Some physical problems in biology

A review is presented within the framework of the theory of evolution, after it has been extrapolated from the population level to the cellular and molecular levels. From Darwin's seminal and persuasive insight - the theory of common descent - we assume, with him, that probably all the organic beings which have ever lived on this earth have descended from some one primordial form, into which life was first breathed [1]. We are now aware that this primordial cell may have been a protocyanobacterium, but it has often been called a last universal ancestor, a breakthrough organism, or a progenote, a term introduced by Woese [2] which has gained wide acceptance. Strictly speaking, in the intermediate period, ranging from the first living cell to the progenote, life may have evolved in the absence of significant diversity, effectively as a single phylum, incorporating organisms whose genetic systems were already based on DNA. Earlier still, prior to the encapsulation of nucleic acids in microspheres, evolution may already have been at work on RNA molecules (the RNA world). This takes our discussion into the period of chemical evolution, a concept first put forward by Oparin [3], whose principal merit is to have formulated the underlying problem in clear scientific terms. This review does not attempt to be comprehensive. It is mainly devoted to the discussion of certain concepts that may have played a relevant role in the pathway that led to the origin and evolution of the progenote. We do not dwell on the main events of the intermediate period. The topics that we have chosen to include are: the origin of chirality of protein amino acids, the origin of translation, and the origin of the genome. We conclude with some comments on one further aspect of the evolutionary process - the development of biodiversity - by considering the origin of the first eukaryotic cell, an event which, according to the fossil record, may have preceded the evolutionary radiation in the early Cambrian by over a billion years.     

Synergistic effects of cellulolytic and pectinolytic enzymes in degrading sugar beet pulp

Three cellulases, one hemicellulase and three pectinases were used, separately or in binary and ternary combinations, to hydrolyze dried beet-pulp, a by-product of the sugar industry. By IE-HPLC the compositions and concentrations of the sugars released were determined. The results obtained by enzymatic saccharification were compared to those obtained by acid hydrolysis. The synergistic action of cellulolytic and pectinolytic enzymes in release of total monosaccharides, and of glucose, arabinose and galacturonic acid was also studied. The combination of cellulase, hemicellulase and pectinase, commercially available, was as effective in degrading the beet pulp as the acid hydrolysis. Pectinase appeared to be the most important enzyme, since by hydrolyzing the pectic surface of the lignocellulosic substrate, it favoured the degradation of cellulose and hemicellulose by the respective enzymes.     

Lack of Correlation between Activation of Jun–NH2-terminal Kinase and Induction of Apoptosis after Detachment of Epithelial Cells

Detachment of epithelial cells from the extracellular matrix leads to induction of programmed cell death, a process that has been termed “anoikis.” It has been reported recently that detachment of MDCK cells from matrix results in activation of Jun–NH₂-terminal kinases (JNKs) and speculated that these stress activated protein kinases play a causal role in the induction of anoikis (Frisch, S.M., K. Vuori, D. Kelaita, and S. Sicks. 1996. J. Cell Biol. 135:1377–1382). We report here that although JNK is activated by detachment of normal MDCK cells, study of cell lines expressing activated signaling proteins usually controlled by Ras shows that stimulation of JNK fails to correlate with induction of anoikis. Activated phosphoinositide 3-OH kinase and activated PKB/Akt protect MDCK cells from detachment-induced apoptosis without suppressing JNK activation. Conversely, activated Raf and dominant negative SEK1, a JNK kinase, attenuate detachment-induced JNK activation without protecting from apoptosis. zVAD-fmk, a peptide inhibitor of caspases, prevents MDCK cell anoikis without affecting JNK activation. p38, a related stress-activated kinase, is also stimulated by detachment from matrix, but inhibition of this kinase with SB 203580 does not protect from anoikis. It is therefore unlikely that either JNK or p38 play a direct role in detachment-induced programmed cell death in epithelial cells.     

Partial purification of a phosphoethanolamine methyltransferase from rat brain cytosol

The conversion of phosphoethanolamine to phosphocholine requires 3 separate N-methyltransferases. We had previously purified the enzyme catalyzing the last methylation, phosphodimethylethanolamine N-methyltransferase. We have successfully purified the enzyme catalyzing the initial methylation of phosphoethanolamine. A 434 fold purified enzyme from rat brain was obtained by the sequential use of ammonium sulfate fractionation, Q-Sepharose fast flow column chromatography and a -aminoethyl agarose column chromatography. The pH optimum was 11 or greater, the Km value for phosphoethanolamine was 167.8±41.7 M and the Vmax was 487.3±85 mmoles/mg/hr. The kinetics for S-adenosyl-methionine, the methyldonor, has characteristics of cooperative binding with a Km of 1.805±0.59 mM and a Vmax of 16.9±3.6 moles/mg/hr. The activity was stimulated 6 fold by 2.5 mM MnCl₂ and inhibited by DZA and S-adenosylhomocysteine. These results reinforce the early in vivo observations which had provided suggestive evidence for the existence of a pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain.Abbreviations used Adomet S-adenosylmethionine - AdoHcy S-adenosyl-homocysteine - CAPS 3-(cyclohexyl)amino-1-propanesulphonic acid - Cho choline - 3-DZA 3-deazaadenosine - Etn ethanolamine - N-MT N-methyltransferase - PEG polyethyleneglycol - PMSF phenylmethanesulphonyl fluoride - PEtn phosphoethanolamine - PCho phosphocholine - PMe₂Etn phosphodimethylethanolamine - PtdCho phosphatidylcholine - PtdEtn phosphatidylethanolamine     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

Role of exercise testing early after myocardial infarction in identifying candidates for coronary surgery.

It has been suggested that ST depression in lead V5 or equivalent on early exercise testing after acute myocardial infarction predicts a high risk of death. To evaluate exercise testing and radionuclide ventriculography in this context 103 consecutive patients with myocardial infarction who were able to undertake a limited exercise test before discharge from hospital were exercised and underwent gated blood pool scanning. No serious complications resulted from exercise testing. Twenty nine patients developed ST depression in lead V5, 19 had exertional hypotension, 31 developed a heart rate of greater than or equal to 130 beats/min, and 15 had complex ventricular arrhythmias. Death during the first year after discharge from hospital was associated with exertional hypotension (p less than 0.001) and a heart rate on exercise testing of greater than or equal to 130 beats/min (p less than 0.05); these two variables identified all nine deaths. Inability to complete the exercise protocol for any reason was also predictive of death (p less than 0.01). Ventricular arrhythmias and ST depression in lead V5 induced by exercise were not significantly associated with an increased risk of death. The mean (SD) radionuclide ejection fraction in the patients who died was 29 (16%) compared with 43 (11)% in the patients who survived (p less than 0.001). ST changes on exercise testing after myocardial infarction appear to be less predictive of later complications than haemodynamic signs, which may indicate left ventricular damage rather than ischaemia.     

Carbohydrate composition of purified serum glycoproteins in Mucolipidosis II and Mucolipidosis III

Summary Mucolipidosis II (I-cell disease) and Mucolipidosis III (ML III) are inherited disorders in which the molecular defect may involve an abnormality in a common post-translational modification step (possibly glycosylation) shared by lysosomal hydrolases. We tested whether such an alteration might be a generalized defect in glycoprotein biosynthesis and, thus, be reflected in an abnormal carbohydrate composition of non-lysosomal glycoproteins. The apoprotein of low density lipoprotein (apo-LDL) and immunoglobulin G (IgG) were purified to apparent homogeneity. Gas liquid chromatographic (glc) analysis of the carbohydrate content of these glycoproteins from ML II, ML III and normal sera revealed no differences in the relative ratios and total amounts of mannose, galactose, N-acetylglucosamine and sialic acid. These results suggest that if the postulated post-translational defect in these disorders involves changes in carbohydrate composition, it is not a general defect in glycosylation and may be specific for lysosomal hydrolases.     

Estimation of the Number of Sex Alleles and Queen Matings from Diploid Male Frequencies in a Population of APIS MELLIFERA

The distribution of diploid males in a population of Apis mellifera was obtained by direct examination of the sexual phenotypes of the larvae. Using these data, estimates are derived for the number of sex alleles and the number of matings undergone by the queen. The number of sex alleles is estimated to be 18.9. The estimate is larger than previous ones, which have ranged between 10 and 12. However, the increase in the number of sex alleles can be explained by the large effective population number for our data. The best estimator of the number of matings by a queen is a maximum likelihood type that assumes a prior distribution on the number of matings. For the data presented here, this estimate is 17.3. This estimate is compared to others in the literature obtained by different approaches.     

The relationship between circulating testosterone levels and male sexual behavior in rats

The relationships between plasma testosterone (T) and various parameters of male sexual behavior were examined in intact and castrated T-treated male rats. Repeated blood sampling and behavioral testing revealed no correlation between any measure of sexual behavior and plasma T in normal untreated sexually active males. T-Filled Silastic capsules, implanted subcutaneously at the time of castration, were found to produce plasma T levels proportional to capsule size. Plasma T titers less than 10% of normal (0.2 ng/ml) maintained ejaculatory behavior near normal levels for the 58 days of the experiment. Measures of sexual behavior which showed androgen dependence were intromission latency, postejaculatory interval, and intromission frequency. The plasma T concentration required to maintain these parameters within the intact range was 0.7 ng/ml, which is less than one-third of the mean intact level (2.6 ng/ml). No significant improvement in the sex behavior measures was seen with plasma T levels between 0.7 and 3.1 ng/ml. It was concluded that the absence of relationships between circulating T and sexual behavior in the normal rat population is due to the androgen requirement for this behavior being less than the amount normally present. Findings on T levels and T treatment in noncopulator males are also presented.