Rates of diffusion of fluorescent molecules via cell-to-cell membrane channels in a developing tissue

Diffusion coefficients for the intercellular movement of fluorescent tracers have been measured in the epidermis of a larval beetle. Fluorescent tracer was injected into a cell and the spread of tracer from cell to cell in this monolayer was recorded by a TV camera. Fluorescence intensities were digitized from the TV images at successive times after the start of injection at various distances from the source by a microcomputer interfaced with a video analyzer. From the relationship between concentration (measured as light intensity), time and distance, an effective diffusion coefficient (De) is calculated for the tracer in the tissue. In newly ecdysed epidermis, De for carboxyfluorescein (CF) is 2.7 X 10(-7) cm2/s, and De for lissamine rhodamine B (LRB) is 1.2 X 10(-7) cm2/s, whereas in intermolt epidermis the De's for CF and LRB are 3.7 X 10(-7) and 1.2 X 10(-7) cm2/s, respectively. These diffusion coefficients are only an order of magnitude lower than their values in water. The ratio of De for the two tracers at these two stages of development differs from the ratio predicted in cytoplasm alone, with the movement of the slightly larger molecule (LRB) being impeded relative to that of the smaller molecule (CF). This suggests that the properties of the membrane channels amplify differences in the rates of movement of molecules of similar size. This may be important during cell patterning in development. De for CF was also monitored as junctional resistance was increased in the epidermis. During 30 min of exposure to 0.25 mM chlorpromazine, De dropped to 20% of its initial value of 5 X 10(-7) cm2/s, implying that the junctional membrane, rather than cytoplasm, is the major barrier to molecular diffusion among the cells.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Patterns of daily flight activity in onitine dung beetles (Scarabaeinae: Onitini)

Different species of African dung beetles emerge from the soil at characteristic times of the day to fly and colonize the freshly-deposited dung of mammalian herbivores. Onitine dung beetles in their natural habitat displayed one of five distinctive daily flight behaviours: dusk crepuscular (Onitis alexis Klug, O. caffer Boheman, O. fulgidus Klug, O. tortuosus Houston, O. vanderkelleni Lansberge, O. westermanni Lansberge); dusk/dawn crepuscular (O. pecuarius Lansberge and O. viridulus Boheman); dusk/dawn crepuscular and nocturnal (O. aygulus (Fabricius), O. mendax Gillet, O. uncinatus Klug); late afternoon-dusk and dawn-early morning [Heteronitis castelnaui (Harold)]; or diurnal flight activity [O. belial (Fabricius), O. ion (Olivier)]. These diagnostic daily flight behaviours span a light intensity range of over 6 orders of magnitude and have been retained in selected species introduced into Australia. Ambient light intensity appears to be the primary determinant of the daily flight period in onitine dung beetles. Because the dung of mobile herbivores is rapidly exploited by onitine species for feeding and breeding purposes, different flight behaviours result in a spatial and temporal partitioning of species in the local dung beetle community. The timing of flight may contribute to, or lead to avoidance of, competition between species which may ultimately affect colonization success. Many onitines show a strong preference for dung of specific herbivores, which may further reduce interspecific competition. All crepuscular-nocturnal species examined raised their thoracic temperatures endothermically to between 35°C and 40°C before the onset of flight. In O. aygulus the thoracic temperature excess was as large as 19.3°C. The thermal threshold below which the frequency of flight onsets drops off rapidly is about 12°C for O. aygulus and 17°C for O. alexis and O. pecuarius. Radiant loss of body heat during cool nights and dawns may explain why smaller species (<0.4 g body weight), in particular, are adapted behaviourally so that they fly only during the day or early dusk.     

Active muscle migration during insect metamorphosis

The R1 abdominal retractor muscles of the insect Tenebrio molitor change position during the course of metamorphosis. These muscles detach from the epidermal tendon cells at their anterior ends, and migrate in a posterior direction, parallel to the body axis, to form completely new attachments shortly before adult emergence. Movement is preceded by the loss of sarcomere structure, and the muscles migrate in a partially dedifferentiated condition, closely accompanied by satellite cells and haemocytes. Movement appears to result from the extension of muscle processes towards the epidermis posterior to the larval attachment sites, which contact reciprocal processes extended from the epidermis. Contacts at the new posterior sites are then reinforced, and relinquished at the anterior. This cycle is subsequently repeated. It is envisaged that migration ceases when the muscles encounter a contour in the epidermal gradient known to specify the position of the adult muscle attachment sites. This positional information may be encoded in the epidermal basal lamina. The muscles then redifferentiate, with concurrent differentiation of new epidermal tendon cells. Development of adult muscle attachments appears to require reciprocal morphogenetic interactions between muscle and epidermis.     

Substrate-stereoselectivity of a high-affinity glutamate transporter cloned from the CNS of the cockroach Diploptera punctata

A cDNA encoding a Na(+)-dependent glutamate transporter has been cloned from the brain of the cockroach Diploptera punctata. The cDNA encodes a transporter protein of 481 amino acids, designated DipEAAT1, which when expressed in baculovirus infected insect cells, resulted in a 40-50 fold increase in [(3)H]L-glutamate uptake. DipEAAT1 mRNA is expressed in the brain, as is the RNA encoding TrnEAAT1, a related transporter recently isolated from the caterpillar Trichoplusia ni. The affinity of these transporters for L-glutamate and several structural analogues was compared. Both have a high affinity for L-glutamate, their presumed primary substrate, but quite different affinities for D-aspartate. TrnEAAT1 was found to be similar to other glutamate transporters in that its ability to transport [(3)H]L-glutamate into cells was inhibited strongly by D- and L- isomers of aspartate and its analogues. DipEAAT1, by contrast, was inhibited weakly by all D- isomers tested. The affinity of DipEAAT1 for [(3)H]D-aspartate was found to be an order of magnitude lower than that of TrnEAAT1, revealing an unusual stereoselectivity for aspartate substrates by the cockroach transporter. The activity of DipEAAT1 was also unaffected by the presence of Zn(++) in the bathing solution, despite the presence of a putative Zn(++)-binding motif conferring Zn(++)-sensitivity on some mammalian glutamate transporters.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.