Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.     

The SUMO isopeptidase Ulp2p is required to prevent recombination-induced chromosome segregation lethality following DNA replication stress

SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress.     

Electric activity of the testicular tunica albuginea during ejaculation: a canine study

We have shown in previous studies that electric waves at rest could be recorded from the testicle and originate from the tunica albuginea (TA) and not from the testicular tissue. In the current study, we investigated the hypothesis that the electric activity of the TA increases during ejaculation. Three electrodes were sutured to the TAs of 11 anesthetized male dogs. The slow waves were recorded at rest and on inducing ejaculation by an ejaculator applied to the glans penis. Basal electric waves were recorded from the testicle. Each wave consisted of a negative followed by a positive deflection with a mean frequency of 6.2 +/- 1.3 cycles/min, an amplitude of 0.59 +/- 0.06 mV, and a conduction velocity of 5.2 +/- 0.8 cm/sec. These wave variables showed a significant intermittent increase (P < 0.05) at intervals of 0.6-1.0 secs and occurred simultaneously with the bouts of ejaculation. The increase remained for 0.8-1.2 secs at each ejaculation bout. The number of bouts of increased electric waves varied from 3 to 5. Apparently, the TA is not an inert covering to the testicle, but it seems to have a functional activity. Recording resting electric waves of the TA presumably denotes that the TA possesses a resting tone that appears to support the testicular tissue. During ejaculation, the increased electric activity of the TA, which coincides with semen spurt episodes, presumably denotes TA contraction. The intermittent TA contractions seem to assist in massaging the testicular secretions to the epididymis and the vas deferens and augment testicular circulation. The effect of pathologic conditions of the TA on ejaculation needs to be studied.     

Dominant negative effects of a Gbeta mutant on G-protein coupled inward rectifier K+ channel

HEK293 cells were transfected with cDNAs for Gbeta1(W332A) [a mutant Gbeta1], Ggamma2, and inward rectifier K+ channels (Kir3.1/Kir3.2). Application of Gbeta1gamma2 protein to these cells activated the K+ channels only slightly. When mu-opioid receptors and Kir3.1/Kir3.2 were transfected, application of a mu-opioid agonist induced a Kir3 current. However, co-expression of Gbeta1(W332A) suppressed this current. Most likely, Gbeta1(W332A) inhibited the action of the endogenous Gbeta. Such a dominant negative effect of Gbeta1(W332A) was also observed in neuronal Kir3 channels in locus coeruleus. The mutant, Gbeta1(W332A) protein, although inactive, retains its ability to bind Kir3 and prevents the wild type Gbeta from activating the channel.     

Chloroplast position and photosynthetic characteristics in two monostromatic species,Monostroma angicava and Protomonostroma undulatum (Ulvophyceae), having a shared ecological niche

Monostroma angicava and Protomonostroma undulatum are monostromatic green benthic algae (Ulvophyceae), which grow together in the same intertidal habitat of Muroran, Hokkaido, Japan, during the spring season. Commonly, both species have a single chloroplast with one pyrenoid per cell. The parietal chloroplast is located on the periphery of the thallus in both species, although the location of the chloroplast differs in the two. In M. angicava , the chloroplast was observed to be arranged on one‐side of the thallus surface, whereas, in P. undulatum , it was dispersed and randomly located on either side of the thallus or on the lateral face. The density of chlorophylls (Chls) assessed from the absorption spectra of the thallus and its solvent extract was higher in M. angicava , which appeared dark‐green in color, than in the light‐green colored P. undulatum . The maximum photosynthetic rate per thallus area (μmol O₂ m^?2 s^?1) was higher in M. angicava , whereas, per total chlorophyll content (μmol O₂ g Chl a + Chl b ^?1 s^?1) was higher in P. undulatum . Both species showed similar efficiency of photosynthesis at light‐limiting conditions. The efficiency of light absorption by photosystem II (PSII ) in P. undulatum was higher than M. angicava , whereas the photoprotective response was higher in M. angicava . This indicates that more energy is utilized in M. angicava to protect its PSII due to the chloroplast position, which has more direct exposure to light and, therefore, lowers the efficiency of light absorption by PSII . The higher density of chlorophylls in M. angicava could explain higher photosynthesis per thallus area, whereas, higher efficiency of light absorption by PSII in P. undulatum could explain higher photosynthesis per total chlorophyll content. The differences in light absorption efficiency and quantum efficiency of PSII might be an important ecological strategy in these two species for their coexistence in the intertidal area.     

Soluble fms-Like tyrosine kinase 1 (sFlt1), endoglin and placental growth factor (PlGF) in preeclampsia among high risk pregnancies

Background

Differences in circulating concentrations of antiangiogenic factors sFlt1 and soluble endoglin (sEng) and the pro-angiogenic growth factor PlGF are reported to precede the onset of preeclampsia weeks to months in low-risk pregnant women. The objective of this study was to investigate whether similar changes can be detected in pregnant women at high-risk to develop the syndrome.

Methods

This study is a secondary analysis of the NICHD MFMU trial of aspirin to prevent preeclampsia in high-risk pregnancies. Serum samples were available from 194 women with pre-existing diabetes, 313 with chronic hypertension, 234 with multifetal gestation, and 252 with a history of preeclampsia in a previous pregnancy. Samples collected across pregnancy were analyzed in a blinded fashion for sFlt1, sEng and PlGF.

Results

The odds of developing preeclampsia were significantly increased among women with multiple fetuses for each 2-fold elevation in sFlt1, sEng and the ratio of angiogenic factors (e.g. OR 2.18, 95% CI 1.46-3.32), and significantly decreased for each 2-fold elevation in circulating PlGF (OR 0.50, 95% CI 0.30-0.82) between 7 and 26 weeks'' gestation. Cross-sectional analysis of the angiogenic factors across gestation showed significant differences during the third trimester in women who develop preeclampsia compared with appropriate controls in all high-risk groups. However, when data were examined in relation to the gestational week when preeclampsia was diagnosed only sFlt1 was significantly higher 2 to 5 weeks before the clinical onset of preeclampsia and only in women with previous preeclampsia.

Conclusions

The pattern of elevated concentrations of sFlt1 and sEng, and low PlGF in high-risk pregnant subjects who develop preeclampsia is similar to that reported in low-risk pregnant women. However, differences in these factors among high-risk women who do and do not develop preeclampsia are modest, and do not appear to be clinically useful predictors in these high-risk pregnant women.