DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

Modificaciones de la estructura ósea del fémur proximal. análisis de una muestra esqueletal / Changes in bone structure of proximal femur. an skeletal sample analysis

Objetivo: Los análisis de osteoporosis en restos óseos cobran relevancia en tanto permiten evaluar el estado de salud ósea de una población. Una de las regiones óseas utilizadas para la estimación de la pérdida de la densidad mineral ósea es la epífisis proximal del fémur. El objetivo del trabajo fue analizar, a partir de restos esqueletarios, la variación de la estructura ósea del fémur proximal en adultos y estudiar su relación con la masa corporal, sexo y edad. Materiales y métodos: Se tomó una muestra de 30 fémures izquierdos pertenecientes a la colección osteológica ®Prof. Dr. Rómulo Lambre¼ de la Facultad de Ciencias Médicas (Universidad Nacional de La Plata), y se estimaron 2 índices radiológicos, el índice córtico metafisario y el índice trabecular. Posteriormente, se estudió la relación entre los valores obtenidos y la masa corporal, el sexo y la edad de los individuos.Resultados: El valor promedio estimado por el índice córtico metafisario señaló una baja calidad ósea, sin diferencias entre sexos, y no presentó correlaciones con la edad ni con el peso de los individuos. El 50% de los fémures presentó un valor de índice trabecular indicativo de osteoporosis,sin una diferencia significativa entre sexos.Conclusión: La baja calidad ósea del material se ajustó a lo esperado, debido a la edad de los sujetos analizados. Esto puede haber interferido en la visibilidad de otros procesos, ocurridos en vida, que podrían haber sido causantes de la pérdida de densidad mineral ósea.

Purpose: Analyses of osteoporosis in human bone remains become relevant because allow us to evaluate the health of past population. An usual area used for estimating the loss of bone mineral density is the proximal epiphysis of the femur. The aim of this paper is to analyze changes in proximal femoral bone structure, in relation to body mass, sex and age of individuals.Materials and methods: The sample consist of 30 left femurs belonging to Prof. Dr. R. Lambre Osteological Collection, housed at the Faculty of Medical Sciences, National University of La Plata. Two indices were estimated radiologically (Trabecular Index and Cortico Metaphyseal Index), and related to body mass, sex and age of individuals.Results: The average value estimated by Cortico Metaphyseal Index pointed to a low quality of bone, with no differences between males and females. This index showed no correlation with age and weight of individuals. A 50% of the individuals showed a Trabecular Index indicative of osteoporosis, with no signifi cant difference between sexes.Conclusion: The poor quality of the bone material observed was adjusted to expectation, given the age of the individuals analyzed, which conspired against the visibility of other processes causing the loss of Bone Mineral Density happened earlier in the life of individuals.

Modificaciones de la estructura ósea del fémur proximal: Análisis de una muestra esqueletal

Objetivo: Los análisis de osteoporosis en restos óseos cobran relevancia en tanto permiten evaluar el estado de salud ósea de una población. Una de las regiones óseas utilizadas para la estimación de la pérdida de la densidad mineral ósea es la epífisis proximal del fémur. El objetivo del trabajo fue analizar, a partir de restos esqueletarios, la variación de la estructura ósea del fémur proximal en adultos y estudiar su relación con la masa corporal, sexo y edad. Materiales y métodos: Se tomó una muestra de 30 fémures izquierdos pertenecientes a la colección osteológica ½Prof. Dr. Rómulo Lambre¼ de la Facultad de Ciencias Médicas (Universidad Nacional de La Plata), y se estimaron 2 índices radiológicos, el índice córtico metafi sario y el índice trabecular. Posteriormente, se estudió la relación entre los valores obtenidos y la masa corporal, el sexo y la edad de los individuos. Resultados: El valor promedio estimado por el índice córtico metafi sario señaló una baja calidad ósea, sin diferencias entre sexos, y no presentó correlaciones con la edad ni con el peso de los individuos. El 50% de los fémures presentó un valor de índice trabecular indicativo de osteoporosis, sin una diferencia signifi cativa entre sexos. Conclusión: La baja calidad ósea del material se ajustó a lo esperado, debido a la edad de los sujetos analizados. Esto puede haber interferido en la visibilidad de otros procesos, ocurridos en vida, que podrían haber sido causantes de la pérdida de densidad mineral ósea.(AU)

Purpose: Analyses of osteoporosis in human bone remains become relevant because allow us to evaluate the health of past population. An usual area used for estimating the loss of bone mineral density is the proximal epiphysis of the femur. The aim of this paper is to analyze changes in proximal femoral bone structure, in relation to body mass, sex and age of individuals. Materials and methods: The sample consist of 30 left femurs belonging to Prof. Dr. R. Lambre Osteological Collection, housed at the Faculty of Medical Sciences, National University of La Plata. Two indices were estimated radiologically (Trabecular Index and Cortico Metaphyseal Index), and related to body mass, sex and age of individuals. Results: The average value estimated by Cortico Metaphyseal Index pointed to a low quality of bone, with no differences between males and females. This index showed no correlation with age and weight of individuals. A 50% of the individuals showed a Trabecular Index indicative of osteoporosis, with no signifi cant difference between sexes. Conclusion: The poor quality of the bone material observed was adjusted to expectation, given the age of the individuals analyzed, which conspired against the visibility of other processes causing the loss of Bone Mineral Density happened earlier in the life of individuals.(AU)

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes.

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 µg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ± 13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 µg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 µg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10(6) cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 µg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10(6) cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 µg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 µg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 µg/mL Zn relative to 0 Zn (IVM alone) and 0.7 µg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.

Metabolic requirements associated with GSH synthesis during in vitro maturation of cattle oocytes.

Glutathione (GSH) concentration increases in bovine oocytes during in vitro maturation (IVM). The constitutive amino acids involved in GSH synthesis are glycine (Gly), glutamate (Glu) and cysteine (Cys). The present study was conducted to investigate the effect of the availability of glucose, Cys, Gly and Glu on GSH synthesis during IVM. The effect of the amino acid serine (Ser) on intracellular reduced/oxidized glutathione (GSH/GSSG) content in both oocytes and cumulus cells was also studied. Cumulus-oocyte complexes (COC) of cattle obtained from ovaries collected from an abattoir were matured in synthetic oviduct fluid (SOF) medium containing 8 mg/ml bovine serum albumin-fatty acid-free (BSA-FAF), 10 microg/ml LH, 1 microg/ml porcine FSH (pFSH) and 1 microg/ml 17 beta-estradiol (17beta-E2). GSH/GSSG content was measured using a double-beam spectrophotometer. The COC were cultured in SOF supplemented with 1.5mM or 5.6mM glucose (Exp. 1); with or without Cys+Glu+Gly (Exp. 2); with the omission of one constitutive GSH amino acid (Exp. 3); with 0.6mM Cys or Cys+Ser (Exp. 4). The developmental capacity of oocytes matured in IVM medium supplemented with Cys and the cell number per blastocyst were determined (Exp. 5). The results reported here indicate (1) no differences in the intracellular GSH/GSSG content at any glucose concentrations. Also, cumulus cell number per COC did not differ either before or after IVM (Exp. 1). (2) Glutathione content in oocytes matured in SOF alone were significantly different from oocytes incubated with SOF supplemented with Cys+Glu+Gly (Exp. 2). (3) Addition of Cys to maturation medium, either with or without Gly and Glu supplementation resulted in an increase of GSH/GSSG content. However, when Cys was omitted from the IVM medium intracellular GSH in oocytes or cumulus cells was less but not significantly altered compared to SOF alone (Exp. 3). (4) Glutathione content in both oocytes and cumulus cells was significantly reduced by incubation with 5mM Ser (Exp.4). (5) There was a significant increase in cleavage and blastocyst rates when Cys was added to maturation medium. In contrast, the cleavage, morula and blastocyst rates were significantly different when 5mM Ser was added to maturation media. There was also a significant difference in mean cell number per blastocyst, obtained from oocytes matured with 5mM Ser (Exp. 5). This study provides evidence that optimal embryo development in vitro is partially dependent on the presence of precursor amino acids for intracellular GSH production. Moreover, the availability of Cys might be a critical factor for GSH synthesis during IVM in cattle oocytes. Greater Ser concentration in IVM medium altered "normal" intracellular GSH in both oocytes and cumulus cells with negative consequences for subsequent developmental capacity.

Evaluation of angiogenesis with the expression of VEGF and CD34 in human non-small cell lung cancer.

Angiogenesis is an essential process in the progression of malignant tumors and the most potent angiogenic factor is the vascular endothelial growth factor (VEGF). On the other hand, the CD34 is an endothelial antigen that has been used to highlight the microvasculature vessel density (MVD) as a direct marker of the degree of neoangiogenesis. In the present study we report the VEGF expression and its relationship with MVD, measured by CD34, in two lineages of non-small cell lung cancer (NSCL): low differentiated adenocarcinomas and epidermoid carcinomas, in order to consider the possibility of using the correlation between both antibodies as a prognostic factor. Tumor sections were stained by immunohistochemistry for CD34 and VEGF. The results showed that the mean value of VEGF for adenocarcinoma was significantly higher than the one for epidermoid carcinoma (p < 0.001). However, the mean of MVD did not show significant differences between both types of tumors. The conventional factors taken into consideration (age over 60, sex, and presence of lymph nodes) was not significantly related to the angiogenic factors examined. In conclusion, we could affirm that CD34 is a better prognostic marker of neoangiogenesis in NSCLC, because both types of tumors have the same clinical prognosis, and so we expected the same behaviour from both markers.

DNA synthesis in tongue keratinocytes of hepatectomized and tumor-bearing mice.

Tongue keratinocytes have high S-phase and mitotic indices with evident circadian variation. Transplanted tumors modify the intensity and temporal structure of the S-phase index in cell populations in tumor-bearing animals; also, partial hepatectomy changes the concentrations of substances involved in cellular proliferation, leading to compensatory liver hyperplasia. The aim of our study was to analyze the interaction between tumor growth and the liver regeneration that follows partial hepatectomy, and the effects of both these processes on lingual keratinocytes. We used 380 adult male mice divided into six groups: tumor-free and tumor-bearing mice without surgery, with sham hepatectomy, and with partial hepatectomy. Each group was divided into six subgroups, which were killed at 4-h intervals until a circadian cycle was completed (from 26 until 50h post-surgery in the operated animals). Each animal was injected with 5-bromodeoxyuridine (50mg/kg) 1h before it was killed, and tongue samples were obtained and processed for histology. The sections were placed on silanized slides and incubated with the primary antibody Bu 20a (1/100 dilution). The reaction was developed using diaminobenzidine and staining was detected visually. SIs were measured as the number of labeled nuclei per thousand cells. The mean+/-S.E. of each group was calculated. Differences among experimental groups were analyzed by ANOVA and the Student-Newman-Keuls Multiple Comparisons Test. The results show that the presence of a tumor alters the normal circadian curve of SI in lingual keratinocytes, irrespective of whether the mice underwent surgery. This finding has to be considered in drug treatments for neoplasms and in experiments related to growth.

Chronobiology of the proliferative events related to angiogenesis in mice liver regeneration after partial hepatectomy.

In liver regeneration the formation of new capillary blood vessels is a fundamental requirement for cellular proliferation. Vascular endothelial growth factor (VEGF) is involved in the events of angiogenesis, the mRNA of which is expressed in both hepatocytes and non-parenchymal cells. In this experimental design we try to establish if during liver regeneration in mouse, the expression of VEGF is produced before or after the hepatocytes proliferation. C3H/S adult male mice were divided in three groups in order to study: VEGF expression; S-phase index (SI); and mitotic activity (MA) of hepatocytes. The results that were analyzed by ANOVA, show that VEGF expression starts to increase 26 h after PH with a peak at 28 h. Furthermore, the DNA synthesis (DNAs) reaches maximal level 42 h after pH, meanwhile the MA of the hepatocytes shows an increase 8h after the DNAs peak. In conclusion, it could be argued that the chronobiology of the events related to liver regeneration in mice started with a release of VEGF by the hepatocytes, followed by its DNAs and mitosis.

Effect of normal and tumor factors on different phases of cell populations cycle.

In the present experiments we studied the effect of extracts from intact liver (LE), ES2 tumor extract (TE), plasmas from intact mice (PI), and from tumor bearing animals (PT) on different phases of hepatocytes and renocytes cell cycles. C3HS 28-day-old male mice, standardized for periodicity analysis, were injected at 16:00 hours and killed every 4 hours during a circadian cycle at 20:00/04; 00:00/08; 04:00/12; 08:00/16; 12:00/20 and 16:00/24 (time of day/hours post treatment). Colchicine (2 microg/g) was injected 4 hours before killing them. Samples of livers and kidneys were processed for histology and mitotic index determinations. The results were expressed as colchicine arrested metaphases per 1000 nuclei. The TE, LE and PI had a promoting effect on the mitotic activity of hepatocytes during the first 12 hours post treatment. During the subsequent 12 hours, not only these treatments but also the PI had an inhibiting effect on the mitotic activity of the same cell population. Also the TE and the PT had a promoting effect on the mitotic activity of the renocytes during the first 12 hours while the effect of all treatments showed a clear inhibition of the mitotic activity studied during the last 12 hours. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.

G2 restriction point within populations of hepatocytes and tongue keratinocytes in young, growing mice.

The authors studied the effect of either extracts from liver (LE) or the malignant tumour ES2 (TE) or plasma from intact mice (PI) or tumour-bearing animals (PT) on the mitotic activity of the hepatocytes and tongue keratinocytes in young, growing C3H/s male mice (28+/-1 days old). Animals standardized for periodicity analysis were injected intraperitoneally with either TE, LE, PI, PT, or saline (S) at 16:00 h with 0.01 ml of sample/g of body weight and were then killed at (time of day/h post-injection) 20:00/04, 00:00/08, and 04:00/12. Colchicine (2 microg/g) was injected 4 h before death. Samples of the liver and tongue from each animal were processed for histology and assessment of mitotic index. The results were expressed as colchicine-arrested metaphases/1000 nuclei. The TE and LE stimulated the mitotic activity of hepatocytes and tongue keratinocytes. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.

Effect of tissue and plasma factors on kidney proliferation.

Liver extract, plasma from intact mice, ES2 tumour extract and plasma from tumour bearing mice has an inhibiting effect on the mitotic activity of hepatocytes and duodenal enterocytes. In the present experiments, the effect of these treatments on the mitotic activity of renal tubular cells was studied. C3HS 28 day-old male mice, standardized for periodicity analysis were used. The determination of normal mitotic circadian curve of the renocytes was done. A second batch of mice were injected with 0.01 ml/gr of either liver extract, plasma from intact mice, ES2 tumour extract or plasma from tumour bearing mice, at 16:00 hours and controlled at 08:00, 12:00 and 16:00 hs during 2 consecutive days post treatment. Colchicine (2 micrograms/gr) was injected 4 hours before killing. Kidneys were processed for histology and mitotic index determinations. Results were expressed as colchicine metaphases per 1000 nuclei, and showed that mitotic activity values of treated animals were significantly lower than those of controls. In conclusion, mitotic activity inhibition of renocytes may be due to some non specific plasmatic and/or tissue factors.

An initial characterization of a mitostatic activity for hepatocytes and renocytes in extracts from adult mouse liver.

We have previously demonstrated that liver extract, when administered at 16:00 hr, inhibits the mitotic activity of hepatocytes and renocytes at 08:00, 12:00 and 16:00 hr. during the following 2 days. The experiments reported here were designed to achieve an initial characterization of this liver mitostatic activity. For these studies we used 28-days-old male C3HS mice injected with either saline, liver extract (LE), mitochondrial-pellet extract (MPE), or postmitochondrial supernatant (PMS) at a dose of 1.9 mg of protein/g body weight. Colchicine (2 microg/g) was injected 4 hrs. before sacrifice and the number of arrested metaphases within the hepatocyte and renocyte population was estimated. The mice treated with LE were killed at (time of day/hr. post injection) 16/08:00, 20/12:00 and 24/16:00; while those injected with MPE or PMS were sacrificed at 20/12:00. We found that both the LE and the PMS inhibited the mitotic activity of hepatocytes and renocytes, while MPE reduced the mitotic activity of the liver cells without affecting that of the renal population.