Critical steps in the isolation and expansion of adipose-derived stem cells for translational therapy.

Since the discovery of adipose-derived stem cells (ASCs), there have been high expectations of their putative clinical use. Recent advances support these expectations, and it is expected that the transition from pre-clinical and clinical studies to implementation as a standard treatment modality is imminent. However ASCs must be isolated and expanded according to good manufacturing practice guidelines and a basic assurance of quality, safety, and medical effectiveness is needed for authorisation by regulatory agencies, such as European Medicines Agency and US Food and Drug Administration. In this review, a collection of studies investigating the influence of different steps of the isolation and expansion protocol on the yield and functionality of ASCs has been presented in an attempt to come up with best recommendations that ensure potential beneficial clinical outcome of using ASCs in any therapeutic setting. If the findings confirm the initial observations of beneficial effects of ASCs, the path is paved for implementing these ASC-based therapies as standard treatment options.

Effects of low-level lead exposure on cell survival and neurite length in primary mesencephalic cultures.

The effects of low-level lead exposure on survival and neurite length of rat E15 primary ventral mesencephalic dopaminergic neurons were studied. Lead acetate (0.001-10 microM) added to primary cultures for 48 h (in serum-free defined media [DM]) caused a loss of tyrosine hydroxylase (TH)-positive neurons only at the highest concentrations (1 and 10 microM). In contrast, significant effects on neurite length were observed at concentrations as low as 0.001 microM. Lead-induced decrease in neurite length became more apparent at concentrations of 0.01 microM (mean 37.9% decrease) and 0.10 microM lead acetate (mean 43.9% decrease). These data show that very low concentrations of lead, well below the level necessary to adversely affect neuronal survival, can have dramatic effects on neurite growth. These results support recent clinical findings of detrimental effects of low-level lead exposure on brain development.

Ethanol induced changes in cyclin-dependent kinase-5 activity and its activators, P35, P67 (Munc-18) in rat brain.

The expression of cyclin-dependent kinase-5 (Cdk5) and its regulators, p35 and p67 was investigated in adult rat cerebral cortex and cerebellum, using an experimental paradigm of in vivo chronic ethanol exposure. In parallel, the activity of Cdk5 kinase was measured using a specific substrate histone-H1 peptide. Western blot analysis revealed no appreciable change in the expression of Cdk5 protein levels while, its regulatory proteins, p35 and p67 showed decreased levels following chronic ethanol treatment. However, ethanol treatment resulted in increased Cdk5 activity in both cortex and cerebellum with relatively high activity in cortex. Given the abundant expression and functions of Cdk5 in neural cells, our data implies a regulatory role for Cdk5 in ethanol mediated cell injury and may contribute to impaired CNS development in brain atrophy associated with alcoholic neurodegeneration.

Effect of prenatal and postnatal ethanol exposure on Ca2+ /calmodulin-dependent protein kinase II in rat cerebral cortex.

The ability of ethanol to influence Ca2+ /calmodulin-dependent protein kinase II (CaM kinase II)-mediated phosphorylation in rat cerebral cortex during prenatal and postnatal ethanol treatment was examined. Ethanol treatment increased protein expression of CaM kinase II alpha-subunit in membrane and cytosolic fractions during development. When specific CaM kinase II stimulators (Ca2+ /calmodulin) and inhibitor (autocamtide-2-related inhibitory peptide) were included during in vitro phosphorylation assays, three putative proteins (65, 50, and 40 kDa) were specifically phosphorylated by CaM kinase II, which might be involved in neurosignaling events associated with chronic ethanol treatment. Given that activation of CaM kinase II is a prerequisite for long-term potentiation induction through N-methyl-D-aspartate receptors, ethanol-induced increase in the levels of CaM kinase II alpha-subunit and selective phosphorylation of specific substrate proteins in cerebral cortex suggest a relation between calcium influx and increased CaM kinase II levels that might be relevant in ethanol-induced central nervous system dysfunction.

Role of DNA-dependent protein kinase in neuronal survival.

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.

Elevated DNA double strand breaks and apoptosis in the CNS of scid mutant mice.

Genetic approaches have provided evidence that DNA end-joining problems serve an essential role in neuronal survival during development of mammalian embryos. In the present study, we tested whether the DNA repair enzyme, DNA dependent protein kinase, plays an important role in the survival of cerebral cortical neurons in mice. DNA-PK is comprised of a DNA-binding subunit called Ku and a catalytic subunit called DNA-PKcs. In mice with the scid mutation, DNA-PKcs is truncated near the kinase domain, which causes loss of kinase activity. We compared the spatial and temporal aspects of neuronal cell death in scid versus isogenic wild-type embryos and found a significant increase in dying cells in scid mice, as assessed by nuclear changes, DNA fragmentation and caspase-3 activity. Additional biochemical and immunocytochemical studies indicated that of several DNA repair enzymes investigated, only PARP was increased in scid mice, possibly in response to elevated DNA strand breaks.

Effect of pre- and postnatal ethanol exposure on protein tyrosine kinase activity and its endogenous substrates in rat cerebral cortex.

The rat brain contains high levels of tyrosine-specific protein kinases (PTKs) that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu4Tyr1). Using this peptide as a substrate, we have measured the protein tyrosine kinase activity in membrane and cytosolic fractions from the cerebral cortices of pre- and postnatal ethanol-exposed rats at time intervals of 8, 30, and 90 days. During the course of development of the cerebral cortex, PTK activity decreased both in the membrane and cytosolic fractions from 8 and 90 days of age. Maximum activity was associated at the age of 8 days and gradually declined in the later ages (30 and 90 days) of postnatal development. However, PTK activity in the ethanol exposed rat cerebral cortex was further decreased when compared to controls in all the ages of postnatal development in membrane as well as in cytosolic fractions. In the presence of vanadate, a specific inhibitor of protein tyrosine phosphatases (PTPs), the PTK activity increased, indicating that the balance between protein tyrosine kinase and protein tyrosine phosphatase might be lost during ethanol exposure. In addition, when using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were identified on an immunoblot of membrane and cytosolic fractions from the ethanol-exposed rat cerebral cortex. The immunoblot showed several phosphotyrosine-containing proteins with molecular weights of 114, 70, 36, 34, 32, 20, and 14 kDa that were present in the cerebral cortex. However, higher levels of immunoreactivity of these proteins were found in the ethanol-exposed membrane fractions when compared to control fractions-particularly at the age of 30 and 90 days. Two phosphotyrosine proteins with molecular weights of 38 and 40 kDa showed decreased immunoreactivity at the age of 90 days in the cytosolic fraction of an ethanol-exposed rat's cerebral cortex. The differences in tyrosine-specific protein kinase activity and in phosphotyrosine-containing proteins observed during pre- and postnatal ethanol exposure may reflect specific functional defects in the cerebral cortex which could possibly underlie the mechanism contributing to fetal alcohol syndrome (FAS).

Selective changes in protein kinase C isoforms and phosphorylation of endogenous substrate proteins in rat cerebral cortex during pre- and postnatal ethanol exposure.

The effect of pre- and postnatal ethanol exposure on protein kinase C (PKC) activity, immunochemical analysis of PKC alpha, betaI, betaII, gamma, delta, epsilon, eta, and zeta by isoform-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins was investigated in rat cerebral cortex. The PKC activity was increased throughout the development. However, the activity at the age of 8 days was significantly high in cytosolic and membrane fractions from ethanol-treated rats. Immunochemical analysis showed increased levels of PKC betaI and betaII at the age of 8 days, and a decrease in delta isoform at 8, 30, and 90 days of age. PKC isoforms alpha, gamma, epsilon, and eta showed no appreciable change in ethanol-treated rats. PKC zeta levels were high in the cytosolic fraction from ethanol-treated samples of 90 days age. In vitro phosphorylation of endogenous substrate proteins in the presence of Ca2+/phospholipid showed increased phosphorylation of selective membrane and cytosolic proteins with 87, 65, 50, 43, 36, and 29 kDa in ethanol-treated rats. The phosphorylation of these proteins decreased in the presence of staurosporine, which also supported PKC-mediated phosphorylation. Increased PKC activity, activation of betaI and betaII isoforms, decreased levels of delta isoform, and phosphorylation of selective substrate proteins in cerebral cortex due to alcohol exposure might be relevant in ethanol-induced central nervous system dysfunction and fetal alcohol syndrome.

Effect of ethanol on chromatin and nonhistone nuclear proteins in rat brain.

Changes in chromatin conformation and nonhistone nuclear protein composition were analyzed in various classes of nuclei from the brain of control and chronic ethanol fed rats. Conformational studies of chromatin by circular dichroism spectrophotometry showed an increased molar ellipticity [theta] of chromatin in neuronal, astrocyte and oligodendroglial nuclei due to ethanol treatment. The increased molar ellipticity directly indicates relaxed state of chromatin in these nuclei, which facilitates ready state of transcription and replication. Further, the circular dichroism spectrum, due to a change over point at approximately 260 nm also indicated the possibility of DNA-protein interactions governing chromatin conformation. In microglial nuclei, the circular dichroism spectrum showed a decrease in molar ellipticity due to ethanol treatment, indicating the existence of chromatin in a condensed state. This type of circular dichroism change points towards the possibility of closed conformation, which renders the gene sequences not accessible due to conformational constrains of the chromatin. Since circular dichroism changes indicated the involvement of DNA-protein interactions, changes in nonhistone nuclear proteins were analyzed in these classes of nuclei by two-dimensional gel electrophoresis. In astrocytes and oligodendrocytes two new proteins appeared in each type of nuclei while in neurons and microglial nuclei four different proteins were either completely missing or showed a decrease. These changes indicate the presence of dynamic flux of nonhistone nuclear proteins in chromatin. Taken together, the changes in chromatin conformation, associated with specific changes in non histone nuclear protein composition suggest the modulation of chromatin as a response to ethanol evoked stimulus and has relevance in the regulation of cellular responses to ethanol crisis in brain.

Ethanol-induced changes in hepatic chromatin and nonhistone nuclear protein composition in the rat.

Excessive ethanol consumption has been shown to affect hepatic nucleic acid and protein synthesis. This study was undertaken to identify the changes in hepatic chromatin and nonhistone nuclear proteins as a consequence of chronic ethanol treatment, because these changes could be contributory to alcoholic cirrhosis. Chromatin conformation was monitored by circular dichroism spectrophotometry. The chromatin from alcoholic rat liver showed decreased molar ellipticity (theta). This change in chromatin conformation influences chromatin functions such as replication and transcription through the regulatory pool of nonhistone nuclear (NHN) proteins. The NHN proteins were analysed by ultrasensitive two-dimensional gel electrophoresis. Specific changes in nuclear proteins were documented in the liver of chronic alcohol-fed rats. This study shows chronic ethanol-induced changes in chromatin conformation and nuclear proteins, which might be critical in the mechanism of alcoholic cirrhosis.

Effect of ethanol on nuclear casein kinase II activity in brain.

Casein kinase II (CK II) plays an important role in serine/threonine dependent protein phosphorylation. In brain it is associated with long term potentiation besides its involvement in DNA, RNA and protein metabolism. Ethanol has been shown to induce cognitive impairment and affects DNA, RNA and protein metabolism at various steps. Since CK II is central in all these events, which are specifically affected by ethanol, the role of nuclear CK II is investigated in the present study. Total nuclear casein kinase activity was unaffected while heparin sensitive nuclear casein kinase II activity showed a 30% decrease in the brain from chronic alcohol fed rats. Cytosolic CK II activity was also unaffected. Immunological detection by western analysis using CK II antibodies showed no alteration in the quantity of enzyme. The decrease in nuclear casein kinase II might be responsible for ethanol induced cognitive impairment in the brain.

Synaptosomal transport of branched chain amino acids in young, adult and aged rat brain cortex.

Uptake of branched chain amino acids (BCAA, leucine and isoleucine) was studied in synaptosomes prepared from the cerebral cortex of rats of 1, 3 and 24 months of age. In addition to the conventional low affinity sodium independent transport system, a high affinity sodium dependent stereospecific transport system for the transport of BCAA was identified in synaptosomes prepared from the cerebral cortex of the above three age groups. There was an overall decrease in Km and Vmax of both high and low affinity transport systems for leucine and isoleucine in the cortical synaptosomes of 24-month-old rats when compared with younger age groups. This study indicates that the non-neurotransmitter essential amino acids are transported by high and low affinity transport systems and these systems undergo age-dependent alterations. These changes might be due to the altered synthesis of these transporter proteins and/or synthesis of transporters with altered conformation and/or changes in the physical properties (fluidity) of the membrane. The decrease in the transport of BCAA is on a par with the decrease in the overall metabolism of BCAA in brain. As food consumption decreases in the older age groups of animals, the availability of essential amino acids to the tissues might also be lowered. Under such conditions, it is suggested that the observed increase in the affinity (decreased Km) of the carrier might be helpful in the supply of essential amino acids.

Differential changes in cell morphology, macromolecular composition and membrane protein profiles of neurons and astrocytes in chronic ethanol treated rats.

Cellular morphology, macromolecular composition, (DNA, RNA and Protein content) marker enzyme activities for neurons [neuron specific enolase (NSE)] and astrocytes [glutamine synthetase (GS)] and plasma membrane protein profiles in the bulk isolated neurons and astrocytes from control and ethanol treated rats were studied. One month aged Wistar rats were given ethanol as sole drinking fluid for 10 weeks. Scanning electron microscopy revealed a characteristic cell surface smoothening in astrocytes due to ethanol treatment. DNA levels were unaltered, while RNA and Protein contents were decreased in astrocytes and neurons. Further, 3H-leucine incorporation into proteins was decreased in neurons and astrocytes derived from ethanol treated rats indicating reduced protein synthesis in neurons and astrocytes. GS activity was affected severely suggesting impairment in astrocytic functions. Plasma membrane protein composition was analyzed by 2-D electrophoresis. The analysis indicated several protein defects in the plasma membranes of neurons and astrocytes, which might be involved in 'membrane disorder' during ethanol challenge. 125I-Wheat Germ agglutinin binding studies showed three prominent proteins (160, 116 and 97 kDa) in astrocyte membrane fraction suggesting the possible involvement of N-terminal glycoproteins in altered astrocyte morphology during ethanol ingestion. Impairment in the astrocyte cell functions, protein changes in plasma membrane and cellular morphology studies suggest that astrocytes may be more vulnerable than neurons for ethanol effects.

Ethanol induced alterations in plasma membrane protein phosphorylation of neurons and astrocytes.

The endogenous protein phosphorylation patterns in plasma membranes of bulk isolated neurons and astroglia from control and chronic ethanol treated rats have been investigated. Chronic ethanol treatment resulted in increased phosphorylation of specific proteins with molecular weights 116, 63 and 60 kDa in both neurons and astrocytes. These proteins were further resolved by 2-DE and the analysis suggested an increased phosphorylation of congruent to 10-15 proteins, of which 116 kDa protein is phosphorylated to a higher extent by ethanol. Further, decreased phosphorylation was noticed in D-95 and D-63 proteins in neurons and D-78 and D-54 proteins in astrocytes. Alkali stability experiments for identifying the phosphoamino acid involved in phosphorylation of 116, 63 and 60 kDa proteins suggested that tyrosine and threonine are not involved and probably serine is the likely site for phosphorylation during chronic ethanol treatment. The phosphorylation of specific membrane proteins during chronic ethanol treatment might contribute to ethanol evoked cellular dysfunction.

Differential effects of 15-HPETE and 15-HETE on BHK-21 cell proliferation and macromolecular composition.

Arachidonate and/or linoleate metabolites have been implicated in modulating cell growth, replication and cell transformations. In studies with BHK-21 cells, we found lipoxygenase and cyclooxygenase inhibitors (NDGA and indomethacin, respectively) to be antiproliferative. Studies on the metabolism of arachidonic acid in BHK-21 cells have demonstrated that prostaglandin D2 is the major cyclooxygenase product, and 15-hydroxyeicosatetraenoic acid (15-HETE) is the major lipoxygenase product. Addition of D2 showed a significant decrease in the BHK-21 cell number showing antiproliferative action. Addition of lipoxygenase products, on the other hand, showed differential effects in that 15-HPETE decreased the cell number while 15-HETE increased. NDGA and 15-HPETE decreased DNA, RNA and protein contents, while 15-HETE significantly increased them. 5-HPETE and 5-HETE also showed similar results but were less potent than 15-H(P)ETEs. The differential effects of 15-HPETE and 15-HETE could be due to the generation of free radicals by the hydroperoxide and mitogenic response by hydroxide.

Recent advances in nuclear matrix function.

The nuclear matrix in eukaryotes is a non-histone proteinaceous nucleoskeleton structure having attachment sites for DNA loops during DNA replication. The nuclear matrix has been implicated in transcription, regulation of gene expression, primary transcription processing and provides a mooring for certain hormone receptors. Moreover, the nuclear matrix protein has linkages to intermediate filaments of the cytoskeleton. This review presents recent advances concerning the involvement of the nuclear matrix in DNA replication, relaxation of the superhelical strain in DNA, processing of hnRNA and snRNP, and RNA transport.

Effect of ethanol on hepatic ribosomal proteins and mRNA.

Levels of RNA, mRNA and separation of ribosomal proteins from control and ethanol treated rat liver, showed no change in total RNA content, but poly(A+)mRNA was reduced significantly in ethanolic rats. Ribosomal proteins S2, S3a, S3b, S4, L3, L4, L4a, L10a and L15 were found substantially reduced in experimental rat livers. This study suggests decrease in poly(A+) mRNA coupled with loss of ribosomal proteins must be responsible for decreased protein synthesis in chronic alcoholism.

Identification of two novel cerebrospinal fluid proteins in non specific mental retardation.

Cerebrospinal fluid (CSF) from twenty three patients with non specific mental retardation and fourteen age matched normal samples was subjected for qualitative analysis of protein profiles by two-dimensional gel electrophoresis (2-DE) and the proteins were visualised by ultra sensitive silver staining. Two proteins designated as mental retardation associated proteins (MRAP-I and MRAP-II) were identified in six male patients out of twenty three patients CSF samples. MRAP-I had an isoelectric point of 7.4 with a relative molecular weight 16.5 kDa, while MRAP-II had an iso-electric point of 7.2 with a relative molecular weight 16.8 kDa. The two proteins are presumed to be originated from brain, as they could not be traced in the serum of patients, nor due to proteolytic degradation. Despite unknown origin and identity, their presence in the CSF of a specific group of mentally retarded male patients suggest their possible clinical utility and to define protein alterations in mental retardation.

Differences in the plasma membrane proteins of chronic alcoholic rat brain.

Plasma membranes were isolated from the cerebral cortex of control and chronic ethanol-treated rat brains. Analysis of protein composition by SDS-PAGE and by two-dimensional gel electrophoresis (IEF-SDS-PAGE) revealed significant differences in the membrane protein patterns between control and ethanol-treated rat cerebral cortices, indicating the loss of several proteins in membranes from ethanol-treated rat brains. Plasma membrane-associated protein species are categorized into ethanol-sensitive and -insensitive proteins, based on their response to ethanol. This study reports that ethanol depletes certain intrinsic proteins of membranes that might be responsible for plasma membrane disruption by ethanol.

Liver plasma membrane proteins in chronic ethanol intoxication.

Plasma membrane proteins were isolated from control and ethanol-treated rat livers by the use of two-dimensional gel electrophoresis (IEF-SDS-PAGE). Two groups of proteins differing in their response to ethanol were analysed and termed as ethanol-sensitive proteins (ESP) and ethanol insensitive proteins (EISP). This study indicates a loss of many membrane-associated proteins and strongly suggests a role for these proteins in the mechanism of plasma membrane disruption by ethanol.