Portable NIR spectroscopy: the route to green analytical chemistry.

There is a growing interest for cost-effective and nondestructive analytical techniques in both research and application fields. The growing approach by near-infrared spectroscopy (NIRs) pushes to develop handheld devices devoted to be easily applied for in situ determinations. Consequently, portable NIR spectrometers actually result definitively recognized as powerful instruments, able to perform nondestructive, online, or in situ analyses, and useful tools characterized by increasingly smaller size, lower cost, higher robustness, easy-to-use by operator, portable and with ergonomic profile. Chemometrics play a fundamental role to obtain useful and meaningful results from NIR spectra. In this review, portable NIRs applications, published in the period 2019-2022, have been selected to indicate starting references. These publications have been chosen among the many examples of the most recent applications to demonstrate the potential of this analytical approach which, not having the need for extraction processes or any other pre-treatment of the sample under examination, can be considered the "true green analytical chemistry" which allows the analysis where the sample to be characterized is located. In the case of industrial processes or plant or animal samples, it is even possible to follow the variation or evolution of fundamental parameters over time. Publications of specific applications in this field continuously appear in the literature, often in unfamiliar journal or in dedicated special issues. This review aims to give starting references, sometimes not easy to be found.

Local gentamicin-collagen sponge reduces cardiovascular implantable electronic device infections and pocket hematoma.

OBJECTIVE: Implantation or replacement of a cardiovascular implantable electronic device (CIED) may be associated with complications, such as pocket hematomas and infections. This study aims to determine whether a lyophilized gentamycin-containing collagen implant (GCCI) reduces major CIED infections and pocket hematomas after implant. SUBJECTS AND METHODS: A retrospective study was conducted among patients who underwent implantation or replacement of CIED at the Tor Vergata Polyclinic (Rome, Italy) between June 2007 and November 2019. The primary combined endpoint was infection and hematoma occurrence through 12 months of follow-up post-procedure. The rate of single infectious complications, pocket hematomas or both were also assessed. RESULTS: We compared 475 patients treated with the GCCI (GCCI group) with 714 patients who did not receive it (control group). Complications occurred in 127 patients (11%); a statistically significant reduction of infections and pocket hematomas in the GCCI group was reported when compared with control patients (1% vs. 17%; p<0.0001). A total of 20 (2%) infectious events were reported, 102 (8%) patients developed a pocket hematoma, and 5 (0.4%) had both. The rate of single complications was significantly lower in GCCI group: infection 0.2% vs. 2.6% (p=0.002), pocket hematoma 0.6% vs. 13.8% (p<0.001). The association between antiplatelet/anticoagulation therapy and hematoma development was not statistically significant. CONCLUSIONS: The GCCI is a medical device that can be used in addition to local hemostasis and prophylactic doses of systemic antibiotics with the aim of reducing infective complications and pocket hematoma after permanent CIED implantation or replacement.

The impact of the COVID-19 pandemic on healthcare-associated infections in intensive care unit patients: a retrospective cohort study.

BACKGROUND: During the intensive care units' (ICUs) reorganization that was forced by the COVID-19 emergency, attention to traditional infection control measures may have been reduced. Nevertheless, evidence on the effect of the COVID-19 pandemic on healthcare-associated infections (HAIs) is still limited and mixed. In this study, we estimated the pandemic impact on HAI incidence and investigated the HAI type occurring in COVID-19 patients. METHODS: Patients admitted to the main ICU of the Umberto I teaching hospital of Rome from March 1st and April 4th 2020 were compared with patients hospitalized in 2019. We assessed the association of risk factors and time-to-first event through multivariable Fine and Grey's regression models, that consider the competitive risk of death on the development of HAI (Model 1) or device related-HAI (dr-HAI, Model 2) and provide estimates of the sub-distribution hazard ratio (SHR) and its associated confidence interval (CI). A subgroup analysis was performed on the 2020 cohort. RESULTS: Data from 104 patients were retrieved. Overall, 59 HAIs were recorded, 32 of which occurred in the COVID-19 group. Patients admitted in 2020 were found to be positively associated with both HAI and dr-HAI onset (SHR: 2.66, 95% CI 1.31-5.38, and SHR: 10.0, 95% CI 1.84-54.41, respectively). Despite being not confirmed at the multivariable analysis, a greater proportion of dr-HAIs seemed to occur in COVID-19 patients, especially ventilator-associated pneumonia, and catheter-related urinary tract infections. CONCLUSIONS: We observed an increase in the incidence of patients with HAIs, especially dr-HAIs, mainly sustained by COVID-19 patients. A greater susceptibility of these patients to device-related infections was hypothesized, but further studies are needed.

Patient-centred dietetic care from the perspectives of older malnourished patients.

BACKGROUND: Governing organisations for health services currently recommend a patient-centred (PC) approach to practice for all health professions, including dietetics. For the vulnerable older malnourished patient, this approach needs to be prioritised to improve outcomes. The paucity of patient experience data likely limits evidence-based, patient-centred care (PCC) from being implemented effectively. The present study aimed to identify quality indicators of dietetic services from the perspectives of older malnourished patients to inform evidence-based PC dietetic care. METHODS: Surveys were completed by a sample of 28 females and 28 males (mean age 81 years) who had been seen by a dietitian for malnutrition assessment. In-depth, face-to-face, semi-structured interviews were undertaken with a sub-sample of four females and six males (mean age 81 years). Interviews were transcribed verbatim. Thematic analysis of transcripts and open-ended survey responses was conducted to determine patient-identified quality indicators. RESULTS: Three structure indicators (continuity of care through regular contact and post-discharge dietetic follow-up; interdisciplinary coordination and collaboration; and high-quality hospital food services), five process indicators (addressing a patient's primary medical concern; involving the patient's family; providing clear and simple dietetic information; providing expert dietary knowledge; utilising interpersonal communication skills) and three outcome indicators (improvement in health status; improvement or maintenance of independence; weight gain) were identified. The experiences of older malnourished patients with dietetic services, as described in the present study, reinforce the importance of ensuring high-quality and tailored dietetic care as a key element of PC dietetic services. CONCLUSIONS: The quality indicators of dietetic services identified in the present study may facilitate dietitians to provide evidence-based PCC for older malnourished patients.

Poor nutritional status of older subacute patients predicts clinical outcomes and mortality at 18 months of follow-up.

BACKGROUND/OBJECTIVES: Older malnourished patients experience increased surgical complications and greater morbidity compared with their well-nourished counterparts. This study aimed to assess whether nutritional status at hospital admission predicted clinical outcomes at 18 months follow-up. SUBJECTS/METHODS: A retrospective analysis of N=2076 patient admissions (65+ years) from two subacute hospitals, New South Wales, Australia. Analysis of outcomes at 18 months, according to nutritional status at index admission, was performed in a subsample of n = 476. Nutritional status was determined within 72 h of admission using the Mini Nutritional Assessment (MNA). Outcomes, obtained from electronic patient records, included hospital readmission rate, total Length of Stay (LOS), change in level of care at discharge and mortality. Survival analysis, using a Cox proportional hazards model, included age, sex, Major Disease Classification, mobility and LOS at index admission as covariates. RESULTS: At baseline, 30% of patients were malnourished and 53% were at risk of malnutrition. LOS was higher in malnourished and at risk, compared with well-nourished patients (median (interquartile range): 34 (21, 58); 26 (15, 41); 20 (14, 26) days, respectively; P<0.001). Hazard rate for death in the malnourished group is 3.41 (95% confidence interval: 1.07-10.87; P = 0.038) times the well-nourished group. Discharge to a higher level of residential care was 33.1%, 16.9% and 4.9% for malnourished, at-risk and well-nourished patients, respectively; P ≤ 0.001). CONCLUSION: Malnutrition in elderly subacute patients predicts adverse clinical outcomes and identifies a need to target this population for nutritional intervention following hospital discharge.

Attachment and emotional understanding: a study on late-adopted pre-schoolers and their parents.

OBJECTIVE: The aim of this study was to characterize the role of attachment in adoption, first by assessing the influence of adoptive parents on their late-adopted children and second by investigating the role of children's attachment on an emotional understanding task. DESIGN: On children's arrival into adoptive families, parents' attachment was evaluated. After 12-18 months, children's attachment towards mothers and fathers was assessed. Twelve months later, children participated in an emotional understanding task. METHOD: Parents' attachment was assessed using the Adult Attachment Interview. Children's attachment and emotional understanding were evaluated respectively using the Manchester Child Attachment Story Task and the Test of Emotion Comprehension. RESULTS: A correspondence of 80% (security vs. insecurity) and 60% (security vs. avoidant or ambivalent insecurity K= 0.40) between mothers' and children's pattern of attachment was found. A secure state of mind in both adoptive parents was a protective factor towards children's attachment disorganization. Finally, there was a significant association between children's security of attachment and their performance on the emotional understanding task. CONCLUSION: Adoption appears to be an intervention that assures the adoptive child an opportunity to catch up on emotional development and to partially resolve prior traumatic attachment experiences; adoptive parents play a central role in the emotional adjustment of their children.

Older rehabilitation patients are at high risk of malnutrition: evidence from a large Australian database.

BACKGROUND/OBJECTIVES: Routine nutrition screening is recommended for all older patients admitted to hospital however data on the prevalence of malnutrition in rehabilitation settings is sparse. This study assessed the nutritional status of older patients admitted to rehabilitation hospitals over a 5 year period and described the association between nutritional status and length of hospital stay (LOS) in this context. The usefulness of a recently revised version of the shortened MNA (MNA-SF) was also investigated. METHODS: A retrospective analysis was conducted of patients aged 65 + y admitted to two rehabilitation hospitals in New South Wales, Australia between 1st March 2003-30th June 2004, and 11th January 2005-10th December 2008. Nutritional status was determined on admission by trained dietitians using the full MNA instrument and the MNA-SF. Information on diagnosis-related grouping and length of stay (LOS) was obtained. RESULTS: Data was available for 2076 patients with a mean age of 80.6 (27.7) y. Thirty-three percent and 51.5% of patients were classified as malnourished and at nutritional risk, respectively. Controlling for date of admission and diagnosis related grouping, LOS was higher in malnourished and at risk groups compared to their well nourished peers (P < 0.001) by 18.5 and 12.4 days, respectively. MNA-SF demonstrated high sensitivity but relatively low specificity against the full MNA. CONCLUSION: The majority of older patients in the rehabilitation setting are nutritionally compromised which adversely influences LOS. In order to encourage more widespread screening, the MNA-SF may be able to replace the full MNA.

Low glucose blood levels are associated with abnormal cardiac sympatho-vagal balance in type 2 diabetic patients with coronary artery disease.

BACKGROUND AND OBJECTIVES: Glycemic control has been suggested to improve prognosis in diabetic patients, but recent trials failed to show benefits from intensive glycemic control. Hypoglycaemic episodes or large variability in glucose blood levels causing a sympatho-vagal imbalance of cardiac autonomic function (CAF) might play a role in this result. In our study we assessed whether blood glucose fluctuation may be related to variations in CAF during daily life in diabetic patients with coronary artery disease (CAD). MATERIALS AND METHODS: Twelve patients with type 2 diabetes mellitus with CAD (65+/-4 years, 2 women) underwent simultaneous 48-hour ECG Holter monitoring and continuous interstitial glucose measurements. The highest and lowest glucose levels for each 3-hour segments of the day were identified and heart rate variability (HRV) parameters were measured on Holter recordings on 5-minute intervals centred on these times. RESULTS: Overall, 294 glucose levels were available for analysis. In the whole population several HRV indices were significantly lower in correspondence of the lowest glucose blood levels and this difference was much more evident in patients who were not taking beta-blockers, than in patients who were taking beta-blockers. A significant, although mild, correlation was found between glucose blood levels and several time-and frequency domain HRV variables in patients not taking beta-blockers, but not in these on beta-blockers therapy. DISCUSSION: Our data suggest that, in type 2 diabetic patients with CAD, hypoglycaemic episodes are associated with depressed HRV and that beta-blocking agents are able to contrast this relation. These interesting results merit to be investigated in a larger population of patients.

Cardiac adrenergic nerve function and microvascular dysfunction in patients with cardiac syndrome X.

OBJECTIVE: To assess whether abnormalities in cardiac uptake of (123)I-metaiodobenzylguanidine (MIBG) correlate with coronary microvascular dysfunction in patients with cardiac syndrome X (CSX). SETTING: University hospital. PATIENTS: 29 patients (aged 59 (SD 7) years, 11 men) with typical CSX and a matched group of 20 healthy subjects (aged 56 (7) years, 8 men) were studied. INTERVENTIONS: Planar and single photon emission computed tomography (SPECT) MIBG myocardial scintigraphy was performed in all subjects. Coronary flow response (CFR) to adenosine and to cold pressor test (CPT) in the left anterior descending (LAD) coronary artery was assessed in all CSX patients and in 12 controls by transthoracic Doppler echocardiography. MAIN OUTCOME MEASURES: Abnormalities in cardiac MIBG scintigraphy were observed in 25 CSX patients (86.2%), but in no healthy control (p<0.001). Compared to controls, CSX patients showed a lower heart/mediastinum (H/M) ratio of MIBG uptake (1.69 (0.24) vs 2.2 (0.3), p<0.001) and a higher cardiac MIBG defect score (25 (22) vs 4 (2), p = 0.002). Both CFR to adenosine (3.31 (1.1) vs 1.94 (0.6), p<0.001) and CFR to CPT (2.35 (0.5) vs 1.63 (0.4), p<0.001) were lower in CSX patients than in controls. In CSX patients, however, no correlation was found between MIBG H/M ratio and CFR to adenosine (r = 0.17; p = 0.38) and to CPT (r = -0.28; p = 0.13), as well as between MIBG uptake score in the LAD territory and CFR to adenosine (r = 0.14; p = 0.47) and to CPT (r = 0.06; p = 0.73). CONCLUSION: Our data show striking abnormalities in cardiac adrenergic nerve function and in coronary microvascular function in CSX patients. However, no significant relation between the two abnormalities was found. Further studies are needed to clarify the mechanisms and the role of MIBG defects in CSX patients.

[Emergence of Salmonella Typhimurium monophasic serovar: determinants of antimicrobial resistance in porcine and human strains]. / Emergenza di una variante monofasica di Salmonella Typhi- murium: caratteri di antibiotico-resistenza in stipiti isolati dalla filiera suinicola e da casi di infezione nell'uomo.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common cause of non-typhoideal salmonellosis in humans and animals; since the end of '90 a monophasic serovar defined by the antigenic formula 4,5:i:- has been emerged. This study shows occurrence of monophasic serovar in Italy (Lombardia Region) and analyses antimicrobial resistance of 147 S. Typhimurium and 45 monophasic strains. Multiple drug resistance (MDR) has been detected in 94,5% and 76,6% of 55 porcine and 137 human isolates, respectively; tetra-resistance pattern AmSSuTe is the most common among in both sources (55 out of 116 strains of MDR S. Typhimurium and 16 out of 41 strains of MDR monophasic variant). Transfer of resistance determinants has been investigated by plasmid conjugation for all the 157MDR strains: only 16 isolates (10,2%) gave positive results, and transfer has almost been partial. Data from our investigation and data reported by other studies show a wide difference in the circulating phenotypes and in polymorphism of S. Typhimurium, confirmed also by the emergence of the monophasic serovar.

Conservative and radical oncoplastic approches in the surgical treatment of breast cancer.

In the attempt to optimise the balance between the risk of local recurrence and the cosmetic outcomes in breast surgery, new surgical procedures, so-called oncoplastic techniques, have been introduced in recent years. The term oncoplastic surgery refers to surgery on the basis of oncological principles during which the techniques of plastic surgery are used, mostly for reconstructive and cosmetic reasons. The advantage of the oncoplastic surgery for breast cancer is the possibility of performing a wider excision of the tumour with a good cosmetic result. Oncoplastic surgery is a broad concept that can be used for several different combinations of oncological surgery and plastic surgery: excision of the tumour by reduction mammoplasty, tumour excision followed by remodelling mammoplasty, mastectomy with immediate reconstruction of the breast and partial mastectomy with reconstruction. Careful patient selection and preoperative planning are key components for the success of any oncoplastic operation for breast cancer. Accurate preoperative evaluation of the clinical and biological features of the tumour as well as of the morphological aspects of the breast allow the surgeon to make a decision if a conservative or radical approach is preferable and select the most effective oncoplastic surgical technique. In this review we summarise the indications, advantages and limitations of several oncoplastic procedures.

Validation of an in vitro screen for phospholipidosis using a high-content biology platform.

Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to 100 micro mol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, non-phospholipidogenic controls were correctly called.

Assessing the older person: is the MNA a more appropriate nutritional assessment tool than the SGA?

OBJECTIVES: A study was undertaken to determine which nutritional assessment tool would be better in assessing changes in nutritional status over time in hospitalised older patients. The two tools used were the Mini Nutritional Assessment (MNA) and the Subjective Global Assessment (SGA). DESIGN: Single blind, prospective study conducted over a 60-day period. SETTING: Five regional hospitals within Southern New South Wales, Australia. SUBJECTS: There were 43 patients at the commencement of the study, then 28 patients at day 30 and 20 patients at day 60. METHODS: All patients over 65 years of age admitted to the five hospitals during May 2001 were eligible. The only exclusions were those patients admitted to palliative care or with severe dementia. Two dietitians saw each patient. Each dietitian assessed the patient using his or her assigned nutritional assessment tool either the SGA or the MNA. All dietitians were randomly assigned the tool at the commencement of the study. They were familiarised with the use of the tool by participating in a workshop prior to the start of the study. RESULT: The MNA was able to detect greater numbers of malnourished subjects when compared to the SGA. This finding was consistent across Day 0, 30 and 60 and statistically significant (p<0.05) at all time periods. CONCLUSIONS: This study has shown the MNA to be a more appropriate nutrition assessment tool for older patients when compared to the SGA. The MNA is better able to identify severely malnourished patients. This study illustrated the potential use of the MNA as an ongoing monitor of nutritional status and hence a measure of the impact of nutrition intervention.

Implementation and pharmacoeconomic analysis of a clinical staff pharmacist practice model.

The implementation and pharmacoeconomic analysis of a clinical staff pharmacist (CSP) practice model are described. Staff pharmacists at a large, tertiary care, academic medical center were selected and trained to perform clinical pharmacy services under the direction of clinical pharmacy specialist mentors. Clinical interventions by these CSP practitioners were evaluated in terms of direct cost savings (the difference in actual acquisition costs between therapies) and cost avoidance (the dollar value of adverse drug events [ADEs] avoided). The CSPs performed a total of 4959 interventions during a 12-month period. The interventions provided direct cost savings of $92,076 and an estimated cost avoidance of $488,436. Comparing cost savings and cost avoidance with the expenses of providing these services indicated a net economic benefit of $392,660. A new model of pharmacy practice that integrates staff pharmacists into existing clinical practice has the potential to minimize the risks, decrease the costs, and improve the outcomes associated with drug therapy.

Effects of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish.

The zebrafish Danio rerio is a new model system for studying the genetics of hematopoiesis. To define naturally occurring viruses which could infect and replicate within hematopoietic precursors of the zebrafish, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) were studied. Infection of whole fish with viral supernatants demonstrated infectious replicants for both viruses, indicating that the virus host range includes the zebrafish. In other species, infection with these viruses leads to prominent hematopoietic necrosis of the head kidney, the major site of adult hematopoiesis. We detected a transient toxicity of the virus to hematopoietic precursors and terminally differentiated red cells after viral infections. The kinetics of hematopoietic defects between IHNV and IPNV infection differed; fish infected with either virus, however, recovered by 6 days postinfection. In contrast to other fish infected with the virus, hematocrit did not change appreciably during this time. These studies are the first to demonstrate IHNV and IPNV infection of the zebrafish and reveal the potential for use of such viruses for gene transfer experiments to infect zebrafish hematopoietic cells.

Distinct biochemical and topological properties of the 31- and 27-kilodalton plasma membrane intrinsic protein subgroups from red beet.

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

Amplification of the ATP-binding cassette 2 transporter gene is functionally linked with enhanced efflux of estramustine in ovarian carcinoma cells.

An estramustine-resistant human ovarian carcinoma cell line, SKEM, was generated to explore resistance mechanisms associated with this agent. Cytogenetic analysis revealed that SKEM cells have a homogeneously staining region (hsr) at chromosome 9q34. Microdissection of the hsr, followed by fluorescence in situ hybridization to SKEM and normal metaphase spreads, confirmed that the amplified region was derived from sequences from 9q34. In situ hybridization with a probe specific for ABC2, a gene located at 9q34 that encodes an ATP-binding cassette 2 (ABC2) transporter, indicated that this gene is amplified approximately 6-fold in the estramustine-resistant cells. Southern analysis confirmed that ABC2 was amplified in SKEM, and Northern analysis indicated that the ABC2 transcript was overexpressed approximately 5-fold. The ABC1 gene located at 9q22-31 was not amplified in the resistant cells, and mRNA levels of several other ABC transporter genes were unaltered. Consistent with the concept that increased ABC2 expression contributes to the resistant phenotype, we observed that the rate of efflux of dansylated estramustine was increased in SKEM compared with control cells. In addition, antisense treatment directed toward ABC2 mRNA sensitized the resistant cells to estramustine. Together, these results suggest that amplification and overexpression of ABC2 contributes to estramustine resistance and provides the first indication of a potential cellular function for this product.

Mercury-induced conformational changes and identification of conserved surface loops in plasma membrane aquaporins from higher plants. Topology of PMIP31 from Beta vulgaris L.

Aquaporins are integral membrane proteins occurring in mammals, plants, and microorganisms, which serve as channels that permit the bidirectional passage of water through cellular membranes. Higher plants contain abundant levels of aquaporins in both the tonoplast and plasma membrane. Aquaporins contain six transmembrane segments with three surface loops located at the apoplastic face of the membrane and two loops at the cytosolic side. In this study, we probed the topology of plasma membrane aquaporins to determine the effects of divalent cations on aquaporin conformation, and to identify structural features that distinguish plasma membrane intrinsic proteins from tonoplast intrinsic proteins. Plasma membrane vesicles from storage tissue of Beta vulgaris L. were subjected to limited proteolysis, and proteolytic fragmentation patterns were detected using affinity-purified antibodies recognizing aquaporins of 31-kDa. In its native membrane-associated state, the 31-aquaporin band, PMIP31, was refractory to proteolysis by trypsin. However, mercuric compounds specifically induced a conformational change resulting in the exposure of a proteolytic cleavage site and formation of a unique 22-kDa proteolytic fragment (p22). N-terminal sequence analysis of p22 established its identity as an aquaporin-derived fragment. Topological studies using sealed right-side-out plasma membrane vesicles established that the proteolytic cleavage site is located at surface loop C, the second apoplastic loop, immediately preceding the sequence Gly-Gly-Gly-Ala-Asn. The Gly-Gly-Gly-Ala-Asn-X-X-X-X-Gly-Tyr motif of loop C and a 14 amino acid motif in apoplastic loop E, Thr-Gly-Ile/Thr-Asn-Pro-Ala-Arg-Ser-Leu/Phe-Gly-Ala-Ala-Ile/Val-Ile/ Val-Phe/Tyr-Asn are completely conserved in all known higher plant aquaporins of plasma membrane origin and are not present in any of the known tonoplast intrinsic proteins. These results demonstrate that the two highly conserved plasma membrane intrinsic protein surface loops are structural features that clearly distinguish plasma membrane from tonoplast aquaporins.

Glutathione-associated enzymes in the human cell lines of the National Cancer Institute Drug Screening Program.

The steady state expression of glutathione S-transferases (GSTs) at both the protein and mRNA level is reported for the 60 tumor cell lines that are used for the National Cancer Institute Drug Screening Program. Individual GST isozymes were separated, identified, and quantified (with reverse-phase calibration curves) through a novel high performance liquid chromatographic procedure. GSTP1 was the predominant isozyme and was found at quantifiable levels in all but two of the cell lines. This isozyme ranged from 0.03% to 2.7% of the total cytosolic protein. For the mu family, 90% of the lines had GSTM2, 68% had GSTM3, but only 28% were positive for the M1 phenotype. The M1 proportion is lower than would be expected from the standard M1 null phenotype for human populations. Isozymes of the alpha family were detected only at very low levels in 35% of the lines. Significant quantitative correlations among enzyme activity, total enzyme protein, and mRNA were shown for GSTP1. However, such relationships were not apparent for the mu or alpha families. Levels of glutathione (GSH), and the transcript levels of other enzymes involved in GSH homeostasis were determined. gamma-Glutamyl cysteine synthetase (gamma-GCS) was present in all cell lines, but did not correlate with levels of intracellular GSH. Glyoxalase-I and gamma-glutamyl transpeptidase, both involved in GSH salvage, were found in 100% and 70% of the cell lines, respectively. Using a pattern-matching computer program, COMPARE, we compared and correlated the arrays of mRNA and protein levels with the pattern of chemosensitivity or chemoresistance of the 60 cell lines with 175 agents constituting a standard agent database. This database is composed of compounds to which a putative mechanism of action has been assigned. Although Pearson correlation coefficients relating the target and drug patterns were generally modest, when the patterns for the enzyme protein and mRNA levels for GST pi were correlated to drug sensitivity patterns, the list of 30 agents most closely matching (for which P < 0.05) was enriched with alkylating agents. gamma-GCS also showed an enrichment of alkylating agents in the COMPARE correlations, indicating that high levels of gamma-GCS may be an important determinant of resistance. In contrast, none of the other enzymes or GSH had patterns of expression that resulted in an obvious correlation to the sensitivity or resistance of alkylating agents.