David W.E. Smith

Is greater female longevity a general finding among animals

(A) There are data showing beyond question a gender gap, with women living longer than men, especially in economically developed societies. There is greater male vulnerability to the major causes of human death.

Specialization of Rabbit Reticulocyte Transfer RNA Content for Hemoglobin Synthesis

The amount of acceptance of each amino acid per absorbancy unit of rabbit reticulocyte transfer RNA was determined. The results were compared with the amino acid composition of rabbit hemoglobin and with a similar determination of the transfer RNA content of rabbit liver. The histidine and isoleucine transfer RNA content of reticulocytes is specialized for the synthesis of hemoglobin, in which histidine is unusually common and isoleucine unusually scarce compared to most proteins.

Does genotypic sex have a direct effect on longevity

Females of the human species live longer than males, and the longevity differential is probably not entirely explained by reasons which are presently obvious. Genotypic sex has long been suspected to affect longevity to the advantage of the female. Several recent findings about the X and Y chromosomes must be reckoned with in considering determinants of longevity which derive from genotypic sex. The advantages of having two X chromosomes are apparent, notwithstanding X-chromosome inactivation. Not only can some cells compensate for biosynthetic deficiencies of others, but also cell selection according to which X chromosome is active can occur during development according to cell viability and proliferative capacity. It has recently been observed that at least some genes on inactive X chromosomes are reactivated late in life. Details of the reactivation process must be studied to determine its significance and the effects of the process on late life survival. The recent mapping of the catalytic polypeptide of DNA-polymerase-alpha to the X chromosome calls attention to a new property of the genotype which could affect the basic ability of cells to proliferate. It is likely that this enzyme, perhaps in concert with DNA-polymerase-delta, is required for DNA replication, suggesting that two alleles for this enzyme and cell selection within the female phenotypic mosaic for DNA replication may provide a sex-linked determinant of cell proliferation which could be advantageous in late life. Much remains to be learned about the Y chromosome, although there are early results consistent with a determinant of longevity on that chromosome which operates to the male disadvantage and probably does not involve sex hormones. The genotype may be a significant determinant of longevity in humans even if it does not appear to be so in non-human animals, because causes of death are different. Determinants of longevity are based on susceptibility or vulnerability to the causes and diseases of mortality, and these differ in different species.

The transfer RNA content of rabbit reticulocytes: Enumeration of the individual species per cell

Abstract Rabbit reticulocytes (immature red blood cells which synthesize hemoglobin) contain approximately 4.19 · 10 5 tRNA molecules capable of amino acid acceptance per cell. Reversed phase column chromatography was used to indicate the number of molecules of the isoaccepting species for each amino acid of reticulocyte tRNA. There are from 800 to more than 30 000 molecules of the individual species per reticulocyte. For most amino acids there are 500–1000 tRNA molecules for each residue of the amino acid in rabbit hemoglobin.

Problems of Translating Heterologous Genes in Expression Systems: The Role of tRNA

tRNA can have large effects on the expression and overexpression of heterologous genes in microbial expression systems through reduced translation and errors in amino acid sequences of protein products. Examples are given of large effects on gene expression related to tRNA content and to tRNA base modifications, both of which differ in heterologous expression systems compared to the cells from which the genes originally came. tRNA should be of greater concern in the expression of heterologous genes.

Effects of post-transcriptional base modifications on the site-specific function of transfer RNA in eukaryote translation☆

The site-specific function in translation of several naturally occurring mammalian transfer RNAs has been studied in a series of investigations with some similarities to studies in other laboratories of tRNAs in suppression. Equal amounts of aminoacyl-tRNA isoacceptors with contrasting isotopes were added in pairs to reticulocyte lysates and allowed to incorporate their amino acids into rabbit globin. Rates of incorporation from unlimiting amounts of each isoacceptor into the corresponding amino-acid-containing sites were determined. The tRNAs of each isoacceptor pair differed as to post-transcriptional base modifications. The natural occurrence of these isoacceptors can be correlated with rates of cellular division, with more rapidly dividing and neoplastic cells containing hypomodified tRNA. The overall incorporation of lysine into globin from a fully modified tRNALys that decodes AAG is faster by 25 to 30% than from the corresponding hypomodified tRNALys. There is considerable scatter in values for incorporation ratios at different lysine-containing sites, with the hypomodified isoacceptor even being preferred at one site. The AAG decoding isoacceptors are capable of translating AAA although much more slowly than AAG. In translating AAA, in contrast to translating AAG, the hypomodified tRNALys isoacceptor is preferred. A Y base-deficient hypomodified tRNAPhe isoacceptor found only in some kinds of rapidly dividing tumor cells donates its phenylalanine preferentially to globin in competition with the fully modified Y-containing tRNAPhe of liver by 15 to 17%. There is a considerable range of incorporation ratios at the different phenylalanine-containing sites of the globin subunits. No correlation can be made between the isoacceptor preferred and the phenylalanine codon being translated. The incorporation of histidine from a fully modified tRNAHis-containing Q base in its anticodon, compared with that from the hypomodified counterpart isoacceptor that lacks Q base and that occurs in rapidly dividing cells, showed no difference in their ability to incorporate overall or into individual histidine-containing sites. There is little evidence that adjacent bases or codons in messenger RNA affect the tRNAs preferred in the translation of most sites. A striking pattern of tRNA preference was observed in three cases in which there are tandem codons, with the same codon appearing twice in succession.(ABSTRACT TRUNCATED AT 400 WORDS)

Pet Ownership by Elderly People: Two New Issues.

Two new issues of pet ownership were considered in a mail questionnaire and an interview survey of a large number of elderly people age sixty and over. First, evidence was obtained that pets are an important determinant of housing choice. Second, many elderly pet owners have made no arrangements for the pet if they predecease it or become unable to care for it. Problems related to these issues are discussed.

A comparison of rabbit liver and reticulocyte transfer RNA: Evidence of unique species in reticulocytes

The comparison of rabbit liver and reticulocyte tRNA has provided evidence that the tRNA content of reticulocytes is specialized for hemoglobin synthesis (Smith, D.W.E. and McNamara, A.L. (1971) Science 171, 577–579 and erratum, 1040). This comparison is extended in the present study to the level of isoaccepting species by reversed-phase chromatography of the tRNAs followed by determination of acceptance of 18 amino acids. Though the elution patterns of isoaccepting species for some amino acids are qualitatively and quantitatively similar, the distribution of tRNA for other amino acids among isoaccepting species is very different in reticulocytes and liver. There are two isoaccepting species each for asparagine and histidine in reticulocytes compared to only one in liver. These unique species were confirmed by a second reversedphase chromatographic technique, and are seen regardless of whether liver or reticulocyte enzymes are used to aminoacylate the tRNA. The single species for each of these amino acids in liver corresponds to the first of the two species in reticulocytes in order of chromatographic elution. The structural differences between the two species found in reticulocytes are not yet clear. Coding studies were done with the partially purified asparagine and histidine species from liver and reticulocytes using trinucleotide diphosphate stimulated binding of the aminoacylated tRNAs to Escherichia coli ribosomes. Consistent with the “Wobble Hypothesis”, all tRNA species responded to both codons for their respective amino acids, though differences in the codon responses were found which may be significant.

The association of histidyl-tRNA synthetase with reticulocyte ribosomes

Abstract Some aminoacyl-tRNA synthetase activity is found attached to ribosomes. This phenomenon was investigated in detail using the histidyl-tRNA synthetase of rabbit reticulocytes, 10–15% of which is attached to ribosomes from cells lysed osmotically. The enzyme can be eluted from ribosomes by KCl solutions of 0.1 to 0.2 M. The enzyme eluted from ribosomes and the enzyme of reticulocyte cytosol can be associated with KCl washed ribosomes. A constant ( K A ) of 5 · 10 8 to 5 · 10 9 M −1 has been determined for the association reaction, indicating a high level of affinity and specificity. One bound enzyme molecule is found per 500 ribosomes from freshly lysed reticulocytes, and one molecule can be associated per 300 ribosomes from a crude enzyme preparation. One enzyme molecule can be associated per two ribosomes using a partially purified enzyme preparation. Thus the number of binding sites approaches a limit, and possibly molecules in addition to His-tRNA synthetase can be attached to the same binding sites. Sucrose density gradient centrifugation indicates that the enzyme is bound both to polysomes and to 80-S ribosome monomers, and that the enzyme is bound by each of the ribosomal subunits. There appear, therefore, to be at least two binding sites per ribosome. The significance of ribosomal binding of aminoacyl-tRNA synthetases, which occurs with high affinity and specificity in the absence of KCl, is considered.

A Conserved Hydrogen-Bonding Network of P2 bis-Tetrahydrofuran-Containing HIV-1 Protease Inhibitors (PIs) with a Protease Active-Site Amino Acid Backbone Aids in Their Activity against PI-Resistant HIV

ABSTRACT In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 μM) and tipranavir (TPV; EC50, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRVRP20), with a 2.6-fold increase in its EC50 (0.097 μM) compared to its corresponding EC50 (0.038 μM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30′ of HIVA02 protease (PRA02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30′ due to an absence of polar groups. The P2′ thiazolyl moiety of RTV showed two conformations in the crystal structure of the PRA02-RTV complex, one of which showed loss of contacts in the S2′ binding pocket of PRA02, supporting RTVs compromised antiviral activity (EC50, >1 μM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30′ most likely contributes to its persistently greater antiviral activity against HIVWT, HIVA02, and HIVDRVRP20.