Insights on Human Small Heat Shock Proteins and Their Alterations in Diseases.

The family of the human small Heat Shock Proteins (HSPBs) consists of ten members of chaperones (HSPB1-HSPB10), characterized by a low molecular weight and capable of dimerization and oligomerization forming large homo- or hetero-complexes. All HSPBs possess a highly conserved centrally located α-crystallin domain and poorly conserved N- and C-terminal domains. The main feature of HSPBs is to exert cytoprotective functions by preserving proteostasis, assuring the structural maintenance of the cytoskeleton and acting in response to cellular stresses and apoptosis. HSPBs take part in cell homeostasis by acting as holdases, which is the ability to interact with a substrate preventing its aggregation. In addition, HSPBs cooperate in substrates refolding driven by other chaperones or, alternatively, promote substrate routing to degradation. Notably, while some HSPBs are ubiquitously expressed, others show peculiar tissue-specific expression. Cardiac muscle, skeletal muscle and neurons show high expression levels for a wide variety of HSPBs. Indeed, most of the mutations identified in HSPBs are associated to cardiomyopathies, myopathies, and motor neuropathies. Instead, mutations in HSPB4 and HSPB5, which are also expressed in lens, have been associated with cataract. Mutations of HSPBs family members encompass base substitutions, insertions, and deletions, resulting in single amino acid substitutions or in the generation of truncated or elongated proteins. This review will provide an updated overview of disease-related mutations in HSPBs focusing on the structural and biochemical effects of mutations and their functional consequences.

Real-time oxide evolution of copper protected by graphene and boron nitride barriers.

Applying protective or barrier layers to isolate a target item from the environment is a common approach to prevent or delay its degradation. The impermeability of two-dimensional materials such as graphene and hexagonal boron nitride (hBN) has generated a great deal of interest in corrosion and material science. Owing to their different electronic properties (graphene is a semimetal, whereas hBN is a wide-bandgap insulator), their protection behaviour is distinctly different. Here we investigate the performance of graphene and hBN as barrier coatings applied on copper substrates through a real-time study in two different oxidative conditions. Our findings show that the evolution of the copper oxidation is remarkably different for the two coating materials.

Mesenchymal stem cells from Shwachman-Diamond syndrome patients display normal functions and do not contribute to hematological defects.

Shwachman-Diamond syndrome (SDS) is a rare inherited disorder characterized by bone marrow (BM) dysfunction and exocrine pancreatic insufficiency. SDS patients have an increased risk for myelodisplastic syndrome and acute myeloid leukemia. Mesenchymal stem cells (MSCs) are the key component of the hematopoietic microenvironment and are relevant in inducing genetic mutations leading to leukemia. However, their role in SDS is still unexplored. We demonstrated that morphology, growth kinetics and expression of surface markers of MSCs from SDS patients (SDS-MSCs) were similar to normal MSCs. Moreover, SDS-MSCs were able to differentiate into mesengenic lineages and to inhibit the proliferation of mitogen-activated lymphocytes. We demonstrated in an in vitro coculture system that SDS-MSCs, significantly inhibited neutrophil apoptosis probably through interleukin-6 production. In a long-term coculture with CD34(+)-sorted cells, SDS-MSCs were able to sustain CD34(+) cells survival and to preserve their stemness. Finally, SDS-MSCs had normal karyotype and did not show any chromosomal abnormality observed in the hematological components of the BM of SDS patients. Despite their pivotal role in the hematopoietic stem cell niche, our data suggest that MSC themselves do not seem to be responsible for the hematological defects typical of SDS patients.

CAG repeat length in androgen receptor gene is not associated with amyotrophic lateral sclerosis.

BACKGROUND: Epidemiological and clinical studies show higher prevalence of amyotrophic lateral sclerosis (ALS) in males than in females and more severe lesions in androgen receptor (AR)-expressing tissues. The AR gene contains a polymorphic CAG trinucleotide repeat, whose expansion over a certain threshold is toxic to motor neurons, causing spinal and bulbar muscular atrophy (SBMA). PURPOSE AND METHODS: We tested the hypothesis that the AR CAG repeat linked to SBMA is a risk factor for ALS. We analyzed AR CAG expansions in 336 patients with ALS and 100 controls. RESULTS: We found a negative association of AR CAG expansions with ALS susceptibility, clinical presentation, and survival. CONCLUSIONS: Our findings do not support a role of the AR CAG repeat length in ALS.

Smad proteins are targets of transforming growth factor beta1 in immortalised gonadotrophin-releasing hormone releasing neurones.

Transforming growth factor beta (TGFbeta) is one of the growth factors involved in the neuroendocrine control of the gonadotrophin-releasing hormone (GnRH) neurones. It is produced and released by the astrocytes surrounding GnRH neurones and directly controls their secretory activity. TGFbeta signalling is based on a complex of two receptors that transduces the signal through peculiar intracellular substrates, the Smad proteins, which, upon activation, move into the nucleus, and modify the transcription of TGFbeta responsive genes. The present study aimed to verify whether TGFbeta1 is able to regulate the Smad pathway in GT1-1 cells (i.e. an immortalised neuronal cell line releasing GnRH). We show that: (i) GT1-1 cells express Smad 2, 3, 4, and 7; (ii) TGFbeta1 enhances the phosphorylation of Smad 2 and 3 at short times of exposure (15-30 min); (iii) TGFbeta1 induces the synthesis of the inhibitory Smad 7 at longer times (60-120-240 min); (iv) the conditioned medium of type 1 astrocytes enhances the phosphorylation of Smad 2 and 3 in GT1-1 cells and a TGFbeta1 neutralising antibody counteracts this effect. The results indicate that Smads are targets of TGFbeta1 and that astrocytes are able to modulate Smads proteins in GT1-1 cells through the release of TGFbeta1. Taken together, the data provide new evidence that glial cells are important regulators of the GnRH neuronal activity.

Role of glial cells, growth factors and steroid hormones in the control of LHRH-secreting neurons.

The mechanisms through which steroid hormones influence the LHRH system are not completely clarified and still represent a crucial and debated field of research in the neuroendocrine control of reproduction. Several data indicate that glial cells influence the activity of hypothalamic LHRH-secreting neurons, via the release of growth factors. It is now well known that glial cells express different kinds of steroid receptors and consequently may be considered as a target for the action of steroid hormones. To this purpose, the possibility that the effects of steroid hormones on LHRH neurons may be mediated by glial elements has been taken in consideration and observations supporting this hypothesis have been reported and discussed here. The results so far obtained strongly suggest that steroid hormones and growth factors, in order to exert their modulatory actions on LHRH dynamic, act in an integrated manner at the level of hypothalamic astrocytes.

Oestrogens, via transforming growth factor alpha, modulate basic fibroblast growth factor synthesis in hypothalamic astrocytes: in vitro observations.

The data presented here show that, in cultures of type 1 astrocytes obtained from the hypothalamus of neonatal female rat, 17beta-oestradiol is able to increase both the mRNA and the protein levels of basic fibroblast growth factor (bFGF). In particular, after 24 h of exposure to 17beta-oestradiol (10(-9) and 10(-10) m), an increase of messenger levels of bFGF appears in hypothalamic type 1 astrocytes. Similarly, an induction of bFGF protein is also evident at this time of exposure. The effect on the mRNA and protein levels of bFGF is blocked by the presence in the medium of an antibody raised against the transforming growth factor alpha (TGFalpha) receptor. This observation indicates that, TGFalpha, whose synthesis is modulated by oestrogens in hypothalamic astrocytes and which is able to increase, both the mRNA and the protein levels of bFGF in our experimental model, may act as the mediator of the oestrogenic induction of bFGF. Hypothalamic astrocytes, together with hypothalamic neurones synthesizing and secreting luteinizing hormone-releasing hormone (LHRH), form the LHRH network in conjunction with other neuronal systems. Gonadal steroids in general, and oestrogens in particular, play an important role in the control of the activity of this network. In addition, bFGF and TGFalpha, two growth factors released from astrocytes, are able to influence the activity of LHRH neurones. The present observations suggest that oestrogens may also act on LHRH neurones in an indirect fashion (i.e. by modulating the expression of bFGF and TGFalpha in glial cells).

Interactions between growth factors and steroids in the control of LHRH-secreting neurons.

How the gene expression and the release of luteinizing hormone releasing hormone (LHRH) are controlled in LHRH-secreting neurons is a very crucial and still debated topic of the neuroendocrinology. Several observations present in literature have recently indicated that glial cells may influence the activity of hypothalamic LHRH-secreting neurons, via the release of growth factors. The present review will summarize data obtained in our laboratory indicating that: (a) type 1 astrocytes, a kind of glial cells, are able to release in vitro growth factors belonging to the transforming growth factors beta (TGFbeta) family (i.e. TGFbeta1 and TGFbeta2) which influence the gene expression and the release of the decapeptide in immortalized LHRH-secreting neurons; (b) glial cells are also able to influence the steroid metabolism occurring in these neurons and in some cases this effect is exerted by TGFbeta1; (c) the mRNA levels of TGFbeta1 and of basic fibroblast growth factor (bFGF), another growth factor involved in the control of LHRH-secreting neurons, are modified in the rat hypothalamus during the different phases of the estrous cycle; (d) steroid hormones are able to modulate the gene expression of TGFbeta1 and bFGF both in vivo (i.e. in the whole hypothalamus of ovariectomized rats) and in vitro (cultures of type 1 astrocytes). On the basis of these results a possible functional correlation in the control of LHRH-secreting neurons between growth factors and gonadal steroids will be discussed and proposed.

Neuroactive steroids and peripheral myelin proteins.

The present review summarizes observations obtained in our laboratories which underline the importance of neuroactive steroids (i.e., progesterone (PROG), dihydroprogesterone (5alpha-DH PROG), tetrahydroprogesterone (3alpha, 5alpha-TH PROG), testosterone (T), dihydrotestosterone (DHT) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol)) in the control of the gene expression of myelin proteins (i.e. glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22)) in the peripheral nervous system. Utilizing different in vivo (aged and adult male rats) and in vitro (Schwann cell cultures) experimental models, we have observed that neuroactive steroids are able to stimulate the mRNA levels of Po and PMP22. The effects of these neuroactive steroids, which are able to interact with classical (progesterone receptor, PR, and androgen receptor, AR) and non-classical (GABA(A) receptor) steroid receptors is further supported by our demonstration in sciatic nerve and/or Schwann cells of the presence of these receptors. On the basis of the observations obtained in the Schwann cells cultures, we suggest that the stimulatory effect of neuroactive steroids on Po is acting through PR, while that on PMP22 needs the GABA(A) receptor. The present findings might be of importance for the utilization of specific receptor ligands as new therapeutical approaches for the rebuilding of the peripheral myelin, particularly in those situations in which the synthesis of Po and PMP22 is altered (i.e. demyelinating diseases like Charcot-Marie-Tooth type 1A and type 1B, hereditary neuropathy with liability to pressure palsies and the Déjérine-Sottas syndrome, aging, and after peripheral injury).

Formation and effects of neuroactive steroids in the central and peripheral nervous system.

This chapter summarizes several observations that emphasize the importance of neuroactive steroids in the physiology of the central and peripheral nervous systems. A new, and probably important, concept is emerging: Neuroactive steroids not only modify neuronal physiology but also intervene in the control of glial cell functions. The data presented here underscore that (1) the mechanism of action of the various steroidal molecules may involve both classical (progesterone and androgens) and nonclassical steroid receptors [gamma-aminobutyric acid type A (GABAA) receptor], (2) in many instances, the actions of hormonal steroids are not due to their native molecular forms but to their 5 alpha- and 3 alpha,5 alpha-reduced metabolites, (3) several neuroactive steroids exert dramatic actions on the proteins proper of the peripheral myelin (e.g., glycoprotein Po and peripheral myelin protein 22), and (4) the effects of steroids and of their metabolites might have clinical significance in cases in which the rebuilding of the peripheral myelin is needed (e.g., aging, peripheral injury).

Steroid effects on the gene expression of peripheral myelin proteins.

The present article summarizes recent observations obtained in our laboratory which clearly indicate that sex steroids exert relevant effects on the peripheral nervous system. In particular, the following important points have emerged: (1) Steroids exert stimulatory actions on the synthesis of the proteins proper of the peripheral myelin (e.g., glycoprotein Po and peripheral myelin protein 22) in vivo and on the Schwann cells in culture; (2) in many cases the actions of hormonal steroids are not due to their native molecular forms but rather to their metabolites (e.g., dihydroprogesterone and tetrahydroprogesterone in the case of progesterone; dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol in the case of testosterone); (3) the mechanism of action of the various steroidal molecules may involve both classical (progesterone and androgen receptors) and nonclassical steroid receptors (GABA(A) receptor); and finally, (4) the stimulatory action of steroid hormones on the proteins of the peripheral myelin might have clinical significance in cases in which the rebuilding of myelin is needed (e.g., aging, peripheral injury, demyelinating diseases, and diabetic neuropathy).

Hypothalamic transforming growth factor beta1 and basic fibroblast growth factor mRNA expression is modified during the rat oestrous cycle.

The present observations show that the mRNA levels of two growth factors, previously described to be involved in the control of neurones synthesizing the luteinizing hormone releasing hormone (LHRH) [i.e. transforming growth factor beta1 (TGFbeta1) and basic fibroblast growth factor (bFGF)], fluctuate in the hypothalamus of adult female rats during the oestrous cycle. In particular, the expression of TGFbeta1-mRNA shows a peak on the morning of the day of proestrus, which precedes the increased secretion of the two gonadotrophins that occurs on that day. In the case of bFGF, the peak is evident in the evening of the same day and is concomitant with that of the gonadotrophins. We evaluated the effects of ovariectomy and of exogenous oestrogens on the mRNA levels of these two growth factors in the hypothalamus. The data indicate that 3 weeks of ovariectomy are not able to change the hypothalamic messenger levels of the two growth factors considered, which remain at the levels found in diestrus 1, and that 17beta-oestradiol is able to induce a significant increase of both TGFbeta1- and of bFGF-mRNA levels in the hypothalamus of the ovariectomized rat. The present in vivo observations support the concept, previously proposed on the basis of in vitro data, that growth factors, such as TGFbeta1 and bFGF, play a role in the hypothalamic control of reproduction, and suggest that the control of LHRH dynamics involves a strict cooperation between gonadal steroids and growth factors.

Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis.

In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers of transgenic Arabidopsis plants for functional genomic studies. A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin). Analysis of herbicide-resistant plants indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting in a preponderance of independently derived T-DNA insertions. T-DNA insertions were usually integrated in a concatenated, rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci. Using pooling strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions. We show the utility of these transgenic lines for identifying insertion mutations using gene sequence and PCR-based screening.

The action of steroid hormones on peripheral myelin proteins: a possible new tool for the rebuilding of myelin?

The present paper summarizes recent results we have obtained while studying the effect of sex steroids on the gene expression of two peripheral myelin proteins, the glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22). In particular, we have analyzed the effect of progesterone (P), testosterone (T) and their 5alpha- and 3alpha-5alpha-reduced derivatives [respectively, dihydrotestosterone (DHTT) and 5alpha-androstan-3alpha, 17beta-diol (3alpha-diol) for T, and dihydroprogesterone (DHP) and tetrahydroprogesterone (THP) for P]. The data obtained, utilizing different in vivo and in vitro experimental models, have indicated that: a) DHP is able to enhance the low messenger levels of Po present in the sciatic nerve of aged male rats; b) P, DHP and THP treatments stimulate the gene expression of Po in the sciatic nerve of adult male rats or in cultures of rat Schwann cells, while only THP is effective on PMP22; c) P and DHP are also able to increase the low messenger levels of Po present in transected sciatic nerve; d) the removal of circulating androgens by castration is able to decrease the mRNA levels of Po in the sciatic nerve, a phenomenon which is counteracted by the consequent treatment with DHT; e) the stimulatory effect of DHT on the gene expression of Po is also evident in cultures of rat Schwann cells, but in this case the effect seems to be due to the interaction of this steroid with the progesterone receptor; f) in cultures of Schwann cells PMP22 mRNA levels are stimulated only by 3alpha-diol treatment. Taken together, these observations showing the positive effects of sex steroid hormones on the gene expressions of Po and PMP22, suggest that a treatment with these molecules or their synthetic agonists may be useful in cases in which the rebuilding of myelin is necessary.

Transforming growth factor beta2 is able to modify mRNA levels and release of luteinizing hormone-releasing hormone in a immortalized hypothalamic cell line (GT1-1).

On the basis of our previous observations which indicated that transforming growth factor beta1 (TGFbeta1) affects the gene expression and the release of luteinizing hormone-releasing hormone (LHRH) in GT1-1 cells, we have presently evaluated whether also TGFbeta2 might be effective on these parameters. The data here reported show that also TGFbeta2 is able to affect LHRH dynamics, and that this action presents a different kinetics than that reported by TGFbeta1. In particular TGFbeta2 is able to facilitate LHRH release and to decrease the mRNA levels of this decapeptide. The present data have also shown that, GT1-1 cells express the messengers for the two most important receptors of the TGFbeta family, namely TGFbetaRI and TGFbetaRII and consequently represent a target for the action of the different isoforms of TGFbeta. Since the two isoforms of TGFbeta are produced and released from astrocytes, the present data add new support to the hypothesis that astrocytes participate in the control of LHRH secretion in a paracrine fashion.

Astrocyte-neuron interactions in vitro: role of growth factors and steroids on LHRH dynamics.

The data here reviewed, obtained with in vitro models, indicate that growth factors and steroids play a significant role in astrocyte-neuron interactions. Different designs have been adopted: (1) GT1-1 cells (a cell line derived from a mouse hypothalamic LHRH-producing tumor) were cocultured with type 1 rat astrocytes; and (2) GT1-1 cells were exposed to the conditioned medium (CM) in which type 1 rat astrocytes had been grown for 24 h. LHRH release and mRNA LHRH levels were measured respectively in the medium and in cell homogenates, at different time intervals (LHRH release, by RIA; LHRH mRNA by Northern blot analysis). The data obtained show that type 1 astrocytes secrete in the medium TGFbeta, which is able to modulate the release and the gene expression of LHRH in GT1-1 cells; and that one or more LHRH-degrading enzymes is/are present in the conditioned medium of type 1 astrocytes. A second part of the experiments have indicated that type 1 astrocytes are also able to affect, in different directions, the metabolism of testosterone and progesterone into their 5alpha-reduced metabolites occurring in the GT1-1 cells. In particular, it has been observed that the conversion of testosterone into DHT is decreased by the coculture with type 1 astrocytes, while the conversion of progesterone into DHP is increased by the same coculture conditions. Moreover, type 1 astrocytes are sensitive to steroid hormones, and in particular to the 5alpha-reduced metabolites of progesterone; this has been shown by analyzing the effects exerted by different steroids on the gene expression of the typical astrocyte marker GFAP.