Scaling-up of dental pulp stem cells isolated from multiple niches

Dental pulp (DP) can be extracted from child's primary teeth (deciduous), whose loss occurs spontaneously by about 5 to 12 years. Thus, DP presents an easy accessible source of stem cells without ethical concerns. Substantial quantities of stem cells of an excellent quality and at early (2-5) passages are necessary for clinical use, which currently is a problem for use of adult stem cells. Herein, DPs were cultured generating stem cells at least during six months through multiple mechanical transfers into a new culture dish every 3-4 days. We compared stem cells isolated from the same DP before (early population, EP) and six months after several mechanical transfers (late population, LP). No changes, in both EP and LP, were observed in morphology, expression of stem cells markers (nestin, vimentin, fibronectin, SH2, SH3 and Oct3/4), chondrogenic and myogenic differentiation potential, even after cryopreservation. Six hours after DP extraction and in vitro plating, rare 5-bromo-2'-deoxyuridine (BrdU) positive cells were observed in pulp central part. After 72 hours, BrdU positive cells increased in number and were found in DP periphery, thus originating a multicellular population of stem cells of high purity. Multiple stem cell niches were identified in different zones of DP, because abundant expression of nestin, vimentin and Oct3/4 proteins was observed, while STRO-1 protein localization was restricted to perivascular niche. Our finding is of importance for the future of stem cell therapies, providing scaling-up of stem cells at early passages with minimum risk of losing their "stemness".     

Cellular and biochemical characteristics of semen obtained from pubertal chimpanzees by masturbation

Semen characteristics were studied in 6 wild-born chimpanzees with dental ages ranging approximately from 6 to 12 years. The animals formed 2 groups, early pubertal (EP, N = 3, 6-9 years) and late pubertal (LP, N = 3, 11-12 years). Mean body weight, testicular volume and serum androgen concentration were significantly lower in Group EP (32.2 +/- 1.6 kg, 34.0 +/- 7.7 cm3, 2.1 +/- 0.1 ng/ml) than in Group LP (55.7 +/- 5.7 kg, P less than 0.01; 100.5 +/- 11.9 cm3, P less than 0.01; 3.6 +/- 0.7 ng/ml, P less than 0.05). Ejaculates were obtained by masturbation in all subjects. The mean ejaculate volume was lower in Group EP (0.56 +/- 0.20 ml) than in Group LP (3.77 +/- 0.73 ml, P less than 0.01). In Group EP, 2 animals were azoospermic while the third produced semen with means of 57.1 x 10(6) spermatozoa per ml, 20% motility and 40% vitality. These values were low when compared with the mean values of Group LP (376 x 10(6) spermatozoa per ml, 67% motility and 78% vitality). Mean total sperm count was correlated with testicular volume (r = 0.84) and serum androgen concentration (r = 0.96). The mean concentrations of L-carnitine, fructose, citrate and acid phosphatase for the two groups were not significantly different; but, related to the differences in ejaculate volumes, their total amounts in total ejaculate were lower in Group EP than in Group LP. These results suggest that, in chimpanzees, mechanisms of seminal plasma production and ejaculation are functional early in the reproductive life and that the emission of spermatozoa occurs later.     

Pyruvate modifies glycolytic and oxidative metabolism of rat embryonic spinal cord astrocyte cell lines and prevents their spontaneous transformation

This study aimed to provide detailed data on mitochondrial respiration of normal astrocyte cell lines derived from rat embryonic spinal cord. Astrocytes in early passages (EP), cultured without pyruvate for more than 35 passages, defined here as late passages (LP), undergo spontaneous transformation. To study initial steps in cell transformation, EP data were compared with those of LP cells. LP cells had reduced glycolysis, fewer mitochondria and extremely low oxidative rates, resulting from a dysfunction of complexes I and II + III of the respiratory chain. Treatment of EP cells with pyruvate until they were, by definition, LP cultures prevented transformation of these cells. Pyruvate-treated EP cells had more mitochondria than normal cells but slightly lower respiratory rates. The increase of mitochondrial content thus appears to act as a compensatory effect to maintain oxidative phosphorylation in these LP 'non-transformed' cells, in which mitochondrial function is reduced. However, pyruvate treatment of transformed LP cells during additional passages did not significantly restore their oxidative metabolism. These data highlight changes accompanying spontaneous astrocyte transformation and suggest potential targets for the control of astrocyte proliferation and reaction to various insults to the central nervous system.     

Regional differences in microsomal lipid peroxidation and antioxidant levels in the guinea pig adrenal cortex

Lipid peroxidation (LP) and antioxidant levels were studied in the chromatically distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea pig adrenal cortex. Ferrous ion (Fe2+) produced a concentration-dependent (10(-5) to 10(-3) M) stimulation of microsomal LP in both zones, but LP, as estimated by malonaldehyde production, was far greater in the inner zone. Although cytosolic ascorbic acid content was similar in the two zones, microsomal tocopherol levels were approx 4 times greater in the outer than inner zone. Subphysiological concentrations of ascorbic acid, like Fe2+, initiated LP to a greater extent in inner than outer zone microsomes; optimal stimulation of LP by ascorbic acid occurred at concentrations of 100-200 microM in both zones. Physiological concentrations of ascorbic acid (1-5 mM), by contrast, did not initiate LP and, in fact, markedly inhibited Fe2+-induced LP in both inner and outer zone microsomal preparations. Outer zone microsomes were more sensitive to the antioxidant effects of ascorbic acid than were inner zone preparations. Addition of alpha-tocopherol to inner zone microsomal suspensions inhibited Fe2+-induced LP. The results indicate that there are regional differences in adrenocortical LP which may be caused by differences in tocopherol content. alpha-Tocopherol may serve important antioxidant functions within the adrenal cortex, thereby contributing to the functional zonation of the gland.     

Effects of peptide factors from right and left brain hemispheres of rats on amplitude-time characteristics of projective and interhemispheric evoked responses

Peptide extracts of the right and left hemispheres were applied to the projective (somatosensory and visual) and temporal associative regions of the left brain hemisphere in cats. In the zones of peptide application, evoked potentials (EP) in response to singular and coupled somatic, visual and transcallosal stimuli were registered. The data obtained showed that right and left peptide extracts had different effects on evoked potentials of the left hemisphere. Thus ipsilateral extract increased the amplitude of projective EP, decreased duration of their cycles and amplitude of transcallosal responses. Contralateral extract, on the contrary, activated interhemispheric inputs to brain cortex, suppressed thalamic inputs and increased multimodal properties of neurons. A differential approach to the problem of specific correction of pathological states of the right and left brain hemispheres is required. Right and left peptide extracts may be used in normalization of interhemispheric activity balance in compensatory-recovering processes.     

PTH-dependent adenylyl cyclase activation in SaOS-2 cells: passage dependent effects on G protein interactions

Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS-2 cells varies as a function of cell passage. In early passage (EP) cells (< 6), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (> 22), PTH exceeded FOR dependent ACA. Potential biochemical mechanisms for this passage dependent change in ACA were considered. In EP, prolonged exposure to pertussis toxin (PT) markedly enhanced ACA activity in response to PTH, Isoproterenol and Gpp(NH)p, whereas ACA in response to FOR was decreased. In contrast, the identical treatment of LP with PT diminished all ACA in response to PTH, Gpp(NH)p, and FOR. The dose dependent effects of PT on subsequent [(32)P]ADP-ribosylation of its substrates, GTPase activity, as well as FOR-dependent ACA, were equivalent in EP and LP. The relative amounts of G(alpha)i and G(alpha)s proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [(32)P]ADP-ribosylation, were quantitatively similar in EP and LP. Western blot levels of G(alpha)s and G(alpha)i proteins were not influenced by prior exposure to PT. Both PT and CT dependent [(32)P]ADP-ribosylation were dose-dependently decreased following exposure to PT. However, the PT-dependent decline in CT-dependent [(32)P]ADP-ribosylation occurred with enhanced sensitivity in LP. The protein synthesis inhibitor cycloheximide partially reversed the PT associated decrease in FOR dependent ACA in EP. In contrast, cycloheximide completely reversed the PT associated decrease in FOR and as well as PTH dependent ACA in LP. G(alpha)s activity, revealed by cyc(-) reconstitution, was not altered either by cell passage or exposure to PT. The results suggest that the coupling between the components of the complex may be pivotally important in the differential responsiveness of early and late passage SaOS-2 cells to PTH.     

Effect of motor deprivation in early ontogeny on evoked potentials of the sensomotor and visual cortex in the rat

The influence of long-term (3 months) locomotor deprivation of rats in a month age, on the evoked potentials (EP) of the sensorimotor and the visual cortex was studied in conditions of presentation of single and paired stimuli. Changes were revealed in both cortical zones. An increase of peak latency of the initial positive EP phase in the sensorimotor cortex, and prolongation of the process of changes in excitability of neural elements, elicited by conditioning stimulus, was revealed both in the sensorimotor and the visual areas. The effect of deprivation on the dynamics of changes in neuronal systems excitability was greater in the visual evoked responses.     

小麦不同蘖位叶片出生的两段线性模式及其品种、播期效应

叶片出生动态是小麦生长发育进程及其协调状况的重要表现,研究发现,小科叶片出生与播后累积ＧＤＤ（ｆｇｒｏｗｉｎｇ ｄｅｇｒｅｅ ｄａｙｓ ａｆｔｅｒ ｓｏｗｉｎｇ）的关系遵循两段（阶段Ⅰ快于阶段Ⅱ）线性模式,护颖分化期为两段模式的分界点,这一规律在正常发育的冬性和春性品种的７主茎及分蘖中表现一致,冬性品种播期１（９月３０日）、播期３（３月２日）的主茎及冬、春性品种各播期的Ｔ３分蘖,因生长发育异常而”     

Lipid peroxidation in the myocardium in experimental infarct

Changes in the content of lipid peroxidation (LP) products and activities of antioxidant enzymes--superoxide dismutase, glutathione peroxidase and catalase in myocardium of rats after experimental infarction as well as after pretreatment with antioxidant ionol, beta-adrenoblocker inderal and verapamil, an inhibitor of slow Ca2+-channels have been studied. In the left ventricles of the control animals decreased levels of LP-products (Schiff bases and lipid hydroperoxides) have been registered as compared with right ventricles, accompanied by increased activity of antioxidant enzymes in the left ventricles. In experimental infarction the level of LP products increases and activity of antioxidant enzymes decreases both in ischemic and nonischemic regions of the heart. In nonischemic zone these changes can be prevented by pretreatment with inderal and ionol but not with verapamil.     

Features of changes in bioelectric reactions of the cerebral cortex in rats with deafferentation pain syndrome]

In rats with pain syndrome after sciatic nerve section the authors studied spontaneous and evoked bioelectric activity in sensomotor cerebral cortex of both hemispheres. Electrocorticogram showed the presence of hyper-synchronic discharges and paroxysmal peak-wave (700-800 mV) activity in contralateral hemisphere. While stimulating the injured limb the threshold of evoked potentials (EP) was observed to decrease, its amplitude to increase and focus maximum EP activity to extend.     

Relation between the parameters of lipid peroxidation and the constant potential of the rat brain

Studies were carried out of the relationship between the constant potential level (CPL) and the intensity of lipid peroxidation (LP) of the brain membranes in rats with different motor asymmetry. Strong correlation was found between CPL and the content of LP products (diene conjugates, hydroperoxides and Schiff bases) in the hemispheres of the brain (R = 0.7-0.8).     

Ontogenetic development of electrocorticogram in the rat

Spontaneous electrocorticogram (ECoG) was recorded in frontal (sensorimotor) temporal (auditory) and occipital (visual) cortical regions of 86 male rats (immobilized with d-tubocurarine) aged from 3 days to adulthood. Activity which could be classified as ECoG was for the first time recorded in 5-day-old rats; it was formed by groups of slow waves with unstable frequency intermingled with periods of isoelectric line. Discontinuous ECoG activity was regularly registered even in 10-day-old rats, exceptionally in 12-day-old rats. During further maturation of the continuous ECoG an increase in frequency and an establishment of a basic rhythmic activity synchronous over both hemispheres took place, so that 25- and 30-day-old rats did not differ from the adult ones. Autocorrelagrams and power frequency spectra demonstrated a broad frequency range of the basic rhythm as well as delay in the development of occipital cortical areas in comparison to frontal areas.     

Conjugation of the neuronal impulse activity in the rabbit neocortex during self stimulation of the lateral hypothalamus

Correlation of impulse activity of visual and sensorimotor neurons of both hemispheres in 10-s interval after self-stimulation of the right and left lateral hypothalamus was studied by plotting cross- and autocorrelation histograms. The number of cell pairs, in which sensorimotor neurons discharged after visual ones, increased after self-stimulation (from 24 +/- 6 to 44 +/- 7%). Frequencies of the alpha- and theta-range in crosscorrelograms increased; the alpha frequency increased mainly in the right hemisphere, while the theta frequency increased in the left hemisphere. The interhemispheric asymmetry in the interaction between cortical neurons was not associated with the side of hypothalamic stimulation.     

Changes in the size and shape of the synaptic vesicles in the sensorimotor cortex of the rat brain in the initial phases of kindling

The sensorimotor area of rat cerebral cortex was subjected to repeated electrical stimulation at 10-min intervals, with resultant formation and progressive lengthening of self-sustained after-discharges (SSAD). One and 60 min after the third SSAD ended, we carried out an electron microscopy morphometric analysis of the agranular synaptic vesicles in type I synapses (after Gray) in the second cortical layer of the homotopic area of the unstimulated hemisphere. One minute after the seizure ended, 5.8% enlargement of the synaptic vesicles compared with the control was demonstrated in zone II of the synapse (0.1-0.2 micron from the active zone of the synapse). Neither the size nor the shape of the synaptic vesicles in the other parts of the synaptic apparatus altered. Sixty min after the seizure ended, a 5.5% enlargement of the synaptic vesicles in zone I (0.0-0.1 micron) and a 5.4% enlargement of those in zone II was found. The synaptic vesicles in zone I in the experimental animals were more oval than in the controls. Our findings support the vesicular theory and testify that hyperfunction, up to temporary exhaustion of the synaptic apparatuses, produces a change in the transmitter content of the synaptic vesicles. A raised amount of transmitter in the synaptic vesicles near the active zone could be one of the factors responsible for continued hyperexcitability of the tissue one hour after the seizure had ended. The results likewise support the concept of two mechanisms of synaptic vesicle formation, and hence of the existence of two different vesicle populations.     

GABA metabolism and the formation of a sensorimotor cortex evoked potential in the presence of excess zinc ions

Changes in GABA content, the enzymatic activity of its metabolism and the formation of sensorimotor cortical evoked potential (EP) were studied following long-term ZnCl2 administration. It has been established that a single ZnCl2 injection at a dose of 0.1 and 1 mg/kg was accompanied by an increase in the amplitude of sensorimotor cortical EP, though GABA accumulation in this brain structure was observed. This might account for the prolongation of the period of the potential appearance. Long-term (for 7, 14, 21 days) ZnCl2 administration at a dose of 0.1 mg/kg produced a sharp depression in the potential appearance and an increase in GABA content by 50% with the enhancement of glutamate decarboxylase activity and the attenuation of GABA-transaminase activity.     

Are extraversion and openness indicators of a slow life history strategy?

Theory and data generally concur that a slower Life History Strategy (LHS) is associated with higher Conscientiousness, Agreeableness and Emotional Stability. Whether Extraversion and Openness are indicators of a slow LH, or whether they include both fast and slow LH components, remains unresolved. I addressed these questions in two studies: one of university students observed via periodic brief audio recordings during 72 h of their daily lives, and the second a re-analysis of data from Block and Block's (2006) longitudinal study. In both studies, I operationalized LHS as the correlation between an individual's California Q-Sort (CAQ) profile and the slow Life History (SLH) CAQ template created by Sherman et al. (2013) and Dunkel et al. (2015). I calculated Five Factor Model dimension scores using McCrae et al's (1986) method. In both samples, individuals whose CAQ profile more closely resembled the SLH template were higher in Conscientiousness, Agreeableness, and Emotional Stability than those pursuing a faster LHS. Extraversion was unrelated to LHS, while Openness in the Block and Block data set was actually associated with a faster LHS. Analysis of individual CAQ items revealed some differences between the studies. Generally, Extraversion-loading items tapping excitement-seeking and self-dramatization, and Openness-loading items tapping nonconformity, were associated with a faster LHS. Participants in the audio recording study also completed a HEXACO personality inventory and the Arizona Life History Battery. LHS as measured by the ALHB was uncorrelated with LHS as measured by the CAQ. Controlling for Extraversion yielded a significant positive relationship between the two LHS measures, suggesting that their poor convergent validity resulted from differences in their coverage of Extraversion-related content.     

Link protein as an enhancer of cumulus cell-oocyte complex expansion

To investigate the specific components involved in regulating cumulus cell-oocyte complex (COC) expansion in an in vitro mouse experiment, freshly-isolated COC were cultured in the presence of various combinations of FSH (1.0 microg/ml), proteins of the inter-alpha-inhibitor (I alpha I) family (a light chain, also known as bikunin, heavy chains [HC1 + HC2] and I alpha I [0.01-2.0 microg/ml]) and link protein (LP) (0.016-10 microg/ml) for 24 h and were observed for expansion of their cumulus cells (percent of COC with + 3 and + 4 expansion and average projected area). The COC were videotaped in real time at the initiation of culture and after 24 h of culture. FSH alone did not stimulate cumulus expansion under serum-free conditions; however, treatment with I alpha I (0.1-2.0 microg/ml) or heavy chains (10 microg/ml), but not bikunin (10 micro g/ml), in the presence of FSH significantly increased COC expansion, with maximal promotion occurring at 1.0 microg/ ml of I alpha I. Addition of LP (2.0 micro g/ml) to the medium containing I alpha I (1.0 microg/ml) and FSH resulted in significantly higher expansion levels than were observed in response to I alpha I alone, although LP alone (10 microg/ml) had no or very little effect by itself. Anti-I alpha I or anti-LP polyclonal antibody, which inhibits binding of I alpha I and LP, respectively, to hyaluronic acid (HA), markedly reduced expansion of the surrounding cumulus cell extracellular matrices. Therefore, in vitro, LP might serve, in part, to enhance the COC expansion possibly by stabilizing HA-I alpha I (or heavy chains) complex on the surrounding cumulus cell matrices.