[First French-speaking days of users of decision support system in clinical pharmacy: Feedback and perspectives]. / Premières journées francophones des utilisateurs de système d'aide à la décision en pharmacie clinique : retour d'expériences et perspectives.

Clinical decision support systems (CDSS) are tools that have been used for several years by clinical pharmacy teams to support pharmaceutical analysis, with a perspective of contributing to the quality of care in collaboration with the other health care team members. These tools require both technical, logistical and human resources. The growing use of these systems in different establishments in France and in Europe gave birth to the idea of meeting to share our experiences. The days organized in Lille in September 2021 aimed at proposing a time of exchange and reflection on the use of these CDSS in clinical pharmacy. A first session was devoted to feedback from each establishment. These tools are essentially used to optimize pharmaceutical analysis and to secure patient medication management. This session outlined the clear advantages and common limitations of these CDSS. Two research projects were also presented to put the use of these tools into perspective. The second session of these days, in the form of workshops, addressed 4 themes that surround the implementation of CDSS: their usability, the legal aspect, the creation of rules and their possible valorization. Common problems were raised, the resolution of which requires close collaboration. This is a first step proposing a beginning of harmonization and sharing that should be deepened in order not to lose the dynamics created between the different centers. This event ended with the proposal to set up two working groups around these systems: the creation and structuring of rules for the detection of risk situations and the common valorization of the work.

Assessment of renal function in the anaesthetised rat following injection of superparamagnetic iron oxide nanoparticles.

A recent study showed that a significant fall in mean arterial pressure (MAP) occurred following intravenous injection of two novel superparamagnetic iron oxide nanoparticles (SPIONs), MF66 and OD15. To assess if this was caused by excessive glomerular clearance, the effect of both particles on renal function was studied. Experiments were performed on sodium pentobarbital anaesthetised male Wistar rats (250-350 g). Twenty-minute urine clearances were taken followed by an i.v. bolus of MF66, OD15 (2 mg·kg-1), or dH2O (0.4 mL·kg-1). MF6 or OD15 injection resulted in a significant transient drop in MAP and renal blood flow by approximately 33% and 50% (P < 0.05). The absolute excretion of sodium was significantly increased (P < 0.05) by almost 80% and 70% following OD15 and MF66, respectively. Similarly, fractional excretion of sodium was increased by almost 80% and 60% following OD15 and MF66, respectively. The glomerular filtration rate was not significantly affected, but urine flow increased nonsignificantly by approximately 50% and 66% following i.v. injection of OD15 and MF66, respectively. SPIONs produce a decrease in blood pressure and a natriuresis; however, the rate of fluid filtration in the kidney was not significantly affected.

False recognition in Lewy-body disease and frontotemporal dementia.

The primary goal of this study was to evaluate the false recognition phenomenon in persons with frontotemporal dementia (FTD) and those with Lewy-body disease (LBD). Patients with LBD (n=10) or FTD (n=15) and their corresponding controls (n=30) were subjected to the Deese-Roediger-McDermott (DRM) paradigm to induce false recognition. Patients were first presented with items semantically related to a nonpresented critical target. The critical target was later included in a word list shown to patients to assess level of recognition. Both groups of patients showed a reduced level of false recognition of the critical target when controlling for their overall level of false alarms. This reduction was greater in persons with LBD than in those with FTD. Correlational analyses of performance on neuropsychological tests and the DRM variables indicated that the reduced DRM effect was associated with inhibition deficits in patients with LBD and with inhibition deficits and verbal memory in those with FTD. Our results support current models suggesting that these cognitive components contribute to the false recognition effect.

Inhibition of L-NAME-induced hypertension by combined treatment with apocynin and catalase: the role of Nox 4 expression.

Reactive oxygen species (ROS) such as superoxide (O2-) generated by NAD(P)H oxidases have emerged as important molecules in blood pressure regulation. This study investigated the effect of apocynin and catalase on blood pressure and renal haemodynamic and excretory function in an L-NAME induced hypertension model. Forty Male Wistar-Kyoto (WKY) rats (n=8 per group) were treated with either: vehicle (WKY-C); L-NAME (WKY-L, 15 mg/kg/day in drinking fluid); WKY-L given apocynin to block NAD(P)H oxidase (WKY-LApo, 73 mg/kg/day in drinking water.); WKY-L given catalase to enhance ROS scavenging (WKY-LCat, 10000 U/kg/day i.p.); and WKY-L receiving apocynin plus catalase (WKY-LApoCat) daily for 14 days. L-NAME elevated systolic blood pressure (SBP), 116+/-1 to 181±4 mmHg, reduced creatinine clearance, 1.69+/-0.26 to 0.97+/-0.05 ml/min/kg and fractional sodium excretion, 0.84+/-0.09 to 0.55+/-0.09 % at day 14. Concomitantly, plasma malondialdehyde (MDA) increased six fold, while plasma total superoxide dismutase (T-SOD), plasma nitric oxide (NO) and plasma total antioxidant capacity (T-AOC) were decreased by 60-70 % and Nox 4 mRNA expression was increased 2-fold. Treatment with apocynin and catalase attenuated the increase in SBP and improved renal function, enhanced antioxidative stress capacity and reduced the magnitude of Nox4 mRNAs expression in the L-NAME treated rats. This study demonstrated that apocynin and catalase offset the development of L-NAME induced hypertension, renal dysfunction and reduced oxidative stress status, possibly contributed by a reduction in Nox4 expression during NOS inhibition. These findings would suggest that antioxidant compounds such as apocynin and catalase have potential in treating cardiovascular diseases.

Radiation therapy for vaginal and perirectal lesions in recurrent ovarian cancer.

The role for localized radiation to treat ovarian cancer (OC) patients with locally recurrent vaginal/perirectal lesions remains unclear, though we hypothesize these patients may be salvaged locally and gain long-term survival benefit. We describe our institutional outcomes using intensity modulated radiation therapy (IMRT) +/- high-dose rate (HDR) brachytherapy to treat this population. Our primary objectives were to evaluate complete response rates of targeted lesions after radiation and calculate our 5-year in-field control (IFC) rate. Secondary objectives were to assess radiation-related toxicities, chemotherapy free-interval (CFI), as well as post-radiation progression-free (PFS) and overall survival (OS). PFS and OS were defined from radiation start to either progression or death/last follow-up, respectively. This was a heavily pre-treated cohort of 17 recurrent OC patients with a median follow-up of 28.4 months (range 4.5-166.4) after radiation completion. 52.9% had high-grade serous histology and 4 (23.5%) had isolated vaginal/perirectal disease. Four (23.5%) patients had in-field failures at 3.7, 11.2, 24.5, and 27.5 months after start of radiation, all treated with definitive dosing of radiation therapy. Patients who were platinum-sensitive prior to radiation had similar median PFS (6.5 vs. 13.4 months, log-rank p = 0.75), but longer OS (71.1 vs 18.8 months, log-rank p = 0.05) than their platinum-resistant counterparts. Excluding patients with low-grade histology or who were treated with palliative radiation, median CFI was 14.2 months (range 4.7 - 33.0). Radiation was well tolerated with 2 (12.0%) experiencing grade 3/4 gastrointestinal/genitourinary toxicities. In conclusion, radiation to treat locally recurrent vaginal/perirectal lesions in heavily pre-treated OC patients is safe and may effectively provide IFC.

Role of norepinephrine & angiotensin II in the neural control of renal sodium & water handling in spontaneously hypertensive rats.

BACKGROUND & OBJECTIVES: A wealth of information concerning the essential role of renal sympathetic nerve activity (RSNA) in the regulation of renal function and mean arterial blood pressure homeostasis has been established. However, many important parameters with which RSNA interacts are yet to be explicitly characterized. Therefore, the present study aimed to investigate the impact of acute renal denervation (ARD) on sodium and water excretory responses to intravenous (iv) infusions of either norepinephrine (NE) or angiotensin II (Ang II) in anaesthetized spontaneously hypertensive rats (SHR). METHODS: Anaesthetized SHR were acutely denervated and a continuous iv infusion of NE (200 ng/min/ kg) or Ang II (50 ng/min/kg) was instigated for 1 h. Three 20-min urine clearances were subsequently collected to measure urine flow rate (UV) and absolute sodium excretion (U(Na)V). RESULTS: Higher UV and U(Na)V (P<0.05) were observed in denervated control SHR as compared to innervated counterparts. The administration of NE or Ang II to innervated SHR produced lower UV and U(Na)V (P<0.05 vs. innervated control SHR). Lower diuresis/natriuresis response to ARD was observed in NE-treated SHR compared to denervated control SHR (P<0.05). Salt and water excretions in denervated NE-treated SHR, however, were significantly higher (P<0.05) relative to the excretion levels in control denervated SHR. Conversely, there was a higher (all P<0.05) diuresis/natriuresis response to ARD when Ang II was administered to SHR compared to denervated control or innervated Ang II-treated SHR. INTERPRETATION & CONCLUSIONS: NE retains its characteristic antidiuretic/antinatriuretic action following ARD in SHR. Typical action of Ang II on salt and water excretions necessitates the presence of an intact renal innervation. Ang II is likely to facilitate the release of NE from renal sympathetic nerve terminals through a presynaptic site of action. Moreover, there is a lack of an immediate enhancement in the renal sensitivity to the actions of NE and Ang II following ARD in a rat model of essential hypertension.

The restorative effect of apocynin and catalase in l-arginine induced hypotension on normotensive subjects - the role of oxidative stress.

L-arginine is a substrate for nitric oxide synthase (NOS) responsible for the production of NO. This investigation studied the effect of apocynin, an NADPH oxidase inhibitor and catalase, an H2O2 scavenger on L-arginine induced oxidative stress and hypotension. Forty Wistar-Kyoto rats were treated for 14 days with vehicle, L-arginine (12.5mg/ml p.o.), L-arginine+apocynin (2.5mmol/L p.o.), L-arginine+catalase (10000U/kg/day i.p.) and L-arginine plus apocynin+catalase respectively. Weekly renal functional and hemodynamic parameters were measured and kidneys harvested at the end of the study for histopathological and renal NADPH oxidase 4 (Nox4) assessments. L-arginine administration in normotensive rats decreased systolic blood pressure (120±2 vs 91±2mmHg) and heart rate (298±21 vs 254±15b/min), enhanced urinary output (21.5±4.2 vs 32±1.9ml/24h , increased creatinine clearance (1.72±0.56 vs 2.62±0.40ml/min/kg), and fractional sodium excretion (0.88±0.16 vs 1.18±0.16 %), caused proteinuria (28.10±1.93 vs 35.26±1.69mg/kg/day) and a significant decrease in renal cortical blood perfusion (292±3 vs 258±5bpu) and pulse wave velocity (3.72±0.20 vs 2.84±0.13m/s) (all P<0.05). L-arginine increased plasma malondialdehyde (by ~206 % P<0.05) and NO (by~51 %, P<0.05) but decreased superoxide dismutase (by~31 %, P<0.05) and total antioxidant capacity (by~35 %, P<0.05) compared to control. Renal Nox4 mRNA activity was approximately 2.1 fold higher (P<0.05) in the L-arginine treated rats but was normalized by apocynin and apocynin plus catalase treatment. Administration of apocynin and catalase, but not catalase alone to rats fed L-arginine, restored the deranged renal function and structure, prevented hypotension and enhanced the antioxidant capacity and suppressed Nox4 expression. These findings suggest that apocynin and catalase might be used prophylactically in states of oxidative stress.

Influence of combined hypertension and renal failure on functional alpha(1)-adrenoceptor subtypes in the rat kidney.

BACKGROUND AND PURPOSE: This study investigated whether the alpha(1)-adrenoceptor responsiveness of the renal vasculature was altered in the state of hypertension combined with renal failure. EXPERIMENTAL APPROACH: Male spontaneously hypertensive rats (SHR) received cisplatin (5 mg kg(-1) i.p.) to induce renal failure. Seven days later, the rats were anaesthetized and the reductions in renal blood flow (RBF) to electrical renal nerve stimulation (RNS) and intrarenal administration of three adrenoceptor agonists (noradrenaline, phenylephrine and methoxamine) were determined before and after amlodipine, 5-methylurapidil, chloroethylclonidine or BMY 7378. KEY RESULTS: In renal failure SHR (RFSHR), RBF and creatinine clearance were significantly reduced (approximately 70%), while urine output and fractional sodium excretion were four and twenty-fold higher, respectively, compared to SHR. Vasoconstrictions induced by RNS or the adrenoceptor agonists were greater in RFSHR than SHR, and these responses were blunted by 5-methylurapidil, BMY 7378 and amlodipine in the SHR, while chloroethylclonidine had no effect. In the RFSHR, all renal vasoconstrictions were reduced by amlodipine and BMY 7378 but 5-methylurapidil attenuated those caused by RNS, noradrenaline and methoxamine while those to phenylephrine were enhanced. Chloroethylclonidine potentiated renal vasoconstrictor responses to methoxamine and phenylephrine but not RNS or noradrenaline in RFSHR. CONCLUSIONS AND IMPLICATIONS: These findings suggest alpha(1A)- and alpha(1D)-adrenoceptors mediated the renal vasoconstrictor responses in SHR and RFSHR. In the RFSHR, other alpha(1)-adrenoceptor subtypes, for example, alpha(1B)-adrenoceptors appeared to play a greater role.

Clonidine and a novel clonidine analog AL-12 cause sympathoinhibition in spontaneously hypertensive and diabetic spontaneously hypertensive rats.

This study examined the sympathoinhibitory effects of clonidine and a novel clonidine analog, AL-12, in rat models of genetic hypertension and a combined state of genetic hypertension and diabetes. Rats in the treatment groups were given either clonidine or AL-12 while the respective control groups received either saline or Tween 80 for 6 days. Physiological data were collected during this period, which was followed by acute studies on day 7 when bolus administrations (i.v.) of graded doses of noradrenaline, phenylephrine and methoxamine were carried out. It was observed that in AL-12-treated nondiabetic spontaneously hypertensive rats (SHR), the pressure responses to all adrenergic agonists were greater (p < 0.05) in the treated group, while in the diabetic SHR rats a larger pressure response was observed only to noradrenaline (p < 0.05). In nondiabetic SHR rats treated with clonidine, a greater (p < 0.05) pressure response was observed only in the case of phenylephrine. In the diabetic SHR rats treated with clonidine, the pressure responses to the adrenergic agonists were similar (p > 0.05) in the treated and its control animals except that methoxamine caused a greater (p < 0.05) pressure response in the control group. The data obtained suggest that clonidine and AL-12 act possibly via vascular alpha1 and alpha2 adrenoceptors present at both pre- and postsynaptic locations.

The effect of superoxide dismutase enzyme inhibition on renal microcirculation of spontaneously hypertensive-stroke prone and Wistar rats.

A significant factor in the development of hypertension may be excessive vasoconstriction within the renal medulla. This study therefore investigated the role of superoxide dismutase (SOD) in the regulation of renal medullary and cortical blood perfusion (MBP and CBP, respectively) in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar rats. CBP and MBP were measured before and after intra-renal infusion of the SOD inhibitor, diethyldithio-carbamic acid (DETC). Under basal conditions, mean arterial pressure was significantly greater in SHRSP than Wistar rats, but both MBP and heart rate (HR) were significantly lower in SHRSP relative to Wistar rats (P<0.05, n=7 in both groups). Infusion of DETC (2 mg/kg/min) into the cortico-medullary border area of the kidney significantly decreased MBP in the SHRSPs (by 28+/-3 %, n=7, P<0.05), indicating a greater vasoconstriction within this vascular bed. However, DETC also significantly decreased MBP in Wistar rats to a similar extent (24+/-4 %, n=7, P<0.05). These results suggest that superoxide anions play a significant role in reducing renal vascular compliance within the renal medulla in both normotensive and hypertensive animals, although the responses are not greater in the hypertensive relative to the control animals.

The innervation of the kidney in renal injury and inflammation: a cause and consequence of deranged cardiovascular control.

Extensive investigations have revealed that renal sympathetic nerves regulate renin secretion, tubular fluid reabsorption and renal haemodynamics which can impact on cardiovascular homoeostasis normally and in pathophysiological states. The significance of the renal afferent innervation and its role in determining the autonomic control of the cardiovascular system is uncertain. The transduction pathways at the renal afferent nerves have been shown to require pro-inflammatory mediators and TRPV1 channels. Reno-renal reflexes have been described, both inhibitory and excitatory, demonstrating that a neural link exists between kidneys and may determine the distribution of excretory and haemodynamic function between the two kidneys. The impact of renal afferent nerve activity on basal and reflex regulation of global sympathetic drive remains opaque. There is clinical and experimental evidence that in states of chronic kidney disease and renal injury, there is infiltration of T-helper cells with a sympatho-excitation and blunting of the high- and low-pressure baroreceptor reflexes regulating renal sympathetic nerve activity. The baroreceptor deficits are renal nerve-dependent as the dysregulation can be relieved by renal denervation. There is also experimental evidence that in obese states, there is a sympatho-excitation and disrupted baroreflex regulation of renal sympathetic nerve activity which is mediated by the renal innervation. This body of information provides an important basis for directing greater attention to the role of renal injury/inflammation causing an inappropriate activation of the renal afferent nerves as an important initiator of aberrant autonomic cardiovascular control.

Antidiuretic and antinatriuretic response to high salt load in normotensive Wistar-Kyoto rats: Role of alpha-1A-adrenoreceptors.

Altered renal adrenergic responses have been recognized as pathophysiological responses to high salt intake. This study aims to investigate the influence of 6 weeks of high salt diet on α1A -adrenoceptor regulation of renal tubular antinatriuretic and antidiuretic response in normal Wistar Kyoto rats. To achieve the above objective, antinatriuretic and antidiuretic response to phenylephrine was measured in the absence and presence of 5-methylurapidil (5-MeU) using the inulin clearance method. Systemic mean arterial blood pressure and renal haemodynamics were also measured simultaneously. Six weeks of high salt intake in Wistar-Kyoto (WKY) rats did not bring any significant increase in mean arterial blood pressure. WKY rat on high salt diet (WKYHNa) showed an exaggerated increase in absolute and fractional sodium excretion. There was a significant involvement of α1A -adrenoceptor in carrying out renal tubular antinatriuretic and antidiuretic response in Wistar Kyoto rats on normal sodium diet (WKYNNa). However, α1A -adrenoceptor played a minimal role in handling the tubular reabsorptive response in WKY rats on high salt diet.

Improvement in baroreflex control of renal sympathetic nerve activity in obese Sprague Dawley rats following immunosuppression.

AIM: This investigation explored the hypothesis that in obesity an inflammatory response in the kidney contributed to a renal nerve-dependent blunting of the baroreflex regulation of renal sympathetic nerve activity. METHODS: Rats received a normal (12% kcal) or high-fat (45% kcal) diet for 8 weeks plus daily injections of vehicle (0.9% NaCl i.p) or tacrolimus (0.25 mg kg-1 day-1 i.p) from weeks 3-8. Following anaesthesia, left renal sympathetic nerve activity was recorded, baroreflex gain curves were generated, by infusing phenylephrine and sodium nitroprusside, and cardiopulmonary baroreceptors challenged by infusing a saline load. RESULTS: The high-fat diet elevated weight gain and adiposity index by 89 and 129% (both, P < 0.001). Mean blood pressure (132 ± 4 vs 103 ± 5 mmHg), fractional noradrenaline excretion and creatinine clearance (5.64 ± 0.55 vs 3.32 ± 0.35 mL min-1 kg-1 ) were 28, 77 and 69% higher (all P < 0.05), but urine flow and fractional sodium excretions were 42 and 72% (both P < 0.001) lower compared to normal rats. Plasma and renal TNF-α and IL-6 concentrations were fourfold to fivefold (P < 0.001) and 22 and 20% higher (both, P < 0.05), in obese rats but normalized following tacrolimus. In obese rats, baroreflex sensitivity was reduced by 80% (P < 0.05) but restored by renal denervation or tacrolimus. Volume expansion reduced renal sympathetic nerve activity by 54% (P < 0.001) in normal and obese rats subjected to renal denervation and tacrolimus, but not in obese rats with an intact renal innervation. CONCLUSION: Obesity induced a renal inflammation and pointed to this being both the origin of autonomic dysregulation and a potential focus for targeted therapy.

Bradykinin receptor blockade restores the baroreflex control of renal sympathetic nerve activity in cisplatin-induced renal failure rats.

AIM: This study investigated the effect of renal bradykinin B1 and B2 receptor blockade on the high- and low-pressure baroreceptor reflex regulation of renal sympathetic nerve activity (RSNA) in rats with cisplatin-induced renal failure. METHODS: Cisplatin (5 mg/kg) or saline was given intraperitoneally 4 days prior to study. Following chloralose/urethane anaesthesia, rats were prepared for measurement of mean arterial pressure (MAP), heart rate and RSNA and received intrarenal infusions of either Lys-[des-Arg9 , Leu8 ]-bradykinin (LBK), a bradykinin B1 receptor blocker, or bradyzide (BZ), a bradykinin B2 receptor blocker. RSNA baroreflex gain curves and renal sympatho-inhibitory responses to volume expansion (VE) were obtained. RESULTS: In the control and renal failure groups, basal MAP (89 ± 3 vs. 80 ± 8 mmHg) and RSNA (2.0 ± 0.3 vs. 1.7 ± 0.6 µV.s) were similar but HR was lower in the latter group (331 ± 8 vs. 396 ± 9 beats/min). The baroreflex gain for RSNA in the renal failure rats was 39% (P < 0.05) lower than the control but was restored to normal values following intrarenal infusion of BZ, but not LBK. VE had no effect on MAP or HR but reduced RSNA by some 40% (P < 0.05) in control but not renal failure rats. Intrarenal LBK infusion in the renal failure rats normalized the VE induced renal sympatho-inhibition whereas BZ only partially restored the response. CONCLUSION: These findings suggest that pro-inflammatory bradykinin acting at different receptors within the kidney generates afferent neural signals which impact differentially within the central nervous system on high- and low-pressure regulation of RSNA.

Enhanced expression of endothelial nitric oxide synthase in the myocardium ameliorates the progression of left ventricular hypertrophy in L-arginine treated Wistar-Kyoto rats.

The present study investigated the role of endothelial nitric oxide synthase (eNOS) enzyme in the development of left ventricular hypertrophy (LVH) in Wistar-Kyoto rats. The effect of L-arginine administration on cardiac structure, arterial stiffness, renal and systemic hemodynamic parameters was studied and the change in expression of eNOS and cystathione γ lyase (CSE) in the myocardium of LVH rats was evaluated. LVH was induced using isoprenaline (5 mg/kg, S.C.) and caffeine (62 mg/L in drinking water) for 14 days. Following to that, L-arginine (1.25 g/L in drinking water) was given for 5 weeks as a donor of NO. eNOS and CSE gene expressions were down regulated in the LVH group by about 35% and 67% respectively when compared to control. However, in the LVH group treated with L-arginine there was up regulation of eNOS by almost 27% and down regulation in CSE by 24% when compared to control (all P < 0.05). Heart index and H2S plasma levels were reduced by almost 53% in the L-arginine treated LVH group compared to the control (all P < 0.05). Mean arterial pressure, heart rate and pulse wave velocity were reduced while renal blood perfusion increased in L-arginine treated LVH rats compared to their untreated counterparts (all P < 0.05). The enhanced expression of eNOS in L-arginine treated LVH rats resulted in the amelioration of oxidative and haemodynamic parameters suggesting that NO system is an important therapeutic target in cardiac and LV hypertrophies.

Are the high mobility group non-histone chromosomal proteins associated with 'active' chromatin?

When 10--20% of the DNA in thymus and liver nuclei is rendered acid-soluble by DNAase I digestion, under conditions which may be expected to specifically degrade away most of the transcribed sequences, very little of the high-mobility-group non-histone proteins are released from the nuclei. Therefore, high-mobility-group proteins are probably not specifically associated with this portion of the genome.

The isolation of three new high mobility group nuclear proteins.

In addition to the four high mobility group non-histone chromosomal proteins HMG 1, 2, 14 and 17 and histone H1, perchloric acid extracts of nuclei contain a number of other smaller low molecular weight proteins. Three of these proteins (HMG 18, 19A, and 19B) have been purified and characterized. Protein HMG 18 has high lysine and alanine contents, resembling histone H1. Proteins HMG 19A and 19B have high contents of basic and acidic amino acids and resemble HMG proteins 1, 2, 14 and 17. N-terminal sequence analyses of the proteins show that they are not degradation products of histones or the other HMG proteins. However, there are sequence similarities between HMG 18 and histone H5, and between HMG 19B and HMG 17, supporting the view that the HMG proteins and the lysine-rich histones are functionally related.

Studies on the degradation of high mobility group non-histone chromosomal proteins.

During the isolation of high mobility group non-histone proteins from calf thymus chromatin by methods described previously (e.g. Goodwin, G.H., Nicolas, R.H. and Johns, E.W. (1975) Biochim. Biophys, Acta. 405, 280--291) protein degradation occurs resulting in a number of proteins appearing in the chromatin extracts which are not present in high mobility group protein preparations in which proteolysis has been completely inhibited. These extra proteins, formerly numbered high mobility group proteins 3, 5, 6 and 8, are thus probably degradation products of other nuclear proteins, produced during the isolation procedure. From the amino acid analyses, tryptic peptides and N-terminal sequences, it is concluded that high mobility group protein 3 is probably a degradation product of high mobility group protein 1. The amino acid analysis of high mobility group protein 8 is very similar to that of the N-terminal half of histone H1 suggesting that high mobility group protein 8 is a degradation product of this histone.

An improved large scale fractionation of high mobility group non-histone chromatin proteins.

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.

Comparisons of the structures of the chromosomal high mobility group proteins HMG1 and HMG2 prepared under conditions of neutral and acidic pH.

The chromosomal proteins HMG1 and 2 have been prepared by salt extraction and phosphocellulose chromatography at neutral pH (Isackson, P.J., Debold, W.A. and Reeck, G.R. (1980) FEBS Lett. 119, 337-342) to minimize protein denaturation. The structures of these phosphocellulose-prepared high mobility group proteins have been compared with those of high mobility group proteins using the previously described acid-extraction conditions which fully denature the proteins. When compared in the same solvent conditions the acid-extracted proteins did not refold to give the same level of alpha-helical and tertiary folded structures as the phosphocellulose-prepared proteins, suggesting that acid treatment can cause some irreversible damage to the proteins. This finding was supported by changes in the structure observed when phosphocellulose-prepared HMG1 was neutralized after exposure to acid. Gel filtration studies reveal no differences in the size of the high mobility group proteins, phosphocellulose-prepared and acid-extracted proteins both being largely monomeric in solution. Little difference was detected in the DNA-binding properties of the two types of protein, nor was there any difference in the oxidation state of the cysteines. However, isoelectric focusing analysis revealed differences in the subfractions of HMG2 prepared by the two methods.