Biomedical subjects

R Hurst

Publications and source records attributed to R Hurst.

At least 37 records · Page 2Linked to original sources

PubMed1996

Spinal dural arteriovenous malformations. Intraoperative evoked potential evidence for pathophysiology. A case report.

STUDY DESIGN: This case report details intraoperative evoked potential changes during surgical removal of a T8 dural arteriovenous malformation. OBJECTIVES: The pattern of changes in somatosensory-evoked responses during surgical correction of spinal dural at arteriovenous malformation can illuminate the pathophysiologic process behind the clinical symptoms. SUMMARY OF BACKGROUND DATA: Arteriovenous malformation of the spinal dura can manifest with multiple symptoms, including progressive myelopathy and pain. The pathophysiologic process behind these symptoms could be either direct compression of the spinal cord by the arteriovenous malformation, ischemia resulting from the cord, or increased venous pressure. METHODS: To investigate these hypotheses further, the results of posterior tibial evoked potentials obtained during surgical removal of a T8 dural arteriovenous malformation were analyzed. RESULTS: At baseline, the cortical (P40) potential was markedly prolonged bilaterally. During surgery, just after the dura was opened, a marked increase was observed in the latencies of the P40 and P60 components of the evoked response on the right, which began to resolve as soon as the arteriovenous malformation was occluded. Only minimal changes were seen on the left. CONCLUSIONS: These results are most consistent with the increased venous pressure hypothesis for the pathogenesis of neurologic symptoms in dural arteriovenous malformations.

Aged↗

PubMed1996

Endorectal color Doppler imaging of primary and recurrent rectal wall tumors: preliminary experience.

OBJECTIVE: The purpose of our study was to assess whether color Doppler imaging is a useful adjunct to endorectal sonography in evaluating rectal wall neoplasms and in distinguishing recurrent or residual tumor from postoperative changes. MATERIALS AND METHODS: Thirty-two patients underwent endorectal sonography with color Doppler imaging for one of two indications: evaluation and staging of a suspected primary rectal wall cancer, or distinguishing tumor recurrence from postoperative scarring in patients with previously resected lesions. Rectal wall lesions were evaluated for hypervascularity, and their corresponding flow patterns were graded from 0 to 4. Local tumor staging with endorectal sonography and color Doppler imaging was performed before the results of pathological staging were available. RESULTS: Ninety-five percent of patients with malignant rectal wall tumors demonstrated hypervascularity during color Doppler imaging. In 61% of patients with rectal cancers, prominent perirectal vessels were identified supplying the tumor and were never identified in patients with benign lesions. Endorectal sonography alone correctly identified perirectal fat invasion with a 100% sensitivity and an 83% specificity. Color Doppler imaging during endorectal sonography correctly identified perirectal fat invasion with a sensitivity of 91% and a specificity of 83%. Of the 8 patients with previously resected lesions (five adenomas and three cancers), two patients had recurrent tumor, and both of these demonstrated grade 3 abnormality. Six patients with biopsy-proven fibrosis only had grades of 0 or 1. CONCLUSIONS: Both benign and malignant rectal wall tumors demonstrate abnormal intratumoral hypervascularity when seen with color Doppler imaging. Advanced rectal cancers (T3 or greater) are also associated with abnormal perirectal vascularity. Color Doppler imaging during endorectal sonography does not appear to improve discrimination of benign from T2 (or less) rectal wall cancers, nor does it appear to improve the ability to locally stage rectal wall cancers during endorectal sonography. However, endorectal sonography with color Doppler imaging may add significant information in distinguishing recurrent tumor from postsurgical scarring.

Female↗

PubMed1996

Rectal carcinoma: are we making a difference?

Evaluation of preliminary results at our institution revealed an improvement in survival of patients treated for rectal cancer from 1950-1988. A study was conducted to analyze responsible factors. A total of 335 patients with accurate staging and follow up for at least 5 years or until death were reviewed retrospectively. Of the 335 patients, 295 (88%) were treated by surgical resection, abdominal perineal resection (APR) (n=179), and anterior resections (n=105), the most common procedures. Operative mortality for patients with curable disease (n=188) was 18 per cent (n=34), which has significantly decreased from 24 per cent (n=28) (1950-1969) to 8 per cent (n=6) (1970-1988), (P < 0.01). Operative mortality for APR and anterior resection was 22 per cent (n=29) and 8 per cent (n=5), respectively. Mortality for APR decreased from 28 per cent (1950-1969) to 10 per cent (1970-1988) (P < 0.02) accounting for most of the improvement. Of the 335 patients, 142 (42%) presented with stage IV disease, which decreased from 54 per cent (n=47) (1950s) to 22 per cent (n=10) (1980s), P < 0.01. The overall 5-year survival was 19 per cent (n=64), with a corresponding increase per decade from 13 per cent (1950s) to 43 per cent (1980s), (P < 0.001). Of the remaining 188 patients, 41 had involved surgical margins and decreased 5-year survival of 8 per cent when compared to patients with clear surgical margins (37%) P < 0.01). Multifactor analysis revealed that stage of disease at presentation, involved surgical margins, and operative mortality were significant independent variables. Earlier stage at presentation and improved operative management increased the survival of patients with rectal cancer at our institution.

Cause of Death↗

PubMed1996

Looking beyond the presenting problem.

General practitioners tend to manage disease within the context of the patient's total life. They also have to deal with illness in its various forms when no overt 'disease' is evident. Looking at the interplay of these two facets of ill health gives a deeper understanding of what general practice is about and how general practitioners deal with the problems of their practice. Two cases are presented from the author's personal practice which display how general practitioners deal with the whole person rather than isolated problems. Analysis reveals that the ability to listen to the patient and the willingness to take adequate time is crucial to successful general practice.

Delivery of Health Care↗

PubMed1995

High-level expression and purification of coffee bean alpha-galactosidase produced in the yeast Pichia pastoris.

alpha-Galactosidase isolated from coffee beans cleaves the terminal alpha-galactose residues from oligosaccharide chains on blood group B red cells, thus generating group O cells. Such enzymatically converted red cells not only maintain full erythrocyte integrity and viability in vitro, but also demonstrate immune tolerance and a normal life span in vivo. In order to produce large quantities of recombinant alpha-galactosidase for use in the study of blood-type conversion, we subcloned the cDNA coding for coffee bean alpha-galactosidase into the EcoRI site of the vector pPIC9 in order to express the enzyme in Pichia pastoris, a methylotrophic yeast strain. After P. pastoris transformation, colonies were screened for high-level expression of alpha-galactosidase, based on enzyme activity. In order to increase enzyme production, the growth conditions in the shake flask culture and fermentor culture were optimized. Under the conditions applied, biologically active alpha-galactosidase was produced and secreted into the culture medium at a level of approximately 0.4 g per liter of the fermentor culture. The protein was purified to apparent homogeneity by a simple chromatography procedure, as suggested by a single band of 41 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its homogeneity was further confirmed by chromatofocusing and N-terminal sequencing. P. pastoris appears to be the choice as host for the large-scale production of recombinant alpha-galactosidase used for blood type conversion.

Amino Acid Sequence↗

PubMed1995

Chloride is required for receptor-mediated divalent cation entry in mesangial cells.

Agonists which stimulate the inositol 1,4,5 trisphosphate ([1,4,5]-IP3)-dependent mobilization of Ca2+ from intracellular stores also stimulate entry of divalent cations across the cell membrane. Under appropriate experimental conditions, divalent cation entry across the cell membrane can be monitored as the rate at which the intracellular fluorescence of divalent cation indicators is quenched by the addition of Mn2+ to the extracellular medium. We report that addition of vasopressin to fura-2-loaded glomerular mesangial cells in culture markedly accelerated the rate at which Mn2+ quenched fura-2 fluorescence at its Ca(2+)-insensitive wavelength in the presence of extracellular NaCl, but that this quench response was attenuated when Cl- was removed from the extracellular medium by equimolar substitution with impermeant anions (gluconate, methanesulfonate, acetate, lactate). Similarly, loss of agonist-induced quench also occurred when Cl- was substituted with gluconate in K(+)-containing media. Addition of the Cl- channel inhibitor, 5-nitro-2-(3-phenylpropylaminobenzoic acid) (NPPB), also inhibited Mn(2+)-induced quench of fura-2 fluorescence following vasopressin addition. In contrast, in the presence of gramicidin to provide an alternate conductance pathway to accompany divalent cation entry, agonist-dependent Mn2+ quench occurred even in the absence of extracellular Cl-, indicating that the requirement for Cl- was not the result of cotransport on a common transporter nor the result of Cl- serving as a necessary cofactor for divalent cation entry. A similar dependence on extracellular Cl- was observed for other Ca(2+)-mobilizing agonists such as endothelin, as well as the intracellular Ca2+ ATPase inhibitor, thapsigargin. Extracellular Cl- dependence for agonist-induced divalent cation entry was also reflected in a corresponding extracellular Cl- dependence for agonist-induced mesangial cell contraction. It has been previously shown by ourselves (Kremer et al., 1992a, Am. J. Physiol., 262:F668-F678) and others that agonist-stimulated calcium mobilization in mesangial cells is accompanied by inhibition of K+ conductance and increased Cl- conductance. Accordingly, we conclude that the current findings suggest that activation of Cl- conductance provides regulated charge compensation for receptor-mediated divalent cation entry in response to Ca(2+)-mobilizing vasoconstrictor agonists in mesangial cells.

Animals↗

PubMed1994

ATP-dependent chaperoning activity of reticulocyte lysate.

We have developed an assay for chaperone-mediated protein renaturation using thermally denatured Firefly luciferase. Dilution of denatured luciferase (> 99% loss of activity) into reticulocyte lysate typically results in recovery of 5-15% activity. Addition of an ATP-regenerating system increases yields to > 60%, while heat shock or the addition of denatured proteins inhibits the chaperoning activity. Reticulocyte lysate contains abundant quantities of the heat shock proteins, hsp90 and hsp70, and a 60-kDa protein homologous to the yeast stress protein, STI1. Immune isolated samples of these three proteins support recovery of up to 35% of luciferase activity in an ATP-dependent manner, suggesting that these or associated proteins are involved in the renaturation of luciferase. Furthermore, we observed a correlation between luciferase renaturation activity and the levels of hsp70 and hsp90 in reticulocyte lysate preparations. Purified hsp90 and hsp70, along with an ATP-regenerating system, are able to renature luciferase to greater than 20% of its original activity. This renaturation is most efficient when hsp90 and hsp70 are at about a 2:1 ratio and at concentrations similar to those found in reticulocyte lysate. This study provides evidence for an ATP-dependent chaperoning activity in reticulocyte lysate that involves a cooperative action of hsp70 and hsp90.

Adenosine Triphosphate↗

PubMed1994

Transfusions to group O subjects of 2 units of red cells enzymatically converted from group B to group O.

BACKGROUND: It has previously been shown that full-unit (200 mL) transfusions of red cells (RBCs) enzymatically converted from group B to group O by treatment with alpha-galactosidase (ECO RBCs) are both safe and efficacious for normal group O or A subjects. STUDY DESIGN AND METHODS: The present study describes the results of a comprehensive clinical and serologic assessment of 2-unit (400 mL) ECO RBC transfusions to each of four normal group O subjects (after each had donated 1 unit of whole blood). RESULTS: Clinical (hematologic tests, chemistry analysis, urinalysis) and serologic analyses revealed no evidence of immediate or delayed transfusion reaction, despite a threefold to fivefold elevation in pre-existing anti-B antiglobulin titer. 51Cr-labeled ECO RBCs were administered to one of the four subjects to allow direct measurement of ECO RBC survival in the circulation, which indicated that it was normal (24-hour survival, 95%; t1/2, 29.5 days). The observed increases in hemoglobin (by 1.3 +/- 0.4 g/dL [13 +/- 4 g/L]) and hematocrit (by 3.2 +/- 0.8% [0.032 +/- 0.008]) in transfused subjects provide further evidence of the efficacy of these cells in vivo. CONCLUSION: These results extend those observed in our earlier 1-unit transfusion studies and suggest that ECO RBCs pose little risk and will be useful in transfusion medicine.

ABO Blood-Group System↗

PubMed1993

Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2 alpha kinase.

The heme-regulated inhibitor (HRI) of protein synthesis becomes activated in rabbit reticulocyte lysates in response to a variety of conditions including heme-deficiency, addition of oxidants, and heat shock. Activated HRI inhibits translation by catalyzing the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2. The molecular nature of the "signal" that leads to the activation of HRI in response to heat shock has not been characterized. We have recently reported that HRI interacts with the 90- and 70-kDa heat shock proteins (hsp) and a 56-kDa protein in hemin-supplemented lysates [Matts, R.L., Xu, Z., Pal, J.K., & Chen, J.-J. (1992) J. Biol. Chem. 267m 18160-18167]. In this report, we demonstrate that addition of denatured proteins, bovine serum albumin (BSA), beta-lactoglobulin, or alpha-lactalbumin, but not the addition of the native proteins, inhibits protein synthesis in hemin-supplemented reticulocyte lysates. The inhibition was reversed upon the addition of 10 mM cAMP or purified eIF-2B, classical criteria for HRI-mediated translational inhibition. Denatured BSA, but not native BSA, stimulated the phosphorylation of the alpha-subunit of eIF-2. This stimulation of eIF-2 alpha phosphorylation was inhibited by a monoclonal antibody to HRI, confirming that denatured BSA was causing the activation of HRI. The concentration of denatured BSA required to inhibit protein synthesis by 50% correlated with the levels of hsp70 present in each lysate preparation. Lysate hsp70 co-immunoadsorbed with denatured BSA, but not with not with native BSA. Hsp70 was co-adsorbed with HRI from lysate in the presence of native BSA, but not in the presence of denatured BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.

Adenosine Triphosphate↗

PubMed1992

The benign course of cavernous carotid artery aneurysms.

Recently, the benign nature of aneurysms of the cavernous carotid artery has been questioned. In a review of cases evaluated from 1980 to 1990 with this developmental aneurysm, the authors found 70 patients with 79 cavernous carotid artery aneurysms. As expected, the great majority (59 patients) had ophthalmoplegia as the initial problem. Retro-orbital pain (three cases) and a carotid-cavernous fistula (five cases) were infrequently the sole manifestation. Mirror-image asymptomatic aneurysms were found in nine patients and asymptomatic cavernous aneurysms were found in three additional patients. Thirty-four patients not surgically treated were followed for a mean of 2.8 years, and 36 surgical patients were followed for a mean of 4.1 years prior to treatment. Of the 79 aneurysms, one (1.3%) ruptured into the subarachnoid space during this period. Other than optic neuropathy or cranial neuropathy, no patient had a permanent neurological deficit; the 12 asymptomatic aneurysms remained asymptomatic. It is concluded that an aneurysm of the cavernous carotid artery is rarely associated with life-threatening complications, and treatment should be considered principally for patients with intolerable pain or problems related to vision.

Aged↗

PubMed1991

Toxic heavy metal ions activate the heme-regulated eukaryotic initiation factor-2 alpha kinase by inhibiting the capacity of hemin-supplemented reticulocyte lysates to reduce disulfide bonds.

Addition of toxic heavy metal ions (Cd2+, Hg2+, and Pb2+) to hemin-supplemented rabbit reticulocyte lysate brings about the activation of the heme-regulated eukaryotic initiation factor 2 alpha kinase (HRI) and the inhibition of protein chain initiation. In this report we examined the effects of monothiol and dithiol compounds, metal ion-chelating agents, and metallothioneins (MT) on metal ion-induced inhibition of protein synthesis. The dithiol compounds dithiothreitol and 2,3-dimercaptopropane sulfonic acid prevented and relieved the inhibition of protein synthesis caused by Cd2+ and Hg2+ in hemin-supplemented lysates, but the monothiol compounds 2-mercaptoethanol, cysteamine, D-(-)penicillamine, and glutathione had no effect. The inhibition of protein synthesis caused by Cd2+ was reversed by the addition of excess EDTA but not by the addition of excess nitrilotriacetic acid. Toxic heavy metal ions inhibited the capacity of hemin-supplemented lysate to reduce disulfide bonds. Addition of excess EDTA to Cd(2+)-inhibited lysates restored the capacity of the lysate to reduce disulfide bonds and inhibited the phosphorylation of eukaryotic initiation factor eIF-2. MTs and their apoproteins (apoMTs) inhibited the activation of HRI and protected protein synthesis from inhibition by Cd2+, Hg2+, and Pb2+. Addition of apoMTs to heavy metal ion-inhibited lysates restored the capacity of lysates to reduce disulfide bonds. The restoration of the lysate's thioredoxin/thioredoxin reductase activity was accompanied by the inactivation of HRI and the resumption of protein synthesis, indicating that apoMTs can "detoxify" metal ions already bound to proteins. Several observations presented in this report suggest that the binding of metal ions to the alpha-domain of MT is responsible for the ability of MT to sequester bound metal in a non-toxic form. Addition of glucose 6-phosphate or NADPH had no effect on protein synthesis in metal ion-inhibited lysates, and NADPH concentrations in Cd(2+)-inhibited and hemin-supplemented control lysates were equivalent. The data suggest that the metal ions cause the inhibition of protein synthesis by binding to vicinal sulfhydryl groups present in some critical protein(s), possibly the dithiols present in the active site of thioredoxin and (or) thioredoxin reductase, which leads to the activation of HRI.

Animals↗

PubMed1991

Single-unit transfusions of RBC enzymatically converted from group B to group O to A and O normal volunteers.

Full-unit transfusions of RBC enzymatically converted from group B to group O by treatment with alpha-galactosidase (ECO RBC) to group O and A normal healthy individuals exhibit excellent in vivo survival times (24-hour survival 95.1% +/- 2.3%, T50 36.9 +/- 4.6 days). These results confirm our earlier findings describing ECO RBC in vitro viability and normal in vivo survival time after small-volume infusions. No significant increase in pretransfusion anti-B titer or score is observed in either group O or A subjects provided that sufficient enzyme is used to treat the cells: Cells transfused to group O recipients require higher levels of enzyme (185 to 200 U/mL RBC) than those infused to group A (90 U/mL RBC). Two separate single-unit transfusions of ECO RBC to one group O recipient (4.5 months apart) also survived normally (24-hour survival 96% and 92%, T50 40 and 36 days) and did not increase preexisting anti-B levels in this subject. ECO RBC were not agglutinated or lysed by recipient sera before or after transfusion. Similarly, no antibody development to the alpha-galactosidase used in cell treatment (and washed from the product before transfusion) could be detected in any subject. The sustained increase in hemoglobin levels after transfusion of ECO RBC suggests that this product will be useful in treatment of acute and chronic anemia.

ABO Blood-Group System↗

PubMed1990

Bladder surface glycosaminoglycans: an epithelial permeability barrier.

Sulfated polysaccharide's ability to modulate the movement of small molecules was examined both in vivo and in vitro. For the in vivo test, the rabbit bladder was utilized and C-14 labeled urea 45-Ca, or 3H2O was placed into the lumen of control bladders, bladders pretreated with protamine sulfate (20 mg./cc) and bladders pretreated with protamine sulfate (20 mg./cc) plus pentosanpoly-sulfate (PPS), 10 mg./cc. After 45 minutes, the controls absorbed 21% of the urea, 16% of the calcium, and 38% of the 3H2O; the protamine treated group 40% urea, 23% calcium, and 51% H2O; the PPS only group 22% urea and the protamine plus PPS group absorbed 24% of urea, 18% calcium, and 44% water. Differences between the control and protamine groups were statistically significant, p less than 0.01 for urea 45-Ca and 3H2O. The bladder mucosa contained a significantly higher concentration of urea and calcium after protamine treatment which were both reversed by PPS (p less than 0.01) while 3H2O content went down significantly (p = 0.03), reflecting a loss of the hydrophilic effect of bladder GAG. The control mucosas had 250 cpm/mg. tissue urea for Ca 64 cpm/mg. and water 262 cpm/mg., the protamine group urea 498 cpm/mg., Ca 190 cpm/mg., and H2O 139 cpm/mg.; the protamine plus PPS group urea 344 cpm/mg., Ca 129 cpm/mg., and water 168 cpm/mg. For the in vitro studies, an Ussing chamber was employed. Normal rabbit bladder membranes were placed in the chambers and the potential difference was zeroed across the membrane. There were three groups, membranes that were treated only with the irrigating solution, membranes pretreated with protamine, and membranes pretreated with protamine plus PPS. At the end of 40 minutes, there was an approximately 1.2% movement of urea across the control membrane, a 3.5% movement across the protamine treated membrane (a significant increase p less than 0.001) and a 1.1% movement across the protamine plus PPS treated membrane. It would appear that the surface polysaccharide may play an important role as a bladder permeability barrier in modulating both charged and uncharged small molecule movement in that its ability to impair such movement can be inhibited by protamine and this protamine effect can be reversed by a treatment with an exogenous sulfated polysaccharide.

Animals↗

PubMed1989

Evidence for the association of the heme-regulated eIF-2 alpha kinase with the 90-kDa heat shock protein in rabbit reticulocyte lysate in situ.

Inhibition of protein synthesis initiation in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation occurs due to the activation of a heme-regulated protein kinase (HRI), which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. How the activation of HRI in hemin-supplemented lysate occurs in response to oxidants and heat stress is not well understood. Recently, the 90-kDa heat shock protein (hsp 90) has been reported to co-purify with HRI activity. In this report, we have used monoclonal antibodies directed against hsp 90 to determine whether HRI and hsp 90 are functionally associated in the reticulocyte lysate in situ. The AC88 antibody recognizes only free hsp 90 and only bound significant amounts of hsp 90 upon prolonged incubation in the absence of heme or upon N-ethylmaleimide treatment of hemin-supplemented lysates. HRI activity is not absorbed by the AC88 antibody. The 8D3 monoclonal antibody, which binds to both free hsp 90 and hsp 90 complexed to steroid hormone receptors, absorbed the hsp 90 present in hemin-supplemented lysates and reduced the HRI activity by 70-95%. Progressively more HRI activity is not adsorbed by the 8D3 antibody the longer the reticulocyte lysate is incubated in the absence of hemin. The HRI that is adsorbed from heme-deficient lysates by the 8D3 antibody is also more active. The sedimentation rate of HRI was analyzed by glycerol gradient centrifugation. HRI present in hemin-supplemented lysate was found to have a sedimentation coefficient of approximately 7.5-8 S and was adsorbed from fractions by the 8D3 antibody in association with hsp 90. A second peak of HRI activity with a sedimentation coefficient of approximately 4.5-5 S was detected upon glycerol gradient centrifugation of heme-deficient lysates. Upon Western blot analysis, heme-deficient lysates were found to have less hsp 90 in the 7.5-8 S region of glycerol gradients than hemin-supplemented lysates. The data suggest that HRI is associated with hsp 90 in an inactive form in hemin-supplemented lysates and dissociates from hsp 90 upon activation. There also appears to be an intermediate of active HRI which is associated with hsp 90 or which can reversibly associate with hsp 90. Similarities between the stages of HRI activation and steroid hormone receptor activation and transformation are discussed.

Animals↗

PubMed1988

Correlation between the distribution of the reversing factor and eukaryotic initiation factor 2 in heme-deficient or double-stranded RNA-inhibited reticulocyte lysates.

The recycling of eukaryotic initiation factor eIF-2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit-bound eIF-2.GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme-deficient and dsRNA-inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF-2(alpha-32P). RF and eIF-2(alpha P) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K+. The association of eIF-2(alpha-32P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F-, indicating the presence of an endogenous ribosome-associated phosphatase activity which is capable of dephosphorylating eIF-2(alpha P) in the absence of F-. These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S-bound eIF-2.GDP complex to promote the dissociation of GDP from eIF-2 and the release of eIF-2 from the 60 S subunit as a complex with RF.

Animals↗

PubMed1988

Gallium 67 uptake in thymic rebound.

We have reported a case of localized thymic enlargement and uptake of gallium 67 in a child who had received antineoplastic chemotherapy. The enlarged thymus showed normal histology, a picture consistent with thymic rebound after nonspecific stress. This case further demonstrates the need to consider thymic rebound as a cause of gallium 67 uptake in children with neoplastic diseases.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1987

Inhibition of rabbit reticulocyte lysate protein synthesis by heavy metal ions involves the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2.

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.

Animals↗