Volumetric Instillation of Fixatives and Inert Substances Into Mouse Lungs

Mouse lungs were instilled with various fixatives to establish the optimum volume necessary to fix the lung without distortion and to compare the efficacy of the fixatives. Fixation with either Stieve's or Bouin's fluid was found preferable to 2.5% and 5% glutaraldehyde, 4% neutral buffered formalin, and to a mixture of formalin and Stieve's fixative. In addition, a comparison was made between diluted Ames O.C.T. Compound and 4% aq. gelatin as supportive substances for unfixed lungs in preparation of cryostat sections and for histochemistry. A 1:2 dilution of O.C.T. was found to be superior to 4% gelatin in preparative, cutting and adhesive properties. The optimal instilled volume for mouse lungs was found to be 0.1 ml for every 7 grams of body weight, introduced at a rate of 0.1 ml per 10 seconds.     

Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca²⁺ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N₃ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC₅₀ = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.     

Micromethods in single muscle fibers. 1. Determination of catalase and superoxide dismutase

Methods have been developed for the measurements of catalase and superoxide dismutase (SOD) in single, isolated muscle fibers. These fibers are also classified according to fiber type. Catalase is determined using a fluorescent method for the measurement of hydrogen peroxide consumed. SOD measurements are carried out using a modification of established techniques whereby the inhibition of oxidation of epinephrine by SOD is assayed fluorometrically. Both enzymes may be determined in submicrogram samples of dried muscle. This approach avoids the complication of the inclusion of nonmuscle tissue with varying enzymatic activities which is frequently experienced when using homogenates of muscle, particularly diseased muscle. In addition, these techniques can be used to determine the inherent variation in SOD and catalase activities within individual fibers of the same fiber type. The Km and Vmax for catalase, determined using homogenates of human muscle, were found to be 12 mM and 1.45 mumol/min/mg dry wt, respectively. Catalase of muscle was inhibited 50% by 2 microM sodium azide. Mn-SOD contributes less than one-fifth of the total SOD activity. Therefore the activity is largely due to the Cu-Zn form of SOD. These methods are applicable to a wide variety of tissues.     

Differential net food utilization by larvae of Sitophilus oryzae and Sitophilus granarius

The net utilization of a meridic diet by Sitophilus oryzae from the newly hatched larva to the pupa was more efficient compared with that of S. granarius. The artificial diet was highly digestible to both species, but S. oryzae converted 24·1 per cent of the ingested food into body substance compared with 12·4 per cent converted by S. granarius. The mean pupal weight of S. granarius (1·402 mg) was greater than that of S. oryzae (1·012 mg); however, larvae of S. granarius consumed 2·6 times as much food and excreted 8·1 times as much faecal material to obtain that weight. Even though S. granarius consumed the diet at a greater rate, the relative growth rate of S. oryzae was faster. The intracellular symbiotes present in S. oryzae may have a rôle in the species' faster development and more efficient utilization of its food.     

The jamming avoidance response in gymnotoid pulse-species: A mechanism to minimize the probability of pulse-train coincidence

Summary Gymnotoid electric fish with pulse-type electric organ discharges (EODs) shorten (lengthen) their EOD intervals as pulses of a slightly slower (faster) train scan their EODs (Figs. 1, 2). They thus minimize the chance of pulse coincidence by transient accelerations (decelerations) of their EOD rate.Studies in curarized preparations demonstrate that this Jamming Avoidance Response (JAR) is controlled by electroreceptive input alone and without reference to an internal electric organ pacemaker-related signal (Fig. 8). A sufficient stimulus input consists of a train of strong, EOD-like stimulus pulses (S₁), which mimic the animal's experience of its own EOD, and a train of small pulses (S₂) of slightly different repetition rate, which mimic EODs of a neighbor. Correct behavioral responses require S₁ pulses of sufficient intensity to recruit pulse-markertype receptors; also spatial and temporal patterns must closely resemble those of the animal's EOD. These features are of little significance for S₂ pulses which, while scanning S₁ pulses, only provide a small perturbation of electroreceptive feedback from S₁ pulses. Inappropriate S₁ stimulation impairs and sometimes reverses (Fig. 7) the behavioral discrimination of scan directions. The JAR is explained in terms of excitatory and inhibitory processes (Fig. 3) which are triggered by S₂ stimulation, at specific phases within the S₁ cycle (Figs. 4–6).The JAR in pulse species strongly resembles the JAR in wave-species (Bullock et al., 1972) and could be considered an evolutionary ancestor of the latter. It is a response to a particular novelty in electroreceptive feedback.We thank Drs. T.H. Bullock, C. Hopkins and an anonymous referee for most helpful criticism. This research was supported by NSF grand BMS74-18640 and NIMH grant PHSMH-2614901 to W.H. and NIH grant/ROI NS 12337-01 to J.B.     

A clonal analysis reveals early developmental restrictions in the Drosophila head.

Head development of Drosophila melanogaster was studied by forming, in a background of Minute (M) cells, clones whose cell division rate was higher (M⁺). By studying such clones true developmental restrictions on clonal growth may be revealed, and not restrictions on clones which are just the result of interactions between neighboring cells. Pigment, bristle, and trichome markers allowed clone detection in both the compound eye and in much of the head cuticle. Clones were formed by X-ray-induced mitotic recombination at three stages in the first and second instar. Initial experiments determined some parameters of cell division in the compound eye and verified the differential division rate of M⁺ cells growing in an M background, and vice versa. The earliest and most striking developmental restriction on clonal growth divides the head into a dorsal and a ventral compartment. No evidence for anterior-posterior compartmentalization was found. By 75 hr of development in Minute flies, several lines of developmental restriction are formed which subdivide these two compartments. Evidence is presented which supports these conclusions: One subdivision may contain cells of different clonal origin, cells derived from the same progenitor blastoderm cell may be in different subdivisions, and each subdivision is formed from a group of contiguous cells.