Epithelial outgrowth from suspension cultures of human prostatic tissue

Summary The primary objective of this study was to obtain pure cultures of prostatic epithelium. Encapsulation by epithelial cells and hypocellularity in stroma occurred when explants of prostatic tissue were maintained in suspension cultures. Twenty per cent fetal bovine serum incorporated into the medium provided optimal conditions for encapsulation and preservation of epithelial cell viability and architecture. Horse serum at the same concentration was less effective. When encapsulated explants were allowed to attach to the substrate, 10 and 20% horse serum favored growth of epithelial cells while fetal bovine serum also stimulated fibroblastic growth. Mechanisms for the induction of hypocellularity, encapsulation, squamous metaplasia and central necrosis in explants were studied. Relationship between the type and concentration of serum and the nature and extent of outgrowth are discussed. This work was supported in part by the American Cancer Society Grants DT-20 and IN-5N, U. S. Public Health Service Grant RR-05357, the Parke Davis Fund, and the John U. White Fund for Urological Research.     

柠檬酸和抗坏血酸对蝴蝶兰叶外植体褐变发生的影响

目的:探究柠檬酸和抗坏血酸对蝴蝶兰叶片外植体褐变发生的影响以及对PPO活性变化影响的作用机理.方法:以褐变率和褐变指数为参考数据,分析柠檬酸和抗坏血酸对外植体PPO活性和PPO反应产物积累的影响以及与外植体褐变发生的关系.结果:分别用100mg/L柠檬酸共培养和50mg/L抗坏血酸浸泡处理叶片外植体,经离体培养3d,褐变率分别比对照降低94.9%和54.9%,离体培养6d,褐变指数低于对照的0.53,分别为0.46和0.36,同时PPO活性降低.结论:推测柠檬酸抑制褐变的原因是直接与酶蛋白作用,抗坏血酸则与新生醌类物质结合.     

Organogenesis of vegetative shoots from in vitro cultured flower buds of Mammillaria albicoma (Cactaceae)

We tested the organogenetic capacity of floral buds of Mammillaria albicoma Böed. (Cactaceae). Buds were incubated on solid MS medium supplemented with 0.1 mg l^?1 α-naphthaleneacetic acid (NAA) and 5.0 mg l^?1 6-benzylaminopurine (BA). Callus growth was observed from the cut explant base and from within the perianth. These calli during subsequent subcultures to the same medium gave rise to adventitious shoots. Shoots formed also directly from the perianth, as confirmed by observations in the light microscope and scanning electron microscope (SEM). On transfer to a fresh medium, the shoots produced proliferating cultures. This is the first report of regeneration of cactus shoots from floral explants.     

Circadian clock rhythms in different adipose tissue model systems

ABSTRACT

Circadian clock-controlled 24-h oscillations in adipose tissues play an important role in the regulation of energy homeostasis, thus representing a potential drug target for prevention and therapy of metabolic diseases. For pharmacological screens, scalable adipose model systems are needed that largely recapitulate clock properties observed in vivo. In this study, we compared molecular circadian clock regulation in different ex vivo and in vitro models derived from murine adipose tissues. Explant cultures from three different adipose depots of PER2::LUC circadian reporter mice revealed stable and comparable rhythms of luminescence ex vivo. Likewise, primary pre- and mature adipocytes from these mice displayed stable luminescence rhythms, but with strong damping in mature adipocytes. Stable circadian periods were also observed using Bmal1-luc and Per2-luc reporters after lentiviral transduction of wild-type pre-adipocytes. SV40 immortalized adipocytes of murine brown, subcutaneous and epididymal adipose tissue origin showed rhythmic mRNA expression of the core clock genes Bmal1, Per2, Dbp and REV-erbα in pre- and mature adipocytes, with a maturation-associated increase in overall mRNA levels and amplitudes. A comparison of clock gene mRNA rhythm phases revealed specific changes between in vivo and ex vivo conditions. In summary, our data indicate that adipose culture systems to a large extent mimic in vivo tissue clock regulation. Thus, both explant and cell systems may be useful tools for large-scale screens for adipose clock regulating factors.     

Long-sized oligogalacturonides inhibit,whereas spermidine enhances,xylogenesis in tobacco leaf explants

Abstract

Long-sized oligogalacturonides (OGs) are plant cell wall fragments involved in defence responses and developmental processes. A hormone/OG interaction in the control of different organogenic processes is known. However, hormones also modulate polyamine (PA) effects on organogenesis. Furthermore, OGs are known to affect mitotic activity leading to specific morphogenic events, and PAs are known to affect mitotic activity leading to xylogenesis. Thus, it may be reasonable to assume that OGs and PAs affect mitotic activity in the same cell types, and in the same hormone-induced morphogenic processes, e.g., xylogenesis. To gain further insight into this aspect, the effects of OGs, and of putrescine (Put) and spermidine (Spd), on auxin (indoleacetic acid, IAA) plus benzyladenine (BA)-induced morphogenesis in tobacco leaf explants were investigated histologically. The effect of PA biosynthetic inhibitors in the culture medium was also monitored, as well as the combined application of the inhibitor with the corresponding PA. Results show that vascular mitoses consistently occurred in the control (IAA+BA-treated) explants, leading exclusively to xylogenic nodule formation. The application of OGs resulted in an inhibition of vascular mitoses, and into a strong reduction of vascular nodule formation. By contrast, Spd enhanced both vascular mitoses and nodule formation, and Put was less effective than Spd on both events. Taken together, the results reveal a new biological activity of OGs and Spd in morphogenesis, obtained under the same hormonal conditions, and in the same tissue (i.e., the vascular parenchyma), namely the inhibition of xylogenesis by OGs, and its promotion by Spd. The fact that the effects of Spd and OGs on this morphogenic event may involve a different relationship with auxin is discussed.     

人体黏膜活组织模型在HIV-1 性传播途径感染中的应用

性传播途径已经成为全球人免疫缺陷病毒1型 (Human immunodeficiency virus type 1,HIV-1) 传播的主要方式。对HIV-1黏膜感染机制的深入理解,将有助于研发新型有效的生物技术阻断其感染和传播。目前,HIV-1黏膜感染机制的研究主要依赖于体外细胞培养和灵长类动物模型。近年来,一种新型黏膜活组织模型 (包括人体生殖道或肠道黏膜等组织) 的建立,可再现HIV-1突破黏膜屏障进入基底侧的生物学过程,适用于HIV-1黏膜感染机制与黏膜局部感染阻断生物技术的临床前有效性评价研究。     

Thiopurine Prodrugs for Plant Chemotherapy Purposes

Thiopurine prodrugs are antiviral chemicals used in medical therapy whose mechanisms of action are associated with inhibition of purine biosynthesis. In terms of plant chemotherapy, previous research of 6‐mercaptopurine (MP) administration in tobacco tissue culture infected by Tobacco mosaic virus (TMV) showed no inhibition of virus activity. Currently, not enough data exist to confirm thiopurine drug ineffectiveness against viruses in the plant kingdom. This paper presents a screening of MP, 6‐methylmercaptopurine riboside (MMPR), 6‐thioguanine (6‐TG) and 1‐amino‐6‐mercaptopurine (1A‐MP) against TMV and Cucumber mosaic virus (CMV) in in vitro tobacco explants and against Grapevine leafroll‐associated virus 3 (GLRaV 3) in in vitro grapevine explants. ELISA and RT‐PCR were used to evaluate antiviral activity. Higher toxicity levels of MP derivatives, compared to MP, were noted in tobacco and grapevine explants. 1A‐MP or 6‐TG treatment resulted CMV and GLRaV 3 virus‐eradicated explants as obtained with Inosine 5′‐monophosphate dehydrogenase inhibitors, whereas TMV was not eradicated by any of the studied drugs.     

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing¹. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas². To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases³, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.     

Daily rhythms are retained both in spontaneously developed sarcomas and in xenografts grown in immunocompromised SCID mice

The circadian clock generates and regulates many daily physiological, metabolic and behavioral rhythms as well as acute responses to various types of stresses including those induced by anticancer treatment. It has been proposed that modulatory function of the clock may be used for improving the therapeutic efficacy of established anti-cancer treatments. In order to rationally exploit this mechanism, more information is needed to fully characterize the functional status of the molecular clock in tumors of different cellular origin; however, the data describing tumor clocks are still inconsistent. Here we tested the status of clock in two models of tumors derived from connective tissue: sarcomas spontaneously developed in p53-deficient mice and human fibrosarcoma cells grown as xenografts in immunocompromised severe combined immunodeficient (SCID) mice. We show that both types of tumors retain a functional clock, which is synchronized in phase with normal tissues. We also show that spontaneously developed tumors are not only oscillating in the context of an organism where they receive hormonal and metabolic signals but continue oscillating ex vivo in tissue explants demonstrating that tumors have functional clocks capable of timing all their functions. We also provide evidence that similar to liver, tumors can be synchronized by food availability independent of the central pacemaker in the suprachiasmatic nuclei (SCN). These data provide the basis for the design of anticancer therapies that take into account the circadian metabolic and physiological patterns of both the tumor and normal tissues.     

Somatic embryogenesis and plant regeneration from stem explants of Moricandia arvensis

Efficient and reproducible plant regeneration has been established from stem internode explants of Moricandia arvensis, a crucifer of special interest due to its C₃-C₄ intermediate photosynthetic activity. Somatic embryogenesis was induced in one-third of explants cultured on Murashige and Skoog based medium containing 9 mm 2,4-dichlorophenoxyacetic acid. High frequencies of plant regeneration (>90%) resulted when somatic embryos were germinated on medium lacking growth regulators. Regenerated plants were diploid, fertile and morphologically similar to seed-derived plants of M. arvensis. This is the first report of somatic embryogenesis in M. arvensis. This plant regeneration system should facilitate gene identification and localisation studies of C₃-C₄ physiology by insertional mutagenesis, a prerequisite for the isolation and transfer of genes involved in C₃-C₄ metabolism from Moricandia to cultivated brassicas. Received: 28 November 1996 / Revision received: 30 January 1997 / Accepted: 15 February 1997