Kidney function and arterial acid-base status during continuous stimulation of the carotid chemoreceptors in vagotomized cats]

In spontaneously breathing, chloralose narcotized and bilaterally vagotomized cats under mannite saline diuresis, the isolated carotid sinus were perfused for 1 hr with arterial and then for 1 hr with venous blood, and subsequently again with arterial blood for 1 hr. Either measure d or calculated were the parameters of arterial acid-base status, the arterial systemic bl-od pressure, the p-aminohippuric acid (PAH), inulin (IN), and osmolar clearance, the filtration fraction, the tubular reabsorption of oxmotically free water, the urinary time volume, and fractional excretions of the kidneys. Stimulation of the carotic chemoreceptors increased respiration, slightly enhanced the CPAH, and caused a minor fall of CIN with little altered filtration fraction. The renal resistance increased. The absolute and fractional excretions of urine, sodium, potassium, and of osmotically active particles rose significantly. Tubular reabsorption of osmotically free water showed a singificant increase. Urinary osmolarity remained constant in general. The changes of water and electrolyte excretion did not correlate with those of renal hemodynamics. The results argue against natriuresis being caused on chemoreceptor stimulation by increased filtrate volume of the whole kidney, or by decreased osmolarity of the renal medulla. The results are discussed with regard to the renal function in arterial oxygen deficiency.     

Kinetic and thermodynamic analysis of the role of start codon/anticodon base pairing during eukaryotic translation initiation

Start codon recognition is a crucial event in the initiation of protein synthesis. To gain insight into the mechanism of start codon recognition in eukaryotes, we used a yeast reconstituted initiation system to isolate the step of Met-tRNA_i•eIF2•GTP ternary complex (TC) binding to the 40S subunit. We examined the kinetics and thermodynamics of this step in the presence of base changes in the mRNA start codon and initiator methionyl tRNA anticodon, in order to investigate the effects of base pairing and sequence on the stability of the resulting 43S•mRNA complex. We observed that the formation of three base pairs, rather than their identities, was the key determinant of stability of TC binding, indicating that nothing is inherently special about the sequence AUG for this step. Surprisingly, the rate constant for TC binding to the 40S subunit was strongly codon dependent, whereas the rate constant for TC dissociation from the 43S•mRNA complex was not. The data suggest a model in which, after the initial diffusion-limited encounter of TC with the 40S subunit, the formation of three matching start codon/anticodon base pairs triggers a conformational change that locks the complex into a stable state. This induced-fit mechanism supports the proposal that initiation codon recognition by the 43S complex induces a conformational change from an open state to a closed one that arrests movement along the mRNA.     

Anti-tumor efficacy of interleukin-2-activated killer cells in human neuroblastoma ex vivo

We studied the ex vivo sensitivity of continuously cultured neuroblastoma cells from 3 different patients towards interleukin-2-induced cell-mediated cytotoxicity. A mean (+/- SD) target cell lysis (4 h 51Cr release) of 49 +/- 11, 46 +/- 8, and 32 +/- 11% in SMS-SAN, LA-N-1, and SK-N-BE2 cell lines, respectively, was achieved when neuroblastoma cells were co-cultured at an effector-to-target (E:T) ratio of 50:1 with peripheral blood mononuclear cells (PBMC) that had been preincubated for 4 days in the presence of recombinant interleukin-2 (rIL-2; 100 U/ml). Under identical conditions, 93 +/- 9% of Daudi cells (a standard target for rIL-2-activated killer cells) were lysed. Preincubation of rIL-2-induced PBMC cultures in the presence of irradiated neuroblastoma targets (LA-N-1, SK-N-BE2) resulted in a significant cytolytic augmentation. At E:T ratios of 50:1 and 10:1, day-4 rIL-2/LA-N-1-stimulated PBMC produced 69 +/- 7 and 41 +/- 11% lysis of LA-N-1 cells, as compared to 46 +/- 8 and 22 +/- (mean +/- SD) 7% lysis by untargeted PBMC that were preincubated with rIL-2 (100 U/ml) in the absence of LA-N-1 target cells (p less than 0.05). Co-incubation of rIL-2-induced PBMC preparations with irradiated LA-N-1 and SK-N-BE2 cells, respectively, did not significantly enhance the cytolytic activity against other neuroblastoma targets and the standard Daudi cell line (p greater than or equal to 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells

The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium ((2)H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4(dim)CD5(bright) (proliferative) fraction contained more (2)H-labeled DNA and hence divided cells than the CXCR4(bright)CD5(dim) (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle-associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4(dim)CD5(bright) and CXCR4(bright)CD5(dim) fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.     

A phase-II study of low-dose cyclophosphamide and recombinant human interleukin-2 in metastatic renal cell carcinoma and malignant melanoma

Summary Recent preclinical and clinical studies that have demonstrated antitumor activity of high-dose recombinant interleukin-2 (rIL-2), and animal models that demonstrated a synergistic effect of low-dose cyclophosphamide, led us to study rIL-2 (Cetus Corp., Emeryville, Calif) in a phase II clinical trial in combination with low-dose cyclophosphamide in 32 patients, 18 with malignant melanoma and 14 with renal cell carcinoma. rIL-2 was given once daily at 3×10⁶ U/m², as a 30-min infusion for 14 days in cycle I and for 2×5 days in cycles II and III respectively; if tolerated, the dose was increased to a maximum of 6×10⁶ U m^–2 day^–1; the cycles, separated by 1 week treatment-free intervals, were preceded each by a single i.v. bolus of cyclophosphamide at 350 mg/m². The most prominent side-effects encountered in this trial consisted of a capillary leak syndrome, myalgia and fever that required dose reduction during the first cycle in one-half of the patients. Given the limit of tolerable toxicities in a standard care unit, the regimen employed achieved minor antitumor activity. No remission was achieved in patients with renal cell carcinoma, and 15% of melanoma patients showed objective responses (partial response + minor response).     

Initiation of protein synthesis by hepatitis C virus is refractory to reduced eIF2.GTP.Met-tRNA(i)(Met) ternary complex availability

A cornerstone of the antiviral interferon response is phosphorylation of eukaryotic initiation factor (eIF)2alpha. This limits the availability of eIF2.GTP.Met-tRNA(i)(Met) ternary complexes, reduces formation of 43S preinitiation complexes, and blocks viral (and most cellular) mRNA translation. However, many viruses have developed counterstrategies that circumvent this cellular response. Herein, we characterize a novel class of translation initiation inhibitors that block ternary complex formation and prevent the assembly of 43S preinitiation complexes. We find that translation driven by the HCV IRES is refractory to inhibition by these compounds at concentrations that effectively block cap-dependent translation in vitro and in vivo. Analysis of initiation complexes formed on the HCV IRES in the presence of inhibitor indicates that eIF2alpha and Met-tRNA(i)(Met) are present, defining a tactic used by HCV to evade part of the antiviral interferon response.     

Similarities and differences between the light and heavy chain Ig variable region gene repertoires in chronic lymphocytic leukemia

Analyses of Ig V(H)DJ(H) rearrangements expressed by B-CLL cells have provided insights into the antigen receptor repertoire of B-CLL cells and the maturation stages of B-lymphocytes that give rise to this disease. However, less information is available about the L chain V gene segments utilized by B-CLL cells and to what extent their characteristics resemble those of the H chain. We analyzed the V(L) and J(L) gene segments of 206 B-CLL patients, paying particular attention to frequency of use and association, mutation status, and LCDR3 characteristics. Approximately 40% of B-CLL cases express V(L) genes that differ significantly from their germline counterparts. Certain genes were virtually always mutated and others virtually never. In addition, preferential pairing of specific V(L) and J(L) segments was found. These findings are reminiscent of the expressed VH repertoire in B-CLL. However unlike the V(H) repertoire, V(L) gene use was not significantly different than that of normal B-lymphocytes. In addition, Vkappa genes that lie more upstream on the germline locus were less frequently mutated than those at the 3' end of the locus; this was not the case for Vlambda genes and is not for V(H) genes. These similarities and differences between the IgH and IgL V gene repertoires expressed in B-CLL suggest some novel features while also reinforcing concepts derived from studies of the IgH repertoire.     

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used a reconstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2 (eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2.GTP.Met-tRNA(i) ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiator-specific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the T loop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.