Neuroendocrine,epigenetic, and intergenerational effects of general anesthetics

The progress of modern medicine would be impossible without the use of general anesthetics (GAs). Despite advancements in refining anesthesia approaches, the effects of GAs are not fully reversible upon GA withdrawal. Neurocognitive deficiencies attributed to GA exposure may persist in neonates or endure for weeks to years in the elderly. Human studies on the mechanisms of the long-term adverse effects of GAs are needed to improve the safety of general anesthesia but they are hampered not only by ethical limitations specific to human research, but also by a lack of specific biological markers that can be used in human studies to safely and objectively study such effects. The latter can primarily be attributed to an insufficient understanding of the full range of the biological effects induced by GAs and the molecular mechanisms mediating such effects even in rodents, which are far more extensively studied than any other species. Our most recent experimental findings in rodents suggest that GAs may adversely affect many more people than is currently anticipated. Specifically, we have shown that anesthesia with the commonly used GA sevoflurane induces in exposed animals not only neuroendocrine abnormalities (somatic effects), but also epigenetic reprogramming of germ cells (germ cell effects). The latter may pass the neurobehavioral effects of parental sevoflurane exposure to the offspring, who may be affected even at levels of anesthesia that are not harmful to the exposed parents. The large number of patients who require general anesthesia, the even larger number of their future unexposed offspring whose health may be affected, and a growing number of neurodevelopmental disorders of unknown etiology underscore the translational importance of investigating the intergenerational effects of GAs. In this mini review, we discuss emerging experimental findings on neuroendocrine, epigenetic, and intergenerational effects of GAs.     

Nucleotide levels in human and bovine lenses: a study on regional postmortem changes

Regional lens adenine nucleotide levels were determined in human and bovine lenses using sensitive HPLC techniques. In adult lenses of both species adenine nucleotide levels were highest in the anterior cortex (plus epithelium and capsule) followed by the posterior cortex (plus capsule) and lowest in the nucleus. With increasing lens age ATP level remained steady in the anterior cortex (plus epithelium and capsule) and nucleus, while those in the posterior cortex showed a tendency toward decrease. In both species, human as well as bovine, adenine nucleotide levels of the anterior cortex (plus epithelium and capsule) underwent early postmortem changes. Thus, immediate postmortem sampling as well as regional determinations are a must for detailed studies of lens nucleotide metabolism.     

Multiclefted nuclei. A helpful feature for identification of intermediate trophoblastic cells in uterine curetting specimens.

Intermediate trophoblast is a distinct form of trophoblast, the presence of which in uterine curettings is considered a reliable indicator of intrauterine pregnancy even in the absence of chorionic villi. However, the appearance of intermediate trophoblastic cells have not been described in sufficiently specific terms to permit their reliable identification, and distinction from decidual cells can be difficult. We have noticed for some time that the intermediate trophoblastic cells often show multiple deep clefts in the nuclei, and the present study was performed to address the issue of whether this nuclear feature is reliable for their identification. We reviewed 242 uterine curettings of intrauterine pregnancy, documented by the presence of chorionic villi, and were able to find a distinct population of cells with large, hyperchromatic, multiclefted nuclei scattered in the decidua in 88% of the cases. In most instances, these cells produced a characteristic variegated pattern that was readily recognizable at low magnification. Positive immunostaining for cytokeratin (CAM 5.2) in these isolated cells within the decidua confirmed their trophoblastic nature. In contrast, multiclefted nuclei were absent in the 51 negative control cases, which included decidualized endocervical polyps (40 cases), uterine curettings from patients with tubal pregnancy (10 cases), and endometriosis with decidual change (one case). We conclude that intermediate trophoblastic cells can usually be reliably identified in curettings of intrauterine pregnancy by their characteristic nuclear multiclefting.     

Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labelled probes and transgenic tissues.

The sensitivity of non-isotopic in situ hybridization (NISH), particularly on formalin-fixed, paraffin-embedded (FFPE) clinical tissues, has been the subject of controversy. Generally, NISH has been regarded as being less sensitive than radiolabelled procedures, although some reports have contradicted this. Accordingly, tissues from mice which were transgenic for variable amounts of the human alpha-1-antitrypsin gene were used to optimize the NISH procedure and to estimate the sensitivity. This approach showed that prolonged incubation of slides in final substrate resulted in high sensitivity--about 13 kb of target DNA. However, this prolonged incubation crucially depended on achieving minimal non-specific background staining. Many factors affected the degree of background staining, but five were particularly important. First, the method of mounting cut sections onto slides. Second, the length of the probe (ideally less than 400 bp). Third, the procedure for proteolytic digestion. Fourth, the denaturation technique, and fifth, the quality of the dextran sulphate used in the hybridization mix. The optimized protocol showed variable patterns of mRNA distribution in the transgenic mouse livers, while DNA distribution appeared uniform.     

Production and characterization of highly specific anti-methotrexate monoclonal antibodies

Spleen cells of a Biozzi HR mouse immunized with a bovine serum albumin-methotrexate conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-three monoclonal antibodies (MAbs), selected by indirect ELISA, were produced and partially characterized. Using methotrexate (MTX) and eight structurally related compounds, binding specificities of the MAbs were assessed by inhibition enzyme immunoassay. All the MAbs had very low affinity for folic acid and its analogs and for the major MTX metabolite 7-hydroxymethotrexate. Using a computer cluster analysis program based on the binding specificities, the MAbs were divided into three groups. The thirteen MAbs in group I recognized primarily the pteridine portion of the MTX molecule; the eight group II MAbs recognized the benzene ring as well as the pteridine structure. The two MAbs in group III poorly distinguished between the different parts of the MTX molecule. The apparent equilibrium association constants of the anti-MTX MAbs in groups I, II, and III ranged from 7 x 10(9) to 3 x 10(8) M-1 (except for 1 MAb), from 5 x 10(7) to 6 x 10(6) M-1 (except for 2 MAbs), and from 1 x 10(6) to 3.5 x 10(5) M-1, respectively.     

A table of senile forms of cataract. Clinical picture, prognosis, biochemistry and etiology

The three important types of age-related senile cataract, namely deep supranuclear cortical cataract, superficial subcapsular cortical cataract (sometimes associated with secondary nuclear cataract), and primary nuclear cataract, are arranged in a table which may be used for quick reference with regard to clinical features - slit lamp findings, age at onset of opacification, rate of progression, and related biochemistry (e.g., ATP content, glutathione content, and the cation pump (K+ greater than Na+] - as well as etiological features. The significance of the table is illustrated on the basis of an important example from the literature ("Sunlight and Human Cataracts").     

Purified 60-kilodalton Legionella protein antigen with Legionella-specific and nonspecific epitopes.

In a previous study, all convalescent-phase sera from patients with culture-confirmed legionellosis reacted on immunoblots with a Legionella genus-wide 58-kilodalton (kDa) protein antigen (J.S. Sampson, B.B. Plikaytis, and H.W. Wilkinson, J. Clin. Microbiol. 23:92-99, 1986). The present study was done to immunologically characterize and determine the diagnostic relevance of this purified antigen. The antigen was precipitated from enriched cell extracts with ammonium sulfate and purified by high-pressure liquid chromatography. High-titered rabbit antiserum produced to the purified protein was used to show its presence on immunoblots in the 60-kDa range in 38 Legionella serogroups, representing 23 species, and in 39 non-Legionella bacteria. The antiserum was made specific for Legionella strains by sequential absorptions with Bordetella pertussis, Pseudomonas aeruginosa, and Pseudomonas fluorescens whole cells. Serum from legionellosis patients reacted with both specific and nonspecific epitopes. Results of indirect immunofluorescence experiments showed that neither specific nor nonspecific epitopes of the 60-kDa protein were surface exposed on Legionella cells and that cross-reactive epitopes were variably exposed on non-Legionella bacteria. The 60-kDa protein antigen should be useful in diagnostic tests for legionellosis if care is taken to expose cryptic epitopes and if the tests use or measure only the Legionella-specific epitopes.