The effect of exchanger inhibitory peptide (XIP) on sodium-calcium exchange current in guinea pig ventricular cells

While the osteopenia associated with oestrogen deficiency is thought to arise from a relative defect in bone formation with respect to resorption, oestrogen administration itself leads to a decrease, rather than an increase, in bone formation. This decrease in bone formation, which arises from oestrogen's inhibitory effect on bone turnover, presumably masks any underlying tendency of oestrogen treatment towards stimulation of bone formation. To investigate this further, we have examined the early effect of discontinuing the administration of oestradiol-17 beta (OE2; 40 micrograms/kg) on bone formation indices in ovariectomized 13-week-old rats, before the turnover-induced increase in formation occurs. Histomorphometric indices were assessed at the proximal tibial metaphysis 0, 7, 10, 13 and 16 days following discontinuation of OE2 treatment. Measurements of body weight, uterine weight and longitudinal growth rate confirmed that there were rapid effects of OE2 deficiency on these parameters. We could detect no significant increase in bone resorption, as measured by osteoclast surface and number, until 16 days after ending treatment with OE2; this was coincidental with a reduction in bone volume. Shorter periods of OE2 deficiency were associated with a marked decrease in bone formation, as assessed by dynamic histomorphometric indices. This inhibition of bone formation was largely due to a reduction in double fluorochrome-labeled trabecular surfaces, which were decreased by approximately 70%. We conclude that ending OE2 administration in ovariectomized rats caused a striking decrease in trabecular bone formation, if such indices are assessed prior to the subsequent turnover-induced increase in formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tetracaine reports a conformational change in the pore of cyclic nucleotide-gated channels

Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide-gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the alpha subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.     

Quenching of chlorophyll a fluorescence in the aggregates of LHCII: steady state fluorescence and picosecond relaxation kinetics

The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).     

Physiologically relevant one-compartment pharmacokinetic models for skin. 1. Development of models

Many studies have used pharmacokinetic (compartment) models for skin to predict or analyze absorption of chemicals through skin. In these studies, several different definitions of the rate constants were used. The purpose of this study was to develop a general procedure for relating compartment model rate constants to dermal absorption parameters, such as permeability and partition coefficients, and to assess whether different definitions of the rate constants produce different results. Rate constant expressions were developed by requiring a one-compartment model to match a one-membrane model at specific conditions. Because a membrane model contains more information than a compartment model, a compartment model cannot match the membrane model in all respects. Consequently, many compartment models (i.e., different definitions of the rate constants) can be developed which match the membrane model for different conditions. Using this procedure, 11 different compartment models were developed and compared to the membrane model for four different dermal absorption scenarios. The compartment model that most closely matches the membrane model depends on the specific exposure scenario and what is to be predicted. One of the new compartment models agrees reasonably well with the membrane model, for the cases considered.     

Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit

Physiologically, a postprandial glucose rise induces metabolic signal sequences that use several steps in common in both the pancreas and peripheral tissues but result in different events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexokinases. In the beta-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase II in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site-specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane. Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose-phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism at the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three different points: first, through NADH transfer to the respiratory chain complex I via the malate/aspartate shuttle; second, by providing FADH2 to complex II through the glycerol-phosphate/dihydroxy-acetone-phosphate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase. As cytosolic and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phosphocreatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in beta-cells. An interplay between the plasma membrane-bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally at the KATP channels, coordinated to the activity of mitochondrial CrP production. Closure of the KATP channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in beta-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cytosolic/mitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP production through mitochondrial nucleoside diphosphate kinase. Pathophysiologically, there are at least two genetically different forms of diabetes linked to energy metabolism: the first example is one form of maturity-onset diabetes of the young (MODY2), an autosomal dominant disorder caused by point mutations of the glucokinase gene; the second example is several forms of mitochondrial diabetes caused by point and length mutations of the mitochondrial DNA (mtDNA) that encodes several subunits of the respiratory chain complexes. Because the mtDNA is vulnerable and accumulates point and length mutations during aging, it is likely to contribute to the manifestation of some forms of NIDDM.(ABSTRACT TRUNCATED)     

Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb PstI fragment and a 4.8 kb PstI/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5' region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.     

Synthesis and biological activities of phenyl piperazine-based peptidomimetic growth hormone secretagogues

A new class of potent, orally active phenyl piperazine-based GH secretagogues have been discovered from attempts to mimic the arrangement of the phenyl substituent in the spiroindanyl piperidine and spiroindoline sulfonamide privileged structures of 4 and 1, respectively. The best of these compounds, 18 (EC50 = 2.8 nM) is nearly as potent as MK-0677 for releasing GH from rat pituitary cells.     

Hodgkin's disease: impact on childbearing

Although Hodgkin's disease is considered one of the "curable" cancers, the high cure rates associated with this disease are not accomplished without risk of potentially severe, immediate, and late side effects. Because the predominant histologic subtype of Hodgkin's disease found in the United States generally affects the 15- to 25-year-old age group, the disease, its treatment, and the side effects of treatment will affect many women of childbearing age. The treatment and its effects on the patient and offspring, including posttreatment fertility, are key issues for perinatal nurses.     

Recycling of disposable medical devices (medical device products)

Modern medicine uses large amounts of one-way-devices. This includes mass articles as well as technically sophisticated ones, all of them in accordance with GMP (Good-manufacturing-practice) guidelines. Recycling of selected one-way devices in technically possible and economically useful. Prerequisite for recycling is the inclusion of legal, economic and ecologic points of view. In addition the validation of processes and agreement on installation of continuous quality control is necessary to minimise risks. We suggest admission of recycling and reuse of one-way devices and suggest to proceed stepwise: 1. Principal decision to permit recycling 2. Appointment of an interdisciplinary Federal Board "Medical Devices" for coordination and channelling of activities 3. Selection of products for recycling 4. Principal decision for the systematics of the process together with: process control, product control during a pre-testing stage, random tests in prospective, multi-centre studies for the control of production and use. 5. Introduction of internal quality control systems to allow internal and external control of routine work.     

Bedwetting and psychopathology in adolescents

OBJECTIVE: To document the incidence, source, and reasons for all complaints received by a large municipal emergency medical services (EMS) program. METHODS: A retrospective review of all complaints received during three consecutive years (1990-1992) in a centralized EMS system serving a large municipality (population 2 million). All cases were categorized by year, source, and nature of the complaint. RESULTS: In the three study years, EMS responded to 416,892 incidents with nearly a half-million patient contacts. Concurrently, 371 complaints were received (incidence of 1.12 per thousand); 132 in 1990, 129 in 1991, and 110 in 1992. Most complaints involved either: 1) allegations of "rude or unprofessional conduct" (34%), 2) "didn't take patient to the hospital" (19%), or 3) "problems with medical treatment" (13%). Only 1.6% (n = 6) were response-time complaints. Other complaints included "lost/damaged property," "taken to the wrong hospital," "inappropriate billing," and "poor driving habits." The most common sources were patient's families (39%) and the patients themselves (30%). Only 7.8% were from health care providers. CONCLUSION: Reviews of complaints provide information regarding EMS system performance and reveal targets for quality improvement. For the EMS system examined, this study suggests a future training focus on interpersonal skills and heightened sensitivities, not only toward patients, but also toward bystanders and family members.