The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

Incubation of subcellular fractions isolated from rat cerebral cortex with [-³²P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.A preliminary account of part of this work has been published (1).     

Visual fields of orb web and single line web spiders of the family Uloboridae (Arachnida,Araneida)

Summary In the family Uloboridae, web reduction is associated with changes in web monitoring posture and prosomal features. A spider must extend its first pair of legs directly forward to monitor the signal line of a reduced web. This posture is facilitated by shifts in prosomal musculature that cause reduced web uloborids to have a narrower anterior prosoma, a reduced or absent anterior eye row, and prominent posterior lateral eye tubercles. The eye tubercles and larger posterior eyes of these uloborids suggest that web reduction may also be accompanied by ocular changes that compensate for reduction of the anterior eyes by expanding the visual fields of the posterior eyes. A comparison of the visual fields of the eight-eyed, orb web species Octonoba octonaria and a four-eyed, reduced web Miagrammopes species was made to determine if this is true. Physical and optical measurements determined the visual angles of each species' eyes and the pattern of each species' visual surveillance. Despite loss of the anterior four eyes, the Miagrammopes species has a visual coverage similar to that of O. octonaria. This is due to (1) an increase in the visual field of each of the four remaining Miagrammopes eyes, accruing from an extension of the retina and an increase in the lens' rear radius of curvature, and (2) a ventral shift of each visual axis, associated with the development of an eye tubercle and an asymmetrical expansion of the retina. Miagrammopes monitor their simple webs from twigs or moss where they are vulnerable to predation. Therefore, maintenance of visual cover may enable them to detect predators in time to assume or maintain their characteristic, cryptic posture. It may also allow them to observe approaching prey and permit them to adjust web tension or prepare to jerk their webs when prey strikes.     

Genetic and Biophysical Study of R Plasmids Conferring Sulfonamide Resistance in Shigella Strains Isolated in 1952 and 1956

The conjugative plasmids determining sulfonamide resistance in five Shigella strains, each isolated from a different patient, have been characterized. One S. flexneri 2a strain, isolated in 1952, harbored an fi(+) plasmid of molecular weight 53 x 10(6), which specified synthesis of F-like pili and bore determinants for sulfonamide resistance (Su) and bacteriocinogeny (Col). This plasmid was compatible with plasmids of groups F(I), F(II), I(alpha), and P. A second S. flexneri 2a strain isolated in 1952 harbored an fi(-) plasmid of molecular weight 59 x 10(6), bearing the Su determinant and compatible with all plasmids tested. This strain also harbored an fi(+) group-F(II) plasmid of molecular weight 42 x 10(6), which bore the Col determinant and specified synthesis of F-like pili. Three S. dysenteriae 2 strains isolated in 1956 carried apparently identical fi(-) plasmids of molecular weight 58 x 10(6), which bore the Su determinant, could form transconjugants in Pseudomonas but not in Proteus, and were incompatible with the P-group plasmid RP4.     

Ultrastructural observations on the kinetosomes,and Golgi bodies during the asexual life cycle ofSaprolegnia

Summary At the onset of zoospore cleavage the centrioles ofSaprolegnia ferax reorientate, develop into kinetosomes and become associated with microtubular roots and a striate fibre. After cytoplasmic cleavage a flagellum, with a hitherto undescribed transition zone structure, develops from each kinetosome. Flagellum axonemes occur inside recently encysted primary spores. In vegetative hyphae and germinating cysts most recognizable Golgi bodies are characteristically associated with a cisternum of the endoplasmic reticulum and a mitochondrion but during sporogenesis they all lie adjacent to nuclei where they are apparently active in vesicle production. The structural details of these changes are described and their significance discussed. We wish to acknowledge the numerous helpful discussions with Dr. J. L. Gay. The senior author held a S.R.C. studentship during the course of this work, part of which was submitted in partial fulfillment of the requirements for the degree of Ph. D. at the University of London.     

Purification of Staphylocidal β-Lysin from Rabbit Serum

A cationic protein of rabbit serum bactericidal for Staphylococcus aureus was purified. The specific activity per unit of protein of the purified staphylocidal preparation was approximately 37,000 times greater than that of the serum from which it was isolated. Similar techniques were used to purify serum beta-lysin active against Bacillus subtilis approximately 24,000 times. The staphylocidal activity cannot be attributed to the same beta-lysin active against B. subtilis, lysozyme, or antibody-complement systems. The concentrations of staphylocidal beta-lysin in the sera of the five mammalian species studied did not correlate with their beta-lysin activities against B. subtilis. The two beta-lysins are similar in that both were heat-stable, sensitive to trypsin digestion, had molecular weights near 6,000, and were found in higher concentrations in serum than in plasma. Furthermore, similar techniques can be used to absorb and elute both substances in highly purified forms using cellulose asbestos filter pads and ion exchange chromatography on carboxymethyl cellulose. In contrast to the beta-lysin against B. subtilis, the staphylocidal beta-lysin was not released from blood platelets, and it was inactive in the presence of heparin, sodium citrate, sodium oxalate, ethylenediaminetetraacetic acid, acidic phospholipids, and acid pH values. A variety of proteins, including those of normal serum, preferentially inhibited the bactericidal activity of staphylocidal beta-lysin but not the beta-lysin against B. subtilis.     

Rapid Schedules for Koh Clearing and Alizarin Red S Staining of Fetal Rat Bone

Various schedules for staining fetal rat skeleton with alizarin red S were tested to determine a procedure that would produce a completely cleared and well-stained specimen in a short period of time. A 2 day procedure is presented which can produce specimens that are satisfactory but not completely transparent. A 7 day procedure produces cleared and stained specimens which can be well visualized with a dissecting microscope (30×). Fetal rats of 21 days gestation were fixed in 10% formalin for at least 1 wk. The specimens were skinned and eviscerated and then dehydrated in 2 changes of acetone for 12 hr (8 ml per gram body weight). The specimens were then placed in 1% KOM-alizarin red S (6 mg/liter) or 3 days, followed by 10% KOH-alizarin red S for 3 days. Finally, the specimens were placed in a mixture of benzyl alcohol, ethanol, and glycerol (1:2:2) (4 ml per gram body weight) for 12 hr, and then transferred to pure glycerol for storage.     

Amino acids as determinants of host preference for the xylem feeding leafhopper,Homalodisca coagulata (Homoptera: Cicadellidae)

Summary Homalodisca coagulata is a highly polyphagous xylem feeder with distinct seasonal patterns in it's selection of host plants. These patterns were examined in relation to the amino acid content of the xylem for four common host species; Lagerstroemia indica, Baccharis halimifolia, Prunus persica, and Prunus salicina. Xylem fluid was collected from each host species at times when numbers of feeding leafhoppers were both low and high. In each case, concentrations of amino acids were greatest when numbers were high. Similarly, comparisons between host species at given times showed that concentrations of amino acids were positively correlated with host selection. In a second study, amino acids of xylem were manipulated by budding scions of a non-preferred host (P. persica) on rootstocks of preferred (P. salinica) and non-preferred (P. persica) hosts. Morphology and phenology of the budded trees were similar to that of the scion species yet the xylem composition of amino acids was primarily dependent on the rootstock. Concentrations of amino acids and the preference of leafhoppers were roughly two-fold greater for scions of the preferred than the non-preferred rootstock. In both studies, amides (glutamine plus asparagine) were the amino acids most highly correlated with host selection. These compounds are the predominant amino acids in xylem fluid, have high nitrogen to carbon ratios, and account for a high percentage of the caloric value in xylem fluid. Many of the less abundant amino acids were positively correlated with host preference, but the correlations were less consistent and correlation coefficients were generally lower.Florida Agricultural Experiment Station Journal Series No. 9672     

Evidence that actin reinforces the extensible hyphal apex of the oomyceteSaprolegnia ferax

Summary Filamentous actin in the apices of growing hyphae of the oomyceteSaprolegnia ferax is distributed such that it could compensate for weakness in the expanding apical cell wall and thus play a role in morphogenesis of the tip. The tapered extensible portion of the hyphal tip where the cell wall is plastic contains a cap of actin which differs in organization from the actin in subapical, inextensible regions of the hypha. Rapidly growing hyphae which are expected to have a longer plastic cell wall region contain longer actin caps. Furthermore, the weakest point in the hyphal apex, demonstrated by osmotic shock-induced bursting, was within the taper where the wall is plastic but never in the extreme apex where actin was most densely packed and presumably the strongest. Treatment of hyphae with cytochalasin E/dimethyl sulphoxide induced rapid changes in actin caps. Cap disruption was accompanied by transient growth rate increases, subsequent rounding and swelling of apices and a shift of osmotically induced burst points closer to the apex. These correlated changes are consistent with a role for the actin cap in tip morphogenesis. The association between regions of plasticity in the apical cell wall, the extent of the actin cap, the location of the weakest point in the apex and the effects of damage to the actin cap suggest that the cap functions to support the apex in regions where the cell wall is weak.Abbrevations CE cytochalasin E - DMSO dimethyl sulphoxide - RP rhodamine phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

Effect of gestational sex steroid exposure on limb development and endochondral ossification in the pregnant C57Bl/6J mouse: I. Medroxyprogesterone acetate

Although data supporting the teratogenic potential of intrauterine progestin exposure is lacking, concern persists among some individuals within the scientific community that these drugs have the potential for nongenital teratogenesis, especially with regard to limb reduction defects. Our laboratory has been interested in the ontogeny of steroid receptors in the developing embryo and in the role of steroid-receptor interactions in limb development, particularly the process of endochondral ossification. Since limb reduction defects can be produced from abnormal processes that are operative during organogenesis or during midgestation (vascular disruption) we have designed an animal study whereby embryos were exposed to sex steroids throughout organogenesis and fetal development. The present study assesses the effects of medroxyprogesterone acetate (MPA) on intrauterine endochrondral bone development specifically, as well as overall embryo-fetal development. Primagravid C57Bl/6J mice were treated via subdermal pellets which deliver MPA at dosages of 5.0, 50.0, and 500.0 mg/kg/day on gestational days 7 through 19. These doses were 25-, 250-, and 2,500-fold higher on a mg/kg basis than the human dose equivalent (HDE). No increases in nongenital malformations were noted at any evaluated MPA dosage level. At 25 X the HDE, MPA did not influence endochondral bone development as evidenced by a lack of significant effects on assessed bone growth parameters. In the 250- and 2,500-fold HDE dosage groups, MPA was shown to exert an embryotoxic effect inducing 48 and 100% resorptions respectively. Mean embryo weights/litter were significantly reduced by MPA exposure at 250 X the HDE. Intrauterine exposure to 250 X the MPA HDE induced reductions in humeral and femoral diaphyseal length in proportion to a reduction in overall growth. The data demonstrate that MPA, administered at dosages of up to several orders of magnitude in excess of the HDE and which permitted embryo survival, did not induce increases in the frequency of nongenital teratogenesis at any dose or gestational stage. Importantly, limb reduction defects were not noted even in instances where the dosage of MPA induced an inhibition of endochondral bone growth.