kDNA minicircles of the major sequence class of C. fasciculata contain a single region of bent helix widely separated from the two origins of replication

The major sequence class of Crithidia fasciculata minicircles is shown to have a single region of bent helical DNA widely separated from the two replication origins located 180 degrees apart on the minicircle map. The position of the bend in the DNA has been mapped both by gel electrophoretic methods and by direct electron microscopic observation of the DNA. This sequence directed bending is apparently the result of homopolymeric dA X dT tracts 4-6 base pairs long repeated in phase with the helix screw. The region of the bend contains nineteen such homopolymeric tracts in a region of about 200 base pairs with sixteen of the tracts oriented in the same direction.     

Zip4 (Slc39a4) Expression is Activated in Hepatocellular Carcinomas and Functions to Repress Apoptosis,Enhance Cell Cycle and Increase Migration

Background

The zinc transporter ZIP4 (Slc39a4) is important for proper mammalian development and is an essential gene in mice. Recent studies suggest that this gene may also play a role in pancreatic cancer.

Methods/Principal Findings

Herein, we present evidence that this essential zinc transporter is expressed in hepatocellular carcinomas. Zip4 mRNA and protein were dramatically elevated in hepatocytes in the majority of human hepatocellular carcinomas relative to noncancerous surrounding tissues, as well as in hepatocytes in hepatocellular carcinomas occurring in farnesoid X receptor-knockout mice. Interestingly, meta-analysis of microarray data in the Geo and Oncomine databases suggests that Zip4 mRNA may also be elevated in many types of cancer. Potential mechanisms of action of ZIP4 were examined in cultured cell lines. RNAi knockdown of Zip4 in mouse Hepa cells significantly increased apoptosis and modestly slowed progression from G₀/G₁ to S phase when cells were released from hydroxyurea block into zinc-deficient medium. Cell migration assays revealed that RNAi knockdown of Zip4 in Hepa cells depressed in vitro migration whereas forced over-expression in Hepa cells and MCF-7 cells enhanced in vitro migration.

Conclusions

ZIP4 may play a role in the acquisition of zinc by hepatocellular carcinomas, and potentially many different cancerous cell-types, leading to repressed apoptosis, enhanced growth rate and enhanced invasive behavior.     

Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization

Spreading depolarization (SD) is a feed‐forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn²⁺ release and intracellular accumulation occurs with SD, and elevated intracellular Zn²⁺ ([Zn²⁺]_i) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn²⁺]_i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn²⁺]_i loading prior to SD, by transient ZnCl₂ application with the Zn²⁺ ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post‐synaptic potentials (fEPSPs) in a concentration‐dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn²⁺]_i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn²⁺ release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn²⁺]_i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn²⁺]_i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn²⁺]_i accumulation in a range of brain injury models.     

Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

A type II DNA topoisomerase (topollmt), purified to near homogeneity from the trypanosomatid C. fasciculata has been shown to be localized to the single mitochondrion of these kinetoplastid protozoa. Immunoblots show at least a 10-fold higher level of topollmt (per milligram of protein) in preparations of partially purified mitochondria as compared with those from whole cells. Analyses of type I and type II topoisomerase activities in both mitochondrial and whole cell extracts show a 4- to 5-fold higher specific activity of topollmt in mitochondrial extracts while a nuclear type I topoisomerase has a 4- to 5-fold lower specific activity in the same extract. Immunolocalizations using anti-topollmt antibodies show the enzyme to be present in close association with the mitochondrial DNA networks (kinetoplast DNA or kDNA). This association appears at two distinct locations on opposite sides of the kDNA network.     

LEUKEMIA AFTER RADIOIODINE THERAPY FOR HYPERTHYROIDISM

In a group of 650 patients with hyperthyroidism treated with radioiodine between 1945 and 1961, two cases of leukemia occurred. In one patient the diagnosis of chronic lymphatic leukemia was made three years after the I(131) therapy. Acute or subacute myelomonocytic leukemia developed in the other patient after an interval of 14 years. However, no conclusions can be drawn regarding a possible causal relationship between such therapy and the subsequent occurrence of leukemia. With passage of time, an ever increasing number of cases of leukemia can be expected in such patients on the basis of natural occurrence alone.     

THYROID PROLIFERATIVE POTENTIAL AS A FUNCTION OF AGE

The effect of a goitrogenic stimulus on thyroid weight and thyroid cell ³HTdR labeling of Sprague-Dawley rats varying from 2 to 40 weeks of age was determined. Propylthiouracil ad libitum in drinking water produced a spurt in follicle cell labeling index and thyroid weight evident after 24 hr for all age groups. The increase in labeling index reached a peak at 5–7 days and then decreased to a level a few times greater than that of the normal unstimulated thyroid. The tritiated thymidine labeling index for thyroid follicle cells and the effect of PTU thereon was determined for August male rats of 3 days to 12 weeks of age. In the older rats, the follicle cell labeling index rose to 5–6% after 4–5 days of PTU treatment and then slowly fell to about 1%, For the unstimulated control rat of comparable age, the labeling index was about 0.1%. At all ages the thyroid showed a rapid response to PTU. Examination of the time sequence of mitotic labeling showed that the DNA synthesis period was 7.5 hr for normal 2-week-old rats and for 10–12-week-old rats that had received PTU for 4 days. There was no second wave of labeled mitoses in either group during the 48-hr interval studied. From the curve of thyroid weight vs time on PTU and from the labeled mitoses curve, inferences regarding the minimum fraction of proliferating follicle cells in the stimulated ‘adult’rat thyroid were made.     

Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations

We examined the impact of intracellular dialysis on fluorescence detection of neuronal intracellular Zn²⁺ accumulation. Comparison between two dialysis conditions (standard; 20 min, brief; 2 min) by standard whole‐cell clamp revealed a high vulnerability of intracellular Zn²⁺ buffers to intracellular dialysis. Thus, low concentrations of zinc‐pyrithione generated robust responses in neurons with standard dialysis, but signals were smaller in neurons with short dialysis. Release from oxidation‐sensitive Zn²⁺ pools was reduced by standard dialysis, when compared with responses in neurons with brief dialysis. The dialysis effects were partly reversed by inclusion of recombinant metallothionein‐3 in the dialysis solution. These findings suggested that extensive dialysis could be exploited for selective detection of transmembrane Zn²⁺ influx. Different dialysis conditions were then used to probe responses to synaptic stimulation. Under standard dialysis conditions, synaptic stimuli generated significant FluoZin‐3 signals in wild‐type (WT) preparations, but responses were almost absent in preparations lacking vesicular Zn²⁺ (ZnT3‐KO). In contrast, under brief dialysis conditions, intracellular Zn²⁺ transients were very similar in WT and ZnT3‐KO preparations. This suggests that both intracellular release and transmembrane flux can contribute to intracellular Zn²⁺ accumulation after synaptic stimulation. These results demonstrate significant confounds and potential use of intracellular dialysis to investigate intracellular Zn²⁺ accumulation mechanisms.