Glutamine synthetase localization in cortisol-induced chick embryo retinas

We report here for the first time, in chick retina, Muller cell localization of glutamine synthetase (GS) activity by an immunohistochemical technique, in agreement with previous reports of glial localization of this enzyme in rat brain and retina. Age-dependent changes in the endogenous enzyme activity as well as cortisol-induced changes in GS activity, both in ovo and in vitro, measured biochemically, reflect the changes observed by staining.     

A rapid procedure for the isolation of lysosomes from kidney cortex by Percoll density gradient centrifugation

Highly pure lysosomes were isolated from buffalo(Bubalus bubalis) kidney cortex by a procedure involving differential and isopycnic Percoll density gradient centrifugations. Arylsulphatase, N-acetyl-Β-glucosamindase and cathepsin D in the lysosomal preparation were 26–45-fold enriched over the homogenate. The purified lysosomes contained less than 0·06% of mitochondrial, microsomal and peroxisomal marker enzymes. In the electron micrographs the particles appeared as large dense granules of size 0·3-1·9 μm with no apparent structural features belonging to mitochondria or microsomes. The isolation procedure was also found to be suitable to obtain highly pure lysosome particles from renal cortex of other sources such as rat, lamb and beef. No ultracentrifugation steps were involved in the procedure     

Changes in testis blood flow,the spermatic artery and prostaglandin F 2α content in testis tissue of heat-stressed rams

Blood flow to the testis, measured by the¹³³-Xenon isotope clearance technique, initially increased after rams were exposed to elevated (32°C) temperature. However, after 5 and 7 days continuous exposure, blood flow decreased significantly. Similar changes in blood flow to the testis were found during whole body exposure to elevated temperature, or when the temperature of the testis was increased by scrotal heating. After one week of heating the wall of the spermatic artery in the middle region of the pampiniform plexus had thickened, and the arterial lumen had decreased significantly. The PGF ₂ content in the testis of rams exposed to elevated temperature increased significantly. It is tentatively postulated that the higher levels of PGF ₂ in the testis of rams exposed to elevated temperature may be responsible for impaired spermatogenic function by constricting the spermatic artery in the pampiniform plexus region and thereby reducing blood flow to the testis.Published with approval of the Director of Kentucky Agricultural Experiment Station as Journal Article 75-5-150.Presented at the Seventh International Biometeorological Congres, 17–23 August 1975. College Park, Maryland, U.S.A.     

Sequential staining of DNA-aldehyde with Schiff's reagent and acriflavine-SO2.

Acid hydrolysed DNA of rat liver was stained with Schiff's reagent at pHs 1.7 or 3.0 followed by staining with acriflavine-SO2 at pH 2.0 as well as with acriflavine-SO2 followed by Schiff's reagent at pH 1.7 or 3.0. Nuclei stained with Schiff's reagent at pH 1.7 were brown-yellow and an analysis of their absorption characteristics revealed two peaks--one at 470 nm and the other at 570 nm. Although nuclei stained with Schiff's reagent at pH 3.0 followed by acriflavine-SO2 were deep magenta in colour, they also showed similar peaks of maximum absorption. Identical peaks were also seen when the sequence of staining was reversed. It is suggested that in the conventional Feulgen-type reactions only some of the DNA-aldehyde molecules are stained; the remaining molecules can be stained by sequential application of another Schiff or Schiff-type reagent such as acriflavine-SO2. The possible mechanism of staining in these cases has been discussed.     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.     

Anaerobic fermentative production of lactic acid using cheese whey and corn steep liquor

Cheese whey was the most suitable substrate for production of lactic acid under anaerobic conditions by Entercoccus flavescens which, on supplementating with corn steep liquor (5% v/v) and 10 mM CaCO₃ at pH 5.5, 37°C, yielded 12.6 g lactic acid/l in 36 h. Production was scaled up to a 10 l bioreactor under controlled pH and continuous CO₂ supply and gave 28 g lactic acid/l in 30 h resulting in a net 8.7-fold increase in production as compared to unoptimized conditions.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic cultures and plant regeneration in <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. (muscadine grape)

A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l⁻¹ in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l⁻¹, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.