Immobilized cells: behaviour of carrageenan entrapped yeast during continuous ethanol fermentation

Summary Data of cell concentration, viability and microscopic observation of cell distribution inside carrageenan immobilized yeast beads are reported. Results were obtained from a continuous packed-bed reactor performing alcoholic fermentation and the main observations made on cell activity are in agreement with the fermentation profiles inside the fermenter.     

The effect of fish predation on Cyclops life cycle

Two populations of Cyclops abyssorum tatricus studied in neighbouring alpine lakes in Tatra Mountains (southern Poland) differed in their life cycles. In the lake with planktivorous fishes, Cyclops was typically monocyclic, with highly synchronized reproduction during two winter months, while in the fishless lake its reproduction was asynchronous and continued for six months. Direct and indirect effects of fish predation on Cyclops life cycles are discussed.     

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. They are usually sporadic but can also occur associated with the neurofibromatosis type 2 (NF2) syndrome. The gene responsible for NF2, recently isolated from chromosome 22, encodes a membrane-organizing protein that shows high sequence homology to a protein family thought to link the cytoskeleton with membrane proteins. Mutations of the NF2 gene have been described in sporadic meningiomas, exclusively in tumors that show loss of heterozygosity (LOH) of 22q. These preliminary results indicate that the NF2 gene is involved in the pathogenesis of at least a subset of meningiomas, where it does indeed behave as a tumor suppressor gene. In order to characterize better the role of the NF2 gene in the genesis of meningiomas we have examined the entire coding sequence of the gene in 125 meningiomas by single-strand conformational polymorphism analysis; furthermore, LOH analysis for markers of 22q has been carried out. Inactivating mutations were identified in 30% of our samples, all of which also showed LOH of 22q. The majority of mutations identified were frameshifts and nonsense mutations, which are predicted to produce a truncated or non-functional protein. We also found two missense and three in-frame deletions that may pinpoint specific regions of the protein critical to its function. Furthermore, the distribution of mutations throughout the gene, suggested that exons 2, 3, 5, 11 and 13 are more frequently involved. Our results reconfirm the importance of the NF2 gene in the pathogenesis of meningiomas and also suggest that there may be a nonrandom clustering of mutations throughout the gene.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

XY gonadal dysgenesis and gonadoblastoma: a study in two sisters with a cryptic deletion of the Y chromosome involving the SRY gene

This paper reports a case of XY gonadal dysgenesis in two sisters. Both patients presented an eunochoid female phenotype with normal external genitalia. At laparotomy, the elder sister was found to have bilateral gonadoblastoma. Cytogenetic studies, which included G and C banding and in situ hybridization, showed that the patients had an apparently normal 46, XY karyotype. PCR analyses revealed absence of the conserved portion (HMG box) of the SRY gene and of the Y chromosome pseudoautosomal boundary region sequence in both patients. The presence of the ZFY sequence was detected by Southern hybridization in the two affected sisters. The patients' father (46, XY, no mosaicism detected in peripheral blood lymphocytes) was positive for SRY and ZFY sequences. The occurrence of gonadoblastoma is discussed in terms of the genetic factors that may lead to tumor development.     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Ectopic ACTH syndrome

Ectopic ACTH syndrome represents a cancer-induced amplification of a property [proopiomelanocortin (POMC) peptides production] normally present in the cells from which the cancer originated but with aberrant posttranslational processing of POMC resulting in a greatly elevated secretion of ACTH precursors. The classic ectopic ACTH-producing tumors described in the 1960s were highly malignant but more recently slowly growing tumors such as carcinoids are reported with increasing frequency. Clinical features of patients with ectopic ACTH were analyzed, including biochemical abnormalities, plasma ACTH, cortisol and urinary steroids. Dynamic tests such as high-dose dexamethasone suppression, metyrapone and ovine-CRH (oCRH) stimulation were explored, as well as inferior petrosal sinus ACTH sampling before and after oCRH. Among the tumor markers examined, elevation of ACTH precursors was uniformly present followed by increased output of calcitonin, gut hormones, oncofetal and placental hormones in decreasing order. Since more than 90% of ectopic ACTH tumors are neuroendocrine in nature exhibiting APUD characteristics, their 2 markers, neuron-specific enolase and chromogranins are very useful. The imaging procedures for localization of the tumor ranged from chest X-rays to computed tomography and magnetic resonance of the chest and abdomen. Abdominal ultrasonography was also useful. Finally somatostatin receptor scintigraphy permitted demonstration of unrecognized tumors and/or metastases, even when the tumors were occult. The ACTH content, immunostaining for APUD markers and altered POMC processing were evaluated in ectopic tumors and/or metastases. Occult ectopic ACTH syndrome of more than 4–6 months of symptoms without the emergence of an obvious source was reviewed. Since the tumors are often clinically and biochemically undistinguishable from pituitary-dependent Cushing's disease, inferior petrosal sinus sampling for ACTH after oCRH stimulation established the diagnosis in over 90% of the cases. 60% of the occult tumors were thoracic carcinoids (3/4 bronchial carcinoids), followed by small cell lung cancer and pancreatic neuroendocrine tumors. In 12% the primary etiology was not detected. The rare syndrome of ectopic CRH syndrome (6 published cases) leading to excessive stimulation of the pituitary which became hyperplastic and secreted excessive amounts of ACTH is discussed. Finally, the 12 published cases and 1 unreported patient with ectopic CRH-ACTH tumors were reviewed, the majority being metastatic small cell lung carcinomas, bronchial and thymic carcinoids.     

Primitive nervous systems: Peripheral habituation in decerebrate polyclad flatworms

The response to a vibration stimulus recorded from the cords of the ventral submuscular plexus of the polyclad flatworm, Notoplana acticola, consists of a burst of action potentials. The response can be abolished by the application of MgCl₂ to the sea water bathing the preparation. With repeated application of the stimulus, decreasing numbers of action potentials can be measured. This waning responsiveness can be dishabituated by applying a more intense vibration stimulus or with electrical shocks applied directly to the ventral nerve plexus. With electrical stimuli a number of shocks have to be applied before the response can be dishabituated. Changes in responsiveness can be measured simultaneously in a number of sites in the plexus even after the nerves between recording sites have been severed. With different interstimulus intervals the extent of habituation changes. As interstimulus intervals increase from 1 to 5 sec, there appears to be a decrease in responsiveness which recovers when interstimulus intervals become longer than 5 sec.