Biomedical subjects

G Guzman

Publications and source records attributed to G Guzman.

At least 19 recordsLinked to original sources

PubMed2006

E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics.

Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca(2+)-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (tau(1)) of myosin heads from thin filaments, 2) rebinding time (tau(2)) of the cross bridges to actin, and 3) dissociation time (tau(3)) of ADP from the active site of myosin. tau(1) was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. tau(2) was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. tau(3) was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. tau(1) was statistically greater in Tg-m than in controls. tau(2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. tau(3) was the same in Tg-m and controls. To determine whether the difference in tau(1) was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in tau(1) was probably caused by myofibrillar disarray. The decrease in tau(2) of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.

Actins↗

PubMed2006

Expression of entry receptor nectin-1 of herpes simplex virus 1 and/or herpes simplex virus 2 in normal and neoplastic human nervous system tissues.

Herpes simplex virus 1 and/or Herpes simplex virus 2 (HSV) are important pathogens of human nervous system (NS) and genetically modified HSV strains have been proposed as vectors for gene therapy targeting the brain and brain tumors. Nectin-1 is an immunoglobulin-like adhesion molecule that participates in the formation of synapses and serves as an entry receptor for HSV. The expression pattern of nectin-1 in normal human NS and brain tumors is not well understood. To better understand the nectin-1 expression in normal and neoplastic human NS, immunohistochemistry was used to detect the nectin-1 expression in sections of normal human brain, spinal cord and trigeminal and dorsal root ganglia (n=10) and in sections of primary NS neoplasms (n=22). In normal human NS, nectin-1 was detected in the soma and processes of central and peripheral neurons, in ependymal cells, choroid plexus epithelial cells, vascular endothelial cells and meningothelial cells. Oligodendrocytes, astrocytes, vascular smooth muscle cells, and Schwann cells showed variable immunoreactivity. Among tumors, schwannoma, fibrous meningioma, and medulloblastoma were nectin-1 negative. Oligodendroglioma, ependymoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma multiforme and meningothelial meningioma showed weak focal nectin-1-positivity. Ganglion cells of ganglioglioma were strongly positive. These studies provide novel information about the expression of nectin-1 in normal and neoplastic NS, and thus may lead to a better understanding of cell targeting by HSV during HSV-induced neurological disease and during a HSV-based gene therapy.

Basal Ganglia↗

PubMed2001

Costs of managing Helicobacter pylori-infected ulcer patients after initial therapy.

BACKGROUND: The objective of this research was to evaluate the outcomes and costs of alternative approaches to managing patients previously treated for peptic ulcer disease and Helicobacter pylori infection. MATERIALS AND METHODS: A decision-analytic model was used to compare (1a) urease breath testing (UBT) for assessment of H. pylori status versus (1b) observation without further testing or treatment, among patients who were symptom-free following initial antimicrobial and antisecretory therapy for endoscopically demonstrated ulcer and H. pylori infection; and (2a) UBT versus (2b) repeat endoscopy with H. pylori testing, and versus (2c) repeat antimicrobial and antisecretory therapy without further testing, among patients who remained symptomatic following initial therapy. RESULTS: Among patients who were symptom free after initial therapy, 6.1% receiving UBT had symptomatic ulcer at one year, compared to 18.2% of those simply observed. The expected first-year cost per symptom-free patient following initial therapy was $591 for UBT compared to $480 for observation. Among patients with persistent symptoms after initial therapy, 21% receiving repeat therapy had symptomatic ulcer at one year, compared to 23.8% receiving repeat endoscopy, and 23.3% receiving UBT. Corresponding medical costs per patient were, respectively, $766, $1787 and $1122. CONCLUSIONS: The optimal approach to managing patients following initial treatment for ulcer and H. pylori infection depends on symptom status following initial therapy. For symptomatic patients, the preferred approach is to prescribe a repeat course of antimicrobial and antisecretory therapy. For patients without symptoms following initial therapy, UBT is the preferred approach because it is associated with a threefold lower risk of symptomatic ulcer at one year, although it costs an additional $110 per patient, compared with observation.

Breath Tests↗

PubMed2000

Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+ binding, and phosphorylation.

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.

Amino Acid Sequence↗

PubMed2000

A Leishmania homologue of receptors for activated C-kinase (LACK) induces both interferon-gamma and interleukin-10 in natural killer cells of healthy blood donors.

Natural killer (NK) cells from individuals unexposed to Leishmania organisms proliferate with high interferon (IFN)-gamma secretion in response to crude Leishmania antigen preparations. In an attempt to identify the molecules that induce blood cells to proliferate and to secrete cytokines, we tested the effect of a 36-kDa Leishmania homologue of receptors for activated C-kinase (LACK) on peripheral blood mononuclear cells from unexposed individuals. Mainly CD8(+) and NK cells proliferated in response to LACK. At both the mRNA and soluble protein level, the main sources for LACK-induced IFN-gamma and interleukin (IL)-10 were T and NK cells. Furthermore, in the presence of anti-major histocompatibility complex (MHC) class II antibody, there was inhibition of LACK responses in both CD4(+) and CD16/56(+) cells, with a marked decrease in IFN-gamma but with an increase in IL-10 production. We conclude that the response to LACK is part of the response to Leishmania organisms in unexposed donors described elsewhere. That this NK-dominated response is MHC class II sensitive, whether through a direct or indirect effect, is discussed.

Animals↗

PubMed2000

Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy.

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G(1) --> A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca(2+) sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca(2+) sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca(2+) sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca(2+) regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.

Amino Acid Sequence↗

PubMed2000

Investigating the role of Ca2+-binding site IV in barnacle troponin C.

Two genetically engineered, recombinant versions of native barnacle troponin C (TnC) (BTnC,) were created from the bacterially expressed, recombinant, wild-type BTnC (BTnCWT) to investigate the role of the Ca(2+)-specific sites in force regulation. The mutant BTnC4- contains a single amino acid mutation in site IV which results in the inactivation of site IV Ca2+ binding; the mutant BTnCTrunc lacks the last II amino acids of the C-terminal, and hence most of site IV. Both mutant proteins, which retain an active site II, bind to native TnC-depleted myofibrillar bundles and restore approximately 40% of the tension-generating capacity, about half that seen with purified native BTnC1 or BTnC2. This observation implies that the Mg(2+)-dependent interaction with troponin I (TnI) is at a location on TnC other than the C-terminal Ca(2+)-binding sites of BTnC2. Replacement with BTnCTrunc increases the sensitivity of the myofibrillar bundle to changes in ionic strength. Decreasing the ionic strength from 0.15 to 0.075 M increased force by 34%, a value much greater that the 8% increase seen in control bundles or bundles substituted with BTnC4-. These findings implicate TnC in determining this fibre characteristic, although this cannot be simply due to the alteration in the numbers of Ca2+ ions bound by the troponin complex since both BTnC4- and BTnCTrunc bind only 1 mol Ca2+/mol protein.

Amino Acid Sequence↗

PubMed1999

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.

Acrylamide↗

PubMed1999

Characterisation of a mutant of barnacle troponin C lacking Ca2+-binding sites at positions II and IV.

This study investigates a mutant barnacle troponin C (TnC) protein (BTnC2-4-) in which the Ca2+-binding sites (sites II and IV) have been rendered non-functional. Eliminating Ca2+ binding at both Ca2+-binding sites of barnacle TnC did not prevent the incorporation of BTnC2-4- into TnC-depleted myofibrillar bundles, although, as expected, the mutant was not able to effect muscle regulation. We conclude that the Mg2+ involved in stabilising the interaction between TnC and TnI in the barnacle must bind at a separate location to the Ca2+-binding sites. Competition experiments between BTnC2-4- and wild-type barnacle TnC (BTnCWT) or the native isoform BTnC2 indicate that BTnC2-4- has an approximately fourfold higher affinity for barnacle TnI than BTnCWT but a lower affinity for TnI compared to BTnC2. These results indicate that disabling sites II and IV changes the affinity of BTnC2-4- for TnC-denuded barnacle myofibrils, altering the stability of the bond formed between TnC and the thin filament.

Animals↗

PubMed1999

Investigation of a genetically engineered mutant of barnacle troponin C containing a central helix deletion.

To examine the importance of the central alpha-helix of troponin C (TnC) we have bacterially expressed one of the isoforms of barnacle TnC (BTnC2), BTnCWT, but without the aspartate residue at position 80 in the central helix (BTnC80-). This manipulation is expected to produce an approximately 100 degrees axial rotation of the C-domain with respect to the N-domain, and a net charge change of -1. BTnC80- mutant was able to restore force to TnC-depleted skinned barnacle myofibrillar bundles to a greater extent than wild-type protein (approximately = 170%). Competition experiments between BTnC80- and BTnC2-4-, a mutant lacking both of the calcium-specific sites (sites II and IV), shows that deletion of a single amino acid in the central helix results in a protein with increased affinity for the thin filament and one that is bound preferentially compared to BTnC2-4- when at equimolar concentrations.

Animals↗

PubMed1999

Gallbladder rupture during laparoscopic cholecystectomy: does it have an effect on postoperative morbidity?

Gallbladder rupture during laparoscopic cholecystectomy is a common event that may lead to increased postoperative morbidity. To evaluate this event, we reviewed 300 cases of laparoscopic cholecystectomy. Duration of surgery and hospitalization, postoperative symptoms, wound infection, and late complications were analyzed by comparing two groups of patients, one without gallbladder rupture (A) and one with rupture (B). Gallbladder rupture was found in 40 cases (13.9%). Duration of surgery averaged 81 min for group A and 96.5 min for group B. Postoperative symptoms in the first 24 hours were present in approximately 10% of patients in both groups. Within the first 24 hours, 92.3% of patients in group A were discharged compared with 85% in group B. One patient (0.4%) in group A developed wound infection compared with 2 patients (5%) in group B (p = 0.05). To date, no patients have developed late abdominal complications associated with the procedure. Although this was a retrospective and uncontrolled study, gallbladder rupture during laparoscopic cholecystectomy was found to be associated with increased wound infections. No other significant effects on postoperative morbidity were detected.

Adult↗

PubMed1996

The role of the four Ca2+ binding sites of troponin C in the regulation of skeletal muscle contraction.

In order to study the role of the Ca2+-specific sites (I and II) and the high affinity Ca2+-Mg2+ sites (III and IV) of TnC in the regulation of muscle contraction, we have constructed four mutants and the wild type (WTnC) of chicken skeletal TnC, with inactivated Ca2+ binding sites I and II (TnC1,2-), site III (TnC3-), site IV (TnC4-), and sites III and IV (TnC3,4C-). All Ca2+ binding site mutations were generated by replacing the Asp at the X-coordinating position of the Ca2+ binding loop with Ala. The binding of these mutated proteins to TnC-depleted skinned skeletal muscle fibers was investigated as well as the rate of their dissociation from these fibers. The proteins were also tested for their ability to restore steady state force to TnC-depleted fibers. We found that although the NH2-terminal mutant of TnC (TnC1,2-) bound to the TnC-depleted fibers (with a lower affinity than wild type TnC (WTnC)), it was unable to reactivate Ca2+-dependent force. This supports earlier findings that the low affinity Ca2+ binding sites (I and II) in TnC are responsible for the Ca2+-dependent activation of skeletal muscle contraction. All three COOH-terminal mutants of TnC bound to the TnC-depleted fibers, had different rates of dissociation, and could restore steady state force to the level of unextracted fibers. Although both high affinity Ca2+ binding sites (III and IV) are important for binding to the fibers, site III appears to be the primary determinant for maintaining the structural stability of TnC in the thin filament. Moreover, our results suggest an interaction between the NH2- and COOH-terminal domains of TnC, since alteration of sites I and II lowers the binding affinity of TnC to the fibers, and mutations in sites III and IV affect the Ca2+ sensitivity of force development.

Animals↗

PubMed1996

Results of the economic evaluation of the first study. A multinational prospective economic evaluation. FIRST Investigators. Flolan International Randomized Survival Trial.

We present the prospective economic evaluation that served as a secondary endpoint for the FIRST study, a randomized international multicenter trial of patients with severe congestive heart failure. Although the clinical results of this study were disappointing, we demonstrated the feasibility of incorporating prospective economic evaluation in phase III clinical trials.

Aged↗

PubMed1994

Cost-effectiveness of screening for hereditary hemochromatosis.

BACKGROUND: Recent studies have estimated the prevalence of hereditary hemochromatosis to be 3 to 8 per 1000. Early detection and treatment can prevent disease manifestations and normalize life expectancy. We used decision analysis techniques to determine whether screening the population at large for hereditary hemochromatosis would be cost-effective. METHODS: We constructed a model to compare the cost and outcome of a strategy of performing screening transferrin saturation tests on cohorts of 30-year old men with that of awaiting symptomatic disease. Baseline estimates of disease prevalence and complication rates were based on the published literature. Costs of treatment were estimated based on prevailing local costs. Sensitivity analyses were then conducted to determine which variables had the most significant impact on the decision to screen. RESULTS: At our baseline estimates, the decision to screen was found to be a dominant strategy and resulted in cost saving. Sensitivity analysis showed that four variables had the most significant impact on the decision to screen: (1) the prevalence of hereditary hemochromatosis, (2) the probability of developing disease manifestations, (3) the cost of the screening test, and (4) the discount rate. Screening was a dominant strategy for asymptomatic men provided that the prevalence of hereditary hemochromatosis was at least 3 per 1000, the probability of developing disease manifestations was greater than 0.4, the test cost was less than $12, and the discount rate was less than 3%. Using more pessimistic estimates, the cost per life year saved was still less than that considered acceptable for many common medical interventions. CONCLUSION: Screening for hereditary hemochromatosis has a favorable cost-effectiveness ratio over a wide range of assumptions. We recommend that practitioners consider including a serum transferrin saturation test in their routine screening for asymptomatic white men.

Adult↗

PubMed1994

Endoscopic techniques in coronal brow lifting.

Laboratory investigations in fresh cadaver heads demonstrated the feasibility of endoscopic techniques applicable to aesthetic surgery of the face, including the cheeks, the forehead, the orbit, the nose, and the nasal septum. Clinical experience with coronal brow lifting in 32 patients using endoscopic techniques is also presented. The elimination of the ear-to-ear coronal incision is an obvious advantage. The endoscopic approach accomplishes division and weakening of the corrugators, procerus, and frontalis muscles, as well as moderate elevation of the eyebrows. Excess skin is accommodated by dissecting the scalp posteriorly toward the occiput.

Adult↗

PubMed1993

Improved visualization of carcinoid liver metastases by indium-111 pentetreotide scintigraphy following treatment with cold somatostatin analogue.

Five patients with hepatic metastases of midgut carcinoid underwent somatostatin receptor scintigraphy with indium-111 pentetreotide before and during treatment with octreotide. Octreotide treatment changed the biodistribution of 111In-pentetreotide significantly. Whereas the radioactivity in liver, spleen and kidney decreased, hepatic metastases showed increased contrast. In one patient, liver metastases could only be detected during octreotide treatment. These data suggest that the diagnostic reliability of somatostatin receptor scintigraphy in carcinoid liver metastases is not necessarily compromised by octreotide therapy. Because of different biodistributions, the detection of liver metastases may even be improved during octreotide therapy.

Carcinoid Tumor↗

PubMed1993

The potential value of somatostatin receptor scintigraphy in medullary thyroid carcinoma.

In a prospective study, ten patients with recurrent medullary thyroid carcinoma (markedly elevated calcitonin levels) were investigated by means of somatostatin receptor scintigraphy (SRS) with 111Inpentetreotide. Scintigraphically, 30 sites of pathological uptake were found, mostly located in the neck and upper mediastinum. So far, 18 suspected tumour sites underwent histological examination and 14 of them could be verified as metastases of medullary thyroid carcinoma (MTC). The remaining four putative tumour lesions turned out to be false positive scintigraphic findings caused by chronic inflammation and somatostatin receptor positive tumours other than MTC. We conclude that SRS is a promising imaging modality for localization of MTC recurrence and may thus make a contribution to better management of this patient group.

Adult↗

PubMed1993

Diagnostic reliability of somatostatin receptor scintigraphy during continuous treatment with different somatostatin analogs.

In order to evaluate the diagnostic reliability of somatostatin receptor scintigraphy (SRS) in patients treated with somatostatin analogs, ten patients with metastatic neuroendocrine tumors were investigated before and during continuous treatment. Different somatostatin analogs were used for therapy: five patients received octreotide (Sandostatin, Sandoz, Switzerland) and five were treated with BIM 23014 (Lanreotide, Ipsen Biotech, France) within the scope of a clinical phase II study. The SRS findings were analyzed in terms of biodistribution of the labeled somatostatin analog and tumor visualization comparing the two studies in each patient. Whereas liver, spleen, and kidney uptake were decreased during octreotide treatment and increased on lanreotide therapy, tumor accumulation was intensified in all but one patient. Our results suggest that the diagnostic value of SRS is not necessarily restricted during treatment with somatostatin analogs. Indeed, tumor visualization may even be enhanced in this therapeutic setting.

Adult↗