Conditions affecting primary cell cultures of functional adult rat hepatocytes

Summary The conditions for obtaining representative, primary adult rat hepatocyte cultures were explored. The methods applied included enzymatic liver perfusion which was nondestructive to hepatocytes, the prevention of aggregation of dissociated cells and the selective attachment of viable cells. These procedures yielded a recovery of 50% of the liver cells which gave rise to cultures representing 14% of the total liver cells. The cultures were composed of homogeneous epithelial-like cells cytologically similar to hepatocytes and possessed a number of liver-specific enzymes. There was virtually no cell division initially and most cells died between 24 and 48 hr. Insulin enhanced the attachment of the liver cells, altered their morphology, but did not prolong cell survival. This study was supported by grant no. BC 133 from the American Cancer Society.     

Hormonal mechanisms for differentiation in plant tissue culture

Summary Cells possess extraordinary powers to organize their molecular processes not only to maintain a cell in a given steady state but also to recognize that state during differentiation. Regulation of these organizational forces appears to be under the control of chemical factors, and a hormonal concept of regulation has evolved. Hormones have been considered to act by reacting with a specific target site. This may be part of their mode of action, but I would like to suggest that a hormone enters and becomes part of a total molecular resonance system. In so doing, the entire molecular system of the cell is modified. Of the known plant hormones, the cytokinins, because of their role in experimentally induced cell division and differentiation, serve as a probe of hormonal involvement in differentiation. Cultured somatic cells of tobacco plants can be induced to undergo differentiation by addition of cytokinin and auxin to the medium. Studies of the cytokinin hormones show a series of diverse molecular involvements. The archetype cytokinin, N⁶-(Δ²-isopentenyl) adenosine (i⁶Ado), occurs in some molecular species of tRNA where it plays a vital role in the codon-anticodon interaction of tRNA and m-RNA. i⁶Ado under-goes extensive metabolism in the tobacco tissue. It is either degraded to adenosine or converted to derivatives that possess biological activity. It is perhaps, therefore, more correct to consider the hormone function as being derived from this total metabolic web. The normal somatic cells of tobacco cultures spontaneously change occasionally into an autonomous form that requires no external growth factors. This line of cells synthesizes i⁶Ado. The metabolic web of the hormone-dependent strain can be perturbed by added auxin but such is not the case in the autonomous strain. These data provide some insight into the altered state of cytokinin activity in which a cell line changes into an autonomous form. Curiously, in become independent of the requirement for exogenous cytokinin, the autonomous tissue becomes sensitive to added cytokinin. i⁶Ado also inhibits the growth of lines of mammalian cancer cells grown in culture. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Next-Generation Sequencing of Connective Tissue Genes in Patients with Classical Ehlers-Danlos Syndrome

Background: Ehlers-Danlos syndrome (EDS) is a common non-inflammatory, congenital connective tissue disorder. Classical type (cEDS) EDS is one of the more common forms, typically caused by mutations in the COL5A1 and COL5A2 genes, though causative mutations in the COL1A1 gene have also been described. Material and methods: The study group included 59 patients of Polish origin, diagnosed with cEDS. The analysis was performed on genomic DNA (gDNA) with NGS technology, using an Illumina sequencer. Thirty-five genes related to connective tissue were investigated. The pathogenicity of the detected variants was assessed by VarSome. Results: The NGS of 35 genes revealed variants within the COL5A1, COL5A2, COL1A1, and COL1A2 genes for 30 of the 59 patients investigated. Our panel detected no sequence variations for the remaining 29 patients. Discussion: Next-generation sequencing, with an appropriate multigene panel, showed great potential to assist in the diagnosis of EDS and other connective tissue disorders. Our data also show that not all causative genes giving rise to cEDS have been elucidated yet.     

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK‐dependent Cyclooxygenase‐2

Obesity is a world‐wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro‐inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS‐modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase‐2 (COX‐2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS‐stimulated gene expression of COX‐2 together with the phosphorylation of p47^phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase‐dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS‐mediated COX‐2 expression and preadipocyte proliferation. Moreover, LPS‐induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX‐2 or PEG₂ receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK‐dependent COX‐2 expression.     

棉铃虫幼虫加单氧酶活性的组织分布

棉铃虫（Ｈｅｌｉｃｏｖｅｒｐａ ａｒｍｉｇｅｒａ）６龄幼虫不同组织的加单氧酶活性的测定结果显示,对－硝基苯甲醚Ｏ－脱甲基酶主要分由于外来物质的入口部位,以中肠和脂肪体的活性较高,在前肠、后肠和马氏管等组织中有相对较低的活性,而在体壁和精巢中未检测到Ｏ－ 甲基作用。体壁表现一定的艾氏剂环氧化作用,但其活性不及中肠的１０％,内源性制剂被证明并非体壁低加单氧酶活性的主要原因。不同组织生物量的差异及其动态     

Local Recurrence of Soft Tissue Sarcoma Revisited: Is there a Role for “Selective” Radiation?

BackgroundRadiation therapy (RT) is often utilized in cases of high-grade soft tissue sarcoma (STS), but there remain situations where treatment is with surgical excision alone. Our goals were to determine (1) the local recurrence (LR) rate with and without perioperative RT and (2) associations between local recurrence, patient, tumor, and treatment variables.MethodsWe performed a retrospective review of 165 consecutive STS patients. A Cox proportional hazards model was used to investigate variables associated with local recurrence.ResultsLR occurred in 15/78 (19%) without RT, 4/29 (14%) with postoperative RT, and 0/58 with preoperative RT (p=0.002). We found increased rates of local recurrence at 24 months for myxofibrosarcoma (p=0.001) and no-RT (p=0.003). Myxofibrosarcoma accounted for 33 (20%) of the study patients and 12 (63%) of the local recurrences.ConclusionThe LR rate in patients treated with surgery alone was disproportionately attributable to myxofibrosarcoma (11/23 cases, 48%). Other subtypes demonstrated a lower rate of LR in the absence of RT (4/55 cases, 7%), and no LR occurred when final margins were >2 mm. In certain circumstances treatment with a negative margin surgical resection followed by close observation is justifiable. RT is effective and should continue to be considered routinely in myxofibrosarcoma or when surgical margins are inadequate. Level of Evidence: III     

High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br.

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2 mg l^-1 BA and 0.01 mg l^-1 NAA or 4 mg l^-1 kinetin and 0.01 mg l^-1 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.Abbreviations BA benzyladenine - 2-4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog     

Matrix Metalloproteinases of Normal Human Tissues

This review considers biochemical properties of the family of matrix metalloproteinases (MMPs) of normal human tissues and the involvement of these enzymes in morphogenesis. Four main MMP subfamilies are characterized, and a group of other MMPs is described. Data on mechanisms of activation and inhibition of MMPs in certain tissues during various physiological processes (embryogenesis, angiogenesis, tissue growth and involution) are considered. Information about tissue inhibitors of MMP is presented, and the ability of these inhibitors to regulate the activity of MMPs is analyzed.     

Detection and Quantification of Fusarium oxysporum f. sp. niveum Race 1 in Plants and Soil by Real-time PCR

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is the most serious soil-borne disease in the world and has become the main limiting factor of watermelon production. Reliable and quick detection and quantification of Fon are essential in the early stages of infection for control of watermelon Fusarium wilt. Traditional detection and identification tests are laborious and cannot efficiently quantify Fon isolates. In this work, a real-time polymerase chain reaction (PCR) assay has been described to accurately identify and quantify Fon in watermelon plants and soil. The FONRT-18 specific primer set which was designed based on identified specific sequence amplified a specific 172 bp band from Fon and no amplification from the other formae speciales of Fusarium oxysporum tested. The detection limits with primers were 1.26 pg/μl genomic DNA of Fon, 0.2 pg/ng total plant DNA in inoculated plant, and 50 conidia/g soil. The PCR assay could also evaluate the relationships between the disease index and Fon DNA quantity in watermelon plants and soil. The assay was further used to estimate the Fon content in soil after disinfection with CaCN₂. The real-time PCR method is rapid, accurate and reliable for monitoring and quantification analysis of Fon in watermelon plants and soil. It can be applied to the study of disease diagnosis, plant-pathogen interactions, and effective management.     

Regeneration of plantlets from embryogenic suspension cultures of pickling cucumber (Cucumis Sativus L. CV. Endeavor)

Summary Procedures have been developed for the initiation and long-term maintenance of embryogenic suspension cultures of pickling cucumber (Cucumis sativus) cultivar Endeavor and for the regeneration of normal plantlets. Embryogenic calluses from petiole explants plated on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), both at 5μM, were used to initiate the embryogenic suspension cultures. Among various growth regulator combinations evaluated for initiation and maintenance of these suspension cultures, only MS medium with 2,4-D and BA, both at 1μM, produced cultures that were yellow, friable, and still regenerable after repeated subculture (every two wk) over a 3- to 15-mo. period. The effects of various concentrations of auxin and cytokinin in the plating medium, the addition of AgNO₃, and various plating procedures were also evaluated. The highest frequency of regeneration of shoots and plantlets was achieved by plating aggregates onto filter paper overlaid on MS medium with naphthalene acetic acid (NAA)/BA at a concentration of 2:1 or 1:1μM. The addition of activated charcoal (0.5%) or AgNO₃ (30μM) in the plating medium did not enhance the frequency of plantlet regeneration. The highest frequency of normal-appearing plantlets recovered was 42 to 46% per petri dish. The procedures described in this study can be used to increase plantlet recovery from individual embryogenic calluses of pickling cucumber.