse testes, respectively. Characterization of the endocrine status of wobbler testis revealed reduced transcript levels of estrogen receptor alpha and reduced intratesticular androgen levels. However, androgen treatment did not affect MSJ-1 protein levels in either frogs or mice. In conclusion, our data in Rana esculenta and the wobbler mouse demonstrate a tight correlation between MSJ-1 protein expression and postmeiotic stages. In particular, the findings in the wobbler testis suggest a role for this protein in acrosomogenesis.