The importance of the negative charge of beta-lactam compounds for the inactivation of the active-site serine DD-peptidase of Streptomyces R61.

The interaction between the Streptomyces R61 penicillin-sensitive DD-peptidase and deacetyl-cephalosporin C or its lactone derivative has been studied at different pH values. The results show the importance of an enzyme group of pK approximately equal to 9 which might form an ion pair with the free carboxylate of the former compound. This electrostatic interaction is shown to contribute to the formation of the first, non-covalent enzyme-inactivator complex by a factor of at least 50.

Structure-activity relationships in the beta-lactam family: an impossible dream.

The difficulty of establishing structure-activity relationships in the beta-lactam family of antibiotics stems from the fact that: (1) The targets in various bacteria exhibit widely different sensitivities. (2) Some bacteria produce beta-lactamases, enzymes capable of destroying the antibiotics. The rates of the reactions with the beta-lactamases and the target enzymes are not necessarily related. (3) In Gram-negative bacteria, the diffusion rate through the outer membrane varies independently from the two other factors.

The ultrastructure of the electric burn in man: a transmission electron microscopy-scanning electron microscopy study.

The authors describe the ultrastructural aspects of the electric burn observed with the transmission electron microscope (TEM) and the scanning electron microscope (SEM). With the TEM the most apparent feature is the homogenization and arrangement of the filamentous cytoplasmic material in elongated and parallel bundles together with elongation of the nuclei and junctional structures. SEM studies present a three-dimensional vision of the filamentous material that confirms and clarifies the cellular deformation as a whole. An hypothesis is advanced suggesting that the phenomenon is due to the effects produced by the extremely rapid dehydration of the tissue or that the electric current and Joule effect can act upon the filaments and contractile proteins, causing the cell deformation.

Scanning electron microscope study of the lung in drowning.

The authors describe the results obtained using the scanning electron microscope (SEM) to study drowned lungs and control lungs. Results are compared with a previous study carried out using laboratory animals. The results from both studies are analogous, in the absence of chronic pulmonary emphysema. The diagnosis of chronic pulmonary emphysema is simple with the SEM even when dealing with autoptic material obtained 24 to 48 h after death, but the diagnosis of drowning becomes difficult in lungs with chronic emphysema.

Scanning electron microscopic ultrastructural alterations of the pulmonary alveolus in experimental drowning.

Scanning electron microscopic structural and ultrastructural alterations of alveolar wall in experimental drowning are described. The results obtained confirm the literature's data. Peculiar microlesions affecting Type I alveolar cells, the basement membrane, and sometimes the capillary endothelium could also be observed. Finally, Type II alveolar cells show alterations which could be related with a possible direct action of the drowning liquid.

Medical-legal issues in headache: penal and civil Italian legislation, working claims, social security, off-label prescription.

Primary headaches can be considered simultaneously as symptom and disease itself, while secondary headaches are expressions of a pathological process that can be systemic or locoregional. Because of its subjective features, headache is often difficult to assess and quantify by severity, frequency and invalidity rate, and for these reasons it has often been implicated in legal controversies. Headache has seldom been considered in the criminal law, except when it represents a typical symptom of a disease whose existence can be objectively assessed (i. e. raised intracranial pressure). Therefore, in civil legislation it is not yet coded to start claiming for invalidity compensation. In particular, one of the most debated medical-legal questions is represented by headaches occurring after head injury. Headache is often the principal symptom at the beginning of several toxic chronic syndromes, with many implications, especially in working claims, and, more recently, it may be referred to as one of the most frequent symptoms by victims of mobbing (i. e. psychological harassment in the workplace). The National Institute for Industrial Accident Insurance (INAIL) scales (instituted by the law 38/2000) mention the "Subjective cranial trauma syndrome" and give an invalidity rate evaluation. With reference to other headache forms, no legislation really exists at the present time, and headache is only considered as a symptom of a certain coded disease. Requests for invalidity social pension and the question of off-label prescriptions (drug prescription for a disease, without formal indication for it) are other controversial matters.

Typogenetics: an artificial genetic system.

Cellular automata are now used to model various natural phenomena. In particular, they provide a logical universe in which artificial cells can be embedded in the form of propagating virtual automata. In this paper, we propose a molecular automaton which is an attempt to implement the "molecular logic of the living state" in an artificial biochemistry. This automaton is an artificial genetic system composed of two classes of interacting artificial molecules. It was shown to be self-replicating and to possess features that are analogous to those of the postulated prebiotic molecular systems.

Studying artificial life with a molecular automaton.

In a previous paper, we proposed a molecular automaton which was an attempt to implement the "molecular logic of the living state" in an artificial biochemistry. This automaton is an artificial genetic system composed of two classes of interacting molecules, the informants and the transformers. In the present paper, we show that such a tangled hierarchy is susceptible to give rise to general hypercycles we called tanglecycles. These tanglecycles are shown to be self-reproducing autocatalytic metabolisms. They can be considered as genuine "acellular beings" in which the information is conserved in a dynamical loop. Our phenomenological study of tanglecycles reveals the emergence of properties usually associated with life, including self-reproduction, selection and chaos. In the field of the autocatalytic networks it represents a new approach in studying the self-organization of the matter with a prebiotic point of view.

An exceptional case of necrophilia.

The authors describe a case of necrophilia in which the corpse of a young girl was disinterred. No lesions were observed on the genitals and breasts, but the aspect of the anus suggested that some object had been introduced in it. The possible modalities of the event are discussed.

Dermal surface in electric and thermal injuries. Observations by scanning electron microscopy.

The dermal surface (after enzymatic digestion of the dermo-epidermal junction) in electric and heat lesions is described. There do not appear to be elements that help in forming a precise differential diagnosis. It is possible, however, to differentiate these lesions from all traumatic cutaneous injuries and some limited nontraumatic lesions (e.g., verrucae). Remarkably good preservation of the dermal surface even in an advanced state of decomposition does suggest the possibility of certain diagnosis of electric and heat lesions even when postmortem phenomena have greatly damaged or even totally removed the epithelial part of the skin.

A case of murder by impalement.

We report here the case of a young oligophrenic man killed by a psychopath by impalement, using a sharp stick which was thrusted into the perineal region, thus causing injuries to abdominal and thoracic viscera.

SEM study of dermal surface. A new approach to forensic traumatology.

We describe a method of enzymatic digestion of the dermo-epidermal junction, that allows an observation with a SEM of the finest details of the dermal surface covered by the reticular fibrils of the basement membrane. By means of this technique, we have made a panoramic review of the traumatology and have observed typical characteristics in all types of examined lesions. It seems that the method can constitute a new approach for medicolegal study, in all those cutaneous lesions that involve, even superficially, the dermis.

Dermal surface morphology in wound healing. An experimental scanning electron microscope study.

The authors observed, with a scanning electron microscope, the dermal surfaces of experimental wounds, following the enzymatic digestion with trypsin of the dermo-epidermal junction. Cut lesions showed an early reconstitution of the basement membrane and progressive convergence of the margins. A peculiar arrangement of the dermal crests was also observed. In wounds with loss of substance, the complete coating by newly formed basement membrane took longer. There was an early deformation of the holes of hair follicles in the undamaged dermis surrounding the lesion, as well as the appearance of creases that moved toward the center. This process was over within a few days; then the lesion became very small and the holes of the hair follicles reappeared. The results suggest a force of traction bound toward the center of the lesion and acting on the surface, the effects of which on the basement membrane are most evident between the eighth and the 15th days.

New observations on cutaneous firearm wounds.

The authors describe the aspect of the dermal surface in firearm wounds. In entrance wounds, the abraded area is rather evident, with loss of the papillary pattern and lacerations of the basement membrane. Exit wounds appear as irregularly lacerated interruptions, with protruding collagen fibers; the surrounding dermis has nearly undamaged papillae. Also described, still on the dermal surface, are the flame effect and the powder tattooing. Evident lesions of the basement membrane, produced by the grains of powder, can be observed. Particles [gunshot residues (GSR)] are often recognizable lying on the dermal surface or entrapped among the collagen fibers of the superficial dermis.

Lung morphology in experimental acute paraquat poisoning. SEM observations.

The authors describe the SEM ultrastructural aspects of rat lungs following acute paraquat poisoning. The results obtained confirmed previous TEM observations and suggest an early inhibition of secretory activity in type II alveolar cells. This morphological aspect can be related to the generally known early development of respiratory difficulties following acute paraquat poisoning. The technique employed allows a precise study of alveolar reepithelization in further stages of poisoning. These last observations agree with the hypothesis of reepithelization by type II alveolar cells.

The pH dependence of the active-site serine DD-peptidase of Streptomyces R61.

Titration of the active-site serine DD-peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac2-L-Lys-D-Ala-D-Ala and enzyme inactivation by the beta-lactam compounds benzylpenicillin, N-acetylampicillin and ampicillin relies on the acidic form of an enzyme's group of pK approximately equal to 9.5. It is proposed that protonation of a lysine epsilon-amino group facilitates initial binding by charge pairing with the free carboxylate of the substrate and the beta-lactam molecules. Lowering the pH from 7 to 5 has no effect on the second-order rate constant of enzyme acylation by benzylpenicillin and N-acetylampicillin but results in a decreased rate constant of acylation by ampicillin and Ac2-L-Lys-D-Ala-D-Ala. Protonation of the side-chain amino group of ampicillin and a decreased efficacy of the initial binding of the peptide to the enzyme seem to be responsible for the observed effects. Whatever the molecule bound to the enzyme, there is no sign for the active involvement of an enzyme's histidine residue of pK 6.5-7.0 in the hydrolysis pathway.

The importance of the negative charge of beta-lactam compounds in the interactions with active-site serine DD-peptidases and beta-lactamases.

The interaction between various penicillins and cephalosporins the carboxylate group of which at C-3 or C-4 had been esterified or amidated and different penicillin-recognizing enzymes was studied. In general, our findings reinforced the common assumption that an anionic group at that position is necessary for the effective acylation of these enzymes. However, the relative activities of the modified beta-lactams as inactivators of the Streptomyces R61 DD-peptidase or as substrates of the Bacillus licheniformis, Streptomyces albus G and Enterobacter cloacae beta-lactamases did not fit a general scheme in which the intrinsic electronic and geometric properties of the beta-lactam compounds would be sufficient to explain their substrate or inactivator properties towards the various types of enzymes investigated.

Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.