Missense mutations in the regulatory domain of PKC gamma: a new mechanism for dominant nonepisodic cerebellar ataxia

We report a nonepisodic autosomal dominant (AD) spinocerebellar ataxia (SCA) not caused by a nucleotide repeat expansion that is, to our knowledge, the first such SCA. The AD SCAs currently comprise a group of > or =16 genetically distinct neurodegenerative conditions, all characterized by progressive incoordination of gait and limbs and by speech and eye-movement disturbances. Six of the nine SCAs for which the genes are known result from CAG expansions that encode polyglutamine tracts. Noncoding CAG, CTG, and ATTCT expansions are responsible for three other SCAs. Approximately 30% of families with SCA do not have linkage to the known loci. We recently mapped the locus for an AD SCA in a family (AT08) to chromosome 19q13.4-qter. A particularly compelling candidate gene, PRKCG, encodes protein kinase C gamma (PKC gamma), a member of a family of serine/threonine kinases. The entire coding region of PRKCG was sequenced in an affected member of family AT08 and in a group of 39 unrelated patients with ataxia not attributable to trinucleotide expansions. Three different nonconservative missense mutations in highly conserved residues in C1, the cysteine-rich region of the protein, were found in family AT08, another familial case, and a sporadic case. The mutations cosegregated with disease in both families. Structural modeling predicts that two of these amino acid substitutions would severely abrogate the zinc-binding or phorbol ester-binding capabilities of the protein. Immunohistochemical studies on cerebellar tissue from an affected member of family AT08 demonstrated reduced staining for both PKC gamma and ataxin 1 in Purkinje cells, whereas staining for calbindin was preserved. These results strongly support a new mechanism for neuronal cell dysfunction and death in hereditary ataxias and suggest that there may be a common pathway for PKC gamma-related and polyglutamine-related neurodegeneration.     

Spleen and cardiovascular function during short apneas in divers.

We investigated the spleen volume changes as related to the cardiovascular responses during short-duration apneas at rest. We used dynamic ultrasound splenic imaging and noninvasive photoplethysmographic cardiovascular measurements before, during, and after 15-20 s apneas in seven trained divers. The role of baroreflex was studied by intravenous bolus of vasodilating drug trinitrosan during tidal breathing. The role of lung volume was studied by comparing the apneas at near-maximal lung volume with apneas after inhaling tidal volume, with and without cold forehead stimulation. In apneas at near maximal lung volume, a 20% reduction in splenic volume (P = 0.03) was observed as early as 3 s after the onset of breath holding. Around that time the heart rate increased, the mean arterial pressure abruptly decreased from 89.6 to 66.7 mmHg (P = 0.02), and cardiac output decreased, on account of reduction in stroke volume. Intravenous application of trinitrosan resulted in approximately 6-mmHg decrement in mean arterial pressure, while the splenic volume decreased for approximately 13%. In apneas at low lung volume, the early splenic contraction was also observed, 10% without and 12% with cold forehead stimulation, although the mean arterial pressure did not change or even increased, respectively. In conclusion, the spleen contraction is present at the beginning of apnea, accentuated by cold forehead stimulation. At large, but not small, lung volume, this initial contraction is probably facilitated by downloaded baroreflex in conditions of decreased blood pressure and cardiac output.     

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Summary In the presence of inhibitors for mitochondrial H⁺-ATPase, (Na⁺+K⁺)- and Ca²⁺-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca²⁺ and Mg²⁺, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg²⁺ and Mn²⁺, but not by Ca²⁺, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H⁺ secretion is electrogenic and requires either exit of a permeant anion (Cl^–) or entry of a cation, e.g., Na⁺ via electrogenic Na⁺/d-glucose and Na⁺/l-phenylalanine uptake. In the presence of Na⁺, ATP-driven H⁺ efflux is stimulated by blocking the Na⁺/H⁺ exchanger with amiloride. These data prove the coexistence of Na⁺-coupled substrate transporters, Na⁺/H⁺ exchanger, and an ATP-driven H⁺ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H⁺ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.     

Elevation in resting blood flow attenuates exercise hyperemia.

These experiments tested the hypothesis that elevating muscle blood flow before exercise would wash out vasoactive substances produced by muscle contraction and reduce the magnitude of exercise hyperemia and/or delay the response. In chronically instrumented dogs (n = 7), hindlimb blood flow was measured with chronically implanted flow probes during mild treadmill exercise. In an anesthetized preparation (n = 8), arterial and venous blood flows of a single hindlimb were obtained during 1-s tetanic contractions evoked by electrical stimulation of the cut sciatic nerve. Elevation of blood flow by intra-arterial infusion of adenosine attenuated the increase in flow during exercise and tetanic contraction by 48 and 47%, respectively. No delay was observed in the latency to peak flow. The attenuated hyperemic response to exercise or contraction is best explained by washout of vasoactive substance(s) produced by contracting muscle, but the residual response suggests that a metabolic mediator may not be the sole explanation for exercise hyperemia.     

Rapid escape reflexes in aquatic oligochaetes: variations in design and function of evolutionarily conserved giant fiber systems

Summary This study provides neuroanatomical and electrophysiological evidence that an arrangement of three dorsal giant fibers, functioning as two distinct and dichotomous conduction pathways, has been evolutionarily conserved within the three major orders of aquatic and terrestrial oligochaetes. The medial giant fiber (MGF), activated by afferents of anterior segments, initiates anterior shortening; whereas, the two lateral giant fibers (LGFs), activated in synchrony by afferents of posterior segments, initiate a different response (usually tail withdrawal). Notwithstanding these common features, the design and function of LGF systems differ considerably in aquatic and terrestrial groups. In posterior segments of aquatic species, LGFs are disproportionately larger and conduct faster than MGFs. This contrasts with posterior segments of earthworms in which LGFs are smaller and conduct slower than MGFs.In addition, in aquatic tubificids, a single LGF spike is sufficient to evoke rapid and complete tail withdrawal, whereas a pair of closely-spaced LGF spikes are needed to elicit posterior shortening in earthworms. The graded nature of earthworm escape seems appropriate for worms that burrow in relatively hard substrates and may frequently encounter inanimate stimuli that evoke meaningless giant fiber spiking. On the other hand, the all-or-none nature of the tubificid escape appears advantageous for relatively sedentary worms that are vulnerable to intense predation but reside in aqueous sediments where triggering of giant fiber spikes by non-threatening stimuli is infrequent.Our studies suggest that anatomical and physiological modifications of giant fiber pathways in aquatic and terrestrial worms have occurred during the evolution of oligochaete nervous systems. We hypothesize that differential predation pressures, together with fundamental differences in lifestyle and habitat, have led to this divergence in the structure and function of evolutionarily conserved sets of homologous giant interneurons.Abbreviations HRP horse raddish peroxidase - LGF lateral giant fiber - MGF medial giant fiber - VNC ventral nerve cord     

Quantitative evaluation of the trapping reaction of copperthiocholine histochemical procedures for localization of cholinesterases

Summary The aim of the present study was to investigate whether the trapping reaction of the histochemical procedure for the localization of ChE of Koelle and Friedenwald (1949) and its modification by Brzin and Pucihar (1976) proceeds quantitatively. The weight of the precipitate formed in the tissue sample during the histochemical procedure was compared with enzyme activity of an equal sample. The differential magnetic microbalance was used for measurements of reduced weight and for previous determination of density of the precipitate. The evidence for the composition of the final product was drawn from the quantitative analysis of copper and iodine and from the infrared spectra.Tsuji's statement (1974) that cuprous copper thiocholine iodide is the final product of histochemical procedures investigated was confirmed. It was found that the trapping reaction of the original as well as of the modified procedure under our experimental conditions in tissue sections proceeds quantiatively which means that one of the basic conditions for reliable localization is fulfilled.This work was supported by the grant of Research Association of Slovenia and NIH Grant No 02-008-1, Z-ZF-6     

Circadian rhythms from multiple oscillators: lessons from diverse organisms

The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.     

Synthesis of hydroquinone-α-glucoside by α-glucosidasefrom baker’s yeast

Hydroquinone-α-glucoside was synthesised from hydroquinone and maltose as glucosyl donor by transglucosylation in a water system with α-glucosidase from baker’s yeast. Only one phenolic –OH group was α-anomer-selectively glucosylated. The optimum conditions for transglucosylation reaction were at 30 °C for 20 h with 50 mM hydroquinone and 1.5 M maltose in 100 mM sodium citrate/phosphate buffer at pH 5.5. The glucoside was obtained at 0.6 mg/ml with a 4.6% molar yield with respect to hydroquinone.