In situ histochemical localisation of alkaloids and acetogenins in the endosperm and embryonic axis of Annona macroprophyllata Donn. Sm. seeds during germination.

Currently, the Annonaceae family is characterised by the production of acetogenins (ACGs), and also by the biosynthesis of alkaloids, primarily benzylisoquinolines derived from tyrosine. The objective of this study was to confirm the presence of alkaloids and acetogenins in the idioblasts of the endosperm and the embryonic axis of A. macroprophyllata seeds in germination. The Dragendorff, Dittmar, Ellram, and Lugol reagents were used to test for alkaloids, and Kedde's reagent was used to determine the presence of acetogenins in fresh sections of the endosperm and embryonic axis of seeds after twelve days of germination. A positive reaction was observed for all the reagents, and the presence of alkaloids and acetogenins was confirmed in the idioblasts of the endosperm and those involved in the differentiation of the embryonic axis of the developing seedling. We concluded that the idioblasts store both metabolites, acetogenins and alkaloids. Beginning at differentiation, the idioblasts of the embryonic axis simultaneously biosynthesise acetogenins and alkaloids that are characteristic of the species during the development of the seedling. The method used here can be applied to histochemically confirm the presence of acetogenins and alkaloids in tissues and structures of the plant in different stages of its life cycle.

Histochemical detection of acetogenins and storage molecules in the endosperm of Annona macroprophyllata Donn Sm. seeds.

Acetogenins (ACGs) are bioactive compounds with cytotoxic properties in different cell lines. They are antitumoural, antiparasitic, antimalarial, insecticidal, antimicrobial, antifungal and antibacterial. These secondary metabolites function in plant defence and are found in specific organelles and specific cells, thereby preventing toxicity to the plant itself and permitting site-specific defence. The aim of this work was to histochemically determine the in situ localisation of ACGs in the endosperm of Annona macroprophyllata seeds using Kedde's reagent. Additionally, the colocalisation of ACGs with other storage molecules was analysed. The seeds were analysed after 6 and 10 days of imbibition, when 1 or 2 cm of the radicle had emerged and metabolism was fully established. The seeds were then transversally cut in half at the midline and processed using different histological and histochemical techniques. Positive reactions with Kedde's reagent were only observed in fresh, unfixed sections that were preserved in water, and staining was found only in the large cells (the idioblasts) at the periphery of the endosperm. The ACGs' positive reaction with Sudan III corroborated their lipid nature. Paraffin sections stained with Naphthol Blue Black showed reactions in the endosperm parenchyma cells and stained the proteoplasts blue, indicating that they might correspond to storage sites for albumin-like proteins. Lugol's iodine, which is similar in chemical composition to Wagner's reagent, caused a golden brown reaction product in the cytoplasm of the idioblasts, which may indicate the presence of alkaloids. Based on these results, we propose that Kedde's reagent is an appropriate histochemical stain for detecting ACGs in situ in idioblasts and that idioblasts store ACGs and probably alkaloids. ACGs that are located in idioblasts found in restricted, peripheral areas of the endosperm could serve as a barrier that protects the seeds against insects and pathogen attack.

Liriodenine alkaloid in Annona diversifolia during early development.

Plants of the Annonaceae family produce a series of alkaloids, including liriodenine oxoaporphine. Its distribution in these primitive angiosperms suggests that it plays an important role, but very little is known about which plant organs it accumulates in, or in which developmental stages it is synthesised. Accordingly, liriodenine production was studied during the early stages of germination and seedling development in Annona diversifolia Saff. Liriodenine samples were obtained from the roots and were characterised on the basis of spectroscopic data. Quantification was done by HPLC in the organs and tissues of newly collected seeds, seeds following 1-, 2-, 5- and 10-day imbibitions, upon emergence of the radicle and at the seedling stage. According to our results, liriodenine could not have originated from the parent plant, nor during embryogenesis because it appears for the first time in the endosperm approximately 5 days after the start of imbibition. Therefore, its synthesis does not depend directly on photosynthesis. During the seedling stage it is found in the root and the stem but it is absent from the cotyledonary leaves and the first true leaves. Liriodenine biosynthesis begins during the early stages of development in the endosperm and seed radicles.

Is it possible to increase pCR in the neoadjuvant treatment with a dose-dense/sequential combination?: results from a phase II Trial combining epirubicin and cyclophosphamide followed by paclitaxel and gemcitabine ± trastuzumab in stage II and III breast cancer patients.

PURPOSE: To evaluate the pathologic complete response (pCR) rate of a combination of epirubicin (E) and cyclophosphamide (C) followed by paclitaxel (P) and gemcitabine (G) (+ trastuzumab[T]) in Her2+ patients) in a sequential and dose-dense schedule as neoadjuvant chemotherapy for stages II and III patients with breast cancer. Secondary endpoints: clinical response rate, disease free survival, safety and correlation between pCR and biologic markers. PATIENTS AND METHODS: Eligible patients were treated with E (90 mg/m²) and C (600 mg/m²) for 3 cycles (first sequence) followed by P (150 mg/m²) and G (2500 mg/m²) (second sequence) for 6 cycles. All drugs were administered on day 1, every 2 weeks, with prophylactic growth factor support. Weekly T (2 mg/kg [4 mg/kg first infusion]) was administered concomitantly with P and G in Her2+ patients. A core biopsy was performed before treatment for biologic markers assessment. Patients underwent surgery, radiotherapy, and adjuvant hormonal therapy according to institutional practice. RESULTS: Seventy-three patients were treated. A pCR was achieved in 27 (37%) patients (32.1%, Her2- and 50%, Her2+). pCR was significantly higher in tumors that were hormonal receptor negative, poorly differentiated and positive for Ki67 and p53. Breast-conserving surgery was performed in 47 patients (64.4%). Most frequent grade 3/4 hematologic and nonhematological toxicities included neutropenia (12%), nausea/vomiting (17%), and transient liver enzymes elevation (7%). One patient suffered an asymptomatic and reversible decrease in left ventricular ejection fraction. CONCLUSIONS: These results show a highly effective regimen in terms of pCR with a good toxicity profile in the neoadjuvant treatment of patients with breast cancer. The addition of trastuzumab increased pCR rate in Her2+ tumors.

Frecuencia relativa de Boophilus microplus (Acari: Ixodidae) en bovinos (Bos taurus y B. indicus) en ocho zonas ecológicas de Costa Rica / Relative frequency of Boophilus microplus (Acari: Ixodidae) in bovines (Bos taurus and B. indicus) in 8 ecological zones of Costa Rica

The research on the relative abundancy of Boophilus microplus took place in ten farms, distributed in eight ecological zones (EZ) and two rainfall systems of Costa Rica. Monthly visits were made to pick up ticks larger than 4 mm from the right side of the animals. The total number of ticks was increased by two and divided between the total of sampled bovines per farm. Farm visits were adjusted to animals bath to allow the recovery of the tick population. B. microplus was detected in all sampled farms throughout the year. Media comparison statistic analyses were made and no differences were found among EZ, farms and seasons. On the contrary, there were important statistical differences between rainfall systems. The rainfall system 1, that included rain seasonality, showed larger tick infestations (p = 0.03). An isothermal and low variability regarding relative humidity situations were present in some of the areas included in this research. Bath intensity was lower to other past studies. Even though no differences were found between production systems, it is important to emphasize that bath intensity was higher in european cattle farms, in comparison to others of the same EZ, but of crossbred. A tick manual removable system was recommended in dual-purpose and specialized dairy farms with no more than 30-35 animals, as an alternative feasible system that significantly reduced acaricide use.

Abundancia relativa de Amblyomma spp. (Acari: Ixodidae) en bovinos (Bos taurus y B. indicus) de Costa Rica / Relative abundance of Amblyomma spp. (Acari: Ixodidae) in bovines (Bos taurus and B. indicus) from Costa Rica

The research describe the big amount of ticks of the Amblyomma genus, found on bovines through monthly samplings carried out in ten farms in eight ecological zones (EZ) of Costa Rica. Ticks larger than 4 mm were picked up from the right side of the animals during the visit. The study compiled meteorological information for some farms located in the experiment, showing that the most fluctuant variable is rainfall. The most important Amblyomma species found was A. cajennense. Amblyomma nymphs were found only from January to May, which coincides with the lower humidity season in the rain seasonality area; as for it is expected only one generation per year. In the lab work Amblyomma nymphs are kept to measure the moulting season and the surviving time under controlled conditions, but no major differences were found between both sexes. The surviving periods show that it is not possible to do a grazing land handling, in order to control this genus species. Adults of the genus Amblyomma are present through all the year, not showing any specific preference for a season. The research divided the investigation areas in rain seasonability and not-seasonality systems. The highest amount of Amblyomma is found given in the rain seasonality system or of pacific influence. A. maculatum is present only in the EZ of Tropical Humid Forest transition to pre-mountainous. Likewise, Ixodes boliviensis is found in the EZ of low mountainous Very Humid Forest.

[Relative frequency of Boophilus microplus (Acari: Ixodidae) in bovines (Bos taurus and B. indicus) in 8 ecological zones of Costa Rica]. / Frecuencia relativa de Boophilus microplus (Acari: Ixodidae) en bovinos (Bos taurus y B. indicus) en ocho zonas ecológicas de Costa Rica.

The research on the relative abundancy of Boophilus microplus took place in ten farms, distributed in eight ecological zones (EZ) and two rainfall systems of Costa Rica. Monthly visits were made to pick up ticks larger than 4 mm from the right side of the animals. The total number of ticks was increased by two and divided between the total of sampled bovines per farm. Farm visits were adjusted to animals bath to allow the recovery of the tick population. B. microplus was detected in all sampled farms throughout the year. Media comparison statistic analyses were made and no differences were found among EZ, farms and seasons. On the contrary, there were important statistical differences between rainfall systems. The rainfall system 1, that included rain seasonality, showed larger tick infestations (p = 0.03). An isothermal and low variability regarding relative humidity situations were present in some of the areas included in this research. Bath intensity was lower to other past studies. Even though no differences were found between production systems, it is important to emphasize that bath intensity was higher in european cattle farms, in comparison to others of the same EZ, but of crossbred. A tick manual removable system was recommended in dual-purpose and specialized dairy farms with no more than 30-35 animals, as an alternative feasible system that significantly reduced acaricide use.

[Relative abundance of Amblyomma spp. (Acari: Ixodidae) in bovines (Bos taurus and B. indicus) from Costa Rica]. / Abundancia relativa de Amblyomma spp. (Acari: Ixodidae) en bovinos (Bos taurus y B. indicus) de Costa Rica.

The research describe the big amount of ticks of the Amblyomma genus, found on bovines through monthly samplings carried out in ten farms in eight ecological zones (EZ) of Costa Rica. Ticks larger than 4 mm were picked up from the right side of the animals during the visit. The study compiled meteorological information for some farms located in the experiment, showing that the most fluctuant variable is rainfall. The most important Amblyomma species found was A. cajennense. Amblyomma nymphs were found only from January to May, which coincides with the lower humidity season in the rain seasonality area; as for it is expected only one generation per year. In the lab work Amblyomma nymphs are kept to measure the moulting season and the surviving time under controlled conditions, but no major differences were found between both sexes. The surviving periods show that it is not possible to do a grazing land handling, in order to control this genus species. Adults of the genus Amblyomma are present through all the year, not showing any specific preference for a season. The research divided the investigation areas in rain seasonability and not-seasonality systems. The highest amount of Amblyomma is found given in the rain seasonality system or of pacific influence. A. maculatum is present only in the EZ of Tropical Humid Forest transition to pre-mountainous. Likewise, Ixodes boliviensis is found in the EZ of low mountainous Very Humid Forest.

Producción endógena de alcohol en pacientes con cirrosis hepática, alteración motora y sobrecrecimiento bacteriano / Endogenous ethanol production in patients with liver cirrhosis, motor alteration and bacterial overgrowth

BACKGROUND: Small intestinal bacterial overgrowth generates endogenous ethanol production both in experimental animals and humans. Patients with cirrhosis have small intestinal bacterial overgrowth, but endogenous ethanol production has not been studied in them. AIM: To investigate endogenous ethanol production in patients with cirrhosis, altered intestinal motility and small intestinal bacterial overgrowth. PATIENTS AND METHODS: Eight patients with cirrhosis of different etiologies and altered gastrointestinal motility, consisting in changes in the migrating motor complex, were studied. All had also small intestinal bacterial overgrowth, measured by means of the H2 breath test with lactulose. Plasma ethanol levels were measured by gas liquid chromatography in fasting conditions and 120 min after a carbohydrate rich meal. RESULTS: In fasting conditions, no patient had endogenous ethanol production. Alter the meal, ethanol in concentrations of 11.3 and 8.2 mg/del were detected in two patients. Negligible amounts of ethanol were detected in 4 patients and two patients had undetectable alcohol levels. CONCLUSIONS: A low endogenous production of ethanol was demonstrated in six of eight patients with cirrhosis.

p27KIP1 is abnormally expressed in Diffuse Large B-cell Lymphomas and is associated with an adverse clinical outcome.

Cell cycle progression is regulated by the combined action of cyclins, cyclin-dependent kinases (CDKs), and CDK-inhibitors (CDKi), which are negative cell cycle regulators. p27KIP1 is a CDKi key in cell cycle regulation, whose degradation is required for G1/S transition. In spite of the absence of p27KIP1 expression in proliferating lymphocytes, some aggressive B-cell lymphomas have been reported to show an anomalous p27KIP1 staining. We analysed p27KIP1 expression in a series of Diffuse Large B-cell Lymphoma (DLBCL), correlating it with the proliferative index and clinical outcome, to characterize the implications of this anomalous staining in lymphomagenesis in greater depth. For the above mentioned purposes, an immunohistochemical technique in paraffin-embedded tissues was employed, using commercially available antibodies, in a series of 133 patients with known clinical outcomes. Statistical analysis was performed in order to ascertain which clinical and molecular variables may influence outcome, in terms of disease-free survival (DFS) and overall survival (OS). The relationships between p27KIP1 and MIB-1 (Ki-67) were also tested. An abnormally high expression of p27KIP1 was found in lymphomas of this type. The overall correlation between p27KIP1 and MIB-1 showed there to be no significant relationship between these two parameters, this differing from observations in reactive lymphoid and other tissues. Analysis of the clinical relevance of these findings showed that a high level of p27KIP1 expression in this type of tumour is an adverse prognostic marker, in both univariate and multivariate analysis. These results show that there is abnormal p27KIP1 expression in DLBCL, with adverse clinical significance, suggesting that this anomalous p27KIP1 protein may be rendered non-functional through interaction with other cell cycle regulator proteins.

Clinical outcome in diffuse large B-cell lymphoma is dependent on the relationship between different cell-cycle regulator proteins.

PURPOSE: The goal of this work was to perform a comprehensive exploration of the relationship between the clinical outcome of diffuse large B-cell lymphoma (DLBCL) and the expression of a panel of tumor suppressor and oncogenic proteins, which includes some cell-cycle regulator proteins involved in the p53 pathway. PATIENTS AND METHODS: To this end, we collected the clinical data of 141 patients with DLBCL and immunohistochemically analyzed diagnostic tumoral tissue from each patient for the presence of Ki67 (MIB1, Immuno-tech, Marseille, France), bcl2, p53, p21/WAF1, MDM2, and retinoblastoma (Rb) proteins. RESULTS: The results show that several proteins are associated with some of the clinical traits analyzed. Multivariate analysis showed that an extended overall survival (OS) time was associated with low growth fraction, high Rb protein, and low MDM2 expression, as well as with known clinical parameters. The probability of inducing a complete remission (CR) was only associated with clinical parameters, although univariate study showed that a low growth fraction was associated with a higher probability of inducing a CR. Univariate study of disease-free survival (DFS) showed that tumors with high bcl2 expression and nodal origin have a shorter DFS time, although multivariate study only confirmed the adverse effect of bcl2 expression. CONCLUSION: Taking all these results into consideration, it seems that although the overall outcome for patients with DLBCL is decided by a combination of different clinical and biologic variables, the expression of some of these cell-cycle regulator proteins appears to be specifically associated with the different clinical features of tumors.

Treatment of advanced gastric cancer with the combination fluorouracil, leucovorin, etoposide, and cisplatin: a phase II study of the ONCOPAZ Cooperative Group.

A phase II study was performed to assess the efficacy and toxicity of the combination of 5-fluorouracil (5-FU), leucovorin (LV), etoposide, and cisplatin (FLEP) in patients with advanced gastric carcinoma. A total of 46 consecutive, previously untreated patients with unresectable, measurable gastric carcinoma were treated with 300 mg/m2 LV, 100 mg/m2 etoposide, 500 mg/m2 5-FU, and 30 mg/m2 cisplatin on days 1-3 every 28 days. All courses were given on an outpatient basis. A total of 169 courses of treatment were given. In all, 18 of the 46 patients (39%) had an objective response [95% confidence interval (CI), 25%-54%] and 2 (4%) patients experienced a clinical complete response. The median duration of response was 5 months. The main side effects were hematological and gastrointestinal. Grade 3-4 toxicity was encountered as follows: leukopenia, in 9.5% of the courses; anemia, in 3%; thrombocytopenia, in 3%; nausea/vomiting, in 4%; and diarrhea, in 5%. Hospitalization due to fever and granulocytopenia was required in 5 patients, 3 of whom died of sepsis. In conclusion, FLEP shows moderate activity in patients with advanced gastric carcinoma, albeit at the cost of a high degree of toxicity. For this reason we do not recommend its use.

Comparison of two antibiotic regimens (piperacillin plus amikacin versus ceftazidime plus amikacin) as empiric therapy for febrile neutropenic patients with cancer.

A total of 170 febrile episodes in neutropenic patients with cancer were randomly assigned to be treated with piperacillin-amikacin or ceftazidime-amikacin. The overall response rates were similar in both groups (68 and 65%, respectively). Response rates for clinically or microbiologically documented episodes were 54.5% for piperacillin-amikacin and 58.8% for ceftazidime-amikacin. Response rates for gram-negative bacillary infections were 65 and 73%, respectively. There was also no difference for gram-positive infections (31 and 50%, respectively). The toxicities were also comparable and consisted of skin rashes, hypokalemia, and diarrhea. Vancomycin was added if the fever persisted 72 h after the beginning of therapy; it increased the response rates to 94% when used with piperacillin-amikacin and 92% when used with ceftazidime plus amikacin. Our results suggest that the combinations show similar global efficacies in the treatment of febrile episodes in cancer patients.

[Comparison of homologous and heterologous standard curves using TR-FIA for the determination of total T4 in the rat]. / Comparación de rectas patrón homóloga y heteróloga mediante TR-FIA, para la determinación de T4 total en rata.

A homologous standard curve for rat T4 measurement has been compared to heterologous standard curves, by a time resolved fluoroimmunoassay (TR-FIA). Homologous standard curves have been carried out starting from rat serum in the absence of thyroid hormone by using Samuel's technique. The security criteria (sensitivity, inter and intraassay precision, and accuracy), were measured and compared between the homologous and heterologous test. Analysis of variance and slopes between homologous and heterologous standard curves showed homogeneous variances, the differences in the slopes being not significant.

Bone marrow examination in small cell lung cancer--when is it indicated?

To assess the usefulness of bone marrow (BM) examination performed as part of the staging procedure in SCLC, 137 patients with this neoplasm were studied. At diagnosis, BM was infiltrated in 23 patients (17%) and in 5 of them (22%) it was the only metastatic site. BM examination changed the previously given stage in 9% of the patients who were supposed to have limited disease before this test was performed. BM involvement correlated with a higher frequency of bone (p less than 0.05) and liver (p less than 0.01) metastasis. Correlations were found neither with blood or serum biochemistry disturbances nor with postchemotherapy haematologic toxicity. The BM positive patients had a survival rate similar to those with extensive disease but without BM infiltration. We conclude that BM examination should be performed only when no other metastatic sites have been found after a complete staging study.

[Genealogic evidence of genetic control in Down syndrome]. / Evidencia genealógica de control genético en el síndrome de Down.

The aim of this work is to reevaluate the hypothesis of the existence of a possible genetic control of nondisjunction of chromosome 21. The population under study was the families trisomy (TLI) as diagnosed in the Medical Genetic Unit of LUZ from 1971 to 1988. The control population was the families of 418 individuals with no apparent genetic pathology (CON), approximately matched in age with the patients. The frequency of consanguineous unions in parents (UCPM), maternal grandparents (UCAM) and paternal grandparents (UCAP) of both patients and controls was determined. The results indicated that UCPM was significantly greater in TLI than CON while the UCAM and UCAP were more frequent in CON. The coefficient of consanguinity in parents of TLI was three times greater than in parents of CON. The results of this investigation suggest that the consanguinity should be considered another risk factor for the nondisjunction of chromosome 21 and it is independent of maternal age and support the hypothesis that there exists an autosomal recessive gene that, in the homologous state, results in a predisposition toward nondisjunction of chromosome 21 in the first mitotic division.

[Usefulness of sialic acid as a tumor marker]. / Utilidad del ácido siálico como marcador tumoral.

In order to analyze the utility of sialic acid as a tumor mass marker, its levels have been determined in 253 neoplastic patients, 57 patients with non-tumoral pathology and 60 healthy controls. Significant differences in the sialic acid values were observed between the control group and the rest (p less than 0.001). When the neoplastic patients were studied according to the extension of their disease, we found that the SA levels in patients under complete remission were significantly lower than in the other stages (p less than 0.01) and that patients in stage IV presented the highest levels (p less than 0.05). Differences between stages I, II and III were not detected. Furthermore, no differences were observed regarding either the type of neoplasia or if the patients had or did not have tumor pathology. We conclude that sialic acid is a tumor marker with poor specificity that grossly relates with the tumor size.