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Biomedical subjects

G Kramer

Publications and source records attributed to G Kramer.

At least 91 records · Page 5Linked to original sources
PubMed1985

Electrostatic effect of trypsin binding on the hydrogen exchange rate of bovine pancreatic trypsin inhibitor beta-sheet NH's.

The changes of H-D exchange rates upon protein-protein interactions are generally interpreted as a result of the changes of the dynamic properties of the proteins. The effect of trypsin binding on the H-D exchange kinetics of some trypsin inhibitor amide H's was reported (Simon et al., 1984). In this paper the electrostatic potential originating from the trypsin molecule is calculated at the positions of the studied amide H's in the trypsin-trypsin inhibitor complex. We conclude that the observed decrease of the exchange rates is mainly due to the electrostatic field of the trypsin molecule.

Amino Acid Sequence↗
PubMed1985

Intracortical glutamate injection produces helpless-like behavior in the rat.

Acute injection of glutamate into frontal neocortex of naive rats produced a subsequent deficit in escape performance behavior that was similar to that produced by exposure to uncontrollable shock. The behavioral deficit was dose-related. The behavioral deficit was similar in time-course to that produced by 15 min (but not 40 min) of exposure to learned helplessness induction. Unlike learned helplessness produced by exposure to inescapable shock, the behavioral deficit produced by intracortical glutamate injection was not prevented by chronic intraperitoneal administration of imipramine.

Animals↗
PubMed1985

The 90-kDa component of reticulocyte heme-regulated eIF-2 alpha (initiation factor 2 alpha-subunit) kinase is derived from the beta subunit of spectrin.

Antibodies from three different lines of monoclonal hybridomas crossreact with both the beta subunit of spectrin and the 90-kDa peptide present in highly purified preparations of the heme-controlled eIF-2 alpha (initiation factor 2 alpha-subunit) kinase from rabbit reticulocytes. Antibodies from two of the three lines enhance the enzymatic activity of the kinase preparation for phosphorylation of the alpha subunit of eukaryotic translational initiation factor 2 (eIF-2) and for phosphorylation of the 100-kDa peptide thought to be a peptide of the kinase that is phosphorylated during its activation. Also, it is shown that both the beta subunit of spectrin and the 90-kDa peptide can be phosphorylated by two protein kinases from reticulocytes, the catalytic subunit of cAMP-dependent protein kinase and a cAMP-independent protein kinase similar to casein kinase II. Furthermore, a phosphorylated 90-kDa peptide can be derived from phosphorylated beta subunit of spectrin by tryptic proteolysis. We conclude that the 90-kDa peptide is derived by proteolysis from the beta subunit of spectrin, probably from its carboxyl terminus, and suggest that the heme-sensitive eIF-2 alpha kinase, like the 56-kDa phosphatase [Wollny, E., Watkins, K., Kramer, G. & Hardesty, B. (1984) J. Biol. Chem. 259, 2484-2492], is associated with an element of the membrane skeleton in intact reticulocytes.

Animals↗
PubMed1984

Purification to homogeneity and partial characterization of a 56,000-dalton protein phosphatase from rabbit reticulocytes.

A 56,000-Da peptide with inherent protein phosphatase activity was isolated from the postribosomal supernatant fraction of rabbit reticulocytes. The peptide appears to form complexes with other proteins that are present in crude fractions. It exhibits atypical retention on steric exclusion columns during high performance liquid chromatography, an unusual characteristic that facilitated its isolation. The protein phosphatase activity of the 56,000-Da peptide is dependent on Mn2+ ions, but is not activated by either the FA, ATP/Mg2+ protein phosphatase activator system or by proteolysis. The protein phosphatase activity of the peptide is increased 3-fold or more by the antigen peptides described in the accompanying paper (Fullilove, S., Wollny, E., Stearns, G., Chen, S.C., Kramer, G., and Hardesty, B. (1984) J. Biol. Chem. 259, 2493-2500).

Amino Acids↗
PubMed1984

Partial characterization of a 230,000-dalton reticulocyte protein and peptides derived from it that affect the activity of a protein phosphatase.

Monoclonal antibodies were raised that recognize a series of highly antigenic, protease-sensitive peptides that modulate protein phosphatase activity in reticulocyte extracts. Purified antigen peptides cause a 3-fold increase in the enzymatic activity of a homogeneous Mr congruent to 56,000 protein phosphatase. The monoclonal antibodies inhibit protein phosphatase activity in crude extracts but do not recognize the protein phosphatase itself. The antigen peptides are associated with the phosphatase throughout its purification from the postribosomal supernatant of rabbit reticulocytes but are separated from it during size exclusion high performance liquid chromatography (see accompanying article: Wollny, E., Watkins, K., Kramer, G., and Hardesty, B. (1984) J. Biol. Chem. 259, 2484-2492). The series of antigenic peptides appears to be derived by proteolysis from a 230,000-Da precursor, which is relatively abundant in undegraded form in the membrane fraction of rabbit reticulocytes and is present in erythrocyte ghosts. Antigen peptides are extracted with spectrin from both sources. The Mr congruent to 230,000 peptide is not the alpha or beta subunit of spectrin or ankyrin and appears not to have been recognized previously. The name "regulin" is proposed.

Animals↗
PubMed1984

Evaluation of short-course preoperative irradiation in the treatment of resectable bronchus carcinoma: long-term analysis of a randomized pilot-study.

A randomized pilot-study on patients with resectable non small-cell lung carcinoma was conducted from December 1971 to May 1976 inclusive. Patients were randomly assigned to receive preoperative irradiation to the mediastinum followed by surgery (RT + S), or to be treated by surgery only (SO). A total of 33 patients clinically staged as T1-2, N0, M0 histologically confirmed bronchus carcinoma were entered onto the study. Sixteen patients were assigned to RT + S and 17 patients received SO. There were 3 operative mortalities, all of them in the SO group. A total of 28 patients, 14 in each group are evaluable, with a minimum period of observation of 7 years. Preoperative irradiation consisted of a Telecobalt photon-beam applied to the mediastinum as anterior and posterior portals. The thoracic spine was protected on the posterior portal by a narrow lead block. A total dose of 20 Gy calculated in the mid plane was given in 5 equal fractions each of 4 Gy administered on 5 consecutive days: Monday through Friday; patients were operated on the following Monday after the week-end. Surgical treatment was similar for both groups and consisted of lobectomy or pneumonectomy, depending on the size and site of the primary tumor. Analysis of the survival data showed an absolute crude 5 years survival rate of 58% for patients who received RT + S versus 43% for SO. The corrected actuarial 5 and 10 years survival rates are 78 and 69% for the group that received RT + S, and 67 and 55% for the group treated by SO, respectively. Nineteen patients were treated more than 10 years ago. Four of 8 (50%) treated by RT + S are alive with no evidence of disease (NED), and 3/11 (28%) treated by SO are alive with NED. The median survival period for the group that received RT + S is 72 months versus 30 months for the group treated by SO. Analysis of the adequacy of surgical resection based on histological examination of the operative specimen showed higher incidence of radical resection in the group that received RT + S (57 versus 28.5%). It is concluded that the treatment protocol of preoperative radiation therapy as outlined is well tolerated and the results are encouraging.

Aged↗
PubMed1984

Role of thermal injury-induced hypoproteinemia on fluid flux and protein permeability in burned and nonburned tissue.

We studied the role of hypoproteinemia, induced by a major burn injury, on the edema process in burned and nonburned tissues including the lung in the adult sheep. We used lymph flow (QL) and the lymph-plasma (L/P) protein ratio as indicators of the rate of fluid and protein flux across the microcirculation and into the interstitium. We compared the response after a full-thickness burn to 30% of total body surface plus resuscitation by means of lactated Ringer's solution (n = 8) with a comparable degree of hypoproteinemia produced by plasmapheresis with vascular hydrostatic pressure and cardiac output kept constant. We measured lung QL and soft tissue (prefemoral) QL from both the burned and nonburned areas. A twofold increase in QL and a decrease in the L/P ratio was seen in both lung and nonburned tissue in both burn and plasmapheresis animals, indicating the postburn response to be due to hypoproteinemia with no increase in protein permeability. The QL in burned tissue was increased five to ten times with an increase in the L/P ratio. Four burned sheep were resuscitated with pooled plasma. Restoration of plasma proteins eliminated the increase in QL in lung and nonburned tissue but had no effect on the burn response. In summary, burn-induced hypoproteinemia plays a major role in the edema process in nonburned tissues and is corrected by restoration of plasma proteins. Edema in burned tissue does not appear to be related to this process.

Animals↗
PubMed1982

Inhibition of peptide initiation by a low molecular weight RNA from rabbit reticulocytes.

A heat-stable inhibitor of protein synthesis has been isolated from the postribosomal supernatant of rabbit reticulocytes. Its activity is not susceptible to protease treatment but is destroyed by incubation with alkali. Inhibitory activity can be quantitatively recovered in the aqueous phase after phenol extraction and has the ultraviolet absorption spectrum of a nucleic acid. It is concluded that the inhibitor is RNA. The inhibitory activity sediments in the range of 3 S, but it has not been demonstrated whether the inhibitor RNA is a single molecular species. The inhibitory RNA does not affect peptide elongation but rather blocks a step of peptide initiation. It does not interfere with the formation of the ternary complex between initiation factor 2, GTP, and methionyl-tRNAMetf and does not activate a protein kinase phosphorylating initiation factor 2. The inhibitory RNA appears to be a novel type of RNA that inhibits polypeptide initiation at a step involving ribosomal subunits.

Animals↗
PubMed1982

Structure and function of peptide initiation factor 2: differential loss of activities during proteolysis and generation of a terminal fragment containing the phosphorylation sites of the alpha subunit.

A procedure is described by which the 38,000-dalton alpha subunit of native eukaryotic peptide initiation factor 2 (eIF-2) can be cleaved by trypsin to yield a 34,000-dalton fragment and a peptide of about 4,000 daltons after elimination of the beta subunit. Under nondenaturing conditions the 4,000-dalton peptide remains bound to the modified eIF-2 and still can be phosphorylated by the heme-controlled eIF-2 alpha kinase from reticulocytes. All of the phosphorylation sites for this protein kinase are located on the 4,000-dalton peptide. The ability of eIF-2 to form a ternary complex with GTP and Met-tRNAf and the ability to promote binding of Met-tRNAf to 40S ribosomal subunits are lost differentially during the proteolysis. Loss of te latter activity occurs rapidly and appears to be correlated with loss of the beta subunit. Loss of activity for ternary complex formation is correlated with the appearance of the 4,000-dalton peptide.

Adenosine Triphosphate↗
PubMed1982

Acute versus sustained hypoproteinemia and posttraumatic pulmonary edema.

We compared the effect of an acute protein depletion versus a sustained protein depletion on pulmonary edema formation. Acute hypoproteinemia was produced either by a rapid plasmapheresis or as the result of acute hemorrhagic shock and resuscitation. Sustained hypoproteinemia was produced by a 24-hour plasmapheresis or as the result of a 50% body burn. Unanesthetized sheep with lung lymph fistulas were used as the experimental model. In the acute depletion groups an early twofold to threefold increase in lymph flow was seen, reflecting an increase in fluid flux, across the microcirculation, with the increase in lymph flow after resuscitation from shock being identical to that seen in a nonshocked animal with a comparable protein depletion. With restoration of the plasma-lymph oncotic gradient, the lymph flow returned to baseline. The lymph protein content always exceeded 2 gm/dl. In the sustained depletion groups the lymph flow also increased twofold to threefold but remained elevated for over 48 hours despite a rapid restoration of plasma-lymph oncotic gradient. The increase in fluid flux after burn was identical to that after protein depletion alone. In these groups the lymph protein content was below 2 gm/dl, indicating a significant interstitial protein depletion. We conclude that a marked increase in fluid flux is seen after sustained protein depletion that is unrelated to oncotic pressure. This process appears to be related to the degree of washout of interstitial protein, possibly decreasing the viscosity of the interstitial matrix, leading to a more rapid edema formation.

Acute Disease↗
PubMed1981

Phosphorylation of glycogen synthase and of the beta subunit of eukaryotic initiation factor two by a common protein kinase.

A protein kinase from rabbit reticulocytes, able to phosphorylate the beta subunit of eukaryotic initiation factor 2 (eIF-2), has been demonstrated to phosphorylate also glycogen synthase. A glycogen synthase kinase (PC0.7) from rabbit skeletal muscle has been shown to phosphorylate the beta subunit of eIF-2. Comparison of highly purified preparations of the two protein kinases has indicated several similarities of properties. 1) Both enzymes were associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000), and exhibited apparent native molecular weights of 176,000-180,000 by gel filtration and 130,000-140,000 by sucrose density gradient sedimentation. 2) Both enzymes phosphorylated glycogen synthase, eIF-2 beta, phosvitin, and casein and were effective in utilizing GTP and ATP as phosphoryl donors. 3) Both enzymes displayed the same chromatographic behavior on phosvitin-Sepharose, phosphocellulose, and DEAE-cellulose. 4) Both enzymes underwent an autophosphorylation of the beta polypeptide when incubated with ATP and Mg2+. On the basis of these and other properties, we propose that the two protein kinases, if not identical, are very similar enzymes.

Animals↗
PubMed1981

Position of transfer ribonucleic acid on Escherichia coli ribosomes. Distance from the 3' end of 16S ribonucleic acid to three points on phenylalanine-accepting transfer ribonucleic acid in the donor site of 70S ribosomes.

Escherichia coli 16S RNA from 30S ribosomal subunits was isolated, oxidized at the 3' end, and labeled with the thiosemicarbazide derivatives of fluorescein or eosin. Labeled 16S RNA was reconstituted into 30S subunits. They were almost fully active compared to 30S subunits reconstituted from unlabeled 16S RNA by using a poly(uridylic acid)-directed polyphenylalanine synthesis assay. Fluorophores were placed at three different positions of tRNAPhe. E. coli and yeast tRNAPhe were oxidized at the 3' end and labeled with the thiosemicarbazide derivative of fluorescein or with the hydrazide of N-methylanthranilic acid. The Y base in the anticodon loop of yeast tRNAPhe was replaced by proflavin or 1-aminoanthracene. Also, E. coli tRNAPhe was photochemically cross-linked between 4-thiouridine at position 8 and cytidine at position 13. After reduction, this site was used as a fluorescent probe. The labeled tRNAs were bound into the peptidyl site of 70S ribosomes, and then the distances from the fluorophore in the modified tRNA to the fluorophore at the 3' end of 16S RNA were measured by nonradiative energy transfer. Calculations were based on measurements of fluorescence lifetimes. The distances to the 3' end of 16S RNA were found to be as follows: 3' end of tRNA, 67-74 A; cross-linked t RNA, 53-60 A; anticodon loop of tRNA, greater than 61 A.

Binding Sites↗
PubMed1981

Localization of 3' ends of 5S and 23S rRNAs in reconstituted subunits of Escherichia coli ribosomes.

Periodate-oxidized 3' ends of 5S, 23S, and 16S rRNAs from Escherichia coli were allowed to react with fluorescein thiosemicarbazide, then labeled rRNAs were reconstituted into active ribosomal subunits. The fluorescein moiety on each of the rRNAs when reconstituted into ribosomal subunits was accessible to anti-fluorescein IgG as determined by fluorescence quenching and by sucrose gradient centrifugation. The region at which an antibody molecule bond to the labeled ribosomal subunits was determined by immunoelectron microscopy. The 3' end of the 5S RNA was localized on the central protuberance of the 50S subunit. The corresponding region for the 3' end of the 23S RNA was below the stalk on the noninterfacing surface. The 3' end of the 16S RNA was localized to the upper edge of the large lobe of 30S subunits, as reported previously.

Escherichia coli↗