Salil K. Niyogi

Analogs of human epidermal growth factor which partially inhibit the growth factor-dependent protein-tyrosine kinase activity of the epidermal growth factor receptor

Three site‐directed mutants of human epidermal growth factor, Leu‐26 → Gly, Leu‐47 → Ala, and He‐23 → Thr, were examined for their ability to stimulate the protein‐tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding affinities of the mutant growth factors were 20‐ to 50‐fold lower, as compared to wild‐type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial ‘uncoupling’ of signal transduction. The mutant analogs were shown to compete directly with the binding of wild‐type, resulting in a decrease in growth factor‐stimulated kinase activity.

Selective effects of metal ions on RNA synthesis rates

The effects of 14 metal ions (chlorides) on the transcription of calf thymus DNA and phage T4 DNA with Escherichia coli RNA polymerase were tested. These assays were conducted under improved conditions of lower pH and in the absence of 2-mercaptoethanol to permit greater stability of the metal ions in solution. Among the divalent metal ions tested, the concentration-dependent order of inhibition of overall transcription is Pb2+ greater than Zn2+ greater than Cu2+ greater than Be2+ greater than Cd2+ greater than Ni2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Mg2+ greater than Sr2+ and is the same with either template. At pH 7.4 and in the absence of 2-mercaptoethanol, considerably lower concentrations of several of the divalent metal ions are needed for inhibition of overall transcription than at pH 8.1 and in the presence of 2-mercaptoethanol. Ca2+, Mg2+, Sr2+, Zn2+, Li+, Na+, and K+--considered to be non-mutagenic and non-carcinogenic--decrease chain initiation (measured with T4 DNA) at concentrations that inhibit overall transcription. Pb2+, Cd2+, Co2+, Be2+, and Mn2+--all mutagenic or carcinogenic--stimulate chain initiation (although at widely different rates) at concentrations that inhibit overall transcription. Cu2+ and Ni2+--both carcinogenic--stimulate initiation only at very low concentrations, followed by a progressive decrease in initiation at concentrations that inhibit overall transcription.

Effect of sigma factor and oligoribonucleotides on the transcription of well-defined templates with the ribonucleic acid polymerase of Escherichia coli☆

Abstract The sigma factor of Escherichia coli RNA polymerase has a substantial stimulatory effect on the transcription of certain well-defined templates, especially single-stranded polymers. This stimulation, dependent on both the base-composition and the secondary structure of the template, is a maximum at low ribonucleoside triphosphate concentrations where chain initiation is presumably rate-limiting. Among the polyribonucleotide templates, poly(U) shows the greatest dependence on sigma factor, whereas poly(C) is completely independent of sigma. The transcription of uridine residues in mixed ribopolymers, however, shows a reduced dependence on sigma. Among the polydeoxynucleotide templates, poly(dA)- and poly(dT)-directed reactions are substantially stimulated by sigma; here, again, the dependence on sigma factor is reduced in mixed deoxypolymers. The dependence on the sigma factor for transcription is removed by oligoribonucleotides complementary to the template. Kinetic studies and experiments with γ- 32 P-labeled ribonucleoside triphosphates indicate that the oligoribonucleotides are presumably bypassing the sigma-dependent initiation step.

Biochemistry and Physiology of Bacteriol Ribonucleases

Publisher Summary This chapter presents the information presently available regarding the various ribonucleases elaborated by bacteria. The chapter focuses mainly to a single species, Escherichia coli , simply because most systematic studies have been carried out on this organism. The increasing number of enzymes whose functions are either to process or degrade polyribonucleotide chains poses a challenge to biochemists and molecular biologists in this area. Most of the knowledge on the mechanism of action of the enzymes comes from studies that involve the isolation of a particular enzyme and studies of its action with specific substrates in vitro , with subsequent extrapolation of these results to in vivo conditions. While progress is being made at an accelerated pace, the answers to many questions of the actual in vivo mechanisms still remain unclear. Recently, through the use of both genetic and biochemical approaches, and in vitro processing systems, rapid progress has been made in elucidating individual steps involved in RNA maturation in a wide variety of systems.

Engineering dynamics of growth factors and other therapeutic ligands.

Peptide growth factors and other receptor-binding cytokine ligands are of interest in contemporary molecular health care approaches in applications such as wound healing, tissue regeneration, and gene therapy. Development of effective technologies based on operation of these regulatory molecules requires an ability to deliver the ligands to target cells in a reliable and well-characterizable manner. Quantitative information concerning the fate of peptide ligands within tissues is necessary for adequate interpretation of experimental observations at the tissue level and for truly rational engineering design of ligand-based therapies. To address this need, we are undertaking efforts to elucidate effects of key molecular and cellular parameters on temporal and spatial distribution of cytokines in cell population and cell/matrix systems. In this article we summarize some of our recent findings on dynamics of growth factor depletion by cellular endocytic trafficking, growth factor transport through cellular matrices, and growth factor production and release by autocrine cell systems. (c) 1996 John Wiley & Sons, Inc.

SV40 Promoters and Their Regulation

Publisher Summary This chapter discusses the complex regulatory region of SV40. It may serve as a model of the regulatory mechanisms that operate in many other eukaryotic systems. The regulatory region of SV40 is 420 bp long and is contained in the HindIII–HpaII fragment located between nucleotide positions 5171 and 346 (25) .This DNA is positioned between the coding sequences of the two sets of genes that are transcribed divergently at early and late times in infection. Most of this region inside the infected cells is generally devoid of nucleosomes. This region contains sequentially a perfect 27-bp palindrome, a 17-bp (A + T)-rich sequence, three copies of a (G + C)-rich 21-bp repeat, and two copies of a 72-bp repeat at the extreme upstream segment. The segment containing the core origin of replication (ori) is characterized by the 27-bp palindromic structure flanked on either side by short segments. The SV40 early promoter contains at least three spatially distinct elements: (1) two 72-bp repeats comprising the enhancer sequence; (2) three 21-bp repeats, each containing two (G + C)-rich motifs; and (3) the TATA box or Goldberg–Hogness box. The functional significance of each element is also discussed in the chapter.

Hydrolysis of oligoribonucleotides by an enzyme fraction from Escherichia coli

This paper reports the isolation from Escherichia coli of an enzyme fraction that rapidly hydrolyzes adenine oligoribonucleotides, yielding 5′-AMP as the major product. Two ribonucleases from E. coli have been described previously. RNase I (Elson, 1959; Spahr and Hollingsworth, 1961) is an endonuclease that hydrolyzes polyribonucleotides and yields nucleoside 3′-monophosphates. RNase II (Spahr and Schlessinger, 1963; Spahr, 1964; Singer and Tolbert, 1964 and 1965; Nossal, Tolbert, and Singer, 1967) is an exonuclease that hydrolyzes polyribonucleotides from the 3′-termini and yields nucleoside 5′-monophosphates. Singer and Tolbert (1965) and Nossal et al. (1967) have reported that short adenine oligoribonucleotides are not hydrolyzed by RNase II.

Utilization of a receptor reserve for effective amplification of mitogenic signaling by an epidermal growth factor mutant deficient in receptor activation.

The idea of a receptor reserve in mediating cellular function is well known but direct biochemical evidence has not been easy to obtain. This study stems from our results showing that L15 of epidermal growth factor (EGF) is important in both EGF receptor (EGFR) binding and activation, and the L15A analog of human EGF (hEGF) partially uncouples EGFR binding from EGFR activation (Nandagopal et al., [1996] Protein Engng 9:781‐788). We address the cellular mechanism of mitogenic signal amplification by EGFR tyrosine kinase in response to L15A hEGF. L15A is partially impaired in receptor dimerization, shown by chemical cross‐linking and allosteric activation of EGFR in a substrate phosphorylation assay. Immunoprecipitation experiments reveal, however, that L15A can induce EGFR autophosphorylation in intact murine keratinocytes by utilizing spare receptors, the ratio of total phosphotyrosine content per receptor being significantly lower than that elicited by wild‐type. This direct biochemical evidence, based on function, of utilization of a receptor reserve for kinase stimulation suggests that an EGF variant can activate varying receptor numbers to generate the same effective response. L15A‐activated receptors can stimulate mitogen‐activated protein kinase (MAPK) that is important for mitogenesis. The lack of linear correlation between levels of receptor dimerization, autophosphorylation, and MAPK activation suggests that signal amplification is mediated by cooperative effects. Flow cytometric analyses show that the percentages of cells which proliferate in response to 1 nM L15A and their rate of entry into S‐phase are both decreased relative to 1 nM wild‐type, indicating that MAPK activation alone is insufficient for maximal stimulation of mitogenesis. Higher concentrations of L15A reverse this effect, indicating that L15A and wild‐type differ in the number of receptors each activates to induce the threshold response, which may be attained by cooperative activation of receptor dimers/oligomers by van der Waals weak forces of attraction. The maintenance of a receptor reserve underscores an effective strategy in cell survival. J. Cell. Biochem. 83: 326–341, 2001.

Differential effects of σ factor, ionic strength, and ribonucleoside triphosphate concentration on the transcription of phage T4 DNA with the ribonucleic acid polymerase of Escherichia coli

The effects of concentration of the σ subunit of Escherichia coli B RNA polymerase (nucleoside triphosphate:RNA nucleotidyltransferase, DNA-dependent, EC 2.7.7.6) on the transcription of phage T4 DNA have been examined under high and low salt and substrate concentrations. The following conclusions were reached: 1. 1. At normal substrate level, KCl stimulates the polymerase reaction with T4 DNA template largely by promoting σ activity, even when the quantity of σ factor present is very small (in the case of calf thymus DNA template, where σ had little stimulatory effect, KCl had no enhancement effect.) 2. 2. σ factor stimulates the initiation of chains beginning with ATP rather than with GTP. This is evident both from substrate-dependence studies and incorporation studies using [γ-32P]ATP and [γ-32P]GTP. 3. 3. The σ-dependent ATP initiations were further promoted by KCl. 4. 4. At extremely low substrate levels an inhibitory effect of KCl is predominant although σ remains stimulatory. This inhibition possibly occurs at the level of chain initiation.

FLUORESCENCE STUDIES WITH HUMAN EPIDERMAL GROWTH FACTOR

Abstract— Steady‐state and time‐resolved fluorescence studies have been performed with human epidermal growth factor, a small globular protein having two adjacent tryptophan residues near its C‐terminus. Based on the relatively red fluorescence and accessibility to solute quenchers, the two tryptophan residues are found to be exposed to solvent. Anisotropy decay measurements show the dominant depolarizing process to have a sub‐nanosecond rotational correlation time indicating the existence of rapid segmental motion of the fluorescing tryptophan residues. From an analysis of the low‐temperature excitation anisotropy spectrum of the protein (and in comparison with that of tryptophan, the peptide melittin, and the dipeptide trp‐trp), it is concluded that homo‐energy transfer and/or exciton interaction occurs between the adjacent tryptophan residues. A thermal transition in the structure of the protein, which is observed by circular dichroism measurements, is not sensed by the steady‐state fluorescence of the protein. This result, in conjunction with the anisotropy decay results, indicates that the two tryptophan residues are in a highly flexible C‐terminus segment, which is not an integral part of the three‐dimensional structure of the protein. Fluorescence measurements with three site‐directed mutants also show very little variation.