BACKGROUND

Chlamydia pneumoniae and the herpes viruses cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1) have been associated with human atherosclerosis in seroepidemiological and separate histopathological studies. We investigated the concurrent presence of these microorganisms in patients undergoing carotid endarterectomy.

RESULTS

Endarterectomy specimens from 76 patients with carotid artery stenosis were stained for C. pneumoniae, CMV, and HSV-1 particles with specific IgG monoclonal antibodies by the avidin-biotin-peroxidase method. IgG antibodies to CMV and C. pneumoniae were also measured in the serum. These were correlated with plaque morphology and the presence of the microorganisms in the atherosclerotic plaques. C. pneumoniae was detected in 54 (71%) (95% confidence interval [CI], 59.5% to 80.9%), CMV was detected in 27 (35.5%) (CI, 24.9% to 47.3%), and HSV-1 was detected in 8 (10.5%) (CI, 4.7% to 19.7%) versus none of 20 (0%) control normal carotid artery and aortic tissue (autopsy) specimens (CI, 0% to 16.8%) (P<.001 for CMV and C. pneumoniae). At least one microorganism was detected in 59 of the specimens (77.6%) (CI, 66.6% to 86.4%), with a single microorganism present only in 35 (46%), two microorganisms present in 18 (23.7%) (CI, 14.7% to 34.8%), and all three present in 6 (7.9%) (CI, 3.0% to 16.4%). Atherosclerotic plaques with thrombosis were more likely to have C. pneumoniae (80.4%) or CMV (57.8%) than were plaques without thrombosis (56.7% and 16.7%, respectively; P=.04 and .007). There was no correlation between the presence of CMV and C. pneumoniae in the atherosclerotic vessels and serum antibody titers.

CONCLUSIONS

C. pneumoniae and CMV are commonly detected in atherosclerotic plaques of the carotid arteries, but their presence cannot be predicted by measuring serum antibodies. The presence of these microorganisms may predispose to a greater risk of thrombosis in the plaques, but further studies are needed to confirm this observation.

It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.

The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans.

Intracerebral infusion of lysed erythrocytes causes brain edema without inducing ischemic cerebral blood flow. Reports have indicated that oxidative damage contributes to secondary brain injury in stroke. In the present study, we investigated whether erythrocyte lysis after intracerebral hemorrhage (ICH) might result in oxidative brain damage. This study had four parts. Male Sprague-Dawley rats received an infusion of autologous lysed erythrocytes into the right striatum. Control rats only had a needle insertion. Neurological deficits, brain water and ion contents were determined in the first part. In the second part, hemoxygenase-1 (HO-1), manganese superoxide dismutase (Mn-SOD), copper/zinc SOD (CuZn-SOD) and protein carbonyl levels were determined by Western blot analysis. In the third part, immunohistochemistry was performed for HO-1. DNA damage was examined using DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) in the fourth part. Infusion of lysed RBCs induced marked edema in the ipsilateral striatum and profound neurological deficits. Western blot analysis and immunohistochemistry indicated that HO-1 was upregulated 24 h after infusion of lysed red blood cells. Both Mn-SOD and CuZn-SOD contents decreased, protein carbonyl levels increased in the ipsilateral striatum, and there was the appearance of PANT- and TUNEL-positive cells suggesting oxidative mechanisms in the erythrocyte-induced brain injury. In conclusion, oxidative stress caused by components of the lysed erythrocytes contributes to the brain injury after ICH.

Clearance of synaptically released dopamine is regulated by the plasmalemmal dopamine transporter (DAT), an integral membrane protein that resides within a complex lipid milieu. Here we demonstrate that cholesterol, a major component of the lipid bilayer, can modulate the conformation of DAT and alter cocaine binding to DAT. In striatal synaptosomes and transfected cells, DAT was in cholesterol-rich membrane fractions after mild detergent extraction. After increasing the membrane cholesterol content by treatment of water-soluble cholesterol (cholesterol mixed with methyl-β-cyclodextrin), we observed an increase in DAT binding B(max) values for cocaine analogs [(3)H]WIN35428 and [(125)I]RTI-55, but similar levels of DAT proteins on the cell surface were shown by surface biotinylation assays. Membrane cholesterol addition also markedly enhanced the accessibility of cysteine sulfhydryl moieties in DAT as probed by a membrane-impermeable maleimide-biotin conjugate. We identified cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT, as the intrinsic residue exhibiting enhanced reactivity. Similar effects on DAT cysteine accessibility and radioligand binding were observed with addition of zinc, a reagent known to promote the outward facing conformation of DAT. Using substituted cysteine mutants on various positions likely to be extracellular, we identified additional residues located on TM1, TM6, TM7, and TM12 of DAT that are sensitive to alterations in the membrane cholesterol content. Our findings in transfected cells and native tissues support the hypothesis that DAT adopts an outward facing conformation in a cholesterol-rich membrane environment, suggesting a novel modulatory role of the surrounding membrane lipid milieu on DAT function.

Acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-CoA, the first intermediate in fatty acid synthesis. We studied the localization of two forms, the prokaryote and the eukaryote forms, of ACCase in pea leaves by comparing the biotin polypeptides of the two ACCases in protein extract from leaves and plastids. We found that the two forms of ACCase were in different cell compartments of pea leaves; the prokaryote form was in the plastids, and the eukaryote form was elsewhere, probably in the cytosol. This result suggested the existence of two sites of malonyl-CoA synthesis. The Gramineae, such as rice and wheat, which lack the accD gene encoding one of the subunits of the prokaryote form of ACCase in their chloroplast genomes, did not have the prokaryote form of the enzyme but had the eukaryote form. The selective grass herbicides of the diphenoxypropionic acid type and the cyclohexanedione type, in vitro, inhibited plastidic ACCase of the eukaryote form from wheat but did not inhibit that of the prokaryote form from pea, suggesting that the origin of the tolerance of intact pea plant toward these herbicides is partly in the insensitivity of the prokaryote form of the enzyme. The origin of the susceptibility of the Gramineae plants toward these herbicides seems to lie in the presence of the herbicide-sensitive eukaryote form and the absence of the insensitive prokaryote form due to the lack of the accD gene in plastid.

Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfer reactions and are potential targets for the treatment of obesity and type 2 diabetes. These enzymes use a tethered biotin cofactor to carry an activated carboxyl group between distantly spaced active sites. The mechanism of this transfer has remained poorly understood. Here we report the complete structure of pyruvate carboxylase at 2.0 angstroms resolution, which shows its domain arrangement. The structure, when combined with mutagenic analysis, shows that intermediate transfer occurs between active sites on separate polypeptide chains. In addition, domain rearrangements associated with activator binding decrease the distance between active-site pairs, providing a mechanism for allosteric activation. This description provides insight into the function of biotin-dependent enzymes and presents a new paradigm for multifunctional enzyme catalysis.

Integrins comprise a family of transmembrane glycoproteins that modulate cell-matrix and cell-cell relationships by acting as receptors to extracellular protein ligands, and also as direct adhesion molecules. The authors studied by immunohistochemistry the distribution of the alpha 1-6,v and the beta 1,3,4 subunits of integrins in samples of normal breast, the spectrum of fibrocystic disease (FCD), and representative benign and malignant neoplasms. Monoclonal antibodies (Mabs) specific for each subunit were applied to cryosections by the avidin-biotin-complex method; selected samples were studied by double immunofluorescence microscopy with the Mabs and a polyclonal antiserum to myosin. The authors found that the alpha 1-3,6,v and the beta 1, integrin subunits were detectable in the normal breast parenchyma; myoepithelial cells were consistently more prominently stained than the basolateral aspect of the luminal cells. This immunoprofile was retained, and in cases enhanced through the spectrum of FCD, in benign tumors and in ductal and lobular carcinomas in situ. In most infiltrating ductal carcinomas, integrin staining tended to decrease except for some cases that reacted strongly for the alpha v subunit. Several mucinous carcinomas reacted strongly for alpha 2,3,6,v and beta 4 subunits, and even more so for the alpha 5 subunit that was not found in the normal breast. Subsets of infiltrating lobular carcinomas stained convincingly for alpha 1,3,6,v and beta 1 subunits in delicate but abundant kinetopodia. Our findings indicate that in hyperplasias and in benign tumors integrin expression patterns parallel those of the normal breast, whereas in carcinomas, variations include decrease, enhancement, and emergence of certain subunits that are not in the normal repertory. Alterations of integrin expression parallel phenotypic changes in breast carcinoma cells; they also reflect their disrupted interaction with the similarly disrupted extracellular matrix. Enhancement of certain integrins in some carcinomas may reflect the selection of subpopulations with increased binding capacity which in turn may impact on their invasive and metastatic properties.

We have demonstrated a requirement for cellular ATP in the receptor-mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric-transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I-BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.

The radical SAM superfamily of metalloproteins catalyze the reductive cleavage of S-adenosyl-l-methionine to generate a 5'-deoxyadenosyl radical (5'-dA*) intermediate that is obligate for turnover. The 5'-dA* acts as a potent oxidant, initiating turnover by abstracting a hydrogen atom from an appropriate substrate. A special class of these enzymes use this strategy to functionalize unactivated C-H bonds by insertion of sulfur atoms. This review will describe the characterization of three members of this class - biotin synthase, lipoyl synthase, and MiaB protein - each of which has been shown to cannibalize itself during turnover.

The radical-S-adenosylmethionine (radical-AdoMet) enzyme MiaB catalyzes the posttranscriptional methylthiolation of N-6-isopentenyladenosine in tRNAs. Spectroscopic and analytical studies of the reconstituted wild-type and C150/154/157A triple variant forms of Thermotoga maritima MiaB have revealed the presence of two distinct [4Fe-4S](2+,1+) clusters in the protein. One is coordinated by the three conserved cysteines in the radical-AdoMet motif (Cys150, Cys154, and Cys157) as previously reported, and the other, here observed for the first time, is proposed to be coordinated by the three N-terminal conserved cysteines (Cys10, Cys46, and Cys79). The two [4Fe-4S]2+ clusters have similar UV-visible absorption, resonance Raman, and Mössbauer properties but differ in terms of redox properties and the EPR properties of the reduced [4Fe-4S]1+ clusters. Reconstituted forms of MiaB containing two [4Fe-4S] clusters are more active than previously reported. Comparison of MiaB with other radical-AdoMet enzymes involved in thiolation reactions, such as biotin synthase and lipoate synthase, is discussed as well as a possible role of the second cluster as a sacrificial S-donor in the MiaB-catalyzed reaction.

The cells of origin, the course, and termination patterns of the ventral, uncrossed component of the rat corticospinal tract (CST) was investigated by using retrograde and anterograde tracing methods. Anterograde tracing with biotin dextran-amine (BDA) revealed the position and detailed morphology of CST fibers in the spinal cord. Cross sections on spinal levels C4, T8, and L4 showed labeled fibers in the ipsilateral ventral funiculus on all levels. Although ipsilateral ventral CST fibers run close to the midline in the cervical cord, they were found to disperse more in the ventromedial funiculus at lower spinal levels. To study the termination patterns of the ipsilateral ventral projection, a dorsal spinal cord hemisection was performed at level T8, severing the crossed dorsomedial and dorsolateral components but leaving ipsilateral ventral running fibers intact. These fibers were observed to have sometimes several collaterals with terminal arbors extending into different spinal segments, innervating mostly laminae III-VI. Structures closely resembling synaptic boutons were identified in these arbors. By retrograde tracing in animals with dorsal spinal cord hemisection, we found labeled cells equally distributed throughout the spinally projecting cortical areas corresponding to the level of tracer injection. Labeled cells were found in layer V. The diameter of the labeled cells was not significantly different from other spinally projecting cortical neurons. In summary, a neuroanatomically complete ipsilateral, ventral corticospinal projection down to low spinal levels was found. The large extension of the terminal arborizations in intermediate laminae of the spinal cord suggests a modulatory role of this CST component.

To expand the electron microscopist's options in localization and visualization, a new and general staining technique has been tested. The avidin-biotin complex serves as a coupling between the electron-dense marker, ferritin, and points of interest in biological samples. When specific cellular components are tagged with biotin, those components may be visualized with ferritin-linked avidin. Because of the remarkably strong affinity of avidin and biotin (characterized by an association constant of 10(15) M(-1)), the staining is rapid and stable. The preparation of ferritin-avidin conjugate is described, and examples are presented of the application of this complex to biotin-tagged membranes. The ghosts of Acholeplasma laidlawii have been treated with biotinyl-N-hydroxysuccinimide ester to label protein amino groups. Erythrocyte membrane oligosaccharides have been oxidized by periodate or by galactose oxidase, and the resulting aldehydes labeled with biotin hydrazide. The avidin-biotin complex in electron microscopy seems especially appropriate for seqential staining procedures, as well as for visualization of reaction sites of biotin-labeled, low-molecular-weight reagents.

The kinetics of binding and endocytosis of 125I-human holotransferrin by isolated human brain capillaries was examined using this system as a model of the human blood-brain barrier (BBB). Both binding and endocytosis of the peptide by human brain capillaries was temperature-dependent and the binding was saturated by holotransferrin, but not by insulin, somatostatin, or vasopressin. Scatchard analysis of the binding reaction revealed a dissociation constant of 448 +/- 110 ng/mL (5.6 +/- 1.4 nmol/L) and a maximal binding constant (Ro) of 8.0 +/- 1.5 ng/mg protein. Thus, the affinity and capacity of the BBB transferrin receptor is within the same order of magnitude as the affinity and capacity of the BBB receptors for insulin, insulinlike growth factor-I, or insulinlike growth factor-II. The human brain capillary transferrin receptor was also detected with a mouse monoclonal antibody to the receptor using the avidin/biotin/peroxidase technique. In conclusion, these studies characterize the human BBB transferrin receptor and support the hypothesis that this receptor acts as a transport system which mediates the transcytosis of transferrin-bound iron through the brain capillary endothelial cell in man.

We investigated the temporal and regional profile of blood-brain barrier (BBB) permeability to both large and small molecules after moderate fluid percussion (FP) brain injury in rats and determined the effects of post-traumatic modest hypothermia (33 degrees C/4 h) on these vascular perturbations. The visible tracers biotin-dextrin-amine 3000 (BDA-3K, 3 kDa) and horseradish peroxidase (HRP, 44 kDa) were injected intravenously at 4 h or 3 or 7 days post-TBI. At 30 min after the tracer infusion, both small and large molecular weight tracers were detected in the contusion area as well as remote regions adjacent to the injury epicenter in both cortical and hippocampal structures. In areas adjacent to the contusion site, increased permeability to the small molecular weight tracer (BDA-3K) was evident at 4 h post-TBI and remained visible after 7 days survival. In contrast, the larger tracer molecule (HRP) appeared in these remote areas at acute permeable sites but was not detected at later post-traumatic time periods. A regionally specific relationship was documented at 3 days between the late-occurring permeability changes observed with BDA-3K and the accumulation of CD68-positive macrophages. Mild hypothermia initiated 30 min after TBI reduced permeability to both large and small tracers and the infiltration of CD68-positive cells. These results indicate that moderate brain injury produces temperature-sensitive acute, as well as more long-lasting vascular perturbations associated with secondary injury mechanisms.

A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.0-8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of the TNP-tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single- and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a variety of tyramide conjugates in bulk amounts at relatively low cost.

Expression of the macrophage antigen CD163 in breast cancer cells is recently shown to be related to early distant recurrence and shortened survival. In this study, 163 patients with rectal cancer, included in the Swedish rectal cancer trial and followed up for a median of 71 months, were examined for the expression of CD163 in the primary tumors. The cancer cells expressed CD163 in the primary tumors in 23% (n = 32) of the patients. In pretreatment biopsies from 101 patients, 10 had CD163-positive cancers and these patients had earlier local recurrence (p < 0.044) and reduced survival time (p < 0.045) compared with those with CD163-negative tumors. When studying surgical specimens from 61 patients randomized to preoperative irradiation (5 x 5 Gy delivered in 1 week), it was found that 31% were CD163 positive whereas the corresponding figure was only 17% for 78 patients who were nonirradiated (p < 0.044), which tentatively may be consistent with X-rays inducing fusion. In CD163-positive tumors there was a reduced apoptotic activity as measured with the Termina deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) technique (p = 0.018). There tended also to be an increased proliferation activity measured as an expression of Ki-67 non significant (NS). It is concluded that primary rectal cancers may express CD-163, and this phenotypic macrophage trait is related to early local recurrence, shorter survival time and reduced apoptosis. Furthermore, the expression of CD163 is more common after irradiation.

The dynamic modification of nuclear and cytoplasmic proteins by the monosaccharide N-acetyl-glucosamine (GlcNAc) continues to emerge as an important regulator of many biological processes. Herein we describe the development of an alkynyl-modified GlcNAc analog (GlcNAlk) as a new chemical reporter of O-GlcNAc modification in living cells. This strategy is based on metabolic incorporation of reactive functionality into the GlcNAc biosynthetic pathway. When combined with the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent visualization of O-GlcNAc and affinity enrichment and identification of O-GlcNAc-modified proteins. Using in-gel fluorescence detection, we characterized the metabolic fates of GlcNAlk and the previously reported azido analog, GlcNAz. We confirmed previous results that GlcNAz can be metabolically interconverted to GalNAz, whereas GlcNAlk does not, thereby yielding a more specific metabolic reporter of O-GlcNAc modification. We also used GlcNAlk, in combination with a biotin affinity tag, to identify 374 proteins, 279 of which were not previously reported, and we subsequently confirmed the enrichment of three previously uncharacterized proteins. Finally we confirmed the O-GlcNAc modification of the ubiquitin ligase NEDD4-1, the first reported glycosylation of this protein.

BACKGROUND

Increased serine elastase activity has been implicated in the vascular remodeling associated with chronic hypoxia-related pulmonary hypertension in rats.

RESULTS

In this study we determined the time course of hypoxia-induced serine elastase activity in the murine lung and related this to initiation of a proteolytic cascade characterized by an increase in matrix metalloproteinases (MMPs). We then used transgenic mice in which overexpression of the selective serine elastase inhibitor elafin was targeted to the cardiovascular system to determine whether upregulation of a naturally occurring serine elastase inhibitor suppresses MMPs and the hemodynamic and structural response to chronic hypoxia (air at 380 mm Hg). In nontransgenic but not in elafin-transgenic mice, we documented a transient increase in serine elastase activity after 12 hours of hypoxic exposure attributed to a 30-kDa protein as determined by elastin zymography and fluorophosphonate/fluorophosphate-biotin labeling. Two days after hypoxia, the pro-forms of MMP-2 and MMP-9 were induced in the nontransgenic mice, but MMP-9 was suppressed in elafin-transgenic mice. Acute hypoxic vasoconstriction was similar in nontransgenic and elafin-transgenic littermates. Chronic hypoxia for 26 days resulted in >1-fold increase in right ventricular pressure (P<0.004) in nontransgenic compared with control or elafin-transgenic littermates. In the latter mice, normalization of the right ventricular pressure was associated with reduced muscularization and preservation of the number of distal vessels (P<0.04 for both comparisons).

CONCLUSIONS

Modulation of the severity of chronic hypoxia-induced pulmonary vascular disease could be a function of endogenously expressed serine elastase inhibitors.

OBJECTIVE

To determine the incidence of DNA fragmentation in human sperm used for intracytoplasmic sperm injection (ICSI) and to correlate any detected DNA damage with semen analysis parameters and fertilization rates in ICSI.

METHODS

Descriptive and correlational clinical study.

METHODS

Tertiary care fertility clinic.

METHODS

A total of 150 semen samples was collected from men in the ICSI program.

METHODS

For each sample, sperm wash and swim-up were performed, and the percentage of recovered sperm with DNA fragmentation was determined with the use of terminal transferase-mediated deoxyuridine triphosphate-biotin end labeling.

METHODS

The percentage of sperm with DNA fragmentation was correlated with semen analysis parameters and ICSI fertilization rates.

RESULTS

The mean (+/- SD) percentage of sperm with fragmented DNA was 14.5% +/- 1.5% and ranged from 0.5% to 75%. A significant negative association was found between the percentage of sperm with DNA fragmentation and the ICSI fertilization rate. We also observed that the motility and morphology of the ejaculated sperm were correlated negatively with the percentage of DNA fragmentation in the washed sperm recovered by the swim-up technique.

CONCLUSIONS

Our results suggest that when poor-quality semen samples are used for ICSI, there is a greater likelihood that some sperm selected for injection, despite appearing normal, contain fragmented DNA. Whether sperm DNA damage may contribute to failure of pronuclear formation and embryo development in some apparently unfertilized ICSI oocytes is unclear.

Monocarboxylate transporter (MCT1) levels in brains of adult Long-Evans rats on a high-fat (ketogenic) diet were investigated using light and electron microscopic immunocytochemical methods. Rats given the ketogenic diet (91% fat and 9% protein) for up to 6 weeks had increased levels of the monocarboxylate transporter MCT1 (and of the glucose transporter GLUT1) in brain endothelial cells and neuropil compared to rats on a standard diet. In ketonemic rats, electron microscopic immunogold methods revealed an 8-fold greater MCT1 labeling in the brain endothelial cells at 4 weeks. Abluminal endothelial membranes were twice as heavily labeled as luminal membranes. In controls, luminal and abluminal labeling was not significantly different. The endothelial cytoplasmic compartment was sparsely labeled (<8% of total endothelial labeling) in all brains. Neuropil MCT1 staining was more intense throughout the brain in ketonemic rats, especially in neuropil of the molecular layer of the cerebellum, as revealed by avidin-biotin immunocytochemistry. This study demonstrates that adult rats retain the capacity to upregulate brain MCT1 levels. Furthermore, their brains react to a diet that increases monocarboxylate levels in the blood by enhancing their capability to take up both monocarboxylates (MCT1 upregulation) and glucose (GLUT1 upregulation). This may have important implications for delivery of fuel to the brain under stressful and pathological conditions, such as epilepsy and GLUT1 deficiency syndrome.

We report here both the range and patterns of reactivity of an IgG1 monoclonal antibody, B72.3, prepared against human, metastatic mammary carcinoma cells. When the avidin-biotin complex (ABC) immunoperoxidase technique was used on tissue sections, monoclonal B72.3 reacted with 19 of 41 (46%) primary mammary carcinomas and 13 of 21 (62%) metastatic lesions, either in axillary lymph nodes or at distal sites. Variable concentrations of antigen, recognized by B72.3, were observed among mammary tumors, as well as among different cell populations of a given tumor mass. Several patterns of antigen distribution were observed: membrane, diffuse cytoplasmic, focal and marginal. No reactivity was observed to normal mammary epithelium, stroma, or lymphocytes of the breast, nor to any cell types in a variety of other normal human tissues, melanomas, and sarcomas. Reactivity with all of four colon carcinomas was also observed. Assay of serial sections of mammary carcinomas with B72.3 and a monoclonal antibody directed against carcinoembryonic antigen demonstrated that these antigens were both distinct and non-coordinately expressed.

The pattern of peptidoglycan (murein) segregation in cells of Escherichia coli with impaired activity of the morphogenetic proteins penicillin-binding protein 2 and RodA has been investigated by the D-cysteine-biotin immunolabeling technique (M. A. de Pedro, J. C. Quintela, J.-V. Höltje, and H. Schwarz, J. Bacteriol. 179:2823-2834, 1997). Inactivation of these proteins either by amdinocillin treatment or by mutations in the corresponding genes, pbpA and rodA, respectively, leads to the generation of round, osmotically stable cells. In normal rod-shaped cells, new murein precursors are incorporated all over the lateral wall in a diffuse manner, being mixed up homogeneously with preexisting material, except during septation, when strictly localized murein synthesis occurs. In contrast, in rounded cells, incorporation of new precursors is apparently a zonal process, localized at positions at which division had previously taken place. Consequently, there is no mixing of new and old murein. Old murein is preserved for long periods of time in large, well-defined areas. We propose that the observed patterns are the result of a failure to switch off septal murein synthesis at the end of septation events. Furthermore, the segregation results confirm that round cells of rodA mutants do divide in alternate, perpendicular planes as previously proposed (K. J. Begg and W. D. Donachie, J. Bacteriol. 180:2564-2567, 1998).

Three classes of HSV-1(F) mutants expressing a resistance phenotype to two highly potent-type common monoclonal antibodies, H126-5 and H233, to glycoprotein B (gB) were selected. Class 1 mutants, selected for resistance to neutralization from nonmutagenized virus stocks, expressed a gB which reacted in biotin-avidin-enhanced surface immunoassays and in immune precipitation tests with the selecting antibodies. Class 2 and 3 mutants were selected for nonreactivity in the biotin-avidin-enhanced surface immunoassay from BUdR-mutagenized, preneutralized virus stocks, but differ in that the selecting antibodies immune precipitated the gB of Class 2 but not that of Class 3. Mutants expressing a resistance phenotype to one monoclonal antibody (H126-5 or H233) invariably retained reactivity in all tests with the heterologous antibody, and recombinants resistant to both antibodies were produced by cotransfection of intact DNA of one mutant with a cloned DNA fragment from another mutant. Class 1 mutations were mapped by marker transfer to a 1734-bp DNA fragment. Class 2 and 3 mutations were mapped to a region defined by a maximum of 377 bp and a minimum of 46 bp, in a biotin-avidin-enhanced surface immunoassay with a panel of DNA fragments of HSV-1(F) BamHI G carrying staggered deletions across the region encoding gB. This region does not overlap the neutralizing antibody determinant site mapped by T.C. Holland, R.M. Sandri-Goldin, L.E. Holland, S.D. Marlin, M. Levine, and J. Glorioso (1983, J. Virol. 46, 649-652) and is located 3' to the ts lesion of HSV-1(HFEM)tsB5 and 5' to the syn3 locus of that virus. It was concluded that (i) inasmuch as the biotin-avidin-enhanced surface immunoassay does not destroy the virus contained in the plaque, it is a rapid and convenient method for both identification and selection of mutants reactive and nonreactive to specific monoclonal antibodies. (ii) gB may contain multiple domains carrying epitopic sites of neutralizing monoclonal antibodies. (iii) The resistance phenotype may arise from mutations which alter the conformation or the amino acid sequence of the epitope. These mutations might be differentiable on the basis of reactivity of mutated gB with selecting monoclonal antibody in nondenaturing and denaturing environments, respectively.