Reduced viability of human vascular endothelial cells cultured on Matrigel™

Optimal vascular homeostasis requires efficient control of both proliferation and elimination of vascular endothelial cells. Programmed cell death, or apoptosis, is the main mechanism controlling cell elimination, and it is an essential component of vascular formation. Human vascular endothelial cells die in vitro, if prevented from obligatory survival factors like growth factors or attachment and cell spreading, but very little is known about the mechanisms controlling endothelial cell elimination. Signaling from the extracellular matrix affects the behavior and functions of human umbilical vein endothelial cells (HUVECs), and we have recently demonstrated the beneficial effects of plating on the reconstituted extracellular matrix Matrigel™, on the inducible nitric oxide production of freshly isolated HUVECs. In this work we observed that cultured HUVECs formed typical capillary-like structures on Matrigel, but unexpectedly, after 24–48 hours their viability was gradually lost. Viability was measured with an assay based on mitochondrial reduction of reagent XTT. No decrease in viability was seen in freshly isolated HUVECs or in cultured fibroblasts during this time. It is known that cells often turn into apoptosis if they receive conflicting information from their surroundings, and apparently signaling from Matrigel to HUVECs, while at their in vitro proliferating phenotype, resulted in launching of the apoptotic machinery. Thus, proliferating and differentiated phenotypes of endothelial cells seemed to have different sensitivity to signals that induce apoptosis. J. Cell. Physiol. 176:92–98, 1998. © 1998 Wiley-Liss, Inc.     

Inducible nitric oxide and prostacyclin productions are differently controlled by extracellular matrix and cell density in human vascular endothelial cells

Both cell-matrix and cell-cell interactions are important regulators of the function of most human cells. In this study we investigated how these interactions controlled the production of vasodilators nitric oxide (NO), and prostacyclin (PGI₂), in freshly isolated human umbilical vein endothelial cells (HUVECs). On the reconstituted extracellular matrix (ECM) Matrigel freshly isolated HUVECs treated with interleukin-1β, lipopolysaccharide, and interferon-γ, produced more NO, but less PGI₂, than on gelatin substratum. High cell density was essential for inducibility of NO production in cells plated on gelatin substratum, but not on ECM. In cells plated on gelatin substratum at low cell density, which mimicked conventional HUVEC culturing conditions, both inducible NO production and the inducible NO synthase (iNOS) mRNA levels, detected by competitive RT-PCR, were low. However, inducible PGI₂ production remained high in these cells. Highest inducible NO productions were observed in HUVECs that presumably had best maintained their original differentiated phenotype. Thus our data imply that the inducible NO and PGI₂ productions of freshly isolated HUVECs were differently controlled by the extracellular matrix and cell density. Our data suggest that both cell-matrix and cell-cell interactions may have a strong influence on the proinflammatory cytokine responses of human vascular endothelial cells. J. Cell. Biochem. 64:538–546. © 1997 Wiley-Liss, Inc.     

An efficient freeze-drying apparatus

Summary A freeze-drying apparatus with a practical temperature regulating system is described. Degassing, drying and embedding all take place in the drying chamber. The drying temperature can be set at a number of fixed temperature levels which may be chosen anywhere between -70° C and +60° C.     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

Indolyl-3-acetic acid from shoots of Salix pentandra

Indolyl-3-acetic acid from vegetative shoots of Salix pentandra L. has been isolated and identified by high performance liquid chromatography and combined gas chromatography-mass spectrometry.     

A new case of an Holarctic element in the Colombian Andes: first record of Cordyla Meigen (Diptera,Mycetophilidae) from the Neotropical region

Three new species of Mycetophilidae – Cordyla monticola sp. n., Cordyla pseudopusilla sp. n. and Cordyla reducta sp. n. – are described from the Colombian Andes, representing the first described species of Cordyla Meigen from the Neotropical region. Colour photos of their habitus, wing and terminalia are provided. The morphological affinities of male terminalia are discussed in a worldwide context. The distributional pattern of the genus clearly indicates a case of northern elements reaching the north-western region of the Neotropics that corresponds to a secondary extension of a Holarctic clade to the south.     

Cd-Specific Mutants of Mercury-Sensing Regulatory Protein MerR, Generated by Directed Evolution

The mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury resistance operons in Gram-negative bacteria, was subjected to directed evolution in an effort to generate a MerR mutant that responds to Cd but not Hg. Oligonucleotide-directed mutagenesis was used to introduce random mutations into the key metal-binding regions of MerR. The effects of these mutations were assessed using a vector in which MerR controlled the expression of green fluorescent protein (GFP) and luciferase via the mer operator/promoter. An Escherichia coli cell library was screened by fluorescence-activated cell sorting, using a fluorescence-based dual screening strategy that selected for MerR mutants that showed GFP repression when cells were induced with Hg but GFP activation in the presence of Cd. Two Cd-responsive MerR mutants with decreased responses toward Hg were identified through the first mutagenesis/selection round. These mutants were used for a second mutagenesis/selection round, which yielded eight Cd-specific mutants that had no significant response to Hg, Zn, or the other tested metal(loid)s. Seven of the eight Cd-specific MerR mutants showed repressor activities equal to that of wild-type (wt) MerR. These Cd-specific mutants harbored multiple mutations (12 to 22) in MerR, indicating that the alteration of metal specificity with maintenance of repressor function was due to the combined effect of many mutations rather than just a few amino acid changes. The amino acid changes were studied by alignment against the sequences of MerR and other metal-responsive MerR family proteins. The analysis indicated that the generated Cd-specific MerR mutants appear to be unique among the MerR family members characterized to date.     

Long Telomeres in Blood Leukocytes Are Associated with a High Risk of Ascending Aortic Aneurysm

Ascending aortic aneurysm is a connective tissue disorder. Even though multiple novel gene mutations have been identified, risk profiling and diagnosis before rupture still represent a challenge. There are studies demonstrating shorter telomere lengths in the blood leukocytes of abdominal aortic aneurysm patients. The aim of this study was to measure whether relative telomere lengths are changed in the blood leukocytes of ascending aortic aneurysm patients. We also studied the expression of telomerase in aortic tissue samples of ascending aortic aneurysms. Relative lengths of leukocyte telomeres were determined from blood samples of patients with ascending aortic aneurysms and compared with healthy controls. Telomerase expression, both at the level of mRNA and protein, was quantified from the aortic tissue samples. Mean relative telomere length was significantly longer in ascending aortic aneurysm blood samples compared with controls (T/S ratio 0.87 vs. 0.61, p<0.001). Expressions of telomerase mRNA and protein were elevated in the aortic aneurysm samples (p<0.05 and p<0.01). Our study reveals a significant difference in the mean length of blood leukocyte telomeres in ascending aortic aneurysm and controls. Furthermore, expression of telomerase, the main compensating factor for telomere loss, is elevated at both the mRNA and protein level in the samples of aneurysmal aorta. Further studies will be needed to confirm if this change in telomere length can serve as a tool for assessing the risk of ascending aortic aneurysm.     

Lkb1 regulates organogenesis and early oncogenesis along AMPK-dependent and -independent pathways

The tumor suppressor Lkb1/STK11/Par-4 is a key regulator of cellular energy, proliferation, and polarity, yet its mechanisms of action remain poorly defined. We generated mice harboring a mutant Lkb1 knockin allele that allows for rapid inhibition of Lkb1 kinase. Culturing embryonic tissues, we show that acute loss of kinase activity perturbs epithelial morphogenesis without affecting cell polarity. In pancreas, cystic structures developed rapidly after Lkb1 inhibition. In lung, inhibition resulted in cell-autonomous branching defects. Although the lung phenotype was rescued by an activator of the Lkb1 target adenosine monophosphate–activated kinase (AMPK), pancreatic cyst development was independent of AMPK signaling. Remarkably, the pancreatic phenotype evolved to resemble precancerous lesions, demonstrating that loss of Lkb1 was sufficient to drive the initial steps of carcinogenesis ex vivo. A similar phenotype was induced by expression of mutant K-Ras with p16/p19 deletion. Combining culture of embryonic tissues with genetic manipulation and chemical genetics thus provides a powerful approach to unraveling developmental programs and understanding cancer initiation.