Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH

The pharmacopoeia protocol for HP-SEC of insulin, using an acidic non-physiological eluent, does not represent insulin's association state in the formulation. This study aimed to evaluate insulin's elution behavior in HP-SEC in a “physiological” (aqueous, neutral pH) eluent, using on-line UV absorption and multi-angle laser light scattering detection. The effect of insulin concentration and association state in the formulation (monitored by circular dichroism) and eluent composition (zinc ion, arginine) on its elution behavior was assessed. We showed that the elution behavior of insulin in “physiological” HP-SEC is affected by both dynamic association–dissociation of insulin molecules and insulin–column interactions. Insulin molecules re-equilibrated in the HP-SEC eluent, making its elution behavior practically insensitive to the association state of insulin in the formulation. Zinc ions in the eluent promoted association of insulin to hexamers, whereas arginine overruled the effect of zinc ions and induced on-column dissociation of insulin to dimers and monomers. Combined results from “physiological” and compendial HP-SEC were shown to provide a better view of the aggregation state of heat-stressed insulin than either of the single methods. The insights obtained with this study are crucial for a proper evaluation of HP-SEC data of insulin.     

Central neural pathway mediating splanchnic osmosensation

To determine the central neural pathway which carries splanchnic osmosensory information to vasopressin (AVP) neurons in the hypothalamus, bilateral electrolytic lesions were placed in the ascending catecholaminergic fiber bundle, the locus coeruleus (LC), the locus subcoeruleus (subLC), the lateral parabrachial nucleus (LPB), the caudal periaqueductal gray (PAG) and the median preoptic nucleus (MPO). Six and seven days later, plasma AVP levels, plasma osmolality, mean arterial pressure and heart rate were measured following gastric infusion of hypertonic (598 mosm/kg; 2ml/4min) or isotonic (290 mosm/kg) saline in conscious rats with indwelling tail artery catheters and nasogastric tubes. The most effective pontine lesions, which were located in the ventral locus subcoeruleus (vsubLC) approximately 1.0 mm below the LC, decreased the AVP response to hypertonic gastric infusion by 59.7% (P < 0.05) as compared to sham-lesioned controls. In addition, unilateral vsubLC lesions dramatically reduced the catecholamine innervation of the ipsilateral paraventricular nucleus (PVN), as qualitatively determined with dopamine β-hydroxylase immunocytochemistry, suggesting that a pathway ascending with catecholaminergic fibers was disrupted. Lesions of the MPO were also very effective, decreasing the AVP response to hypertonic saline infusion by 60.3% (P < 0.05), suggesting that the MPO is an integral relay center in this pathway. On the other hand, LC, LPB and PAG lesions were ineffective. Systemic plasma osmolality or cardiovascular factors did not mediate the AVP response. These results demonstrate, for the first time, that splachnic osmotic information is transmitted to the hypothalamus via pathways within the ascending catecholaminergic fiber bundles, the MPO is a relay center where peripheral and central osmotic information may be integrated, and the LC, LPB and PAG are not part of the splanchnic osmotic pathway.     

Effects of calcium on frequency-modulated secretion of vasopressin from isolated murine neural lobes

The relationship between external calcium and frequency-facilitated arginine vasopressin (AVP) secretion from the murine neurointermediate lobe was examined in vitro. We evaluated the calcium-dependency of frequency-dependent release in this system, and found that log AVP secretion versus log external calcium plots gave slopes of 0.71, 0.92 and 1.2 for 5, 10 and 20 Hz stimulation, respectively. These slopes are considerably lower than the slopes of 3–4 Hz found at conventional synaptic junctions.     

Yeast arginine permease: nucleotide sequence of the CAN1 gene

Summary The yeast CAN1 gene, thought to encode arginine permease, has found use in genetics as a selectable locus. We have sequenced the cloned CAN1 gene, which contains an open reading frame of 1770 nucleotides, encoding a polypeptide of calculated molecular weight 65,766. Disruption of this open reading frame largely abolishes CAN1 gene expression, while subcloned fragments of the open reading frame hybridize strand —specifically to a 2.3 kb yeast RNA message. The encoded protein has no leader signal sequence, and is highly hydrophobic, with a possible twelve membrane-spanning domains, several of which have the high hydrophobic moments seen in channel-forming or permease proteins. This protein structure is consistent with the CAN1 product being the plasma membrane arginine permease.     

Recurrent hypertonic dehydration due to selective defect in the osmoregulation of thirst

A 6-year-old girl with recurrent episodes of hypertonic dehydration was studied. She denied thirst even with a plasma osmolality as high as 421 mosmol/kg. The hypernatremia was associated with an ability to concentrate urine (854 mosmol/kg). Volume expansion with water corrected hypernatremia (162 to 148 mEq/l) and resulted in an increased urine flow and urinary dilution (137 mosmol/kg) because of suppression of endogenousvasopressin (AVP) release (5.1 pg/ml). Hypertonic saline infusion raised the plasma AVP level (25.6 pg/ml) in response to changes in plasma osmolality (305 to 330 mosmol/kg) and led to a maximal urine osmolality of 818 mosmol/kg. With chronic forced fluid intake, the patient maintained a normal resum sodium concentration (range, 135–145 mEq/l) with a urine osmolality as low as 65 mosmol/kg. These findings are consistent with an isolated defect in the osmoregulation of thirst as the cause of the chronic hypertonic dehydration without deficiency in AVP secretion.     

Poly(ethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties

Some tumors, such as melanomas and hepatocellular carcinomas, have a unique nutritional requirement for arginine. Thus, enzymatic degradation of extracellular arginine is one possible means for inhibiting these tumors. Arginine deiminase is an arginine degrading enzyme (ADI) that has been studied as an anti-cancer enzyme. However, ADI has a short serum half-life and, as a microbial enzyme, is highly immunogenic. Formulation of other therapeutic proteins with poly(ethylene glycol) (PEG) has overcome these problems. Here, ADI–PEGs were synthesized using PEGs of varying size, structure (linear or branched chain) and linker chemistries. All ADI–PEGs retained 50% of enzyme activity when PEG was covalently attached to 40% of the primary amines irrespective of the PEG molecular weight or attachment chemistry used. However, it was observed that, as the PEG size increases to 20 kDa, there was a corresponding increase in the pharmacokinetic (pK) and pharmacodynamic (pD) properties of the formulation. Variation in PEG linker or structure, or the use of PEGs >20,000 mw, did not affect the pK or pD. As has been shown with other therapeutic proteins, repeated injection of ADI–PEG into experimental animals resulted in significantly lower titers of antibodies against this protein than unmodified ADI. These data suggest that formulation of ADI with PEG of 20,000 mw results is the optimal method for formulating this promising therapeutic agent.     

L-精氨酸对严重腹腔感染大鼠肠屏障功能的影响

目的　探讨L 精氨酸对严重腹腔感染大鼠肠屏障功能的影响。方法　SD大鼠 90只 ,随机分成对照组、感染组和精氨酸组 ,每组 30只。对照组仅行单纯剖腹手术 ;感染组采用盲肠结扎加穿孔 (CLP)手术制作严重腹腔感染模型 ;精氨酸组行CLP后每天添加L 精氨酸饮食 [0 2 6 4g/ (kg·d) ]。各组术后 1、2、 4d分别取肠黏膜行病理学观察、增殖细胞核抗原 (PCNA)指数测定、血细菌培养和血浆内毒素水平测定。结果　精氨酸组较感染组小肠黏膜病理性损害明显减轻。感染组术后 2、 4d血浆内毒素水平明显高于精氨酸组 ,分别是 (1 4 2± 0 10 )EU/mlvs (1 14± 0 11)EU/ml,(1 94± 0 0 7)EU mlvs (1 5 8± 0 14 )EU/ml,P <0 0 1。精氨酸组术后 2d血细菌阳性率明显低于感染组 (10 %vs 30 % ,P <0 0 1)。感染组PCNA指数先升高 ,后下降 ,而精氨酸组 4d内下降不明显。结论　当严重腹腔感染导致肠屏障功能障碍时 ,L 精氨酸有促进肠黏膜损伤修复、维护肠屏障功能的作用     

Proliferation of mitogen-stimulated human peripheral blood mononuclear cells is inhibited by extracellular arginine deiminase of <Emphasis Type="Italic">Granulicatella elegans</Emphasis> isolated from the human mouth

Granulicatella elegans is a member of normal human oral flora and is thought to be a potent pathogen in endocarditis, especially so-called “culture-negative” endocarditis. To elucidate the pathogenicity of this microorganism in inflammatory diseases, the effect of the extracellular products of this bacteria on human peripheral blood mononuclear cells (PBMCs) was examined. Culture supernatants produced by oral isolates of G. elegans strongly inhibited the proliferation of PBMC stimulated by the T-cell mitogens phytohemagglutinin-P, phorbol 12-myristate 13-acetate, concanavalin A, and staphylococcal enterotoxin B. Purification of the active extracellular product revealed that a fraction containing proteins of approximately 47 kDa showing arginine deiminase activity contributed to the inhibition of PBMC proliferation.     

Clinical outcome of immunonutrition in a heterogeneous intensive care population

Objective To study the effect of a high-protein enteral formula enriched with arginine, glutamine, and antioxidants and containing 3 fatty acids and a mixture of fibers, on the clinical outcome of a heterogeneous intensive care (ICU) population.Design and setting A randomized, prospective, double blind, controlled, two-center clinical trial in two intensive care units in The Netherlands.Patients and participants A total of 597 adult ICU patients expected to require enteral tube feeding for more than 2 days were randomized to receive immunonutrition or an isocaloric control formula. Interventions Patients received either the immunonutrition or the control feed.Measurements and results Intention-to-treat and per-protocol analyses showed no statistically significant difference in clinical outcome parameters between the two groups. Results of the intention-to-treat analysis in control vs. immunonutrition were: median ICU length of stay in days, 8.0 (IQR 5.0–16.0) vs. 7.0 (4.0–14.0); median hospital length of stay in days, 20.0 (IQR 10.0–34.0) vs. 20.0 (10.0–35.0); median days of ventilation, 6.0 (IQR 3.0–12.0) vs. 6.0 (IQR 3.0–12.0); ICU mortality, 26.8% vs. 28.2%; in-hospital mortality, 36.4% vs. 38.5%; infectious complications, 41.7% vs. 43.0%Conclusions The results of this largest randomized, controlled trial found that in the general ICU population immunonutrition has no beneficial effect on clinical outcome parameters. These results are consistent with the literature that is currently available.This revised version was published online in February 2005 with corrections to the heading of a subsection of the Results (A priori subgroup analyses).     

穹窿切断并摘除肾上腺后大鼠下丘脑AVP表达变化

目的:探讨海马参与肾上腺(HPA)轴调节的机制,观察精氨酸加压素(arginine vasopressin,AVP)与糖皮质激素的关系。方法:建立大鼠穹窿切断模型,采用酶联免疫吸附试验(ELISA)方法检测穹窿切断0、4、7、10 d血中糖皮质激素的浓度;建立大鼠穹窿切断同时摘除肾上腺7 d的模型,采用免疫组织化学方法检测AVP的表达变化。结果:穹窿切断7 d后血中糖皮质激素的浓度升高,穹窿切断同时摘除肾上腺7 d后,下丘脑AVP表达升高。结论:海马通过穹窿对HPA轴发挥负反馈调节作用,糖皮质激素负反馈调节下丘脑AVP。