Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen

This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise.     

Response of enzymes and storage proteins of maize endosperm to nitrogen supply

To examine the effects of N nutrition upon endosperm development, maize (Zea mays) kernels were grown in vitro with either 0, 3.6, 7.1, 14.3, or 35.7 millimolar N. Kernels were harvested at 20 days after pollination for determination of enzyme activities and again at maturity for quantification of storage products and electrophoretic separation of zeins. Endosperm dry weight, starch, zein-N, and nonzein-N all increased in mature kernels as N supply increased from zero to 14.3 millimolar. The activities of sucrose synthase, aldolase, phosphoglucomutase, glutamate-pyruvate transaminase, glutamate-oxaloacetate transaminase, and acetolactate synthase increased from 1- to 2.5-fold with increasing N supply. Adenosine diphosphate-glucose pyrophosphorylase and both ATP- and PPi-dependent phosphofructokinases increased to lesser extents, while no significant response was detected for hexose kinases and glutamine synthetase. Nitrogen-induced changes in enzyme activities were often highly correlated with changes in final starch and/or zein-N contents. Separation of zeins indicated that these peptides were proportionately enhanced by N supply, with the exception of C-zein, which was relatively insensitive to N. These data indicate that at least a portion of the yield increase in maize produced by N fertilization is induced by a modification of kernel metabolism in response to N supply.     

Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm

Soluble starch synthase (SSS) I was purified 361-fold from hand-dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min⁻¹ mg⁻¹. A key to this purification protocol was the introduction of a size-exclusion chromatography step, a size-based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl-Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.     

Rhythmic swimming activity in neurones of the isolated nerve cord of the leech.

1. Repeating bursts of motor neurone impulses have been recorded from the nerves of completely isolated nerve cords of the medicinal leech. The salient features of this burst rhythm are similar to those obtained in the semi-intact preparation during swimming. Hence the basic swimming rhythm is generated by a central oscillator. 2. Quantitative comparisons between the impulse patterns obtained from the isolated nerve cord and those obtained from a semi-intact preparation show that the variation in both dorsal to ventral motor neurone phasing and burst duration with swim cycle period differ in these two preparations. 3. The increase of intersegmental delay with period, which is a prominent feature of swimming behaviour of the intact animal, is not seen in either the semi-intact or isolated cord preparations. 4. In the semi-intact preparation, stretching the body wall or depolarizing an inhibitory motor neurone changes the burst duration of excitatory motor neurones in the same segment. In the isolated nerve cord, these manipulations also change the period of the swim cycle in the entire cord. 5. These comparisons suggest that sensory input stabilizes the centrally generated swimming rhythm, determines the phasing of the bursts of impulses from dorsal and ventral motor neurones, and matches the intersegmental delay to the cycle period so as to maintain a constant body shape at all rates of swimming.     

Selenium Species and Their Distribution in Freshwater Fish from Argentina

The distribution and speciation of selenium (Se) in freshwater fish (muscle and liver tissue) from lakes in Argentina was investigated. Three introduced species, brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis), and one native species, creole perch (Percichthys trucha), were investigated. Values for total selenium in muscle ranged from 0.66 to 1.61 μg/g, while in the liver, concentrations were much higher, from 4.46 to 73.71 μg/g on a dry matter basis. Separation of soluble Se species (SeCys₂, selenomethionine (SeMet), SeMeSeCys, selenite and selenate) was achieved by ion exchange chromatography and detection was performed by inductively coupled plasma–mass spectrometry. The results showed that in fish muscle, from 47 to 55 % of selenium was soluble and the only Se species identified was SeMet, which represented around 80 % of soluble Se, while in the liver, the amount of soluble Se ranged from 61 to 76 % and the percentage of species identified (SeMet and SeCys₂) was much lower and ranged from 8 to 17 % of soluble Se.     

Shift work,circadian gene variants and risk of breast cancer

Circadian (clock) genes have been linked with several functions relevant to cancer, and epidemiologic research has suggested relationships with breast cancer risk for variants in NPAS2, CLOCK, CRY2 and TIMELESS. Increased breast cancer risk has also been observed among shift workers, suggesting potential interactions in relationships of circadian genes with breast cancer. Relationships with breast cancer of 100 SNPs in 14 clock-related genes, as well as potential interactions with shift work history, were investigated in a case–control study (1042 cases, 1051 controls). Odds ratios in an additive genetic model for European-ancestry participants (645 cases, 806 controls) were calculated, using a two-step correction for multiple testing: within each gene through permutation testing (10,000 permutations), and correcting for the false discovery rate across genes. Interactions of genotypes with ethnicity and shift work (<2 years vs ≥2 years) were evaluated individually. Following permutation analysis, two SNPs (rs3816360 in ARNTL and rs11113179 in CRY1) displayed significant associations with breast cancer and one SNP (rs3027188 in PER1) was marginally significant; however, none were significant following adjustment for the false discovery rate. No significant interaction with shift work history was detected. If shift work causes circadian disruption, this was not reflected in associations between clock gene variants and breast cancer risk in this study. Larger studies are needed to assess interactions with longer durations (>30 years) of shift work that have been associated with breast cancer.     

Reconstructive management of contralateral breast cancer in patients who previously underwent unilateral breast reconstruction

When a patient who has had unilateral breast reconstruction presents with a new cancer on the opposite side, the reconstructive management of the second breast can be unclear. This study was performed to determine whether reconstruction of the second breast is oncologically reasonable and to evaluate the reconstructive options available to these patients.Patients who had mastectomy with unilateral breast reconstruction between 1988 and 1994 and who had a minimal follow-up of 5 years from the initial breast cancer were reviewed. Of 469 patients reviewed, 18 patients (4 percent) were identified who developed contralateral breast cancer. Mean age at the initial breast cancer presentation was 43 years (range, 26 to 57 years), and mean age at presentation with contralateral breast cancer was 48 years (range, 36 to 67). The mean interval between the initial and contralateral breast cancer presentations was 5 years (range, 1 to 10 years). Mean follow-up from the time of contralateral breast cancer was 5 years (range, 1 to 9 years). In most cases, contralateral breast cancer presented at an early stage (13 of 18 patients; 72 percent), and a shift to an earlier stage at presentation of the contralateral cancer was evident compared with the initial breast cancer. Of the 18 patients who developed contralateral breast cancer, 16 (89 percent) had no evidence of disease, one was alive with disease, and one died. Reconstructive management after the initial mastectomy included 16 transverse rectus abdominis myocutaneous flaps (seven free and nine pedicled), one latissimus dorsi myocutaneous flap with implant, and one superior gluteal free flap. Surgical management of the second breast after contralateral breast cancer included breast conservation in two patients, mastectomy without reconstruction in four, and mastectomy with reconstruction in 12. Reconstruction of the second breast included one free transverse rectus abdominis myocutaneous flap, three extended latissimus dorsi flaps, two latissimus dorsi myocutaneous flaps with implants, three implants alone, two Rubens flaps, and one superior gluteal free flap. No major complications were noted after the reconstruction of the second breast. The best symmetry was obtained when similar methods and tissues were used on both sides.The incidence of contralateral breast cancer after mastectomy and unilateral breast reconstruction is low. In most cases, contralateral breast cancer presents at an earlier stage compared with the initial breast cancer, and the prognosis is good. In patients who develop a contralateral breast cancer after mastectomy and unilateral breast reconstruction, the reconstruction of the second breast after mastectomy is oncologically reasonable and should be offered to provide optimal breast symmetry and a better quality of life. The best result is obtained when similar methods and tissues are used on both sides.     

Implications of axillary sentinel lymph node biopsy in immediate autologous breast reconstruction

For patients with invasive breast cancer, if the results of an axillary sentinel node biopsy are determined to be positive after permanent pathologic examination, the current recommendation is to perform a complete axillary node dissection. Subsequent axillary surgery may compromise the blood supply to an immediate autologous breast reconstruction. The purpose of this study was to determine which clinicopathologic factors in clinically node-negative breast cancer patients may be associated with an increased risk of positive axillary nodes. Identification of these factors will allow surgeons to modify their approach to immediate autologous breast reconstruction in these high-risk patients. The relationship between presenting clinicopathologic characteristics and the incidence of axillary metastases was analyzed by chi-square test and multivariate analysis in 167 patients with invasive breast cancer and a clinically negative axilla who underwent modified radical mastectomy with an immediate free transverse rectus abdominis musculocutaneous (TRAM) flap reconstruction. Axillary nodal metastases were found in 35 percent of clinically node-negative breast cancer patients. Multivariate analysis showed that patient age of 50 years or younger (p = 0.019), T2 tumor stage or greater (p = 0.031), and presence of lymphovascular invasion on the initial biopsy specimen (p < 0.001) were independent predictors of axillary metastases in clinically node-negative patients. Based on these results, the authors propose an algorithm for decision making in clinically node-negative breast cancer patients who desire autologous breast reconstruction and sentinel lymph node biopsy. Options for immediate autologous breast reconstruction in patients undergoing mastectomy and axillary sentinel lymph node biopsy that may minimize the risk of vascular damage on reoperation include the use of the internal mammary artery and vein as recipient vessels for a free TRAM flap or a pedicled TRAM flap. If an axillary-based blood supply is used, the authors are considering the use of cadaveric dermis to isolate the pedicle of the flap away from the remaining axillary contents. New developments in breast cancer diagnosis and treatment necessitate a team approach, with increased communication between the breast surgeon and the plastic surgeon in planning surgery for these patients.     

Site-dependent angiogenic cytokine production in human tumor xenografts

Tumor microenvironment plays a critical role in tumor growth, angiogenesis, and metastasis. Differences in site of tumor implantation result in differences in tumor growth, metastasis, as well as response to chemotherapy. We hypothesized that tumor-induced angiogenic growth factor production into the plasma will also be influenced by site of tumor implantation. We evaluated the site-dependent production of angiogenic growth factors in the plasma of tumor bearing animals at two different sites of implantation. Plasma levels of tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were evaluated in nude mice bearing A2780, SKOV-3, or OVCAR-3 human ovarian tumors, as well as Panc-1, AsPC-1, or BxPC-3 human pancreatic tumors grown as subcutaneous (SC) xenografts or in the intraperitoneal (IP) cavity. Plasma VEGF and bFGF levels produced by two ovarian tumor lines and two pancreatic tumor lines were substantially higher when the tumors were implanted in the IP cavity than in the SC space. These studies indicated that the site of tumor implantation was an important determinant in the production of plasma VEGF and bFGF levels. As more and more anti-angiogenic agents are developed, the need for appropriate animal models becomes apparent. These results suggest the demand for an appropriate model for the in vivo evaluation of anti-angiogenesis.     

Are mice calorically restricted in nature?

An important question about traditional caloric restriction (CR) experiments on laboratory mice is how food intake in the laboratory compares with that of wild mice in nature. Such knowledge would allow us to distinguish between two opposing views of the anti-aging effect of CR--whether CR represents, in laboratory animals, a return to a more normal level of food intake, compared with excess food consumption typical of laboratory conditions or whether CR represents restriction below that of animals living in nature, i.e. the conditions under which house mice evolved. To address this issue, we compared energy use of three mouse genotypes: (1) laboratory-selected mouse strains (= laboratory mice), (2) house mice that were four generations or fewer removed from the wild (= wild-derived mice) and (3) mice living in nature (= wild mice). We found, after correcting for body mass, that ad libitum fed laboratory mice eat no more than wild mice. In fact, under demanding natural conditions, wild mice eat even more than ad libitum fed laboratory mice. Laboratory mice do, however, eat more than wild-derived mice housed in similar captive conditions. Therefore, laboratory mice have been selected during the course of domestication for increased food intake compared with captive wild mice, but they are not particularly gluttonous compared with wild mice in nature. We conclude that CR experiments do in fact restrict energy consumption beyond that typically experienced by mice in nature. Therefore, the retarded aging observed with CR is not due to eliminating the detrimental effects of overeating.