Charge immobilization of the voltage sensor in domain IV is independent of sodium current inactivation

Recovery from fast inactivation in voltage-dependent Na⁺ channels is associated with a slow component in the time course of gating charge during repolarization (i.e. charge immobilization), which results from the slow movement of the S4 segments in domains III and IV (S4-DIII and S4-DIV). Previous studies have shown that the non-specific removal of fast inactivation by the proteolytic enzyme pronase eliminated charge immobilization, while the specific removal of fast inactivation (by intracellular MTSET modification of a cysteine substituted for the phenylalanine in the IFM motif, ICM_MTSET, in the inactivation particle formed by the linker between domains III and IV) only reduced the amount of charge immobilization by nearly one-half. To investigate the molecular origin of the remaining slow component of charge immobilization we studied the human cardiac Na⁺ channel (hH1a) in which the outermost arginine in the S4-DIV, which contributes ∼20% to total gating charge ( Q _max), was mutated to a cysteine (R1C-DIV). Gating charge could be fully restored in R1C-DIV by exposure to extracellular MTSEA, a positively charged methanethiosulphonate reagent. The RIC-DIV mutation was combined with ICM_MTSET to remove fast inactivation, and the gating currents of R1C-DIV-ICM_MTSET were recorded before and after modification with MTSEA_o. Prior to MTSEA_o, the time course of the gating charge during repolarization ( off -charge) was best described by a single fast time constant. After MTSEA, the off -charge had both fast and slow components, with the slow component accounting for nearly 35% of Q _max. These results demonstrate that the slow movement of the S4-DIV during repolarization is not dependent upon the normal binding of the inactivation particle.     

Keratoameloblastoma: a tumor sui generis or a chimera?

The term keratoameloblastoma has been used to describe a histologically heterogeneous group of ameloblastoma variants which have in common the formation of keratin by the ameloblastomatous epithelium. The English language literature contains reports of only 12 cases of keratoameloblastoma, of which 4 cases exhibited a papilliferous component. We report a unique tumor that we believe falls within the broad histopathologic spectrum of keratoameloblastoma. We review the key clinical and histopathologic features of the previously reported cases of keratoameloblastoma and present an additional case that presented as an expansile, radiolucent lesion with internal opacification between the roots of teeth in the left anterior maxillary alveolar ridge of a 45-year-old white male. There is wide variation in the histopathologic appearance of cases reported under the appellation keratoameloblastoma. Our case exhibited a histopathologic feature shared by only 2 of the previously reported cases, notably islands and anastomosing cords of epithelium forming lamellated, pacinian-like stacks of parakeratin that extruded into the collagenous tumor stroma without eliciting a foreign body response. Due to the small number of reported cases, we are unable to accurately assess whether the biologic behavior of keratoameloblastoma differs from other histologic types of ameloblastoma.     

How Zebrafish Can Drive the Future of Genetic-based Hearing and Balance Research

Over the last several decades, studies in humans and animal models have successfully identified numerous molecules required for hearing and balance. Many of these studies relied on unbiased forward genetic screens based on behavior or morphology to identify these molecules. Alongside forward genetic screens, reverse genetics has further driven the exploration of candidate molecules. This review provides an overview of the genetic studies that have established zebrafish as a genetic model for hearing and balance research. Further, we discuss how the unique advantages of zebrafish can be leveraged in future genetic studies. We explore strategies to design novel forward genetic screens based on morphological alterations using transgenic lines or behavioral changes following mechanical or acoustic damage. We also outline how recent advances in CRISPR-Cas9 can be applied to perform reverse genetic screens to validate large sequencing datasets. Overall, this review describes how future genetic studies in zebrafish can continue to advance our understanding of inherited and acquired hearing and balance disorders.     

Endotoxin-induced suppression of erythropoiesis: the role of erythropoietin and a heme synthesis stimulating factor

The regulation of erythropoiesis is primarily controlled by erythropoietin (Ep). Recently, however, other factors that both stimulate and inhibit erythropoiesis have been reported. Using an in vitro liquid culture of bone marrow cells, a factor in normal mouse serum was demonstrated that markedly stimulated heme synthesis by marrow erythroid cells. In this study, the role of this heme synthesis stimulating factor (HSF) and Ep in the erythropoietic suppression caused by endotoxin administration to mice was examined. Although HSF levels did not alter appreciably after endotoxin injection, marrow erythroid cells from these animals became unresponsive to the factor. This could be reversed if Ep was added to the culture in vitro or if the hormone was injected into the mice 18 hr prior to harvesting the marrow. This marrow erythroid cell response is identical to that seen in animals in whom Ep levels are markedly reduced, such as that found in exhypoxic polycythemia, and suggest a decrease in the hormone following endotoxin administration. Additional studies demonstrated that when Ep was injected into mice 6 hr after endotoxin administration, an increase in femoral erythroid colony-forming units (CFU-E), proerythroblast number, and 59 Fe incorporation into femoral marrow cells could be demonstrated. These findings, together with the marrow erythroid cell response to the hormone, suggest that the mechanism for suppression of erythropoiesis after endotoxin injection is a reduction in the level of circulating Ep.     

Clinical investigation of human alpha interferon in chronic myelogenous leukemia

Fifty-one patients with previously untreated or minimally treated chronic myelogenous leukemia in chronic phase received human alpha interferon 3 to 9 X 10(6) units intramuscularly (IM) daily until complete hematologic remission, then at doses ranging from 3 X 10(6) units every other day to 9 X 10(6) units daily. Forty-one (80%) patients achieved a hematologic response, 36 (71%) of them attaining a complete hematologic remission with normal peripheral WBC and differential counts. Responding patients showed continuous but slow normalization of several other blood and marrow parameters including platelet counts, serum lactic dehydrogenase and B12 levels, and marrow cellularity and maturation index. Suppression of the Philadelphia chromosome on serial cytogenetic studies of marrow metaphases was documented in 20 of the 36 patients who achieved complete hematologic remission (56%; 39% of total group), eight of whom (22%) had a decrease of the Philadelphia chromosome-positive metaphases to less than 35%. These changes were persistent for 6 months or longer in 18 patients, seven of whom had continuous suppression of the Philadelphia chromosome to less than 90% for a median of 30+ months (range 21+ to 39+ months). After a median follow-up period of 37 months, 25 patients remain in continued disease control with interferon therapy. The projected 3-year survival rate is 76%, with a yearly death rate of 6%, 9%, and 9% in the first 3 years. Response, Philadelphia chromosome suppression, and survival were significantly better among patients in the low-risk category compared to intermediate- and high-risk categories, as defined by a multivariate analysis-derived prognostic model. The projected 3- year survival rate was 94% for patients who achieved a complete hematologic remission on interferon therapy and 45% for those who did not. Thirteen patients have developed blastic crisis, six with lymphoid and three with undifferentiated morphology. We conclude that human leukocyte alpha interferon effectively controls chronic myeloid leukemia and allows reappearance of diploid hemopoietic cells in some patients.     

Mass screening for colorectal cancer

A voluntary community colorectal cancer screening project to detect occult blood in the stool of asymptomatic individuals was undertaken; 49,353 Hemoccult^® II kits were distributed. A total of 23,674 completed kits were returned to a central repository and processed (compliance rate, 48 percent); 851 participants had positive results (3.6 percent). Of the 640 who underwent further medical evaluation, 299 participants (46.7 percent) who had adequate follow-up had no evidence of disease. Diverse disease entities were detected in 341 participants, which was 1.4 percent of those enrolled. Forty-one patients (0.17 percent) showed significant findings that included 29 cancers (0.12 percent) and 12 (0.05 percent) noninvasive malignant polyps. Of the cancers, there were 27 colorectal, one nonHodgkin's lymphoma, and one carcinoma of the vocal cord. In addition, 107 patients (0.45 percent) had benign polyps and 193 patients (0.82 percent) had various diseases of the gastrointestinal tract and other medical conditions. The cost of the program was modest and the results conformed to those found in previous screening surveys. The heightened public awareness of testing for colorectal disease and the detection of early lesions justifies the guaiac test screening program for mass survey.     

Nephrocolic fistula

A patient with nephrocolic fistula secondary to perinephric abscess was treated successfully with nephrectomy, colonic resection, and colocolostomy. Supported in part by the Dorothy Rider Pool Health Care Trust Fund.     

Evaluation of drugs for elimination of leukemic cells from the bone marrow of patients with acute leukemia

Relatively nonmyelotoxic drugs and drug combinations were investigated for their ability to eliminate malignant cells from human bone marrow. In vitro 90% inhibitory concentration (IC90) doses were established on granulocyte macrophage colony-forming units (GM-CFU) in culture of bone marrow by using the GM-CFU assay for the following drugs: 4- hydroperoxycyclophosphamide (4-HC), Adriamycin, L-asparaginase, bleomycin, hydrocortisone, VP-16, spirogermanium, Taxol, and vincristine. The leukemic cell kill efficiency of these drugs at IC90 doses was compared with that of 4-HC on acute lymphoid leukemia (ALL) cell lines by using the limiting-dilution assay. Under these conditions, no single drug was superior to 4-HC. To increase the in vitro effect in leukemic cell kill, combinations of vincristine with hydrocortisone, Adriamycin, VP-16, and 4-HC were investigated. Vincristine at 1 to 5 micrograms/mL increased the marrow cytotoxicity of hydrocortisone, Adriamycin, and VP-16, but it was protective (subadditive) with 4-HC. Vincristine and 4-HC in combination was additive to supraadditive on ALL cell lines, increased the leukemic cell kill by one to two logs above 4-HC alone at IC90 doses (P less than .05), and was not affected by the addition of excess marrow cells. The recommended doses for chemopurging in clinical studies are vincristine, 1 to 5 micrograms/mL, plus 4-HC, 5 micrograms/mL.     

Use-dependent block of sodium current by ethmozin in voltage-clamped internally perfused canine cardiac Purkinje cells

Block of sodium current (INa) by ethmozin (moricizine), an antiarrhythmic drug, was investigated in isolated, voltage-clamped, canine cardiac Purkinje cells. Initial block of INa by ethmozin (2 microM) in noninactivated cells (held at -150 mV) was 9.3 +/- 1.2% (S.D.). Additional "use-dependent" block developed in response to repetitive depolarization. This block was both frequency-dependent and dose-dependent with the fall in peak INa greater at increasing depolarization frequencies (0.625 to 4 Hz) and with increasing dose (2 microM to 20 microM). Use-dependent block was modeled according to the guarded receptor hypothesis assuming ingress to the channel binding site during the open state of the channel, and egress from the channel independent of the kinetic state of the channel. The rate constants (on-rate = 2100 +/- 100 (S.D.)/M/ms and off-rate = 1.7 +/- 0.3 (S.D.) 10(-5)/ms) were used to predict the time course of INa block in response to repeated depolarizations and the dose-response relationship of steady-state used-dependent block measured in independent experiments. We conclude that ethmozin blocks INa in Purkinje cells in both a non-use-dependent and a use-dependent manner and that the guarded receptor model is useful in describing the use-dependent block.