Common mechanism of toxicity: a case study of organophosphorus pesticides

Despite the fact that glutamine is not considered to be an essential amino acid, it is the amino acid found in the greatest concentration both in plasma (26%) as in skeletal muscle (75%). These levels may decrease in post-operative, trauma, or critical patients. Glutamine performs many functions in which its demand may be increased, such as: it is a precursor of the synthesis of nucleotides; it is an activator of the protein synthesis and at the same time it inhibits the degradation; it is an activator of glycogen synthesis; it is a metabolic substrate for rapidly replicating cells; it is an energy source for the enterocyte which is so important for maintaining the integrity and the function of the intestinal barrier, and the consumption thereof may be increased under conditions of stress. The administration of glutamine intravenously leads to two physical-chemical problems; the first is its low solubility in water; at 20 degrees C this is only 36 g/l, and the second problem is its low chemical stability in an aqueous solution at 22-24 degrees C, this being 11 days. This problem has led the industry to research two dipeptides of glutamine; L-alanyl-glutamine, and L-glycyl L-glutamine, both of which are much more soluble and much more stable. At present there is still a controversy regarding the dosage of glutamine and its dipeptides, with the dose being 0.19-0.29 g/kg/day of L-glutamine or its dipeptide forms, in surgical post-operative periods or to prevent bacterial translocation, and in patients who are candidates for bone marrow transplants, the administered dose has been 0.37-0.57 g/kg/day. The purpose of this study is to review the existing bibliography regarding the efficacy of L-glutamine or its dipeptides in four possible indications for its application in the daily clinical practice, such as: a) In post-operative surgical patients of major or medium surgery, glutamine or its dipeptides reduces the losses of muscular glutamine and its catabolism, showing a less negative nitrogen balance. b) Whether it avoids bacterial translocation. c) Whether it favors the response of the immunological system. d) Whether in patients who are candidates for bone marrow transplants this decreases the side effects due to chemotherapy and radiotherapy such as mucositis, or whether it decreases the number of days of neutrophil recovery. At present, on the European market there are two commercially available brands of glutamine dipeptides: Dipeptiven, by Fresenius Laboratories, Germany. A 100 ml vial which corresponds to 20 g of L-alanyl L-glutamine (8.2 g of alanine + 13.46 g of L-glutamine). This is added to the standard amino acid solution. Glamin, Pharmacia and Upjohn Laboratory, Sweden. This is an amino acid solution with 13.4% essential and non-essential amino acids which are equivalent to 22.4 g of nitrogen/l, and which contain 30.27 g L-glycyl-L-glutamine (10.27 g of glycine + 20 g of L-glutamine).     

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines

BACKGROUND: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. METHODS: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. RESULTS: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r = 0.87, p < 0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r = 0.68, p < 0.05) only in fetal bovine serum in the absence of galactose. CONCLUSION: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.     

Threonine and methionine are limiting amino acids for protein synthesis in patients with AIDS

This study was conducted to identify the most rate-limiting amino acids for whole-body protein synthesis in acquired immunodeficiency syndrome (AIDS) patients. We postulated that an essential amino acid that would be rate limiting in AIDS should have a low basal plasma concentration and should remain at a low level during amino acid infusion. Seven male AIDS patients (median age 37 y, CD4 cell count: 76 mm-3) without any clinically active opportunistic infection during the month before the experiment were infused intravenously with a complete amino acid-glucose mixture for 2.5 h. Eight healthy volunteers were used as controls. Before the infusion, the concentrations of most free essential amino acids (methionine, threonine, histidine, isoleucine, leucine and tryptophan) were significantly lower (P < 0.05) in AIDS patients than in controls. Most plasma free essential amino acids increased significantly during infusion. However, the absolute increase above basal levels for threonine, valine, lysine, (P < 0.05) and methionine (P < 0.073) was smaller in AIDS patients than in control subjects. Thus, threonine and possibly methionine may be rate limiting for whole-body protein synthesis in AIDS patients, suggesting that there are selective amino acid requirements in patients with AIDS.     

Nutritional control of amino acid supply to the mammary gland during lactation in the pig

The paucity of data relating to lactation physiology of the sow has frustrated researchers in estimating nutrient needs for production and mammary maintenance functions. The nutritional control of amino acid supply for milk synthesis is influenced by factors that have yet to be measured, such as blood flow and amino acid contribution from the body protein pool. The interaction or role of hormones such as insulin, glucagon or prolactin in amino acid dynamics and inter-organ exchange during lactation in the sow, are not well understood. The discrepancy existing between milk and mammary amino acid uptake profiles relative to lysine may be indicative of mammary metabolism and possibly maintenance requirements for specific amino acids. Hence, amino acid metabolism in the mammary gland, regardless of arterial blood substrate supply, may play an important role in a factorial approach to determining requirements. Mammary amino acid uptake ratios rather than milk amino acid ratios should provide a better tool to estimate amino acid requirements relative to lysine. Although lysine has typically been limiting in maize-soyabean-meal-based diets fed to lactating sows, current production trends are bringing a new dimension to the formulation of lactating-sow diets. Other amino acids may become limiting if dietary crystalline lysine is added without concern for the whole essential amino acid profile. Formulations based on an ideal amino acid profile for the lactating sow will, therefore, become critical.     

Stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line in batch and continuous cultures

Batch and continuous cultures were carried out to study the stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line, with particular attention to the metabolism at low levels of glucose and glutamine. The apparent yields of cells on glucose and glutamine, lactate on glucose, and ammonium on glutamine were all found to change significantly at low residual concentrations of glucose (< 5 mmol/L) and glutamine (< 1 mmol/L). The uptake rates of glucose and glutamine were markedly reduced at low concentrations, leading to a more effective utilization of these nutrients for energy metabolism and biosynthesis and reduced formation rates of lactate and ammonium. However, the consumption of other amino acids, especially the essential amino acids leucine, isoleucine, and valine and the nonessential amino acids serine and glutamate, was strongly enhanced at low glutamine concentration. Quantitatively, it was shown that the cellular yields and rates associated with glucose metabolism were primarily determined by the residual glucose concentration, while those associated with glutamine metabolism depended mainly on the residual glutamine. Both experimental results and analysis of the kinetic data with models showed that the glucose metabolism of BHK cells is not affected by glutamine except for a slight influence under glucose limitation and glutaminolysis not by glucose, at least not significantly under the experimental conditions. Compared to hybridoma and other cultured animal cells, the recombinant BHK cell line showed remarkable differences in terms of nutrient sensitivity, stoichiometry, and amino acid metabolism at low levels of nutrients. These cell-line-specific stoichiometry and nutrient needs should be considered when designing an optimal medium and/or feeding strategy for achieving high cell density and high productivity of BHK cells. In this work, a cell density of 1.1 x 10(7) cells/mL was achieved in a conventional continuous culture by using a proper feed medium.     

Metabolic role of glutamine and its importance in nutritional therapy

The advances in the field of nutritional support have made certain nutrients very relevant, which, although they have been known for a long time, at present represent an important chapter in nutrition, entering into what is known as "nutritional pharmacology". Among these nutrients is glutamine, an amino acid classified as non-essential, but which in certain circumstances may become to be considered as an "essential nutrient". In the present review, a review is made of its metabolic role, synthesis and degradation, metabolic routes and functions under normal conditions as well as under critical conditions. It is known that glutamine stimulates the synthesis and inhibits the degradation of proteins, it is an important vehicle for the transport of nitrogen and carbon within the tissues, it stimulates the synthesis of hepatic glycogen, it is an energy source for cell division, for the growth of different cells of rapid replication, such as enterocytes, colonocytes, and fibroblasts, as well as for other cells of the immune system, such as lymphocytes and macrophages. Thus its role in the maintenance of structure, in metabolism and function of the intestinal mucosa, and in dysfunctions of the immune system. In parenteral nutrition, at present there are no preparations which include it, given the stability problems which it presents, although attempts have been made to resolve this, using different possibilities, such as di-peptides. However, in enteral nutrition, the diets tend to include it, although in a small proportion. Nevertheless, having recognized its beneficial role in a certain type of patients, at present there are diets which contain glutamine in higher doses, with the object of attempting to cover the increased demands of glutamine which shall arise in these situations. The inclusion of glutamine in nutritional therapy is supported by multiple studies which reflect the beneficial effect of this nutrient, both in enteral nutrition as in parenteral nutrition.     

Utilization of spent hen meal in diets for broiler chickens

Studies were conducted to evaluate spent hen meal (SHM) produced in commercial rendering plants as a nutrient source in diets for broiler chickens. Utilizing previously determined nutrient composition values, including digestible amino acid and TMEn content, diets were formulated to include 0, 5, 10, and 15% of SHM from three different locations. In the first experiment, conducted in battery pens from 1 to 21 d posthatch, diets were formulated with digestible amino acid requirements set at 90, 95, or 100% of NRC (1994) total amino acid requirements. In the second experiment, conducted in floor pens from 1 to 49 d posthatch, diets were formulated with digestible amino acid requirements set at 95% of NRC (1994) total amino acid requirements. Samples of birds from the second experiment were processed to determine the possible influence of SHM inclusion on carcass yield. Results of the present studies indicate that SHM from commercial rendering facilities can be utilized in diets for growing broiler chickens provided adjustments are made in nutrient content and digestibility. When formulated on the basis of digestible amino acid content, levels of SHM up to 10% appear acceptable based upon body weight, feed conversion, bone ash, and carcass yield. Higher inclusion rates generally reduced performance. It is apparent that differences in nutritional quality may exist among products produced by different rendering facilities; however, evaluation of products to assess nutrient quality may be difficult under commercial conditions. As more information is generated regarding typical amino acid content and digestibility of rendered SHM, the product may be used with greater confidence in commercial diets.     

In vitro mucosal digestion of synthetic gliadin-derived peptides in celiac disease

Two celiac-active synthetic peptides derived from the A-gliadin structure corresponding to residues 8-19 (LQPQNPSQQQPQ) and to 11-19 were digested in vitro with small intestinal mucosa from children with celiac disease in remission and from normal children. The products of digestion were separated into two fractions on the basis of M(r) < 400 and M(r) > 400 by gel permeation chromatography and subjected to amino acid analysis. After digestion of the dodecapeptide with celiac mucosa, 71 +/- 14% (molar) of the total digestion products remained in the M(r) > 400 fraction. Glutamine, proline, serine, and asparagine were the major amino acids present. Glutamine, proline, and leucine were the major amino acids in the M(r) < 400 fraction. The M(r) > 400 fraction from the celiac mucosal digestion of the nonapeptide was of similar composition to the corresponding fraction from the dodecapeptide and represented 78 +/- 15% of the total products. Digestion of the two peptides with normal mucosa gave lower amounts of products in the M(r) > 400 fraction, but they were of similar composition to the corresponding fractions from the celiac mucosal digestion. Peptides such as NPSQQP and QNPSQQQ may be present in the M(r) > 400 fractions since glutamine and proline are present in the approximate ratio of 2:1, respectively. The results indicate a defect in the mucosal digestion of peptides which are active in an animal model of celiac disease.     

RNA and protein requirements for eukaryotic selenoprotein synthesis

Selenium has been recognized as an essential nutrient in animals since the 1950s. Demonstration of the role of dietary selenium in protection from oxidative stress followed in the early 1970s, and was largely attributed to its presence as an integral part of cellular glutathione peroxidase. However, the functions of this enzyme did not explain many of the other effects of selenium deficiency. The identification of other mammalian selenoproteins during the last few years has provided new insights into the functions of this trace nutrient. The discovery that type 1 deiodinase (D1) is a selenoenzyme, in addition to unveiling an essential role for selenium in thyroid hormone action, has had more far-reaching implications. Studies of this protein opened the door for investigation of the requirements for eukaryotic selenoprotein synthesis, and the features that distinguish this pathway from the corresponding prokaryotic pathway. Selenium is present in a number of prokaryotic and eukaryotic proteins in the form of the unusual amino acid, selenocysteine. Incorporation of selenocysteine into these proteins requires a novel translation step in which UGA specifies selenocysteine insertion. Since UGA codons are typically recognized as translation stop signals, an intriguing question is raised: How does a cell recognize and distinguish a UGA selenocysteine codon from a UGA stop codon? In this review, we will focus on what is known about selenocysteine incorporation in eukaryotes, briefly summarizing initial studies and discussing a few recent advances in our understanding of this unique "recoding" process.     

Glutamine dipeptides in clinical nutrition

Glutamine is a conditional indispensable amino acid during stress. However, limited solubility and instability of glutamine prevent its addition to presently available nutritional preparations. To overcome these drawbacks, we propose the dipeptide concept by which stable and highly soluble synthetic glutamine containing dipeptides are used. The synthetic dipeptides fulfill all chemical/physical properties to be considered as parenteral substrates. Numerous experimental studies show rapid clearance of parenteral supplied glutamine containing dipeptides without accumulation in tissues; the loss via the urine being inconsequential. Differences related to the dipeptide structure are not observed. There is overwhelming evidence existent that a nutritional support with supplemental glutamine dipeptide positively influences nitrogen excretion, immune status, gut integrity, morbidity, rehabilitation and outcome. Consequently, omission of glutamine from conventional TPN and its subsequent administration should be considered as a replacement of a deficiency rather than a supplementation. It might thus be conceivable that the beneficial effects observed with glutamine nutrition are simply a correction of disadvantages produced by an inadequacy of conventional amino acid solutions. The availability of stable glutamine containing preparations will certainly facilitate an adequate amino acid nutrition in routine clinical setting during episodes of stress and malnutrition.     

Determination of pyridinium crosslinks in serum an optimization of sample preparation

Urinary pyridinoline (UPD) and deoxypyridinoline (UDPD) are selective markers in kinetic studies of mature collagen degradation in connective tissue, especially in bone. In patients with renal dysfunction, the determination of UPD and UDPD is not entirely reliable, while in anuretic patients it is impossible. As renal dysfunction is considered a risk factor for bone diseases, it is essential to determine both markers directly in the serum (SPD and SDPD). Due to the high serum concentrations of proteins, which during acid hydrolysis are converted to amino acid hydrochlorides, the system butanol-water is sometimes separated into two phases during sample preparation. Should this fact not be taken into account, the usual sample processing on a cellulose sorbent could yield substantially lower false results. This calls for some preventive measures: to ensure the homogeneity of the system containing n-butanol it is recommended to add an appropriate third component, e.g. methanol.     

Regulation of pyruvate dehydrogenase activity and glucose metabolism in post-ischaemic myocardium

The realm of laparoscopic surgery has extended to include the neonate as well as the pediatric patient. The advent of new and smaller instrumentation has facilitated this goal. Previous procedures exclusively relegated to laparotomy can now be accomplished as outpatient procedures. Removal of the acute appendix, correction of torsion of an adnexa, as well as the appropriate diagnosis and initial treatment of acute pelvic inflammatory disease are now well established laparoscopic procedures. This article provides insight into the laparoscopic evaluation and management of a number of challenging clinical problems for the endoscopic surgeon, thus providing a minimally invasive approach for patients ranging from neonates to adults.     

Nitrogen balance, 3-methylhistidine excretion, and plasma amino acid profile in infants after cardiac operations for congenital heart defects: the effect of early nutritional support

OBJECTIVE: The objective of this study was to evaluate the effect of nutritional support on proteolysis and plasma amino acid profile in infants early after cardiac operations for congenital heart defects. METHODS: Thirty-seven patients, 2 to 12 months old, were randomized on postoperative day 1 for 24-hour isocaloric metabolic study. Group STANDARD (18 patients) received glucose as the maintenance fluid, and group PN (19 patients) received glucose and crystalloid amino acid solution at a dosage of 0.8 +/- 0.1 gm/kg per day. The nonprotein caloric intake in the two groups was 25 +/- 15 and 33 +/- 9 kcal/kg, respectively (p = not significant). RESULTS: The nitrogen balance was markedly less negative in group PN than in group STANDARD (-114 +/- 81 vs -244 +/- 86 mg/kg, respectively, p = 0.001). There was a highly significant inverse correlation between the nitrogen balance and urinary 3-methylhistidine excretion in both groups, but the muscle proteolysis was blunted more effectively in patients receiving amino acids. Concentrations of the plasmatic branched-chain amino acids, alanine, glycine, and proline, decreased significantly in group STANDARD but not in group PN on postoperative day 2. Glutamine and threonine levels declined significantly on postoperative day 2 in both groups. Low levels of arginine were observed in our patients before operation and in the early postoperative period. The amino acid concentrations normalized on postoperative day 7 in all patients. CONCLUSION: Significant proteolysis and hypoaminoacidemia were observed in infants early after cardiac operations. This hypercatabolic response was blunted by parenteral nutritional support.     

The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria

The urokinase plasminogen activator (uPA) is a proteolytic enzyme which converts the proenzyme plasminogen to the active serine protease plasmin. A cell surface receptor for uPA (uPAR) is attached to the cell membrane by a glycosyl-phosphatidylinositol anchor. Binding of uPA to uPAR leads to an enhanced plasmin formation and thereby an amplification of pericellular proteolysis. We have shown previously that uPAR is expressed on normal blood monocytes and granulocytes, but is deficient on affected blood monocytes and granulocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH), and that uPAR is present in plasma from these patients. In this study a newly established sensitive enzyme-linked immunosorbent assay (ELISA) has been applied for quantitation of uPAR in plasma. Unexpectedly, we found that uPAR is not only present in PNH plasma but also in plasma from healthy individuals. In 39 healthy individuals the mean plasma-uPAR value +/- SD was 31 +/- 15 pM, median 28 (range 11-108), and the corresponding value for six PNH patients was 116 +/- 67 pM, median 90 (range 61-228). The elevated uPAR-level in PNH patients was highly significant (Mann-Whitney test; P < 0.0001), and may possibly contribute to the propensity for thrombosis in PNH by inhibition of the fibrinolytic system. Binding of pro-uPA by uPAR in plasma may interfere with the appropriate binding of pro-uPA to cell-bound uPAR and therefore inhibit cell-associated plasmin generation and fibrinolysis. It is likely that the uPAR in normal plasma reflects the overall level of activity of the uPAR-mediated cell surface proteolysis. The present ELISA may be used for studies of uPAR levels in plasma from patients with conditions in which this activity might be increased, such as cancer and inflammatory disorders. Future studies will determine if uPAR in plasma is a parameter of clinical importance in these diseases.     

Role of hydrophobic amino acids at position 74 of DRB1 chain in rheumatoid arthritis

We have analyzed HLA class II alleles in a group of 153 Czech children with rheumatoid arthritis by PCR and hybridization with oligonucleotide probes. When we try to find a common sequence for all DRB1 alleles involved in juvenile and adult arthritis, we can notice hydrophobic amino acid at position 74, which is present in all these alleles, but not in nonsusceptible alleles, where is the hydrophilic amino acid at position 74. In our model, we speculate that the hydrophilic amino acid at position 74 creates a such kind of epitope which is not suitable for rheumatoid-associated peptides or T cells, and only hydrophobic amino acid can permit binding of these peptides or recognition by certain T cells. Analyses of the DPB1 sequences have shown that alleles which have a negatively charged amino acid at position 69, are more frequent in pauciarticular patients while those with a positively charged amino acid are more frequent in polyarticular patients. A positively charged amino acid at position 69 might present the same rheumatoid associated peptide as susceptible DRB1 alleles. The presence of more rheumatoid-associated peptide on the cell surface may cause conversion to more severe polyarticular forms. A negatively charged amino acid at position 69 could not present this peptide and a low concentration of the peptide on the cell surface presented just by DRB1 molecules keeps disease in a relatively benign condition of pauciarticular forms.     

Growth requirements of endothelial cells in culture: variations in serum and amino acid concentrations

Endothelial cell growth in vitro is limited to the availability of nutrients from commercially available media and added serum. Nutrients, such as amino acids, are chiefly derived from the cell culture medium, rather than from added serum, and optimal endothelial cell growth may be dependent on amino acid levels in the culture media. To test this hypothesis, porcine pulmonary artery-derived endothelial cells were exposed to culture medium 199 (M199), amino acid-deficient M199 (dM199), as well as dM199 supplemented with amino acids. Cell protein was similar in cells cultured for 3 d in M199 supplemented with 1, 3, 5 or 10% bovine calf serum, respectively. Addition of amino acid solutions (L-amino acids [Laa], DL-amino acids [DLaa], 2Laa, or Laa+glutamine) to dM199 demonstrated a cell dependence for optimal growth on the type of amino acids as well as on the total available nitrogen in the media. Compared with M199, dM199 supplemented with Laa only partially supported long-term growth of endothelial cells in culture. On the other hand, dM199 supplemented with either 2Laa, DLaa, or Laa+ glutamine was superior over M199 with regard to endothelial cell growth. The addition of Laa+glutamine to dM199 was most growth-supporting, with an increase of over 2.6-fold in total cell protein compared with cells cultured with M199. These results suggest that, in addition to the presence of essential amino acids, total available nitrogen in culture media may be a critical factor for optimal endothelial cell growth.     

Medically induced gingival hyperplasia

Gingival hyperplasia or gingival overgrowth is a common occurrence in patients taking phenytoin, cyclosporine, or calcium channel blockers. Speech, mastication, tooth eruption, and aesthetics may be altered. Controlling the inflammatory component through an appropriate oral hygiene program may benefit the patient by limiting the severity of the gingival overgrowth. In patients in whom gingival overgrowth is present or may be anticipated, recognition of this condition and referral to a general dentist or periodontist are appropriate steps to management. The physician's awareness of the potential for development of overgrowth and the dental practitioner's role in attempting to prevent or minimize this problem are important aspects. In this article, we discuss the medications associated with gingival hyperplasia and describe appropriate recommendations.     

Harbor seal (Phoca vitulina) C-reactive protein (C-RP): purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum C-RP concentrations

C-reactive protein (C-RP) was purified from harbor seal (Phoca vitulina) serum by calcium dependant phosphoryl-choline and protein A affinity chromatography. Polyacrylamide gel electrophoresis under reducing conditions revealed a single protein moiety with a molecular weight of approximately 25 kDa. An internal peptide derived from this purified protein was subjected to N-terminal amino acid sequencing. A high amino acid sequence similarity was obtained with other published mammalian C-RP molecules confirming that the purified protein was a C-RP homologue. Eight specific monoclonal antibodies (P13, P51, P87, P101, P106, P130, P157 and P219) were raised against this purified protein. All 8 monoclonal antibodies immunoblotted with the 25 kDa C-RP subunit under reducing conditions. A competitive immunoassay was developed identifying elevated C-RP concentrations in harbor seal serum samples with clinical evidence of inflammatory disease. Application of this immunoassay for the measurement C-RP may provide valuable information for the clinical assessment of harbor seal health.     

Identification of a structural determinant for resistance to beta-lactam antibiotics in Gram-positive bacteria

Streptococcus pneumoniae is the main causal agent of pathologies that are increasingly resistant to antibiotic treatment. Clinical resistance of S. pneumoniae to beta-lactam antibiotics is linked to multiple mutations of high molecular mass penicillin-binding proteins (H-PBPs), essential enzymes involved in the final steps of bacterial cell wall synthesis. H-PBPs from resistant bacteria have a reduced affinity for beta-lactam and a decreased hydrolytic activity on substrate analogues. In S. pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is located in the cell division cluster (DCW). We present here structural evidence linking multiple beta-lactam resistance to amino acid substitutions in PBP2x within a buried cavity near the catalytic site that contains a structural water molecule. Site-directed mutation of amino acids in contact with this water molecule in the "sensitive" form of PBP2x produces mutants similar, in terms of beta-lactam affinity and substrate hydrolysis, to altered PBP2x produced in resistant clinical isolates. A reverse mutation in a PBP2x variant from a clinically important resistant clone increases the acylation efficiency for beta-lactams and substrate analogues. Furthermore, amino acid residues in contact with the structural water molecule are conserved in the equivalent H-PBPs of pathogenic Gram-positive cocci. We suggest that, probably via a local structural modification, the partial or complete loss of this water molecule reduces the acylation efficiency of PBP2x substrates to a point at which cell wall synthesis still occurs, but the sensitivity to therapeutic concentrations of beta-lactam antibiotics is lost.