The influence of the src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation

The src‐family kinases p56^lck (Lck) and p59^fyn (Fyn) are expressed in T cells and are among the first signaling molecules to be activated downstream of the T cell receptor (TCR). Evidence is emerging that although closely related, these signaling molecules have discrete functions during development, maintenance and activation of peripheral T cells. For example, during thymopoiesis Lck is uniquely able to provide all the signals required for pre‐TCRβ selection, although Fyn can substitute for a subset of these. Positive selection of CD4 single‐positive (SP) cells is also critically dependent on the expression of Lck but not Fyn, while differentiation of CD8 SP cells proceeds relatively efficiently in the absence of Lck. In naïve peripheral T cells either Lck or Fyn can transmit TCR‐mediated survival signals, and yet only Lck is able to trigger TCR‐mediated expansion signals under conditions of lymphopenia. Stimulation of naïve T cells by antigenic stimuli is also severely compromised in the absence of Lck, but more subtly impaired by the absence of Fyn. We discuss recent experiments addressing how these two src‐kinase family members interface with downstream signaling pathways to regulate these diverse aspects of T cell behavior.     

Effect of UDP-glucuronyltransferase induction on zearalenone metabolism

Experiments were conducted to determine the UDP-glucuronyltransferase (UDP-GT) isoenzyme which catalyzes zearalenone (Z) conjugation, and the effect of increased enzyme activity on Z metabolism. In competitive enzyme assays, the activity of rat liver UDP-GT towards Z was inhibited by 1-naphthol (NA), a GT1 substrate, and 4-hydroxybiphenyl (HB), a GT2 substrate. When enzyme activity was induced with either 3-methylcholanthrene (MC), a GT1 inducer, or phenobarbital (PB), a GT2 inducer, increased UDP-GT activity towards Z, NA and HB was observed. UDP-GT induction by PB increased urinary excretion of conjugated alpha-zearalenol. These results indicate that UDP-GT isoenzymes have overlapping substrate specificities, and that Z detoxification may be enhanced by UDP-GT enzyme induction, resulting in increased urinary excretion of conjugated alpha-zearalenol.     

Molecular and cytogenetic analysis of tumors in von Recklinghausen neurofibromatosis

Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder mapped to 17q11.2 and typically characterized by the occurrence of neural crest-derived tumors. The gene has recently been cloned using reverse genetics or "positional cloning" approaches. Its function, however, remains unknown. We have performed cytogenetic and molecular analyses on 9 malignant tumors from NF1 patients to look for loss of alleles or chromosome rearrangements involving chromosome 17 to test the hypothesis that the NF1 gene acts as a recessive "tumor suppressor" gene. Loss of alleles on this chromosome was detected for 3 of 9 malignant tumors. Two peripheral nerve sheath tumors showed allele loss at informative loci on both the long and short arms of chromosome 17. In contrast, a glioblastoma with focal gliosarcoma showed loss of heterozygosity on the short arm of chromosome 17 only, and not at loci on the long arm. One nerve sheath tumor was previously shown by direct sequence analysis to have a point mutation at the TP53 locus at 17p13. These data support a role for the TP53 gene or other genes on the short arm of chromosome 17 in at least some malignancies in NF1. Six other neurofibrosarcomas showed no allele loss at informative loci on chromosome 17. Cytogenetic analysis was performed on 7 tumors, including 2 with allele loss. The two tumors with allele loss showed abnormal karyotypes while all others were normal. Southern blot and pulsed-field gel analysis using probes within or closely linked to the NF1 locus detected no gross deletions or rearrangements in the tumors studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic prenatal aqueductal stenosis with hydrocephalus in rat

A recessive mutation which arose in Wistar albino rats was variably expressed in the homozygous state as prenatal stenosis of the aqueduct with resultant hydrocephalus. The condition was often compatible with survival to adulthood and with successful reproduction. Mildly sparse hair was the constant gene marker. Eye defects and sometimes foot deformities occurred. The first observable ultrastructural alteration was a disruption of the integrity of the neuroepithelial basal lamina in the cephalic neural tube of affected embryos as early as the 11th fetal day (16-24 somite pairs). The hydrocephalic syndrome closely resembled that produced by giving folic acid analogs to, or producing vitamin B12 deficiency in, pregnant rats in the period including the 11th day. Neither vitamin B12 nor folate, nor certain metabolites closely related to their metabolism, prevented the gene's expression. Homozygote mutants mated with homozygote mutants produced 70% hydrocephalic (dome-shaped heads) offspring, but if the mother was heterozygote, there was a "protective" effect and the number of hydrocephalic young was disproportionately smaller.     

The Transport Barrier of Epithelia: A Comparative Study on Membrane Permeability and Charge Selectivity in the Rabbit

The transport barrier of the epithelia presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal nasal bronchial tracheal > vaginal rectal > corneal > buccal > skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium. These results suggest that, for effective delivery purposes, permeation enhancing methods, by either increasing tissue permeability or modifying drug-membrane charge selectivity, are generally required. The permeation data also suggest that the respiratory epithelia represent good alternate routes for drug delivery, particularly for those that are orally ineffective, i.e., due to extensive gastrointestinal tract degradation or first-pass metabolism.     

HPV-16 viral load in oropharyngeal squamous cell carcinoma using digital PCR

Conclusions: We did not identify any strong associations between HPV-16 viral load and any of the clinical or lifestyle factors.

Objective: The epidemiology of oropharyngeal SCC is changing, with an increasing proportion of HPV-positive cases seen in the last decade. It is known that a high viral load is linked to the development of cervical cancer, the relation between viral load and oropharyngeal SCC is less clear. We sought to determine HPV-16 viral load in HPV-positive oropharyngeal SCCs using highly sensitive digital PCR and to identify clinical and lifestyle factors associated with viral load.

Subjects and methods: We analysed 45 HPV-16 positive oropharyngeal SCCs diagnosed between 2013 and 2015. All patients completed a lifestyle questionnaire and clinical data were extracted from medical charts. Viral load was determined using digital PCR assays for HPV-L1 and RNAseP.

Results: We found large variations in HPV-16 viral load from 1 to 930 copies per cell (median 34 copies per cell).     

Sex-Specific Effects of Chronic Creatine Supplementation on Hippocampal-Mediated Spatial Cognition in the 3xTg Mouse Model of Alzheimer’s Disease

The creatine (Cr) energy system has been implicated in Alzheimer’s disease (AD), including reductions in brain phosphoCr and Cr kinase, yet no studies have examined the neurobehavioral effects of Cr supplementation in AD, including the 3xTg mouse model. This studied investigated the effects of Cr supplementation on spatial cognition, plasticity- and disease-related protein levels, and mitochondrial function in the 3xTg hippocampus. Here, 3xTg mice were fed a control or Cr-supplemented (3% Cr (w/w)) diet for 8–9 weeks and tested in the Morris water maze. Mitochondrial oxygen consumption (Seahorse) and protein levels (Western blots) were measured in the hippocampus in subsets of mice. Overall, 3xTg females exhibited impaired memory as compared to males. In females, Cr supplementation decreased escape latency and was associated with increased spatial search strategy use. In males, Cr supplementation decreased the use of spatial search strategies. Pilot data indicated mitochondrial enhancements with Cr supplementation in both sexes. In females, Cr supplementation increased CREB phosphorylation and levels of IκB (NF-κB suppressor), CaMKII, PSD-95, and high-molecular-weight amyloid β (Aβ) species, whereas Aβ trimers were reduced. These data suggest a beneficial preventative effect of Cr supplementation in females and warrant caution against Cr supplementation in males in the AD-like brain.