Carbon and hydrogen metabolism of xylitol and various sugars in human erythrocytes.

1) Erythrocytes are able to metabolize D-ribose, D-xylitol, D-xylulose, D-fructose and D-glucose; the rates of metabolism increase in that order from 2430 to 26200 ng atom C/ml packed cells per 120 min of incubation. 2) The utilization of the carbon of these substrates and its recovery in the products were found to be in balance, when the change in the 2,3-bisphosphoglycerate concentration was taken into account. 3) The metabolic rates strongly affected the 2,3-bisphosphoglycerate level. Without addition of substrate the decomposition rate of this intermediate was found to be 1030 nmol/ml packed cells per 120 min. 4) The net decrease of the 2,3-bisphosphoglycerate concentration and the conversion of this compound into lactate provides a NAD regeneration system which enables the red blood cells to utilize xylitol. 5) The rate of carbon metabolism via the pentose phosphate cycle is determined by the NADPH requirement of the erythrocytes which was found to be 160 nmol/ml packed cells per 120 min under the experimental conditions employed.     

The vocal repertoire of chipmunks (Genus Eutamias) in California

A study of the vocalizations of chipmunks was conducted in the field and laboratory, utilizing sonagrams made from tape recordings, and observational data. Most observations were made on Eutamias townsendii, but interspecific variation in alarm calls was studied in all ten California species of Eutamias. The vocal repertoire of Eutamias includes several alarm calls: the chip, chuck, chippering, and in some species also the trill, and in others the whistle. Other calls include the agonistic chatter, courtship chatter, growl, and squeal. The chip is the most common call. There is consistent interspecific variation in structure and temporal arrangement of chips. Most of this variation correlates with body size.     

The H+/site ratio of mitochondrial electron transport.

The number of H+ ejected during passage of 2e- through each energy-conserving site of the mitochondrial respiratory chain (the H+/site ratio) was measured in three ways. In each case transmembrane movements of endogenous phosphate were minimized. (1) Measurement of the uptake of weak acids during loading of mitochondria with Ca2+ demonstrated that 2.0 weak acid anions were accumulated per Ca2+ ion. Since 1.7 to 2.0 Ca2+ ions were were taken up per site, these data correspond to an H+/site ratio of 3.5 to 4.0. (2) More direct measurement of H+ ejection using the oxygen pulse technique demonstrated that the H+/site ratio was 3.0. In these experiments phosphate movements were prevented by addition of N-ethylmaleimide to inhibit phosphate-hydroxide antiport, by washing the mitochondria to remove endogenous phosphate, or by working at 5 degrees C to reduce the rate of phosphate transport. When phosphate movements were allowed, H+/site ratios of 2.0 were observed. (3) Measurement of the initial steady rates of oxygen consumption and H+ ejection following addition of substrate to aerobic, substrate-limited mitochondria yielded H+/site ratios of 2.0, which were elevated to 4.0 when phosphate transport was prevented as described above. Previous determinations of the H+/site ratio were thus underestimates due to the unrecognized movements of endogenous phosphate; our results show that the H+/site ratio is at least 3.0 andmay be as high as 4.0.     

Tritosol: A new scintillation cocktail based on Triton X-100

A scintillation cocktail consisting of 3.0 g PPO, 257 ml Triton X-100, 106 ml ethanol, 37 ml ethylene glycol, and 600 ml xylene is described. A linear relationship between counting efficiency and the external standard ratio could be demonstrated over a wide range of quenching. The counting efficiencies (unquenched) for³H are about 47%, for¹⁴C about 87%, and for⁴⁵Ca about 80%.     

Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria.

The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP.     

Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements.

The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0.     

Zur Spezifität des histochemischen Carboanhydratase-Nachweises im inselorgan der Bauchspeicheldrüse

Zusammenfassung Die enzymatische Aufspaltung von Kobalthydrogenkarbonaten durch das Ferment CAH führt beim Häuslerschen Fermentnachweis über eine sekundäre Visualisation der Kobaltkarbonatniederschläge durch Umwandlung in Kobaltsulfid zur Markierung der Fermentaktivität am Schnitt. Untersuchungen am Pankreas zeigen, daß die Aussagekraft fermentmarkierender Niederschläge durch eine unspezifische Fällung zweiwertiger, nichtfermentgebundener Zinkionen beeinträchtigt wird. Das inkretorische Parenchym des Pankreas enthält keine CAH, wird aber intensiv imprägniert durch seinen Reichtum an nichtfermentgebundenen Zinkionen. Nach Abfangen dieser Metallionen durch Metallchelatbildner (Dithizon, NDDC) fehlt eine Kobaltsulfidschwärzung der Inselzellen.Im exkretorischen Parenchym führt dagegen die Bebrütung von Kryostatschnitten zu spezifischer Fermentmarkierung. Aus sterischen Gründen (tertiäre Bindung des zweiwertigen Metalls im Enzymmolekül) ist das CAH-Zink einer Chelatbindung mit Dithizon oder NDDC nicht zugänglich, die Aktivität der CAH in den Gangepithelien der Bauchspeicheldrüse wird durch Chelatbildner nicht beeinflußt.Die Spezifität des Häuslerschen Fermentnachweises ist unbestritten. Zu fordern ist aufgrund der vorliegenden Ergebnisse aber eine Kontrolle der Fermentreaktion durch Chelatbildner, um eine gleichzeitige unspezifische Imprägnation zweiwertiger Metallionen (insbesondere des Zinks) im Kryostatschnitt auszuschließen.

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Summary In the Häusler incubation method the demonstration of the catalytic activity of carbonic anhydrase in tissue sections involves an enzymatic splitting of cobalt hydrogencarbonates. The resulting carbonate precipitates are secondarily visualized by being transformed into cobalt sulfide. Examination of the pancreas by the modified Häusler reaction indicates that the specificity of the precipitates marking the enzyme is masked by an unspecific precipitation of bivalent zinc ions that are not bound to the enzyme. The endocrine parenchyma of the pancreas does not contain any carbonic anhydrase, but is intensely impregnated because of its abundance of zinc ions bound to insulin or glucagon. If these metallic ions are sequestered by the use of metal chelating agents (dithiozone, sodium diethyldithiocarbamate), the unspecific precipitates in the islets of Langerhans are eliminated.In the exocrine parenchyma, however, the incubation of cryostat sections results in a specific demonstration of the enzyme. The steric arrangement of zinc ions within the enzymic molecules (trivalent complexes of bivalent metal ions) prevents a complex linkage of dithiozone or sodium diethyldithiocarbamate to carbonic anhydrase. The activity of carbonic anhydrase in the epithelium of the ducts is not at all inhibited by chelating agents.The specificity of the Häusler incubation method for demonstrating carbonic anhydrase is uncontested. The results show, however, that in histochemical studies of the enzyme the use of chelating agents is necessary as a control to exclude a simultaneous unspecific precipitation of bivalent metal ions (especially zinc) in cryostat sections.

     

Non-condensation of one segment of a chromosome No. 2 in a male with an otherwise normal karyotype (and severe hypospadias)

Summary A child with severe hypospadias is presented, whose karyotype showed in about 11% of mitoses of peripheral blood one member of chromosome pair No. 2 with a non-condensed region near the centromere. The non-condensed segment does not show late replication, however, it is situated very close to the late replicating segment of the long arms of chromosome No. 2. The nature and possible implications of this kind of aberration are discussed. It is held that non-condensation can produce localized chromosome breaks by a mechanism possibly different from any of the classical breakage mechanisms.