Identification of crystalline material found in the thallus of the lichen, Myelochroa leucotyliza

Lichens are symbiotic associations of fungi and algae (or cyanobacteria). Fungal cells produce large amounts of lipid, assisted by algae, and secrete them out of the cells. Some of the secreted lipids crystallize in the thallus of the lichen. The crystalline materials sometimes occupy 30% of total dry weight of the thallus. This unusual amount of lipid crystal led to our interest in investigating the mechanism of lipid secretion. To begin the cell biological study of lipid secretion and to know the significance of the existence of such crystals in the thallus, it is essential to identify the crystal. The lipid crystal extracted from the thallus of a lichen, Myelochroa leucotyliza, was studied by EM observations, TLC analysis, and EM and X-ray diffraction methods. Atranorin is the predominant component of the crystalline materials in the lipids extracted.     

Differences in intramembrane particle distribution in young and old human erythrocytes

The distribution of intramembrane particles in human erythrocytes was studied by freeze-fracture on young and old cells and compared to that obtained after ATP depletion or following addition of a clustering agent. It was shown that intramembrane particles became aggregated and the mean particle density increased as the cells aged. Likewise, both particle aggregation and increased density were found in young cells after moderate ATP depletion. In contrast, mean particle density was markedly reduced in both cell types after exhaustive depletion. Paradoxically, Zn treatment led to decreased particle density in young cells, whilst producing the opposite effect in aged cells. The results suggest that their low ATP content may account for the increased particle density of senescent cells.     

Late type of daughter cell wall synthesis in one of the Chlorellaceae, <Emphasis Type="Italic">Parachlorella kessleri</Emphasis> (Chlorophyta,Trebouxiophyceae)

Autosporulation is a common mode of propagation for unicellular algae. Autospore-forming species of Chlorellaceae, Chlorella vulgaris Beijerinck, C. sorokiniana Shihira et Krauss, C. lobophora Andreyeva, and Parachlorella kessleri (Fott et Nováková) Krienitz et al. have glucosamine as the main constituent of their rigid cell wall. Recent phylogenetic analyses have showed that the Chlorellaceae divided into two sister groups: the Chlorella-clade and the Parachlorella-clade. We compared the cell wall structure and synthesis of the daughter cell wall in the four species by electron microscopy using rapid freezing and freeze substitution methods. The cell wall of C. vulgaris, C. sorokiniana, and C. lobophora consisted of an electron-dense thin layer with an average thickness of 17–20, 22, and 19 nm, respectively. In these three species, daughter cell wall synthesis occurred on the outer surface of the plasma membrane in the early cell-growth phase. The cell wall of P. kessleri, however, was electron-transparent and 54–59 nm in thickness. Ruthenium red staining of P. kessleri indicated that ruthenium-red-specific polysaccharides accumulated over the outer surface of the plasma membrane. Immunoelectron microscopic observation with an anti--1, 3-glucan antibody and staining with wheat germ agglutinin (WGA) indicated that the cell wall contained -1, 3-glucan and WGA specific N-acetyl--D-glucosamine. In P. kessleri, daughter cell wall synthesis began after successive protoplast division. The daughter cell wall synthesis during autosporulation in the four species of Chlorellaceae can be classified into two types—the early and the late types.     

The overwintering physiology of the emerald ash borer, Agrilus planipennis fairmaire (coleoptera: buprestidae)

Ability to survive cold is an important factor in determining northern range limits of insects. The emerald ash borer (Agrilus planipennis) is an invasive beetle introduced from Asia that is causing extensive damage to ash trees in North America, but little is known about its cold tolerance. Herein, the cold tolerance strategy and mechanisms involved in the cold tolerance of the emerald ash borer were investigated, and seasonal changes in these mechanisms monitored. The majority of emerald ash borers survive winter as freeze-intolerant prepupae. In winter, A. planipennis prepupae have low supercooling points (∼−30 °C), which they achieve by accumulating high concentrations of glycerol (∼4 M) in their body fluids and by the synthesis of antifreeze agents. Cuticular waxes reduce inoculation from external ice. This is the first comprehensive study of seasonal changes in cold tolerance in a buprestid beetle.     

Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems

Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (−5 to −40 °C), and cells were thawed followed by return to 37 °C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue™ (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At −15 °C, PC-3 yielded 55% viability versus 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze–thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching −40 °C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze–thaw cycles, and passive thawing to provide maximum cell destruction.     

Claudin-3 and Claudin-5 Protein Folding and Assembly into the Tight Junction Are Controlled by Non-conserved Residues in the Transmembrane 3 (TM3) and Extracellular Loop 2 (ECL2) Segments

The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) that form the TJ strands are unclear. This limits the molecular understanding of paracellular barriers and strategies for drug delivery across tissue barriers. Cldn3 and Cldn5 are both common in the blood-brain barrier but form TJ strands with different ultrastructures. To identify the molecular determinants of folding and assembly of these classic claudins, Cldn3/Cldn5 chimeric mutants were generated and analyzed by cellular reconstitution of TJ strands, live cell confocal imaging, and freeze-fracture electron microscopy. A comprehensive screening was performed on the basis of the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment 3, TM3 (Ala-127/Cys-128, Ser-136/Cys-137, Ser-138/Phe-139), and the transition of TM3 to extracellular loop 2, ECL2 (Thr-141/Ile-142) and ECL2 (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were identified to be involved in claudin folding and/or assembly. Blue native PAGE and FRET assays revealed 1% n-dodecyl β-d-maltoside-resistant cis-dimerization for Cldn5 but not for Cldn3. This homophilic interaction was found to be stabilized by residues in TM3. The resulting subtype-specific cis-dimer is suggested to be a subunit of polymeric TJ strands and contributes to the specific ultrastructure of the TJ detected by freeze-fracture electron microscopy. In particular, the Cldn5-like exoplasmic face-associated and particle-type strands were found to be related to cis-dimerization. These results provide new insight into the mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to the formation of TJ strands with differing ultrastructures.     

Cold tolerance and freeze-induced glucose accumulation in three terrestrial slugs

Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about − 4 °C when cooled under dry conditions, but freezing of body fluids was readily induced at − 1 °C when in contact with external ice crystals. All three species survived freezing for 2 days at − 1 °C, and some A. rufus and A. lusitanicus also survived freezing at − 2 °C. ¹H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22 μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates.     

Comparison of dual- and triple-freeze protocols for pulmonary cryoablation in a Tibet pig model

The purpose of this study was to compare a dual-freeze protocol with a triple-freeze protocol for pulmonary cryoablation in a porcine lung model. Five dual- (10-5-10-5) and five triple-freeze (5-5-5-5-10-5) cryoablations were performed on an exposed operation field in normal porcine lung. Changes in the temperature of the cryoprobes and the diameter of the iceballs were measured during the ablation and pathologic changes in the cryozones (zones of tissue destruction) were reviewed 7 days after the procedure. The diameter of the iceball surface differed between the two protocols. Pathologically, the triple-freeze protocol was associated with a longer complete necrosis zone than the dual-freeze protocol, though the two protocols produced cryolesions and cryozones of similar length, and in both cases there were five areas of tissue destruction. With the same duration of freezing (20 min), the triple-freeze protocol may be better for pulmonary cryoablation than the dual-freeze protocol.     

春季-冻融交替时期9种落叶及半常绿小灌木抗寒性综合评价

在春季—冻融交替时期,以北京地区迎春、金叶女贞、小花溲疏等9种落叶及半常绿小灌木一年生枝条为试材,通过人工模拟降温,测定其在4 ~ -20℃低温胁迫下的6个生理响应指标,并对各指标进行相关性分析。结果表明,相对电导率、半致死温度、游离-脯氨酸含量和相对含水量这4个生理指标可作为9种小灌木抗寒性的评价指标。利用主成分分析、隶属函数法进行抗寒性综合评价,发现9种小灌木的抗寒性强弱顺序为：小花溲疏＞ 紫叶小檗＞ 太平花＞ 红王子锦带＞ 迎春＞ 连翘＞ 金叶女贞＞ 棣棠＞ 金山绣线菊。通过聚类分析可将9种小灌木分为3类,小花溲疏和紫叶小檗归为一类,其抗寒性相对较好;太平花、红王子锦带、迎春和连翘聚成一组,抗寒性一般;金叶女贞、棣棠和金山绣线菊聚在一起,抗寒性相对较差。