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A large body of evidence indicates that vasopressin (AVP) and steroid hormones are frequently secreted together and closely cooperate in the regulation of blood pressure, metabolism, water-electrolyte balance, and behavior, thereby securing survival and the comfort of life. Vasopressin cooperates with hormones of the hypothalamo-pituitary-adrenal axis (HPA) at several levels through regulation of the release of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and multiple steroid hormones, as well as through interactions with steroids in the target organs. These interactions are facilitated by positive and negative feedback between specific components of the HPA. Altogether, AVP and the HPA cooperate closely as a coordinated functional AVP-HPA system. It has been shown that cooperation between AVP and steroid hormones may be affected by cellular stress combined with hypoxia, and by metabolic, cardiovascular, and respiratory disorders; neurogenic stress; and inflammation. Growing evidence indicates that central and peripheral interactions between AVP and steroid hormones are reprogrammed in cardiovascular and metabolic diseases and that these rearrangements exert either beneficial or harmful effects. The present review highlights specific mechanisms of the interactions between AVP and steroids at cellular and systemic levels and analyses the consequences of the inappropriate cooperation of various components of the AVP-HPA system for the pathogenesis of cardiovascular and metabolic diseases.

In the presented review, we analyzed the physiology of male sexual arousal and its relation to the motivational aspects of this behavior. We highlighted the distinction between these processes based on observable physiological and behavioral parameters. Thus, we proposed the experimentally applicable differentiation between sexual arousal (SA) and sexual motivation (SM). We propose to define sexual arousal as an overall autonomic nervous system response leading to penile erection, triggered selectively by specific sexual cues. These autonomic processes include both spinal and supraspinal neuronal networks, activated by sensory pathways including information from sexual partner and sexual context, as well as external and internal genital organs. To avoid misinterpretation of experimental data, we also propose to precise the term "sexual motivation" as all actions performed by the individual that increase the probability of sexual interactions or increase the probability of exposition to sexual context cues. Neuronal structures such as the amygdala, bed nucleus of stria terminalis, hypothalamus, nucleus raphe, periaqueductal gray, and nucleus paragigantocellularis play crucial roles in controlling the level of arousal and regulating peripheral responses via specific autonomic effectors. On the highest level of CNS, the activity of cortical structures involved in the regulation of the autonomic nervous system, such as the insula and anterior cingulate cortex, can visualize an elevated level of SA in both animal and human brains. From a preclinical perspective, we underlie the usefulness of the non-contact erection test (NCE) procedure in understanding factors influencing sexual arousal, including studies of sexual preference in animal models. Taken together results obtained by different methods, we wanted to focus attention on neurophysiological aspects that are distinctly related to sexual arousal and can be used as an objective parameter, leading to higher translational transparency between basic, preclinical, and clinical studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bogacki-Rychlik, Gawęda and Bialy.)

Major Depressive Disorder (MDD) with Peripartum Onset was classified in 2013 by the Diagnostic and Statistical Manual, Fifth Edition (DMS-5) and approved in 2019 by the World Health Organization (WHO). These diagnostic revisions call for the development of new animal models of maternal depression, emphasizing the pregnancy period. We have recently described a novel rat model of maternal MDD with a Peripartum Onset. Exposure to pre-gestational chronic mild stress (CMS) with repeated restrain resulted in maternal depressive-like behavior and impacted offspring's neurodevelopment. The present study examined gender differences in short- vs. long-term neurodevelopmental impact of pre-gestational maternal stress. Stress response was assessed in Sprague Dawley CMS-exposed dams (n=7) by metabolic, hormonal, and behavioral changes and compared to controls dams (n=7). Short-term impact of maternal stress on offspring was examined in terms of metabolic, neurodevelopmental, and behavioral tests in male (n=40) and female (n=35) adolescent offspring on a postnatal day (PD) 48; the long-term impact was assessed in adult male (n=13) and female (n=12) offspring on PD 225. Brain tissue was collected from adolescent and adult offspring for biochemical analysis. Maternal stress was associated with decreased body weight and increased urinary corticosterone during the pre-pregnancy period, but depressive-like behavior was delayed until later in pregnancy. No significant neurodevelopmental changes in suckling male or female offspring derived from the stress-exposed dams were observed. However, adolescent male and female offspring of stress-exposed dams displayed an increased depressive-like behavior and gender-dependent increase in anxiety-like behavior in female offspring. These changes were associated with a brain-region-specific increase in brain-derived neurotrophic factor (BDNF) protein and BDNF receptor (TrkB) mRNA in males. Behavioral changes observed in the adolescents receded in adult male and female offspring. However, plasma BDNF was elevated in stress-exposed adult female offspring. These results suggest that pre-gestational maternal stress is associated with gender-dependent short- vs. long-term neurodevelopmental impact in the offspring. Presented data are of significant public health relevance, and there is an urgent need for further research to confirm these findings and probe the underlying mechanisms., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ultrasonic vocalization (USV) is one of the measurable behavioral parameters of sociosexual interactions in rats. To precisely and accurately describe the neurobehavioral properties of USV and the potentially related specific emotional responsiveness of animals, we need to know which animals vocalize and what is their exact behavioral and physiological response. To this end, we modified the non-contact cage [non-contact erection model (NCE)] by adding a modification [vocalization-non-contact erection (VOC-NCE)] that makes it possible to assign emitted ultrasonic signals to a particular animal. Typically, the NCE cage consists of two compartments separated by perforated baffles. A male is placed in one section, and a receptive female is placed in the other section. This makes possible the accurate description of sexual parameters related to the cues controlled by the experimenter. In VOC-NCE, we completely separated the male USV from the female USV by three appropriately perforated baffles and located microphones combined with ultrasonic screening. We recorded emission in both typical bands, the so-called 22- and 50-kHz bands, with various subtypes, thus highlighting the utility of our protocol to investigate the sexual dimorphism of vocalization. Similar to the anticipatory model, we showed that emission can occur without acoustic feedback from concomitants during the VOC-NCE test. Therefore, we propose a relatively simple method for assigning individual vocalization. We discuss its usefulness and limitations in assessing vocal differentiation related to sexual parameters, adaptive changes during conditioning procedures, and further applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bogacki-Rychlik, Wrona and Bialy.)

Purpose: Flexible bronchoscopy (FB) causes airway narrowing and may cause respiratory failure (RF). Noninvasive mechanical ventilation (NIV) is used to treat RF. Until recently, little was known about noninvasive mechanical ventilation assisted flexible bronchoscopy (NIV-FB) risk and complications., Materials and Methods: A retrospective analysis of NIV-FB performed in 20 consecutive months (July 1, 2018-February 29, 2020) was performed. Indications for: FB and NIV, as well as impact of comorbidities, blood gas results, pulmonary function test results and sedation depth, were analyzed to reveal NIV-FB risk. Out of a total of 713 FBs, NIV-FB was performed in 50 patients with multiple comorbidities, acute or chronic RF, substantial tracheal narrowing, or after previously unsuccessful FB attempt., Results: In three cases, reversible complications were observed. Additionally, due to the severity of underlining disease, two patients were transferred to the ICU where they passed away after >48h. In a single variable analysis, PaO 2 69 ​± ​18.5 and 49 ​± ​9.0 [mmHg] (p ​< ​0.05) and white blood count (WBC) 10.0 ​± ​4.81 and 14.4 ​± ​3.10 (p ​< ​0.05) were found predictive for complications. Left heart disease indicated unfavorable NIV-FB outcome (p ​= ​0.046)., Conclusions: NIV-FB is safe in severely ill patients, however procedure-related risk should be further defined and verified in prospective studies., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2021 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

We verified the hypothesis of the existence of forms of individual-specific differences in the emission of anticipatory precontact vocalization (PVs) indicating individualization related to sexual experience and motivation in male rats. Long-Evans males were individually placed in a chamber and 50-kHz ultrasounds were recorded during 5-min periods. In experiment 1, PVs were recorded before the introduction of a female in four consecutive sessions during the acquisition of sexual experience. In experiment 2, PVs were analyzed in three groups of sexually experienced males: with the highest, moderate, and the lowest sexual motivation based on previous copulatory activity. In both experiments, the total number of ultrasounds, as well as 14 different specific subtypes, was measured. The ultrasound profiles for each male were created by analyzing the proportions of specific dominant subtypes of so-called 50-kHz calls. We decided that the dominant ultrasounds were those that represented more than 10% of the total recorded signals in a particular session. The number of PVs was positively correlated with the acquisition of sexual experience and previous copulatory efficiency (measured as the number of sessions with ejaculation). Furthermore, PVs showed domination of the frequency modulated signals (complex and composite) as well as flat and short with upward ramp ultrasounds with some individual differences, regardless of the level of sexual motivation. The results show a characteristic pattern of PVs and confirm the hypothesis that the number of PVs is a parameter reflecting the level of sexual motivation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bogacki-Rychlik, Rolf and Bialy.)

Normal or dysfunctional sexual behavior seems to be an important indicator of health or disease. Many health disorders in male patients affect sexual activity by directly causing erectile dysfunction, affecting sexual motivation, or both. Clinical evidence indicates that many diseases strongly disrupt sexual motivation and sexual performance in patients with depression, addiction, diabetes mellitus and other metabolic disturbances with obesity and diet-related factors, kidney and liver failure, circadian rhythm disorders, sleep disturbances including obstructive sleep apnea syndrome, developmental and hormonal disorders, brain damages, cardiovascular diseases, and peripheral neuropathies. Preclinical studies of these conditions often require appropriate experimental paradigms, including animal models. Male sexual behavior and motivation have been intensively investigated over the last 80 years in animal rat model. Sexual motivation can be examined using such parameters as: anticipatory behavior and 50-kHz ultrasonic vocalizations reflecting the emotional state of rats, initiation of copulation, efficiency of copulation, or techniques of classical (pavlovian) and instrumental conditioning. In this review article, we analyze the behavioral parameters that describe the sexual motivation and sexual performance of male rats in the context of animal experimental models of human health disorders. Based on analysis of the parameters describing the heterogeneous and complex structure of sexual behavior in laboratory rodents, we propose an approach that is useful for delineating distinct mechanisms affecting sexual motivation and sexual performance in selected disease states and the efficacy of therapy in preclinical investigations., (Copyright © 2019 Bialy, Bogacki-Rychlik, Przybylski and Zera.)

This study examined low-frequency ultrasonic vocalizations (lUSVs) in rats during two types of sexual interactions; postejaculatory interval (PEI) and barrier - noncontact (NC) test. We report distinct classes of lUSVs that can be assigned to different emotional states; relaxation vs. frustration. Totally flat, 22-kHz calls (Class A), were observed during the relaxation state following ejaculation; characterized by immobilization or grooming during the PEI. On the other hand, two-three component lUSVs (Class B) that start at a higher frequency (45-kHz: flat, upward or short signal) and then shift to 35-23-kHz (mostly to 28-23-kHz), correspond as we assume, to arousal and frustration - active states associated with sniffing a hole or exploration during the NC test. We suggest that momentary, abrupt decreases of arousal during the frustration state correspond to Class B lUSVs. The detailed spectral analysis of the high-frequency component of two-component lUSVs is crucial for establishing the relationship between such lUSVs and the corresponding behavior and emotional states. Our studies indicate that while the two-component Class B 22-kHz lUSVs may relate to the frustration state, a single component, flat, Class A lUSV relates to the relaxation state. The results of these studies support a notion that rats emit distinct vocalization patterns, reflecting their emotional states.

Background: The literature data pertaining to the significance of day and time of ICU admission for outcomes of patients are inconsistent. The issue has not been analysed in Poland to date. The aim of the study was to gather information about differences between patients admitted to ICU outside regular working hours (off-hours) and those admitted during working hours (on-hours)., Methods: Analysis involved 20,651 patients from the Silesian Registry of Intensive Care Units carried out since 2010. The findings demonstrated that 34.8% of patients were admitted to ICUs during on-hours (between 8.00 a.m. and 3 p.m. on weekdays) and 65.2% were admitted during off-hours (outside regular working hours). The incidence of admissions and data of patients in both groups were compared in terms of the population characteristics and treatment outcomes., Results: The incidence of admissions (calculated per each 24 hours of treatment) was found to be almost twice as high during on-hours, as compared to off-hours (14.5 vs. 6.9 patients/day). Patients admitted to the ICU during on-hours were less likely to be admitted from the surgical department (19.1% vs. 31.0%, P < 0.001), and more likely to be admitted from the emergency department (25.3% vs. 14.2%, P < 0.001). The incidence of off-hours admissions of cancer patients was lower (5.3% vs. 10.8%, P < 0.001), as compared with patients with alcohol dependence syndrome (10.3% vs. 6.9%, P < 0.001). Patients admitted during off-hours were in more severe conditions and had higher APACHE II scores (on average, 23.8 ± 8.8 vs. 21.8 ± 8.8, P < 0.001); their mortality rates were higher compared to the remaining population (46.8% vs. 39.4%, P < 0.001)., Conclusions: Patients admitted to ICUs during off-hours are in more severe general condition and their treatment outcomes are worse, as compared to patients admitted to ICU during on-hours.

Background: The REGENT-VSEL trial demonstrated a neutral effect of transendocardial injection of autologous bone marrow (BM)-derived CD133+ in regard to myocardial ischemia. The current sub-analysis of the REGENT VSEL trial aims to assess the effect stem cell therapy has on quality of life (QoL) in patients with refractory angina., Methods: Thirty-one patients (63.0 ± 6.4 years, 70% male) with recurrent CCS II-IV angina, despite optimal medical therapy, enrolled in the REGENT-VSEL single center, randomized, double-blinded, and placebo-controlled trial. Of the 31 patients, 16 individuals were randomly assigned to the active stem cell group and 15 individuals were randomly assigned to the placebo group on a 1:1 basis. The inducibility of ischemia, (≥ one myocardial segment) was confirmed for each patient using Tc-99m SPECT. QoL was measured using the Seattle Angina Questionnaire. Each patient completed the questionnaire prior to treatment and at the time of their outpatient follow-up visits at 1, 4, 6, and 12 months after cell/placebo treatment., Results: The main finding of the REGENT-VSEL trial sub-analysis was that transendocardial injection of autologous BM-derived CD133+ stem cells in patients with chronic refractory angina did not show significant improvement in QoL in comparison to the control group. Moreover, there was no significant difference between cell therapy and placebo in a number of patients showing improvement of at least 1 Canadian Cardiovascular Society class during the follow-up period., Conclusions: Intra-myocardial delivery of autologous CD133+ stem cells is safe and feasible but does not show a significant improvement in the QoL or angina pectoris symptoms in patients with chronic myocardial ischemia.

Rationale: New therapies for refractory angina are needed., Objective: Assessment of transendocardial delivery of bone marrow CD133 + cells in patients with refractory angina., Methods and Results: Randomized, double-blinded, placebo-controlled trial enrolled 31 patients with recurrent Canadian Cardiovascular Society II-IV angina, despite optimal medical therapy, ≥1 myocardial segment with inducible ischemia in Tc-99m SPECT who underwent bone marrow biopsy and were allocated to cells (n=16) or placebo (n=15). Primary end point was absolute change in myocardial ischemia by SPECT. Secondary end points were left ventricular function and volumes by magnetic resonance imaging and angina severity. After 4 months, there were no significant differences in extent of inducible ischemia between groups (summed difference score mean [±SD]: 2.60 [2.6] versus 3.63 [3.6], P =0.52; total perfusion deficit: 3.60 [3.6] versus 5.01 [4.3], P =0.32; absolute changes of summed difference score: -1.38 [5.2] versus -0.73 [1.9], P =0.65; and total perfusion deficit: -1.33 [3.3] versus -2.19 [6.6], P =0.65). There was a significant reduction of left ventricular volumes (end-systolic volume: -4.3 [11.3] versus 7.4 [11.8], P =0.02; end-diastolic volume: -9.1 [14.9] versus 7.4 [15.8], P =0.02) and no significant change of left ventricular ejection fraction in the cell group. There was no difference in number of patients showing improvement of ≥1 Canadian Cardiovascular Society class after 1 (41.7% versus 58.3%; P =0.68), 4 (50% versus 33.3%; P =0.63), 6 (70% versus 50.0%; P =0.42), and 12 months (55.6% versus 81.8%; P =0.33) and use of nitrates after 12 months., Conclusion: Transendocardial CD133 + cell therapy was safe. Study was underpowered to conclusively validate the efficacy, but it did not show a significant reduction of myocardial ischemia and angina versus placebo., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01660581., (© 2016 American Heart Association, Inc.)

It has been assumed that the 22-kHz ultrasonic vocalizations (USVs) are emitted by adult rats as a result of a negative emotional state. However, emission of the 22-kHz vocalizations by male rats has been also observed following ejaculation, which has a high rewarding value as shown by a conditioned place preference test. These observations suggest that 22-kHz USVs may also occur in response to a positive emotional state. The aim of this study was to determine whether the postejaculatory 22-kHz USVs are related to conditioning processes. The 22 kHz USVs were recorded in Sprague-Dawley males in the postejaculatory refractory period during conditioning processes to a new chamber unrelated to copulation. During the first session in the clean recording chamber, males vocalized marginally and exhibited intensive rearing behavior. From the second to fourth sessions, vocalization duration increased and the number of rearing decreased. Following established conditioning process, odor cues from foreign males, but not the familiar ones, resulted in decreased duration of 22-kHz USVs and increased the number of rearing. On the other hand, in the presence of mating cues (copulatory chamber and presence of the female), males exhibited increased duration of postejaculatory 22-kHz USVs and reduced number of rearing. These results demonstrated that the conditioning to the cues, both unrelated and related to copulation, is important for evoking postejaculatory 22-kHz USVs as well as the relaxation state. Furthermore, these results confirmed the postejaculatory 22-kHz USVs' involvement in expression of the positive emotional state. (PsycINFO Database Record, ((c) 2016 APA, all rights reserved).)

Unlabelled: The prevalence of diseases of the cardiovascular system is the cause of their frequent use, and thus, easy availability of drugs acting on the cardiovascular system, including people they do not require. In Poland, annually are taken from 4.5 to 5.5 thousand suicide attempts, about 3.7% of them due to excessive consumption of pharmaceuticals. The aim of this study was to analyze patients who for suicidal attempts ingested drugs acting on the cardiac conduction system, and to study performed diagnostic and therapeutic procedures and obtained results., Material and Methods: The study included all hospitalized in the years 1995-2010 in the Department of Anesthesiology and Intensive Care patients after excessive suicidal ingestion of drugs acting on the heart conductive system. The study group comprised a total of 40 patients aged from 15 to 70 years., Results: Suicide attempts in the study group were mostly taken by the lonely people and by the subjects with disturbed adaptation. The patients were unconscious mainly after ingestion of psychotropic drugs. From the 40-person group of patients after suicidal ingestion of drugs affecting the conduction system of heart eight patients (20%) required respirator. It has not been shown that any group of drugs significantly more frequent induced respiratory distress requiring mechanical assistance. Rhythm and conduction disturbances during hospitalization occurred in 15 patients (38%), of which 12 (30%) required temporary endocardial stimulation. The study revealed the longest ventricular stimulation need in patients after consumption of calcium channel blockers. Blood pressure in half of the patients was depressed, and 15 of them (38%) required the use of pressor amines. From the 40-person group 2 people died (one patient after ingestion of 4000 mg of propranolol and the other after eating 6000 mg of verapamil). Patients who died differed from the other patients with in younger age, longer time elapsed since the consumption of drugs to their hospitalization, during admission they had undetectable blood pressure, bradycardia, and decreased serum potassium levels. The remaining 38 patients (95%) were discharged from the Department of Anesthesiology and Intensive Care., Conclusions: Attempts to take his own life with the use of drugs in the cardiac conduction system in the study group were taken by a lonely and adaptive disturbances. The most commonly used preparations for suicide were beta-adrenergic receptors blockers, whereas hemodynamic abnormalities, arrhythmias and conduction disturbances were observed more frequently after the drugs that block calcium channels. In patients with life-saving use in suicide attempts important role in addition to medication dosage plays an elapsed time of their consumption and speed of action taken to remove and prevent absorbtion the poison", as well as close supervision in the intensive care unit.

Unlabelled: Calcium channel blockers are drugs often used in circulatory system diseases. Their overdose can cause asystoly, III grade of atrioventricular dissociation, heart insufficiency and cardiogenic shock. Due to wide availability of these drugs access to them for subjects who want commit suicide is easy. In a specialist literature predominantly you can find case reports about calcium channel blockers suicidal overdose. Our experience includes 12 such cases. The aim of the study was to analyze patients after suicidal calcium channel blockers overdose., Material and Methods: In all cases medical interview, physical examination, neurological consultation, ecg, echocardiography, laboratory tests and in most cases--gasometry and toxicology test were performed., Results: In analyzed 12 subjects group during admission, 7 patients were conscious, 2 had consciousness disturbances and 3 subjects were unconscious. Due to significant hypotonia 10 patients were treated with pressive amines. Arrhythmia and conduction disturbances were treated with endocavitary heart stimulation. There were 2 patients who needed mechanical respiratory support and one person, who due to refractory on farmacologic treatment, cardiogenic shock - needed intra aortal balloon pumping assistance. One person died during the first day of hospitalization. All other patients were discharged from the ward with improvement., Conclusions: Calcium channel blockers intoxications are rare, but usually are characterized by serious course and uncertain prognosis. Temporary endocavitary heart stimulation during calcium channel blockers intoxication usually needs electrode repositions due to ineffective stimulation depending on stimulation threshold increase. Our results show, that extremely fundamental for intoxicated patient are, easy to perform, activities which limit gastro-intestinal drug absorbtion.

Background: Therapeutic hypothermia is currently the best-documented method of improving neurological outcomes in patients after cardiac arrest and successful resuscitation. There is a variety of methods for lowering body temperature. However, there are no data showing that any specific method of cooling improves the results or increases survival. A simple method involving surface cooling and ice-cold intravenous fluids, as well as more technologically advanced methods, are used in clinical practice. One of the more advanced methods is intravascular hypothermia, during which cooling is carried out with the use of a special catheter located in the central vein., Aim: To compare cooling with the use of intravascular hypothermia and cooling using the traditional method., Methods: A prospective study was performed in 41 patients with acute coronary syndromes who did not regain consciousness after out-of-hospital or in-hospital cardiac arrest and restoration of spontaneous circulation. Therapeutic hypothermia (32-34°C) was obtained with the use of an intravascular method (group A, n = 20) or a traditional method (group B, n = 21) for a period of 24 hours. Intravascular cooling involved the use of a catheter inserted in the femoral vein connected to a heat exchanger (Alsius Coolgard, Zoll, Chelmsford, MA, USA). Traditional cooling was carried out using uncontrolled surface cooling, ice-cold intravenous fluids and ice-cold gastric lavage. Nasopharyngeal and urinary bladder temperatures were recorded hourly. The main analysed temperature was the urinary bladder temperature, as the heat exchanger in the intravascular hypothermia group was controlled by the readings taken from this site. Temperature profiles were compared., Results: Temperature < 34°C was reached in 19 (95.0%) patients in group A and in 11 (52.4%) patients in group B (p = 0.004). Stable temperature profile (temperature in the range 32-34°C during the final 12 h of cooling) was reached in 16 (80%) patients in group A and in three (14.3%) patients in group B (p < 0.001). Periods of inadequate cooling (temperature > 34°C) and temperature overshoots (temperature < 32°C) were significantly more frequent in group B. Temperature profiles were significantly different in both groups in the readings taken from both sites., Conclusions: The presented technique of intravascular hypothermia provides more precise temperature control in comparison with the traditional method.

OLIGO performs a range of functions for researches in PCR and related technologies such as PCR and sequencing primer selection, hybridization probe design, inverse and real-time PCR, analysis of false priming using a unique priming efficiency (PE) algorithm, design of consensus and multiplex, nested primers and degenerate primers, reverse translation, and restriction enzyme analysis and prediction; based on a protein sequence, oligonucleotide database allows fully automatic multiplexing, primer secondary structure analysis, and more. OLIGO allows for sequence file batch processing that is essential for automation. This chapter describes the major functions of OLIGO version 7 software.

The epidermal growth factor receptor (EGFR) has been reported to be expressed in high levels in primary breast cancer by immunohistochemistry. In the present study, a reverse transcription (RT)-PCR assay using EGFR primers was developed and evaluated for the detection of circulating micrometastases in the blood of breast cancer patients. Total RNA was extracted from breast cancer cell lines and from the blood of 23 control individuals and 37 breast cancer patients. After reverse transcription, outer and nested primers for EGFR were used for cDNA amplification. RNA integrity was confirmed with parallel RT-PCR amplification using beta2-microglobulin primers. PCR products were electrophoresed on agarose gels containing ethidium bromide and visualized by UV photography. Southern blotting was used to confirm EGFR specificity. The nested EGFR RT-PCR assay was capable of detecting a lower limit of 100 fg of total RNA from the A431 cell line. EGFR RNA was identified from the blood of 4 of 18 (22%) metastatic breast cancer patients, 0 of 6 locally recurrent breast cancer patients, 0 of 13 adjuvant breast cancer patients, and 0 of 23 controls (P = 0.03, metastatic versus control). The 18 metastatic breast cancer patients all had progressive disease at the time of blood sampling. The identity of the four EGFR-positive bands was confirmed by Southern blotting. The presence of RT-PCR positivity for EGFR was not a treatment-related phenomenon, because three of the four EGFR-positive patients were not receiving treatment at the time of blood collection. RT-PCR for EGFR is a sensitive and specific method for the detection of circulating micrometastases in a proportion of patients with metastatic breast cancer.

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the chance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too low Tm of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.

One of the most important factors affecting the quality of polymerase chain reaction (PCR) is the choice of primers. Several rules should be observed when designing primers and, in general, the more DNA sequence information available, the better the chance of finding an "ideal" primer pair. Fortunately, not all primer selection criteria need be met in order to synthesize a clean, specific product, since the adjustment of PCR conditions (such as composition of the reaction mixture, temperature, and duration of PCR steps) may considerably improve the reaction specificity. Amplification of 200-400-bp DNA is the most efficient and, in these cases, one may design efficient primers simply by following a few simple rules described in this chapter. It is more difficult to choose primers for efficient amplification of longer DNA fragments, and use of an appropriate primer analysis software is worthwhile.

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome. Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity. In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu [Kb = (3 +/- 1.2) X 10(6) M-1] causing a decrease of CPM fluorescence. Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected. Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu. The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.