Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons

Cotyledons of developing pea seeds (pisum sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies, The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulse-labeled (1-2 h), newly synthesized vicilin was present as a series of polypeptides with M(r) 60,000-65,000, and newly synthesized vicilin was present as series of polypeptides with M(r) 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al., 1982, J Cell Biol., 93:5- 14). During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides. Between 1 and 20 h later, radioactive legumin subunits (M(r) 40,000 and 19,000) and smaller vicilin polypeptides (M(r) 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies. The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm). Newly synthesized legumin and vicilin polypeptides were assembled into oligomers of 8S and 7S, respectively, in the ER. They appeared in the protein bodies in these oligomeric forms before the appearance of the smaller polypeptides (M(r) less than 49,000). These results indicate that the smaller vicilin polypeptides (M(r) less than 49,000) arise delayed posttranslational processing of some or all of the larger vicilin polypeptides. The precursors of legumin are completely processed in the protein bodies 2-3 h after their synthesis. The processing of the vicilin precursors is much slower (6-20 h) and only a fraction of the precursor molecules are processed. As a result both large (M(r) more than 49,000) and small polypeptides of vicilin accumulate in the protein bodies, whereas legumin accumulates only as polypeptides of M(r) 40,000 and 19,000.     

Stabilization and purification of ornithine transcarbamylase from Neisseria gonorrhoeae.

Ornithine transcarbamylase was stabilized in cell-free extracts by the presence of either carbamyl phosphate or glycerol. The enzyme was rapidly purified by a procedure consisting of ion-exchange chromatography and electrofocusing. The native molecular weight of the enzyme was determined by gel filtration to be 110,000. A subunit molecular weight of 36,000 was determined by polyacrylamide electrophoresis under dissociating conditions. These findings indicated a trimeric quaternary structure for the enzyme. The isoelectric point of the purified enzyme was 4.75, and no evidence of multiple forms of active enzyme was found in either crude or purified preparations. An inactive form of the enzyme appeared upon storage in the absence of stabilization buffer.     

Alterations in number of protofilaments in microtubules assembled in vitro

Tubulin from bovine brain was polymerized in vitro using a variety of assembly conditions. Many of the formed microtubules were shown to contain 14 wall protofilaments. The number of microtubules containing 14 protofilaments increased with consecutive repetitions of cold-dissociation followed by reassembly in vitro.     

Stature after 18 months of treatment with synthetic growth hormone in 12 patients with Turner's syndrome

The increased availability of recombined human growth hormone (rhGH) allows its possible use in clinical situations not classically recognized as regular indications. Among these, the Turner's short stature is presently under experimental evaluation for its responsiveness to rhGH. Twelve patients, 10 with a 45, X karyotype, 1 46 XXiq, and 1 mosaicism, have been given rhGH at a dosage of 0.15 U/kg per injection six times a week. Mean age at onset of treatment was 12.8, mean growth retardation was 4.1 SDS according to Sempé. After 18 months of treatment mean growth catch-up was 0.9 SDS. Maximal velocity was reach during the first trimester of treatment and decreased thereafter but was above normal for bone age in all but 2 after 18 months. The bone age increased less than structural age. No side effects were reported. At the present time the efficacy of rhGH in increasing final height in Turner's patients is likely but not demonstrated by any studies. The exact place of ovarian substitution, even during the prepubertal period, is still matter of discussion. Since the velocity response to rhGH was maximal among the youngest patients an early diagnosis of the syndrome will likely be necessary to improve final stature.     

Remote assessment of ovarian response and follicular status using visual analysis of ultrasound images

Computer assisted evaluation of ultrasonographic image attributes indicative of viability and atresia of ovarian follicles has the potential to become an integral part of ovarian superstimulation protocols. However, in many cases, animal handling facilities, laboratories providing image analysis services and the individual making clinical decisions are geographically separated. The feasibility of remote assessment of follicular status and ovarian response to superstimulation is demonstrated using internet and video conferencing techniques. A cohort of heifers (n = 6) was subjected to ovarian superstimulation. Ultrasound images of the ovarian responses were digitally acquired and transmitted to a distant laboratory for quantitative assessment. Images from follicles which ovulated in response to luteolysis and GnRH treatments were visually and quantitatively different from follicles committed to atresia. Two types of atresia were observed; images of non-ovulatory follicles were characterized as being reflective of the potential to develop into either follicular or luteal cysts. It is probable that the response of individual follicles to the pharmacologic agents used for ovarian superstimulation and ovulation induction may be as important as the total number of follicles recruited. Thus, assessment of the progress and fates of individual follicles may be used to tailor ovarian stimulation to individual donor animals to increase the probability of successful ovarian stimulation and embryo production. The use of the internet for data transfer, image analysis and clinical evaluation places the prospects of providing useful information within the grasp of practitioners who wish to have access to the biological information, but do not wish to invest in the equipment required to make quantitative assessments of visual data.     

Phototrophs in high-iron-concentration microbial mats: physiological ecology of phototrophs in an iron-depositing hot spring

At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [(14)C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.     

The phylogenetic affinities of the chaetognaths: a molecular analysis

The chaetognaths, or arrowworms, constitute a small and enigmatic phylum of marine invertebrates whose phylogenetic affinities have long been uncertain. A popular hypothesis is that the chaetognaths are the sister group of the major deuterostome phyla: chordates, hemichordates, and echinoderms. Here we attempt to determine the affinities of the chaetognaths by using molecular sequence data. We describe the isolation and nucleotide sequence determination of 18S ribosomal DNA from one species of chaetognath and one acanthocephalan. Extensive phylogenetic analyses employing a suite of phylogenetic reconstruction methods (maximum parsimony, maximum likelihood, evolutionary parsimony, and two distance methods) suggest that the hypothesized relationship between chaetognaths and the deuterostomes is incorrect. In contrast, we propose that the lineage leading to the chaetognaths arose prior to the advent of the coelomate metazoa.