OpenUCT :: Browsing by Subject "Physiology"

Browsing by Subject "Physiology"

Now showing 1 - 20 of 26

Open Access
Aspects of structure-activity relationships and physiology of gastrin and related peptides
(1978) Napier, Beverley Jean; Vinik, Aaron I
When, in 1973, I embarked upon this study, the hormone gastrin seemed a suitable hormone to study with regard to investigation of structure-activity relationships and physiology of a peptide hormone. Gastrin was best known at that stage as a linear peptide comprising 17 amino acids, which was stable at room temperature and was resistant to degradation by vigorous treatments such as boiling {Gregory and Tracy, 1964; Berson and Yalow, 1971; Yalow and Berson, 1972) and pH extremes (Piszkiewicz, 1974). Such a molecule could thus be studied at room temperature, using conventional biochemical techniques, without the need for any special precautions to preserve its integrity.
Open Access
Cardioprotective role of signal transducer activator of transcription 3 (STAT-3) against ischaemai reperfusion injuries
(2011) King, Jonathan Chan; Lecour, Sandrine; Opie, Lionel H
Introduction: Sphingosine 1 phosphate (S1P) is a major constituent of high density lipoprotein (HDL) cholesterol. Both S1P preconditioning and ischaemic postconditioning reduce myocardial damage following an ischaemia-reperfusion insult but the mechanisms involved remain unclear. Janus kinase/Signal transducer and activator of transcription 3 (JAK/STAT-3) form part of a recently discovered powerful prosurvival path termed as the Survivor Activating Factor Enhancement (SAFE) pathway. The SAFE pathway plays a critical role in ischaemic preconditioning to promote cell survival but whether activation of STAT-3 is required for S1P preconditioning and ischaemic postconditioning induced cardioprotection is unknown. Hypothesis: Activation of the STAT-3 is required for S1P preconditioning and ischaemic postconditioning.
Open Access
Corticotropin-releasing factor and acute post-operative gut function in truamatic abdominal injury and elective abdominal surgery
(2009) Hill, Lauren; Kidson, Sue
Shocked trauma patients in the Intensive Care Unit undergo a powerful, neuro-endocrine stress response driven by cytokine release and the hypothalamic-pituitary-adrenal axis. The response is activated under stress by corticotropin-releasing factor (CRF), the well-known 41 amino acid peptide neuro-hormone. Evidence from animal and human studies suggests that peripheral CRF is present in the gastrointestinal tract and associated with inflammatory changes. Critically ill patients frequently display somewhat unexplained gastrointestinal dysfunction including delayed gastric emptying, ileus and increased bowel permeability. The aim of the study was to investigate the role of CRF in critically ill adults with traumatic abdominal injury compared with elective surgical patients, and describe any association of CRF levels with alterations in acute post-operative gastrointestinal function. Eight patients with haemorrhagic shock following penetrating abdominal injury and seventeen patients undergoing elective surgery for hepato-biliary disease were studied for serial plasma and intestinal tissue CRF levels using radio-immunoassay. A RT-PCR technique was used to detect mRNA for CRF in intestinal tissue. Light microscopy was used to determine the quantity and distribution of mast cells in intestinal tissue. Post-operative gastric emptying was assessed using the paracetamol absorption test and intestinal permeability by measuring urinary lactulose:mannitol ratios following a bolus of these sugars. The study was approved by University of Cape Town Human Research Ethics Committee. Informed consent (retrospectively in the case of the trauma patients), was obtained from all subjects.
Open Access
The crossed mesostriatal pathway and circling behaviour in rats
(1987) Van Wageningen, Gerhard Derek; Douglas, Rodney J
Rats with unilateral 6-OHDA lesions of the nigrostriatal (NS) projection display motor asymmetry in the form of rotational behaviour. The rotation is in the direction ipsilateral with respect to the lesioned side (Ungerstedt 1979). The nett ipsilateral rotations decrease with time, from 1 week to about a month. This decrease has been interpreted as recovery from the lesion-induced motor asymmetry (Glick and Cox 1978). Pritzel et al. (1983) have ascribed the recovery from motor asymmetry to increased activity of a crossed NS projection, which is spared by the ipsilateral lesion. The present study has defined the size and anatomical path of this crossed projection, and has examined its involvement in the behavioural recovery of rats from lesion-induced motor asymmetry. The anatomy of the crossed projection was investigated in male Long-Evans rats using retrograde HRP tract tracing from deposition sites in the striatum.
Open Access
The effects of magnesium treatment on short-term changes in heart rate variability, ventricular function and lipid profile in streptozotocin-induced diabetic rats
(2017) Amoni, Matthew; Gwanyanya, Asfree; Kelly-Laubscher, Roisin
INTRODUCTION: Diabetes mellitus is a major and rapidly growing worldwide health problem, causing mortality largely in developing countries such as South Africa. Diabetes induces life threatening cardiovascular complications including cardiac autonomic neuropathy, ventricular dysfunction and dyslipidaemia, which are dependent on the duration and severity of the diabetes. Most complications are identified at a late, irreversible stage following long-standing diabetes; therefore, early detection and treatment of cardiovascular complications may reverse impairments and improve outcomes. The early treatment of diabetic complications remains ineffective, as the associated underlying features, such as electrolyte disturbances, are poorly understood. A key electrolyte disturbance in diabetes is hypomagnesaemia, which is also an independent cardiovascular risk factor. However, the effects of magnesium (Mg²⁺) supplementation are unclear. Therefore, this study investigated the effects of Mg²⁺ treatment on the early manifestations of streptozotocin (STZ)-induced diabetic cardiac complications. METHODS: Adult male Wistar rats were treated once with STZ (50 mg/kg, i.p.) or vehicle (citrate), and daily for seven days with MgSO4 (270 mg/kg, i.p.) or saline. Blood glucose and body weight were monitored daily. On the eighth day, in vivo tail-pulse plethysmography was recorded for analysis of heart rate variability (HRV), a marker of cardiac autonomic function. Ex vivo, Langendorff-based left ventricular (LV) pressure-volume parameters were measured using an intraventricular balloon. Other hearts were stained with Masson's trichrome and haematoxylin and eosin for histological analysis. Cardiac tissue Mg²⁺ concentration as well as plasma lipid- and Mg²⁺ levels were measured by colorimetric assays. RESULTS: Diabetes reduced heart rate and increased the low-frequency (LF)/high-frequency (HF) power ratio. Mg²⁺ treatment prevented theses diabetes-induced changes in heart rate and in the low-frequency (LF)/high-frequency (HF) power ratio (p < 0.05, n = 9/group). In addition, Mg²⁺ restored orthostatic stress induced changes in heart rate, and LF/HF ratio in diabetic rats (p < 0.05, n = 9/group). In isolated hearts, Mg²⁺ reversed the diabetes-induced decrease in LV end-diastolic elastance (p < 0.05, n = 6/group) and the right shift of end diastolic equilibrium volume intercept from 49 ± 6 μ L to 25 ± 5 μL (p < 0.05, n = 6/group), without altering LV developed pressure or end systolic elastance. Diabetes significantly increased plasma triglyceride, total cholesterol and blood glucose (p < 0.05, n = 7/group), and significantly decreased body weight (p < 0.05, n ≥ 16/group) compared to control, but these changes were not prevented by Mg²⁺ treatment. Neither diabetes nor Mg²⁺ treatment altered plasma- and tissue Mg²⁺ levels. Histologically, diabetes and Mg²⁺ treatment also did not alter cardiomyocyte size or the amount of interstitial collagen in myocardial tissue. CONCLUSION: These results show that Mg²⁺ treatment attenuates diabetes-induced autonomic dysfunction and improves LV diastolic distensibility in short-term diabetes. However, the diabetic metabolic disturbances of hyperglycaemia and dyslipidaemia, the changes in cardiac microstructure or the plasma- and cardiac tissue Mg²⁺ levels were uninfluenced by Mg²⁺ treatment. This suggests that Mg²⁺ exerted its beneficial effects independent of these factors, highlighting the underling mechanisms remain to be clarified. The Mg²⁺ levels not measured in this study by which changes could have been mediated was intracellularly; an aspect that should be further explored in future studies. Furthermore, whether these effects would be translatable to chronic diabetes is an important next question. Thus, the results of this study suggest that Mg²⁺ may have a modulatory role in treating early diabetic cardiovascular complications, but future studies will need to clarify the underlying mechanisms.
Open Access
Effects of Mg²⁺ pretreatment and the modulation of Mg²⁺-sensitive cardiac ion channels on Ca²⁺ paradox phenomenon in the heart
(2016) Alatrag, Fatma; Gwanyanya, Asfree; Kelly-Laubscher, Roisin
The aim of this study was to investigate the effects of Mg²⁺ pretreatment and of pharmacological inhibitors of TRPM7 channels on CP-induced cardiac injury in the isolated rat heart.
Open Access
The effects of pulsed static and oscillating magnetic fields on rat pineal serotonin N-acetyltransferase activity
(1997) Isaacs, Ian J; Beardwood, C J
Concerns about the possible hazardous effects of electromagnetic fields (EMF ' s) has resulted in attempts to address this issue. Several authors have shown that EMF exposure affects biological systems and more specifically the pineal enzyme, serotonin N-acetyltransferase (SNAT), inhibiting the enzyme and thus melatonin production. The enzyme assay required, for performing this type of work is crucial, as the natural decay of the enzyme could bias experimental results. An assay for measuring SNAT activity was assessed and then improved. The Ca²⁺ ion chelator, EGTA (2mM), preserved enzyme activity during pineal homogenisation. The enzyme is heat sensitive and thus keeping the homogenates on ice facilitated enzyme preservation. Not only was preservation of the enzyme crucial, but optimum substrate concentrations were required to measure maximum levels of SNAT activity. Maximum levels of activity were measured when I/10th of a pineal gland was incubated with 12mM tryptamine. At the specified tryptamine concentration, SNAT activity increased as the concentration of Acetyl CoA increased. The enzyme efficiency, as determined from its Km (8.19x10⁻⁴M), and the level of activity exceeded those measured in other laboratories. This assay was used to determine SNAT activity following magnetic field exposure. The magnetic field studies investigated the effects of pulsed static and oscillating magnetic fields on SNAT activity. Male Long Evans rats were housed in short and long photoperiods and were exposed for either 60mins., 30mins. or 15mins. to magnetic fields at different times during the dark phase. Rats were exposed to regularly repeated inversion of either the vertical or horizontal component of the earth' s magnetic field or to regularly repeated horizontal or vertical lOOμT DC field inversions. Pulsed static magnetic field exposure in all conditions had no significant inhibitory effect on SNAT activity. Oscillating magnetic field studies investigated the effect of exposure of rats to magnetic fields tuned to ion parametric resonance conditions for Ca²⁺. The ion parametric resonance model of Blanchard and Blackman was used to determine the exposure parameters. Rats were thus exposed for an hour to a vertical AC field, of strength 14.3μT (rms), frequency was 17.2Hz while the strength of the vertical DC field was 22.55μT, the existing vertical geomagnetic field strength. Under these conditions oscillating magnetic field exposure also had no significant inhibitory effect on SNAT activity.
Open Access
An electrophysiological study of ion transport across the isolated perfused malpighian tubule of Onymacris plana
(1993) Fisher, David Willis; Isaacson, Leon
A knowledge of the ionic and electrical gradients across the Malpighian tubules of Onymacris plana is fundamental to the understanding of the transport mechanisms of fluid across the basolateral and apical membranes. Until now no one has investigated the isolated perfused Malpighian tubule of Onymacris plana and simultaneously measured basolateral potentials. Malpighian tubule segments (about lmm length) were dissected out and perfused in vitro according to the method of Burg et. al. (1966), while the basolateral membrane potential (Vbl) was measured by means of an intracellular microelectrode. Using cable analysis, the effects of DNP, chloride free Ringers, BaCl² (a K⁺-channel blocker), low (25 mM) and high (130 mM) pntassium Ringers (bathing medium), cyclic-AMP and a corpora cardiaca homogenate (CCH) on. the transepithelial resistance and the fractional resistances of the basolateral and apical membranes were investigated.
Open Access
Ethnic specific associations between body composition and metabolic risk and the role of sex hormones and aromatase among black and white South African women
(2015) Tootla, Mehreen; Goedecke, Julia; Keswell, Dheshnie
Background: Previous evidence has demonstrated ethnic differences in the relationship between body fat distribution and metabolic risk between black and white women. However, the reasons for these differences are not known and may be explained in part by differences in sex hormones. The overall aim of this thesis was therefore to i) examine ethnic-specific associations between body fat and its distribution and cardio-metabolic outcomes (study 1) and ii) examine the associations between sex hormones and subcutaneous adipose tissue (SAT) expression of oestrogen receptors (ERα and ERβ) and aromatase (CYP19A), and body fat distribution and insulin resistance (IR) among black and white women (study 2).Methods: Study 1: In 288 black and 197 white premenopausal women, dual-energy X-ray absorptiometry (DXA) and computed tomography (CT) derived measures of body fat distribution and cardio-metabolic factors including IR (HOMAIR) and lipid levels were measured. Study 2: In a subsample consisting of 13 normal-weight and 15 obese black and 15normal-weight and 12 obese white women, HOMAIR and SI (frequently sampled intravenous glucose tolerance test) and ERα, ERβ and CYP19A gene expression were measured in abdominal and gluteal SAT. Results: Study 1: Compared to white women, black women had less central and greater lower body fat, but had similar IR and lower serum lipid concentrations. Despite these differences, the associations between body fat distribution and measures of IR, as well as TG and HDL-Concentrations were similar in black and white women. Notably, central and peripheral fat deposition was independently associated with IR in both the black and white women, and with TG in the black women. In contrast, the associations between body composition and fasting plasma glucose, TC and LDL-C concentrations differed between black and white women. Fasting glucose concentrations were associated with centralisation of body fat in black but not white women, whereas TC and LDL-C concentrations were associated with centralisation of body fat in white but not black women. In addition to body fat distribution, MVPA was associated with IR in the white women, and contraception use was associated with lipid levels in the black and white women. Study 2: CYP19A expression was positively associated with increased adiposity in black and white women in all three depots. Gluteal ERα was significantly higher and ERβ was significantly lower in the black compared to the white women, irrespective of BMI. Gluteal ERα was negatively associated with trunk fat mass (FM) and HOMAIR in the black women only. Gluteal ERα was significantly lower in obese white compared to normal weight white women. Additionally oestradiol (E2) levels were lower in obese compared to normal-weight white women, but did not differ by ethnicity. Conclusion: Our results indicate that it is important in both black and white populations, to decrease centralisation of body fat. Modifiable risk factors such as MVPA and contraception use should be used as therapeutic targets to prevent and manage CVD. Additionally, oestrogen receptors may be an important determinant of body fat distribution and risk in the black women.
Open Access
Factors affecting energy expenditure and the efficiency of fuel utilization : feeding and exercise models
(1993) Lambert, Estelle Victoria; Koeslag, Johan; Noakes, Timothy D
The first aim of this dissertation was to monitor both rat and human responses to short-term perturbations in energy balance brought about through food energy restriction and refeeding, exercise training and the cessation of exercise training or surgical lipectomy. The second aim of this dissertation was to identify factors which might explain differences in food energy intake in weight-matched, weight-stable "large and small eaters". The final aim of this dissertation was to identify factors which might explain differences in resting energy expenditure in a large sample of weight-stable men and women, including exercising and non-exercising persons, and including persons who may be regarded as "restrained eaters".
Open Access
Histone/Serum Protein interactions, A cause of Pseudoimmunological reactions
(1971) Worthington, Robert Walter
Histones have been described by Murray (1964) as basic nuclear proteins which are at some time associated with DNA. Reviews on the biochemistry of histones have been written by Phillips (1962), Busch (1965 ), Butler, Johns and Phillips (1968), Hnilica (1967) and Bonner, Dabrnus, Fambrough, Huang, Marushige and Tuan (1968). These proteins are at present the subject of considerable interest because they are probably involved in the permanent repression of part of the genetic information of the DNA. If this is so, they must be basically involved in the control of cell differentiation.
Open Access
Mechanisms underlying the development of weakness in idiopathic inflammatory myopathies: an in vitro single muscle fibre contractility study
(2018) Henning, Franclo; Kohn, Tertius A; Carr, Jonathan A
Introduction: Polymyositis (PM), dermatomyositis (DM) and necrotising autoimmune myopathy (NAM) form part of the spectrum of idiopathic inflammatory myopathies (IIMs). Although the pathogenic mechanisms are different, the unifying feature is that of weakness caused, in some way or another, by an inflammatory attack on muscle. The mechanism by which weakness develops is still unclear, but experimental animal data suggest that dysfunction of the contractile apparatus might contribute to muscle weakness in these conditions. This study investigated the contractile function of single muscle fibres from patients with IIMs in vitro. Methods: Muscle biopsies obtained from patients with IIMs and healthy controls were dissected and chemically permeabilised. Single muscle fibres were dissected out and subjected to contractility measurement based on standard protocols utilising a permeabilised single fibre system. Specific force (SF; maximum force normalised to cross-sectional area), was calculated for each fibre and compared between the two groups. In addition, maximum shortening velocity and power output were assessed in some of the fibres, and calcium sensitivity in the rest. The myosin heavy chain composition of each fibre was determined by means of gel electrophoresis. Results: A total of 178 fibres from IIM cases and 174 fibres from controls were studied. Specific (normalised) force was 23%, 24% and 29% lower in the IIM group for all fibre types combined, type I fibres, and type IIa fibres, respectively. Shortening velocity and maximum power output were significantly higher in the IIM group for both type I and IIa fibres, compared to controls, while calcium sensitivity was higher in type IIa fibres from IIM cases than controls. Discussion: The findings from this study suggest that weakness in IIMs may, at least in part, be caused by dysfunction of the contractile apparatus leading to impaired contractile force. The higher shortening velocity, power output and calcium sensitivity in fibres from IIM cases probably represents compensatory mechanisms. Although the mechanism by which contractile function is affected has not been investigated, animal studies suggest a role for TNF-α. The findings of this study provide a basis for further investigation into the mechanisms underlying weakness in IIMs.
Open Access
Nerves in micturition
(2014-12-04) Slater, Charles
This video is intended for medical students starting off their anatomy and physiology studies. It is an overview of the peripheral innervation for the control of urination (micturition) and of male sexual function.
Open Access
Non-selective, calcium-permeable channels in the apical membrane of rabbit renal tubules and in the basolateral membrane of human renal tubules : an exploratory patch clamp study
(1992) Saunders, James; Isaacson, Leon
The presence of calcium (Ca) channels has been investigated in the apical membrane of various segments of rabbit renal tubules and in the basolateral membrane of human tubules, using the patch clamp technique. The rabbit tubule segments comprised proximal straight tubules (PST), thick ascending limbs (TAL), distal convoluted tubules (OCT), and cortical collecting ducts (CCD). The human tubule segments could not be identified, but were probably of proximal origin. The luminal surfaces of the individual tubule segments were accessed by perfusing the tubule and inserting the patch pipette through the open end or, more frequently, by tearing open the tubule to allow direct access by a patch electrode. Either Ba (90 mM) or Ca (70 mM) was used in the pipettes. Where possible, channel activity was sought in voltage clamped (30 to -60 m V) excised patches. The data were digitized at l kHz, and filtered (200- 500 Hz) by a six-pole Bessel filter.
Open Access
Oesophageal pressure and motility with special reference to the mechanism of deglutition
(1958) Botha, Gideon Stephanus Muller
This thesis is primarily concerned with oesophageal motility, but certain aspects of the mechanism of deglutition were also studied in detail, either deliberately or by chance. · All dissections, experiments and microscopical and other examinations were conducted by the author and some specimens personally blocked, Out and stained. The liberal use of illustrations clarifies many points which otherwise would not have been so convincingly described; they also ,emphasise strongly the wide variation of normal, which unfortunately we only too often lose sight of in medicine. The literature on this wide subject is so vast that it cannot be completely covered or referred to in a thesis of this nature. The references at the end of this thesis are mainly recent and should, give the·reader·some indication of the divergence of opinion and current concepts on this complex subject.
Open Access
A pathway-based approach investigating DNA sequence variants to implicate the inflammatory pathway in the predisposition to Achilles tendinopathy
(2011) Nell, Erica-Mari; September, Alison
The aims of this dissertation were therefore (i) to follow a pathway-based approach investigating genes encoding proteins involved in the ECM degradation and apoptosis signalling cascade for associations with AT and (ii) to identify a polygenic risk model, comprised of several genetic markers within genes encoding proteins involved in the inflammatory pathway, to predict risk of AT.
Open Access
Pontomedullary reticular formation neurones : a study of microanatomy, transmitter sensitivity and connections from the substantia nigra pars reticulata
(1990) Kellaway, Lauriston Arthur; Douglas, Rodney J
This investigation examines certain aspects of the medial pontomedullary reticular formation (PMRF) microanatom y and neurotransmission and also the connections between the substantia nigra pars reticulata (SNr) and the PMRF in the rat. The anatomical distribution of the population of PMRF neurones was determined by combining physiological identification with electrical stimulation and retrograde HRP tract-tracing. A dual stimulating/deposition electrode was used to combine antidromic stimulation of PMRF cells with optimal retrograde labelling. 139 PMRF neurones were identified by means of their stereotaxic location and physiological criteria, namely; spontaneous discharge, polymodal sensory responses and large receptive fields.
Open Access
Relation of crystalline style function to food availability and environmental conditions in South African mussels
(1987) Fielding, Peter John; Hemsted, W R T
The mussels Choromytilus meridionalis, Mytilus galloprovincialis, Perna perna and Aulacomya ater are important and often dominant organisms on the littoral and sublittoral shores of the South Africa. M. galloprovincialis has only recently been identified as a separate species, but a cons1derable body of information exists on the physiology and energetics of c. meridionalis, P. perna and A. ater. However, it is not clear what factors determine the different intertidal and geographic distributions of these mytilids. Work in the kelp beds has shown that the energy budgets of mussels depends on the utilisation of particulate material from both kelp and phytoplankton production, which have very different structural complexities and biochemical compositions. There is very little information on the digestive enzymes of South African mussels. The activity of these enzymes will be an important factor affecting the ability of the mussels to effectively utilise a food resource, and this may be a mechanism which partially accounts for their differing distributions. This work examines the quantitative and qualitative nature of the suspended particulate food resource at the boundary of the east and west coast mussel ranges, the differences in the digestive enzyme activities of the four mussel species that might utilise the food resource, and 'the animals' abilities to digest this particulate material.
Open Access
The role of the arterial glycocalyx in sphingosine-1- phosphate induced cardioprotection in the isolated heart of the Wistar rat
(2018) Araibi, Hala; Kelly-Laubscher, Roisin; Gwanyanya, Asfree; Van der Merwe, Elizabeth Lael
Background: Ischemic heart diseases (IHD) are a leading cause of death among cardiovascular diseases. Unfortunately, the myocardial damage due to ischemia in IHD may be worsened by reperfusion therapy, a phenomenon called ischemic-reperfusion (I/R) injury. Coronary vascular damage is a key feature of I/R injury. Among the coronary vascular structures, the endothelial glycocalyx is a delicate polysaccharide and protein-rich layer that plays an important role in the regulation of vascular permeability, and is easily damaged during I/R. Sphingosine-1-phosphate (S1P) is a membrane phospholipid metabolite that has been shown to protect the heart against I/R. It has also been shown to regulate the synthesis of glycocalyx, but its effects on coronary endothelial glycocalyx damage and possible mechanism during I/R are unknown. Therefore, we hypothesized that S1P-induced cardioprotection is mediated by modulation of the glycocalyx during I/R in the isolated rat heart. Methods: Isolated male Wistar hearts were perfused on a Langendorff system with Krebs-Henseleit buffer via retrograde perfusion at constant temperature and pressure. The hearts were stabilized and pre-treated with S1P (10 nM for 7 minutes) before inducing 20 minutes of global ischemia, followed by 60 minutes reperfusion. Functional parameters were recorded throughout the protocol, including left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP), heart rate (HR) and coronary flow (CF). Ventricular infarct size was measured by using triphenyltetrazolium chloride stain. Coronary net filtration rate (NFR) was calculated as a ratio of the amount of transudate to CF. Cardiac edema was assessed by calculating the heart wet/dry weight ratio and histologically quantifying size of the interstitial compartment. The shedding of the glycocalyx was estimated by measuring the release of the glycocalyx component syndecan-1 in the coronary effluent using enzyme-linked immunosorbent assay (ELISA) and determining relative syndecan-1 staining intensity between groups in immuno-stained wax sections of perfusion-fixed hearts. In addition, the histo-morphology of the myocardium was assessed using hematoxylin and eosin staining. Results: The cardiac performance was depressed after I/R, as was reflected by decreased LVDP (P=0.02 vs. control), and an increased LVEDP (P<0.0001 vs. control). I/R also significantly increased infarct size (P=0.04 vs. control). Treatment with S1P before I/R significantly decreased infarct size (P=0.01 vs. I/R), but did not improve the post-ischemic decrease in LVDP or stabilize the LVEDP, and had no effect on CF. I/R significantly increased release of syndecan-1 in the coronary effluent (P=0.0002 vs. control). Immunohistochemically-stained imaging also revealed syndecan-1 staining intensity was significantly decreased or absent in ischemic hearts (P≤0.001 vs. control). Pretreatment with S1P had neither effect on syndecan-1 level in the coronary effluent nor on the intensity of syndecan-1 signal in immuno-stained sections (P=n.s vs. I/R). Histological analysis of cardiac edema revealed an increase in the extracellular area in ischemic hearts compared to the control hearts (P≤0.001 vs. control), and S1P treatment decreased the extracellular area (P≤0.01 I/R+S1P vs. I/R). The NFR, and heart wet/dry ratio were not significantly different post-reperfusion between the groups and S1P had no effect on these parameters. Conclusion: This study showed that pretreatment with S1P protects the heart against I/R injury, as was indicated by the decreased infarct size, and decreased extracellular cardiac edema. S1P had no effect on hemodynamic performance or the shedding of syndecan-1. These results suggest that S1P-induced cardioprotection is not mediated by protection of the glycocalyx via stabilization of syndecan-1. However, it is possible that S1P may stabilize other minor glycocalyx components which were not measured in this study, such as heparan sulphate and hyaluronic acid. This is the first study that evaluated syndecan-1 in the cardiac effluent of the isolated heart of rats with global ischemia, and the study opens up prospects for further investigation of the role of the glycocalyx in other models of I/R injury, such as the more clinically-relevant regional ischemia disease model.
Open Access
Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape
(2017) Wibmer, Constantinos Kurt; Gorman, Jason; Ozorowski, Gabriel; Bhiman, Jinal N; Sheward, Daniel J; Joyce, M Gordon; Asokan, Mangai; Burton, Dennis R; Connors, Mark; Abdool Karim, Salim S; Mascola, John R; Robinson, James E; Ward, Andrew B; Kwong, Peter D; Morris, Lynn; Moore, Penny L
A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design.