Improvement in Ovarian Tissue Quality with Supplementation of Antifreeze Protein during Warming of Vitrified Mouse Ovarian Tissue

Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.

Evaluation of the ability to metabolize 1, 2-propanediol by heterofermentative bacteria of the genus Lactobacillus

Background: New directions of research on lactic acid bacteria include investigation of metabolic pathways for the synthesis and/or metabolism of 1,2-propanediol, commonly used in the food and chemical industry, medicine, pharmacy and cosmetology as well as agriculture. The objective of this study was to compare the capacity of strains representing three diverse heterofermentative species belonging to the genus Lactobacillus to synthesize and/or transform 1,2-PD as well as to suggest new directions of research aimed at commercial use of this metabolite. Results: The novel strain of Lactobacillus buchneri A KKP 2047p, characterized as exhibiting an unusual trait for that species in the form of capacity to metabolize 1,2-PD, grew poorly in a medium containing 1,2-PD as a sole carbon source. The supplementation with glucose facilitated rapid growth of bacteria and use of 1,2-PD for the synthesis of propionic acid. A similar observation was noted for Lactobacillus reuteri. On the other hand, Lactobacillus diolivorans effectively metabolized 1,2-PD which was the sole carbon source in the medium, and the addition of glucose inhibited the synthesis of propionic acid. The experiments also investigated the effect of cobalamin as a diol dehydratase coenzyme involved in the propionic acid synthesis from 1,2-PD whose addition promoted the yield of the reaction in the case of all tested strains. Conclusions: All tested isolates showed the ability to effectively metabolize 1,2-PD (in the presence of cobalamin) and its conversion to propionic acid, which reveals that investigated bacteria meet the essential requirements of microorganisms with a potential application.

Effects of supplementation with antifreeze proteins on the follicular integrity of vitrified-warmed mouse ovaries: Comparison of two types of antifreeze proteins alone and in combination / 대한생식의학회지

OBJECTIVE: The aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries. METHODS: Ovaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. RESULTS: The proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups. CONCLUSION: Supplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.

Advances in cryopreservation of organs / 华中科技大学学报（医学）（英德文版）

Organ transplantation is an effective approach for the treatment of end-stage organ failures. Currently, the donor organs used for clinical transplantation are all preserved at above-zero temperatures. These preservation methods are well-established and simple but the storage time lasts for only 4-12 h. Some researchers tried to extend the organ storage time by improving protectant and HLA matching to raise the use of stored organs and prolong the long-term survival of organs. These efforts still fall short of the clinical demand for organ transplantation. Moreover, a great many organs were wasted due to limited storage time, HLA mismatch, patients' conditions or distance involved. Therefore, preserving organs for several weeks or even months and establishing Organ Bank are the tough challenges and have become a shared goal of global scholars. This article reviews some issues involved in the cryopreservation of organs, such as use of cryoprotecting agents, freezing and thawing methods in the cryopreservation of hearts, kidneys and other organs.

Effect of Antifreeze Protein on Mouse Ovarian Tissue Cryopreservation and Transplantation

PURPOSE: To investigate the effect of antifreeze protein (AFP) supplementation on ovarian vitrification and transplantation. MATERIALS AND METHODS: In this experimental study, we researched a total of 182 ovaries from 4-week-old ICR mice. The equilibration solution included 20% ethylene glycol (EG), and the vitrification solution included 40% EG, 18% Ficoll, and 0.3 M sucrose. Intact ovaries were first suspended in 1 mL of equilibration solution for 10 min, and then mixed with 0.5 mL of vitrification solution for 5 min. Ovaries were randomly assigned to 3 groups and 0, 5, or 20 mg/mL of type III AFP was added into the vitrification solution (control, AFP5, and AFP20 groups, respectively). The vitrified ovaries were evaluated after warming and 2 weeks after autotransplantation. The main outcome measurements are follicular morphology and apoptosis assessed by histology and the TUNEL assay. RESULTS: A significantly higher intact follicle ratio was shown in the AFP treated groups (control, 28.9%; AFP5, 42.3%; and AFP20, 44.7%). The rate of apoptotic follicles was significantly lower in the AFP treated groups (control, 26.6%; AFP5, 18.7%; and AFP20, 12.6%). After transplantation of the vitrified-warmed ovaries, a significantly higher intact follicle ratio was shown in the AFP20 group. The rate of apoptotic follicles was similar among the groups. CONCLUSION: The results of the present study suggest that supplementing AFP in the vitrification solution has beneficial effects on the survival of ovarian tissue during cryopreservation and transplantation.

Antifreeze proteins enable plants to survive in freezing conditions.

Overwintering plants secrete antifreeze proteins (AFPs) to provide freezing tolerance. These proteins bind to and inhibit the growth of ice crystals that are formed in the apoplast during subzero temperatures. Antifreeze activity has been detected in more than 60 plants and AFPs have been purified from 15 of these, including gymnosperms, dicots and monocots. Biochemical characterization of plant antifreeze activity, as determined by the high ice recrystallization inhibition (IRI) activities and low thermal hysteresis (TH) of AFPs, showed that their main function is inhibition of ice crystal growth rather than the lowering of freezing temperatures. However, recent studies showed that antifreeze activity with higher TH also exists in plants. Calcium and hormones like ethylene and jasmonic acid have been shown to regulate plant antifreeze activity. Recent studies have shown that plant AFPs bind to both prism planes and basal planes of ice crystals by means of two flat ice binding sites. Plant AFPs have been postulated to evolve from the OsLRR-PSR gene nearly 36 million years ago. In this review, we present the current scenario of plant AFP research in order to understand the possible potential of plant AFPs in generation of freezing-tolerant crops.

Effect of antifreeze or antioxidant at different cryopreservation conditions on activity of adipose cells / 中华医学美学美容杂志

Objective To explore the effect of different antifreeze or antioxidant at different cryopreservation conditions on the activity and the morphology of adipose cells.Methods After purification,fat granules were divided into 5 groups; the same volume of antifreeze or antioxidants were added in each group,stored at-20℃ (refrigerator),-80℃ (ultra-low-temperature refrigerator) and-196 ℃ (liquid nitrogen) ; the amount of glucose transfer was determined and the morphological changes were observed for each group after two weeks,two months,and three months.Results Fat activity of the group adding both dimethyl sulfoxide (DMSO) and trehalose was significantly higher than that in other groups at the same temperature and the same time point.The decreased amount of glucose transfer of 80℃ group and 196℃ group was significantly lower than that of 20℃ group,and that in groups of each 196℃ were greater than that in groups of each 80℃.Conclusions Antifreeze can obviously keep activity of fat cells.Preservative effect of DMSO is better than trehalose,and that of both combination is much better.Liquid nitrogen storage of adipose tissue for three months and fat granules stored at 80℃ for two months are still suitable for clinical use.

Methemglobinemia from Antifreeze Containing Sodium Nitrite

Sodium nitrite is commercially used as a coloring agent, food preservative, and corrosion inhibitor. Accidental poisoning with sodium nitrite from contaminated food and water causes gastrointestinal irritation, vasodilatation, and methemoglobinemia with subsequent tissue hypoxia. We describe an outbreak case of sodium nitrite-induced methemoglobinemia following the ingestion of noodles contaminated with industrial antifreeze. The eEight patients involved initially complained that their noodles tasted 'unpleasant' and soon afterwards experienced nausea, vomiting, dizziness, and fatigue. Some of them noted cyanosis on their lips and fingers. Subsequent investigations demonstrated a high methemoglobin concentration which was corrected by the intravenous administration of methylene blue three hours after the onset of symptoms. The patients made a prompt, uncomplicated recovery and were discharged home 4 four days later. Industrial antifreeze contains sodium nitrite and calcium nitrite. Because an accidental poisoning of industrial antifreeze causes fatal methemoglobinemia, emergency physicians should promptly identify its symptoms and institute treatment with methylene blue promptly. In addition, industrial agencies must caution construction businesses against such contamination events.

Fish antifreeze proteins: Computational analysis and physicochemical characterization.

Antifreeze proteins (AFPs) protect organisms from freezing and shows great diversity in structure, and they have been found in a variety of organisms. In this study, a total of 15 antifreeze proteins of fish were selected where they represent distinct physicochemical and structural features. The present paper uses bioinformatics approach to describe the physiochemical, functional and structural properties of Antifreeze proteins. Several Physico-chemical properties such as pI, EC, AI, GRAVY and instability index are computed and provide data about these proteins and their properties. The result of primary structure analysis infers that, fish antifreeze proteins are mostly hydrophobic. Disulfide bridges and secondary structures were analyzed using CYS_REC and SOPMA respectively. The three dimensional structure of Antifreeze proteins is predicted by using three homology modeling server Geno3D, Swiss-model and CPHmodels. The model was evaluated with PROCHECK, WHAT IF, and ProSA programs. Model visualization and analysis was done with Pymol. These structures will provide a good foundation for functional analysis of experimentally derived crystal structures.

De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS / SÍNTESIS Y DISEÑO De novo DE UN POLIPÉPTIDO DENDRÍMERO CON UNIÓN AL HIELO BASADO EN LAS PROTEINAS ANTICONGELANTES DE INSECTOS / Síntes e desenhoo de novo de um polipetídeos dendrímero com uniâo ao gelo baseado nas proteínas anticongelantes de insetos

A new strategy is presented for the design and synthesis of peptides that exhibit ice-binding and antifreeze activity. A pennant-type dendrimer polypeptide scaffoíd combining an a-helical backbone with four short (β-strand branches was synthesized in solid phase using Fmoc chemistry in a divergent approach. The 51-residue dendrimer was characterized by reverse phase high performance liquid chromatography, mass spectrometry and circular dichroism. Each (β-strand branch contained three overlapping TXT amino acid repeats, an ice-binding motif found in the ice-binding face of the spruce budworm (Choristoneura fumiferana) and beetle (Tenebrio molitor) antifreeze proteins. Ice crystals in the presence of the polypeptide monomer displayed fiat, hexagonal plate morphology, similar to that produced by weakly active antifreeze proteins. An oxidized dimeric form of the dendrimer polypeptide also produced fiat hexagonal ice crystals and was capable of inhibiting ice crystal growth upon temperature reduction, a phenomenon termed thermal hysteresis, a defining property of antifreeze proteins. Linkage of the pennant-type dendrimer to a tri-functional cascade-type polypeptide produced a trimeric macromolecule that gave flat hexagonal ice crystals with higher thermal hysteresis activity than the dimer or monomer and an ice crystal burst pattern similar to that produced by samples containing insect antifreeze proteins. This macromolecule was also capable of inhibiting ice recrystallization.

Una nueva estrategia se presenta para el diseño y síntesis de péptidos que se unen al hielo y evidencian actividad anticongelante. Un polipéptido dendrímero del tipo banderín, que combina en su estructura un núcleo a-hélice con cuatro ramificaciones cortas de hojas β, se sintetizó en fase sólida utilizando la química Fmoc con una estrategia divergente. El dendrímero de 51 residuos se caracterizó por cromatografía líquida de alta resolución, espectrometría de masas y dicroís-mo circular. Cada ramifcación de hoja β contiene tres repeticiones de los motivos de aminoácidos TxT sobrelapados, un motivo de unión al hielo presente en la cara de unión de las proteínas anticongelantes del gusano de brotes de abeto (Choristoneura fumiferana) y en el escarabajo (Tenebrio molitor). Los cristales de hielo en presencia del polipéptido monomérico presentan una morfología hexagonal plana, similar a la producida por las proteínas anticongelantes con una débil actividad. Un dímero oxidado del polipéptido también produce cristales de hielo hexagonales planos que fueron capaces de inhibir el crecimiento de los cristales de hielo a medida que se disminuía la temperatura, un fenómeno conocido como histéresis térmica, esto es, una propiedad que define las proteínas anticongelantes. La vinculación del dendrímero tipo banderín a un polipéptido tipo cascada trifuncional produjo una macro-molécula trimérica que generó cristales de hielo hexagonales planos con una mayor actividad de histéresis térmica que los dímeros y los monómeros y un patrón de estallido del cristal de hielo muy similar al producido por las muestras que contienen proteínas anticongelantes de insectos. Estas moléculas además fueron capaces de inhibir la recristianización del hielo.

Uma nova estratégia é apresentada para o desenho e síntese de peptídeos que se unem ao gelo e apresentam atividade anticongelante. Um polipeptídeo dendrímero do tipo pennant que combina em sua estrutura um núcleo a-hélice com quatro ramifcacoes curtas de folhas β foi sintetizado em fase sólida utilizando a química Fmoc com uma estratégia divergente. O dendrímero de 51 resíduos foi caracterizado por cromatografa líquida de alta resolução, espectrometria de massas e dicroísmo circular. Cada ramifcacao de folha (β contém três repeticoes dos motivos de aminoácidos TxT sobrepostos, um motivo de união ao gelo presente na cara de união das proteínas anticongelantes do verme de Choristoneura fumiferana e no escaravelho (Tenebrio molitor). Os cristais de gelo, em presença do polipeptídeo monomérico, apresentam urna morfologia hexagonal plana, similar à produzida pelas proteínas anticongelantes com uma atividade fraca. Um dímero oxidado do polipeptídeo também produz cristais de gelo hexagonais planos e fo-ram capazes de inibir o crescimento dos cristais de gelo à medida que a temperatura diminuia, um fenômeno conhecido como histerese térmica una propriedade que def ne as proteínas anticongelantes. A vinculação do dendrímero tipo pen-nant a um polipeptídeo tipo cascata tri-funcional produziu uma macromolécula trimérica que gerou cristais de gelo hexa-gonais planos com uma maior atividade de histerese térmica que os dímeros e os monómeros e um padrão de estouro do cristal de gelo muito similar ao produzi-do pelas amostras que contêm proteínas anticongelantes de insetos. Estas moléculas, aliás, foram capazes de inibir a recristalização do gelo.

Fish antifreeze proteins: Computational analysis and physicochemical characterization.

Antifreeze proteins (AFPs) protect organisms from freezing and shows great diversity in structure, and they have been found in a variety of organisms. In this study, a total of 15 antifreeze proteins of fish were selected where they represent distinct physicochemical and structural features. The present paper uses bioinformatics approach to describe the physiochemical, functional and structural properties of Antifreeze proteins. Several Physico-chemical properties such as pI, EC, AI, GRAVY and instability index are computed and provide data about these proteins and their properties. The result of primary structure analysis infers that, fish antifreeze proteins are mostly hydrophobic. Disulfide bridges and secondary structures were analyzed using CYS_REC and SOPMA respectively. The three dimensional structure of Antifreeze proteins is predicted by using three homology modeling server Geno3D, Swiss-model and CPHmodels. The model was evaluated with PROCHECK, WHAT IF, and ProSA programs. Model visualization and analysis was done with Pymol. These structures will provide a good foundation for functional analysis of experimentally derived crystal structures.

Sarcoma mieloide: Caso clínico con trisomia 11 / Myeloid sarcoma: Report of one case with trisomy 11

Myeloid sarcoma is a form of extra-medullary myeloid neoplasia. Cytogenetic characterization is hampered in the absence of invasion to the bone marrow, origin of cells that are usually studied in cytogenetic studies. We report a 13years old mole presenting with a mass in the right shoulder. A biopsy of the tumor disclosed a Myeloid Sarcoma. A conventional cytogenetic study of a bone marrow aspirate did not show t (8;21) translocation. A fluorescent in situ hybridization (FISH) performed in the paraffin embedded biopsy of the tumor, detected a chromosome 11 trisomy.

Recent Advances in Research of Antifreeze Proteins / 中国生物化学与分子生物学报

Many overwintering organisms produce antifreeze proteins (AFPs) that can be adsorbed onto the surface of ice crystals and modify their growth. These proteins show great diversity in structures, and they have been found in a variety of organisms. AFPs from insects have higher thermal hysteresis activity than other organisms. Recent studies revealed the structures of AFPs and put forward different ice-binding models. No mechanism, however, can apply to all antifreeze proteins and the molecular interaction between AFPs and ice are not accurately resolved. AFPs can be applied extensively to agriculture, aquaculture and low temperature storage of organs, tissues, as well as cells. To confer transgenic plant cold resistance application of AFPs is essential, while the expression and regulation of antifreeze gene need to be elucidated.