Extracellular matrix metabolism by chondrocytes 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

THe incorporation of [³H]glycine into acid-insoluble protein and of [³H]acetate into glysoaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gardients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of poly-ribosomes that was reversed by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence on L-glutamine was also demonstrated for other avain connective tissues.     

Rehabilitation of Barbarine cull ewes using rosemary residues and linseed: effect on weight gain,carcass characteristics and meat quality

The cull ewes represent an important part of sheep flock. However, this category of animal is often submitted to under nutrition leading to poor BW and skeletal carcasses. Their rehabilitation using a high energy diet can be an alternative to improve their body condition. The objective of this experiment was to study the BW gain and carcass characteristics of Barbarine cull ewes using rosemary (Rosmarinus officinalis L.) distillation residues (RR) and extruded linseed. For this, 28 ewes above 6 years old and 33±0.5 kg of BW were divided into four groups: CCC was fed 500 g of barley-straw with concentrate, RCC received 300 g of straw and 200 g of RR as basal diet with concentrate; whereas two other groups received the experimental concentrate, containing 10% of linseed, with 500 g of straw for CLC and 300 g of straw plus 200 g of RR for RLC group. At the end of experiment (90 days), all animals were slaughtered. For all ewes, the daily concentrate intake averaged 700 g; the average daily gain was 131 g and the slaughter BW 43.4 kg without significant difference between groups. Neither basal diet nor concentrate type did affect the carcass’ weight, yield and composition. In addition, the organ’s proportions were similar for all groups. The RR intake slightly improved muscle’s protein content (P=0.03) and tended to decrease initial pH (P=0.06) and to increase meat redness (P=0.06), whereas linseed concentrate had no effect on meat color and its chemical composition. The subcutaneous fat color and firmness score relived a good quality trade for carcasses from all diets, in spite of higher yellowness and lower firmness recorded for linseed diet (P<0.05), which were moderately improved by rosemary combination with linseed. To conclude, the Barbarine cull ewes could gain up to 120 g/day in BW. The used diets permitted this BW gain without undesirable effects on carcass characteristics and meat quality. However, the study of meat fatty acid profile and antioxidant status should continue.     

K+(Rb+) and Ca2+ fluxes in young winter wheat plants exposed to sub-zero temperatures

Ice crystal formation temperature was determined in the region of the crown in one group of 7-day-old intact unhardened high-salt plants of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) with TA (Thermal Analysis) and DTA (Differential Thermal Analysis) methods. After exposure of another group of plants, grown for the first 7 days in the same way as the first group, to various sub-zero temperatures (-1 to 5°C), influx in roots of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) and contents of K⁺ and Ca²⁺ were determined at intervals during 7 days of recovery. Ice crystal formation in the crown tissue was probably extracellular and took place at about -4°C. There was a large loss of K⁺ from the roots after treatment at sub-zero temperatures. This loss increased as the temperature of the sub-zero treatment decreased. During recovery, roots of plants exposed to -1, -2 and -3°C gradually reabsorbed K⁺. Reabsorption of K⁺ in roots of plants exposed to -4°C was greatly impaired. Rb⁺ influx decreased and Ca²⁺ influx increased after sub-zero temperature treatments of the plants. Active Rb⁺ influx mechanisms and active extrusion of Ca²⁺ were impaired or irreversibly damaged by the exposure. While Rb⁺ influx mechanisms were apparently repaired during recovery in plants exposed to temperatures down to -3°C, Ca²⁺ extrusion mechanisms were not. The temperature for ice crystal formation in the region of the crown tissue coincides with the temperature at which the plants lost the ability to reabsorb K⁺ and to repair Rb⁺ influx mechanisms during the recovery period. Plants were lethally damaged at temperatures below ?4°C.     

Isolation and characterization of highly purified mosquito oostatic hormone

Oostatic hormone, the hormone that inhibits vitellogenesis in mosquitoes, was purified 7,000-fold with a recovery of 70% from the ovaries of the mosquito Aedes aegypti. The purification procedure included heat treatment and chromatography on ion exchange and gel filtration columns. The hormone is a small peptidelike molecule of molecular weight 2,200 at pH 4.5, which aggregates into larger molecular species of trimer and octamer at pH 7.0 as determined by gel filtration. The hormone is positively charged at pH 7.8 and has a low Rf at pH 9.4 on disc gel electrophoresis. Injection of purified oostatic hormone (9 ng) into female mosquitoes inhibited yolk deposition and vitellogenin synthesis. Activity of the oostatic hormone in the mosquito ovary increased rapidly following blood feeding and reached a maximum after 48 h. Oostatic hormone of A. aegypti injected into autogenous Aedes taeniorhynchus inhibited egg development. Repeated injections of dilute oostatic hormone at 24 h intervals partially arrested egg development, resulting in 60% reduction in the number of eggs laid. This hormone does not block release of egg development neurosecretory hormone (EDNH) from the mosquito brain but rather appears to act on the ovary.     

Direct somatic embryogenesis from axes of mature peanut embryos

Summary Plant regeneration via somatic embryogenesis was obtained in peanut (Arachis hypogaea L.) from axes of mature zygotic embryos. The area of greatest embryogenic activity was a 2-mm region adjacent to and encircling the epicotyl. Somatic embryogenesis was evaluated on Murashige and Skoog media supplemented with a variety of auxin treatments. Maximum production occurred on medium supplemented with 3 mg · liter⁻¹ 4-amino-3,5,6-trichloropicolinic acid. Explant cultures were transferred to half-strength medium supplemented with 1 mg · liter⁻¹ gibberellic acid for somatic embryo germination and early plantlet growth. Plantlets, transferred to soil, were placed in a greenhouse and grown to maturity.     

Three-dimensional perfused cell culture

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell–cell and cell–extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review.     

De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

Cardiovascular and respiratory responses associated with limb autotomy in the blue crab, Callinectes sapidus

Cardiovascular and respiratory variables were recorded in the blue crab, Callinectes sapidus, during injury and subsequent autotomy of a chela. Cardiac function and haemolymph flow rates were measured using a pulsed-Doppler flowmeter. Oxygen uptake was recorded using an intermittent flow respirometry system. Crabs reacted to the loss of a chela with a rapid increase in heart rate, which was sustained for 2 h. Stroke volume of the heart also increased after the chela was autotomized. A combined increase in heart rate and stroke volume led to an increase in cardiac output, which was maintained for an hour after the loss of a chela. There was also differential haemolymph perfusion of various structures. There was no change in perfusion of the anterolateral arteries or posterior and anterior aortae, during injury of the chela or subsequent autotomy. Haemolymph flow rates did increase significantly through the sternal artery during injury and immediately following autotomy of the chela. This was at the expense of blood flow to the digestive gland: a sustained decrease in haemolymph flow through the hepatic arteries occurred for 3 h following autotomy. Fine-scale cardiac changes associated with the act of autotomy included a bradycardia and/or associated cardiac pausing before the chela was shed, followed by a subsequent increase in cardiac parameters. Changes in the cardiovascular physiology were paralleled by an increase in oxygen uptake, which was driven by an increased ventilation of the branchial chambers. Although limb loss is a major event, it appears that only acute changes in physiology occur. These may benefit the individual, allowing rapid escape following autotomy with a subsequent return to normal activity.     

Optimization of biotin and thiamine requirements for somatic embryogenesis of date palm (Phoenix dactylifera L.)

Summary This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic embryogenesis of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured on hormone-free MS medium containing biotin at 0, 0.1, 1, or 2 mg l⁻¹ combined with thiamine at 0.1, 0.5, 2, or 5 mg l⁻¹. Embryogenic callus weight, number of resultant embryos, and embryo length were significantly influenced by thiamine and biotin concentration. The optimum callus growth treatment consisted of 0.5 mg l⁻¹ thiamine and 2 mg l⁻¹ biotin. This treatment also gave the highest number of embryos. Embryo elongation was greatest at 0.5 or 2 mg l⁻¹ thiamine combined with 1 mg l⁻¹ biotin. Embryos from all treatments germinated and regenerants exhibited normal growth in soil. This study provides an insight into the importance of optimizing various culture medium components to overcome in vitro recalcitrace of date palm.