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Biomedical subjects

S Altman

Publications and source records attributed to S Altman.

At least 73 records · Page 4Linked to original sources
PubMed1994

Gel retardation analysis of the interaction between C5 protein and M1 RNA in the formation of the ribonuclease P holoenzyme from Escherichia coli.

C5 protein binds specifically and with high affinity to M1 RNA to form the ribonuclease P holoenzyme of Escherichia coli. The interactions between the two subunits of the enzyme have been studied in vitro by a gel retardation assay. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant (Kd) for the specific interactions of the subunits in the holoenzyme complex is < or = 0.4 nM. C5 protein also has nonspecific affinity for M1 RNA and a variety of other RNA molecules with Kd values in the order of 10-40 nM. Scatchard analysis of binding data suggests the existence of two modes of interaction between C5 protein and M1 RNA--one high-affinity and one low-affinity mode. Regions of M1 RNA essential for formation of the specific complex with C5 protein have been defined by deletion analysis and footprinting methods. Our data show that regions of M1 RNA that interact with C5 protein are clustered into three main areas that are localized between nucleotides 41-99, 168-198, and 266-287.

Bacterial Proteins↗
PubMed1994

Kinetic and thermodynamic analysis of RNA-protein interactions in the RNase P holoenzyme from Escherichia coli.

A gel retardation assay has been used to examine the kinetic and equilibrium properties of the interaction between C5 protein and M1 RNA in the formation of the ribonuclease P holoenzyme from Escherichia coli. The interaction is relatively insensitive to the identity of the monovalent anions present and to pH in the range 7.0-9.0, but it has a more critical requirement for specific monovalent and divalent cations: NH4+, K+, Mg2+, Ca2+, and Mn2+ all promote efficient formation of the complex. A positive delta S (+6.4 cal mol-1 deg-1) and a negative delta H (-11.3 kcal mol-1) combine to give a delta G equal to -13.3 kcal mol-1 at 37 degrees C in 0.42 M salt. The binding reaction is sensitive to the concentration of monovalent and divalent cations, with the affinity increasing with increasing ionic strength (delta log Ka/delta log [NH4+] = +2.7 +/- 0.1). The dependence of Kd on the ionic strength and the positive delta S suggests that hydrophobic and stacking interactions contribute significantly to the formation of the RNase P holoenzyme.

Amino Acid Sequence↗
PubMed1994

Stimulation of vascular glycosaminoglycan synthesis by subpressor angiotensin II in rats.

The vascular trophic effects of angiotensin II (Ang II) in small doses may precede its hypertension-producing effect, and de novo synthesis of components of extracellular matrix may be a requirement for Ang II-stimulated growth. In the present study, therefore, the incorporation of 35SO4 into glycosaminoglycans (synthesis) of aorta and bladder wall of young adult, male Sprague-Dawley rats was measured ex vivo after 48 hours of Ang II administration at two dose levels, 100 and 200 ng.kg-1.min-1 IP. Vehicle-infused rats served as controls. Compared with controls, systolic blood pressure was unchanged in rats receiving 100 ng.kg-1.min-1 Ang II and rose by 13 mm Hg (P < .05) in rats receiving the 200-ng.kg-1.min-1 dose. In Ang II-treated rats, glycosaminoglycan synthesis of the aorta was increased by 20% (P < .05) and 52% (P < .005) at the two dose levels, respectively. Glycosaminoglycan synthesis of bladder smooth muscle was also increased in Ang II-treated rats (P < .01), but the response was not dose dependent. By 7 to 10 days of Ang II administration (200 ng.kg-1.min-1), glycosaminoglycan synthesis of aorta returned toward baseline (P < .10, > .05). The rate of synthesis of subtypes of glycosaminoglycans in the aorta was proportionately increased by Ang II. The early occurrence, magnitude, and arterial pressure independence of Ang II-induced glycosaminoglycan synthesis suggest that restructuring of extracellular matrix may play an important role in both the trophic and hypertension-producing action of Ang II.

Amino Acid Sequence↗
PubMed1994

Health system reform: let's not miss our chance.

Support for reform is at its highest level, but criticism of President Bill Clinton's plan is widespread. Nevertheless, the core components of the Clinton plan should be supported. These include (1) building on existing employer-based health insurance with a mandate that employers offer coverage to all workers and pay a substantial portion of the premium; (2) requiring that coverage be universal and benefits comprehensive; and (3) controlling total health care spending via a national expenditure limit. This paper does, however, suggest three changes to strengthen the plan: developing a broader base than Medicare and Medicaid to pay for reform; setting the national health care spending limit at a higher level (gross domestic product plus 1 percent); and strengthening the powers of the regional health alliances.

Cost Control↗
PubMed1994

Diagnostic reassessment and treatment response in schizophrenia.

BACKGROUND: The reasons for poor treatment response in some patients with schizophrenia remain unclear. It is possible that misdiagnosis of nonschizophrenic disorders as schizophrenia could result in suboptimal pharmacotherapy in some patients. METHOD: To assess this possibility, 110 severely ill, chronic patients with a referral diagnosis of schizophrenia were comprehensively assessed and rediagnosed according to DSM-III-R criteria. Global Assessment of Function (GAF) and Clinical Global Impressions (CGI) ratings were made at admission and at discharge from the ward, after the implementation of individualized treatment plans. RESULTS: The diagnosis of schizophrenia was confirmed in 80 patients (73%) and revised to another type of psychotic illness in 30 patients (27%). The GAF and CGI ratings were similar at admission in patients with confirmed and revised diagnoses. All patients improved by the time of discharge (p = .0001); however, patients with a revised diagnosis improved more than those with confirmed schizophrenia (p = .02). Patients with a revised diagnosis were less likely to require continued hospitalization on chronic care wards (p = .004). At admission, medication regimens were similar in the two groups of patients. At discharge, patients with a revised diagnosis were less likely to have received neuroleptics (p = .007) and more likely to have received antimanic drugs (p = .0002) or electroconvulsive therapy (p = .0004). CONCLUSION: These findings from a clinical sample suggest that diagnostic reassessment is an important first step in the management of apparently refractory schizophrenia.

Adolescent↗
PubMed1993

A physical assay for and kinetic analysis of the interactions between M1 RNA and tRNA precursor substrates.

A gel-shift assay was devised to detect stable enzyme-substrate (E-S) complexes between M1 RNA, the catalytic subunit of RNase P from Escherichia coli, and its tRNA precursor substrates. The use of deletion derivatives of M1 RNA in the gel-shift assay has allowed us to identify regions of the enzyme that are involved in the binding of the substrate or that are necessary for catalytic activity. Fragments of substrates that contain the 3' CCA sequence bind preferentially to regions in the 5' half of M1 RNA, while 5' leader sequences interact primarily with regions in the 3' half of M1 RNA. The 5' leader sequence present in the precursor to tRNA(Tyr)su3 from E. coli plays an important role in the formation of stable E-S complexes with M1 RNA. The CCA sequence at the 3' end of precursor tRNA substrates is involved in the product-release step of the reaction that is catalyzed by M1 RNA. Direct measurements of the concentrations of all the components in the reaction catalyzed by M1 RNA facilitated a new approach to the kinetic analysis of the action of the enzyme.

Base Sequence↗
PubMed1993

Pathway of activation by magnesium ions of substrates for the catalytic subunit of RNase P from Escherichia coli.

The pathway is described for activation by Mg2+ of substrates for M1 RNA, the catalytic subunit of the RNase P from Escherichia coli. The dissociation constants are reported for binding of Mg2+ to the substrate and for the binding of the metal ion-substrate complex to the enzyme. The enzyme binds the substrate with the same affinity whether or not Mg2+ is already bound to the substate. However, only substrates with bound Mg2+ can make a productive ternary complex when combined with the enzyme. The presence of certain 2'-hydroxyl groups in the substrate is required to stabilize the binding of Mg2+ and, thereby, to increase the lifetime of the ternary complex. An energy profile for the reaction of M1 RNA with a small model substrate is presented and the role of Mg2+ bound to the substrate is discussed.

Base Sequence↗
PubMed1993

Nucleotide sequences of the RNA subunit of RNase P from several mammals.

Sequences of the RNA subunit of RNase P from five primate and two rodent species have been determined. The extent of the differences among these sequences and the corresponding RNA from human tissue correlates to known phylogenetic relationships. All the sequences can be drawn in a secondary structure with common features.

Animals↗
PubMed1993

Recent studies of ribonuclease P.

RNase P is an essential enzyme that is required for the biosynthesis of tRNA. It is composed of RNA and protein subunits. The RNA subunit of the enzyme derived from eubacterial sources can carry out the catalytic function by itself in vitro. Current studies of RNase P focus on structure-function relationships with respect to interactions of the RNA subunit with its substrates and with respect to the determination of the kinetic parameters of the reaction, the role of the protein component, and the rules governing recognition of substrates.

Base Sequence↗
PubMed1992

A trinucleotide can promote metal ion-dependent specific cleavage of RNA.

Nucleotide sequence and metal ion requirements for Mn(2+)-dependent self-cleavage of an RNA 31 nucleotides long [Dange, V., Van Atta, R. B. & Hecht, S. M. (1990) Science 248, 585-588] were examined by analysis of the site-specific cleavage activity of substitution and deletion mutants as well as complexes assembled from fragments of this RNA. A complex of UUU and GAAACp allows specific cleavage between G and A at 37 degrees C and pH 7.5. Additional nucleotides flanking the oligonucleotides in the minimal complex are not necessary for the cleavage reaction to take place but can affect the rate of the reaction. The 2'-OH groups of uridine residues do not participate in catalysis since both poly(U) and poly(dU) can promote the specific cleavage reaction in trans. Cd2+ ions can also promote the specific cleavage reaction and Mg2+ ions (which are inactive alone), under certain conditions, can enhance the Mn(2+)-induced cleavage of RNA.

Animals↗
PubMed1992

Targeted cleavage of mRNA by human RNase P.

Ribonuclease P from Escherichia coli can cleave RNAs in simple, hydrogen-bonded complexes of two oligoribonucleotides that resemble the aminoacyl stem and 5' leader sequence of tRNA precursors. RNase P from human (HeLa) cells cannot catalyze the cleavage in vitro of the 5'-proximal oligoribonucleotide that contains the leader sequence in such simple complexes but can do so when the 3'-proximal oligoribonucleotide (external guide sequence) is altered to resemble three-quarters of a tRNA molecule. In such a complex, the efficiency of cleavage of the mRNA for chloramphenicol acetyltransferase, as the 5'-proximal oligoribonucleotide, depends on the structural details of the external guide sequence and on the choice of target site within the mRNA. The presence of the appropriately designed external guide sequence in cells in tissue culture reduces chloramphenicol acetyltransferase activity and the level of the corresponding intact mRNA in the cells. Thus, it appears that the use of such external guide sequences may provide a general technique for gene inactivation.

Base Sequence↗
PubMed1992

Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli.

The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.

Base Sequence↗
PubMed1992

Targeted cleavage of mRNA in vitro by RNase P from Escherichia coli.

External guide sequences (EGSs) complementary to mRNAs that encode beta-galactosidase from Escherichia coli and nuclease A from Staphylococcus aureus can target these RNAs for cleavage in vitro by RNase P from E. coli. Specific cleavage occurs at locations predicted by the nucleotide sequences of the EGSs. EGSs with regions complementary to the mRNAs that are as short as 13 nucleotides function efficiently and turn over slowly during incubation with the target substrate and the enzyme. EGSs composed of deoxyribonucleotides as well as those composed of ribonucleotides are effective, but cleavage of the targeted substrate with DNA as an EGS is about 10-fold less efficient than that with RNA as an EGS. An RNA EGS inhibited the formation of beta-galactosidase activity in a crude extract (S30) of E. coli that was capable of catalyzing coupled transcription-translation reactions.

Base Sequence↗
PubMed1992

Reconstitution of enzymatic activity from fragments of M1 RNA.

Certain fragments of M1 RNA, the catalytic subunit of RNase P from Escherichia coli, either have no enzymatic activity at all or have altered substrate specificity compared with that of the intact catalytic RNA. After simple mixing in vitro, many of these fragments of M1 RNA can reassociate with other fragments to form complexes that have enzymatic activity typical of wild-type M1 RNA. Furthermore, inactive M1 RNA molecules with internal deletions can be complemented in vitro by other inactive derivatives of M1 RNA that have nonoverlapping deletions. Thus, two inactive molecules of M1 RNA can interact to form an active RNA enzyme. Functional attributes can be assigned to various regions of M1 RNA when the reconstitution process is combined with assays for activity with different substrates.

Base Sequence↗
PubMed1992

Subpressor angiotensin II is a bifunctional growth factor of vascular muscle in rats.

OBJECTIVE: The proposition that angiotensin II in subpressor does stimulates vascular growth in vivo was tested. DESIGN: Young adult, male Sprague-Dawley rats received angiotensin II, 200 ng/kg per min intraperitoneally by osmotic minipump, for 24 h or 7-10 days. Sham-infused rats served as controls. METHODS: Protein (35S-methionine) synthesis in aortic media, portal vein, bladder wall and diaphragm; proteoglycan (35S-sulfate) synthesis in aorta and bladder and synthesis of DNA (3H-thymidine) in aortic media were all measured ex vivo in the rat. RESULTS: The systolic blood pressure of angiotensin II-treated rats was unchanged at 24 h and increased at 7-10 days. At 24 h in angiotensin II-treated rats the protein synthesis in aortic media, portal vein and bladder wall but not in the diaphragm was increased, indicating that the hypertrophic effect of angiotensin II was independent of the arterial pressure. The rate of 35S-methionine washout from angiotensin II- and sham-treated aorta was the same. At 24 h there was also an increase in proteoglycans synthesis of the aorta and bladder wall of angiotensin II-treated rats. In contrast to protein synthesis, the incorporation of 3H-thymidine into aortic muscle DNA was reduced in angiotensin II-treated rats at 24 h, suggesting the inhibition of DNA synthesis. At 7-10 days angiotensin II administration the protein synthesis of aortic media returned to baseline, and DNA synthesis was bimodal: in 53% of rats (n = 10) inhibition continued, and in 26% (n = 5) it was increased by two- to threefold. CONCLUSIONS: The present findings confirm in vivo the bifunctionality of the trophic vascular action of angiotensin II. Vascular hypertrophy may play a role in the slow pressor action of angiotensin II.

Angiotensin II↗
PubMed1992

The incorporation of acupuncture into a small animal practice.

Successful use of acupuncture in small animal practice on a day-to-day basis depends on an organized approach. The staff must be trained to answer telephone inquiries and client questions and to obtain a good history before appointments are scheduled. Colleagues must be ethically and professionally assured of a veterinarian's acupuncture credentials and capabilities, so that they will become a source of patient referrals. A proper examination must be conducted that incorporates traditional western veterinary practices with acupuncture, and the results must be properly recorded. A diagnosis should be established before therapy is initiated, and a plan of treatment set up. The prognosis, projected costs, length of treatment regimen, other means of treating the condition, and any possible sequelae should be discussed with the client. The patient should be monitored during the course of treatment, and reevaluated to determine whether or not changes in acupuncture prescription or additional modes of therapy or supportive care are needed. Communication with the client and the referring veterinarian should be a priority.

Acupuncture Points↗
PubMed1992

Techniques and instrumentation.

There are many methods used to stimulate acupuncture points to achieve therapeutic objectives. Among the most frequently employed are physical pressure or vacuum (acupressure and cupping), temperature variation (cauterization, moxibustion, electronic heat devices, infrared lamps, ultraviolet lights, ice, dry ice, and surface coolants such as ethyl chloride), ultrasound (sonapuncture), injection therapy (aquapuncture), implantation of various materials into points, laser stimulation (laserpuncture), and electrostimulation of the acupuncture points, with or without needles (electroacupuncture). This chapter describes these techniques, their uses, and place in acupuncture therapy.

Acupuncture Therapy↗