Native human platelet factor 4 (PF4) is a homotetrameric protein (70 residues/subunit) known for its anticoagulant heparin binding activity. 2D 15N--1H HSQC NMR experiments of native PF4 in solution show the presence of conformational heterogeneity consistent with the formation of asymmetric homo-tetramers as observed in the X-ray crystal structure of both human and bovine PF4. A chimeric mutant of PF4 (called PF4-M2) which substitutes the first 11 N-terminal residues for the first eight residues from homologous interleukin-8 forms symmetric homo-tetramers with essentially the same heparin binding activity as native PF4. The solution structure of PF4-M2 has been investigated by using two- and three-dimensional 1H- and 15N-NMR spectroscopy and NOE-restrained simulated annealing molecular dynamics. As with other members of the CXC chemokine family whose structures are known, the PF4-M2 subunit monomer consists of a mostly hydrophobic, triple-stranded antiparallel beta-sheet onto which is folded an amphipathic C-terminal helix and a less periodic N-terminal domain. Although N-terminal substitution with the less acidic interleukin-8 sequence most affects the quarternary structure relative to native PF4 at the AC and AD dimer interfaces, AB dimer stability is weakened as reflected in reduced equilibrium association binding constants.

Amino Acid Sequence↗

PubMed1995

Effects of the selective protein kinase C inhibitor Ro 31-7549 on human angiotensin II receptor desensitisation and intracellular calcium release.

The mechanism underlying type I angiotensin II (Ang II) receptor (AT1 receptor) desensitisation is unknown. Structural features suggest it may be a substrate for protein kinase C (PKC). The effects of a selective PKC inhibitor, Ro 31-7549, on receptor desensitisation were investigated in CHO cells expressing the human AT1 receptor. Desensitisation was demonstrated with respect to the calcium response to Ang II in Fura-2-loaded cells. Ro 31-7549 had no effect on desensitisation. However, pretreatment with Ro 31-7549 caused a dose-dependent reduction in calcium release from intracellular stores. PKC may therefore act at a locus distal from the receptor itself.

Angiotensin II↗

PubMed1995

Cloning, characterization and expression of a functional mouse ACTH receptor.

A polymerase chain reaction-generated mouse ACTH-R probe was used to screen a mouse genomic library, and a clone of 13kb containing the entire coding sequence was isolated. The coding sequence shows 84% homology with the human gene at the DNA level and encodes a peptide with 89% homology to the human ACTH-R. This gene is expressed as a major transcript of 1.8kb in the mouse adrenal gland. The gene was expressed in HeLa cells and cAMP production in response to either ACTH or alpha-MSH was measured. cAMP increased in an ACTH dose dependent manner suggesting an EC50 of 7 x 10(-10)M ACTH. alpha-MSH was without effect on this receptor. In conclusion we have cloned a mouse ACTH receptor gene and demonstrated for the first time its expression and functional effect in HeLa cells.

Amino Acid Sequence↗

PubMed1995

Quarternary structure amplification of protein folding differences observed in 'native' platelet factor-4.

Platelet factor 4 (PF4), a protein of 70 residues, exists in solution as a distribution of monomer, dimer and tetramer (M-D-T) states in slow exchange on a 600 MHz 1H-NMR chemical shift time scale. Well-resolved Y60 ring proton resonances in each aggregate state allow derivation of M-D-T populations. Under the same set of solution conditions, M-D-T aggregate state distributions vary from prep to prep or with repeated freeze-drying or raising/lowering the solution pH. These variations are not the result of chemical modifications and reflect differences in the strength of subunit associations and therefore folding. Variations are greatest at pH values at or below the pKas of carboxylate groups, supporting the idea that electrostatic interactions modulate PF4 subunit interactions. Treating these distributions as true equilibria results in free energy differences of about 0.5 kcal/mol subunit. Quarternary structure amplifies free energy differences among various folded states of monomer PF4.

Humans↗

PubMed1995

The relationship between the adrenal tissue renin-angiotensin system, internalization of the type I angiotensin II receptor (AT1) and angiotensin II function in the rat adrenal zona glomerulosa cell.

Many data suggest that the elements of the tissue renin-angiotensin system (RAS) in the adrenal cortex are mostly located in the zona glomerulosa. The relationship of this paracrine/autocrine system with the cellular localization of the angiotensin II (AII) receptor has not bee clarified. Using a specific monoclonal antibody (6313/G2) to the first extracellular domain of the type 1 receptor (AT1), we show here that most of the receptor is internalized in the rat glomerulosa cell. This may result from tonic stimulation by the tissue RAS, and consequent permanent receptor occupancy. When viable glomerulosa cells are incubated with 6313/G2, the receptor is transiently concentrated on the cell surface, and aldosterone output is stimulated. This stimulated output is enhanced by neither threshold nor maximal stimulatory concentrations of AII amide, although the antibody does not inhibit AII binding to the receptor. The antibody directly stimulates inositol trisphosphate (IP3) generation, but, while having no intrinsic action on protein kinase C (PKC) activation, significantly inhibits the PKC response to angiotensin II. The data suggest that although the receptor is mostly internalized, recycling to the plasma membrane is constitutive, or regulated by unknown factors. Retention of the AT1 receptor in the membrane is alone enough to allow sufficient G protein interaction to generate maximal steroidogenic effects, through IP3 generation. PKC activation induced by angiotensin II has no bearing on steroidogenesis in the dispersed glomerulosa cell system.

Adrenal Glands↗

PubMed1995

A formula to calculate blood cross-match requirements for early burn surgery in children.

Excessive bleeding from the burn site accompanies burn surgery, and blood transfusions are therefore an essential and expensive ingredient. A formula, based on the total surface area to be involved in surgery (i.e. burn plus donor area) and the patient's total blood volume, has been in use in our hospital for calculating blood cross-match volumes, but has not been scientifically evaluated. Prospectively the predicted blood loss, based on the formula in 111 consecutive paediatric burns undergoing surgery, was compared with the actual blood loss as measured using a gravimetric method. Provided that an accurate surface area to be involved in surgery is interposed into the formula, there was a statistically highly significant correlation between the predicted and actual blood loss. Invoking this simple formula may help in decreasing unnecessary cross-matching, and discarding, of blood products.

Blood Grouping and Crossmatching↗

PubMed1995

Type 1 angiotensin II receptors in rat and human sperm.

The physiological factors which induce and maintain mammalian sperm maturation and motility generally remain unclear, although several agents are known to be involved. We describe here the application of immunocytochemical and immunoblotting methods to identify the angiotensin II type 1 (AT1) receptor in the tails of ejaculated rat and human sperm. Motility data on stimulated and unstimulated sperm from volunteers and patients attending fertility clinics showed that angiotensin II may increase both the percentage of motile sperm and their linear velocity, while the specific AT1 receptor antagonist DuP753 inhibited the action of angiotensin II on the percentage of motile sperm. In rat seminiferous tubules, AT1 receptors were present in primary spermatogonia and in spermatid tails, but immunoreactivity was not seen in sperm contained in caput or cauda epididymis, showing that AT1 receptor function is regulated during transit through the reproductive tract. Since local tissue reninangiotensin systems are present in both male and female tracts, the data suggest that angiotensin II has a role in the maintenance of sperm function and fertility.

Angiotensin II↗

PubMed1994

A community ability scale for chronically mentally ill consumers: Part II. Applications.

The authors describe uses for a 17-item instrument that efficiently measures the functioning level of chronically mentally ill persons living in the community. The Multnomah Community Ability Scale is designed to be completed by case managers who work with chronically mentally ill consumers. The instrument is sensitive to differences among individuals within this special population of consumers and is easy to complete. Community mental health program staff can be trained to use the scale reliably. The scale has been used to compare levels of severity between urban and rural community mental health program clients. The authors discuss the application of the Multnomah Community Ability Scale to a capitated payment system for severely mentally ill, involuntary clients.

Activities of Daily Living↗

PubMed1994

A community ability scale for chronically mentally ill consumers: Part I. Reliability and validity.

The authors describe the development, reliability testing, and validation of a 17-item instrument that measures the level of functioning of chronically mentally ill persons living in the community. The Multnomah Community Ability Scale is designed to be completed by case managers. The instrument provides a measure of the consumer's severity of disability which can, in turn, be used to: (a) describe an agency's "case mix" of clients; (b) measure consumer progress; (c) assign clients to different levels of service; and (d) assist payors in determining reimbursement. The Multnomah Community Ability Scale is aimed specifically at persons with chronic mental illness, is sensitive to differences among individuals within this special population, and is quick and easy to complete. The scale's reliability and validity have been examined in detail. Inter-rater and test-retest reliability are good. Criterion variables such as length of psychiatric hospitalization and clinicians' global ratings correlated highly with scale scores. Finally, the instrument predicts subsequent state and local hospital admissions.

Activities of Daily Living↗

PubMed1994

Effects of two formats of informed consent on knowledge amongst persons with advanced HIV disease in a clinical trial of didanosine.

To determine subjects' perception of the purpose of informed consent, 113 subjects were recruited from a dose-controlled clinical trial of didanosine (ddI). Subjects were surveyed regarding how they made decisions regarding their medical care in general, about how they obtained information about this trial in particular, and several aspects of the informed consent procedure. Subjects were then randomly allocated to receive information about the trial by either a written only format or a written and verbal format 1 week before commencement of the trial. An eight-item instrument assessed knowledge of ddI prior to and subsequent to receiving information. Most subjects obtained information about HIV-related issues from their specialist (70%) or general (51%) medical practitioner. A large proportion of subjects (88%) reported that they believed their specialist medical practitioner always acted in their best interest. The majority of subjects (79%) believed that subjects should be allowed the choice between participating in the clinical trial and receiving the drug outside the trial mechanism. Of the subjects, 96% believed that informed consent was necessary in clinical trials; however, their opinions of the purpose of informed consent varied widely. Although they signed the informed consent, 44% of the subjects stated that they did not understand 'all' of the information that was provided. We found that the provision of information by written mode alone, or written and verbal modes were both associated with significant increases in knowledge levels and that there was a significant interaction in the degree of change between the two methods, with the written plus verbal method showing the most improvement over time. There was an interaction between degree of improvement in knowledge of didanosine in subjects who received written information versus those who received written and verbal knowledge and time (pre- versus post-consent) and a significant main effect for time. All subjects were relatively well-informed about the drug and stated that specialist and general medical practitioners were their major source of knowledge for all aspects of their HIV health care.

Adult↗

PubMed1994

Vascular complications of elective lower lumbar disc surgery.

Lower lumbar disc surgery is an extremely common operation from which vascular complications are reported rarely. However vascular injury can occur with perforation of the anterior longitudinal ligament. External haemorrhage is uncommon and the true cause of the patient's hypotension may be misdiagnosed. If the hypotension is severe or prolonged laparotomy is indicated, to prevent exsanguination and to effect a repair of the vascular injury. Alternatively, if the injury is missed, an arteriovenous fistula may develop; this diagnosis may be delayed for months or years. One case of severe haemorrhage following lower lumbar disc surgery is reported and a further 69 cases of similar arterial injury have been reviewed from the world literature.

Arteriovenous Fistula↗

PubMed1994

Herpes simplex encephalitis: long term magnetic resonance imaging and neuropsychological profile.

The first comprehensive in vivo documentation of the long term profile of pathological and spared tissue is described in a group of 10 patients with a diagnosis of herpes simplex encephalitis, who were left with memory difficulties as a major residual sequel of their condition. With a dedicated MRI protocol, which included high resolution images of temporal lobe and limbic system areas, data are provided on structures that have recently gained importance as anatomical substrates for amnesia. The major features of the lesion profile were: (1) unilateral or bilateral hippocampal damage never occurred in isolation, and was often accompanied by damage to the parahippocampus, the amygdala, specific temporal lobe gyri, and the temporal poles; (2) the insula was always abnormal; (3) neocortical temporal lobe damage was usually unilateral or asymmetric. It never occurred in isolation, and was invariably associated with more medial pathological changes; (4) anterior and inferior temporal lobe gyri were damaged more often and more severely than posterior and superior temporal lobe gyri; (5) pronounced abnormality was often present in the substantia innominata (region of the basal forebrain/anterior perforated substance); (6) there was evidence of significant abnormality in the fornix; (7) there was evidence of damage to the mammillary bodies; (8) thalamic nuclei were affected in around 50% of cases, with damage usually unilateral; (9) frontal lobe damage was present in a few patients, and affected medial areas more than dorsolateral areas; (10) there was some involvement of the striatum, although this was usually unilateral and mild; (11) there was usually limited involvement of the cingulate gyrus and of the parietal and occipital lobes; (12) the cerebellum and brain stem were never damaged. Lesion covariance analysis indicated a close relation between the presence of abnormalities in temporal lobe and limbic-diencephalic regions. Unlike severe head injury, lesions in the temporal pole were not associated with the presence of lesions in the orbitofrontal cortex. Long term neuropsychological impairments were characterised by a dense amnesia in 60% of cases, and a less serve but noticeable anterograde memory impairment in the others. Naming and problem solving deficits were found in a small number of cases. Only two patients were able to return to open employment. Severity of amnesia showed a significant relation with severity of damage to medical limbic system structures such as the hippocampus, with bilateral damage being particularly important. By contrast, there was a minimal relation between memory loss and severity of damage to the thalamus, to lateral temporal lobe areas, or to the frontal lobes.

Adult↗

PubMed1994

Transient memory loss for people.

A patient had transient memory loss for close family members. She could not even recognise their names as familiar. Her everyday memory was relatively preserved and she retained a clear recollection of the episode. Standard and sleep deprived EEG showed a mild abnormality of the left temporal lobe. Neuropsychological testing found evidence for a mild verbal memory impairment. The findings provide further evidence for the fractionation of transient forms of amnesia, support the dissociation of semantic/retrograde amnesia from episodic/anterograde amnesia, and offer evidence in favour of a left temporal lobe site for retrieval of past memories relating to the identification of people.

Adult↗

PubMed1994

Angiotensin II receptor subtypes in eel (Anguilla anguilla).

Previous studies have shown the effects of angiotensin II (Ang II) in teleosts, and Ang II-binding sites have also been localized in tissues from rainbow trout. The purpose of this study was to extend these findings and to provide an analysis of Ang II receptor (Ang II-R) isoforms in three tissues obtained from European eel (Anguilla anguilla). Ang II-Rs were identified in eel liver, kidney and intestine membranes by the binding of either 0.5 nmol human 125I-labelled Tyr4-Ile5-Ang II/l or increasing concentrations (1-120 nmol/l) of [3,5-3H]Tyr4-Ile5-Ang II. Using an isoelectric focusing technique, two Ang II-binding sites were identified in liver membranes. These migrated to isoelectric points (pI values) 6.5 and 6.7. Seventy per cent of binding to both sites was displaced by a 10,000-fold excess of unlabelled human Ang II. In both whole plasma membranes and brush border membranes from intestine, only one form of the Ang II-R was found, with pI 6.5 and high affinity (Kd = 3.4 nmol/l) for the [3,5-3H]Tyr4-Ile5-Ang II. Similarly, only the isoform focusing at pI 6.5 was observed in renal tubular epithelial brush border membranes. Reduction of disulphide bridges with dithiothreitol significantly enhanced Ang II binding to the isoform at pI 6.5 in liver (P < 0.05) and kidney (P < 0.01), while in liver the binding to the isoform of pI 6.7 was significantly reduced (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Anguilla↗