Attachment of acetylcholinesterase to structures of the motor endplate

Summary The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.     

Monoclonal antibodies for the structural analysis of the Na+/H+ antiporter NhaA from Escherichia coli

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand. Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins. The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.     

Detection of impurities in dietary supplements containing l-tryptophan

Amino Acids - Impurities in nine dietary supplements containing l-tryptophan were evaluated using an HPLC methodology. In five tested products, the total impurities were higher than the thresholds...     

Predictive markers in early research and companion diagnostic developments in oncology

Predictive biomarkers are discovered and used in oncology research to formulate hypotheses aimed at the identification of patients benefiting from specific therapeutic intervention(s). They pave the way to the development of companion diagnostic tests which are tools readily implemented in the clinic and serve to qualify a patient for treatment with a particular targeted drug or the continued use of a particular drug, thus maximizing the benefit to risk ratio of the medical intervention to the patient. Predictive biomarkers are defined by biological characteristics of the patient's or tumor status that can be measured objectively and correlated with clinical outcome: these can be molecular, cellular or biochemical features. Predictive markers need extensive analytical validation - specific for the tool utilized for their assessment - as well as rigorous clinical qualification in the context of the drug treatment for which they define clinical utility. The process of companion diagnostic development is a highly interdisciplinary and complex one, driven by key crucial milestones and accompanying the same and typical process of a whole drug discovery and development continuum, from marker discovery and validation, assay development, clinical qualification until test approval and commercialization.     

Intranuclear arrangement of human chromosome 12 is reflected in metaphase chromosomes as non-random bending

We have found a high correlation of non-random bending of human metaphase chromosome 12 with the intranuclear arrangement deduced by Nogami et al. (Chromosoma 108 (2000) 514), providing further evidence of the relation of non-random bending and the interphase organization of the nucleus.     

War, mental disorder and suicide

War as a human disaster of major significance has led to an increase in the number of suicides committed by people suffering from mental disorders. Considering the results of similar research, we were particularly interested in the effect that war has on the incidence of suicide among of people with mental disorders. The research included 16,362 patients with mental disorders, treated at the Clinic for Psychiatry at the Clinical Hospital Split during the nine-year timeframe which were divided into pre-war (April 6th 1988- April 7rh 1991), wartime (April 6th 1991 -April 7rh 1994) and post-war (April 6th 1997 - April 7th 2000) periods. We studied the effects of how wartime events upon people with mental disorders in terms of their suicide rates, taking into account gender, age group, and the diagnosis under which they were treated. In our research, we found a statistically significant difference in suicide incidence between three observed periods (prewar April 6th 1988 April 7th 1991; wartime April 6th 1991 -April 7th 1994; and postwar April 6th 1997 -April 7th 2000) with the incidence being the highest during the wartime period (chi2 =9.98: p=0.007). Out of 16,362 patients treated at the clinic during the observed timeframe, a total of 78 people committed suicide. Twenty-two patients committed suicide during the first three year pre-war period; 36, during the three year wartime period; and 20, during the third three year post-war period. With this research we intended to offer a better understanding of the complexity of the suicide problem of mental patients as a phenomenon.     

GSM radiocellular telephones do not disturb the secretion of antepituitary hormones in humans

It is known that the endocrine system of experimental animals is susceptible to perturbation by radiofrequency (RF) radiation. Because of the recent interest in health and safety issues of cellular telephones, an experiment was designed to evaluate the effect of a 900 MHz RF radiation emitted by a Global System for Mobile radiotelephone (217 Hz impulses, one-eighth duty cycle, 2 W peak power) on human endocrine functions. Twenty healthy male volunteers aged from 19 to 40 were inducted in the present experiment. Each subject was exposed to RF radiation through the use of a cellular phone 2 h/day, 5 days/wk, for 1 month. Subjects were their own control. End points were serum adrenocorticotropin, thyrotropin, growth hormone, prolactin, luteinizing hormone, and follicle stimulating hormone concentrations. These end points were determined in nine weekly blood samples obtained starting 3 weeks before the commencement of the exposure and ending 2 weeks after exposures. All but one blood sample was drawn 48 h after each weekly session. The seventh drawing was performed the morning after the last weekly exposure. Within each individual, the preexposure hormone concentration was used as a control. Results indicated that all hormone concentrations remained within normal physiologic ranges. A difference was not noted among the nine weekly samples in five of six hormones studied. There was a significant change only in thyrotropin concentration, showing a 21% decrease on the seventh sampling. Because this change recovered fully during the postexposure period, it is concluded that 1 month of intermittent exposures to RF radiation from a cellular telephone does not induce a long-lasting or cumulative effect on the hormone secretion rate of the anterior pituitary gland in humans. Bioelectromagnetics 19:271–278, 1998. © 1998 Wiley-Liss, Inc.     

Leptin Acts via Leptin Receptor-Expressing Lateral Hypothalamic Neurons to Modulate the Mesolimbic Dopamine System and Suppress Feeding

The lateral hypothalamic area (LHA) acts in concert with the ventral tegmental area (VTA) and other components of the mesolimbic dopamine (DA) system to control motivation, including the incentive to feed. The anorexigenic hormone leptin modulates the mesolimbic DA system, although the mechanisms underlying this control have remained incompletely understood. We show that leptin directly regulates a population of leptin receptor (LepRb)-expressing inhibitory neurons in the LHA and that leptin action via these LHA LepRb neurons decreases feeding and body weight. Furthermore, these LHA LepRb neurons innervate the VTA, and leptin action on these neurons restores VTA expression of the rate-limiting enzyme in DA production along with mesolimbic DA content in leptin-deficient animals. Thus, these findings reveal that LHA LepRb neurons link anorexic leptin action to the mesolimbic DA system.     

THE APPEARANCE OF ACETYLCHOLINESTERASE IN THE MYOTOME OF THE EMBRYONIC RABBIT : An Electron Microscope Cytochemical and Biochemical Study

Acetylcholinesterase (AChE) activity has been studied in the myoblast of skeletal muscle of the 9–13 day fetal rabbit. Cytochemical activity is present in the nuclear envelope and the endoplasmic reticulum, including its derivatives the subsurface reticulum and the sarcoplasmic reticulum. End product is also found in the Golgi complex of the more differentiated myoblasts. The formation of reticulum-bound acetylcholinesterase in the myoblast appears to be independent of nerve-muscle contact, since the enzyme is present before the outgrowth of the spinal nerve. The nerve lacks cytochemical end product until the myoblast is well differentiated. Possible mechanisms of spontaneous muscle contraction have been discussed. A second type of myotomal cell, which exhibits a poorly localized end product of AChE activity, has been described. The ready solubility of the enzyme or diffusibility of its end product suggests that the enzyme may be a lyoesterase. This cell may be the precursor of the morphologically undifferentiated cell which is closely apposed to the myotubes in later stages of skeletal muscle development. Biochemical studies show a significant increase in AChE activity in the dermomyotome by day 12, when many of the myoblasts are well differentiated and the second type of myotomal cell is prominent. Cytochemical studies have indicated that many of the cells in the sample lack reaction product of enzymic activity, whereas others are very active. Biochemical values, therefore, reflect the amount of enzyme in the dermomyotome as a whole, but give little information on the enzymic content of individual cells.