Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.NRCC Publication No. 33519     

Junction formation in aggregated embryonal carcinoma cells

Under the action of supplemental calcium, H6 mouse embryonal carcinoma cell aggregates undergo compaction, a morphological phenomenon similar to mouse embryonic compaction. Formation of various types of cell junctions, especially gap junctions, is associated with compaction of the embryo and we sought to analyze the pattern of junction formation during aggregation and compaction of H6 cells. At 24 hr of aggregation, gap junctions were abundant in both uncompacted and compacted aggregates but quantitative analysis of freeze fracture replicas of these junctions showed a 20-fold increase in the size of the largest gap junctions in compacted aggregates. Such a difference in size could even be detected at 12 hr of aggregation. Tight junctions were not normally formed in 12 hr aggregates but initial stages of tight junction formation could be noticed in 12 hr compacted aggregates. More definitive tight junctions and desmosomes were evident only after 48 hr of aggregation. Thus we have observed that both uncompacted and compacted aggregates can form gap junctions at similar frequencies, suggesting that cell flattening, which contributes to the compacted morphology, is not a requisite for gap junctions. Likewise, generation of the compacted morphology seems to be independent of gap junction formation. This supports the idea that compaction in embryonal carcinoma cells results from calcium-induced cell flattening, probably through the mobilization of cytoskeletal elements. Calcium-dependent features of H6 cell aggregation and compaction enables the independent analysis of separate steps in compaction.     

Regeneration of pea (Pisum sativum L. cv. Century) plants by in vitro culture of immature leaflets

In vitro regeneration of plants from immature leaflets of 3 day-old pea (Pisum sativum L. cv. Century) seedlings was studied under defined nutritional, hormonal and environmental conditions. Immature leaflets isolated from the second and third apical leaves of aseptically germinated seeds were cultured on MS medium containing vitamins as in B5 medium, 3% sucrose, 0.8% agar and supplemented with 0.1, 1, and 10 M concentrations of naphthaleneacetic acid (NAA) and 1 and 10 M levels of benzyladenine (BA) in various combinations. Shoot regeneration from the primary callus occurred within 45 to 90 days of culture in most of the hormone combinations. Although the number of calli producing shoots was maximal at 10 M levels of NAA and BA, multiple shoot regeneration was predominant at a combination of 0.1 M NAA and 10 M BA. Indoleacetic acid (IAA) and kinetin (K), both at 10 M, also induced shoot regeneration. No shoots were regenerated when 10 day-old leaflets were used as explants. Root production generally occurred on non-shoot regenerating calli. Roots were induced to differentiate by transferring the regenerated shoots onto half-strength B5 medium supplemented with 1 M NAA.NRCC # 19712Visiting Scientist, supported by a research fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (Rep. Argentina). Permanent address: Facultad de Ciencias Agrarias, IBONE, Casilla de Correos 209, Corrientes (3400), Argentina.     

Vibrational spectroscopy studies of formalin-fixed cervix tissues

Optical histopathology is fast emerging as a potential tool in cancer diagnosis. Fresh tissues in saline are ideal samples for optical histopathology. However, evaluation of suitability of ex vivo handled tissues is necessitated because of severe constraints in sample procurement, handling, and other associated problems with fresh tissues. Among these methods, formalin-fixed samples are shown to be suitable for optical histopathology. However, it is necessary to further evaluate this method from the point of view discriminating tissues with minute biochemical variations. A pilot Raman and Fourier transform infrared (FTIR) microspectroscopic studies of formalin-fixed tissues normal, malignant, and after-2-fractions of radiotherapy from the same malignant cervix subjects were carried out, with an aim to explore the feasibility of discriminating these tissues, especially the tissues after-2-fractions of radiotherapy from other two groups. Raman and FTIR spectra exhibit large differences for normal and malignant tissues and subtle differences are seen between malignant and after-2-fractions of radiotherapy tissues. Spectral data were analyzed by principal component analysis (PCA) and it provided good discrimination of normal and malignant tissues. PCA of data of three tissues, normal, malignant, and 2-fractions after radiotherapy, gave two clusters corresponding to normal and malignant + after-2-fractions of radiotherapy tissues. A second step of PCA was required to achieve discrimination between malignant and after-2-fractions of radiotherapy tissues. Hence, this study not only further supports the use of formalin-fixed tissues in optical histopathology, especially from Raman spectroscopy point of view, it also indicates feasibility of discriminating tissues with minute biochemical differences such as malignant and after-2-fractions of radiotherapy.     

Long-term Survival From Muscleinvasive Bladder Cancer With Initial Presentation of Symptomatic Cerebellar Lesion: The Role of Selective Surgical Extirpation of the Primary and Metastatic Lesion

A 59-year-old man was diagnosed with urothelial carcinoma involving an isolated cerebellar metastasis after presenting to the emergency department for headache complaints. After selective surgical excision of the symptomatic brain lesion and delayed cystectomy due to intractable hematuria, he survived 11 years without evidence of recurrence or subsequent systemic chemotherapy. He eventually expired after delayed recurrence in the lung, supraclavicular lymph node, and brain. To our knowledge, this is the only case of prolonged survival from urothelial carcinoma after selective surgical extirpation of the primary and metastatic lesion without subsequent systemic chemotherapy.Key words: Bladder cancer, Cystectomy, Metastasis, Urothelial carcinomaUsually, brain metastasis of bladder urothelial carcinoma is associated with widespread systemic disease and/or multiple brain lesions. It is exceedingly rare to have bladder cancer metastasize to the brain without evidence of additional systemic manifestations.¹ As with other forms of distant urothelial carcinoma metastasis, brain metastasis is associated with poor prognosis, with survival often less than 14 months in those with solitary brain lesions.² We report an isolated bladder urothelial carcinoma metastasis to the cerebellum with an 11-year survival fol-lowing extirpative therapy of both the primary lesion and brain metastasis.