Profound hypoglycemia in starved, ghrelin-deficient mice is caused by decreased gluconeogenesis and reversed by lactate or fatty acids

When mice are subjected to 7-day calorie restriction (40% of normal food intake), body fat disappears, but blood glucose is maintained as long as the animals produce ghrelin, an octanoylated peptide that stimulates growth hormone secretion. Mice can be rendered ghrelin-deficient by knock-out of the gene encoding either ghrelin O-acyltransferase, which attaches the required octanoate, or ghrelin itself. Calorie-restricted, fat-depleted ghrelin O-acyltransferase or ghrelin knock-out mice fail to show the normal increase in growth hormone and become profoundly hypoglycemic when fasted for 18-23 h. Glucose production in Goat(-/-) mice was reduced by 60% when compared with similarly treated WT mice. Plasma lactate and pyruvate were also low. Injection of lactate, pyruvate, alanine, or a fatty acid restored blood glucose in Goat(-/-) mice. Thus, when body fat is reduced by calorie restriction, ghrelin stimulates growth hormone secretion, which allows maintenance of glucose production, even when food intake is eliminated. In humans with anorexia nervosa or kwashiorkor, ghrelin and growth hormone are known to be elevated, just as they are in fat-depleted mice. We suggest that these two hormones prolong survival in starved humans as they do in mice.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

Differential regulation of Notch signal transduction in leukaemia and lymphoma cells in culture

The transduction of Notch signal plays an intricate role in cell differentiation and pathogenesis of haematological malignancies as well as in certain congenital conditions. We found no genomic changes in either gene in 34 leukaemic samples and 25 leukaemia and lymphoma cell lines. The functionality of Notch signalling was tested using HES1 gene activation. We show that Notch signalling is differentially regulated in T-acute lymphoblastic leukaemia (ALL) and B-lymphoma cells. The Notch pathway is intact in a majority of B-lymphoma cell lines, but EBNA2, which mimics notch function, can occasionally activate the pathway. In contrast, the Notch pathway is constitutively active in T-ALL. This is the first demonstration of a distinction between B-lymphomas and T-cell leukaemias in the functioning of the Notch-signalling pathway. This might be related to their pathogenesis.     

Weekly vs. tri-weekly cisplatin based chemoradiation in carcinoma cervix: a prospective randomized study of toxicity and compliance

BackgroundAddition of chemotherapy to radiation has improved 5-year survival by 6%. However, the optimal dose and schedule of concurrent cisplatin is not well defined, though widely accepted practice is the weekly schedule of 40 mg/m² for 5 weeks. Repeated admissions for weekly cisplatin drain the limited resources in high volume centres. We intended to study the compliance and toxicity of two cisplatin schedules in our patients diagnosed with carcinoma cervix.Materials and methodsBetween 2007–2011, 212 patients, histologically proven squamous cell carcinoma with stages IIB to IIIB were randomized into two arms. All patients were planned for external beam radiotherapy 45 Gy/25 frs over 5 weeks followed by Intracavitary or Interstitial brachytherapy to a total BED dose of 75–85 Gy. Single agent cisplatin given concomitantly, was scheduled weekly (40 mg/m²/cycle, 5 cycles) in an arm A and three weekly (100 mg/m²/cycle, 2 cycles) in an arm B. Toxicity and compliance were evaluated weekly according to the RTOG guidelines. Analysis of the compiled data was done using SSPS version 20.ResultsOf the evaluable 212, 109 patients received weekly cisplatin chemotherapy and 103 patients received three weekly cisplatin. The most common acute toxicity observed was grade I–II leucopoenia. The upper and lower gastrointestinal reactions were high in three weekly arms, which was statistically significant (57% and 42.7%, p < 0.05). Proctitis was observed in 10% of patients in both of the arms and only two patients had Gr1 Cystitis after 6 months of treatment.ConclusionsTri-weekly cisplatin based concurrent chemoradiation can be adopted in high volume centres with manageable haematological and gastrointestinal acute toxicities.     

Genetic diversity and relationships in mulberry (genusMorus) as revealed by RAPD and ISSR marker assays

Background  

The genus Morus, known as mulberry, is a dioecious and cross-pollinating plant that is the sole food for the domesticated silkworm, Bombyx mori. Traditional methods using morphological traits for classification are largely unsuccessful in establishing the diversity and relationships among different mulberry species because of environmental influence on traits of interest. As a more robust alternative, PCR based marker assays including RAPD and ISSR were employed to study the genetic diversity and interrelationships among twelve domesticated and three wild mulberry species.     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

Characterisation of Escherichia coli cell surface by isoelectric equilibrium analysis

A characterisation of the lipopolysaccharide (outermost) layer of Escherichia coli cells has been made by isoelectric equilibrium analysis. Unmodified E. coli cells show a surface isoelectric point (pI) of 5.6. Cells treated with ethyleneimine in order to esterify the carboxyl groups are isoelectric at pH 8.55. When amino groups are blocked the bacterial surface has a pI of 3.85. An analysis of these results suggests that the ionisable groups occurring in the isoelectric zone i.e. the zone amenable to investigation by the isoelectric equilibrium method are: carboxyl groups and amino groups of polysaccharide and protein components. The carboxyl groups have a pK between 3.2 and 4.5 and the amino groups have a pK of 7.5. ε-Amino groups, phenolic hydroxyl groups and guanidyl groups do not occur, and phosphate and amino groups of the phospholipid complex are not detected. The number of thiol groups in the isoelectric zone has been determined using 6,6′-dithiodinicotinic acid. The number of anionogenic and cationogenic groups has been determined. From the density of the negative charges on the surface it is estimated that the isoelectric zone might extend up to 60 Å below the cell surface. The data discussed in this paper relate to the outermost layer of the bacterial cell wall composed of lipopolysaccharide-phospholipid-protein complex. Since reactive groups of the phosphilipid component of the complex have not been detected in the isoelectric zone, it is suggested that the arrangement of lipopolysaccharide phospholipid protein complex is such that the phospholipids are located at a depth of more than 60 Å from the bacterial surface.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Characterisation of ionogenic groups and estimation of the net negative electric charge on the surface of cells using natural pH gradients

The technique of isoelectric focusing has been extended to the study of the cell surface. A few tumour cell types and normal liver cells have been examined and are found to have characteristic isoelectric points. The isoelectric point of a cell, it is shown, provides information about the ionogenic groups present on its surface. The net electric charge borne by cells at their isoelectric points can be used to predict their electrophoretic mobilities in buffers at physiological pH and ionic strengths.