High-resolution mass spectrometry for thymosins detection and characterization

Objectives: The aim of this study was to characterize β and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies.

Methods: A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides.

Results: In addition to thymosin β₄ (Tβ₄) and β₁₀ (Tβ₁₀), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tβ₄ and Tβ₁₀ together with other proteolytic fragments. The sulfoxide derivative of both Tβ₄ and Tβ₁₀ and the acetylated derivatives at lysine residues of Tβ₄ were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α₁ and thymosin α₁₁ together with diverse proteolytic fragments were identified too.

Conclusion: The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.     

4-Methylumbelliferyl-beta-glucosidase in cultured human fibroblasts from controls and patients with Gaucher's disease

β-Glucosidase activity was measured in cultured fibroblasts from controls and patients with Gaucher's disease using 4-methylumbelliferyl-β-glucopyranoside as substrate.K_m against the substrate used was higher for the enzyme in fibroblasts as compared to that in liver. For optimal results higher substrate concentrations than for liver were therefore used.It could be shown that the activity at around pH 4.0, but not at around pH 5.0–5.5, was dependent upon the ionic strength of the buffer. This finding may explain some of the discrepancies between results of earlier authors.Good separation without overlap was obtained between the patients with Gaucher's disease and the controls, the Gaucher patients having much lower activity at pH 4.0–5.5.Using 4-methylumbelliferyl-glycosidic substrates for the assay of β-xylosidase and α-arabinosidase it could be shown that both these activities were markedly reduced in fibroblasts from patients with Gaucher's disease. The conclusion is drawn that all these three activities in fibroblasts are caused by the same enzyme.     

Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders.In amniotic fluid β-galactosidase and α-mannosidase decreased moderately towards term, while β-glucosidase decreased markedly. N-Acetyl-β-glucosaminidase and β-glucuronidase were relatively unchanged.In chorionic villi N-acetyl-β-glucosaminidase, β-galactosidase, and α-mannosidase were substantially decreased towards term, while β-glucosidase was unchanged and β-glucuronidase markedly increased.In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study.The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.     

Gargoylism: hydrolysis of β-galactosides and tissue accumulation of galactose- and mannose-containing compounds

The sugars present in hydrolyzed extracts of human liver and brain were analyzed by gasliquid chromatography after conversion to their alditol acetates. The samples analyzed were obtained from control subjects, patients with gargoylism, and patients with a few other kinds of storage disorders. Accumulation of galactose was demonstrated in the liver and the brain of two patients with gargoylism, and in the liver samples, high levels of mannose were found too. We also studied the hydrolysis of a number of galactosides by homogenates from different tissues in the control subjects and in the patients. Separation methods and kinetic studies demonstrated the presence in normal human tissues of two different beta-galactosidases, which we call enzyme A and enzyme B, respectively. Enzyme A hydrolyzed all the beta-galactosides tested. Enzyme B hydrolyzed the synthetic substrates tested (4-methylumbelliferyl-, p-nitrophenyl-, o-nitrophenyl-, and phenyl-beta-galactoside) but not the natural substrates tested (ceramide-beta-galactoside, ceramide lactoside, transferrin glycopeptide, and keratan sulfate). Enzyme B also exerted beta-glucosidase activity. In various tissues from patients with gargoylism, deficiency of beta-galactosidase A could be demonstrated.It is suggested that the high level of galactose found in the hydrolyzed extracts of tissues from gargoylism patients is due to storage of galactose-rich glycosaminoglycans and glycopeptides, and that this storage is a result of the deficiency of beta-galactosidase A.The high level of mannose in the liver from gargoylism patients seems to indicate storage of glycopeptide, adding a new group of substances to those known to be stored in gargoylism.     

Biochemical activities of lysosomal acid hydrolases in leukemic cells

The biochemical activities of 8 lysosomal acid hydrolases in leukemic cells from 48 patients were examined. Characteristic alterations were found in α-mannosidase, β-galactosidase and N-acetyl-β-glucosaminidase activities of leukemic cells. The level of α-mannosidase activity was much higher in myelo(mono)genous leukemias (AML, AMoL, AMMoL, CML and CMMoL) than in lymphogenous ones (ALL, T-cell leukemia, hairy cell leukemia and CLL) without exception. The β-galactosidase activity also differed as a result of α-mannosidase, except in T-cell leukemia. In T-cell leukemia it was within the range of normal lymphocytes, but in the other lymphogenous leukemias it was significantly below normal. N-acetyl-β-glucosaminidase activity in myelo(mono)genous leukemic cells was above the range of normal granulocytes. The changes in these enzyme levels were consistent. The lymphocytic or myelocytic nature of three cases of acute undifferentiated leukemia could be determined by enzyme studies. In two cases it was lymphocytic and in one it was myelocytic. The enzymatic abnormalities were also found in morphologically mature neutrophils from patients with not only chronic types (CML, CMMoL) but also acute types (AMoL, AMMoL) of leukemias, and were similar to those of their respective leukemic cells. Analysis of lysosomal enzymes (at least three of those mentioned above), can elucidate one of the biochemical properties of leukemic cells and may be valuable in the differentiation of leukemias.     

Effects of thymosin β4 and its N-terminal fragment Ac-SDKP on TGF-β-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis

Thymosin β4 (Tβ4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tβ4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis.

Objectives: In this study, the putative anti-proliferative and anti-fibrogenic effects of Tβ4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tβ4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis.

Methods: We utilized both control and TGF-β-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tβ4 were assessed in CD1 mice treated with bleomycin.

Results: In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-β-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-β. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tβ4 failed to prevent fibrosis induced by the drug at 14 and 21 days.

Conclusion: We conclude that, compared to Tβ4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.     

Variation in oil content and tree size between six geographically separate Santalum spicatum families,established near Narrogin,Western Australia

Six geographically separate sandalwood (Santalum spicatum) tree families were established together in a trial near Narrogin, Western Australia to compare oil content and tree size variation at ages 10 and 18 years. The trial site contained a total of 300 sandalwood trees with 41–55 trees from each family. All 300 sandalwood trees were measured for tree size: height, stem diameter and bole length. Wood core samples (heartwood + sapwood) were also taken at 300 mm above the ground from 71 to 72 separate trees, at ages 10 and 18 years. Each wood core sample was ground separately and then a sub-sample was analysed for oil concentration, and α- and β-santalol concentration within the oil.

The mean extractable oil concentrations within the sandalwood stems at 300 mm above the ground were significantly greater from the Borden and Dumbleyung families (1.1–1.6%) than the Ravensthorpe family (0.5–0.8%), at both ages 10 and 18 years. However, the oil quality (mean concentration of α- and β-santalol) was not significantly different between the six families at ages 10 or 18 years. Instead, oil quality was highly variable within each family.

Between sandalwood ages 10 and 18 years, the overall mean extractable oil concentration from the six different families increased significantly from 0.8 to 1.3%. During the same time period, mean α-santalol concentration increased significantly from 8.6 to 13.8%, and mean β-santalol concentration also increased significantly from 3.3 to 4.5%.

Mean tree height, stem diameter and bole length varied significantly between the six families at age 18 years. The Borden family appeared to be more tree-like in form, with a significantly longer bole length, than some of the other families which were more shrub-like in appearance.     

Leukocyte glucocerebrosidase deficiency diagnostic in adult Gaucher's disease with negative bone marrow biopsy. Some properties of the enzyme in leukocytes and spleen

Abstract. A sixty-one year-old man with Gaucher's disease is described. The disease was manifested by splenomegaly, hypersplenism and elevations of serum acid phosphatase and immunoglobulin but no bone lesions were roentgenologically demonstrable and bone marrow biopsy failed to reveal Gaucher cells. The diagnosis was established prior to splenectomy by the finding of decreased β-D-glucoside glucohydrolase (β-D-GGH) activity in his peripheral leukocytes. Examination of the surgically removed spleen revealed Gaucher cells, a large excess of glucocerebroside and decreased β-D-GGH activity. Splenectomy was followed by normalization of haemoglobin, white cell and platelet counts and of the serum acid phosphatase and immunoglobulin levels. The patient's leukocyte β-D-GGH activity showed an optimum pH similar to that of normal leukocyte enzyme, about 5.4, using both glucocerebroside (Glc-Cer) and 4-methylumbelliferyl-β-D-glucopyranoside (MU-Glc) as substrate. The patient's spleen β-D-GGH activity showed a pH optimum of about 4.5, which differed slightly from that of normal spleen enzyme. The Km values of β-D-GGH in the patient's leukocytes for Glc-Cer and MU-Glc were 1.35 × 10^?4 M and 3.6 × 10^?3 M, respectively, i.e. in the normal range. The patient's splenic tissue Km values were for Glc-Cer and MU-Glc 0.52 × 10^?4 M and 5.8 × 10^?3 M, respectively, as compared to 0.30 × 10^?4 M and 5.0 × 10^?3 M in a normal control.     

Lysosomal enzymes in cerebral atrophy

In seven patients with cerebral atrophy due to pre-senile dementia and/or cerebrovascular disease, the activity of acid phosphatase in lumbar cerebrospinal fluid (CSF) was higher (p < 0.05) than in six controls. The activity of arylsulphatase and β-galactosidase in CSF was the same in the two groups. In the serum, the activities of acid phosphatase and arylsulphatase were the same in the two groups but the activity of β-galactosidase was lower (p < 0.02) in patients with cerebral atrophy.     

Electrophoretic forms of human liver alpha-L-fucosidase and their relationship to fucosidosis (mucopolysaccharidosis F)

1. Human liver α-L-fucosidase was studied by starch gel electrophoresis and isoelectric focusing both before and after treatment with neuraminidase. These studies revealed the presence of 5–6 bands on starch gels and six isoelectric forms with pI's of 6.9, 6.5, 6.1, 5.9, 5.7 and 5.5. The activity of the two most acidic forms was reduced after neuraminidase treatment.2. Liver tissue from a patient with fucosidosis had approximately 4% of normal α-l-fucosidase activity. Starch gel electrophoresis revealed that all electrophoretic forms of the enzyme appear to be deficient.3. Apparent Michaelis constants (K_m 's) determined for both the normal and fucosidotic liver α-l-fucosidase for 4-methylumbelliferyl-α-l-fucopyranoside were found to be 0.085 mM and 0.058 mM, respectively.4. The pH activity profile of normal and fucosidotic liver α-l-fucosidase were very similar with major pH optima at 5.4 and 5.3, respectively.     

Identification of PDGF-BB binding to thymosin β4 by chemical cross-linking

Introduction: The purpose of our work was to identify unknown interaction partners of thymosin β₄ (Tβ₄). It was suggested that Tβ₄ could be an antifibrotic drug for treatment of liver fibrogenesis, because Tβ₄ prevents the platelet-derived growth factor-BB (PDGF-BB)-induced activation of hepatic stellate cells (HSCs). Very little information is available how Tβ₄ counteracts the PDGF-BB-induced activation of HSCs. We propose the hypothesis that Tβ₄ could bind directly to PDGF-BB and thereby reduce the concentration of free PDGF-BB available for binding to the PDGF-β receptor.

Methods: To prove our suggestion of a direct interaction between Tβ₄ and PDGF-BB, we carried out chemical as well as photochemical cross-linking experiments between the two pure proteins in vitro.

Results: We identified an interaction between Tβ₄ and PDGF-BB by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking as well as through biotin label transfer using a bifunctional photoactivatable derivative of Tβ₄. In an in vitro system, PDGF-BB was identified as the first extracellular partner interacting with Tβ₄. This interaction could influence PDGF-BB binding to its receptor and abolish PDGF-BB-related effects.

Conclusion: Direct interaction of Tβ₄ with extracellular factors should be considered as a potential mechanism to explain the pleiotropic effects of β-thymosins.     

Beta-asarone protects against MPTP-induced Parkinson’s disease via regulating long non-coding RNA MALAT1 and inhibiting α-synuclein protein expression

ObjectiveNumerous long non-coding RNAs (lncRNA) have been identified in neurodegenerative disorders including Parkinson’s disease (PD). Emerging evidence demonstrates that β-asarone functions as neuroprotective effects in both in vitro and in vivo models. However, the role of β-asarone and its potential mechanism in PD remain not completely clear.MethodsMPTP-induced PD mouse model and SH-SY5Y cells subjected to MPP+ as its in vitro model were used to evaluate the effects of β-asarone on PD. LncRNA MALAT1 and α-synuclein expression were determined by real-time PCR and western blot methods.Resultsβ-Asarone significantly increased the TH+ cells number and decreased the expression levels of MALAT1 and α-synuclein in midbrain tissue of PD mice. RNA pull-down and immunoprecipitation assays confirmed that MALAT1 associated with α-synuclein, leading to the increased stability of α-synuclein and its expression in SH-SY5Y cells. β-asarone elevated the viability of cells exposed to MPP+. Either overexpressed MALAT1 or α-synuclein could canceled the protective effect of β-asarone on cell viability. In PD mice, pcDNA-MALAT1 also decreased the TH+ cells number and increased the α-synuclein expression in PD mice with treatment of β-asarone.Conclusionβ-Asarone functions as a neuroprotective effect in both in vivo and in vitro models of PD via regulating MALAT1 and α-synuclein expression.     

Gargoylism (mucopolysaccharidosis type II). Accumulation of glycosaminoglycans, gangliosides and glycoproteins and activity of some related glycolytic enzymes in liver, spleen and brain

For a case of gargoylism (Hunter's disease, mucopolysaccharidosis, type II) the effect of enzyme deficiencies on the sugars of glycosaminoglycans, gangliosides and glycoproteins were compared. To study this the glycoproteins and glycosaminoglycans after lipid extraction, were first solubilized by proteolytic digestion with pronase or papain. Subsequently both classes of compounds were separated on DEAE-Sephadex A-50. Compared to the normal values the glycosaminoglycans were elevated about 100-fold, 10-fold and 3-fold in liver, spleen and brain grey matter, respectively. The gangliosides, as measured by their sialic acid content, were much less affected. In liver, spleen, brain grey and white matter respectively 2.90-, 3.98-, 1.16- and 2.16fold values were obtained. The glycoproteinic sugars were hardly influenced, except for sialic acid from liver and brain white matter (1.87- and 1.92-fold respectively). The lipid content of brain white matter was about half the normal value. Both for grey and white matter the ganglioside pattern was abnormal. In both cases G₁ and G₂ were diminished. In grey matter G₃a, G₄, G_s5 and G₆ were elevated. In white matter G₃ and G₃a. β-Galactosidase activity was very low in liver (4.7%) and spleen (11.0%) and moderately affected in brain grey (41%) and white (24%) matter.β-Galactosaminidase, β-glucosaminidase and β-glucuronidase activity were enhanced, especially in liver.     

Molecular and Clinical Aspects of Inherited Cardiomyopathies

Hypertrophic cardiomyopathy (HCM) is phenotypically and genotypically a heterogeneous disease. Since 1989, four chromosomal loci have been identified for HCM and the genes residing on three of these have been identified as β-myosin heavy chain (β-MHC), cardiac troponin-T and α-tropomyosin. These genes code for sarcomere proteins and exhibit the same phenotype, suggesting that HCM is a disease of the sarcomere. Over 40 missense mutations and one deletion of the β-MHC gene have been identified. Similarly, missense mutations in the α-tropomyosin gene and the cardiac troponin-T gene have been identified. From genetic studies, including de novo mutations, it is established that these mutations are indeed responsible for HCM. The molecular basis of the pathogenesis of the cardiac hypertrophy appears to be a compensatory response to the primary defect. In addition to providing a definitive presymptomatic diagnosis, studies correlating β-MHC mutations with clinical prognosis suggest they have significant predictive value and can be helpful in genetic counselling and medical management.

Dilated cardiomiopathies (DCM), the most common form of cardiomyopathies, have an estimated prevalence of nearly 40 per 100000 individuals, and are the most common cause for cardiac transplantation in the United States. Familial dilated cardiomyopathy is thought to account for approximately 20% of the so-called cases of idiopathic DCM.     

Effects of β-glucan pretreatment on acetylsalicylic acid-induced gastric damage: An experimental study in rats

Background: NSAIDs have been found to induce gastrointestinal tract damage. Recently, it has been suggested that this might be mediated by lipid peroxidation.Objective: The aim of this study was to assess the potential protective effects of β-glucan against acetylsalicylic acid (ASA-induced gastric damage by means of its antioxidant capacity in an experimental rat model.Methods: Thirty-two male Wistar albino rats (200–250 g) were randomized into 4 groups consisting of 8 rats each. The β-glucan group received 50 mg/kg β-glucan once a day for 10 days and 30 minutes before anesthesia. The ASA group received saline once a day for 10 days and 300 mg/kg (20 mg/mL) ASA as a single dose, 4 hours before anesthesia. The ASA+β-glucan group was administered 50 mg/kg β-glucan once a day for 10 days and 30 minutes before anesthesia. Additionally, 300 mg/kg (20 mg/mL) ASA was administered as a single dose, 4 hours before anesthesia. The control group received saline once a day for 10 days and 30 minutes before anesthesia. All medications were administered by intragastric gavage. The stomach from each rat was dissected and divided into 2 parts for histologic and biochemical analysis. Gastric tissue malondialdehyde (MDA), nitric oxide (NO) levels, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were determined for oxidative parameter analysis.Results: The gastroprotective and antioxidant effects of β-glucan appeared to attenuate the ASA-induced gastric tissue damage. Compared with the control group, MDA and NO levels and CAT and GSH-Px activities were significantly increased in the stomachs of ASA-treated rats (MDA, 4.12 [0.44] to 13.41 [1.05] μmol/L; NO, 8.04 [7.25–9.10] vs 30.35 [22.34–37.95] μmol/g protein; CAT, 0.050 [0.004] to 0.083 [0.003] k/g protein; GSH-Px, 0.57 [0.42–0.66] to 1.55 [1.19–1.76] U/L; all, P < 0.001), whereas SOD activity was significantly decreased in the same group (291 [29] to 124 [6] U/mL; P < 0.001). In the ASA+β-glucan group, MDA and NO levels and CAT and GSH-Px activities were found to be significantly lower, while SOD activity was found to be significantly higher, in comparison with the ASA-treated group (all, P < 0.001).Conclusion: β-Glucan appeared to attenuate the gastric damage caused by ASA in these rats.     

TGF—β1诱导肾小管上皮细胞转分化过程中对细胞骨架的影响

目的观察TGF-β1在诱导肾小管上皮细胞转分化过程中对细胞骨架的调节作用。方法选择正常大鼠肾小管上皮细胞株NRK52E,经TGF-β1（15μg／L）体外诱导3天,RT-PCR检测细胞廿平滑肌动蛋白（α-SMA）评价细胞的转分化状态,观察细胞形态,同时检测细胞骨架成分β-actin、α—tubulin mRNA的表达;免疫荧光染色观察上述细胞骨架蛋白在细胞内的排列。结果培养的NRK52E细胞经TGF-81体外诱导3天后,细胞中α—SMAmRNA的表达水平明显增多,高于相应的对照组细胞（P〈0．001）,说明NRK52E上皮细胞出现了表型转化,此时培养细胞的形态也发生了改变,由典型的鹅卵石样变为成纤维细胞样,并有多个突起;细胞骨架蛋白β-actinmRNA的表达也明显增多（P〈0．05）;β—actin蛋白的排列也发生了改变,其在胞膜下形成的厚的板样结构消失,转而在胞浆中形成粗大的束状应力纤维,沿细胞长轴纵行排列,此外,β-actin尚在某些细胞边缘形成了片层样伪足和丝状伪足。微管成分α-tubulinmRNA的表达和分布与对照组比较无明显不同。结论TGF-β1能够诱导体外培养的NRK52E细胞转分化,在此过程中细胞形态发生了改变,骨架蛋白β-actin的表达增多并发生了重建,同时骨架蛋白α—tubulin未见明显变化。     

尿α1微球蛋白和β2微球蛋白对2-4期慢性肾病患者的诊断价值

目的：探讨尿α1、β2微球蛋白在2～4期慢性肾病患者中诊断价值。方法解放军总医院2011年12月至2013年5月肾内科的134例住院患者和50例健康体检者的尿α1、β2微球蛋白和其他肾功能指标进行检测分析,采用MDRD公式进行肾小球滤过率计算。结果慢性肾病组尿α1、β2微球蛋白水平明显高于健康对照组,差异有统计学意义（P＜0．01）。2～4期慢性肾病患者中尿α1、β2微球蛋白结果呈明显升高趋势,其差异有统计学意义（P＜0．05）。尿α1、β2微球蛋白在3a和3b期中结果有明显差异,3a期尿α1微球蛋白（20．00±18．9）mg/L、3b期（40．5±28．5）mg/L ;3a期β2微球蛋白（4．1±4．0）mg/L、3b期（10．0±8．7）mg/L。相关分析显示,尿α1、β2微球蛋白与肌酐呈显著正相关,与eGFR呈显著负相关。结论尿α1、β2微球蛋白是用于评价慢性肾脏疾病肾功能较好的实验室指标。     

An improved procedure for diagnosis of Gaucher disease using cultured skin fibroblasts and the chromogenic substrate, 2-hexadecanoylamino-4-nitrophenyl-β-d-glucopyranoside

A procedure has been developed for the determination of glucocerebrosidase activity using the substrate analogue, $\text{2-N-}\text{hexadecanoylamino}\text{-4-}\text{nitrophenyl}\text{-β-}\text{d}\text{-glucopyranoside}$ (HNGlu) with sodium taurocholate and oleic acid as activators. Cultured skin fibroblasts and amniotic fluid cells have been used as the enzyme source. It has been used successfully to confirm the diagnosis of two Type I and two Type II Gaucher patients.The procedure shows approximately a 15-fold increase in sensitivity over other procedures using HNGlu as substrate. Compared with 4-methylumbelliferyl-β-d-glucoside, HNGlu proves to be a highly specific substrate for glucocerebrosidase with little or no hydrolysis by the other β-glucosidases present in fibroblast extracts. It is therefore the chromogenic substrate of choice for determining a glucocerebrosidase deficiency.     

Lysosomal enzyme variations in thirteen cell strains cultured from one amniotic fluid

Fifteen primary amniotic fluid cultures were established from a single sample of amniotic fluid. Three different methods were used to set up these cultures which yielded 13 cell strains. Nine lysosomal enzymes (acid phosphatase, β-glucuronidase, β-galactosidase, α-galactosidase, α-glucosidase, α-mannosidase, α-arabinosidase, $\text{N-}\text{acetyl}\text{-ß-}\text{d}\text{-}\text{glucosaminidase}$ and arylsulphatase A) were assayed in these 13 cell strains. The coefficients of variation of these enzyme levels were less than the coefficients for enzyme levels in cell strains grown from different samples of amniotic fluid but greater than those for the combined culture and assay system used. No assay values were found which could have suggested a possible enzyme deficiency disease.     

Piperacillin–tazobactam: a β-lactam/β-lactamase inhibitor combination

Piperacillin–tazobactam is a β-lactam/β-lactamase inhibitor combination with a broad spectrum of antibacterial activity that includes Gram-positive and -negative aerobic and anaerobic bacteria. Piperacillin–tazobactam retains its in vitro activity against broad-spectrum β-lactamase-producing and some extended-spectrum β-lactamase-producing Enterobacteriaceae, but not against isolates of Gram-negative bacilli harboring AmpC β-lactamases. Piperacillin–tazobactam has recently been reformulated to include ethylenediaminetetraacetic acid and sodium citrate; this new formulation has been shown to be compatible in vitro with the two aminoglycosides, gentamicin and amikacin, allowing for simultaneous Y-site infusion, but not with tobramycin. Multicenter, randomized, double-blinded clinical trials have demonstrated piperacillin–tazobactam to be as clinically effective as relevant comparator antibiotics. Clinical trials have demonstrated piperacillin–tazobactam to be effective for the treatment of patients with intra-abdominal infections, skin and soft tissue infections, lower respiratory tract infections, complicated urinary tract infections, gynecological infections and more recently, febrile neutropenia. Piperacillin–tazobactam has an excellent safety and tolerability profile and continues to be a reliable option for the empiric treatment of moderate-to-severe infections in hospitalized patients.